· 01-01-2017 · Acanthamoeba spp. yang diuji diperolehi daripada Makmal Acanthamoeba, Fakulti...

Jilid Volume 1 Bilangan

Number 1 ISSN 2250-1852 Disember December 2017

Aktiviti Anti-Acanthamoeba Mukus Epidermis Clarias batrachus

terhadap Sista Acanthamoeba Pencilan Klinikal Mohamed Kamel Abd Ghani, Asfarrieza Arsad, Ahmad Zorin Sahalan, Anisah Nordin, Yusof Suboh, & Noraina Ab Rahim

Cytotoxicity and Oxidative Profile of 1,4 Benzoquinone-Exposed Chinese Hamster Lung Fibroblasts V79 Cells Lina Adnan Fadhel Al-Ani, Zariyantey Abd Hamid, Elda Surhaida Latif & Ramya Dewi A/P Mathialagan

Incidence of Fatal Road Traffic Accidents Involving Motor Vehicles During Festive Seasons in Klang Valley 2010-2015 Muhamad Hafizan Harun, Sri Pawita Albakri Amir Hamzah & Noor Hazfalinda Hamzah

Nilai sub perencatan Polymyxin B (PMB) dan kesan morfologi permukaan Pseudomonas aeruginosa sub perencat PMB dengan kaedah TEM Ahmad Zorin Sahalan, Noorsana Hosni & Hing Hiang Lian

Kajian Kes Kawad Kebakaran: Jangka Masa Bergerak di Ambil Bagi Pengusian Bangunan Dalam Kalangan Warga UKM Kampus Kuala Lumpur Anuar Ithnin

The Chromagen Lens: Its Effects on Colour Vision Performance in Congenital Colour Vision Deficients. Koo Sio Ching, Nurul Farhana Sharifudin, Mizhanim Mohamad Shahimin & Sharanjeet Kaur

Perencatan Bakteria Escherichia coli oleh Mukus Epidermis Clarias batrachus Ahmad Zorin Sahalan & Nazahiyah Sulaiman

Evaluation of Hulu Langat River Water Qualities Using Heavy Metals and Microbial Indicators Nur Sakinah I., Hukil S, Kaswandi, M.A., Sahalan, A.Z. & H.L. Hing

1 – 7

8 – 14

15 – 27

28 – 33

34 – 44

45 – 54

55 – 59

60 – 66

SIDANG EDITOR/ EDITORIAL BOARD

Ketua Editor/ Editor-in-Chief

MOHAMED KAMEL ABD GHANI

Pembantu Ketua Editor / Assistant Editor-in-Chief

AHMAD ZORIN SAHALAN

Setiausaha Editorial / Editorial Secretary

RAHAIDA RAMLI

Editor / Editors

Kesihatan Persekitaran dan Sains Forensik/

Environmental Health and Forensics

Science

HING HIANG LIAN

HUKIL SINO

MUHAMMAD IKRAM ABD WAHAB

Ditetik dan Nutrisi/ Dietetic and Nutrition

HASLINA ABDUL HAMID

WONG JYH ELIN

Penjagaan Kesihatan/ Healthcare

BADRULZAMAN ABD HAMID

BASHIRA ISHAK

NOH AMIT

NOOR ALAUDIN ABD WAHAB

Sains Perubatan dan Diagnostik/ Medical

Science and Diagnostic

MAZLYZAM ABDUL LATIF

NURUL FARHANA JUFRI

Rehabilitasi/ Rehabilitation

NOORAFIFI RAZAOB@RAZAB

NOOR NAJWATUL AKMAL ABD

RAHMAN

Jawatankuasa Editorial Teknikal /

Technical Editorial board

AMALINA SYAZANA ADNAN

ATIAH AYUNNI ABD GHANI

AZEEDA SHAMSUDIN

MAIMUNAH JAIS

MARISAZAM SALLEH

MAZLIN AMAN

NOR AYUSLIWATI CHE SIDIK

RAHAIDA RAMLI

ROGAYAH ABU HASSAN@MOHAMAD

Hak Cipta Universiti Kebangsaan Malaysia, 2017

Copyright Universiti Kebangsaan Malaysia, 2017

Lembaga Penasihat Antarabangsa /

International Advisors Board

KHAIRUL OSMAN, UNIVERSITI

KEBANGSAAN MALAYSIA,

MALAYSIA

SUKUMAL CHONGTHAMMAKUN,

MAHIDOL UNIVERSITY, THAILAND

BAY BOON HUAT, NATIONAL

UNIVERSITY OF SINGAPORE,

SINGAPORE

Buletin FSK 1(1)(2017): 1-7

Aktiviti Anti-Acanthamoeba Mukus Epidermis Clarias batrachus

terhadap Sista Acanthamoeba Pencilan Klinikal (Anti-Acanthamoeba Activity of Clarias batrachus Epidermal Mucus against the

Clinical Isolate Cysts of Acanthamoeba)

MOHAMED KAMEL ABD GHANI* ASFARRIEZA ARSAD, AHMAD ZORIN

SAHALAN, ANISAH NORDIN, YUSOF SUBOH, & NORAINA AB RAHIM

ABSTRAK

Insiden keratitis Acanthamoeba yang berkaitan penggunaan kanta sentuh semakin

meningkat akibat amalan penjagaan alatan kanta sentuh yang kurang higenik. Kerintangan

Acanthamoeba terhadap terapi antimikrob semakin berleluasa. Banyak bahan semulajadi

khasnya daripada protein haiwan telah diuji keberkesanannya dalam mengawal serta

membasmi jangkitan. Mukus epidermis ikan merupakan sumber protein yang dikenalpasti

mempunyai peptida antimikrob. Kajian ini dilakukan untuk menentukan aktiviti anti-

Acanthamoeba mukus epidermis Clarias batrachus terhadap sista 4 isolat klinikal

Acanthamoeba, iaitu (HKL 102, HS 5, HTH 73 dan HUKM 38). Mukus C. batrachus

berkepekatan 20% dan 100% ditindakkan terhadap sista Acanthamoeba. Setiap campuran

dipindahkan ke agar bukan nutrien yang dilapisi Escherichia coli. Plat agar diinkubasi

selama 14 hari pada 30oC dan diperhatikan setiap hari sehingga hari ke-14 untuk mengesan

kehadiran trofozoit Acanthamoeba di bawah mikroskop. Kehadiran trofozoit menunjukkan

mukus C. batrachus tidak mempunyai aktiviti anti-Acanthamoeba. Hasil kajian

menunjukkan mukus C. batrachus yang diuji ke atas semua isolat klinikal adalah tidak

berkesan sebagai agen anti-Acanthamoeba.

Kata kunci: Mukus epidermis Clarias batrachus; anti Acanthamoeba; Malaysia

ABSTRACT

Incidence of Acanthamoeba keratitis related to contact lens usage is increasing due to the

lack of hygienic care in handling contact lenses. There is a widespread resistance of

Acanthamoeba towards antimicrobial therapy. Many natural substances, especially animal

proteins, have been tested on their effectiveness in controlling and eradicating infection.

The epidermal mucus of fish is a good source of protein and has been identified to have

antimicrobial peptides. This study was conducted to determine the effectiveness of Clarias

batrachus epidermal mucus as anti-Acanthamoeba on 4 clinical isolates of Acanthamoeba

cysts, (HKL 102, HS 5, HTH 73 and HUKM 38). C. batrachus mucus with the

concentrations of 20% and 100% was tested against the Acanthamoeba cysts. Each mixture

was transferred onto non-nutrient agar laid with Escherichia coli. The agar plates were

incubated for 14 days at 30oC and monitored daily until day 14 to detect the presence of

Acanthamoeba trophozoites under the microscope. The presence of trophozoites, indicates

the ineffectiveness of C. batrachus mucus. The result showed that C. batrachus epidermal

mucus tested on all clinical isolates was ineffective as an anti-Acanthamoeba agent.

Key words: Clarias batrachus epidermal mucus; anti Acanthamoeba; Malaysia

2

PENGENALAN

Acanthamoeba spp. merupakan protozoa

hidup bebas yang wujud dalam bentuk

trofozoit yang aktif atau sista yang dorman.

Ia menyebabkan jangkitan pada mata yang

boleh menyebabkan kebutaan dan

jangkitan ini dikenali sebagai keratitis

Acanthamoeba.

Kebanyakkan kes keratitis

Acanthamoeba yang dilaporkan berkait

dengan pemakaian kanta sentuh (Stapleton

et al. 2009). Di Malaysia, kes keratitis

Acanthamoeba yang pertama dilaporkan

berlaku pada seorang pesakit wanita yang

telah memakai kanta sentuh selama 15

tahun (Kamel & Norazah, 1995) dan lebih

banyak lagi kes yang melibatkan pengguna

kanta sentuh dilapurkan kemudian.

Keratitis Acanthamoeba jarang berlaku

tetapi jika berlaku, rawatannya adalah

sangat sukar kerana Acanthamoeba

semakin rintang terhadap terapi antimikrob

(Moore et al. 1987).

Kini terdapatnya usaha untuk

mengawal jangkitan keratitis

Acanthamobea. Banyak bahan semulajadi

khasnya daripada protein haiwan telah

diuji keberkesanannya dalam mengawal

serta membasmi jangkitan. Protein haiwan

yang ingin diketengahkan ialah daripada

ikan keli kayu atau nama saintifiknya

Clarias batrachus. Mukus daripada

epidermis ikan keli diuji akan

keberkesanannya dalam merencat

pertumbuhan dan perkembangan

Acanthamoeba spp.

C. batrachus merupakan jenis

ikan keli yang tidak mempunyai sisik dan

bergantung sepenuhnya kepada mukus

epidermis sebagai rintangan mekanikal

pertama terhadap patogen akuatik (Su

2011). Rintangan mekanikal adalah mukus

epidermis ikan yang mempunyai

komponen sistem imun seperti lgM dan

lisozim dan mempunyai peptida

antibakteria yang dapat menghalang

kolonisasi parasit akuatik, bakteria dan

fungus (Ebran et al. 2000). Lazimnya

mukus epidermis ikan mengandungi bahan

bioaktif yang merupakan molekul protein

dan terdiri daripada peptida antimikrob

(Zassloff 2002). Peptida antimikrob

merupakan salah satu komponen imun

semulajadi yang bertindak sebagai sistem

pertahanan utama bagi kebanyakkan

organisma hidup. Peptida antimikrob

adalah agen yang berupaya memusnahkan

kedua-dua bakteria gram positif dan

negatif, fungus, virus dan parasit dengan

sedikit atau tanpa sebarang kesan toksik

terhadap sel perumah (Hancock & Scott

2000). Komponen antimikrob semulajadi

ikan dilaporkan berkesan sebagai agen

anti-parasit pada kajian yang telah

dijalankan oleh Vizioli & Salzet (2002).

Penemuan peptida antimikrob boleh

dianggap sebagai salah satu penyelesaian

masalah kerintangan mikroorganisma

terhadap antibiotik yang semakin

berleluasa (Park et al. 1997). Spektrum

aktiviti antimikrob yang terdapat di dalam

mukus epidermis ikan boleh digunakan

untuk merawat jangkitan parasit.

BAHAN DAN KAEDAH

Sebanyak empat isolat klinikal (HKL 102,

HS 5, HTH 73 dan HUKM 38)

Acanthamoeba spp. yang diuji diperolehi

daripada Makmal Acanthamoeba, Fakulti

Perubatan, Universiti Kebangsaan

Malaysia. Mukus ikan keli kayu

dikumpulkan dan ditapis dengan penapis

membran 0.45 μm, manakala 10 ml mukus

yang lain tidak ditapis dengan penapis

membran. Seratus peratus daripada mukus

ini dilakukan pencairan dengan air suling

steril sehingga membentuk 20% pencairan

yang digunakan dalam kajian ini. Suspensi

3

sista Acanthamoeba divorteks selama satu

minit supaya sista bertaburan sama rata di

dalam salin PAGE. Sebanyak 10 μl

suspensi sista dipipet masuk ke dalam

telaga mikrotiter yang telah mengandungi

100 μl ekstrak mukus. Sista dimasukkan ke

dalam semua telaga kecuali telaga untuk

kawasan negatif. Terdapat dua kawalan

positif dan dua kawalan negatif. Kawalan

positif yang pertama adalah terdiri

daripada larutan PAS dan sista, manakala

kawalan positif yang kedua terdiri daripada

sista dan larutan 3% hidrogen peroksida.

Kawalan negatif yang pertama hanya

mengandungi larutan PAS dan kawalan

negatif kedua hanya mengandungi larutan

ekstrak mukus. Campuran dibiarkan

selama 24 jam. Selepas dieram selama 24

jam di dalam inkubator bersuhu 30oC,

campuran di setiap telaga dipipet masuk ke

dalam vial 1.5 ml yang berlainan.

Mendapan yang mengandungi sista di

bawah vial akan dipindahkan terus ke atas

agar bukan nutrien yang telah dititiskan

dengan suspensi E. coli matian haba pada

hari sebelumnya. Setelah itu, semua piring

petri ditutup dan dieramkan pada suhu

30oC selama 48 jam. Agar diperhatikan

setiap hari selama 14 hari di bawah

mikroskop songsang untuk mengesan

kehadiran trofozoit Acanthamoeba yang

muncul daripada peringkat sista. Jika tiada

pertumbuhan trofozoit Acanthamoeba

berlaku dalam masa 14 hari, maka larutan

ekstrak mukus yang digunakan adalah

berkesan dalam membunuh sista

Acanthamoeba.

KEPUTUSAN

Hasil Ujian Penentuan Aktiviti Anti-

Acanthamoeba Mukus Epidermis

Clarias Batrachus

Kehadiran trofozoit diperhatikan selama

14 hari, selepas 48 jam tempoh inkubasi.

Keputusan positif dicatatkan jika trofozoit

tidak hadir selepas 14 hari. Dalam kajian

ini, Jadual 1., menunjukkan hasil ujian

keberkesanan aktiviti anti-Acanthamoeba

mukus epidermis C. batrachus terhadap

sista Acanthamoeba yang terdiri daripada

empat isolat klinikal. Mukus epidermis C.

batrachus yang dihasilkan diuji secara

langsung tanpa siri pencairan. Mukus

epidermis C. batrachus yang diuji

terbahagi kepada dua, iaitu mukus yang

ditapis dengan menggunakan penapis

membran 0.2 µm dan mukus yang tidak

melalui ujian penapisan. Berdasarkan

jadual tersebut, didapati mukus epidermis

C. batrachus tidak berkesan sebagai agen

anti-Acanthamoeba ke atas keempat-empat

isolat Acanthamoeba di dalam kajian ini.

Kawalan Positif dan Negatif Ujian

Penentuan Aktiviti Anti-Acanthamoeba

Mukus Epidermis C. Batrachus

Dalam kajian ini, terdapat dua kawalan

positif dan kawalan negatif. Jadual 2.,

menunjukkan keputusan ujian kawalan

positif dan negatif untuk menguji

keberkesanan aktiviti anti-Acantamoeba

mukus epidermis C. batrachus. Kedua-dua

kawalan positif dan negatif memberikan

keputusan seperti yang dijangkakan.

Penentuan Kumpulan Isolat Sista

Acanthamoeba spp

Penentuan kumpulan isolat Acanthamoeba

dilakukan secara mikroskopi. Jadual 3

menunjukkan kumpulan isolat

Acanthamoeba di mana kesemuanya terdiri

daripada kumpulan II iaitu polyphagids.

4

JADUAL 1. Keputusan ujian keberkesanan aktiviti anti-Acanthamoeba mukus epidermis

C. batrachus terhadap sista Acanthamoeba isolat klinikal

Isolat 20% mukus

(ditapis

menggunakan

penapis

membran)

100% mukus

(ditapis

menggunakan

penapis

membran)

20% mukus

(tidak ditapis

menggunakan

penapis

membran)

100% mukus

(tidak ditapis

menggunakan

penapis

membran)

Isolat

Klinikal

HKL 102

HS 5

HTH 73

HUKM 38

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

Petunjuk: X - Tidak berkesan (Trofozoit hadir)

JADUAL 2. Keputusan kawalan ujian penentuan aktiviti anti-Acanthamoeba mukus

epidermis C. batrachus

Strain Kawalan

Positif Negatif

Suspensi

sista

% H202 Larutan PAS Mukus

C. batrachus

HKL 102 + - X X

HS 5 + - X X

HTH 73 + - X X

HUKM 38 + - X X

Petunjuk: + Kehadiran sista dan trofozoit Acanthamoeba

- Tiada trofozoit Acanthamoeba

X Tiada kehadiran sista dan trofozoit Acanthamoeba

H202 Hidrogen Peroksida

PAS Page Amebic Saline

JADUAL 3. Pengkelasan kumpulan Acanthamoeba yang dipencilkan daripada isolat

klinikal.

Isolat Kumpulan Sista

HKL 102 Kumpulan II (Polyphagids)

HS 5 Kumpulan II (Polyphagids)

HTH 73 Kumpulan II (Polyphagids)

HUKM 38 Kumpulan II (Polyphagids)

5

PERBINCANGAN

Kajian mengenai potensi mukus epidermis

Clarias batrachus sebagai sumber

antimikrob ke atas Acanthamoeba

dijalankan untuk menambahbaik rawatan

yang sedia ada. Diketahui daripada lapuran

terdapatnya keupayaan kerintangan

Acanthamoeba terhadap kemoterapi

antimikob yang semakin meningkat (Lloyd

et al. 2001).

Berdasarkan analisis morfologi

sista, genus Acanthamoeba dalam kajian

ini tergolong dalam kumpulan dua iaitu

kumpulan polyphagids. Isolat sista dari

kumpulan ini bersaiz kurang daripada 18

µm. Kumpulan polyphagids mempunyai

ektosista dan endosista yang berkedut,

berbentuk bintang, poligon, segitiga atau

bujur (Pens et al. 2008) dan selalunya

diketahui bersifat patogenik.

Merujuk kepada hasil ujian

keberkesanan mukus epidermis Clarias

batrachus terhadap sista Acanthamoeba

isolat klinikal (Jadual 1.), didapati bahawa

mukus epidermis C. batrachus tidak

berkesan sebagai agen anti Acanthamoeba.

Ini adalah kerana terdapatnya pertumbuhan

trofozoit setelah sista dirawat atau

didedahkan kepada mukus C. batrachus.

Menurut Whyte (2007), komponen protein

pada mukus ikan seperti glikoprotein,

lisozim dan protease adalah efektif pada

suhu 15-25 0C dan boleh bertahan sehingga

suhu 0-4oC. Suhu optimum bagi

pertumbuhan Acanthamoeba adalah pada

30oC (Schuster & Visvesvara 2004).

Komponen protein yang bertindak sebagai

antimikrob mungkin telah termusnah

semasa proses inkubasi Acanthamoeba

pada suhu 30oC.

Selain itu, mukus C. batrachus

ditindakkan secara langsung ke atas sista

Acanthamoeba melalui penapisan

membran 0.2 µm dan tanpa penapisan

membran 0.2 µm. Tujuan penapisan mukus

menggunakan membran 0.2 µm adalah

untuk menghalang partikel seperti alga,

Giardia lamblia, dan sista

crypotosporidium dan bakteria lain yang

boleh menyebabkan kontaminasi ketika

ujian dijalankan. Namun, tidak semua

protein dapat melepasi penapis membran

0.2 µm kerana struktur protein pada mukus

boleh berubah dan membentuk kelompok.

Maka mukus yang tidak ditapis digunakan

secara langsung sebagai agen anti-

Acanthamoeba.

Oleh kerana komponen protein

aktif yang dihasilkan adalah sedikit, maka

mukus yang diambil dari ikan telah

ditindakkan secara langsung ke atas sista

Acanthamoeba tanpa dilakukan sebarang

pengekstrakkan protein. Mukus yang

diperolehi dari epidermis Clarias

batrachus ditindakkan secara langsung

seratus peratus dan daripada mukus ini

dilakukan pencairan dengan air suling

sehingga membentuk 20% pencairan.

Perbandingan dibuat untuk melihat kesan

antimikrob pada mukus yang pekat dan

mukus yang telah dicairkan. Menurut

Grinde dan rakan-rakan (1988),

glikoprotein, lisozim dan protease

merupakan komponen yang boleh larut di

dalam air. Selain daripada glikoprotein

sebagai komponen utama mukus, peptida

antimikrob seperti pelteobagrin juga boleh

didapati (Su 2011).

Walaupun hasil kajian kurang

memberangsangkan terhadap ekstrak

mukus C. batrachus, namun masih banyak

lagi ujian terperinci yang perlu dilakukan

kerana menurut Su (2011), mukus

epidermis ikan telah dibuktikan

mempunyai aktiviti antibakteria,

berpotensi sebagai sumber peptida

antimikrob. Di dalam kajian Hancock &

Scott (2000), peptida antimikrob kation

mempunyai spektrum aktiviti luas yang

berkesan ke atas bakteria gram positif dan

negaif, kulat, virus dan parasit. Ia juga

6

dapat membunuh atau meneutralkan

parasit termasuk planaria dan nematod.

Acanthamoeba dikatakan semakin rintang

terhadap antimikrob. Berdasarkan kajian

yang dilakukan, sumber Clarias batrachus

tidak dapat membunuh sista

Acanthamoeba sepenuhnya. Menurut

Walker (1996), banyak spesies

Acanthamoeba telah dilaporkan rintang

terhadap antimikrob, perubahan suhu dan

kekeringan.

Di Malaysia, insiden keratitis

Acanthamoeba dilaporkan semakin

meningkat. Penemuan empat (9.1%)

daripada 44 kes keratitis yang dilaporkan

adalah positif jangkitan Acanthamoeba

(Kamel et al. 2005).

Keratitis Acanthamoeba

merupakan jangkitan okular yang sukar

dirawat kerana Acanthamoeba peringkat

sista rintang terhadap agen-agen

antimikrob pada kepekatan yang boleh

diterima oleh kornea (Kilvington et al.

2002).

Pencarian agen atau kompaun baru

yang berkesan sebagai agen anti-

Acanthamoeba perlu diteruskan dalam

usaha untuk merawat penyakit ini.

KESIMPULAN

Hasil kajian menunjukkan bahawa mukus

epidermis Clarias batrachus didapati tidak

berkesan sebagai agen anti-Acanthamoeba

terhadap kesemua isolat klinikal

Acanthamoeba yang diuji.

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invertebrates. Trends Pharmacology

Sciences. 23(11): 494-496.

Walker, C.W.B. 1996. Acanthamoeba:

ecology, pathogenicity and laboratory

detection. British Journal of

Biomedical Sciences. 53: 146-151.

Whyte, S.K. 2007. The innate immune

response of finfish – A review of

current knowledge. Fish and Shellfish

Immunology. 23(6): 1127-1151.

Zassloff, M. 2002. Antimicrobial peptides of

multicellular organisms. Nature. 415:

389-395.

Mohamed Kamel Abd. Ghani*

Asfarrieza Arsad

Ahmad Zorin Sahalan

Biomedical Science Programme,

School of Diagnostic & Applied Health Sciences,

Faculty of Health Sciences,

Universiti Kebangsaan Malaysia,

Jalan Raja Muda Abdul Aziz,

50300 Kuala Lumpur,

Malaysia.

Anisah Nordin,

Yusof Suboh,

Noraina Ab Rahim

Department of Medical Parasitology

Faculty of Medicine,

Universiti Kebangsaan Malaysia

50300 Jalan Raja Muda Abdul Aziz

Kuala Lumpur,

Malaysia

*Corresponding author; email: [email protected]

Buletin FSK 1(1)(2017): 8-14

Cytotoxicity and Oxidative Profile of 1,4 Benzoquinone-Exposed Chinese

Hamster Lung Fibroblasts V79 Cells

LINA ADNAN FADHEL AL-ANI, ZARIYANTEY ABD HAMID*, ELDA SURHAIDA

LATIF & RAMYA DEWI A/P MATHIALAGAN

ABSTRACT

1,4 Benzoquinone is a highly toxic, and mutagenic metabolite of benzene. The effects of 1,4

Benzoquinone (1,4 BQ) on Chinese Hamster lung fibroblasts V79 cells was investigated for

cytotoxicity and oxidative profiles. Cell viability was determined using MTT assay following

exposure to 1,4 BQ at 12.5, 25, 50, 100, 200, and 400µM for 2, 14 and 24 hours. The Reactive

Oxygen Species (ROS) was assayed following 2 hours of 1,4 BQ exposure at 12.5, 25, 50, 100,

151µM. The oxidative profiles were studied using the Glutathione (GSH) and Superoxide

Dismutase (SOD) assays after exposure to 1,4 BQ at 2 and 24 hours using pre-determined IC25,

IC50 and IC75 for respective incubation period. Results showed that 1,4 BQ significantly reduced

the cell viability (p<0.05) as early as 2 hours following exposure to higher concentrations (100µM,

200µM, and 400 μM). It was notable that longer exposure to 1,4 BQ at 24 hours promotes

significant reduction of cell viability (p<0.05) initiated at 1,4 BQ concentrations as low as 12.5µM.

Meanwhile, oxidative profile showed that 1,4 BQ induced ROS level with significant elevations

noted at 50 and 100µM as compared to control. Moreover, exposure to 1,4 BQ suppressed the level

of antioxidants (GSH and SOD) with remarkable (p<0.05) reduction being noted following 24

hours’ exposure. In conclusion, 1,4 BQ exposure to Chinese Hamster lung fibroblasts V79 cells

promotes cytotoxicity which could be mediated by oxidative stress in which the effects were found

to be dependent on concentration and exposure time.

Keywords: Benzene; 1,4 Benzoquinone; V79 cells; Cytotoxicity; Oxidative Stress

INTRODUCTION

Benzene is an organic chemical naturally

found in the environment and widely used as

industrial solvent. To date, occupational and

environmental exposure to benzene remains a

major health concern (Davidson et.al 2001).

Benzene is proven to be toxic and carcinogenic

agent both in vivo and in vitro models.

Benzene metabolites were identified to be

responsible for all damaging effects produced

in the body after exposure to benzene (Snyder

et.al 1996). The metabolism mechanism is

complex, involving various enzymes and

various organs in the body. Three major sites

for benzene metabolism has been identified

with liver and/or lungs being the primary sites

while bone marrow as a secondary site. Such

discrepancies are explained by the nature of

enzymes found in each tissue that are needed

for benzene metabolisms to occur (Rapapport

et.al 2009; M.Mchale et.al 2012).

Benzene exposure has been widely

associated with haematological disorders as

the secondary site for its metabolism occurs in

bone marrow where a reactive and toxic

metabolite namely, 1,4-Benzoquinone (1,4

BQ) is produced (Yardely-Jones et.al 1991).

Meanwhile, it was reported that exposure to

benzene can also induce non-haematological

malignancies; such as lung cancer (Powley

et.al 2002). Although benzene toxicity is

9

widely studied, but efforts to understand the

toxicity effects and their underlying

mechanisms continues to evolve. Such

findings are important to ensure continuous

biomonitoring and enforcement activities

towards allowable exposure limit of benzene;

particularly in occupational setting.

Kohen et al, 2002 previously reported

the relationship between oxidative stress and

cytotoxicity however their involvement in 1,4

BQ-mediated toxicity still remain unclear.

Thus, the potential role of oxidative stress in

mediating 1,4 BQ-induced cytotoxicity was

further investigated in this study. To

demonstrate this objective, we conducted

cytotoxicity and oxidative profiles assessment

on Chinese Hamster lung fibroblasts V79

following their exposure to a benzene

metabolite, namely 1,4 BQ. Moreover, the role

of concentrations and time points in governing

the effects of 1,4 BQ exposure were also

determined in this study. MTT assay, Reactive

Oxygen Species (ROS) along with

antioxidants glutathione (GSH) and

Superoxide Dismutase (SOD) assays were

employed to address cytotoxicity and oxidative

profiles.

MATERIALS & METHODS

Cell Culture

V79 cell line was obtained from the American

Type Culture Collection ATCC, USA, and

maintained in growth media (DMEM)

supplemented with 10% fetal bovine serum,

1% Pen-Strep antibiotic, 3.7g/L of sodium

bicarbonate and 0.11g of sodium pyruvate.

Cells were grown at 37°C in a humidified

atmosphere of 5% CO2.

Assessment of Cell Viability

Briefly, V79 cells were seeded at 5x104

cells/ml (Peng et.al 2009) into the 96-well

plate, and exposed to 1,4 BQ at 12.5, 25, 50,

100, 200 and 400µM for 2, 14 and 24 hours.

Meanwhile untreated cells represent control

group. Following incubation at the respective

time-points, 20µl of 5mg/ml MTT solution

was added into each well; and the plate was

further incubated for additional 4 hours. Next,

150µl of the supernatant was removed from

each well followed by addition of 150µl

DMSO. Plate was incubated for 15 minutes

which were then measured at 570nm

absorbance using a microplate reader.

Assessment of Reactive Oxygen Species

(ROS) Level

Determination of ROS level in cells was

performed following method as described by

Abd Hamid et.al (2014). Cells were exposed to

1,4 BQ at 12.5, 25, 50, 100 and 151µM for 2,

14 and 24 hours. Meanwhile untreated cells

represent control group. Briefly, 5x104 cells/ml

were centrifuged and loaded with 1µl of

hydroethidine (HE) (10mM) for 30 minutes at

37 0C in 5 % CO2 incubator. ROS production

was quantified by measuring the intensity of

HE-fluorescence using flow cytometry. ROS

production levels of the cells were presented

by the fluorescence intensity and expressed as

percentages of ROS-producing cells.

