Subtyping and Surveillance of Salmonellosis in Malaysia.

Surveillance and Subtyping of 

Salmonella spp. spp. 

in Malaysia. 

Kwai Lin Thong, PhD 

University of Malaya 

Kuala Lumpur, Malaysia

Outline 

Overview of Surveillance system in 

Malaysia 

Incidence of Salmonellosis in Malaysia 

Subtyping by PFGE of predominant 

Salmonella enterica serovars

Foodborne Disease Surveillance 

Carried out by different units, sections, departments and 

institutions within the Ministry of Health 

Passive surveillance 

Data collected through physician based surveillance and 

outbreak investigation 

Notification is received from government health facilities 

& private hospitals & GP 

Foodborne diseases monitored: cholera, typhoid & 

paratyphoid, hepatitis A, dysentery, food poisoning 

(nontyphoidal 

nontyphoidal Salmonella & other etiologic agents) 

Acute gastroenteritis (AGE) also reported but not 

mandatory

Overview of Surveillance Food Borne 

Laboratory 

surveillance 

Microbiology 

Labs 

IMR/ 

PHLs 

diseases in Malaysia 

Mandatory 

Notification 

Dis. Surv. 

Public: 

Health Ctr 

Hospitals 

Private: 

GP clinics 

Hospitals 

District Heath Office 

State Health Dept 

Clinical Based 

(sentinel/national 

Syndromic 

Sentinel selected 

Clinics 

National (hospitals) 

Syndromic A/E wards 

National : Div. Disease Control, Ministry of Health 

Community based 

surveillance 

Community/Media 

International sources 

Other 

agencies 

Veterinary Dept 

(Zoonoses)

Incidence rates in Malaysia (1995-2003) 

DISEASES 1995 1996 1997 1998 1999 2000 2001 2002 2003 

Food poisoning 7.08 15.29 31.08 31.45 38.04 36.61 29.99 28.63 14.72 

Typhoid/ 

paratyphoid 

Salmonella infection 

-major foodborne disease in Malaysia 

per 100.000 population 

4.46 4.50 3.24 3.53 3.57 3.45 2.92 3.48 1.74 

Source: Disease Control Div, Ministry of Health, Malaysia 2005

Burden of Foodborne diseases 

True incidence –difficult difficult to determine 

Passive surveillance: reported cases 

*67% of reported outbreaks occurred in public schools, schools, 

residential 

colleges,factories 

*any outbreak cases or sporadic cases involved any any 

food outlets – 

difficult to detect 

Grossly underestimated 

A local study done in the State of Kelantan 

(2001) showed a higher ( 2x) estimated 

annual IR of typhoid cases (Wan ( Wan Mansor, Mansor, 

unpublished 

data)

Laboratory Based Surveillance for 

Introduced in 2002 

Infectious Diseases 

an initiative between Surveillance Section & Institute for Medical 

Research (IMR), Ministry of Health 

Complements the mandatory notifiable disease surveillance system. 

This system requires participating hospitals to submit isolates isolates 

to IMR 

for re-culture re culture and subtyping. subtyping 

Database may not be 100% representative as it involves only 

government hospital laboratories 

Currently 6 types of bacteria being monitored: 

Vibrio cholerae, cholerae, 

Haemophillus influenzae, influenzae, 

Salmonella enterica 

S. Typhi/Paratyphi, 

Typhi/Paratyphi, 

Neisseria meningitidis

Laboratory based surveillance flowchart 

Microbiology labs 

of public/private 

hospitals & private labs 

notification 

Isolates with laboratory 

IMR/NPHL and other 

Reference Labs 

Notification form ( confirmation, typing, etc 

notification 

District Health Officer 

State Health Office 

results 

results 

Communicable Disease Surveillance 

Section, Ministry of Health 

Data notification Information feedback

Infectious Disease Bulletin 

Based on the lab- 

surveillance system, the 

Disease Surveillance 

Section, Disease Control 

Division, MOH, 

publishes a Bulletin 

Aims: 

