Surveillance and Surveillance and SubtypingSubtyping of of SalmonellaSalmonella sppspp. in Malaysia.. in Malaysia.

KwaiKwai Lin Thong, PhDLin Thong, PhDUniversity of MalayaUniversity of Malaya

Kuala Lumpur, MalaysiaKuala Lumpur, Malaysia

OutlineOutline

Overview of Surveillance system in Overview of Surveillance system in MalaysiaMalaysia

Incidence of Incidence of SalmonellosisSalmonellosis in Malaysiain Malaysia

SubtypingSubtyping by PFGE of predominant by PFGE of predominant Salmonella Salmonella entericaenterica serovarsserovars

FoodborneFoodborne Disease SurveillanceDisease Surveillance

Carried out by different units, sections, departments and Carried out by different units, sections, departments and institutions within the Ministry of Healthinstitutions within the Ministry of Health

Passive surveillancePassive surveillanceData collected through physician based surveillance and Data collected through physician based surveillance and outbreak investigationoutbreak investigation

Notification is received from government health facilities Notification is received from government health facilities & private hospitals & GP& private hospitals & GP

FoodborneFoodborne diseases monitored: cholera, typhoid & diseases monitored: cholera, typhoid & paratyphoid, hepatitis A, dysentery, food poisoning paratyphoid, hepatitis A, dysentery, food poisoning ((nontyphoidalnontyphoidal SalmonellaSalmonella & other etiologic agents)& other etiologic agents)Acute gastroenteritis (AGE) also reported but not Acute gastroenteritis (AGE) also reported but not mandatorymandatory

Laboratory surveillance

Mandatory NotificationDis. Surv.

Clinical Based(sentinel/national

Syndromic

Community basedsurveillance

Otheragencies

MicrobiologyLabs

Public:Health CtrHospitalsPrivate:GP clinicsHospitals

Sentinel selectedClinicsNational (hospitals)Syndromic A/E wards

Community/MediaInternational sources

District Heath Office

State Health Dept

National : Div. Disease Control, Ministry of Health

Veterinary Dept(Zoonoses)IMR/

PHLs

Overview of Surveillance Food Borne diseases in Malaysia

Incidence rates in Malaysia (1995-2003)DISEASESDISEASES 19951995 19961996 19971997 19981998 19991999 20002000 20012001 20022002 20032003

Food poisoningFood poisoning 7.087.08 15.2915.29 31.0831.08 31.4531.45 38.0438.04 36.6136.61 29.9929.99 28.6328.63 14.7214.72

Typhoid/Typhoid/paratyphoidparatyphoid

4.464.46 4.504.50 3.243.24 3.533.53 3.573.57 3.453.45 2.922.92 3.483.48 1.741.74

Source: Disease Control Div, Ministry of Health, Malaysia 2005per 100.000 population

Salmonella infection -major foodborne disease in Malaysia

Burden of Burden of FoodborneFoodborne diseasesdiseasesTrue incidence True incidence ––difficult to determinedifficult to determinePassive surveillance: reported casesPassive surveillance: reported cases

*67% of reported outbreaks occurred in public scho*67% of reported outbreaks occurred in public schools, residential ols, residential colleges,factoriescolleges,factories*any outbreak cases or sporadic cases involved an*any outbreak cases or sporadic cases involved any food outlets y food outlets ––difficult to detectdifficult to detect

Grossly underestimatedGrossly underestimatedA local study done in the State of A local study done in the State of KelantanKelantan(2001) showed a higher ( 2x) estimated (2001) showed a higher ( 2x) estimated annual IR of typhoid cases (annual IR of typhoid cases (Wan Wan MansorMansor, unpublished , unpublished data)data)

Laboratory Based Surveillance for Laboratory Based Surveillance for Infectious DiseasesInfectious Diseases

Introduced in 2002Introduced in 2002

an initiative between Surveillance Section & Institute for Medical Research (IMR), Ministry of Health

Complements the mandatory Complements the mandatory notifiablenotifiable disease surveillance system.disease surveillance system.This system requires participating hospitals to submit isolaThis system requires participating hospitals to submit isolates to IMR tes to IMR for refor re--culture and culture and subtypingsubtyping..