Assessment of Glutathione (GSH) and

Superoxide Dismutase (SOD) Levels

Briefly, cells were exposed to 1,4 BQ for 2

hours at IC25 (45µM), IC50 (151µM) and IC75

(340µM) and for 24 hours at IC25 (10µM), IC50

(110µM) and IC75 (285µM) as pre-determined

by MTT assay. Meanwhile untreated cells

represent control group. Then, cell lysate was

prepared and the total protein was determined

using Bradford assay prior to antioxidants

determination. GSH level was measured using

modified method of Ellman (1959).

Quantification of GSH was achieved using

spectrophotometric assay, which involves

oxidation of GSH by the sulfhydryl reagent

10

5,5′-dithio-bis (2-nitrobenzoic acid) (DTNB)

to form the yellow derivative 5′-thio-2-

nitrobenzoic acid (TNB) that was measurable

at 412 nm. As for SOD, the level was

determined based on the inhibition of

nitroblue-tetrazolium (NBT) reduction based

on method as described previously (Beyer and

Fridovich, 1987). The reduction of NBT by

superoxide radicals to blue coloured formazan

was measured at 560 nm. One unit (1U) of

SOD activity is defined as that amount of SOD

required to inhibit the reduction of NBT by

50% under the specified conditions. SOD

activity was expressed as U/min/mg.

Statistical Analysis

Data were expressed as mean ± standard error

mean, and statistical significance of effect was

determined by analysis of variance (ANOVA),

and Pearson correlation test. The level of

statistical significance was set at p < 0.05.

RESULTS

Effects of 1,4 BQ on Cellular Viability

Overall, exposure to 1,4 BQ decreased cell

viability at every concentration and time-point

(Figure 1). A significant reduction in cell

viability (p<0.05) was evident as early as 2

hours following exposure to 1,4 BQ at higher

concentrations (100µM, 200µM and 400µM)

compared to control group. At 14 hours,

significant reduction in cell viability (p<0.05)

was noted at 200µM and 400µM compared to

control group. Meanwhile, it was also notable

that longer exposure to 1,4 BQ at 24 hours

promotes significant reduction of cell viability

(p<0.05) initiated at 1,4 BQ concentrations as

low as 12.5µM.

FIGURE 1. Effect of 1,4 BQ on V79 viability following exposure at various concentrations for 2,

14 and 24 hours. Significant difference (p < 0.05) as compared with control group at (a) 2 hours,

(b) at 14 hours, and (c) at 24 hours. Data is presented as mean ± S.E.M of triplicates.

11

FIGURE 2. Effect of 1,4 BQ on ROS levels following exposure at various concentrations for 2

hours. (a) indicates significant difference (p<0.05) compared to control group. Data is presented

as mean ± S.E.M of triplicates.

FIGURE 3: Effects of 1,4 BQ on GSH (A) and SOD (B) levels. Cell lysates were obtained from control

and treated groups at respective pre-determined concentrations according to exposure time-points as

follows: IC25:45, 10µM, IC50:151, 110µM, and IC75: 340, 285µM for 2 and 24 hours, respectively.

Significant difference (p < 0.05) as compared with control group at (a) 2 hours and (b) 24 hours. Data is

presented as mean ± S.E.M of triplicates.

12

Effect of 1,4 BQ on ROS Levels

Results showed that 1,4 BQ exposure promotes

greater ROS production in V79-treated cells

than control. Across the tested concentrations,

exposure to 1,4 BQ for 2 hours at 50 and

100µM induce significant (p<0.05) increment

in ROS production as compared to control

group (Figure 2).

Effect of 1,4 BQ on GSH and SOD Levels

Overall, 1,4 BQ exposure suppresses the

antioxidants level of V79-treated cells as

compared to control. It was noted that the

levels of GSH (Figure 3A) and SOD (Figure

3B) were significantly reduced (p<0.05)

following exposure to 1,4 BQ for 24 hours at

IC25 (10µM), IC50 (110µM) and IC75 (285µM).

Meanwhile, the levels of GSH (Figure 3A) and

SOD (Figure 3B) were significantly reduced

(p<0.05) following exposure to 1,4 BQ for 2

hours at IC75 (340µM) as compared to control

group.

DISCUSSION

In the present study, the cytotoxic effect of 1,4

BQ was investigated and the notable reduction

in cellular viability was observed at as early as

2 hours’ exposure. The effect was found to be

dependent on concentrations and time-points.

Shorter time-point exposure requires higher

concentrations of 1,4 BQ to exert cytotoxicity,

while exposure to longer time-points can

initiate cytotoxicity following exposure to

lower concentrations. This finding is in line

with previous study (Yang et.al 2010) which

reported the occurrence of cytotoxicity

following long-term 1,4 BQ exposure at low

doses.

1,4 benzoquinone toxicity can be

mediated through a number of mechanisms

including oxidative stress. Previous study had

pointed out that 1,4 BQ possessed the ability to

bind with macromolecules, such as DNA,

leading to DNA damage and indirectly

triggered cellular damage by generating free

radicals (Fiola et al. 2004, Winn 2003). Hence,

oxidative profile were conducted in this study

to evaluate potential mechanism of toxicity

throughout different time points of exposure.

ROS level assessment revealed that 1,4 BQ

induced ROS formation, significantly at high

concentrations of 50-100µM. This finding is in

line to previous researchers who confirmed

that 1,4 BQ compound is able to generate ROS,

specifically the superoxide anion (Kolachana

et.al 1993; Yang et.al 2010), which could

suggest the potential role of ROS in mediating

1,4 BQ cytotoxicity.

To further elucidate the role of

oxidative stress in the cytotoxicity of 1,4 BQ,

levels of antioxidants, namely GSH and SOD

were measured. GSH levels were found to be

reduced following exposure to 1,4 BQ at as

early as 2 hours. Meanwhile, longer incubation

time-point of 24 hours was employed and

significantly greater reduction of GSH was

noted at every tested concentration (100µM,

200µM and 400µM). This observation is in

agreement with a study done by Ludewig et al.

(1989), who concluded that 1-hour incubation

of 1,4 BQ was able to significantly reduce the

cellular GSH level at cytotoxic concentrations

(100µM, 200µM and 400µM). The decrease in

GSH can be explained in part by a reduction

reaction of 1,4 BQ with GSH, forming

oxidized Glutathione disulfide (GSSG)

(Ludewig et al. 1989). Moreover, the major

explanatory reason for low GSH levels

measured at this present study can be attributed

to conjugation of GSH with 1,4 BQ.

Superoxide dismutase enzyme is one of

the main antioxidants found in cells, thus

evaluation of its level in response to 1,4 BQ

exposure is addressed in this study. Results

have shown that, SOD level is reduced

following exposure to 1,4 BQ in a responsive

manner similar to GSH finding. This

observation suggests that exposure to 1,4 BQ

could promote oxidative stress as

13

demonstrated by suppressed level of

antioxidants and enhanced ROS production in

treated cells. The low level of antioxidants can

be attributed to the action of 1,4 BQ itself on

cells as this compound is identified to be

mutagenic at doses as low as ≤2.5µM

(Ludewig et.al 1989), and genotoxic against

various genes including oxidative stress-

related genes (Hirabayashi et al. 2004; Yoon et

al. 2003). Alteration in gene expression can

lead to low activity of SOD enzymes, leading

to impaired responses against oxidative stress

and subsequent cytotoxicity of treated cells. In

conclusion, 1,4 BQ induces oxidative stress

mediating cytotoxicity with the effects being

dependent upon its concentration and exposure

time-points.

ACKNOWLEDGEMENT

This work was supported by FRGS (UKM)

FRGS/1/2016/SKK13/UKM/03/1. We thank

the Program of Biomedical Science and

Bioserasi Laboratory for providing the

facilities throughout this research.

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Lina Adnan Fadhel Al-Ani

Zariyantey Abd Hamid*

Elda Surhaida Latif

Ramya Dewi a/p Mathialagan






50300 Kuala Lumpur,

Malaysia.


Buletin FSK 1(1)(2017): 15-27

Incidence of Fatal Road Traffic Accidents Involving Motor Vehicles During

Festive Seasons in Klang Valley 2010-2015

MUHAMAD HAFIZAN HARUN, SRI PAWITA ALBAKRI AMIR HAMZAH* & NOOR

HAZFALINDA HAMZAH

ABSTRACT

Road traffic injuries are one of the most common causes of death in Malaysia. Even though motor

vehicle accidents occur every day, the rate is higher during festive seasons in this multiracial and

multi-religious country. The aim of this study is to analyse the incidence of fatal road traffic

accidents in Klang Valley involving motor vehicles during festive seasons for the period of 6 years.

Four festive seasons (Eid al-Fitr, Eid al-Adha, Chinese New Year and Deepavali) in Malaysia

represent three main celebrations in this country. Demographic data of age, gender, race, and

religion, time of death and cause of death are collected retrospectively from the post mortem record

in four hospitals. There are 233 cases (200 males and 33 females) involving 42% Malays, 12%

Chinese, 28% Indians and 18% others. The trend in the rate of fatal road accidents shows a

decreasing value from 2011 until 2014 before it increased in 2015. Deepavali holiday has the

highest percentage of fatalities in 2011 It was also noted, head injury is the most common injury

that causes death, followed by multiple injuries and almost 50% of fatal road accidents happened

during the evening or at night. Therefore, this study suggests all road users to be extra careful while

driving their vehicles on the road, especially during festive seasons.

Keywords: fatal road traffic accidents; motor vehicle accident; festive seasons; Klang Valley

INTRODUCTION

Celebration of the festive seasons seems

incomplete without news on road accidents in

mass media, either involving death or serious

injury. Kareem (2003) noted that about

400,000 people in Asia have died on the roads

every year and more than 4,000,000 are

injured. In the most recent safety data by the

International Transport Forum (2015), there

were 6,674 road deaths in 2014 a decrease of

3.5% from 2013 according to the provisional

data provided by the Royal Malaysian Police

(RMP). These numbers are the biggest

reduction in the past ten years. Evidently,

based on the statistics by the Road Transport

Department of Malaysia (n.d), the total of

registered vehicles in Malaysia from 2010 to

2015 is 139,514 and the total number of

vehicles involved in road accidents has

increased steadily from 414,421

(2010)to489,606 (2015). Of all states in

Malaysia, Selangor has the highest number of

road accidents from 2010 until 2015. It also has

the biggest increase of total road accidents

which is 25,392 accidents throughout six years

as compared to other states in Malaysia,

according to statistics released by Ministry of

Transport Malaysia (2015).

Based on these statistics, motor vehicle

users ‘safety has become a top priority to the

relevant authorities. Since 1997, the Road

Safety Research Centre under the Faculty of

Engineering, Universiti Putra Malaysia has

been given authority to carry out research

about motorcycle safety (Rohayu et al. 2012).

Besides that, since 2001, the RMP has been

conducting a road safety operation known as

Ops Selamat (formerly known as Ops Sikap) to

ensure the safety of road users in Malaysia

16

during festive seasons such as Eid al-Fitr, Eid

al-Adha, Chinese New Year and Deepavali.

Even though multiple preventive actions have

been implemented, the casualties of road

accidents during festive seasons are still a

concern annually, either in urban or rural areas

and a variety of causes may have contributed

to this scenario.

In the present study, data regarding

fatal road traffic accidents (RTAs) involving

motor vehicles during festive seasons in Klang

Valley are collected from four different

government hospitals. The collected data

composed of demographic, types of injuries,

time of death and the cause of death. Thus, at

the end of this research, it is hoped that it will

help in identifying the demographic data and

cause of deaths of fatal road accidents during

festive seasons.

METHOD

The data collection was based on fatal RTAs

involving motor vehicles during festive

seasons, which was Eid al-Fitr, Eid al-Adha,

Chinese New Year and Deepavali, from three

days before and three days after the festive

seasons in Klang Valley for a six-year period

from 2010 to 2015.

The data was collected from four different

governmental hospitals which were Pusat

Perubatan Universiti Kebangsaan Malaysia,

Hospital Tengku Ampuan Rahimah, Hospital

Sungai Buloh and Hospital Serdang. The data

was analysed based on age, gender, race,

religion, time of death and cause of death. The

data covered areas in Klang Valley, including

Kuala Lumpur, as well as cities and towns

surrounding Kuala Lumpur, Selangor and

Putrajaya, Malaysia. Klang Valley was chosen

for its highest number of road accidents during

festive seasons (Nor & Abdullah 2014).

RESULTS

A total of 233 cases of RTAs was collected in

this study. Figure 1 shows gender distribution

among the RTA victims in the study. There

were 86% fatalities involving male road users

and only 14% are females.

Figure 1: Distribution of gender among the RTA victims

0

20

40

60

80

100

120

140

160

180

200

Male Female

200

33

NU

MB

ER

OF

FA

TA

LIT

IES

GENDER

17

Figure 2: Distribution of age groups among the RTA victims

Figure 2 shows the age distribution among the

RTA victims in the study. The age group

between 15-19 years with 47 fatalities (20%)

was the biggest contribution to the RTA.

Table 1: Distribution of race among the RTA

victims

Race Number of

fatalities

Percentage

(%)

Malay 97 42

Chinese 28 12

Indian 66 28

Others 42 18

Total 233 100

Table 1 shows the racial distribution among the

RTA victims. Malay is the biggest contributor

with 97 (42%) fatalities while only 28 (12%)

fatalities are Chinese.

Table 2: Distribution of religion among RTA

victims

Religion Number of

fatalities

Percentage

(%)

Islam 117 50

Buddhism 34 15

Hinduism 73 31

Christianity 9 4

Total 233 100

Table 2 shows the religion distribution among

the RTA victims. Almost half of the cases are

Muslim with 117 fatalities while only 34

(15%) fatalities are Buddhist. Table 3 shows

the injuries distribution among the RTA

victims. A total of 93 (40%) fatalities are due

to head injuries. Table 4 and Figure 3 show the

time of death among the RTA victims. Early

morning (22.3%), evening (24.6%) and night

(25%) have higher percentage of fatalities

while midnight has the lowest percentage of

fatalities (3.12%).

0

5

10

15

20

25

30

35

40

45

50

1 2

6

47

37

3234

21

710

129 9

1 2 20 1

NU

MB

ER O

F FA

TALI

TIES

AGE (YEARS)

18

Table 3: Distribution of injuries among the victims of RTA

Injury 2010 2011 2012 2013 2014 2015 Total

Head 26 18 17 14 7 11 93

Chest 2 11 3 3 1 1 21

Abdominal 2 3 2 3 1 - 11

Neck - - 1 - 2 - 3

Spinal - - - 1 - - 1

Pelvic - 1 - 1 - - 2

Cervical - 1 - - - - 1

Thoracic aorta - 1 - - - - 1

Internal bleeding - 1 - - - - 1

Multiple injuries 6 8 5 7 8 11 45

Crush injuries 1 - - - - - 1

Head and Abdominal 1 - - - - 1 2

Head and Neck 1 1 2 - 1 - 5

Head and Chest 2 4 4 1 3 5 19

Head and Spinal - - 1 1 - - 2

Chest and Abdominal 2 2 3 2 1 1 11

Chest and Spinal - 1 - 1 - - 2

Chest and Pelvic - - - - - 1 1

Chest and crush injuries - - 1 - - 1

Neck and Abdominal - 1 - - - 2 3

Neck and Chest - - - 1 - - 1

Thoracic and Abdominal - - - - - 1 1

Myocardial Infarction - - - 1 - - 1

Ischemic Heart Disease

(IHD)

- - - 1 1 - 2

Drowning secondary to RTA - - - 1 - - 1

Severe burn - - - - - 1 1

Total 43 53 39 38 25 35 233

Table 4: Distribution of time of death among the RTA victims

Time Number of

fatalities

Percentage

Early morning (0100 - 0659) 50 22.3

Morning (0700 - 1159) 35 15.6

Noon (1200 - 1359) 21 9.40

Evening (1400 - 1859) 55 24.6

Night (1900 - 2359) 56 25.0

Midnight (0000 - 0059) 7 3.12

Total 224 100

*Unidentified time of death in 9 cases

19

Figure 3: Distribution of time of death among the RTA victims according to hours

Figure 4: Distribution of time of death and gender among the RTA victims

Figure 4 shows the distribution of time of death

and gender among the RTA victims. Male has

the highest number of fatalities at nights with

50 (25.9%) fatalities while female has the

highest fatalities during the evenings with 14

(45.2%) fatalities. Figure 5 shows the time of

death and race distribution among the RTA

victims. Time of death with the highest

percentage of fatalities (26%) among Malays

was early morning and evening while among

Indians was in the early morning.

0

2

4

6

8

10

12

14

16

00

00

–0

05

9

01

00

–0

15

9

02

00

–0

25

9

03

00

–0

35

9

04

00

–0

45

9

05

00

–0

55

9

06

00

–0

65

9

07

00

–0

75

9

08

00

–0

85

9

09

00

–0

95

9

10

00

–1

05

9

11

00

–1

15

9

12

00

–1

25

9

13

00

–1

35

9

14

00

–1

45

9

15

00

–1

55

9

16

00

–1

65

9

17

00

–1

75

9

18

00

–1

85

9

19

00

–1

95

9

20

00

–2

05

9

21

00

–2

15

9

22

00

–2

25

9

23

00

–2

35

9

NU

MB

ER O

F FA

TALI

TIES

TIME (HOURS)

0

5

10

15

20

25

30

35

40

45

50

0100 - 0659(Early

Morning)

0700 - 1159(Morning)

1200 - 1359(Noon)

1400 - 1859(Evening)

1900 - 2359(Night)

0000 - 0059(Midnight)

48

29

18

41

50

7

26

3

14

6

0

NU

MB

ER O

F FA

TALI

TIES

TIME

Male Female

20

Figure 5: Distribution of the time of death and race among the RTA victims

*Unidentified time of death in head injury is five cases.

*Unidentified time of death in multiple injuries is one case.

Figure 6: Distribution of time of death, head injury and multiple injuries among the RTA

victims

Figure 6 shows the distribution of time of death

and head injury among the RTA victims. Head

injury is most likely to occur at nights (28.4%),

while multiple injuries mostly happen in the

evening and night.

0

5

10

15

20

25

0100 - 0659(Early

Morning)

0700 - 1159(Morning)

1200 - 1359(Noon)

1400 - 1859(Evening)

1900 - 2359(Night)

0000 - 0059(Midnight)

NU

MB

ER O

F FA

TALI

TIES

TIME

Malay Chinese Indian Others

0

5

10

15

20

25

0100 - 0659(Early

Morning)

0700 - 1159(Morning)

1200 - 1359(Noon)

1400 - 1859(Evening)

1900 - 2359(Night)

0000 - 0059(Midnight)

1716

8

21

25

1

8

54

13 13

0

NU

MB

ER O

F FA

TALI

TIES

TIME

Head injury Multiple injuries

21

Table 5: Distribution of the RTA victims

based on the total accidents per festive season

Festive

Seasons

Number of

fatalities

Percentage

(%)

Chinese New

Year

55 24

Eid al-Fitr 60 26

Eid al-Adha 50 21

Deepavali 68 29

Total 233 100

Table 5 shows the distribution of total

accidents per festive seasons among the RTA

victims. Deepavali has the highest number of

fatalities among the festive seasons (29%).

Figure 7 shows the distribution of festive

seasons and religion among the RTA victims.

Malay shows the highest percentage of

fatalities in all festive seasons except for

Deepavali. Indian has the highest fatal road

accidents during Deepavali (36.8%). Figure 8

shows the distribution among the RTA victims

according to the total accidents per year. There

are 53 (18%) fatalities occurring in 2011, and

it is the highest number of fatalities throughout

the six years. Meanwhile, in 2014, least

number of fatalities of 11% was recorded.

Figure 9 shows the distribution of total

accidents per year and festive seasons among

the RTA victims, Chinese New Year in 2010

has the highest number of fatalities with

29.1%, while Eid al-Adha has the lowest

number of fatalities in 2014 with 4 (8%)

fatalities.

Figure 7: Distribution of festive seasons and race among the RTA victims

0

5

10

15

20

25

30

Chinese New Year Eid al-Fitr Eid al-Adha Deepavali

NU

MB

ER O

F FA

TALI

TIES

FESTIVE SEASON

Malay Chinese Indian Others

22

Figure 8: Distribution of the total accidents per year among the RTA victims

Figure 9: Distribution of the total accidents per year and festive seasons among the RTA

victims

DISCUSSION

RTA ranks at 9th place as the leading causes of

death worldwide in 2004 and it is expected to

be at 5th place by 2030 (Lee at al. 2016; World

Health Organization 2008). World Health

Organization (WHO) also reported that RTA

will become the second leading cause of death

in developing countries by 2020 (Wong et al.

2009). Furthermore, over a million people

around the world had died due to RTAs. Over

50% of RTAs involving motorcyclists or two-

wheel vehicles dominating in developing

countries such as Vietnam, Hong Kong and

0

10

20

30

40

50

60

2010 2011 2012 2013 2014 2015

NU

MB

ER O

F FA

TALI

TIES

YEAR

0

2

4

6

8

10

12

14

16

18

2010 2011 2012 2013 2014 2015

NU

MB

ER O

F FA

TALI

TIES

YEAR

Chinese New Year Eid al-Fitr Eid al-Adha Deepavali

23

Malaysia. Meanwhile, fatal RTAs were

expected to be an issue in high-income

countries because of an expanded motorisation

level (Ziyad & Akter 2011).

In Malaysia, during festive seasons, the

number of vehicles on the roads can increase

quite significantly. For example, in the North-

South Expressway, the number of vehicles

could rise to 1.6 million users on peak days in

conjunction with Eid al-Adha celebration

while the normal capacity was approximately

1.2 million vehicles per day, as said by

Director General of Road Safety Department,

Datuk Arifin Che Mat (Utusan Online 2016).

Thus, the increasing number of vehicles on the

roads might also be associated with the

increasing rate of road accidents. However,

there was no published data regarding fatal

RTAs during festive seasons involving motor

vehicles in Klang Valley.

RTAs, can be defined as a collision of

one motor vehicle with another one and can

cause injury or fatality (Segen's Medical

Dictionary 2011). In this retrospective study,

the total number of fatalities due to RTAs

during festive seasons was 233 cases, 86%

were male and only 14% were female. Thus, it

shows that male is more likely to get involved

in fatal RTAs. According to Massie &

Campbell (1993), men were 1.5 times at the

risk of getting into a fatal RTAs per mile driven

as compared to women because male drivers

were more likely to drive at high speed, not

stopping when yellow lights appeared, made a

shorter gap in the traffic stream or turning left

before oncoming traffic, more aggressive in

driving, did not always wear restraints and tend

to drive under the influence of alcohol (Massie

& Campbell 1993; Finn and Bragg 1986;

Veevers 1982). Al-Balbissi (2003) had

observed higher male rates in traffic violations,

especially in disobeying stop signs, using the

wrong lane, ignoring the yield sign, neglecting

the traffic signs and incorrect overtaking.

Hence higher accident rates was in male

drivers compared to female drivers.

Ferguson (2003) stated that young

drivers were at higher risk than older drivers.

In the current study, it shows that age group of

15-19 years old was the biggest contributor to

fatal road accidents since they represent 20%

fatalities among the age groups. Meanwhile,

group with age above 65 years old had less

than 3% fatalities in road accidents as

compared to young people, by Cammisa et al.

(1999) and Ferguson (2003), pointed out that,

teenagers with their own vehicles are likely to

drive a very long way and have higher risk of

getting involved in more crashes, due to their

immature and aggressive behavior as they are

still in the learning process. According to

Eman et al. (2014) those aged between 20-24

years old are the age with the second highest

percent of having frequent accidents while

those between 16-24 years old is the major

traffic rules violator. This might indicate that

young people are not concerned of the danger

around them.

The statistics in the current study also

showed that Malays are the biggest contributor

to fatal RTAs (42%) during festive seasons

from 2010 to 2015. Moe (2008) also noted

similar results in which; Malays had the

highest number of severe RTAs injuries among

the documented 145 cases. This is probably

due to Malays constituting approximately half

of the population in Malaysia which is about

63% (Department of Statistics Malaysia 2010).

Similarly since more than half (about 61%) of

the population was Muslim in Malaysia

(Department of Statistics Malaysia 2010), this

might explain why Islam had the biggest

percentage (50%) of fatalities in RTAs.

The current study showed that fatal

RTAs occurred mostly in early mornings,

evenings and nights. This is worsened by the

festive moods and ambient as road users might

travel far on the road to visit their relatives

especially in the evening, to avoid traffic

congestion. Meanwhile, limited vision at

nights and driving after drinking might also

lead to serious traffic consequences. Figure 4

24

showed male had the highest number of

fatalities at night with 25.9%. When

comparing the sex differences to percentage of

fatalities at night, males showed 89% fatalities

as compared to females which were only 11%.

Hagen (1975) and Veevers (1982) had

mentioned that men might have over-

confidence regarding driving performance as

compared to women.

In addition, Malay had the most

number of fatalities during the early morning

and evening as compared to other races based

on Figure 5. Early morning, 0100 until 0659,

also showed the highest percentage of fatalities

among Indians. Kortteinen (2008) reported

that Indians are, especially those residing in

estates by far the heaviest drinkers in Kuala

Selangor, with an annual drinking levels of

absolute alcohol more than14 liters as

compared to Chinese which is only about 5

liters.

Kareem (2003) and Abdelfatah (2016)

noted that severe head injury is the main cause

of death in RTA, and this is in accordance to

the findings of this study where head injuries

were the commonest injury seen almost every

year (52%). Head injury was highest at night

while multiple injuries was most commonly

seen in the evening, as shown in Figure 6.

Chavali et al. (2006) stated that single-vehicle

crash may occur more frequently in young

adults (both males and females) that results in

head injury related deaths. Amir et al. (2013)

concluded that young males riding two-

wheeled motorized vehicles were more at risk

of getting head injury when involved in RTA.

Meanwhile, according to Kulanthayan et al.

(2000), nearly half of the total death rate

(49.2%) among motorcycle riders in Malaysia

is because of head injuries.

In contrast to the head injury, multiple

injuries were more common in evenings and at

nights periods. Multiple injuries or polytrauma

can be defined as a situation where multiple

organ systems were injured (Pepe 2003). Payal

et al. (2013) stated that 72% of RTA victims

had polytrauma. This is because, since festive

seasons usually means longer holidays people

might travel a lot on the road in the evening

and nightswhich adds to the traffic volume

(Jamilah et al. 2012), causing a higher vehicle-

kilometre traveled among road users, which

would ultimately lead to a higher risk of RTA.

Approximately 1% of RTA fatalities

were due to IHD and myocardial infarction in

this study. IHD caused by coronary artery

dissection following blunt trauma is unusually

rare (Mubang et al. 2016; Harada et al. 2002;

Unterberg et al. 1989; Oliva et al. 1979).

Meanwhile, for myocardial infarction to

happen due to to blunt trauma, it might be

associated with coronary artery injury

(Mubang et al. 2016; Harada et al. 2002).

According to Lai et al. (2006), coronary artery

injury is usually related to rib and sternal

fractures. The fractures might be due to RTA

since it was directly associated with blunt chest

trauma (Lima et al. 2009). Besides, if an

unrestrained driver is involved in a high-speed

motor vehicle collision, his/her chest would be

damaged by the steering wheel and axis when

the car stops abruptly (Behzadnia et al. 2007).

However, Petch (1998) suggested that those

who died in RTA due to heart diseases might

already been previously diagnosed with the

disease and they are at high risk of collapse and

sudden death during driving (New Zealand

Transport Agency 2014).

Deepavali had the highest number of

fatalities in RTAs with 68 (35%) fatalities.

Moreover, Indian was the biggest contribution

to fatal RTA during Deepavali with 36.8%.

Since alcohol drinking culture has long been

associated with Indians (Aradhana 2015),

driving on the road under influence of alcohol

during Deepavali could be a contributing

factor to the high number of fatalities in RTAs

in Deepavali. In fact, there were a few studies

that showed that Malaysian Indians spent

roughly RM150 million for alcohol alone

during the festive seasons, quoted by the

Malaysian Indian Congress Youth, Chief C.

25

Sivarajah to the Astro Awani (Suparmaniam

2015). In fact, there have been a nationwide

campaign such as Alcohol-Free Deepavali

Campaign that was created to avoid wasteful

expenses on liquor on Deepavali and

emphasize the importance of Deepavali. There

was a decreasing trend of the RTAs from 2011

until 2014, On Deepavali, the most number of

fatalities was recorded in 2011 and 2014. On

Chinese New Year and Eid al-Adha in 1998,

Malaysia achieved the highest number of road

accident in one holiday season with 9,901

accidents, killing 274 victims as well as 240

were fatal (Kareem 2003). Liew et al. (2016),

reported that, among 202 respondents in Klang

Valley that used the North-South Expressway,

more than 50% of them had involved in a crash

once, followed by 41% having been involved

in 2 to 5 crashes and 1% had an accident of

more than 5 times. More studies need to be

done to determine the causes of the high

number of fatalities during festive seasons

especially Deepavali. Finally, active

information reinforcement is very important to

maintain safe practice among road users and it

must be maintained at the highest standard

(Masuri et al. 2012).