•To To diseminate/share 

diseminate/share 

information on 

communicable disease 

and to stimulate 

exchanges of ideas 

•To To update and inform 

health professionals 

http://dph.gov.my

Top 10 Nontyphoidal Salmonella Serotypes identified and 

reported to the Laboratory Based Surveillance database, 2003-5 

2003 2004 2005 

Serotypes total % Serotypes total % Serotypes total % 

Enteritidis 244 26.7 Enteritidis 206 25.0 Enteritidis 155 28.1 

Weltevreden 200 21.9 Weltevreden 165 20.0 Weltevreden 142 25.7 

Corvallis 115 12.6 Corvallis 117 14.2 Corvallis 57 10.3 

Typhimurium 49 5.4 Typhimurium 43 5.2 Typhimurium 37 6.7 

Stanley 32 3.2 Albany 37 4.5 Limete 9 3.3 

Tshongwe 29 3.2 Limete 18 2.2 Stanley 8 1.4 

Biegdam 19 2.1 Braenderup 15 1.8 Agona 7 1.3 

Albany 17 1.9 Tshongwe 15 1.8 Albany 5 0.9 

Braenderup 12 1.3 Stanley 11 1.3 Rissen 5 0.9 

Newport 10 1.1 Bovismorfibic 

ans 

10 1.2 Virchow 5 0.9 

Total 915 682 460 

Source: Lab Surv. Bulletin, Disease Control Division, Ministry of 

Health Malaysia (October 2005 )

Application of PFGE as a 

Subtyping tool 

Still not fully implemented in all PHLs 

Designated 4 PHLs 

- availability of infrastructure: PFGE 

equipment 

-lack lack of technical expertise 

-no no dedicated staff to perform PFGE 

Institute of Medical Research (IMR) : 

National Reference Centre: Centre: 

routinely does 

serotyping, serotyping, 

AST, and perform PFGE on a case to case 

basis

Salmonella Typhi 

most common serotype 

Typhoid fever 

A public health problem & endemic in 


Annual incidence of 10.2 to 17.9 per 

100,000 population but can be as high as 

50.3 /100,000 in certain hyper-endemic 

state ( eg Kelantan)

Example of application of PFGE in a 

recent outbreak of typhoid fever in 


In 2005, an outbreak of typhoid fever in 

Kelantan 

As of 8 May, 2005- 2005 State Health Dept of 

Kelantan reported 517 confirmed typhoid 

cases. Included 2 deaths 

19 (2.14%) food samples, 

12 ( 2.9%) drinking water samples 

2 (0.2%) food handlers 

–tested tested positive for Salmonella spp

Week 

Number of notified cases of Typhoid Fever 

in Kelantan, Malaysia (May-June 2005) 

No cases ( 

no death) 

Cumulative cases ( death) 

(throughout the country) 

2005 2004 

Wk 18 ( 1-7 May 2005 397 1408 (0) 146 (0) 

Wk 19 (8-14 May,2005 120 (2) 1533 (2) 152 

Wk 20 (15-21 May,2005) 156 1718 (2) 158 

Wk 21 (22- 28.5.2005) 99 1822 (2) 160 (0) 

Wk 22( 29.5 - 4.6.2005 84 1919 (2) 164 (0) 

Wk 23 (5 -11.6.2005) 61 1985 (2) 170 

Wk 24 (12 -18.6.2005) 48 2013(2) 183 

Wk 25(19- 25.6.2005) 23 2045 185 

Source: Infect Dis Bull. Disease 

Control Div, Min Health, 


988

Action 

(Ministry of Health) 

State Response Team 

Rapid Response Team (RRT) from Min.Health

Action 

Intensive health education activities 

carried out 

Public awareness on personnel hygiene 

was actively promoted 

Typhim Vi vaccination given to food 

handlers 

A total of 528 food premises were closed 

under PCID Act 1988 and Food Act 1983

Source: Infectious Dis. Bulletin, MOH, August,2005

Laboratory Analysis 

Strains were submitted to IMR for phage-typing, 

antimicrobial susceptibility test, and PFGE 

subtyping. 

- mostly phage B1 type 

- All antibiotic sensitive strains 

A total of 103 S. Typhi isolates with Vi-Phage 

Type B1, isolated in 2004 until May 2005 were 

picked randomly, and analyzed by PFGE at the 

Bacteriology Unit, IMR

A B C D E F G I 

Fig. 1: PFGE patterns of Salmonella Typhi 

(vi-Phage type B1) involved in outbreak in 

Kelantan in the year 2004 till May 2005. 