Database may not be 100% representative as it involves only Database may not be 100% representative as it involves only government hospital laboratoriesgovernment hospital laboratories

Currently 6 types of bacteria being monitored:Currently 6 types of bacteria being monitored:VibrioVibrio choleraecholerae, , HaemophillusHaemophillus influenzaeinfluenzae, , Salmonella Salmonella entericaentericaS. S. Typhi/ParatyphiTyphi/Paratyphi, , NeisseriaNeisseria meningitidismeningitidis

Laboratory based surveillance flowchartLaboratory based surveillance flowchart

Microbiology labsof public/private hospitals & private labs

IMR/NPHL and other Reference Labs( confirmation, typing, etc

District Health Officer

State Health Office

Communicable Disease SurveillanceSection, Ministry of Health

Data notification Information feedback

Notification form

Isolates with laboratory

notification

results

notification

results

http://dph.gov.my

Infectious Disease Bulletin

Based on the labBased on the lab--surveillance system, the surveillance system, the Disease SurveillanceDisease SurveillanceSection, Disease Control Section, Disease Control Division, MOH, Division, MOH, publishes a Bulletinpublishes a Bulletin

Aims:Aims:••To To diseminatediseminate/share /share information on information on communicable disease communicable disease and to stimulate and to stimulate exchanges of ideasexchanges of ideas••To update and inform To update and inform health professionalshealth professionals

2003 2004 2005

Serotypes total % Serotypes total % Serotypes total %Enteritidis 244244 26.726.7 Enteritidis 206206 25.025.0 Enteritidis 155155 28.128.1Weltevreden 200200 21.921.9 Weltevreden 165165 20.020.0 Weltevreden 142142 25.725.7Corvallis 115115 12.612.6 Corvallis 117117 14.214.2 Corvallis 5757 10.310.3Typhimurium 4949 5.45.4 Typhimurium 4343 5.25.2 Typhimurium 3737 6.76.7Stanley 3232 3.23.2 AlbanyAlbany 3737 4.54.5 LimeteLimete 99 3.33.3Tshongwe 2929 3.23.2 LimeteLimete 1818 2.22.2 StanleyStanley 88 1.41.4Biegdam 1919 2.12.1 Braenderup 1515 1.81.8 AgonaAgona 77 1.31.3

Albany 1717 1.91.9 Tshongwe 1515 1.81.8 AlbanyAlbany 55 0.90.9

Braenderup 1212 1.31.3 StanleyStanley 1111 1.31.3 RissenRissen 55 0.90.9

Newport 1010 1.11.1 BovismorfibicBovismorfibicansans

1010 1.21.2 VirchowVirchow 55 0.90.9

Top 10 Nontyphoidal Salmonella Serotypes identified and reported to the Laboratory Based Surveillance database, 2003-5

Source: Lab Surv. Bulletin, Disease Control Division, Ministry of Health Malaysia (October 2005 )

Total 915 682 460

Application of PFGE as a Application of PFGE as a SubtypingSubtyping tooltool

Still not fully implemented in all Still not fully implemented in all PHLsPHLsDesignated 4 Designated 4 PHLsPHLs

-- availability of infrastructure: PFGE availability of infrastructure: PFGE equipmentequipment--lack of technical expertiselack of technical expertise--no dedicated staff to perform no dedicated staff to perform PFGEPFGEInstitute of Medical Research (IMR) : Institute of Medical Research (IMR) :

National Reference CentreNational Reference Centre: : routinely does routinely does serotypingserotyping, AST, and perform PFGE on a case to case , AST, and perform PFGE on a case to case basisbasis

Salmonella Salmonella TyphiTyphimost common serotypemost common serotype

Typhoid feverA public health problem & endemic in MalaysiaAnnual 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 Example of application of PFGE in a recent outbreak of typhoid fever in recent outbreak of typhoid fever in

MalaysiaMalaysiaIn 2005, an outbreak of typhoid fever in

KelantanAs of 8 May, 2005As of 8 May, 2005-- State Health Dept of State Health Dept of KelantanKelantan reported 517 confirmed typhoid reported 517 confirmed typhoid cases. Included 2 deathscases. Included 2 deaths19 (2.14%) food samples, 19 (2.14%) food samples, 12 ( 2.9%) drinking water samples12 ( 2.9%) drinking water samples2 (0.2%) food handlers 2 (0.2%) food handlers

––tested positive for tested positive for SalmonellaSalmonella sppspp

Week No cases ( no death)

Cumulative cases ( death)(throughout the country)

2005 2004Wk 18 ( 1-7 May 2005 397 1408 (0) 146 (0)Wk 19 (8-14 May,2005 120 (2) 1533 (2) 152Wk 20 (15-21 May,2005) 156 1718 (2) 158Wk 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) 170Wk 24 (12 -18.6.2005) 48 2013(2) 183Wk 25(19- 25.6.2005) 23 2045 185Source: Infect Dis Bull. Disease Control Div, Min Health, Malaysia