CONCLUSION

As a conclusion, several crucial findings were

discovered regarding the incidence of fatal

RTA in the Klang Valley during the festive

periods from 2010 until 2015. More safety

precautions and campaigns on road accidents

need to be done especially focusing on teen age

group since they had the highest percentage of

fatal RTAs. Furthermore, night time is still the

peak time period for fatalities during festive

seasons compared to daytime. Although the

trend of fatal road accidents on Deepavali had

shown a declining pattern from 2011 to 2015,

Deepavali still held the highest number of

fatalities during festive seasons in Klang

Valley. Therefore, , the government and

related law enforcement agencies in Malaysia

should also focus more towards preventing

fatal RTAs during Deepavali and Eid al-Adha

as well as Chinese New Year and Eid al-Fitr.

ACKNOWLEDGEMENT

The authors would extend their gratitude to the

Forensic Departments of Pusat Perubatan

Universiti Kebangsaan Malaysia, Hospital

Tengku Ampuan Rahimah, Hospital Sungai

Buloh and Hospital Serdang for data

collection.

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Muhamad Hafizan Harun,

Sri Pawita Albakri Amir Hamzah*

Noor Hazfalinda Hamzah

Forensic Science Program

School of Diagnostic & Applied Health Science

Faculty of Health Sciences


Jalan Raja Muda Abdul Aziz

50300, Kuala Lumpur, Malaysia


http://english.astroawani.com/malaysia-news/indians-spend-rm150-mil-year-deepavali-mic-youth-79911



http://www.utusan.com.my/berita/nahas-bencana/jumlah-kenderaan-kecuaian-punca-nahas-musim-perayaan-1.350064



Buletin FSK 1(1)(2017): 28-33

Nilai sub perencatan Polymyxin B (PMB) dan kesan morfologi permukaan

Pseudomonas aeruginosa sub perencat PMB dengan kaedah TEM (The sub Minimum Inhibitory Concentration (Sub-MIC) of Polymyxin B (PMB) against

Pseudomonas aeruginosa and Its Cell Surface Changes Analyzed by TEM)

AHMAD ZORIN SAHALAN*, NOORSANA HOSNI & HING HIANG LIAN

ABSTRAK

Polymyxin B merupakan antibiotik peptida yang berkesan terhadap jangkitan Pseudomonas

dengan mengganggu ketelapan dinding sel dan akhirnya menyebabkan kematian bakteria.

Kepekatan aktif PMB boleh mengakibatkan kesan nefrotoksik yang serius. Oleh itu kesan sub

perencat (Sub-MIC) boleh membantu dalam perawatan dan mengurangkan kesan toksik PMB. Di

dalam kajian ini, kepekatan sub perencatan PMB di kaji dengan mengunakan TEM. Nilai MIC

ditentukan dahulu untuk mendapatkan nilai sub perencatan di mana kepekatan yang diuji untuk

PMB termasuklah 0.05 mg/ml, 0.025 mg/ml, 0.0125 mg/ml, 0.00 625 mg/ml, 0.003 125 mg/ml

dan 0.001 5625 mg/ml. Nilai kepekatan MIC terhadap Pseudomonas aeruginosa di dalam kajian

ini dicatat pada 0.00 625 mg/ml dan nilai sub perencatan PMB dikenalpasti sebagai kepekatan

terendah PMB selepas MIC iaitu 0.003 125 mg/ml. Pemerhatian TEM pula menunjukkan

perubahan signifikan pada membran bakteria selepas P. aeruginosa di rawat dengan dos sub

perencatan PMB selama tiga puluh minit. Pembentukan “bleb” dilihat terbentuk pada lapisan luar

membran P. aeruginosa. Hasil penemuan ini membuktikan bahawa PMB masih mampu mengubah

membran luar P. aeruginosa pada kepekatan sub perencatan untuk menembusi sel bakteria.

Kata kunci: Polymyxin B; Pseudomonas aeruginosa; Sub-perencatan; Sub-MIC; Elektron

Mikroskop Transmisi.

ABSTRACT

Polymyxin B (PMB) is an effective peptide antibiotic to treat Pseudomonas infection. It

disorganizes the bacterial outer membrane component which leads to increase in membrane

permeability and eventually results in the bacterial death. Unfortunately, PMB has serious

nephrotoxic and neurotoxic effect and is a dose dependent effect. The purpose of this study is to

determine a much lower dose for PMB, i.e. sub MIC concentration which would help to decrease

the toxic effect. To study the effect, TEM will be used to analyze the sub inhibitory effect of PMB.

Pseudomonas aeruginosa were cultured to mid logarithmatic phase and were treated with a range

of PMB concentration of 0.05 mg/ml, 0.025 mg/ml, 0.0125 mg/ml, 0.006 25 mg/ml, 0.003 125

mg/ml and 0.001 5625 mg/ml. MIC dose for PMB against Pseudomonas aeruginosa is 0.006 25

mg/ml, therefore the sub-inhibitory dose is confirmed at one concentration below MIC level which

is 0.003 125 mg/ml. TEM has revealed that there has been a significant change on the bacterial

cell membrane after P. aeruginosa was treated with sub inhibitory dose for thirty minutes.

Formation of bleb was observed on the outer membrane of P. aeruginosa. The finding suggests

that at sub inhibitory dose, PMB were able to disorganize P. aeruginosa outer membrane.

Keywords: Polymyxin B; Pseudomonas aeruginosa; Sub-Inhibitory Concentration (Sub –MIC);

Transmission Electron Microscope

29

PENGENALAN

Kepekatan sub perencatan ialah kepekatan

antibiotik yang lebih rendah berbanding

kepekatan MIC tetapi menghasilkan kesan

antibakterial yang signifikan kepada

mikroorganisma dari segi morfologi ataupun

kuantitatif (Rolinson 1977). Istilah sub-MIC

digunakan untuk merujuk kepada kepekatan

antibiotik di bawah MIC (<1MIC). Ramai

penyelidik menggunakan istilah kepekatan

subperencatan apabila merujuk kepada

kepekatan sub-MIC antibiotik. Walau

bagaimanapun, istilah ini tidak tepat kerana ia

khusus merujuk kepada kepekatan antibiotik

yang tidak memberi kesan kepada

pertumbuhan organisma yang diuji (Davies et

al. 2006).

Namun dalam kajian oleh Zhanel et al.

(1992) melaporkan kesan subperencatan boleh

merencat pertumbuhan bakteria dan ini

digambarkan pada perubahan morfologi dan

ultrastruktur pada bakteria. Kaplan (2011)

dalam kajiannya menunjukkan bahawa

kepekatan subprencatan sesetengah antibiotik

mampu merencat pertumbuhan bakteria,

meskipun tidak dapat membunuhnya. Dalam

satu kajian melibatkan jangkitan

Pseudomonas, sel bakteria yang terbenam jauh

ke dalam biofilm boleh terdedah kepada

kepekatan subperencatan sahaja kerana

perbezaan kecerunan difusi (Singh et al. 2010),

iaitu sel di bahagian atas atau paling luar akan

terkena kesan antibiotik sepenuhnya manakala

yang jauh kedalam hanya terdedah pada

antibiotik sedikit sahaja.

Kesan subperencatan antibiotik juga

dilihat mampu mengubah ultrastruktur dan

sifat antigenik bakteria; pelekatan bakteria

kepada sel epitelium; proses sintesis dan

sekresi enzim dan toksin baktera; serta

pengaktifan profaj (Deneve et al. 2009).

Di dalam kajian ini, kesan sub

perencatan Polymyxin B (PMB), iaitu sejenis

antibiotik peptida yang kuat, di kaji akan

keberkesanannya terhadap Pseudomonas

aeruginosa. Bakteria ini rintang kepada

pelbagai jenis antibiotik dan seringkali

dikaitkan dengan kes morbiditi dan kematian

di hospital, terutamanya infeksi bakteria gram

negatif Pseudomonas aeruginosa (Tam et al.

2005). Kesan sub perencatan terhadap PMB

hanya dapat di analisa menerusi mikrograf

TEM sahaja kerana kaedah ini sensitif dan

segala perubahan morfologi permukaan

bakteria dapat dianalisa dengan lebih jelas

(Sahalan et al. 2008).

BAHAN DAN KAEDAH

STOK BAKTERIA DAN POLYMYXIN B

Kultur bakteria ujian iaitu isolat klinikal

Pseudomonas aeruginosa diperoleh daripada

kultur stok makmal Sains Bioperubatan,

Fakulti Sains Kesihatan, Universiti

Kebangsaan Malaysia, Kuala Lumpur.

Subkultur bakteria dibuat setiap dua minggu

sekali untuk memastikan bakteria berada

dalam keadaan baik dan aktif sepanjang sesi

ujian dilakukan. Kultur bakteria disimpan di

dalam peti sejuk pada suhuh 4°C.

Antibiotik peptida yang digunakan

iaitu Polymyxin B diperolehi daripada stok

makmal Sains Bioperubatan, Fakulti Sains

Kesihatan, Universiti Kebangsaan Malaysia,

Kuala Lumpur. Cara penyediaan larutan stok

dan larutan kerja PMB adalah berdasarkan

garis panduan Clinical and Laboratory

Standard Institute (CLSI) 2015.

PENENTUAN KEPEKATAN

PERENCATAN MINIMUM (MIC) DAN

SUB PERENCATAN (SUB-MIC) PMB

TERHADAP Pseudomonas aeruginosa

Kepekatan perencatan minimum (MIC) untuk

polymyxin B ditentukan melalui ujian

mikropencairan menggunakan 96 telaga plat

mikrotiter. MIC ditakrifkan sebagai kepekatan

antibiotik yang paling rendah yang mampu

merencat pertumbuhan bakteria sepenuhnya

30

selepas 24 jam pengeraman pada suhu 37°C.

Kekeruhan dalam telaga dalam plat mikrotiter

ditafsirkan sebagai pertumbuhan signifikan

mikroorganisma.

MIC dilakukan dengan memasukkan

sebanyak 100 µl dan 50 µl kaldu Mueller-

Hinton masing-masing dimasukkan ke dalam

telaga mikrotiter. Kemudian 50 µl PMB

ditambah ke dalam telaga pertama dan 50 µl

dipindahkan ke telaga kedua dan seterusnya.

Pada telaga terakhir, 50 µl campuran pada

kolum terakhir tadi dibuang, dan akhir sekali

50 µl bakteria yang telah dicairkan

dimasukkan ke dalam setiap telaga mikrotiter.

Plat mikrotiter kemudiaannya ditutup dengan

penutup plat mikrotiter atau parafilm sebelum

diinkubasi pada suhu 37°C selama lebih

kurang 18 hingga 20 jam; (vii) Setelah itu, plat

mikrotiter dititiskan dengan 20 µl 1%

Triphenyl tetrazolium chloride (TTC) dan

dinkubasi pada suhu 37°C selama setengah

jam. Perubahan warna merah jambu pada plat

mikrotiter menunjukkan pertumbuhan bakteria

manakala plat yang jernih menunjukkan

terdapat aktiviti perencatan bakteria. Bacaan

MIC diambil pada kepekatan minimum yang

merencat pertumbuhan bakteria sepenuhnya.

Kepekatan subperencatan minimum

bagi PMB ditentukan dengan memperoleh

kepekatan PMB dibawah MIC (1 < MIC). Pada

kepekatan ini, terdapat pertumbuhan bakteria

pada telaga plat mikrotiter. Kekeruhan dalam

telaga dalam plat mikrotiter ditafsirkan sebagai

pertumbuhan signifikan mikroorganisma.

KAEDAH PENYEDIAAN BAKTERIA

UNTUK TEM

Sampel untuk pemerhatian dibawah

mikroskop elektron trasmisi disediakan sehari

sebelum pemerhatian dilakukan. Oleh itu,

sampel P. aeruginosa dari plat induk dikultur

ke dalam 5 ml kaldu agar nutrien pada suhu

37°C selama 18 hingga 20 jam. Kemudian, 200

µl suspensi bakteria dipipet ke dalam 5 ml

kaldu nutrien yang baru dan dieramkan selama

2 jam pada suhu 37°C. Setelah itu, suspensi

bakteria diempar pada kelajuan 3000 r.p.m.

selama 10 minit. Suspensi bakteria dibasuh

sebanyak tiga kali menggunakan air suling.

Akhir sekali, supernatan dibuang dan

pallet bakteria disuspensi di dalam 10 µl air

suling sebagai kawalan manakala untuk ujian,

17.5 µl larutan PMB dicampurkan kedalam

12.5 µl suspensi bakteria. Larutan PMB pada

kepekatan subperencatan minimum

dicampurkan ke dalam suspensi bakteria dan

dibiarkan selama 30 minit. Campuran

kemudiannya diletakkan ke atas grid kuprum

yang telah disaluti formvar lalu dibiarkan

kering. Sampel dirawat dengan pewarnaan

negatif menggunakan 0.1% asid fototungstik

dan diperhatikan di bawah TEM.

HASIL

Nilai MIC yang dicatatkan seperti didalam

jadual 1 dimana MIC dicatat sebagai

0.00625mg/ml manakala sub-perencatan

adalah 0.003125 mg/ml.

Jadual 1: Penentuan kepekatan sub perencatan

PMB terhadap Pseudomonas

aeruginosa menggunakan teknik

mikropencairan

Kepekatan

PMB (mg/ml)

Pertumbuhan

bakteria

0.05 -

0.025 -

0.0125 -

0.00625 -

0.003 125 +

0.001 5625 +

0.000 781 25 ++

0.000 390 625 ++

Petunjuk: - Tiada pertumbuhan

+ Ada pertumbuhan

Gambar 1 menunjukkan struktur morfologi

Pseudomonas aeruginosa yang tidak dirawat

31

dengan PMB. Pemerhatian menggunakan

TEM mendapati struktur morfologi luar

bakteria masih kelihatan utuh dan tiada

kerosakan. Lapisan struktur peptidoglikan juga

jelas kelihatan.

Gambar 1: Pemerhatian TEM terhadap

Pseudomonas aeruginosa tanpa

rawatan PMB magnifikasi x100

000.

Gambar 2 menunjukkan struktur morfologi

Pseudomonas aeruginosa selepas dirawat

dengan kepekatan sub perencatan PMB iaitu

0.003 125 mg/ml selama 30 minit.

Pemerhatian dibawah TEM menunjukkan

terdapat kerosakan minima pada struktur

morfologi bakteria dimana pembentukan

“bleb” dilihat pada membran luar sel manakala

struktur flagella masih kelihatan utuh.

Walaupun dalam kepekatan yang rendah, PMB

masih mampu menyebabkan kerosakan pada

membran sel bakteria.

Gambar 2: Pemerhatian TEM terhadap

Pseudomonas aeruginosa yang

dirawat dengan 0.000 1566 25

mg/ml PMB selama 30 minit

dengan magnifikasi x100 000

PERBINCANGAN

Kepekatan sub perencatan antibiotik adalah

kepekatan di bawah paras MIC, maka

kepekatan sub perencatan PMB terhadap P.

aeruginosa dalam kajian ini adalah pada

kepekatan 0.003 125 mg/ml. Keputusan ini

menyokong kajian oleh Zhanel et al. (1992)

dan Rolinson (1977) dimana kepekatan

subperencatan mampu merencat pertumbuhan

bakteria dan menyebabkan perubahan

morfologi dan ultrastruktur pada bakteria. Ini

membuktikan bahawa meskipun pada

kepekatan yang lebih rendah, PMB mampu

untuk menyebabkan kerosakan pada sel

membran luar bakteria.

Walaupun Pseudomonas aeruginosa

rintang terhadap kebanyakan kumpulan agen

antimikrob, namun PMB berkesan mengawal

jangkitan bakteria ini (Berditsch et al. 2015).

PMB bertindak dengan mengikat pada

membran sel bakteria dan mengganggu

kenyahtelapan membran dengan cara

memindahkan jambatan kalsium dan

magnesium yang menstabilkan LPS bakteria

(Sahalan et al. 2013). Keadaan ini seterusnya

mengakibatkan komponen intraselular keluar

dari sel dan akhirnya sel bakteria mengalami

pembengkakan dan lisis (Berditsch et al.

32

2015). Walaupun kepekatan PMB rendah

sehingga subperencatan, ianya mampu untuk

menyebabkan kerosakan pada sel membran

luar bakteria.

Mikroskop elektron transmisi (TEM)

menunjukkan kesan kepekatan sub perencatan

PMB terhadap morfologi membran luar

bakteria diaman pembentukan ‘bleb’ berlaku

pada membran luar permukaan bakteria dan

kajian ini signifikan dengan kajian oleh Koike

et al. (1971). Selain itu, Sahalan & Dixon

(2008) juga turut melaporkan bahawa pada

kepekatan yang lebih rendah, PMB mampu

menyebabkan kebocoran protein periplasmik

daripada bakteria. Kesan sub perencatan ini

juga menunjukkan bahawa kaedah gabungan

sub perencatan PMB dengan antibiotik lain

boleh membantu lagi dalam pemusnahan

bakteria dengan cepat dan lebih banyak.

PENGHARGAAN

Penghargaan dan ucapan terima kasih di atas

bantuan geran penyelidikan Universiti

Kebangsaan Malaysia dan kakitangan makmal

Program Sains Bioperubatan termasuk Encik

Faisal Ariffin dan Mariahyati Abu Bakar.

RUJUKAN

Berditsch, M., Jager, T., Strempel, N., Schawartz,

T., Overhage J., Ulrich A.S.. 2015. Synergistic

Effect of Membrane-Active Peptides

Polymyxin B and Gramicidin S on Multidrug-

Resistant Strains and Biofilms of

Pseudomonas aeruginosa. Antimicrobial

Agents and Chemotherapy. 59 (9): 5288-5296.

Davies, J., Spiegelman, G.B., Yim, G. 2006. The

world of subinhibitory antibiotic

concentrations. Current Opinion in

Microbiology. 9: 445-453.

Deneve, C., Bouttier, S., Dupuy, B., Barbut, F.,

Collignon, A., Janoir, C. 2009. Effects of

subinhibitory concentrations of antibiotics on

colonization factor expression by

moxifloxacin-susceptible and

moxifloxacinresistant Clostridium difficile

strains. Antimicrobial Agents and

Chemotherapy. 53(12):5155-5162.

Kaplan, J.B. 2011. Antibiotic-induced biofilm

formation. International Journal Artificial

Organs. 34(9): 737-751

Koike, M., Iida, K. 1971. Effect of Polymyxin

on the Bacteriophage Receptors of the Cell

Walls of Gram-Negative Bacteria. Journal of

Bacteriology. 108(3): 1402-1411.

Rolinson, G.N. 1977. Subinhibitory concentration

of antibiotics. Journal of Antimicrobial

Chemotherapy. 3(2): 111-113.

Sahalan, A.Z., Abd. Aziz A.H., Hing H.L., Abd.

Ghani M.K. 2013. Divalent Cations (Mg2+,

Ca2+) protect bacterial outer membrane

damage by polymixin B. Sains Malaysiana.

42(3):301-306.

Sahalan, A.Z. & Dixon, R.A. 2008. Role of the cell

envelope in the antibacterial activities of

polymyxin B and polymyxin B nonapeptide

against Escherichia coli. International Journal

of Antimicrobial Agents. 31 (1): 224–227.

Singh, R., Ray, P., Das, A., Sharma, M. 2010.

Penetration of antibiotics through

Staphylococcus aureus and Staphylococcus

epidermidis biofilms. Journal of Antimicrobial

Chemotherapy. 65: 1955-1958.

Tam, V.H., Schilling, A.N., Vo, G, Kabbara, S.,

Kwa, A.L., Wiederhold, N.P., Lewis, R.E.

2005. Pharmacodynamics of Polymixin B

against Pseudomonas aeruginosa.

Antimicrobial and Agents Chemotherapy. 49:

3624-3640.

Zhanel, G.G., Hoban, D.J., Harding, G.K.M. 1992.

Subinhibitory antimicrobial concentrations: A

review of in vitro and in vivo data. Canadian

Journal of Infectious Disease. 3(4):193-201.

33

Ahmad Zorin Sahalan*

Noorsana Hosni

Hing Hiang Lian






50300 Kuala Lumpur,

Malaysia.


Buletin FSK 1(1)(2017): 34-44

Kajian Kes Kawad Kebakaran: Jangka Masa Bergerak di Ambil Bagi

Pengusian Bangunan Dalam Kalangan Warga UKM Kampus Kuala Lumpur (Case Study of Fire Evacuation Drill: Emergency Evacuation Time Taken During Fire

Emergency Among Staff UKM Campus Kuala Lumpur)

ANUAR ITHNIN*

ABSTRAK

Jangkamasa bergerak semasa pengusian bangunan dalam kejadian kecemasan bergantung kepada

faktor seperti situasi persekitaran bangunan, pengetahuan keselamatan dan kemahiran responden

berkaitan sesuatu bangunan. Objektif kajian untuk mengkaji jangkamasa bergerak yang diambil

bagi penghuni bangunan dalam kalangan warga UKM kampus K. Lumpur semasa kejadian

kecemasan kebakaran. Kajian ini melibatkan 139 responden berdasarkan kes kawad kebakaran

yang telah dijalankan bermula loceng pengera kecemasan dibunyikan sehingga responden berada

di kawasan tempat berkumpul selamat yang ditetapkan. Majoriti responden adalah perempuan

(59.7 %), Melayu (87.1 %) dan mempunyai tahap pendidikan ijazah ke atas (46.8%). Majoriti

responden telah berkhidmat kurang dari 5 tahun (46%), 5 hingga 10 tahun (32.4%) dan melebihi

10 tahun (21.6%). Dari aspek masa bergerak, seramai 58 responden (54%) mengambil masa 3

hingga 5 minit, 41% responden kurang dari 3 minit dan 4.3% responden mengambil masa lebih

dari 5 minit untuk bergerak sampai ke tempat berkumpul yang ditetapkan. Terdapat perbezaan

yang signifikan (p=0.001) di antara masa bergerak bagi responden yang berumur melebihi 30

tahun dan kurang 30 tahun. Manakala, terdapat perbezaan yang signifikan (p=0.030) di antara

responden yang telah berkahwin berbanding bujang dari segi masa yang diambil untuk ke tempat

selamat berkumpul. Terdapat perbezaan yang signifikan (p=0.001) antara responden yang telah

berkhidmat kurang dari 5 tahun berbanding lebih 5 tahun masa diambil bagi tujuan pengusian

bangunan. Responden yang telah pernah menghadiri taklimat dan latihan berkaitan, faham

prosedur pengusian bangunan dan mempunyai kefahaman dan pengetahunan berkaitan

keselamatan menunjukkan peratusan yang lebih tinggi iaitu melebihi 5 minit jangka masa yang

diambil bagi tujuan pengusian bangunan. Dari aspek faktor yang menghalang pergerakan,

menunjukkan seramai 26.6% responden menyatakan terdapat halangan dan laluan sempit, tidak

tahu laluan kecemasan dan tempat berkumpul (23.7%), berjalan perlahan dan tidak tergesa-gesa

(22.1%), tiada penunjuk ke tempat berkumpul (12.9%) dan tangga terlalu curam dan lampu tidak

terang di laluan (5%) yang menyebabkan mereka mengambil masa untuk bergerak ke tapak

berkumpul yang ditetapkan.

Kata kunci: kecemasan kebakaran; pengusian bangunan; jangkamasa bergerak; tempat selamat

berkumpul

ABSTRACT

The duration of move for evacuation building during emergencies depends on factors such as

building environment situation, safety knowledge and respondent's skills related to a building. The

objective of the study was to examine the duration of the movements taken by the residents of the

UKM campus K. Lumpur during a fire emergency event. This study involved 139 respondents

based on the case of fire drill which had been conducted in 2011. The time movement taken started

35

since the emergency alarm bells were ringed until the respondents were in the designated safe area.

Respondents consisted mostly of women (59.7%) and Malay (87.1%). The majority of respondents

had a higher degree (46.8%), Diploma / STPM (30.9%) and SPM / SRP was 22.3%. The majority

of respondents have served less than 5 years (46%), 5 to 10 years (32.4%) and over 10 years

(21.6%). From the aspect of time movement for evacuation, 58 respondents (54%) took 3 to 5

minutes, 41% respondents were less than 3 minutes and 4.3% respondents were more than 5

minutes took their moves to the designated assembly point. There was a significant difference (p

= 0.001) between the movements of respondents aged over 30 years and less than 30 years old. On

the other hand, there were significant differences (p = 0.030) among respondents who had married

compared to bachelors took their moves to the designated safe area. There was a significant

difference (p = 0.001) between respondents who had served less than 5 years compared to over 5

years taken for the purpose of building evacuation. Thus respondents who have attended briefings

and related exercises, understand the building maintenance procedures and have understanding

and safety related knowledge show a higher percentage of less than 5 minutes of time taken for

the purpose of evacuation building. In terms of slow down the movement during the evacuation

building, showed a total of 26.6% respondents stated that there were obstacles and narrow routes,

not knowing the emergency routes and assembly point (23.7%), walk slowly and no sense of

urgency (22.1%), 12.9%) and the stairs are too steep and the lights are not bright on the path (5%)

which cause them to take the time to move to the designated safe area.

Keywords: fire emergency; evacuation building; moving time; assembly point

PENGENALAN

Faktor yang memberi kesan terhadap

keselamatan kebakaran di bangunan adalah

faktor persekitaran bangunan itu sendiri.

Faktor penting untuk ciri bangunan dalam

menentukan tindakan semasa berlakunya

kebakaran adalah berdasarkan faktor situasi

dan ciri-ciri tersendiri kejuruteraannya (Kobes

et al. 2010). Ciri-ciri tersendiri situasi

persekitaran bangunan termasuk kepadatan

penghuni, kemudahan untuk pencarian,

kewujudan tempat selamat berkumpul,

kewujudan pasukan tindakan kecemasan dan

penyenggaraan kemudahan pencegah

kebakaran. Kepadatan penghuni merujuk

kepada bilangan orang yang berada di dalam

bangunan. Terdapat hubungan langsung antara

tahap kepadatan penghuni dengan

kemungkinan berlakunya kes kematian (Tubbs

2004).

Pencarian jalan keluar menjelaskan

bagaimana penghuni menyesuaikan diri

mereka di dalam bangunan (Raubal &

Egenhofer 1998). Ini berkait dengan

pengetahuan ruang dan kepelbagaian

kebolehan kognitif mereka. Kebolehan

pencarian jalan keluar ditentukan oleh persepsi

penghuni dan pengetahuan kedua-dua aspek

persekitaran dan situasi. Lima kategori faktor

persekitaran yang memberi kesan dalam

pencarian jalan keluar (Raubal & Egenhofer

1998) iaitu, akses penglihatan, tahap perbezaan

aspek arkitek contohnya ciri unik bangunan di

mana penghuni mengunakannya untuk tujuan

orientasi, paparan, kebiasaan dengan bangunan

dan kewujudan papan tanda dan tanda lokasi.

Komponen yang relevan kepada paparan

adalah papan tanda jalan keluar, rekabentuk

jalan keluar, serta lokasi bagi laluan

kecemasan dan tangga kecemasan. Di antara

elemen penting keselamatan kebakaran adalah

menyelenggara kemudahan berkait kebakaran

supaya berada dalam keadaan yang baik.

36

METODOLOGI

Kajian rentas ini menggunakan soalselidik

yang mengandungi 3 bahagian iaitu

sosiodemografi, ciri pekerjaan dan jangkamasa

diambil untuk pengusian bangunan.

Responden terdiri dari 139 warga kampus yang

terlibat dalam kawad kebakaran yang telah

dilakukan di kampus UKM Kuala Lumpur

pada tahun 2011. Warga penghuni bangunan

terlibat bergerak keluar dari bangunan apabila

loceng kecemasan dibunyikan ke arah kawasan

berkumpul berhampiran yang telah ditetapkan.

Masa penghuni mula bergerak bermula apabila

loceng kecemasan berbunyi sehingga

penghuni berada di kawasan perhimpunan

dicatatkan. Borang soalselidik diberikan

kepada responden yang terlibat setelah mereka

berada di tempat berkumpul untuk diisi oleh

responden dan kemudiannya diserahkan

kepada pegawai insiden di setiap tempat

selamat berkumpul. Data kajian dianalisis

menggunakan SPSS.

HASIL

Jadual 1 menunjukkan data sosiodemografi

responden yang terdiri daripada 139 warga

UKM Kampus K. Lumpur, di mana majoriti

adalah perempuan (59.7 %) dan Melayu (87.1

%) dan sudah berkahwin (66.2%). Dari aspek

pendidikan, majoriti mempunyai ijazah ke atas

(46.8%), Diploma/ STPM (30.9%) dan di

peringkat SPM/SRP adalah 22.3 peratus.

Majoriti responden telah berkhidmat di antara

kurang dari 5 tahun adalah 46 peratus, antara 5

hingga 10 tahun (32.4%) dan melebihi 10

tahun seramai 21.6 peratus.

Berdasarkan Jadual 1, majoriti

responden (54%) mengambil masa 3 hingga 5

minit selepas bunyi amaran kebakaran

dibunyikan bergerak ke tapak berkumpul yang

telah ditetapkan. Manakala, 41% responden

mengambil masa kurang dari 3 minit dan 4.3%

responden mengambil masa lebih 5 minit

untuk bergerak sampai ke tempat berkumpul

yang ditetapkan.