A : Major outbreak strain in 

2005 (84% of the total strains 

tested) 

B – F : Closely related with 

outbreak strain (8 % of the 

strains tested) 

G – I : Possibly part of the 

outbreak strain (8% of the 

strains tested) 

9 PFGE patterns were 

identified from the 103 

outbreak related strains 

studied

2005 Typhoid outbreak 

•Earliest isolate: 

8/05; 15/1/2005; KBR; Muhamad Safuan; 14 

yrs; Male; Malay; 8/12/2004 

•estimated that 90% having similar pattern to 

8/05 clone 

•Trace back: 

In 2004, there were 18 cases of S.Typhi with 

phage B1 type in Kelantan. 

PFGE profile same as predominant profile of 

8/05

Retrospective subtyping of Salmonella spp isolated 

from environmental sources in the outbreak area 

(Kelantan) 

M 1 5 10 

Representative gel images 

of strains from well water, tap water 

•Suspected well waters 

100 Salmonella spp, 

None of the gel images matches 

Typhi profiles 

Serotyping was carried out 

After PFGE analysis 

No Typhi serotype was identified

Conclusion 

•Persistence strain (first isolated in Dec 2003, 

throughout in 2004 and again in 2005) 

•Person-to-person spread of infection 

•Peak of the outbreak coincided with a festival 

Mostly Mostly PT B1 

Various Various PFGE XbaI Xba subtypes, one predominant 

subtype 

Yet Yet to determine the source and linkage 

Poor Poor sanitary condition 

A A lack of treated quality water 

Involvement Involvement of carriers who are food handlers

Future Direction 

Stimulus for further action from Authority 

to set up a coordinated system and 

increased awareness of PulseNet activity 

Need to improve communication between 

MOH, Epidemiologists, IMR, NPHL and 

relevant parties 

Focus training of more staff 

Need better inter-laboratory 

inter laboratory 

standardization in PFGE

S.Typhi .Typhi 

PFGE performed on more than 300 isolates fo S.Typhi from sporadic and outbreak 

cases from different parts of Malaysia 

M M M 

M M M 

NB camera : technical 

problem with `over-exposure 

Multiple pulsotypes among representative S.Typhi strains

ice (Tol 0.1%-0.1%) (H>0.0% S>0.0%) [0.0%-100.0%] 

FGE 

100 

95 

90 

85 

80 

75 

70 

65 

60 

PFGE 

30.00 

40.00 

50.00 

80.00 

100.00 

120.00 

140.00 

160.00 

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350.00 

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600.00 

18 

15 

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14 

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1 

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5 

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8 

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1 

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15 

66 

3 

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9 

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X29 

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X72a 

Cluster analysis of S.Typhi

Some findings 

Aprox. Aprox. 

115 different XbaI Xba Pulsed-Field Pulsed Field Profiles (PFPs ( PFPs) ) were observed 

among the 300 isolates analyzed (F:0.4-1.0; (F:0.4 1.0; 14-22 14 22 bands; 22-600 22 600 kbp). kbp). 

The present observation concurred with the previous report (Thong (Thong 

et 

al.,JCM 1994) that strains associated with outbreaks are genetically 

more homogeneous as compared with those from sporadic cases of 

typhoid fever. 

Each of the outbreaks was associated with strains with 1-3 closely related 

profiles (F:0.74-1.0) while strains isolated from sporadic cases are more 

diverse (F:0.4-1.0) 

Six out of the 11 outbreaks (between 1987-2003) occurred in a northeastern 

state of Malaysia. This area is particularly in highly endemic for Salmonella 

Typhi. 

The exact reasons are not clear. However, the population is generally 

poorer. Poor hygiene and contaminated foods/water are most probable 

reasons. 

High population of carriers? 

Use of well water?

S. . Enteritidis-2 Enteritidis nd Most Common Serovar 

M M 

♠ 


problem with `over-exposure’ 

* * 

Most common non-typhoidal 

Salmonella serovar since 2000 

Since 1994, subtyped more than 

300 strains 

Very `clonal’ : one to 4 bands 

difference 

♠ Most common pattern 

* Non SE

Application of PFGE in distinguishing SE isolated 

from imported Almond Nut 

Indigenous SE 

SE from Imported Almond Nut, USA 

M 

•Officer from US embassy requested 

Imported almond nut to be tested. 