988

Number of notified cases of Typhoid Fever in Kelantan, Malaysia (May-June 2005)

ActionAction(Ministry of Health)(Ministry of Health)

State Response TeamState Response TeamRapid Response Team (RRT) from Rapid Response Team (RRT) from Min.HealthMin.Health

ActionAction

Intensive health education activities Intensive health education activities carried outcarried outPublic awareness on personnel hygiene Public awareness on personnel hygiene was actively promotedwas actively promotedTyphimTyphim Vi vaccination given to food Vi vaccination given to food handlershandlersA total of 528 food premises were closed A total of 528 food premises were closed under PCID Act 1988 and Food Act 1983under PCID Act 1988 and Food Act 1983

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

Laboratory AnalysisLaboratory AnalysisStrains were submitted to IMR for phage-typing, antimicrobial susceptibility test, and PFGE subtyping.

-- mostly phage B1 typemostly phage B1 type-- All antibiotic sensitive strainsAll 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 : 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)

Fig. 1: PFGE patterns of Salmonella Typhi(vi-Phage type B1) involved in outbreak in Kelantan in the year 2004 till May 2005.

A B C D E F G I

9 PFGE patterns were identified from the 103 outbreak related strains studied

2005 Typhoid outbreak2005 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

Representative gel imagesof strains from well water, tap water

•Suspected well waters100 Salmonella spp,

None of the gel images matchesTyphi profiles

Serotyping was carried out After PFGE analysis

No Typhi serotype was identified

M 1 5 10

Retrospective subtyping of Salmonella spp isolated from environmental sources in the outbreak area(Kelantan)

ConclusionConclusion•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 festivalMostly PT B1Mostly PT B1

Various PFGE Various PFGE XbaXbaII subtypes, one predominant subtypes, one predominant subtypesubtype

Yet to determine the source and linkageYet to determine the source and linkage

Poor sanitary conditionPoor sanitary condition

A lack of treated quality waterA lack of treated quality water

Involvement of carriers who are food handlersInvolvement of carriers who are food handlers

Future DirectionFuture DirectionStimulus for further action from Authority Stimulus for further action from Authority to set up a coordinated system and to set up a coordinated system and increased awareness of increased awareness of PulseNetPulseNet activityactivityNeed to improve communication between Need to improve communication between MOH, Epidemiologists, IMR, NPHL and MOH, Epidemiologists, IMR, NPHL and relevant partiesrelevant partiesFocus training of more staff Focus training of more staff Need better interNeed better inter--laboratory laboratory standardization in PFGEstandardization in PFGE

SS.Typhi.Typhi

M M MM M M

PFGE performed on more than 300 isolates fo S.Typhi from sporadic and outbreakcases from different parts of Malaysia

NB camera : technicalproblem with `over-exposure

Multiple pulsotypes among representative S.Typhi strains

Dice (Tol 0. 1% -0.1%) (H>0. 0% S>0.0%) [0. 0%-100.0%]PFGE

100

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Cluster analysis of S.Typhi

Some findingsSome findingsAproxAprox. . 115 different 115 different XbaXbaII PulsedPulsed--Field Profiles (Field Profiles (PFPsPFPs) were observed ) were observed among the 300 isolates analyzed (F:0.4among the 300 isolates analyzed (F:0.4--1.0; 141.0; 14--22 bands; 2222 bands; 22--600 600 kbpkbp).).

The present observation concurred with the previous report (ThonThe present observation concurred with the previous report (Thong et g et al.,JCMal.,JCM 1994) that strains associated with outbreaks are genetically 1994) that strains associated with outbreaks are genetically more homogeneous as compared with those from sporadic cases of more homogeneous as compared with those from sporadic cases of typhoid fever.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?

SS. . EnteritidisEnteritidis--22ndnd Most Common Most Common SerovarSerovarM M

* *Most common non-typhoidalSalmonella serovar since 2000

Since 1994, subtyped more than300 strains

Very `clonal’ : one to 4 bandsdifference

♠

♠ Most common pattern

NB camera : technicalproblem with `over-exposure’

* Non SE

SE from Imported Almond Nut, USAIndigenous SE

M

Application of PFGE in distinguishing SE isolatedfrom imported Almond Nut

•Officer from US embassy requestedImported almond nut to be tested.•Chemistry Dept ( Food Analysis Unit)-Isolated Salmonella spp and tested withPCR ( inv) ( Commercial Kit)

•Results not Accepted

•PFGE was performed `blind’•: Pattern indicated Salm. Enteritidis•Different from local strains

Another publicity for promotion PulseNetActivity!