Jadual 2 dan Rajah 1 menunjukkan,

jangka masa bergerak sehingga ke tapak

berkumpul setelah bunyi amaran kebakaran

dibunyikan berdasarkan sosiodemografi

responden. Dari aspek umur, terdapat

perbezaan yang signifikan (p=0.001) di antara

masa bergerak responden yang berumur

melebihi 30 tahun dan di bawah umur 30

tahun. Hasil dapatan juga menunjukkan

terdapat perbezaan yang signifikan (p=0.030)

mengikut status perkahwinan di antara yang

telah berkahwin dan bujang. Terdapat

perbezaan yang signifikan antara responden

yang telah berkhidmat kurang dari 5 tahun

berbanding dengan lebih 5 tahun berkhidmat

untuk jangka masa bergerak yang diambil

sehingga ke tapak berkumpul setelah bunyi

amaran kebakaran dibunyikan.

JADUAL 1. Jangka masa bergerak keseluruhan responden selepas bunyi amaran kebakaran

dimulakan sehingga berkumpul di tapak berkumpul yang ditetapkan.

Jangkamasa yang diambil bergerak

sehingga ke tapak berkumpul

Frekuensi Peratusan, %

< 3minit 58 41.7

> 3 minit < 5 minit 75 54.0

> 5 minit 6 4.3

Jumlah 139 100.0

37

JADUAL 2. Jangka masa bergerak sehingga ke tapak berkumpul setelah bunyi amaran

kebakaran dibunyikan berdasarkan sosiodemografi responden.

Faktor Sosiodemografi

Jangka Masa Bergerak

(minit)

>5 min <5 min 2 p

Umur >30 Tahun 32 (86.5) 25 (24.5) 43.11 0.001**

<30 Tahun 5 (13.5) 77 (75.5)

Jantina Perempuan 23 (62.2) 60 (58.8) 0.13 0.72

Lelaki 14 (37.8) 42 (41.2)

Bangsa Bukan-

Melayu

2 (5.4) 16 (15.7) 2.55 0.11

Melayu 35 (94.6) 86 (84.3)

Status Perkhawinan Berkhawin 7 (18.9) 40 (39.2) 5.00 0.03**

Bujang 30 (81.1) 62 (60.8)

Pendidikan Rendah 6 (16.2) 25 (24.5) 1.08 0.30

Tinggi 31 (83.8) 77 (75.5)

Jangkamasa di Bangunan <5 Tahun 8 (21.6) 56 (54.9) 12.11 0.001**

>5 Tahun 29 (78.4) 46 (45.1)

Jangkamasa berkhidmat <5 Tahun 7 (18.9) 53 (52.0) 12.08 0.001**

>5 Tahun 30 (81.1) 49 (48.0)

Petunjuk: *nilai p < 0.005

Berdasarkan Rajah 2, hasil kajian

menunjukkan peratusan yang lebih tinggi

jangka masa yang diambil sekiranya responden

telah pernah menghadiri taklimat dan latihan

berkaitan, faham prosedur pengusian

bangunan dan mengetahui bidang tugas

masing-masing terutama pengawai insiden.

Berdasarkan Rajah 3, menunjukkan peratusan

lebih tinggi jangka masa bergerak sehingga ke

tapak berkumpul setelah bunyi amaran

kebakaran dibunyikan sekiranya mempunyai

kefahaman dan pengetahunan berkaitan

keselamatan dan kesihatan pekerjaan seperti

AKKP 1994, AKJ 1967, Akta Bomba 1988

dan sebagainya.

38

RAJAH 1. Jangka masa bergerak sehingga ke tapak berkumpul setelah bunyi amaran kebakaran

dibunyikan berdasarkan sosiodemografi responden.


dibunyikan.

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

100.0%

>30

<30

Pe

rem

pu

an

Lela

ki

ber

khaw

in

bu

jan

g

Ren

dah

Tin

ggi

<5 t

ahu

n

>5 t

ahu

n

Umur Jantina status perkhawinan pendidikan jangka masa dibangunan

>5 min

<5

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

100.0%

Tidak Ya Tidak Ya Tidak Ya Tidak Ya Tidak Ya Tidak Ya Tidak Ya

Taklimat bertindakprosidur

kecemasan

selamat diri tugaspegawaiinsiden

organisasimampu

fahamiprosidur

latihankhusus

>5 min

<5

39


dibunyikan berdasarkan kefahaman dan pengetahunan berkaitan keselamatan dan kesihatan

pekerjaan.

Rajah 4 dan Rajah 5, menunjukkan faktor-

faktor yang menghalang pergerakan sehingga

ke tapak berkumpul. Seramai 26.6%,

responden menyatakan laluan terdapat

halangan dan sempit, tidak tahu tempat

berkumpul dan laluan kecemasan (23.7%),

kebanyakannya berjalan perlahan dan tidak

tergesa-gesa (22.1%) menyebabkan masa

diambil untuk bergerak sehingga ke tapak

berkumpul. Manakala, 12.9% responden

menyatakan bahawa tiada penunjuk ke tempat

berkumpul dan ianya tidak sesuai. Disamping

itu, 5 peratus responden menyatakan tangga

terlau curam dan lampu tidak terang di laluan

menyebabkan mereka mengambil masa

bergerak ke tapak berkumpul yang ditetapkan.

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

Tidak Ya Tidak Ya Tidak Ya Tidak Ya Tidak Ya Tidak Ya

aktaparit1974 UKBS1984 bomba1988 OSHA1994 akta149 EQA1974

>5 min

<5

40

RAJAH 4. Menunjukkan faktor-faktor yang menghalang jangka masa bergerak sehingga ke

tapak berkumpul setelah bunyi amaran kebakaran dibunyikan.

RAJAH 5. Menunjukkan faktor-faktor yang menghalang jangka masa bergerak (kurang 5 minit

dan lebih 5 minit) sehingga ke tapak berkumpul setelah bunyi amaran kebakaran dibunyikan.

26.6%

23.7%

20.1%

12.9%11.5%

5.0%

0.0%

5.0%

10.0%

15.0%

20.0%

25.0%

30.0%

laluan terdapathalangan dan sempit

tidak tahu tempatberkumpul dan

laluan kecemasan

semua orangberjalan perlahan-

lahan,tidak tergesa-gesa,no urgency

tiada penunjuk ketempat berkumpul

tempat berhimpunjauh dan tidak sesuai

tangga curam,lamputidak terang di laluan

0.0%

5.0%

10.0%

15.0%

20.0%

25.0%

30.0%

35.0%

40.0%

45.0%

50.0%

laluan terdapathalangan dan sempit

semua orang berjalanperlahan-lahan,tidak

tergesa-gesa,nourgency

tangga curam,lamputidak terang di laluan

tempat berhimpunjauh dan tidak sesuai

tiada penunjuk ketempat berkumpul

tidak tahu tempatberkumpul dan laluan

kecemasan

>5 min

<5

41

PERBINCANGAN

Bangunan kampus UKM Kuala lumpur

merupakan sebuah bangunan yang sudah

dibina lebih daripada 40 tahun yang

menempatkan 3 buah fakulti iaitu Fakulti

Pergigian, Fakulti Farmasi dan Fakulti Sains

Kesihatan. Di samping itu, terdapat

kemudahan lain seperti perpustakaan, kafeteria

dan sebagainya. Selain warga kerja UKM,

terdapat pelajar dan pelawat berada dalam

kawasan kampus UKM Kuala Lumpur dalam

sesuatu masa tertentu. Oleh pihak pengurusan

kampus UKM K. Lumpur sangat

mengutamakan isu keselamatan bagi

menjamin keselamatan pekerja, pelajar dan

pelawat yang berada dalam kawasan

pentadbirannya.

Jenis bangunan di kampus UKM Kuala

Lumpur adalah dikelaskan kepada kelas A

yang merupakan pembinaan berunsur struktur,

lantai, dan dinding yang tidak mudah terbakar

serta mempunyai sokongan bata atau konkrit.

Masa yang dicadangkan diterima bagi

kesemua penghuni mengosongkan bangunan

tersebut semasa kejadian kecemasan

kebakaran 3 minit. Berdasarkan hasil kajian,

41% responden berjaya mengambil masa

kurang dari 3 minit untuk melakukan

pengosongan bangunan semasa kajian ini.

Manakala, 54% mengambil masa 3 hingga 5

minit dan hanya 4.3% yang mengambil masa

lebih 5 minit semasa kawad pengusian

bangunan dilakukan.

Keselamatan bangunan merupakan

komponen bangunan berpotensi mempunyai

bahaya yang mungkin boleh berlaku yang

terdiri daripada beberapa komponen seperti

struktur bangunan, tangga, laluan kecemasan,

pintu dan tingkap berkaca. Berdasarkan hasil

kajian menunjukkan 5% responden

menyatakan laluan tangga yang curam dan

pencahayaan tidak terang juga faktor jangka

masa yang diambil melebihi masa yang

sepatutnya diambil semasa pengusian

bangunan semasa kecemasan kebakaran terjadi

iaitu kurang dari pada 3 minit. Tangga

merupakan salah satu komponen bangunan

yang penting yang sepatutnya dibina di

kedudukan yang sesuai dan selamat. Bilangan

tangga memainkan peranan yang besar dalam

mempengaruhi keselamatan penghuni untuk

menampung jumlah penghuni yang ramai

terutama apabila berlakunya kecemasan.

Kelebaran tangga yang dibina hendaklah

berdasarkan Uniform Building by Law 1984

(UBBL 1984), Seksyen 168, sub-seksyen (2)

iaitu mempunyai lebar yang bersesuaian yang

boleh menampung beban pendudukan tertinggi

bagi maksud melepaskan diri. Tangga perlu

mempunyai saiz dan bentuk pemegang tangan

yang berciri ergonomik agar mudah untuk

dicengkam dan dicapai supaya bahaya

kegelinciran dapat dielakkan dan tangga

hendaklah diterangi dan tidak terhalang

dengan apa-apa objek yang boleh

menyebabkan penghuni terhalang daripada

menggunakannya terutama waktu kecemasan

dan menpunyai pengudaraan baik (UBBL

1984).

Berdasarkan hasil kajian juga

menunjukkan bahawa seramai 26.6%

responden menyatakan laluan terdapat

halangan dan sempit. Laluan susur koridor

sangat penting bagi keselamatan untuk

bergerak serta mengelakkan risiko kemalangan

terutama apabila berlaku kecemasan. Keadaan

laluan yang sempit mampu meningkatkan

pelanggaran dan penolakan yang seterusnya

mengakibatkan berlakunya kemalangan dan

kecederaan. Seramai 12.9% responden

menyatakan tiada penunjuk ke tempat

berkumpul, manakala seramai 23.7%

menyatakan tidak tahu tempat berkumpul

selamat yang telah ditetapkan. Tanda tempat

keluar kecemasan hendaklah ditanda dengan

tanda menunjukkan arah hendaklah diterangi

berterusan sepanjang tempoh pendudukan

yang dapat dilihat dengan mudah dan

hendaklah tidak dilindungi oleh apa-apa

perhiasan, perabot atau kelengkapan lain.

42

Faktor Persekitaran Bangunan

Pasukan Tindakan Kecemasan atau

Emergency Response Team (ERT) merupakan

sekumpulan penghuni bangunan yang

bertindak dalam keadaan kecemasan dan

sentiasa berada di lokasi. Mereka ini telah

dilatih dalam latihan pencegahan kebakaran

serta koordinasi dalam pemindahan penghuni

kebakaran. Ahli ERT yang terlatih dalam

tindakan kecemasan merupakan pengaruh

positif terhadap kepantasan proses

pemindahan penghuni sesuatu bangunan

(Purser 2003). Kajian menunjukkan responden

yang telah menghadiri taklimat dan latihan

berkaitan serta fahami prosedur kecemasan

hanya mengambil masa kurang dari 5 minit

untuk sampai ke tampat berkumpul yang

ditetapkan.

Galangan fizik, seperti pembahagian

fizik petak kebakaran dan asap, jarak

maksimum perjalanan ke pintu kecemasan dan

pemasangan alat kebakaran adalah komponen

utama jalan keluar dan sistem keselamatan

hidup (Kobes et al. 2010). Pembahagian petak

kebakaran berkait dengan galangan fizik perlu

diambil kira seperti kemerebakan api dan asap

di dalam bangunan. Kes kematian dalam

kebakaran pada bangunan yang mempunyai

banyak bilangan tingkat menunjukkan

kemungkinan besar kes tersebut adalah akibat

daripada kesan api ataupun kesan asap di

tangga bangunan (Kobes 2008). Di antara

berikut adalah faktor halaju pemindahan

penghuni di dalam tangga kecemasan (Proulx

2007) adalah saiz tangga, kepadatan orang di

dalam tangga, pemindahan penghuni segera

penghuni dari tingkat tertentu, penghuni cuba

memasuki kumpulan yang berada di tangga

yang menuju ke bawah, percakapan di antara

penghuni semasa menuruni tangga, penghuni

yang lebih berat badan, terlalu tinggi dan

terlalu rendah dan penggunaan kasut yang

tidak sesuai (kasut ketat, kasut tumit tinggi dan

lain-lain).

Faktor Kemanusian Terhadap Tindakan

Semasa Kebakaran

Secara umumnya, tiga faktor yang utama

terlibat dalam menentukan tahap pencapaian

tindakan kecemasan semasa bangunan

terbakar. Kobes (2008) telah mengenalpasti

faktor-faktor tersebut, di antaranya adalah ciri

kebakaran, ciri manusia dan ciri bangunan.

Ciri kebakaran dan bangunan memberi

pengaruh secara langsung terhadap tahap

pencapaian tindakan kecemasan dan juga

memberi kesan langsung kerana secara

fiziknya ia wujud di persekitaran di mana

manusia berada di dalamnya untuk melakukan

aktiviti. Manakala, ciri manusia iaitu

perwatakan semulajadi manusia secara

individu dan berkumpulan juga merupakan

pengaruh langsung terhadap tahap pencapaian

tindakan kecemasan (Kobes et al. 2010).

Manakala ciri manusia termasuk keberkesanan

perancangan kecemasan bagi sesebuah

organisasi pasukan tindakan kecemasan,

pemahaman persekitaran bangunan, kejiranan,

penghuni dan kebiasaan perlakuan penghuni

bangunan. Keperluan penghuni bangunan

perlu dititikberatkan oleh pengurus fasiliti

terutamanya terhadap golongan kurang upaya

seperti kecacatan penghuni bangunan.

Seterusnya adalah latihan yang berterusan

kerana dengan mengadakan latihan, kecekapan

penghuni dan kecekapan ahli pasukan tindakan

kecemasan akan meningkat di samping

pendidikan dan penyebaran maklumat

dikalangan penghuni bangunan.

Semasa kebakaran, tekanan psikologi

mungkin meningkat disebabkan peningkatan

kapasiti untuk memproses sesuatu maklumat

(Proulx 1993), ataupun untuk mereka

berkonfrantasi dengan keadaan yang tidak

biasa bagi mereka (Verwey 2004). Tekanan

psikologi yang terlalu banyak boleh

membantutkan proses kognitif serta memberi

satu keadaan bagaimana individu tersebut

perlu bertindak dalam keadaan kecemasan

tersebut (Proulx 1993). Peningkatan tahap

43

tekanan adalah tidak sama. Sebagai contoh,

tahap cemas seseorang. Ini kerana ia boleh

didefinasikan sebagai tidak rasional, tidak

logik akal, dan tingkah-laku luar kawalan

(Sime 1990). Kawalan dalaman ini adalah

berdasarkan tahap pengetahuan seseorang,

perasaan dan ciri-ciri biologi serta pengaruh

keadaan persekitaran yang memainkan

peranan tersendiri (Sillem 2005).

Ciri-ciri sosial yang penting adalah

interaksi dengan manusia setempat, tahap

komitmen terhadap tugasan, peranan dan

tanggungjawab ke atas bangunan. Penilaian

insidens telah menunjukkan bahawa semasa

situasi kecemasan, manusia akan lebih

cenderung untuk bekerjasama berbanding

dengan bertindak secara sendirian (Galea et al.

2007). Ciri kebiasaan terhadap susun atur

bangunan. Kebiasaan susun atur bangunan

adalah berhubungkait dengan tingkah laku

pemilihan jalan keluar dalam keadaan

kecemasan. Penghuni kebiasaannya akan

memilih pintu keluar utama. Penghuni

biasanya akan mengungsikan bangunan

berdasarkan jalan yang biasa dilalui dan laluan

keluar utama iaitu sebagaimana mereka

memasuki bangunan (Kobes et al. 2010).

KESIMPULAN

Aspek keselamatan bangunan sangat penting

terutamanya berkaitan bangunan yang dibina

secara beringkat-tingkat tingginya. Dalam

aspek keselamatan bangunan jika berlakunya

kebakaran, kebolehupayaan penghuni untuk

menyelamatkan diri dengan selamat adalah

penting. Secara umumnya, tiga faktor yang

utama terlibat dalam menentukan tahap

pencapaian tindakan kecemasan semasa

bangunan terbakar iaitu ciri-ciri kebakaran,

manusia dan bangunan. Manakala tiga faktor

keberkesanan dalam perancangan kecemasan

bagi sesebuah organisasi pasukan tindakan

kecemasan adalah, pertama, pemahaman

persekitaran bangunan, luar dan dalam, kedua

ialah keperluan penghuni dan faktor ketiga

adalah latihan yang berterusan untuk

meningkatkan kecekapan ahli pasukan

tindakan kecemasan di samping penyebaran

maklumat untuk mendidik dalam kalangan

penghuni bangunan berkaitan bahaya

kebakaran.

PENGHARGAAN

Penghargaan kepada semua warga kampus

Kuala Lumpur di atas kerjasama yang terlibat

dalam menjayakan kajian ini.

RUJUKAN

Benthorn, L. & Frantzich, H. 1996. Fire Alarm in

a Public Building: How Do People Evaluate

Information and Choose Evacuation Exit?

Department of Fire Safety Engineering.

Sweden: Lund University.

Bruck, D. 2001. The who, what, where and why of

waking to fire alarms: A Review. Fire Safety

Journal 36: 623–639.

Galea, E.R., Shields, J., Canter, D., Boyce, K., Day,

R., Hulse, L., Siddiqui A., Summerfield, L.,

Marselle, M. & Greenall, P.V. 2007. The UK

WTC 9/11 Evacuation study: Methodologies

ued in the elicitation and storage of human

factors data; In: Interflam 2007, Conference

Proceedings, vol. 1. 11th International Fire

Science and Engineering Conference.

Interscience, London. pp. 169–181.

Kobes, M. Heslot, I. de Vries, I & Post, G,J. 2010.

Building safety and behavior in fire: A

literature review. Fire Safety Journal. 45: 1-11.

Tubbs, J.S. 2004. Developing trends from deadly

fire incidents: A preliminary assessment,

ARUP: Westborough, MA.

Purser, D.A. 2003. Data benefits: Fire prevention.

Fire Engineers Journal 21: 21–24.

Proulx, G. 1993. A stress model for people facing

a fire. Journal of Environmental Psychology

13: 137–147.

Raubal, M. & Egenhofer, M.J. 1998. Comparing

the complexity of way finding tasks in built

environments. Environment and Planning B

25: 895–913.

44

Sime, J.D. 1990. The concept of ‘panic’: Fires and

human behaviour. 2nd Ed. London: David

Fulton Publishers Ltd.

UBBL 1984, Uniform Building by Law 1984.

Anuar Ithnin*

Environmental Health & Industrial Safety Programme,





50300 Kuala Lumpur,

Malaysia.


Buletin FSK 1(1)(2017): 45-54

The Chromagen Lens: Its Effects on Colour Vision Performance in

Congenital Colour Vision Deficients.

KOO SIO CHING, NURUL FARHANA SHARIFUDIN, MIZHANIM MOHAMAD

SHAHIMIN* & SHARANJEET KAUR.

ABSTRACT

The ChromaGen lens is a coloured filter lens designed to enhance colour perception and

contrast in colour vision deficiency (CVD) subjects. The study is aimed to determine the

effects of the ChromaGen lens on colour perception, tested with the Ishihara plates and the

D-15 test on participants with congenital colour vision deficiency. Ten CVD participants,

all males (aged=26.6±3.3 years) participated in this study. This experiment takes form of

pre and post design. Pre-examination includes patients’ visual status (VA, binocular vision,

contrast sensitivity, colour vision status using the Ishihara plates and the D-15 test).

Participants were grouped based on the type of colour vision deficiencies using the Neitz

anomaloscope. All participants had optimum correction with visual acuity of at least 6/6 or

better on both eyes. During post-examination, all participants were required to wear the

best subjectively-chosen ChromaGen lenses that gave optimum effect on participant’s

colour perception. All tests in pre-examination session were repeated. Data were collected

and analysed using Török11 and SPSS 20.0 softwares. Participants were grouped into

deutan and protan CVD groups. In pre-examination session, all participants failed the

Ishihara test and only deuteranomaly group passed the D-15 test initially. In post-

examination session (wearing the ChromaGen lens), there were significant reduction in the

error rates in the Ishihara test (p<0.005, Wilcoxon Signed Rank test), especially for the

deutan CVD group. However, only 5 out of 8 deutan participants (3 deuteranomaly and 2

deuteranopia) passed the Ishihara test with the use of the ChromaGen lens. While for the

D-15 test, overall there was significant reduction of error score (p<0.005) reported

especially in the group of deuteranopia with the ChromaGen lens wear, although only two

out of seven participants (2 deuteranopia) who initially failed the D-15 test passed the test

with the ChromaGen lens. There were no significant differences reported in contrast

sensitivity and visual acuity with the ChromaGen lens. The ChromaGen lens can enhance

colour vision performance of the Ishihara and the D-15 test in CVD patients without

affecting participant’s visual acuity and contrast sensitivity. Therefore, the ChromaGen

lens could serve as an option to help increase colour vision perception in CVD patients.

Keywords: ChromaGen lens; congenital colour vision deficiency; Ishihara test plate;

Farnsworth Munsell D-15

46

INTRODUCTION

Colour vision deficiency (CVD) is an

inability to perceive certain colour. Colour

vision deficiency can be classified either as

congenital or acquired form. Usually,

colour deficiency is an inherited condition

caused by a common X-linked recessive

gene, which is passed from a mother to her

son/daughter. However, disease or injury

damaging the optic nerve or retina can also

result in loss of colour recognition

(Fletcher & Voke 1985, Simunovic 2010).

Previous studies have shown that 8% of

male and 0.5% of female with congenital

colour vision deficiency (Simunovic 2010).

This colour vision defect can be examined

by using colour vision test instrument

including the Ishihara plate, the

Farnsworth Munsell D-15 and the

Farnsworth-Munsell 100 Hue.

Unfortunately, there is no known

treatment to cure this colour vision

deficiency (Hovis 1997) but there are

several ways in helping people with this

problem in their routine activities. People

with CVD especially children, may

experience difficulty during their study

years. Therefore, their parents and teachers

should help these children by giving

counselling advices and give more

attention to them so that they are not losing

interest in their studies. Recently, there are

special coloured lenses available in the

market known as the ChromaGen lens that

may help to improve contrast and enhance

colour perception in CVD patients (Harris

1997).

The ChromaGen lens is a coloured

filter lens that used to enhance colour

perception as well as help in improving

contrast and colour differentiation in CVD

patients. The ChromaGen lens system

comprises of eight colours in the form of

filter and contact lens, in which each of the

colour has a specific colour wavelength

(Harris 1997, Oriowo & Alotaibi 2011,

Swarbick et al. 2001). The selection for the

most suitable ChromaGen lens to be worn

is depends on the subjective response of the

participant. The colour vision performance

with ChromaGen is tested using the

Ishihara test plates, the Farnsworth

Munsell D-15 or the Farnsworth Lantern

test. A study by Swarbrick et al. (2001)

found that the rate of errors in the Ishihara

plate test can be reduced significantly with

the use of the ChromaGen lenses especially

in participants with deutan colour defect. In

addition, the error rate was reduced in the

test Farnsworth Munsell D-15. However,

the application of the ChromaGen lens

does not have a significant impact on the

Farnsworth Lantern test.

A study by Oriowo and Alotaibi

(2011) also suggested that subjectively, 85%

reported some enhancement in colour

perception with the ChromaGen lens,

while 15% of participants did not showed

any difference. Therefore, this study seeks

to find the effect of the ChromaGen lens on

congenital deutan and protan colour vision

deficiencies through Ishihara and D-15

tests.

METHODOLOGY

PARTICIPANTS

Ten participants with previously diagnosed

congenital colour vision defect (deutan or

protan) participated in this study.

Participants were all male, age ranged

between 17 and 48 years old

(aged=26.6±3.3 years). All participants

gave written informed consent prior to

participation. Participants had VA of 6/6 or

better on both eyes and free from any

binocular vision problems, ocular and

systemic diseases.

47

PROCEDURES

For the preliminary examination, history

taking and preliminary test were conducted

to confirm that the participant fulfilled all

the fixed criteria. Participants were

questioned on history of previous systemic

or ocular disease or long-term use of

medication to rule out any acquired colour

defects. Preliminary test includes entrance

tests, refraction, binocular vision test, slit-

lamp examination and ophthalmoscopy.

PRE-TEST

After participants had been confirmed to

have CVD, contrast sensitivity was tested

using Pelli-Robson contrast sensitivity

chart. Type and severity of colour vision

defect were tested using the Ishihara plate

and the D-15 test. The Ishihara plate and

the D-15 test are colour discrimination

tasks and were selected as both of the

colour vision tests are widely used for

colour vision screening purposes. The

Ishihara test was tested at a reading

distance of approximately 60 to 70cm from

participant’s eyes. Participants were tested

on their ability to correctly determine the

number or pattern from a series of plates

with different coloured dots. The D-15 test

consists of 15 different coloured caps.

Participants would have to arrange the

coloured caps according to the reference

cap’s hue. Both colour vision tests were

conducted under binocular vision and

under non-flickering fluorescent light. The

Neitz anomaloscope was used to reconfirm

and subdivided the type of colour vision

defects into dichromat and anomalous

trichromat (Fletcher & Voke 1985).

CHOOSING THE SUITABLE

CHROMAGEN LENS TINT

After the type of colour vision defect was

diagnosed, ocular dominance test, i.e. Mile

test was conducted to determine the

dominant and non-dominant eye of each

participant. Different ChromaGen trial

lenses were first placed in front of the non-

dominant eye of the participant. The most

suitable type of the ChromaGen lens was

chosen based on subjective response of

participant to the Ishihara plate, by

counting the total number of error plates.

The dominant eye was tested again with the

same procedure. If there is no improvement

with the ChromaGen lens placed in front of

the dominant eye, no lens will be added in

front of it.

POST TEST

For the post examination, the Ishihara test

and the D-15 test was conducted with the

ChromaGen lens on, with the pre-selected

tint chosen during the first visit. Both the

Ishihara and the D-15 test were conducted

under binocular viewing condition. Visual

acuity test and contrast sensitivity test were

also conducted with the ChromaGen lens

on.

ANALYSIS

Results of the D-15 test were analysed

using Török system11. The improvement of

colour vision status in this research was

based on changes of error score in the

Ishihara test and the D-15. All data were

collected and analysed using SPSS 20.

48

RESULTS

LENS TINT SELECTION

The ChromaGen lens selected for each

participant is summarized in Table 1. Nine

out of ten participants preferred a binocular

use of the ChromaGen lenses. Only one

participant (deuteranomaly) reported no

benefit from a second lens during lens

selection. For the non-dominant eye, five

of the participants selected magenta (M),

four participants chose pink (P) and one of

them selected blue (B) tint. All participants

requested the darkest tint density.

TABLE 1. The ChromaGen lens tints selected by each participant for non-dominant and

dominant eye.

Participant CVD Dominant eye ChromaGen lens selected

Non-dominant

eye

Dominant eye

1 Deuteranomaly R M3 M3

2 Deuteranomaly R P3 M3

3 Deuteranomaly L M3 No lens

4 Deuteranopia R M3 M3

5 Deuteranopia L M3 M3

6 Deuteranopia R P3 M3

7 Deuteranopia L M3 M3

8 Deuteranopia L P3 M3

9 Protanopia L B1 M3

10 Protanopia R P3 P1

M=magenta, P=pink, B=blue; 1=lightest density, 3=darkest density

COLOUR VISION TEST

PERFORMANCE.

ISHIHARA PLATE

Pre-test revealed all participants failed the

Ishihara test with 8 participants failed all

plates (80%), one participant made 12

mistakes (10%) and another one participant

had 10 errors (10%). These errors were

significantly reduced on the post-test using

the ChromaGen lenses. The errors on

Ishihara test after wearing ChromaGen lens

were between 0 to 12 errors. Although

there was a decline in the rate of error in

the Ishihara test after wearing the

ChromaGen lens, only five out of 10

participants (50%) passed the test with the

use of the ChromaGen lens as shown in

Figure 1.

49

FIGURE 1. Error rate out of maximum 17 for Ishihara test (1990 edition) before and after

wearing ChromaGen lens. The horizontal dash line represents the pass and fail of the test.

TABLE 2. Effect of ChromaGen lens on Ishihara test in overall and based on each type

of colour vision deficiency by using Wilcoxon Signed Rank test.