•Chemistry Dept ( Food Analysis Unit)- 

Isolated Salmonella spp and tested with 

PCR ( inv) ( Commercial Kit) 

•Results not Accepted 

•PFGE was performed `blind’ 

•: Pattern indicated Salm. Enteritidis 

•Different from local strains 

Another publicity for promotion PulseNet 

Activity!

Salmonella Typhimurium-4 Typhimurium th most 

M 

* 



common 

37 Strains isolated between 

2002-2005 subtyped 

20 different patterns 

46% of strains subtyped belong 

to 2 patterns which differed 

by only one band 

* non-Typhimurium

M 

Salmonella Weltevreden 



•2 nd most common nontypoidal 

Salmonella 

•37 strains isolated between 

2002-2005 subtyped into 

32 patterns 

•No predominant profile

Dice (Tol 0.3%-0.3%) (H>0.0% S>0.0%) [0.0%-100.0%] 

PFGE 

85 

90 

95 

PFGE 

100 

3000 

2500 

2000 

1500 

1200 

1000 

700.00 

600.00 

500.00 

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60.00 

40.00 

30.00 

20.00 

TA679/02 S. Corvallis SXT.STR.TET MKAK 

Stool 

TA555/03 S. Corvallis STR.TET 

Hosp. KL Stool 


Klang 

Stool 


Selayang Stool 


Klang 

Stool 

TA660/02 S. Corvallis SXT.STR.TET Alor Setar Stool 


Sg. Petani Sputum 


Johore Bahru Stool 




Seremban Stool 



TA608/03 S. Corvallis AMP.STR.TET Hosp. UKM Stool 


Hosp. UKM Stool 


Melaca 

Stool 


Johore Bahru Blood 

TA683/03 S. Corvallis STR.TET.(NA) Kaji Kuman Stool 






MKA 

Stool 


Kuala Terengganu Stool 



TA683/02 S. Corvallis STR.TET.NA Alor Setar Stool 



TA635/03 S. Corvallis AMP.STR.TET.(NA) Hosp. KL Stool 




MKA 

Stool 


Kuala Terengganu Stool 



HSA 

Blood 


Klang 

Stool 


Sg. Petani Stool 


Sg. Petani Stool 






Salmonella Corvallis 

3 rd most common 

nontyphoidal Salmonella 

37 strains ( 2002-2005) subtyped 

into 17 PFGE patterns 

55% of strains belong to 

3 common patterns, which 

differ in 1-3 bands only 

All the strains resistant to 

Streptomycin, Tetracyclin

Future Work……… 

Work……….. 

.. 

Improvement in surveillance of FD 

MyfoodNet ( Malaysian 

( Malaysian Foodborne Diseases Network) 

Further improvement in Laboratory skill 

PulseNet activities 

Increase human resources 

Workshop, training

Malaysian Foodborne Diseases 

Network ( MyFoodNet) 

MyFoodNet 

Idea mooted by Dr Wan Mansor, Mansor, 

Disease Surveillance, 

Ministry of Health, Malaysia 

Lack of reliable data to describe the actual burden of 

diseases 

Infrastructure, facilities, resources available but 

uncoordinated. 

Hence, MyFoodNet was proposed to coordinate and 

improve resources and capabilities in control and 

prevent of foodborne diseases 

To establish an active surveillance system of FD 

To precisely estimate the actual burden of FD 

PulseNet activities will placed also be coordinated

Future Work……… 

Work……….. 

.. 

Improvement in surveillance of FD 

MyfoodNet ( Malaysian 

( Malaysian Foodborne Diseases Network) 

Further Further improvement in Laboratory 

skill 

Capacity Capacity building: Workshop, training 

Improvement in communication and 

enhanced collaborative efforts between 

different agencies in developing 

PulseNet Activities

Acknowledgements 

Funding support: 

• University of Malaya 

• MOSTI, Malaysia 

• National Institute of Infectious Disease, 

Japan ( Dr Haruo Watanabe) 

Meeting Support: Support: 

APHL, CDC Atlanta 

Colleagues in the PulseNet Asia Pacific 

Institute for Medical Research, Malaysia 

Ministry of Health, Malaysia

Subtyping and Surveillance of Salmonellosis in Malaysia.

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