SalmonellaSalmonella TyphimuriumTyphimurium--44thth most most commoncommon

M*

* non-Typhimurium

37 Strains isolated between 2002-2005 subtyped

20 different patterns

46% of strains subtyped belong to 2 patterns which differed by only one band

NB camera : technicalproblem with `over-exposure’

SalmonellaSalmonella WeltevredenWeltevreden

M

NB camera : technicalproblem with `over-exposure’

•2nd most common nontypoidalSalmonella

•37 strains isolated between2002-2005 subtyped into 32 patterns

•No predominant profile

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

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S. CorvallisS. Corvallis

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MKAKHosp. KL

Klang

SelayangKlang

Alor Setar

Sg. Petani

Johore Bahru

Johore Bahru

Seremban

Seremban

Hosp. UKM

Hosp. UKM

Melaca

Johore Bahru

Kaji KumanJohore Bahru

Hosp. KL

MKAKuala Terengganu

Seremban

Alor Setar

Hosp. UKM

Hosp. KL

Hosp. UKM

MKA

Kuala Terengganu

Kaji Kuman

HSA

Klang

Sg. PetaniSg. Petani

Hosp. KL

Hosp. KLKaji Kuman

StoolStool

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Salmonella Corvallis

3rd most commonnontyphoidal Salmonella

37 strains ( 2002-2005) subtypedinto 17 PFGE patterns

55% of strains belong to 3 common patterns, which differ in 1-3 bands only

All the strains resistant toStreptomycin, Tetracyclin

Future WorkFuture Work………………....Improvement in surveillance of FDImprovement in surveillance of FD

MyfoodNetMyfoodNet ( Malaysian ( Malaysian FoodborneFoodborne Diseases Network)Diseases Network)

Further improvement in Laboratory skillFurther improvement in Laboratory skillPulseNetPulseNet activitiesactivities

Increase human resourcesIncrease human resourcesWorkshop, trainingWorkshop, training

Malaysian Malaysian FoodborneFoodborne Diseases Diseases Network ( Network ( MyFoodNetMyFoodNet))

Idea mooted by Dr Wan Idea mooted by Dr Wan MansorMansor, Disease Surveillance, , Disease Surveillance, Ministry of Health, MalaysiaMinistry of Health, Malaysia

Lack of reliable data to describe the actual burden of Lack of reliable data to describe the actual burden of diseasesdiseasesInfrastructure, facilities, resources available but Infrastructure, facilities, resources available but uncoordinated.uncoordinated.

Hence, Hence, MyFoodNetMyFoodNet was proposed to coordinate and was proposed to coordinate and improve resources and capabilities in control and improve resources and capabilities in control and prevent of prevent of foodbornefoodborne diseasesdiseases

To establish an active surveillance system of FDTo establish an active surveillance system of FDTo precisely estimate the actual burden of FDTo precisely estimate the actual burden of FDPulseNetPulseNet activities will placed also be coordinatedactivities will placed also be coordinated

Improvement in surveillance of FDImprovement in surveillance of FDMyfoodNetMyfoodNet ( Malaysian ( Malaysian FoodborneFoodborne Diseases Network)Diseases Network)

Further improvement in Laboratory Further improvement in Laboratory skillskillCapacity building: Capacity building: Workshop, trainingWorkshop, training

Improvement in communication and Improvement in communication and enhanced collaborative efforts between enhanced collaborative efforts between different agencies in developing different agencies in developing PulseNetPulseNet ActivitiesActivities

Future WorkFuture Work………………....

AcknowledgementsAcknowledgementsFunding support:Funding support:

•• University of Malaya University of Malaya •• MOSTI, MalaysiaMOSTI, Malaysia•• National Institute of Infectious Disease, National Institute of Infectious Disease,

Japan ( Dr Haruo Watanabe)Japan ( Dr Haruo Watanabe)

Meeting SupportMeeting Support: APHL, CDC Atlanta: APHL, CDC Atlanta

Colleagues in the PulseNet Asia PacificColleagues in the PulseNet Asia Pacific

Institute for Medical Research, MalaysiaInstitute for Medical Research, MalaysiaMinistry of Health, MalaysiaMinistry of Health, Malaysia