N z p-value

Overall 10 -2.807 0.005

Deuteranomaly 3 -1.604 0.109

Deuteranopia 5 -2.032 0.042

Protanopia 2 -1.342 0.180

The highest percentage of improvement in

Ishihara test after wearing the ChromaGen

lens is deuteranomaly with 94.1% while

the lowest percentage of improvement with

29.4% is in one deuteranopia participant

and another one is protanopia. Overall,

deutan colour vision defect shows higher

percentage of improvement than protan.

The overall results shown in Table 2

presents the effect of the ChromaGen lens

on participant of colour vision deficiency

were statistically significant (p<0.05).

However, based on classification of colour

vision deficiency, deuteranopia showed

significant reduction in error rate (p=0.042)

after wearing ChromaGen lens.

50

FARNSWORTH MUNSELL D15

Results of D-15 colour vision test were

recorded as the cap order on the standard

scoring template, taking into account the

maximum number of crossing as 12. The

order of the cap’s number was then

interpreted using Torok system23.

Comparison was made before and after

wearing ChromaGen lens which includes

number of crossings, total error score

(TES), total colour differences score

(TCDS), confusion index (CI) and

selective index (SI). The value of angle

was used for classifying the types of colour

vision defect with and without ChromaGen

system. All results were analysed using

Wilcoxon Signed-Rank test. All

deuteranomalous participants passed the

D-15 test at baseline, while the rest of

participants failed the test (>1 crossing)

(the fail rate in D-15 test depends on the

number of crossings).

Overall, there are significant

differences between with and without

wearing the ChromaGen contact lens

(p<0.05; see Table 3), although only two

participants who initially failed in D-15

test passed with wearing ChromaGen

contact lens (2 deuteranopia). However, for

the results according to types of colour

vision defect, only deuteranopia showed a

significant improvement with ChromaGen

contact lens. Although there is reduction in

error rates in D-15 test for deuteranomaly

and protanopia, the changes are not

significant. Both protanopia participants

showed change of the type of colour vision

defect from protan to deutan which

evaluated based on the circular diagram

and angle after wearing the ChromaGen

lens. These findings are summarized in

Table 4 and 5.

TABLE 3. Wilcoxon Signed Rank test: Overall effect of ChromaGen lens to the D-15

test

Comparison between

before and after

wearing ChromaGen

lens

Evaluation of D-15 N z P value

Number of crossing 10 -2.201 0.028

TES 10 -2.521 0.012

TCDS 10 -2.521 0.012

Angle 10 -0.421 0.674

CI 10 -2.521 0.012

SI 10 -2.380 0.017

TES = Total error score, TCDS = Total colour differences score, CI = Confusion Index,

SI = Selective Index

51

TABLE 4. Wilcoxon Signed Rank test: Effect of ChromaGen lens to the D-15 test based

on types of colour vision defect.

Comparison

before and after

wearing

ChromaGen lens

Types of colour

vision defect

Evaluation of D-15 N Z P value

Deuteranopia Number of crossing 5 -2.023 0.043

TES 5 -2.023 0.043

TCDS 5 -2.023 0.043

Angle 5 -0.677 0.498

CI 5 -2.023 0.043

SI 5 -2.023 0.043

Deuteranomaly Number of crossing 3 0.000 1.000

TES 3 -1.000 0.317

TCDS 3 -1.000 0.317

Angle 3 -1.000 0.317

CI 3 -1.000 0.317

SI 3 -1.000 0.317

Protanopia Number of crossing 2 -1.000 0.317

TES 2 -1.342 0.180

TCDS 2 -1.342 0.180

Angle 2 -1.342 0.180

CI 2 -1.342 0.180

SI 2 -1.342 0.180



RESULT OF VISUAL ACUITY AND

CONTRAST SENSITIVITY

Our findings showed that there were no

significant effect of the ChromaGen lens

wear on participants’ visual acuity and

contrast sensitivity (Table 6).

DISCUSSION

PERFORMACE ON ISHIHARA PLATE

Our study proved that the ChromaGen

lenses can improve colour vision

performance on the Ishihara plate test,

especially for participants with deutan

colour vision defect. These finding is

supported by the previous research which

is done by Swarbrick et al. 2001, that

ChromaGen lens significantly reduce

Ishihara error rate, particularly for deutan

participants.

Comparison between deutan and

protan CVD showed that percentage of

improvement in the Ishihara test after using

ChromaGen lenses is higher in deutan by

almost 90% while protan is about 40%.

Oriowo and Alotaibi 2011 demonstrated

that deuteranopia improved by 70%,

followed by deuteranomaly with 33% and

protanopia by 24% on Ishihara test using

ChromaGen lens.

52

TABLE 5. Error rates of the D-15 test (mean ± SD) before and after wearing ChromaGen

lens.

Overall

(n=10)

Deuteranopia

(n=5)

Deuteranomaly

(n=3)

Protanopia

(n=2)

Before ChromaGen lens

Number of

Crossing

6.60±1.507 10.00±0.707 0.00±0.000 8.00±1.00

TES 29.950±3.973 37.820±1.1634 12.067±0.667 37.100±3.100

TCDS 299.56±40.29 388.900±16.008 121.600±4.600 343.15±32.55

Angle 7.850±9.383 -9.940±1.454 37.333±24.667 8.100±4.500

CI 3.099±0.461 4.0320±0.13691 1.020±0.0200 3.885±0.305

SI 4.209±0.751 6.0820±0.59159 1.2767±0.1033 3.925±0.325

After ChromaGen lens

Number of

crossing

3.20±1.031 3.60±1.327 0.00±0.000 7.00±0.00

TES 21.110±2.988 23.02±3.927 11.40±0.00 30.900±1.100

TCDS 203.70±27.696 121.60±4.600 117.00±0.00 295.90±1.900

Angle 10.63±14.398 -10.260±18.879 62.00±0.00 -14.200±9.900

CI 2.102±0.321 2.322±0.454 1.00±0.00 3.205±0.145

SI 2.451±0.350 2.746±0.518 1.3800±0.00 3.320±0.350



TABLE 6. The effect of ChromaGen lens on visual acuity and contrast sensitivity.

PERFORMANCE ON D-15 TEST

Performance of colour vision was tested

using the D-15 test and the improvement

was evaluated by taking into account the

reduction in error rates for number of

crossing, TES, TCDS, CI and SI (Vingrys

& King-Smith 1988). The angle of axis of

confusion, was used to classified the types

of colour vision defects only. Overall,

results for the D-15 test showed that there

was improvement in performance of colour

vision especially in the group of

deuteranopia with ChromaGen contact lens

wear, although only two out of seven

participants (2 deuteranopia) who initially

failed the D-15 test passed the test with

ChromaGen lens. While for the

deuteranomaly group (in which the whole

group passed the D-15 test initially; <2

crossings) and protanopia (in which the

whole group failed in D-15 test initially; >2

crossings), the reduction of error rates was

not significant. These finding was in

accordance to a research by Swarbrick et

al. (2001) which showed that deutan

improved more in the performance of

Comparison before

and after wearing

ChromaGen lens

Test N z P-value

Visual acuity 10 0.00 1.00

Contrast sensitivity 10 0.00 1.00

53

colour vision with the ChromaGen lens

compared to protan.

In other perspective, the results for

the angle of the axis of confusion, which

used to indicate the types of colour vision

defect showed overall shift in the

confusion axis towards the deutan defect

(including deuteranope and the protanope

participants). This situation has been

reported previously with the use of red

tinted filter by Mutalib et al. (2012) on

FM100 Hue test, X-Chrome lens (Paulson

1980) on Hardy-Rand-Rittler

Classification (Paulson 1980) and long

wavelength bandpass filter by Hovis (1997)

on D-15 test.

Results suggest that deuteranope

participants are likely to show greater

effect than protanopes from the D-15

colour vision test. A study performed with

red filter on D-15 test found that deutan

participants might derive more useful

luminance information from the use of red

filters, while protan more reliant on

chromaticity cue which causes poorer

performance due to confusion (Richer &

Adam 1984).

EFFECT ON CONTRAST

SENSITIVITY AND VISUAL ACUITY

The Pelli-Robson chart was used to test the

contrast sensitivity for the participants

while the Snellen chart was used to test

visual acuity. Results showed that there

were no changes in contrast sensitivity and

visual acuity with and without wearing the

ChromaGen lenses under normal lighting

condition. These findings are similar to the

research from Utine et al. (2012) which

uses contrastometer to test the effect of

ChromaGen lens on contrast sensitivity.

However, in terms of subjective response,

four out of ten participants (1 deuteranopia,

1 deuteranomaly and 2 protanopia)

complained of blurring vision after

wearing ChromaGen lens. Swarbrick et al.

(2001) noted there was slight difficulty

with vision in dim light especially in

deutan group, although the difference was

not statistically significant.

CONCLUSION

In generally, the ChromaGen lens system

improves the performance of colour vision

by reducing the error rates in the Ishihara

plate and the D-15 especially in

deuteranope participants. In addition of

enhancing colour vision perception, results

showed that ChromaGen system does not

give any effect in terms of contrast

sensitivity and visual acuity under normal

lighting condition. The ChromaGen

system may be worn by congenital CVD

patients, especially deuteranopes to

increase colour vision performance in daily

living activities which required colour

discrimination task.

REFERENCES

Fletcher R, Voke J. ‘Defective Colour Vision,

Fundamentals, Diagnosis, and

Management’. Hilger, Institute of

Physics, Bristol. 1985.

Harris D. ‘Colouring Sight: a study of Contact

Lenses fittings with colour-enhancing

lenses’. Optician 1997; 213(5604): 38-

41.

Hovis JK. ‘Long Wavelength Pass Filters

Designed for the Management of

Color Vision Deficiencies’. Optometry

& Vision Science 1997; 74: 222-230.

Mutalib HA, Sharanjeet K, Keu LK, Choo PF.

‘Special Tinted Contact Lens on

Colour-Defects’. La Clinica

Therapeutica 2012; 163(3): 199-204.

Oriowo OM, Alotaibi AZ. ‘ChromaGen lenses

and abnormal colour perception’. The

South African Optometrist 2011;

70(2): 69-74.

Paulson HM. ‘The X-Chrome Lens for

Correction of Color Deficiency’.

54

Military Medicine 1980; 145: 557-

560.

Richer S, Adam AJ. ‘An Experimental Test of

Filter-Aided Dichromatic Color

Discrimintation’. American Journal of

Optometry c& Physiological Optics

1984; 61: 256-264.

Simunovic, M.P. ‘Colour vision deficiency’.

Eye. (2010)

https://doi.org/10.1038/eye.2009.251

Stresing D. ‘Color Blindness (Color Vision

Problems; Color Vision Deficiency)’.

EBSCO Publishing. 2011 [cited 2012

September 17] Available from:

http://healthlibrary.epnet.com/print.as

px?token=de6453e6- 8aa2-4e28-

b56c-

5e30699d7b3c&ChunkIID=102895

Swarbick HA, Nguyen P, Nguyen T, Pham P.

‘The ChromaGen Contact Lens

System: Colour Vision Test Results

and Subjective Responses’.

Ophthalmic Physiological Optics

2001; 21(3): 182-196.

Török B. Panel D-15 Test (Version: December

2006). Available from:

http://www.torok.info/colorvision/d15

.htm.

Utine CA, Dinç UA, Çiftçi F. ‘Assessment of

Contrast Sensitivity Changes

Secondary to ChromaGen Contact

Lens Application in Patients with

Congenital Colour Deficiency’.

Yeditepe University, Department of

Ophthalmology, Istanbul, Turkey.

2012.

Vingrys AJ, King-Smith PE. ‘A Quantitative

Scoring Technique for Panel Tests of

Color Vision’. Investigative

Ophthalmology & Visual Science

1988; 29: 50-63.

Koo Sio Ching

Nurul Farhana Sharifudin

Mizhanim Mohamad Shahimin*

Sharanjeet Kaur.

Optometry and Vision Sciences Programme

Centre of Health Care Sciences



Kuala Lumpur,

Malaysia.


Buletin FSK 1(1)(2017): 55-59

Perencatan Bakteria Escherichia coli oleh Mukus Epidermis Clarias

batrachus (Inhibition of Escherichia coli by the epidermal mucus extract of Clarias batrachus)

AHMAD ZORIN SAHALAN* & NAZAHIYAH SULAIMAN

ABSTRAK

Mukus epidermis ikan kini telah dikenalpasti sebagai suatu sumber biologi yang kaya dengan

pelbagai bahan bioaktif terutamanya peptida antibakteria. Kajian ini dilakukan bagi mengenalpasti

potensi mukus epidermis Clarias batrachus (ikan keli kayu) sebagai sumber kepada bahan

antibakteria. Sampel mukus dikumpul dari epidermis C. batrachus dan diekstrak menggunakan

kaedah pengekstrakan akues. Ujian penentuan aktiviti antibakteria ekstrak kasar mukus epidermis

C. batrachus dilakukan dengan menggunakan kaedah spektrofotometrik. Hasil kajian

menunjukkan terdapatnya aktiviti antibakteria oleh ekstrak kasar mukus dengan kepekatan 20

mg/ml terhadap pertumbuhan kedua-dua strain bakteria kajian, iaitu Escherichia coli ATCC

25922. Hasil kajian ini menyokong peranan mukus sebagai salah satu sistem pertahanan badan

serta komponennya, iaitu peptida antibakteria sebagai komponen utama dalam sistem pertahanan

badan semulajadi. Justeru, penemuan ini boleh dijadikan sebagai salah satu langkah pertama ke

arah pembangunan antibiotik kelas baru.

Kata kunci: Clarias batrachus; ikan keli kayu; mucus; epidermis; antibakteria.

ABSTRACT

Fish epidermal mucus have been identified as a potential biological source enriched with various

bioactive materials, especially antibacterial peptides. This research is carried out to determine the

potential of Clarias batrachus mucus as a source of antibacterial component. The mucus from the

cat fish was collected from its epidermal skin surface and extracted by using aqueous extraction

methods. The antibacterial activity was determined by using spectrophotometric method. This

study showed the presence of antibacterial activity from crude mucus epidermal extract at the

concentration of 20 mg/ml against Escherichia coli ATCC 25922. This result supports the role of

mucus as part of fish immune system with the antibacterial peptide acting as the main component

in innate immunity system. Hence, these findings offer a step towards the development of a new

class antimicrobial as a panacea of the resistance towards antibiotics in market.

Key words: Clarias batrachus; cat fish; epidermal mucus; antibacterial

PENDAHULUAN

Haiwan marin khasnya ikan mempunyai

potensi tinggi sebagai sumber agen

antimikrob kerana ianya mempunyai sistem

pertahanan perumah yang teguh bagi

membolehkan ia kekal hidup di dalam

persekitaran akuatik yang kaya dengan

patogen bawaan air (Elis 2001). Sebagai

barisan pertahanan yang pertama, mukus

epidermis ikan merupakan salah satu

komponen sistem imun semulajadi.

Beberapa kajian saintifik mendapati bahawa,

di dalam mukus epidermis ikan terdapat

bahan bioaktif imun semulajadi seperti,

enzim proteolitik, lektin, lisozim,

56

flavoenzim, immunoglobulin, protein C-

reaktif dan peptida antibakteria yang

melindungi ikan daripada risiko jangkitan

patogen akibat pendedahan yang berterusan

terhadap mikroorganisma (Safari et al.,

2016; Whyte 2007; Subramaniam et al.

2008). Ikan keli kayu atau Clarias batrachus

(Yusri Atan, 2012) merupakan spesies ikan

keli yang banyak ditemui di rantau Asia

Tenggara. Ia merupakan spesies ikan yang

tidak mempunyai sisik, justeru ia bergantung

sepenuhnya kepada mukus epidermis sebagai

pelindung mekanikal pertama terhadap

patogen akuatik.

Potensi ikan keli kayu sebagai sumber

produk bioaktif dari segi industri hiliran

bioteknologi masih kurang dikaji serta

diusahakan. Adalah menarik jika mukus ikan

keli kayu di kaji akan keberkesanannya

dalam mengawal serta menghindari infeksi

bakteria dan hasil daripada kajian akan

mengenengahkan bahan bioaktif yang novel

untuk kajian selanjutnya dalam bidang

perubatan mahupun jagaan kesihatan. Oleh

kerana itu, kajian terhadap kesan mukus

daripada epidermis Clarias batrachus

terhadap bakteria Escherichia coli di kaji.

Bakteria ini merupakan patogen daripada air

tercemar dan mengakibatkan jangkitan

intestinal serta STEC atau E. coli penghasil

shiga toxin (Karlowsky et al. 2013).

BAHAN DAN KAEDAH

Sampel Kajian dan Kultur Bakteria

Ujian

Clarias batrachus atau ikan keli kayu

diperolehi daripada penternak ikan keli di

Ulu Langat. Umur ikan keli yang digunakan

adalah antara 4-5 bulan.

Bakteria Escherichia coli (ATCC 29522)

yang digunakan di dalam kajian ini diperoleh

daripada Makmal Sains Bioperubatan,

Fakulti Sains Kesihatan,

Persampelan Ikan dan Pengumpulan

Sampel Mukus (Nagashima et al. 2001)

Ikan keli kayu yang masih hidup dibersihkan

terlebih dahulu dengan air suling steril.

Sebanyak 500 ml air suling steril dimasukkan

ke dalam bekas berisi ikan. Ikan dibiarkan

semalaman di dalam bilik sejuk (4°C).

Permukaan badan ikan yang telah mati

dibilas dengan air suling dan mukus

dikumpulkan dengan menggunakan spatula.

Mukus yang telah dikumpulkan

kemudiannya diempar pada 2500 rpm selama

10 minit pada suhu 4°C. Supernatan

dikumpulkan dan seterusnya diempar pada

60 000 rpm selama 10 minit pada 4°C.

Supernatan disimpan pada suhu -40°C bagi

mengelakkan kerosakan pada sampel yang

disebabkan oleh enzim.

Fasa Pertumbuhan Bakteria Yang

Digunakan Dalam Ujikaji

Bakteria yang digunakan untuk ujikaji ini

telah diselaraskan hanya kepada bakteria

pada pertumbuhan fasa log awal sahaja. Pada

peringkat fasa ini umur bakteria masih lagi

muda dan baru di mana ianya sesuai untuk

dijalankan kajian anti-bakteria (Ahmad Zorin

et al. 2000). Untuk mendapat pertumbuhan

fasa log awal ini, bakteria perlulah di kultur

terlebih dahulu di dalam medium kaldu

nutrient serta dieram pada suhu 37oC.

Kemudian pertumbuhan bakteria akan di

catat setiap 10 minit dengan mengunakan

spektrofotometer pada jarak gelombang

625nm.

Penentuan Aktiviti Antibakteria (Patton

et al. 2006)

Satu suspensi kaldu bakteria disediakan dan

dieram selama 3 hingga 4 jam pada suhu

37°C untuk mendapatkan pertumbuhan

bakteria pada fasa pertumbuhan log awal.

Kemudian, kaldu ini diempar pada kelajuan

57

4000 rpm selama 10 minit dengan

menggunakan tiub ependorf. Supernatan

disingkirkan dari tiub pengempar dan palet

bakteria diampaikan dengan memasukkan 10

ml larutan penimbal salin berfosfat (PBS) 0.1

M steril. Ampaian bakteria yang terhasil

disetarakan kepekatannya pada bacaan 0.5

OD625 .

Ampaian bakteria yang disediakan

kemudian dibahagikan kepada tiga bahagian

iaitu kawalan negatif, kawalan positif dan

ujian. Kawalan positif mengandungi 40ug

Gentamycin bersama bakteria ujian,

manakala untuk kawalan negatif hanya

ampaian bakteria sahaja yang ada di dalam

kelalang. Kelalang ujian pula mengandungi

mukus bersama bakteria ujian. Kesemua

kelalang tersebut dimasukkan ke dalam

mandian air yang bergoncang pada 50 psm

(pusingan seminit). Pada selang masa 10, 20,

30, 40, 50 dan 60 minit, sebanyak 1 ml

sampel akan diambil pada setiap kelalang

bagi setiap masa tersebut. Sampel yang di

ambil akan dimasukkan ke dalam kuvet dan

diuji akan kepekatan bakteria yang ada pada

jarak gelombang 625 nm.

Analisa Kehadiran Antibakteria di

Dalam Mukus

Cerapan daripada data spektrofotometer

(OD625) pada selang masa 10, 20, 30, 40, 50

dan 60 minit (termasuklah untuk ujian,

kawalan positif dan kawalan negatif) di

plotkan membentuk graf kepekatan bakteria

pada penyerapan OD625 melawan masa.

Maka sebarang penurunan atau kenaikan

terhadap kepekatan atau jumlah bakteria

dianalisa sebagai kesan perencatan,

bakterisidal ataupun tiada kesan bakterisidal.

HASIL

Ujian Penentuan Aktiviti Antibakteria

Ekstrak Mukus Epidermis C. batrachus

ke atas Escherichia coli.

Bacaan OD bagi kawalan negatif

menunjukkan bacaan antara 0.80 pada minit

0 kepada 0.833 pada minit ke-60. Manakala

kawalan positif ddengan antibiotic

Gentamycin pula memberikan penurunan

bacaan penyerapan secara mendadak, iaitu

daripada 0.80 pada minit ke-0 kepada 0.502

pada minit ke-60.

Seterusnya, bagi ekstrak mukus

epidermis C. batrachus, bacaan OD yang

diperolehi didapati mengalami penyusutan

daripada minit ke-0 sehingga minit ke-60,

iaitu 0.80 dan akhirnya menurun 0.6 (Rajah

1).

PERBINCANGAN

Mukus epidermis Clarias batrachus sebagai

sumber kepada bahan antibakteria telah

dikaji. Mukus yang telah dipencil melalui

kaedah pengesktrakan akues (Nagashima et

al. 2001) ini diuji akan aktivit antimikrobnya

dengan kaedah ujian mengunakan

spektrofotometer. Kaedah ini dipilih kerana

ujian ini menyediakan kaedah yang lebih

ringkas, cepat dan sensitif (Giardino et al.

2016, berbanding dengan kaedah

konvensional iaitu resapan telaga atau

resapan cakera (Patton et al. 2006).

58

RAJAH 1. Kepekatan bakteria OD625 melawan masa (minit) menunjukkan aktiviti antibakteria

ekstrak mukus epidermis C. batrachus terhadap terhadap E. coli. Kawalan negatif mengandungi

suspensi bakteria sahaja manakala kawalan positif adalah campuran suspensi bakteria dengan 40ug

Gentamycin.

Hasil utama kajian ini mendapati

bahawa kadar pertumbuhan E. coli telah

menurun apabila didedahkan terhadap mukus

C. batrachus berbanding dengan kawalan

negatifnya iaitu bakteria tanpa mukus. Kesan

penurunan pertumbuhan atau penurunan

bilangan bakteria ini bermakna bahawa sel

bakteria telah termusnah atau lisis sel berlaku

(Ahmad Z. Sahalan et al., 2008) yang

mengakibatkan bacaan penyerapan

ketumpatan optikal spetrofotometer atau OD

(“optical density”) itu menurun secara

berperingkat atau berurutan. Ianya berlaku

sebaik sahaja bakteria yang diuji ini terdedah

kepada mukus dalam sela masa satu jam.

Kesimpulan daripada pemerhatian ini

menunjukan bahawa adanya komponen

bioaktif di dalam mukus yang menyebabkan

kesan bakterisidal. Selain itu, kesan ini juga di

sokong oleh kawalan positif ujikaji di mana

Gentamycin (40 ug) memberikan gambaran

lengkuk penurunan terhadap pertumbuhan atau

bilangan bakteria yang hampir sama dengan

mukus, walaupun kesan penurunan yang

disebabkan oleh Gentamycin memberikan

penurunan yang sangat ketara atau lebih

bakterisidal. Ujian untuk kawalan positif ini

menjadi rujukan kepada mukus ikan C.

Batrachus bahawa kehadiran bahan bioaktif

seakan antibiotik yang mampu menglisiskan

sel bakteria Gram negatif khasnya E. coli dan

seterusnya mampu mengawal infeksinya.

KESIMPULAN

Kajian ini menilai keberkesanan antibakteria

mukus ikan keli Clarias batrachus terhadap

E. coli. Sebagai kesimpulannya, hasil ujikaji

menunjukkan bahawa terdapat kehadiran

bahan antibakteria di dalam mukus ikan keli

Clarias batrachus yang memberikan kesan

bakterisidal terhadap bakteria yang diuji.

PENGHARGAAN

Penghargaan kepada Gran No. Industri 2013-

049 dan juga Makmal Sains Bioperubatan

PPSDKG, Fakulti Sains Kesihatan, UKM,

khasnya pada Pn Mariahyati Abu Bakar dan

En. Faisal Ariffin.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 20 40 60 80

Ke

pe

kata

n b

akte

ria

OD

62

5

Masa (minit)

kawalan -ve

kawalan +ve

Mukus ikan

59

RUJUKAN

Ahmad Z. Sahalan, Ronald A. Dixon. (2008) Role

of the cell envelope in the antibacterial

activities of polymyxin B and polymyxin

B nonapeptide against Escherichia coli.

International Journal of Antimicrobial

Agents. 31 224–227.

Ahmad Zorin, S., Mohd. Zaki, S., Nihayah, M. &

Ridzwan, B.H. 2000. Kesan perencatan

Holothuris edulis terhadap Staphylococcus

aureus dan Escherichia coli. Pascasidang

Simposium Sains Kesihatan Kebangsaan

Malaysia Ke-3, Fakulti Sains Kesihatan

Bersekutu, Universiti Kebangsaan

Malaysia.

Ellis, A.E. 2001. Innate host defense mechanisms

of fish against viruses and bacteria.

Developmental and Comparative

Immunology. 25: 827-839.

Giardino L, Estrela C, Generali L, Mohammadi Z,

Asgary S. 2015. The in vitro Effect of

Irrigants with Low Surface Tension on

Enterococcus faecalis. Iran Endod J.

Summer;10(3):174-8.

Karlowsky JA., denisuikAJ., Legence-Wiens PR.,

Adam HJ., Baxter MR., Hoban DJ., In

Vitro activity of Fosfomycin against E.

coli isolated from patient with urinary tract

infection (UTI) in Canada. Antimicrobial

Agent Chemotherapy. 12:14-16.

Nagashima, Y., Sendo, A., Shimakura, K.,

Kobayashi, T., Kimura, Fujii, T., 2001.

Antibacterial factors in skin mucus of

rabbit fishes. Journal of Fish Biology. 58:

1761–1765.

Patton, T., Barrett, J., Brennan, J. & Moran, N.

2006. Use of a spectrophotometric

bioassay for determination of microbial

sensitivity to manuka honey. Journal of

Microbiological Methods. 64(1): 84-95.

Safari R, Hoseinifar SH, Nejadmoghadam S, Jafar

A. 2016. Transciptomic study of

mucosal immune, antioxidant and growth-

related genes and non-

specific immune response of common carp

(Cyprinus carpio) fed dietary Ferula

(Ferula assafoetida). Fish Shellfish

Immunol.

Subramaniam, S., Ross, N.W. & MacKinnon, S.L.

2008. Comparison of antimicrobial

activity in the epidermal mucus extracts of

fish. Comparative Biochemistry and

Physiology, Part B. 150: 85-92.

Whyte, S.K. 2007. The innate immune response of

finfish – A review of current knowledge.

Fish and Shellfish Immunology. 23(6):

1127-1151.

Yusri Atan. 2012. Ikan di Malaysia. Glosari

Nama sahih Speseis . Buku Perikanan.

Terbitan Institute Jabatan Perikanan

Malaysia.

Ahmad Zorin Sahalan*






50300 Kuala Lumpur,

Malaysia.

Nazahiyah Sulaiman

Jabatan Perkhidmatan Veterinar,

Kementerian Pertanian dan Industri Asas Tani,

62624 Presint 4

Wilayah Persekutuan Putrajaya.

*Corresponding author; email:

[email protected]

Buletin FSK 1(1)(2017): 60-66

Evaluation of Hulu Langat River Water Qualities Using Heavy Metals and

Microbial Indicators

NUR SAKINAH I., HUKIL S, KASWANDI, M.A., SAHALAN, A.Z. & H.L. HING*

ABSTRACT

Drinking polluted water may cause water borne disease like cholera and other serious illnesses.

Water quality can be determined by using physio-chemical and biological parameters of water

and used to assess the water quality related to health of ecosystems, safety of human contact and

drinking water. The physio-chemical parameter determined were pH, temperature, turbidity and

heavy metals. Meanwhile, microbial parameters determined were Total coliform, Fecal coliform,

Escherichia coli and Streptococcus faecalis. Heavy metals such as Al, Fe, Mn and K were

present in all sample of surface water and soil river samples. But at A sampling point of Mersing

River, Mn was present in surface water and soil river. The water pH value of 6.5 was detected at

all sampling points. The temperature range and level of turbidity at all samplings were lower

than INWQS standard. Levels of bacterial contamination at all sampling points were lower than

INWQS standard (Class III/Class IV) except for sampling point A of Mersing River. Correlation

analysis showed that there were significantly strong correlations of heavy metals of Mn between

sample of surface water and soil river at sampling point of Mersing River, rs= 0.756. In

conclusion, composition of heavy metals and microbial indicator at selected branch of Hulu

Langat River was safe for recreational purposes.

Keywords: Heavy metals; microbial indicator; INWQS standard

INTRODUCTION

Water covers up to 71% of the earth’s surface.

On earth, mostly it is found in oceans and

other large water bodies, with 1.6% of water

below ground in aquifers and 0.001% in the

air as vapor. Oceans hold 97% of surface

water, glaciers and polar ice caps 2.4% and

other land surface water such as rivers, lakes

and ponds about 0.6% (Gleick 2000). To

maintain good quality potable water is very

important in order to prevent disease and

improve quality of life especially in rural

populations of the developing countries

(Rengel 2000). In facts, natural water contains

different types of impurities that are

introduced in to the aquatic system by

different ways. These include leaching of

soils and weathering of rocks, dissolution of

aerosol particles from the atmosphere and

from several human activities including

mining, processing and the use of metal based

materials (Adefemi et al. 2010.). In this

research, physio-chemical and biological

parameters of surfaces water samples were

measured at Hulu Langat River by choosing 4

branches of the river; Mersing River, Lopo

River, Congkak River and Joint Branch River

to determine its heavy metal and microbial

indicators using physio-chemical parameters.

All the data and results from this study were

used to determine either water quality at Hulu

Langat River is in safe level based on INWQS

standards or not.

MATERIALS AND METHODS

Physio-chemical parameters such as

temperature, pH of water, turbidity, type as

well as composition of heavy metals were

61

determined. While, biological parameters

include Total coliform, Fecal coliform,

Escherichia coli and Streptococcus faecalis.

These test parameters were conducted using

surface water and soil samples at Hulu Langat

River with 2 different concentrations (0.1ml

and 1.0ml) at each sampling points from 4

main rivers. Type and composition of heavy

metals was analyzed using SEM-EDX while

bacterial level contamination was measured

through several test like hydrogen sulphide,

membrane filtration and Colilert. The purpose

of using different types of parameters is to

ensure water quality evaluation in Hulu

Langat River would give comprehensive

results outcome for this research project.

STATISTICAL ANALYSIS

Experimental results are analyzed statistically

using the Statistical Package for Social

Sciences (SPSS) version 22. Descriptive data

shows level of physio-chemical and biological

parameters. Correlation analysis shows the

correlation between type of heavy metals in

surface water and soils samples.

RESULTS

Samples were collected on 23/09/2015 and

14/10/2015 at four main rivers of Hulu Langat

around 10am until 12pm under same weather

condition. The level of turbidity (Figure 1.1A)

and temperature of water (Figure 1.1 B) at all

samplings were lower than INWQS standard.

The pH of the water at all sampling points

was detected at 6.5 (Figure 1.1 C).

Heavy metals such as Al, Fe, Mn and K were

present in the collected samples of surface

water and soils river samples particularly in

the Mersing River (Figure 1.2 A and B).

Significantly, a strong correlation between

Mn in soil and water was detected at point A

of Mersing River where the rs= 0.756 (Table

1.1). Although level of bacterial

contamination at all sampling points were

lower than INWQS standard (Class III/Class

IV), the Mersing river at point A has recorded

to be the highest of bacterial concentration

compared to other sites (Figure 1.1A). Figure

1.3 until figure 1.6 shows number of bacteria

colonies of Total Coliform, Fecal Coliform,

Escherichia coli and Streptococcus faecalis.

Point A has the highest number bacterial

colonies of Total coliform at 4.59

log10cfu/100 ml. Based on figure 1.4, the

number of Fecal coliform is highest at point A

(4.43 log10cfu/100 ml) but decreased

gradually until point D (3.39 log10cfu/100

ml). Figure 1.5, showed Escherichia coli

were present at all sampling point which is

highest at A sampling point with 3.63

log10cfu/100 ml. Streptococcus faecalis

(Figure 1.6) was also detected at point A, B

and C sampling points except D. It is highest

at sampling point B with 3.55 log10cfu/100

ml. By comparing to INWQS standard, the

number of bacterial in each sampling point

fell under Class III/ Class IV based on the

aspect of total bacterial level. Figure 1.7

showed the ratio of fecal coliform with fecal

Streptococcal or known as FC: FS ratio. If

the ratio is above 4.0 its showed that the water

is mainly contaminated by human fecal waste

but if the ratio is less than 0.7, it is animal

wastes. But if the ratio falls in between 4.0 to

0.7, it showed to be due to both human and

animal wastes. As a result, the ratio of

contamination at Mersing River and Congkak

River were showed to be of human and

animal contamination, while at Lopo River

sampling point showed contamination of

animal wastes.

62

6.5 6.5 6.5 6.5 6.5 6.5 6.5 6.5

-1

1

3

5

7

9

A1 B1 C1 D1 A2 B2 C2 D2

Stan

dar

dp

H

pH

Sampling points

28.25/

(0.267)

28/

(0.134)

27.25/

(0.134)27/

(0.134)

28.25/

(0.134)

28.25/

(0.134)

27.25/

(0.134)

27.5/

(0)

24

25

26

27

28

29

30

A1 B1 C1 D1 A2 B2 C2 D2

Tem

pe

ratu

re o

C

Sampling points

StandardoC

4.52/

( 0.379)

4.22/

( 0.339)2.95/

(0.195)

4.27/

(0.361)2.68/

(0.102)

5.92/ (0.163)

3.65/

(3.65)

5.24/

(0.259)

0

1

2

3

4

5

6

7

AI B1 CI D1 A2 B2 C2 D2Sampling points

Standard below 1000 NTU

Turb

idit

y(N

TU)

(A) (B)

(C)

FIGURE 1.1 Turbidity of water (A) Temperature of water (B) and

pH value of surface water (C).

A. B.

FIGURE 1.2 Spectrum SEM-EDX shows present of Al, Fe, Mn and K at A sampling point in

surface water river (A) and soil (B) (Mersing River)

63

TABLE 1.1 Correlation Mn (water) has strong positive correlation with Mn (soil), rs = 0.756

Al water Fe water Mn water K water

Al soil -0.286 0.119 0.577 -0.412

Fe soil -0.405 0.000 0.082 -0.167

Mn soil -0.733 0.234 0.756** -0.653

K soil 0.571 -0.357 -0.412 -0.571

FIGURE 1.3 Bacterial number of Total

coliform

FIGURE 1.4 Bacterial number of Fecal

coliform

FIGURE 1.5 Bacterial number of Escherichia

coli

FIGURE 1.6 Bacterial number of

Streptococcus faecalis

4.59/

(2.34) 4.45/

(1.86)

4.5/

(1.95)

4.01/

(2.32)

0123456789

10

A B C D

Total

coliform

Type of bacteria

(log10cfu/100ml)

Standardrange (4.7 cfu/100 ml)

4.43/

(1.69) 4.32/

(1.82)

4.17/

(1.77) 3.39/

( 0.99)

0

1

2

3

4

5

6

7

8

A B C D

Fecal

coliform

Type of bacteria

(log10cfu/100ml)


3.63/

(1.67)

3.46/

(1.79)3.28/

(1.07)

2.9/

(1.07)

0

1

2

3

4

5

6

7

A B C D

Escherichia

coli

Type of bacteria

(log10cfu/100ml)

Standardrange (3.7 cfu/

100 ml)

3.47/ (1.99)

3.55/ (2.21)

2.65/ (1.07)

0

-2

0

2

4

6

8

A B C D

Streptococcusfaecalis

Type of bacteria

(log10cfu/100ml)


64

FIGURE 1.7 Ratio of fecal Coliform with

fecal Streptococcal (FC:FS)

DISCUSSION

The data obtained for pH, turbidity and

temperature for four branch rivers in Hulu

Langat River showed good water quality

control of Class III/Class IV based on

INWQS. The parameters were influenced by

many factors including the increase rate of

chemical reaction and nature of biological

activities (Obire et al. 2008). Temperature is

also one of the factors that govern the

assimilative capacity of the aquatic system.

Discharge from industrial sources can also

increase river water temperature

(Environmental Protection Agency 1976).

Hence, The Ministry of Health has set a

threshold level of raw water turbidity at 1000

NTU. Based on Gada (2011), there are many

factors contributing to changes in pH level of

water bodies which includes, waste water

discharge, logging activity, runoff from

mining operations, acid rains and the types of

rock naturally found in that area.

As surface water pH is acidic, the land

is dominated by Al, Fe, and Mn. These heavy

metals will bind nutrients needed by plants,

especially the elements of P (phosphorus), K

(potassium), S (sulfur), Mg (magnesium) and

others (Asaolu et al. 1997) These effects

caused plant unable to absorb nutrients

properly, even though the content of nutrients

is high in the soil with pH value less than 7.

There were also present of Al, Fe and K at all

sampling point except for point A (Mersing

River) with present of Mn is surface water

and soils sample. Based on correlation test, it

also shows there were significant strong

correlations of heavy metals of Mn between

sample of surface water and soil river at

sampling point of Mersing River, rs= 0.756.

These manganese compounds is

usually present the atmosphere and it is the

results of suspended manganese particulates

originated from industrial emissions, volcanic

emissions, soil erosion and the burning of

MMT-containing petrol (IPCS 1999). While

manganese in surface waters occurs due to

both suspended and dissolved forms. Its

presence depended much on other factor such

as pH. Anaerobic groundwater often contains

elevated levels of dissolved manganese. The

divalent form (Mn2+) predominates in most

water at pH 4–7, but more highly oxidized

forms may occur at higher pH values or result

from microbial oxidation (WHO, 2011). It is

supported when pH is decreased, the amount

of exchangeable manganese mainly Mn2+

form will increase in the soil solution

(Marschner, 1991). This Mn form is available

for plants and it is readily transported into the

root cells and translocated to the shoots,

where it is finally accumulated. In contrast,

based on other forms of Mn predominate at

higher pH values (alkaline) such as Mn (III)

and Mn (IV) which are not available and

cannot be accumulated in plants (Rengel

2000). Manganese can be adsorbed onto soil,

the extent of adsorption depending on the

organic content and cation exchange capacity

of the soil. Furthermore, it can also be

bioaccumulated in lower organisms like

phytoplankton, algae and some fish but no

reports in higher organisms (Oluduro et al.

2007).

While for the total number of bacterial

contamination, it showed the presence of

many types of bacteria but fortunately the

concentration is under control based on the

Class III/Class IV level of INWQS (Rezania

et al. 2016). . E. coli is a widely used

0.91 0.6

3.3

00

1

2

3

4

A B C D

FC:FS ratio

FC:FS

< 0.7 animals

> 4.0

human

65

indicator of fecal contamination in water

bodies. These fecal coliform bacteria indicate

or determine the presence of human wastes or

excrement in a water bodies. External contact

and subsequent ingestion of bacterial

contamination can lead to harmful effects.

The risk of water contamination by fecal

bacteria may be increased if a watershed

includes areas where feces accumulate as

result of specific land uses.

The reasons for the presence of

coliforms is due to the discharged of water

from the industrial sector and the construction

sector (houses development) which runs along

the Hulu Langat River and Semenyih River

(Fawaz et al. 2013). This has given an impact

to the water quality of the river. Furthermore

other factors such as rain distribution, runoff

water, recreational activities and human waste

have also contributed to bacterial pollution.

Presence of septic tanks and containers on the

property near the sampling site were found to

contribute to the detection of coliforms

mainly in groundwater. These water may

flows, runoff or leeched into river estuaries

and ended up in contaminating water

(Ouedraogo et al. 2017).

The source of pollution at point A in

Mersing River mostly comes from logging

activity, industrial activity and crowded

houses development compared to another

source of water pollution at different

sampling sites (Asraf et al. 2017). As a

conclusion, overall results indicated that

Therefore, composition of heavy metals and

microbial indicator at selected branch in Hulu

Langat River was safe for recreational

purposes.

ACKNOWLEDGEMENT

The author would like to acknowledge the

Jabatan Pengairan Negeri Selangor (JPS) for

contributing to the research and also would

like to greet appreciation to Mr. Hilmi Baba

and Mr. Faisal Ariffin for their kind

assistance in laboratory processing.

REFERENCES

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Determination of physico-chemical

parameters and heavy metals in water

samples from Itaogbolu area of Ondo-

State, Nigeria, African. Journal of

Environmental Science and Technology

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Asaolu, S.S., Ipinmoroti, K.O., Adeyinowo, C.E.

& Olaofe, O. 1997. Interrelationship of

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coastal Area Nigeria. African Journal of

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Ashraf A, Saion E, Gharibshahi E, Yap CK,

Kamari HM, Elias MS, Rahman SA.2017.

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Malaysia by instrumental neutron

activation analysis and inductively

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Criteria for Water Use. E.P.A, Environ

Agency. 440 1a-76-023.

Fawaz Al-Badaii, Mohammad Shuhaimi-Othman,

and Muhd Barzani Gasim. Water Quality

Assessment of the Semenyih River,

Selangor, Malaysia. Journal of Chemistry.

Volume 2013 (2013), Article ID 871056,

10pages.http://dx.doi.org/10.1155/2013/8

71056.

Gada, M. 2011. Water Quality Tests.

http://www.grc.nasa.gov/www/l-

2/fenlewis.htmX [21 October 2011]

Gleick P.H. 2000 A look at the 21st Century

Water Resources Development.

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Volume 25, Number 1, March 2000.

IPCS. 1999. Manganese and its compounds.

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66

Marschner, H. 1991. Mechanisms of adaptation of

plants to acid soils. Plant-soil interactions

at low pH: Kluwer Academic Publisher

Obire, M. A. and Puthetti, R.R. 2008. The impact

of human activities on drinking water

Quality. Journal of Basic and Applied

Biology. 52-58

Oluduro, A.O., Adewoye, B.I. 2007. Efficiency

of moringa Oleifera Seed extract on the

microflora of surface and ground water.

Journal of Plant Science. 453-438.

Ouedraogo I, Defourny P, Vanclooster M. 2017.

Validating a continental-scale

groundwater diffuse pollution model

using regional datasets. Environmental

Science and Pollution Research

International 2017 Dec 11.

Rengel, Z. 2000. Uptake and transport of

manganese in plants. Journal of Soil

Science. Plant Nutrition. 57-87.

Rezania S, Din MF, Taib SM, Dahalan FA,

Songip AR, Singh L, Kamyab H. 2016.

The efficient role of aquatic plant (water

hyacinth) in treating domestic wastewater

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Journal of Phytoremediation.

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Organization.

Hukil Sino

Forensic Science Program

School of Diagnostic & Applied Health Science

Faculty of Health Sciences


Jalan Raja Muda Abdul Aziz

50300, Kuala Lumpur, Malaysia

Mohd Ambia Kaswandi

Ahmad Zorin Sahalan

Nur Sakinah Ismail


School of Diagnostic & Applied Health

Sciences, Faculty of Health Sciences,



50300 Kuala Lumpur. Malaysia

Hing Hiang Lian*

Environmental Health & Industrial Safety

Programme,

School of Diagnostic & Applied Health

Sciences, Faculty of Health Sciences,



50300 Kuala Lumpur,

Malaysia.

*Corresponding author; email:

[email protected]

Buletin FSK 1(1)(2017): 67-71

Mechanical Stretch Application in Identifying Vascular Related Diseases

NURUL FARHANA JUFRI*

ABSTRACT

Hypertension and estrogen deficiency are known risk factors that contribute to the incidence

of cardiovascular and cerebrovascular disease. It is thought that hypertensive risk due to

excessive stretching of vascular walls and the loss of a protective effect from estrogen causes

injury and weakness of the vascular structure that may lead to atherosclerosis, coronary heart

disease, aneurysm and/or stroke. Previously, estrogen has been demonstrated to exert a

protective effect on the vascular system as it has anti-inflammatory and anti-atherogenic

properties, reducing reactive oxygen species production and increasing mitochondria efficiency.

Excessive mechanical tensile stretch, as occurs during hypertension, is associated with cellular

injury and endothelial dysfunction. However, the results of the combination of these positive

and negative effects from both stimuli are still unclear and warrant further investigation. The

model may reflect protein regulation that may occur during hypertension and has potential in

developing estrogen-like compounds in future therapeutic approach.

Keywords: cerebrovascular diseases; endothelial cells; estrogen; mechanical stretch; vascular

ABSTRAK

Tekanan darah tinggi dan kekurangan estrogen merupakan faktor risiko yang menyumbang

kepada insiden penyakit kardiovaskular dan serebrovaskular. Risiko tekanan darah tinggi

dianggap berpunca dari regangan berlebihan pada dinding salur darah and kehilangan kesan

perlindungan dari estrogen yang menyebabkan kecederaan serta kelemahan pada struktur

vaskular. Ini boleh membawa kepada aterosklerosis, penyakit jantung, aneurism dan strok.

Estrogen telah menunjukkan kesan perlindungan pada sistem vaskular dengan kesan anti

keradangan dan anti aterogenik, mengurangkan penghasilan radikal oksigen reaktif serta

meingkatkan keefisienan mitokondria. Regangan berlebihan yang berlaku semasa tekanan

darah tinggi dikaitkan dengan kecederaan sel dan ketidakfungsian endothelial.

Walaubagaimanapun, kesan gabungan kedua-dua faktor ini masih tidak jelas dan memerlukan

penyelidikan lanjut. Model kajian dapat menunjukkan regulasi protein yang mungkin berlaku

semasa tekanan darah tinggi dan potensi dalam membangunkan bahan seperti estrogen dalam

kajian teraputik akan datang.

Kata kunci: penyakit serebrovaskular; sel endothelial; estrogen; regangan mekanikal; vaskular

INTRODUCTION

The vascular system is a dynamic

environment as it is constantly exposed to

various stimuli such as hemodynamic forces

and vasoactive substances. Hemodynamic

forces in a physiological setting are

important in maintaining vascular

homeostasis as it involved in vessel

remodeling, angiogenesis and regulating

vascular tone (Anwar et al., 2012). In the

early stages of the stretch stimuli, the

vascular remodeling process involves cell

migration, proliferation, apoptosis and

extracellular matrix (ECM) synthesis that

can potentially counteract any effects of

mechanical stress. However, excessive

mechanical cyclic stretch, such as in the case

68

of hypertension (blood pressure reading

above of 140/90 mmHg), may initiate

endothelial dysfunction (ED), neointima

formation, an inflammatory cascade and

ECM remodeling (Anwar et al., 2012). This

is supported by the finding of excessive

stretch could increase the endothelial

adhesion molecule ICAM1 and release of its

soluble form together with inflammatory

cytokine IL-8 (Tian et al., 2016). Meanwhile,

the low stretch magnitude could up-regulate

nuclear NF-κB, which suggests that

disturbances to wall stretch can be

atherogenic similar to disturbed flow

(Pedrigi et al., 2017). Several vascular

diseases, such as atherosclerosis, coronary

heart disease and aneurysm formation may

result from these processes. Thus,

understanding the mechanism of action due

to the excessive mechanical cyclic stretch

may benefit to prevent vascular disease.

In addition to hemodynamic forces,

the vascular system is exposed to vasoactive

substance such as blood proteins, peptides

and hormones. For instance, one of the

hormones thought to be crucial in the

vascular system is estrogen that is a sexual

hormone important for female sexual

characteristics. This hormone circulates at

physiological levels (118-914 pmol/L) in

premenopausal women (Stricker et al., 2006).

Previous studies have demonstrated that

estrogen has many beneficial and protective

effects on the human vascular system and

that many of these are operational during

cardiovascular diseases. For example,

estrogen has anti-inflammatory and anti-

atherogenic property (Cid, Schnaper, &

Kleinman, 2002), reduces reactive oxygen

species (ROS) production (Stirone et al.,

2005), increases mitochondria efficiency

(Duckles & Krause, 2007) and releases nitric

oxide (NO) for vascular dilatation (Cid et al.,

2002; Duckles et al., 2007, 2012; Guo et al.,

2010; Tostes et al., 2003). However, as

women undergo menopause, the

physiological level of estrogen starts to

decline, and the incidence of cardiovascular

and cerebrovascular disease starts to rise

(Regitz-Zagrosek, 2006). For example,

premenopausal women have a lower risk

(odd ratio 0.24) for aneurysmal subarachnoid

hemorrhage compared to postmenopausal

women (Longstreth et al., 1994; Okamoto et

al., 2001). In addition, it has been found that

coronary heart disease develops 10 years

later in women who are in the

postmenopausal stage compared to men at

the same age (Xing et al., 2009). These

examples might be attributed to the loss of

any protective effects of estrogen occurring

after menopause and highlight estrogen as a

potentially important hormone in women.

Figure 1 suggested the processes

involved as a foundation in this study. As any

blood vessel is exposed to mechanical forces

such as tensile stretch or shear stress,

vascular remodeling is activated to maintain

the structure and to counteract any

mechanical stress experienced. The

remodeling processes involve cell migration,

proliferation, apoptosis and extracellular

matrix degradation/synthesis to sustain

vascular homeostasis. However, prolonged

cyclic stretch (such as occurs in chronic

hypertension), leads to several vascular

pathologies that can eventually lead to

atherosclerosis and/or aneurysm. Several

processes involved such as ED, neointima

formation, inflammation and ECM

imbalances that eventually contribute to

these anomalies. However, estrogen has

been reported to be protective against these

deleterious processes. Given that the

endothelial cells (EC) layer is the innermost

cell layer in tissues that are in active contact

with blood flow, this represents a promising

starting point to focus on disease

development. Furthermore, as the

endothelial layer has direct contact with

circulating estrogen, it can lead to the

initiation of the cellular responses affecting

the downstream signaling pathway in cells.

Given these reason, ECs can be the main

interest in further research.

69

Figure 1: The relationship between blood vessels, mechanical stretch and estrogen exposure.

Mechanical forces such as shear stress and tensile stress against vessel wall will

induce vascular remodeling involving cells migration, proliferation, apoptosis and

ECM degradation/synthesis. However, prolonged hypertension can cause ED,

neointima formation, inflammation, hypertrophy and ECM imbalances. These could

promote atherosclerosis development and weakness of the arterial wall could initiate

aneurysm formation. As these processes involve molecular signaling activation,

many downstream proteins may be activated or inactivated due to the stimuli. The

specific proteome changes due to mechanical tensile stretch, estrogen and the

combination of both stimuli can be elucidated by proteomics approach.

Previous studies have focused on the effect

of shear stress and its associated pathological

implications on ECs. However, the effect of

cyclic stretch specifically on ECs and its

molecular changes have not been

investigated in depth and require further

investigation (Chiu et al., 2009; Lacolley,

2004; Ngai et al., 2010; Shyu, 2009). In

addition, although many studies have

focused on human umbilical vein endothelial

cells (HUVEC), HUVEC is derived from

immune fetal tissue that exhibit functional

differences from adult vascular endothelia

(Tan et al., 2004). Thus, the experimental use

and application of adult endothelial cell is

better as they represent adult cells and more

accurate model worthy of investigation

(Felty, 2011; Shoajei et al., 2014).

Application of mass spectrometry-

based “shotgun” proteomics is useful to

elucidate protein changes caused by these

stimuli. Proteomics is a powerful tool used to

map the cellular and molecular responses to

specific stimulus. The proteomics approach

enables quantification and ranking of altered

proteins in any biological system under

defined spatial, temporal and environmental

conditions. This information can enhance

understanding of the pathophysiology of

disease, enable development of disease

biomarkers as diagnosis tools and contribute

to the development of targeted protein in

novel therapeutic approaches.

As excessive mechanical cyclic

70

stretch is associated with pathological

development in vascular endothelium and

estrogen has been reported to exhibit its

protective effects against cardiovascular

disease, the effect of the combination of these

stimuli is unknown and the new information

could inform therapeutic approaches,

especially in cardiovascular diseases.

REFERENCES

Anwar, M. A., Shalhoub, J., Lim, C. S., Gohel, M.

S., Davies, A. H. 2012. The effect of

pressure-induced mechanical stretch on

vascular wall differential gene

expression. J Vasc Res 49: 463-478.

Chiu, J. J., Usami, S., & Chien, S. 2009. Vascular

endothelial responses to altered shear

stress: pathologic implications for

atherosclerosis. Ann Med 41(1): 19-28.

Cid, M. C., Schnaper, H. W., & Kleinman, H. K.

2002. Estrogens and the vascular

endothelium. Ann N Y Acad Sci 966:143-

157.

Duckles, S. P., & Krause, D. N. 2007.

Cerebrovascular effects of oestrogen:

multiplicity of action. Clin Exp

Pharmacol Physiol 34: 801-808.

Felty, Q. 2011. Proteomic 2D DIGE profiling of

human vascular endothelial cells

exposed to environmentally relevant

concentration of endocrine disruptor

PCB153 and physiological concentration

of 17β-estradiol. Cell Biol Toxicol 27:

49-68.

Guo, J., Krause, D. N., Horne, J., Weiss, J. H., Li,

X., & Duckles, S. P. 2010.

Estrogenreceptor-mediated protection of

cerebral endothelial cell viability and

mitochondrial function after ischemic

insult in vitro. J Cereb Blood Flow

Metab 30:545-554.

Lacolley, P. 2004. Mechanical influence of cyclic

stretch on vascular endothelial cells.

Cardiovasc Res 63(4): 577-579.

Longstreth, W. T., Lorene, M. N., Thomas, D. K.,

& Gerald, B. 1994. Subarachnoid

hemorrhage and hormonal factors in

women. Ann Intern Med 121:168-173.

Ngai, C. Y., & Yao, X. 2010. Vascular responses

to shear stress: the involvement of

mechanosensors in endothelial cells.

Open Circ Vasc J 3: 85-94.

Okamoto, K., Horisawa, R., Kawamura, T., Asai,

A., Ogino, M., Takagi, T., & Ohno, Y.

2001. Menstrual and reproductive

factors for subarachnoid hemorrhage risk

in women: A case-control study in

Nagoya, Japan. Stroke 32:2841-2844.

Pedrigi, R.M., Papadimitriou, K. I., Kondiboyina,

A., Sidhu, S., Chau, J., Patel, M. B.,

Baeriswyl, D. C., Drakakis, E. M. &

Krams, R. 2017. Disturbed Cyclical

Stretch of Endothelial Cells Promotes

Nuclear Expression of the Pro-

Atherogenic Transcription Factor NF-κB.

Ann Biomed Eng 45(4): 898–909.

Regitz-Zagrosek, V. 2006. Therapeutic

implications of the gender-specific

aspects of cardiovascular disease. Nat

Rev Drug Discov 5: 425-438.

Shoajei, S., Tafazzoli-Shahdpour, M.,

Shokrgozar, M. A., & Haghighipour, N.

2014. Alteration of human umbilical vein

endothelial cell gene expression in

different biomechanical environments.

Cell Biol Int 38(5): 577-581.

Shyu, K. G. 2009. Cellular and molecular effects

of mechanical stretch on vascular cells

and cardiac myocytes. Clin Sci (Lond)

116(5): 377-389.

Stirone, C., Duckles, S. P., Krause, D. N., &

Procaccio, V. 2005. Estrogen increases

mitochondrial efficiency and reduces

oxidative stress in cerebral blood vessels.

Mol Pharmacol 68: 959-965.

Stricker, R., Eberhart, R., Chevailler, M. C.,

Quinn, F. A., Bischof, P., & Stricker, R.

2006. Establishment of detailed

reference values for luteinizing hormone,

follicle stimulating hormone, estradiol,

and progesterone during different phases

of the menstrual cycle on the Abbott

ARCHITECT analyzer. Clin Chem Lab

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Tan, P. H., Chan, C., Xue, S. A., Dong, R.,

Ananthesayanan, B., Manunta, M.,

Kerouedan, C., Cheshire, N. J., Wolfe, J.

H., Haskard, D. O., Taylor, K. M., &

George, A. J. 2004. Phenotypic and

functional differences between human

saphenous vein (HSVEC) and umbilical

vein (HUVEC) endothelial cells.

Atherosclerosis 173(2): 171-183.

Tian, Y., Gawlak, G., O'Donnell, J.J.,

Mambetsariev, I., & Birukova, A. A.

2016. Modulation of endothelial

inflammation by low and high

magnitude cyclic stretch. Plos One 11(4):

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e0153387.

Tostes, R. C., Nigro, D., Fortes, Z. B., &

Carvalho, M. H. C. 2003. Effects of

estrogen on the vascular system. Braz J

Med Biol Res 36: 1143-1158.

Xing, D., Nozell, S., Chen, Y.F., Hage, F., &

Oparil, S. 2009. Estrogen and

mechanisms of vascular protection.

Arterioscler Thromb Vasc Biol 29: 289-

295.

Nurul Farhana Jufri*

Programme of Biomedical Science,

School of Diagnostic and Applied Health Sciences,




50300 Kuala Lumpur,

Malaysia.


Buletin FSK 1(1)(2017): 72-79

Visual Perceptual Skills Performance Among a Sample of Year-2 Students

in Klang Valley

SUMITHIRA NARAYANASAMY, HUI YEE TEY, YIN YIN WONG & SHARANJEET-

KAUR*

ABSTRACT

Children with visual-perceptual deficits are associated with difficulty in learning process. One

of the most commonly-used visual-perceptual skills test is the Test of Visual-Perceptual Skills

(Non-Motor)-Revised (TVPS-R). This study was conducted to determine the normative data

of TVPS-R test for a sample of Year-2 students in Klang Valley and to determine the

relationship between visual-perceptual skills and reading performance. Visual-perceptual

functions of Year-2 students from two primary schools in Klang Valley were tested. Reading

performance was determined by measuring the reading speed using a Malay Language Related

Reading Text. A total of 202 (93 male and 109 female) students with mean age 7.33 ± 0.47

were recruited. Results showed that the mean standard scored obtained for visual

discrimination, visual memory, visual spatial-relationships, visual form constancy, visual-

sequential memory, visual figure ground and visual closure subtests were 109.02 ± 13.32,

110.22 ± 11.74, 115.34 ± 10.57, 107.83 ± 12.54, 116.34 ± 11.03, 113.31 ± 10.90 and 112.58 ±

11.62 respectively. The performance on all the subtests exceeded that reported for age-matched

children in US. In addition, a positive correlation between all visual perceptual skills subtests

with reading performance except for visual closure. In conclusion, the performance of

Malaysian children outperformed their peer in US in TVPS-R test. This study provided

normative data of TVPS-R test for Malaysian children for reference of future studies. The

results also suggested the necessity to develop population-based normative data for TVPS-R

test.

Keywords: Visual-perceptual skills; TVPS-R; Malaysian children

INTRODUCTION

It is widely believed that a child's vision is an

important factor for optimising their

educational experience given that a

substantial amount of learning materials are

presented visually. Good vision promotes

well acquisition of information from the

environment and sends them to the higher

visual system for interpretation (Schneck

2005). However, there is a misconception

that visual acuity is the only relevant visual

function and that a visual acuity of 6/6

obtained with a standard high contrast

distance letter chart adequately represents the

overall functioning of the visual system. In

reality, a wider range of visual factors have

been associated with learning-related

problems such as visual acuity (O’Grady

1984, Ygge et al. 1993), refractive error

(Grisham and Simons 1986), visual

efficiency (Simons and Grisham 1987) and

visual perceptual skills (Chen, Bleything,

and Lim 2011, Kulp 1999). Visual efficiency

in these studies refers to processes such as

accommodation, vergence and ocular

motility.

Visual perceptual skills refers to a

wide range of skills such as visual spatial,

visual analysis (also known as visual

information processing) and visual motor

integration, that are required for interpreting

and understanding visual information

(American Optometric Association 2008).

Poorly developed visual perceptual skills

have been proposed to be associated with

poorer learning outcomes (Chen, Bleything,

and Lim 2011). Visual perceptual skills are

divided into three broad categories; visual

spatial, visual analysis and visual motor

73

integration (Borsting 2006). Visual spatial

skills relate to directional concepts which are

important for navigating through the

environment (differentiating left and right,

up and down, front and back), while visual

analysis skills relate to the ability to

recognise, recall and organise visual

information. Visual motor integration skills

are related to the ability to coordinate

perceptual skills with fine motor skills

(Borsting and Rouse 1994). Therefore, visual

perception assessment is important because

children with visual-perceptual function

deficits usually demonstrate difficulty in

learning process such as reading, writing,

spelling and doing mathematics and may

influence their daily living activities, work,

play, leisure and social participation (Kulp

1999, Sortor, Kulp, and Taylor 2003, Kavale

1982). In addition, the test results can also be

an indicator of academic performance when

they enter primary school (Kalb and

Warshowsky 1991, Todd 1993).

One of the most commonly-used

visual-perceptual skills test is the Test of

Visual-Perceptual Skills (Non-Motor)-

Revised (TVPS-R). TVPS-R is a test that has

been revised to determine subject’s visual-

perceptual strengths and weaknesses based

on non-motor response. It consists of 7

subtests including visual discrimination,

visual memory, visual spatial-relationships,

visual form-constancy, visual sequential

memory, visual figure-ground and visual

closure (Gardner 1996). Visual-perceptual

skills have been shown to be influenced by

culture, language (Lai and Leung 2012),

geographical region (Xuechuan 1995),

academic performance (Chen, Bleything,

and Lim 2011), race and socioeconomic

status (Dunn, Loxton, and Naidoo 2006). To

date, the normative database for the TVPS-R

test is only available for the Caucasian

(Gardner 1996) and Chinese population (Ho

et al. 2015). There is no normative data of

visual-perceptual skills available for children

in Malaysia. This is surprising given that the

prevalence of visual-perceptual problems in

Malaysian children was reported to be

between 3% and 48% (Chen, Bleything, and

Lim 2011). Therefore, this study was

indicated to: (1) determine the normative

data of all the sub-tests of TVPS-R for a

sample of Year-2 students in Klang Valley,

(2) compare the data with the normative data

from US, and (3) to investigate the

relationship between visual-perceptual skills

and reading performance.

METHODS

Participants

Two primary schools in Klang Valley were

randomly chosen. A consent form was

distributed to the parents or legal guardians

of all Year-2 students in both primary

schools. Subjects who fulfilled the following

criteria were recruited: (1) informed consent

given by the parents or legal guardians, (2)

habitual monocular distance visual acuity of

at least 6/9 or better at 6m, (3) habitual

monocular near visual acuity of at least N6 or

better at subject’s reading distance, (4) no

any learning disabilities, expressive or

receptive language disturbances, visual-

perceptual handicaps and not emotionally

disturbed. The study followed the tenets of

the Declaration of Helsinki and was

approved by the Universiti Kebangsaan

Malaysia (UKM) Ethical Committee for

medical research (Reference number: UKM

PPI/111/8/NN-093-2015), Malaysia

Education Ministry and Department of

Education Federal Territory of Kuala

Lumpur.

Procedures

Phase I

Questionnaires

A questionnaire was distributed to the

parents or legal guardians to determine

whether subjects have hearing problems,

expressive or receptive language

disturbances, visual-perceptual handicaps,

emotionally disturbed or learning

disabilities. Subjects with any of these

problems were excluded.

74

Visual Function Assessments

Monocular distance and near visual acuities

were measured at 6m and 40cm respectively,

using Snellen chart and a UKM near reading

chart. The smallest line and letters that could

be read by the subject were recorded. Near

visual acuity was recorded with N notation.

Phase II

Visual-Perceptual Skills Assessment

Visual-perceptual skills were tested with the

Test of Visual-Perceptual Skills (Non-

Motor)-Revised (TVPS-R). This test consists

of seven individual subtests namely visual

discrimination, visual memory, visual

spatial-relationships, visual form constancy,

visual-sequential memory, visual figure

ground and visual closure. The test was

conducted in a well-illuminated, quiet and

well-ventilated room that was free from any

auditory and visual distractions following the

instruction given on the manual on a one-to-

one basis. In the visual discrimination

subtest, subjects were needed to determine

and match similar forms. In the visual

memory subtest, subjects were required to

remember and recall a single shape. In the

visual spatial-relationships subtest, subjects

were required to look for the shape that is

different from others. In the form constancy

subtest, subjects were required to find the

exact form shown although it might be

different in orientation and size. In the visual-

sequential memory, subjects were required to

remember and recall the shapes in a correct

sequence. In visual figure ground, subjects

were required to match and find the form

from a complicated background. Lastly in

visual closure, the subjects were required to

determine a complete shape from the

fragments given. Each correct answer was

scored as one point, and each subtest was

ended when three consecutive questions

were answered incorrectly. The raw score,

which was the total number of correct

responses was then converted to a ‘‘scaled

score’’ with reference to the manual. The

sum of the scaled scores for all the subtests

was then converted to a standard score with

reference to the look-up table in the manual.

Reading Performance

Reading performance evaluation was carried

out using a Malay Language Related Reading

Text for grade 1 (Omar et al. 2015). The

formula for measuring the reading speed in

unit of words per minutes (wpm) was as

follows:

The Malay Language Related Reading Text

for grade 1was used for children with age

from 7 to 9 years old. The text was shown in

Figure 1.

FIGURE 1. Malay Language Related

Reading Text for grade 1

Data Analysis

All the data were analyzed using SPSS

version 21.0. One sample t-test was used to

test for any significant difference in visual

perceptual skills of children in this study

against the US normative data (i.e. 100

standard scores for all subtests) (Gardner

1996). Pearson correlation test was also

performed to determine the relationship

between visual-perceptual skills and reading

performance. A p-value of < 0.05 was

considered statistically significant.

RESULTS

A total of 202 subjects, consisting of 93 male

and 109 female, met the inclusion criteria and

were recruited in this study. The mean age of

75

the participants was 7.33 ± 0.47 years. Of the

202 subjects, 191 (95%) passed the visual

discrimination subtest, 196 (97%) passed the

visual memory subtest, 193 (96%) passed the

visual spatial-relationships subtest, 174

(86%) passed the visual form-constancy

subtest, 166 (82%) passed the visual

sequential memory subtest, 179 (89%)

passed the visual figure-ground subtest and

139 (69%) passed the visual closure subtest.

Visual-Perceptual Functions

The mean and standard deviation of the

standard scores of each subtest are

summarized in Table 1. The mean of

standard scores for visual discrimination

subtest was 109.02 ± 13.32, which exceeded

the standardized score of 100 suggesting that

the overall performance of Malaysian

children was better than that of age-matched

US children. Similar results were also shown

by the other subtests. One sample t-test

indicated that there was a significant

difference between the normative data of this

study compared with the US normative value

for all the subtests as summarized in Table 2.

TABLE 1. Summary of mean and standard deviation of the standard scores by subtest

Subtest N Mean Standard

Deviation (SD) Minimum Maximum

VD 191 109.02 13.32 68 132

VM 196 110.22 11.74 73 136

VSR 193 115.34 10.57 74 129

VFC 174 107.83 12.54 79 136

VSM 166 116.34 11.03 85 139

VFG 179 113.31 10.90 77 133

VC 139 112.58 11.62 81 137

Legends: *VD=Visual Discrimination, VM=Visual Memory, VSR=Visual Spatial-

Relationships, VFC=Visual Form-Constancy, VSM=Visual Sequential-Memory, VFG=Visual

Figure-Ground, VC=Visual Closure

TABLE 2. Results of one sample t-test according to subtest.

Test Value = 100

Subtest t df p value MD

VD 9.36 190 <0.05 9.02

VM 12.19 195 <0.05 10.22

VSR 20.16 192 <0.05 15.34

VFC 8.24 172 <0.05 7.83

VSM 19.10 164 <0.05 16.34

VFG 16.35 177 <0.05 13.31

VC 12.76 137 <0.05 12.58



Figure-Ground, VC=Visual Closure, df=degree of freedom, MD=Mean Difference

76

Relationship Between Visual Perceptual

Skills And Reading Performance

The mean reading speed for the subjects in

this study was 71.63 ± 26.38 wpm. Pearson

correlation test showed that all TVPS-R

subtests were significantly associated with

reading performance except for visual

closure (R=0.016, p>0.05). However, the

coefficient of correlation (R) indicated a

weak relationship (approximately 0.1 to 0.3)

between visual-perceptual skills and reading

performance. Coefficient of determinant (R²)

also showed that visual perceptual skills

accounted for approximately 3% to 7% of the

variance in the prediction of reading

performance. Table 3 summarises the

correlation results between each visual-

perceptual skills and reading performance.

TABLE 3. Correlation between visual-perceptual skill and reading performance.

Reading Performance

VP Skill N R R² p

VD 191 0.232 0.054 <0.05

VM 196 0.198 0.039 <0.05

VSR 193 0.174 0.030 <0.05

VFC 174 0.255 0.065 <0.05

VSM 166 0.255 0.024 <0.05

VFG 179 0.213 0.045 <0.05

VC 139 0.016 0.026 >0.05



Figure-Ground, VC=Visual Closure, R=Coefficient of Correlation, R²=Coefficient of

Determinant

DISCUSSION

The visual perceptual skills performance on

a sample of Year 2 children and its

association with reading performance was

evaluated in this study. The results indicated

that the visual-perceptual skills performance

measured using TVPS-R test among Year-2

students in Klang Valley was significantly

better than their age-matched peers in the US.

These results are in accord with studies

conducted on Hong Kong and Chinese

preschool children, who demonstrated

significantly better performance in visual

perceptual skills compared to the US peers

(Ho et al. 2015). A plausible reason for this

could be due to the differences in the school

curriculum between the two countries.

Education system in Malaysia emphasises on

various aspects such as communication

skills, reading, writing, mathematics,

science, art, leisure and moral values as early

as pre-primary and primary levels

(Kementerian Pelajaran Malaysia 2001),

while children in the US of the same age are

mainly taught social and cognitive skills

(Department of Health and Services 1996).

In fact children in Malaysia are usually

expected to be able to be able to read and

recall alphabets, copy simple or complex

pictures or characters even in the pre-primary

levels (ages of 4 to 5).

In addition, the differences in visual

perceptual skills performance could also be

attributed to the cultural differences between

the two countries as development of these

77

skills has been argued to be affected by

culture (Josman, Abdallah, and Engel-Yeger

2006, Williams and Williams 1987, Katz,

Kizony, and Parush 2002, Levine 1987).

The current study also found a

positive association between the visual-

perceptual skills and reading performance,

which suggested that the better the visual-

perceptual skills, the better the reading

performance. This significant result may

indicate that visual-perceptual skills could

possibly be a predictor of children’s reading

performance. This is supported by Kavale

(1982), who reported that visual-perceptual

skills contributed to about 6% to 20% of the

variation in the prediction of reading

performance. In this current study, it was

found that the visual-perceptual skills

accounted for 3% to 7% of the variations in

the prediction of reading performance. Other

factors that could also have an impact on the

reading performance of children includes

interest, motivation, practice, teaching

methods in school and at home and other

visual functions (Widyana 2009).

The results of this study should be

considered in light of some limitations. The

schools selection in this study may not

represent the overall children population in

Malaysia as only two randomly selected

national schools. In addition, only 8 years old

children were recruited for this current study.

It would be of interest to investigate visual

perceptual skills performance in children

with a wider age range and from different

schools (national and vernacular schools)

given that these factors has been suggested to

impact on visual perceptual skills

performance.

CONCLUSIONS

In summary, Malaysian children had a better

visual perceptual skills performance than

their US peers, which can be attributed to

differences in the education systems between

the countries. This result suggests that it is

necessary to develop individual normative

data of TVPS-R for each population. This

study provided preliminary normative data

for all the TVPS-R subtests for Malaysian

children. The association between visual-

perceptual skills and reading performance

also suggested that visual-perceptual skills

could be considered as one of the predictors

of reading performance.

REFERENCES

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79

Sumithira Narayanasamy

Hui Yee Tey

Yin Yin Wong

Sharanjeet-Kaur*

Optometry & Vision Science Programme,




50300 Kuala Lumpur.,

Malaysia.

Corresponding author; email: [email protected]

Buletin FSK 1(1)(2017): 80-88

Detection of Antibacterial Activity from the Crude Stalk Extracts of Alocasia

denudata Engler

MAZLYZAM ABDUL LATIF, WAFA’ ZAHARI, AHMAD ZORIN SAHALAN, SITI

AISYAH ARSHAD & ASMAH HAMID*

ABSTRACT

Keladi candik or its scientific name Alocasia denudata Engler also known as keladi ular, keladi

harimau or keladi merbah by the local community. Previous studies have shown that A. denudata

have abilities accelerating skin wound healing in mice. A good wound healing agent may be more

beneficial if it’s also display potential to inhibit bacterial growth. Therefore, this study was

conducted to detect the presence of antibacterial activity from the crude stem extracts of A.

denudata. The stalk of A. denudata was extracted using three different solvents comprising

dichloromethane, methanol and distilled water. These three extracts were tested for antibacterial

activity using disc diffusion method against Staphylococcus aureus (ATCC 25923),

Staphylococcus epidermidis, Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae

and Proteus sp. The findings showed that aqueous extracts of A. denudata produced the largest

inhibition zone diameter on the growth of K. pneumonia of 7:26 ± 0.63 mm at a concentration of

100 mg/ml. Meanwhile, dichloromethane extract showed the largest inhibition zone diameter on

the growth of K. pneumoniae, S. aureus, and an average of S. epidermidis measuring 7.0 ± 0.0 mm

at a concentration of 100 mg/ml. Methanol extract showed the greatest inhibition zone diameter

on the growth of K. pneumoniae of 7.0 ± 2.0 mm at a concentration of 100 mg/ml. The positive

result of antibacterial activity was continued with Minimum Inhibitory concentration (MIC) test.

However, each type of extract was not able to inhibit the growth of bacteria. Therefore, no MIC

values generated in this study. In conclusion, Alocasia denudata Engler crude extracts tested in

this study showed less significant effect on the bacteria tested.

Keywords: Alocasia denudata Engler; wound healing; antibacterial activity

INTRODUCTION

From a microbiological perspective, the

primary function of normal, intact skin is to

control microbial populations that live on the

skin surface and to prevent underlying tissue

from becoming colonized and invaded by

potential pathogens. Exposure of subcutaneous

tissue following a loss of skin integrity (i.e., a

wound) provides a moist, warm, and nutritious

environment that is conducive to microbial

colonization and proliferation (Bowler 2002).

However, the abundance and diversity of

microorganisms in any wound will be

influenced by factors such as wound type,

depth, location, and quality, the level of tissue

perfusion, and the antimicrobial efficacy of the

host immune response.

When infection occurs, a wound fails to

heal, the patient suffers increased trauma and

treatment costs rise. Thus, concern among

health care practitioners regarding the risk of

wound infection is justifiable not only in terms

of increased trauma to the patient but also in

view of its burden on financial resources and

the increasing requirement for cost effective

management within the health care system.

Topical antimicrobial therapy is one of the

81

most important methods of wound care

(Ranjith et al. 2006). The goal of topical

antimicrobial therapy in wound care is to

control microbial colonization and subsequent

proliferation thus promoting the healing of the

wounds (Veerapur et al. 2004). Some

medicinal plants are commonly used in folk

medicine for wound care (Rathi et al. 2004).

The usage of traditional medicine

involving herbal plants have been used by

almost 60% of the worldwide population

(World Health Organization 2003). In

developing countries, people prefer natural

products because they believe that natural

products have fewer side effects besides of

cheaper price (Modak et al. 2007; Shibabrata

et al. 2016; Maver et al. 2015). The potential

of some herbal plants to be developed into a

wound healing agent has been proven using

relevant in vitro antimicrobial activity test

(Adetutu et al. 2011; Kalayou et al. 2012)

Therefore, in wound healing, a wound healing

agent must be present in each treatment. In the

treatment of wound healing, anti-microbial

agent is important to ensure the wound heals

quickly and perfectly. There are many local

and foreign herbs that have been studied on its

effectiveness as antibacterial and antimicrobial

agents (Ette Okon Ettebong et al. 2017).

Among the main compounds that have

antibacterial properties are quinones,

anthraquinone, flavones, flavonoids,

flavonols, catechol and pyrogallol (Marjorie

1999).

Alocasia denudata Engler also known

as Keladi Ular or Keladi Harimau by local

community (Musa et al. 2009) have been used

by local communities to heal skin wounds.

Previous study has shown that A. denudata has

potential in accelerating wound healing in

mice (Mohd Zaki et al. 2011). Aqueous extract

of A. denudata can provide a good curative

effect on open wound in rat (Abdul Latif et al.

2015). Therefore, in this study, A. denudata

will be tested to screen for the antibacterial

effects rather than it’s beneficial in

accelerating wound healing process.

MATERIALS AND METHODS

Collection and Preparation of Plant

Material

Fresh plant materials of A. denudata were

collected from Kampung Gerik, Perak for

research purpose. Specimen sample was kept

at Herbarium, Faculty of Science and

Technology, National University of Malaysia

with reference number of UKM 29848.

Preparation of Aqueous Extracts

Distilled water was added to A. denudata stalks

with ratio of 1 ml distilled water and 1 g weight

of A. denudata stalks. The plant sample was

then grounded using a blender and the mixture

was filtered. The filtrate was then included in

1.5 ml appendorf tubes and centrifuged at a

speed of 13000 rpm for 30 minutes at 4°C. The

volume of supernatant was measured using a

measuring cylinder. The mixture was placed

into a round-bottomed flask before storing in

the freezer at -40 °C for 24 hours. After 24-

hour, the round-bottomed flask containing the

sample was subjected to the process of drying

(lyophilized) by using a freeze dryer until

powder is formed. When the powder is

obtained, it was stored in an airtight container

and stored in a freezer at -20 °C until use.

Preparation of Non-Polar and Polar

Extracts

Extraction of plant sample was conducted

using 2 different types of solvent which were

dichloromethane and methanol using cold

soaking method. 500 g of fresh plant sample

was extracted by soaking in 1 L

dichloromethane in a round bottom flask,

stirred for 6 minutes, closed tight using rubber

cork and left overnight at room temperature.

82

Thereafter, the solution was filtered using filter

paper (Whatman No. A-1) and the extract was

freeze dried and carefully stored at -18 °C in

labeled sterile bottles. The procedure was

repeated for methanol extraction.

Bacterial Culture

Two gram-positives i.e. Staphylococcus

epidermis and Staphylococcus aureus and

three gram-negatives i.e., Pseudomonas

aeruginosa, Klebsiella pneumoniae and

Proteus sp. were the bacterial species used

throughout this experiment. They were

obtained from the culture collection of the

Biomedical Science Programme, Faculty of

Health Sciences UKM. These bacteria were

chosen due their predominant cause of

infection on wound and skin.

Screening for Antibacterial Activity

The agar disc diffusion method was employed

for the determination of antibacterial activities

of the extracts of A. denudata Engler. All

bacterial cultures were first grown on nutrient

agar at 37 °C for 24 h. Few colonies (2 to 3)

of similar morphology of the respective

bacteria were transferred to a liquid medium

(Mueller Hinton Broth) and incubated until

adequate growth of turbidity equivalent to

McFarland 0.5 turbidity standard was

obtained. The inoculum of the respective

bacteria was streaked on to the Mueller Hinton

plates. The powdered plant extracts were

dissolved in 10% aqueous dimethyl sulfoxide

(DMSO) and sterilized by filtration through a

0.22 µm membrane filter. Sterile filter paper

discs (5 mm) (Whatman no. 1) were punched

and impregnated with 10 μl of the DCM,

MeOH and aqueous extracts (corresponding to

100, 50, and 25 and 12.5 mg/ml) and allowed

to dry at room temperature. These were placed

on the Mueller-Hinton agar plates inoculated

with the test strains. The plates were then

allowed to stay for 1 h at room temperature and

finally incubated at 37 °C for 24 hours. The

assessment of antibacterial activity was based

on the measurement of diameter of inhibition

zone (mm) formed around the disc. The

experiment was run in triplicate and the mean

values were presented. Gentamicin (30 μg)

was used as a positive control while 10%

DMSO acted as a negative control.

Determination of Minimum Inhibitory

Concentration (MIC)

The minimum inhibitory concentration (MIC)

of the crude extracts of A. denudata was

determined by a microdilution method, using

culture media of Mueller-Hinton broth. The

inoculum was prepared as described

previously. Methanol and dichloromethane

extraction were diluted with culture broth to a

concentration of 100 mg/mL. Further 1:2 serial

dilutions were performed by addition of

culture broth to reach concentrations ranging

from 100 to 0.098 mg/mL. A total of 50 μL of

each dilution were distributed in 96-well

plates, as well as a sterility control and a

growth control (containing culture broth plus

DMSO, without antimicrobial substance).

Each test and growth control well was

inoculated with 50 μL of a bacterial suspension

(108 CFU/mL or 105 CFU/well). All

experiments were performed in triplicate and

the microdilution trays were incubated at 36 ºC

for 18 h. MIC values were defined as the

lowest concentration of antibacterial

substances, which completely inhibited

microbial growth. The results were expressed

in milligrams per milliliters (19).

RESULTS

Aqueous extract of A. denudata showed the

greatest inhibition zone diameter on the growth

of K. pneumoniae which was is 7:26 ± 0.63

mm at a concentration of 100 mg/ml followed

by S. epidermidis of 7.0 ± 0.0 mm at a

concentration of 100 mg / ml. While P.

83

aeruginosa showed the smallest inhibition

zone with an average of 6.0 ± 0-41 mm at all

concentrations. Proteus sp. did do not show

any inhibition zone (Table

1). Dichloromethane extract solution showed

the largest inhibition zone diameter on the

growth of K. pneumoniae, S. aureus, and S.

epidermidis with the average measurement of

7.0 ± 0.0 mm at a concentration of 100 mg /

ml. While P. aeruginosa and Proteus sp.

showed the smallest diameter of the inhibition

zone between 0.0 ± 0.0 mm to 6.0 ± 1.0 mm

(Table 2).

Based on Table 3, methanol extract

solution showed the greatest inhibition zone

diameter on the growth of K. pneumoniae of

7.0 ± 2.0 mm at a concentration of 100

mg/ml. While P. aeruginosa. S. aureus, S.

epidermidis and Proteus sp. showed the

smallest inhibition zone with which the

average diameter of inhibition zones ranging

from 5.0 ± 0.0 mm to 6.0 mm ±

0:58. Minimum inhibitory concentration

(MIC) was conducted involving samples that

had a positive effect on the presence of

antibacterial activity screening test. Based on

Table 4, it was found that from the lowest

concentration of extract (0195 mg / ml) to the

highest concentration of 100 mg/ml for the

three types of extracts, no inhibition of growth

for all types of bacteria was

recorded. Therefore, no minimum inhibitory

concentration (MIC) was recorded in this

study.

DISCUSSION

Krishnaraju and Ganesan (1995) reported that

the process of plant extraction using different

solvents have potential to extract active

substances. Selection of solvent for the

extraction process is essential for the

extraction of active compounds found in plants

that are capable of curing illnesses

(Muthuvelan & Balaji, 2008). Three types of

solvents were used in this study comprising

methanol, dichloromethane and distilled water

(aqueous extraction). The solvents used have

different polarities where the highest polarity

solvent begins with distilled water followed by

methanol. Dichloromethane is the lowest

polarity solvent used in this study. Different

solvents will dissolve the active compound

depending on the polarity of the active

compound itself (Rohana 1997)

TABLE 1. Inhibition zone diameter of aqueous extract of A. denudata at different

concentrations.

Bacteria Concentration of extract (mg/ml) Positive

control

Negative

control 12.5 25 50 100

Inhibition zone (mm)

P.aeruginosa 6.0±0.41 6.0±0.41 6.0±0.41 6.0±0.41 21 0

S.aureus 5.5±0.29 5.5±0.29 5.5±0.29 5.5±0.29 25 0

K.pnemoniae 6.26±0.48 6.76±0.25 6.76±0.25 7.26±0.63 33 0

Proteus sp 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 25 0

S.epidermidis 6.76±0.25 6.76±0.25 6.76±0.25 7.0±0.0 31 0

84

TABLE 2. Inhibition zone diameter of dichloromentane extract of A. denudata at different

concentrations.


control

Negative

control 12.5 25 50 100


P.aeruginosa 0.0±0.0 5.26±0.25 6.0±1.0 5.5±0.5 21 0

S.aureus 5.26±0.25 6.0±0.58 7.0±0.0 7.0±0.0 25 0

K.pnemoniae 7.0±0.0 7.0±0.0 7.0±0.0 7.0±0.0 33 0

Proteus sp 5.26±0.25 5.5±0.5 0.0±0.0 0.0±0.0 25 0

S.epidermidis 7.5±0.36 5.83±0.5 7±0.82 7±0.82 31 0

TABLE 3. Inhibition zone diameter of methanol extract of A. denudata at different

concentrations.


control

Negative

control 12.5 25 50 100


P.aeruginosa 0.0±0.0 0.0±0.0 0.0±0.0 5.5±0.5 21 0

S.aureus 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 25 0

K.pnemoniae 5.5±0.5 6.5±1.5 6.5±1.5 7.0±2.0 33 0

Proteus sp. 6.0±0.58 6.0±0.58 6.0±0.58 6.0±0.58 25 0

S.epidermidis 6.0±0.58 6.0±0.58 6.0±0.58 6.0±0.58 31 0

Dichloromethane has high extraction

efficiency for most of the non-polar

compounds to polar compounds (Rossberg et

al. 2006). Dichloromethane is suitable for

simultaneous analysis and has the following

advantages: low boiling point of 39.6 °C and

easy to concentrate after the extraction process.

Also, easy to separate dichloromethane

solution containing the target compounds from

the water because of its specific gravity, and it

is not flammable.

Bacteria used in this study were obtained from

the American Type of Cell Culture (ATCC)

and used as the standard screening test for

antibacterial activity of plants. These bacteria

have been known not to exhibit any form of

resistance against any of the commercial

antibiotics today. Result demonstrated that the

aqueous extract of A. denudata showed no

inhibition zone on Proteus sp. while the

dichloromethane extract showed inhibition

against all the bacteria strains used in this

study.

85

TABLE 4. Minimum inhibition concentration (MIC) of extracts A. denudata

Bacteria Type of extracts

Aqueous Dichloromethane Methanol Positive

Control*

Negative

Control#

P. aeruginosa + + + + -

S. aureus + + + + -

S. epidermidis + + + + -

Proteus sp. + + + + -

K. pneumoniae + + + + -

Legends: * Positive control (50µL Muller Hinton broth + 50µL bacteria inoculum)

# Negative control (50µL Muller Hinton broth + 50µL extracts)

+ Presence of pellet or bacterial growth

- Absence of pellet or bacterial growth

However, the inhibition effect of

dichloromethane extract was not significant,

and this might be due to the active compound

that contains antibacterial activity was not

found in the stalk of A. denudata on that

polarity. Methanol extract did not show any

inhibition on S. aureus. However, inhibitory

effects on other bacteria were also not

significant. To fully assess the antimicrobial

activity of medicinal plant, other study using

standardized method is needed as additional

test (Ncube et al. 2008).

All extracts showed poor inhibitory

effect on all tested strains of bacteria which

produced inhibition zone much less than the

positive control of Gentamicin. This can be

associated with findings of previous studies

which indicated that the crude extract derived

from plant usually has low antibacterial

activity. Antibacterial substances have been

isolated from crude extract that contains less

bioactive substances (Akinyemi et al. 2006). In

addition, previous studies also showed that the

crude extract of bioactive herbaceous plants

have inhibitory zone around that value

(Olukoya et al. 1993). The lack of antibacterial

activity displayed by the results can be caused

by low solubility of the active compounds in

the solvents used for the studies. Further

studies using solvents with different polarity

may produce better methods to extract active

compounds (Romero et al. 2005). Eventough

the results displayed poor antibacterial

activity, it doesn’t mean that the plant itself do

not contain any antimicrobial active

compounds. This can be caused by small

concentration of antimicrobial active

compounds in the extract used at experimental

dosage (Taylor et al. 2001). This small

concentration can be increased by increasing

the dosage of extracts. Extracts also may not

have any effects on the bacteria used in this

study but may have effects on other bacteria

(Shale et al. 1999).

Previous preliminary screening on 12

traditionally used medicinal plants showed that

methanol extract had remarkable antibacterial

activity compared to aqueous extract (Parekh

et al. 2006). It may be caused by two main

factors which are the production of

biologically active components naturally

found in plants such as saponins, tannins,

86

alkaloids and anthraquinone which can be

enhanced by the presence of methanol

(Akinyemi et al. 2005). Saponins and alkaloids

are metabolites found to have antibacterial

activity (Tschesche 1971). The second factor is

a high extraction capacity by methanol

allowing more antibacterial active components

to be recovered (Akinyemi et al. 2005).

Another factor that influenced the

sensitivity test results were temperature and

period of incubation. In this study, agar plates

were incubated at 37 °C for 24 hours. The

incubation time should be adjusted as long

incubation period would result in a smaller

zone of inhibition and vice versa. Thickness of

the agar used was about 5 mm. Inhibitory zone

size would be increased when the agar used

was too thin and would give false positive

results (NCCLS 2000).

In this study, the MIC test was

performed to determine the minimum

concentration of the three extracts of A.

denudata that can inhibit the growth of test

bacteria. Among the factors that influenced the

MIC value are the size of the inoculum, agar

medium, incubation time, pH and temperature

(Jeniffer 2001). MIC values obtained from the

three extracts against the five types of bacteria

were negative. All extracts could not inhibit

the growth of bacteria studied. This has proven

that the aqueous solvent, methanol and

dichloromethane were not capable of

producing enough antibacterial substances

found from the stalk of A. denudata.

CONCLUSION

This study has found that the aqueous,

methanol and dichloromethane extract of A.

denudata showed insignificant inhibition on

the tested bacteria. The study also found no

visible effect on the inhibition of Proteus. sp

by aqueous extract. Methanol extracts also

showed no inhibition zone on Staphylococcus

aureus. The minimum inhibitory concentration

test showed no MIC values resulting from all

the extracts tested on each type of bacteria. In

conclusion, aqueous, methanol and

dichloromethane extraction of A. denudata

exhibited insignificant antibacterial effect

against bacteria that cause skin diseases tested

in this study.

ACKNOWLEDGEMENT

This study was made possible with fundings

from the research grants UKM-GUP-2011-121

and FRGS/ 1/2014/SKK01/UKM/02/1,

Programme of Biomedical Science, School of

Diagnostic & Applied Health Science, Faculty

of Health Sciences, Universiti Kebangsaan

Malaysia.

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Mazlyzam Abdul Latif

Wafa’ Zahari

Ahmad Zorin Sahalan

Siti Aisyah Arshad

Asmah Hamid*.





50300 Jalan Raja Muda Abdul Aziz,

Kuala Lumpur,

Malaysia


Buletin FSK 1(1)(2017): 89-94

Pemencilan Acanthamoeba spp. daripada Paip Air Domestik (Isolation of Acanthamoeba spp. from Domestic Water Taps)

MOHAMED KAMEL ABD GHANI*, NURULHUDA SHARIF, ANISAH NORDIN,

YUSOF SUBOH, NORAINA ABD RAHIM & NORAZAH AHMAD

ABSTRAK

Acanthamoeba sp. merupakan sejenis ameba hidup bebas yang boleh menyebabkan

keratitis dan infeksi sistem saraf pusat. Keratitis biasanya terjadi akibat penggunaan kanta

sentuh yang terkontaminasi atau pemakaian kanta sentuh semasa berenang di kawasan

tercemar. Kajian ini dijalankan untuk memencilkan Acanthamoeba sp. daripada paip air.

Sebanyak 90 sampel swab paip air domestik telah diambil. Sampel swab dimasukkan ke

dalam botol Bijou yang steril yang mengandungi 10 ml larutan salin Page. Kesemua sampel

dituras menggunakan membran turas dengan liang bersaiz 5 m. Sedimen yang diperolehi

diletakkan di atas agar bukan nutrien yang telah dilapisi dengan Escherichia coli matian

haba. Plat kemudian dieram pada suhu 30C dan diperiksa setiap hari di bawah ‘inverted

microscope’ selama 14 hari untuk melihat jika ada sebarang pertumbuhan Acanthamoeba

sp. Morfologi Acanthamoeba peringkat trofozoit dan sista digunakan sebagai kriteria

taksonomi untuk pengenalpastian. Secara keseluruhannya, sampel swab paip air

menunjukkan kehadiran Acanthamoeba (1.1%). Hasil penemuan ini menunjukkan paip air

berupaya menyediakan habitat untuk organisma ini membiak. Oleh itu, paip air berpotensi

menyalurkan air yang terkontaminasi oleh ameba dan ia amat berbahaya terutamanya

kepada pengguna kanta sentuh yang mencuci kanta sentuh dengan menggunakan air paip.

Penemuan organisma ini daripada sampel paip air menjadi peringatan berguna khususnya

kepada pengguna kanta sentuh agar mereka tidak menggunakan air paip untuk mencuci

kanta sentuh atau peralatannya dan tidak memakai kanta sentuh semasa berenang.

Kata kunci: Acanthamoeba; pemencilan; swab paip air

ABSTRACT

The pathogenic, free living amoeba, Acanthamoeba sp. is the causative agent of keratitis

and infection of central nervous system. Individuals become infected with keratitis because

of wearing contaminated contact lens or swimming in contaminated water. This study was

carried out to isolate Acanthamoeba sp. from domestic water taps. A total of 90 domestic

water taps were swabbed. Swabs obtained from water taps were put into steriled Bijou

bottle containing 10 ml of Page saline solution. All samples were filtered through a

membrane filter of 5 m pore size. Heat-killed Escherichia coli was pipetted as nutrient

source before the sediment was inoculated on the surface of non-nutrient agar (NNA). The

plates were incubated at 30C and examined daily using inverted microscope for 14 days

for growth of Acanthamoeba sp. Morphology of the Acanthamoeba trophozoites and cysts

were used as the taxonomic criteria for identification. Overall, the result showed that only

1.1% of the water tap swab was positive for Acanthamoeba. This study suggested the

90

possibility of water taps acting as reservoir for Acanthamoeba sp. multiplication.

Therefore, domestic water supply running through contaminated water taps might become

a potential source of Acanthamoeba sp. infection to human especially among contact lens

wearers who do not practice good hygienic care while cleaning their contact lenses. The

presence of this organism in water taps will be a reminder to contact lens users not to use

tap water to clean their contact lens paraphernalia or wear them while swimming.

Keywords: Acanthamoeba; isolation; water tap swab

PENGENALAN

Acanthamoeba sp. merupakan sejenis

ameba hidup bebas yang tersebar luas di

persekitaran termasuk air, udara dan tanah.

Pada manusia ia boleh hidup dalam rongga

hidung dan tekak (Visvesvera & Stehr-

Green 1990). Organisma ini hadir di

persekitaran dan tisu manusia dalam 2

peringkat hidup iaitu peringkat sista yang

sangat rintang terhadap keadaan

persekitaran dan peringkat trofozoit yang

mampu berensistasi dalam keadaan

persekitaran yang tidak sesuai untuk

kemandiriannya (Ma et al. 1990).

Terdapat beberapa spesies

Acanthamoeba yang bersifat patogenik

terhadap manusia dan berupaya

menginfeksi sistem saraf, mata, hidung

dan kulit (Szenasi et al. 1998).

Walaubagaimanapun, infeksi

Acanthamoeba sp. pada kornea telah mula

mendapat perhatian penyelidik sejak

kebelakangan ini kerana ia boleh

menyebabkan keratitis yang boleh

membawa kepada kebutaan (Seal et al.

1995).

Kes pertama keratitis Acanthamoeba

sp. dikesan di Texas pada 1973 daripada

spesimen kikisan kornea pesakit yang

mengalami trauma dan terdedah kepada air

yang terkontaminasi. Sejak kes pertama

dilaporkan sebanyak 10 kes lagi dilaporkan

dalam tempoh beberapa dekad.

Kebanyakan kes-kes awal keratitis

berasosiasi dengan insidens trauma pada

mata atau pendedahan mata kepada air

terkontaminasi (Kilvington & White

1994).

Namun, bilangan kes keratitis semakin

meningkat secara dramatik sejak 1985

berikutan peningkatan penggunaan kanta

sentuh terutamanya kanta sentuh lembut

sebagai alternatif bagi mengatasi masalah

penglihatan dan tujuan kosmetik

disebabkan ciri-cirinya yang fleksibel dan

selesa apabila dipakai (Schaumberg et al.

1998; Kilvington 1993).

Di Malaysia, kes pertama keratitis

Acanthamoeba sp. telah dilaporkan pada

1995 melibatkan seorang wanita yang

mempunyai sejarah penggunaan kanta

sentuh yang terkontaminasi dalam jangka

masa yang lama (Mohamed Kamel &

Norazah 1995).

Kanta sentuh yang terkontaminasi

bertanggungjawab menjadi alat untuk

menularkan Acanthamoeba sp. pada

kornea dan seterusnya menyebabkan

keratitis (Kilvington 1993). Sumber

kontaminasi boleh diperolehi daripada

persekitaran apabila pengguna membasuh

kanta sentuh dengan air paip atau salin

pencuci yang tidak steril atau pemakaian

kanta sentuh semasa mandi atau berenang

(Kilvington & White 1994; Schaumberg et

al. 1998).

Di Malaysia, laporan mengenai

kehadiran ameba hidup bebas ini di

persekitaran akuatik masih amat kurang.

Oleh itu bagi memahami dengan lebih

jelas, hubungan antara infeksi

Acanthamoeba sp. terhadap manusia

dengan sumber penyebab infeksi, kajian ini

91

dijalankan bagi menilai kehadiran

Acanthamoeba sp. daripada sumber paip

air. Dengan ini, diharapkan pemahaman

mengenai sumber infeksi ameba ini akan

diketahui dengan lebih jelas dan langkah-

langkah pencegahan boleh diambil.

BAHAN DAN KAEDAH

Sampel

Bagi sampel swab paip air, sebanyak 90

sampel telah dipilih. 60 sampel terdiri

daripada swab saluran air paip yang

disalurkan daripada tangki simpanan

manakala 30 sampel lagi terdiri daripada

swab ke atas saluran air paip yang

disalurkan terus daripada paip utama.

Sampel swab seterusnya dimasukkan ke

dalam botol Bijou yang mengandungi 10

ml larutan salin Page sebagai media

pengangkut. Sampel diambil di pelbagai

lokasi berbeza iaitu di Kolej Tun Syed

Nasir, Kampus UKM Cawangan Kuala

Lumpur, Kampung Boyan dan rumah

pengguna di sekitar daerah Kluang.

Pemprosesan Sampel

Sampel swab bersama media pengangkut

divorteks untuk menanggalkan organisma

yang melekat pada swab. Membran turas

diletakkan di atas set penuras dan larutan

tersebut kemudian dituras menggunakan

pam vakum Apabila proses penurasan

selesai, membran turas dipindahkan

daripada set penuras dan diletakkan secara

terbalik di atas plat agar bukan nutrien yang

mengandungi ‘heat killed' E. coli. Sampel

tersebut kemudian dieram pada suhu 30C.

Sampel kemudian diperiksa di bawah

‘inverted microscope’ selama 14 hari untuk

mencerap kehadiran trofozoit dan sista

sebelum sampel disahkan negatif.

Hasil Kajian

Sebanyak 1 daripada 90 (1.1%) sampel

swab paip air menunjukkan hasil positif

kehadiran Acanthamoeba sp. Bilangan

sampel positif daripada setiap lokasi yang

diambil ditunjukkan dalam Jadual 1

manakala Jadual 2 menunjukkan peratusan

sampel yang positif.

PERBINCANGAN

Dalam kajian ini, pemencilan

Acanthamoeba sp. hanya sedikit diperolehi

daripada sampel swab paip air iaitu

mewakili 1.1% daripada keseluruhan

sampel. Ini mungkin disebabkan sumber

makanan bagi ameba ini adalah kurang

pada kawasan paip air. Anisah et al. 2003

juga telah menemui kehadiran

Acanthamoeba daripada swab paip air

domestik di sekitar Kuala Lumpur

sebanyak 2.4%. Teknik swab ke atas paip

air dipilih dalam kajian ini kerana ia lebih

sensitif berbanding menggunakan sampel

air paip (Stapleton et al. 1991). Ini adalah

kerana akumulasi bahan organik pada

muncung paip air berupaya menyediakan

habitat yang sesuai untuk pelekatan dan

pembiakan ameba ini (Anisah et al. 2003).

Sista Acanthamoeba sp. sangat

resistan terhadap klorin dan sedimen lain

(Radford et al. 1995). Oleh itu, penemuan

ameba daripada swab paip air dalam kajian

ini tidaklah mengejutkan. Dalam kajian

yang dijalankan oleh Jonckheere dan

Voorde (1976), mereka mendapati

kepekatan klorin pada aras 4 g/ml tidak

berupaya memusnahkan sista ameba ini

selepas interaksi selama 3 jam.

92

JADUAL 1. Pemencilan Acanthamoeba sp. daripada sampel swab paip air.

Jenis sampel Bilangan sampel yang positif

Acanthamoeba sp./ jumlah keseluruhan

sampel yang diuji

Swab paip air

Sumber air paip daripada paip utama

Sumber air paip daripada tangki simpanan

0/30

1/60

JADUAL 2. Peratus sampel yang positif

Jenis sampel

Bilangan sampel yang

positif/jumlah keseluruhan

sampel

Peratus sampel yang positif

(%)

Swab Paip Air

1/90

1.1

Ia hanya boleh dimusnahkan jika air paip

dirawat dengan klorin dalam suatu jangka

masa yang lama. Hasil penemuan ini

menunjukkan tabiat mencuci kanta sentuh

dengan menggunakan air paip yang

terkontaminasi berupaya menyebabkan

penularan ameba ini pada permukaan kanta

sentuh yang seterusnya boleh

menyebabkan keratitis Acanthamoeba

(Larkin et al. 1990; Mohamed Kamel et al.

2000). Salah seorang pesakit keratitis

Acanthamoeba di Malaysia, yang juga

seorang pengguna kanta sentuh, mendapat

jangkitan ini kerana telah menggunakan air

paip terkontaminasi semasa mencuci kanta

sentuh beliau (Mohamed Kamel et al.

2000).

Bagi persampelan swab paip air,

larutan salin Page digunakan sebagai media

pengangkut. Larutan salin Page merupakan

larutan yang mempunyai tekanan osmotik

yang rendah yang dapat mengekalkan

struktur dan morfologi trofozoit. Kaedah

pengkulturan sampel secara terus ke atas

agar bukan nutrien adalah kaedah yang

terbaik. Namun, kadang kala dalam

keadaan tertentu media pengangkut boleh

digunakan. Dalam kajian ini, media

pengangkut digunakan kerana keadaan

persekitaran kawasan persampelan tidak

sesuai untuk pengkulturan terus kerana

persekitarannya tidak steril. (Gradus et al.

1989).

Ameba ini telah dibuktikan wujud

di persekitaran akuatik dalam kajian yang

dilakukan di luar negara. Keputusan yang

diperolehi melalui kajian ini menunjukkan

Acanthamoeba sp. memang wujud di

persekitaran akuatik di Malaysia.

KESIMPULAN

Hanya 1.1% daripada sampel swab paip air

domestik yang diambil menunjukkan

kehadiran Acanthamoeba. Kehadiran

ameba ini pada swab paip air membuktikan

bahawa paip air boleh menjadi habitat bagi

organisma ini untuk membiak. Oleh itu,

paip air berpotensi untuk menyalurkan air

paip yang dikontaminasi oleh ameba dan

93

ini amat berbahaya terutamanya kepada

pengguna kanta sentuh yang tidak

mengamalkan aspek kebersihan semasa

mencuci kanta sentuh.

RUJUKAN

Anisah, N., Yusof, S., Wan Norliana, A. &

Norhayati, M. 2003. Isolation of

Acanthamoeba sp. from domestic

water tap. Tropical Biomedicine

20(1):87-89.

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Decarlo, J. 1989. Filter culture

technique using amoeba saline

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Pathology 92: 682-685.

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Differences in destruction of cyst of

pathogenic and nonpathogenic

Acanthamoeba by chlorine. Applied

and Environmental Microbiology

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Kilvington, S. & White, D.G. 1994.

Acanthamoeba: biology, ecology and

human disease. Reviews in Medical

Microbiology 5(1): 12-20.

Kilvington, S. 1993. Acanthamoeba

trophozoite and cyst adherence to four

types of soft contact lens and removal

by cleaning agents. Eye 7: 535-538.

Larkin, D.F.P., Kilvington, S. & Easty, D.L.

1990. Contamination of contact lens

storage cause by Acanthamoeba and

bacteria. British Journal of

Ophthalmology 74: 133-135.

Ma, P., Visvesvera, G.S., Martinez, A.J.,

Theodore, F.H., Daggett, P.M. &

Sawyer, T.K. 1990. Naegleria and

Acanthamoeba infections: Review.

Reviews of Infectious Diseases 12(3):

490-510.

Mohamed Kamel, A.G. & Norazah, A. 1995.

First case of Acanthamoeba keratitis in

Malaysia. Transactions of The Royal

Society of Tropical Medicine and

Hygine 89: 652.

Mohamed Kamel, A.G., Faridah Hanom, A.,

Norazah, A., Noor Rain, A., Hay, J. &

Seal, D. 2000. A case of waterborne

contact lens associated Acanthamoeba

Keratitis from Malaysia: successful

treatment with Chlorhexidine and

Propamidine. International Medical

Journal 7(1): 63-65.

Radford, C.F., Bacon, A.S., Dart, J.K.G. &

Minassian, D.C. 1995. Risk factors for

Acanthamoeba keratitis in contact lens

users: A case control study. British

Medical Journal 310: 1567-1570.

Schaumberg, A.D., Snow, K.K. & Dana, M.R.

1998. The epidemic of Acanthamoeba

keratitis: Where do we stand. Cornea

17(1): 3-10.

Seal, D.V., Hay, J. & Kirkness, C.M.. 1995.

Acanthamoeba keratitis: a water borne

disease. Dlm. Betts, W.B., Casemore,

D., Fricker, C., Smith, H. & Watkins,

J. (pnyt). Protozoan Parasites and

Water. hlm. 223-225. Britain: The

Royal Society of Chemistry.

Stapleton, F., Seal, D.V. & Dart, J. 1991.

Possible environmental sources of

Acanthamoeba species that cause

keratitis in contact lens wearers.

Reviews of Infectious Diseases 13(5):

S392.

Szenasi, Z., Endo, T., Yagita, K. & Nagy, E.

1998. Isolation , identification and

increasing importance of free living

amoeba causing human disease.

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94

Mohamed Kamel Abd Ghani*

Nurulhuda Sharif






50300 Kuala Lumpur,

Malaysia.

Anisah Nordin

Yusof Suboh

Noraina Abd Rahim

Jabatan Parasitologi,

Fakulti Perubatan,


Norazah Ahmad

Institut Penyelidikan Perubatan,

Jalan Pahang,

Kuala Lumpur

*Corresponding author; email : [email protected], [email protected]

BULETIN SAINS KESIHATAN (BSK)

Panduan Kepada Penyumbang

SKOP

Buletin Sains Kesihatan (BSK) ialah sebuah jurnal ilmiah berwasit yang komited kepada

perkembangan dapatan penyelidikan dalam pelbagai bidang sains kesihatan dan perubatan serta

teknologi berkaitan. Ia meliputi makalah dalam pelbagai aspek sains kesihatan, penyelidikan klinikal

dan juga pra klinikal. Antara bidang tersebut termasuklah audiologi, biokimia, pergigian, dietetik,

pengimejan perubatan, sains bioperubatan, sinaran perubatan, pemakanan, optometri, farmakologi,

farmasi, fisiologi, fisioterapi, terapi carakerja, sains forensik, kesihatan masyarakat, psikologi

kesihatan, kesihatan persekitaran, sains pertuturan dan sains sukan. Buletin ini menerbitkan kertas asli,

komunikasi pendek, laporan kes dan nota penyelidikan yang menarik minat ramai para sarjana. BSK

diterbitkan oleh Sidang Pengarang daripada Fakulti Sains Kesihatan, Universiti Kebangsaan Malaysia.

Selain itu para sarjana terkenal daripada universiti dalam dan luar negara dilantik sebagai ahli lembaga

penasihat dan penilai artikel yang dikemukakan.

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Segala surat-menyurat mengenai makalah serta perkara yang berkenaan hendaklah dialamatkan

kepada:

Editor-in-Chief

BULETIN SAINS KESIHATAN


Universiti Kebangsaan Malaysia.


Kuala Lumpur, Malaysia.

E-mail: [email protected]

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Para penulis bertanggungjawab sepenuhnya bagi memastikan manuskrip mereka tidak melanggar

mana-mana hak cipta yang sedia ada. Para penulis seharusnya mendapat keizinan untuk menerbitkan

semula atau mengubahsuai bahan-bahan yang mempunyai hak cipta, dan menunjukkan bukti keizinan

tersebut semasa menyerahkan naskah akhir manuskrip.

PROSES PENILAIAN

Sesebuah manuskrip akan dinilai oleh Sidang Editor dan sekurang-kurangnya seorang pewasit bebas.

Keputusan tentang penerbitan sesebuah manuskrip didasarkan kepada saranan ahli-ahli lembaga ini.

Sesebuah manuskrip akan dinilai berasaskan kesesuaiannya dengan Buletin Sains Kesihatan,

sumbangan kepada disiplin ilmu, kejituan analisis, keluasan konsepsual, persembahan yang jelas, dan

kesempurnaan teknikal. Nama penuh dan afiliasi semua penulis manuskrip hendaklah dinyatakan pada

halaman depan yang dibuat secara berasingan dengan manuskrip. Manuskrip yang diserahkan oleh

mana-mana anggota Sidang Editor juga tertakluk kepada prosedur penilaian yang sama.

NASKAH SEMAKAN

Satu set pruf akan dihantar kepada penulis bagi tujuan penyemakan kesilapan percetakan. Adalah

menjadi tanggungjawab penulis untuk memaklumkan sebarang pembetulan kepada Sidang Editorial

BULLETIN OF HEALTH SCIENCES (BHS)

Guide to Contributors

SCOPE

The Bulletin of Health Sciences (BHS) is a refereed journal committed to the advancement of scholarly knowledge

and research findings of health and medical sciences and the related technology. It contains articles on every aspect

of health sciences, pre clinical and clinical related research. These include audiology, biochemistry, dentistry,

dietetic, medical imaging, biomedical science, radiotherapy, nutrition, optometry, pharmacology, pharmacy,

physiology, physiotherapy, occupational therapy, forensic science, public health, health psychology, environmental

health, speech science and sports science. The bulletin publishes original articles, short communications, case

reports and research notes whose content and approach are of interest to a wide range of scholars. BHS is published

by an autonomous Editorial Board drawn from the Faculty of Health Sciences, Universiti Kebangsaan Malaysia. In

addition, distinguished scholars from local and foreign universities are appointed to serve as referees and advisory

board members.

SUBMISSION PROCEDURE

The bulletin publishes manuscripts written in the Malay and English language. Manuscript submitted to the journal

for publication should be original contribution and must not have been previously published or is under

consideration simultaneously by any other publication.

The manuscript should be typed with double spacing, single column and font size 12 Times New Roman on A4

paper not exceeding 15 pages for original articles (6 pages for research notes, short communications and case reports)

and should be submitted using the online submission system.

All correspondence pertaining to articles and related matters should be addressed to:

Editor-in-Chief

BULLETIN OF HEALTH SCIENCES






FORMAT AND STYLE

The title of a manuscript should be concise, descriptive and preferably not exceeding 15 words. The manuscript

must include an abstract, describing its main points within 150 - 250 words in the Malay and English language. If

the manuscript is written in malay, it must also have an english abstract and title.

In general, the contents should comprise of Introduction, Materials and Methods, Results and Discussion,

Conclusion, Acknowledgement and References. The manuscript should be supplied with 3-5 keywords.

All illustrations including figures, charts and graphs, must be labelled and supplied on pages separate from the text.

The desired placement in the text should be clearly indicated. These illustrations should be referred to and numbered

serial, as figures. All illustrations should be clearly drawn in permanent ink or photographed in sharp black and

white and reproduced in the form of high - contrast glossy prints or digital images and provided in camera ready

form.

1

References in the text should be denoted by giving the name(s) of the author(s). All alphabetically ordered

references list should be included at the end of the manuscript. All references cited in the text must appear in the

reference list. Authors are responsible for the accuracy and completeness of all information in the reference.

Manuscripts must conform to the references in Penerbit UKM style or the Chicago Manual of Style (University of

Chicago Press). The references style adopted should be consistent throughout the manuscript.

COPYRIGHT

It is the author's responsibility to ensure that his or her submitted work does not infringe any existing copyright.

Authors should obtain permission to reproduce or adapt copyrighted material and provide evidence of approval upon

submitting the final version of a manuscript.

REVIEW PROCESS

Manuscripts will be reviewed by the Editorial Board and at least one independent referee. Decisions regarding the

publication of a manuscript will be based on the Board's recommendations. The manuscript will be evaluated based

on its appropriateness for the Bulletin of Health Sciences (BHS), contribution to the discipline, cogency of analysis,

conceptual breadth, clarity of presentation and technical adequacy. Manuscripts submitted by members of the

journal's Editorial Board are subjected to the same review procedure.

PROOFS

One set of proofs will be sent to the author(s) to be checked for printer’s errors and it is the responsibility of the

author(s) to submit corrections to the Editorial Board.

BULETIN SAINS KESIHATAN (BSK) ialah sebuah jurnal ilmiah berwasit yang diterbitkan dua kali

setahun secara atas talian oleh Fakulti Sains Kesihatan, Universiti Kebangsaan Malaysia. Ia menerbitkan

makalah dalam bidang sains kesihatan dan perubatan serta teknologi yang berkaitan. Makalah ditulis

dalam bahasa Melayu atau Inggeris dan boleh berbentuk kertas asli, komunikasi pendek, laporan kes atau

nota penyelidikan. Tujuan utama jurnal ini ialah untuk menyediakan saluran bagi menerbitkan karya

penyelidikan yang dijalankan dalam bidang sains kesihatan di Universiti Kebangsaan Malaysia dan

mengalu-alukan sumbangan karya dari dalam dan luar negara.

Segala surat menyurat mengenai makalah serta perkara yang berkenaan hendaklah dialamatkan

kepada:

BULLETIN OF HEALTH SCIENCES (BHS) is a peer reviewed online journal published biannually by

the Faculty of Health Sciences, National University of Malaysia. It publishes articles in the field of

medical and health sciences and the related technology. Articles are published in Malay or English and

can be written as original articles, short communications, case reports and research notes. The primary

purpose of this journal is to act as a channel for the publication of research work on health sciences

undertaken at Universiti Kebangsaan Malaysia and contribution of articles from within and outside the

country is most welcomed.

All correspondence pertaining to articles and related matters should be addressed to:

Ketua Editor / Editor-in-Chief

BULETIN SAINS KESIHATAN

Fakulti Sains Kesihatan,





Mechanical Stretch Application in Identifying Vascular Related Diseases Nurul Farhana Jufri

Visual Perceptual Skills Performance Among a Sample of Year-2 Students in Klang Valley Sumithira Narayanasamy, Hui Yee Tey, Yin Wong & Sharanjeet-Kaur

Detection of Antibacterial Activity from the Crude Stalk Extracts of Alocasia denudata Engler Mazlyzam Abdul Latif, Wafa’ Zahari, Ahmad Zorin Sahalan, Siti Aisyah Arshad & Asmah Hamid

Pemencilan Acanthamoeba spp. daripada Paip Air Domestik Mohamed Kamel Abd Ghani, Nurulhuda Sharif, Anisah Nordin, Yusof Suboh, Noraina Abd Rahim & Norazah Ahmad

67 – 71

72 – 79

80 – 88

89 – 94

Diterbitkan Oleh

Fakulti Sains Kesihatan Universiti Kebangsaan Malaysia

Jalan Raja Muda Abdul Aziz 50300, Kuala Lumpur

Malaysia

www.ukm.my/fsk

Dicetak Oleh UKM Cetak 2017

· 01-01-2017 · Acanthamoeba spp. yang diuji diperolehi daripada Makmal Acanthamoeba, Fakulti...

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