1. HAND FOOT AND MOUTH DISEASE (HFMD) EPIDEMIOLOGY AND SAMPLING

2. Clinical Case Definition
Clinical Case of HFMD
Any child with:
Mouth/tongue ulcer and
Maculopapular rashes and/ or vesicles on palms and soles
With OR without history of fever
2. Laboratory criteria
Any case that has clinical symptom and positive for HFMD virus either through virus isolation or PCR detection
Case Classification:
Suspected
Confirmed

6. OBJECTIVES OF LAB SURVEILLANCE
Monitor Pathogen spectra of HFMD
Molecular epidemiology of EV71, Distribution of genetic type and change, and correlations between prevalence virus strain and severity of symptoms in different outbreak spots

7. KATEGORI PENSAMPELAN SURVELAN MAKMAL HFMD
SURVELAN SENTINEL KLINIK
SURVELAN KES TERUK
SURVELAN WABAK

8. SURVELAN SENTINEL
Survelan makmal untuk enterovirus melalui klinik sentinel bagi mengesan circulating enterovirus dan virus genotype telah bermula sejak tahun 2007.
Bermula Ogos 2012, sembilan (9) negeri telah dikenalpasti untuk untuk memilih dua (2) klinik sentinel bagi pelaksanaan survelan makmal bagi enterovirus penyebab HFMD melalui klinik sentinel.
Jumlah klinik sentinel adalah 19 (Tahun 2014).
Pengambilan spesimen dilakukan secara rawak sistematik sehingga maksimum 5 sampel setiap bulan untuk setiap klinik sentinel yang dipilih. (iaitu kes 1, 2, 7, 12 dan 17) setiap bulan untuk setiap klinik sentinel yang dipilih. [The first two cases and subsequently every 5th case up to a maximum of 5 cases per month per sentinel clinic.]
Rujukan: (7)dlm.KKM-171/BKP/02/10/0142 Jld 3 bertarikh Ogos 2013

9. Klinik Sentinel Survelan Makmal HFMD
Negeri
Klinik Sentinel
P.Pinang
KK Bayan Baru
KK Bukit Panchor
Perak
KK Taiping
JPL Hosp Seri Manjung
Melaka
KK Masjid Tanah
KK Merlimau*
WPKL & Putrajaya 
Kiddicare Child Specialist Centre
KK Putrajaya Presint 9
Johor
JPL KK Mahmoodiah
KK Batu Pahat
Selangor
KK Ampang
KK Pandamaran
Kelantan
KK Bandar Kota Bharu
KK Bandar Pasir Mas
Sarawak
KK Jalan Masjid
KK Oya
KK Miri
Sabah
KK Luyang
KK Sandakan
Malaysia
Jumlah
Catatan:
Target penghantaran sampel = 5/bulan
* Menggantikan KK Tengkera

11. SURVELAN KES TERUK
Pemilihan kes untuk penghantaran sampel adalah kes yang disyaki HFMD atau memenuhi kriteria klinikal kes HFMD dan merupakan kes yang teruk, kes yang dimasukkan ke hospital atau kematian.

13. SURVELAN WABAK
Pemilihan sampel adalah 10% daripada jumlah kes yang memenuhi kriteria klinikal HFMD daripada satu wabak tetapi tidak melebihi lima (5) sampel bagi setiap wabak.
Pemilihan wabak adalah merujuk kepada wabak baru dan tiada pengesahan makmal bagi agen penyebab wabak dilakukan dalam lokaliti yang sama.
Manakala bagi lokaliti yang telah dikenalpasti agen penyebab kes HFMD dalam tempoh 14 hari daripada tarikh onset kes terakhir, sampel tidak perlu dihantar ke makmal untuk ujian diagnostik
Rujukan: Minit Mesyuarat Teknikal (Epidemiologi) Sektor Zoonosis Bil. 2/2013

15. Taburan Enterovirus (EW1-18/2015)

16. LABORATORY REQUEST FORM

20. PPE FOR SAMPLING SPECIMEN
Gove
Mask
Disposable gown

21. Specimen Collection Kits
1. Dacron swab recommended 2. Viral Transport Media (VTM) 3. Ice pack for °C maintainance (2-8 °C). 4. Three layer packaging
*Provided by NPHL upon request

22. Viral Transport Media (VTM)
Sterile
containing ml volume
can be stored in freezer at -20 °C until use
4. can be stored for short periods of time at 4-6°C
5. check expiry date and turbidity

23. SWAB STICK SHOULD BE USED : √ Dacron √ Rayon √ Polyesterfiber swabs
DO NOT USE
X calcium alginated
X cotton swabs
X Wooden sticks
*Provided by NPHL upon request

24. CASE SELECTION CRITERIA
Sample should be collected from patient
below 10 years with
HFMD clinical criteria

25. STATISTIC ANALYSIS

27. Important Note :
One Suitable Specimen From One Patient Is Recommended.
2. Specimen Selection Highly Recommended From Site Of
Infection Or Symptoms.

28. SPECIMEN STORAGE Refrigerate at 2- 4°C ( DO NOT FREEZE)
Possible send ASAP
Within 24 – 48 hours after collection

29. PACKAGING Send with ice pack (4o - 8oC) Triple layer packaging.
Specimen tube
Container
Insulator
Send with ice pack (4o - 8oC)
Triple layer packaging.
First layer should be water tight.
Use absorbent material in all layers
Do not FREEZE

30. Triple Packaging System

31. TRANSPORTATION Transport specimens at 2-8ºC.
Fill with ENOUGH ice packs or coolant packs.
Packaging of specimens + complete + Correct request form
Clearly state on surface of box - HFMD
Call & Inform before sending
Office Hour : Tel no
After Office Hour : H/P No. : 011 –

32. VITAL TO MAKE SURE Specimen must maintain (2 -8 °C)
NPHL measures temperature upon receiving
Sender to consider :
1. Cold box size
2. Specimen Volume
3. Transportation time from sender to NPHL
4. Ice pack quantity
5. Ice pack material (gel / water)

33. SUMMARY : SPECIMEN HANDLING & TRANSPORTATION
Step 1 : Specimen selection 
Step 2 : Specimen collection 
Step 3 : Fill in form and label the specimen
Step 4 : Store the specimens in 2-8°C as soon as possible
Step 5 : Pack individually in biohazard plastic bag accompanied with complete Request FORM
Step 6 : Transportation of the specimen by maintaining 2-8°C until reach NPHLwithin 48hours of specimens collection

34. NPHL TEST METHOD Virus Isolation technique Cell line
viability of the virus is vital
2. Molecular technique – PCR
Specific primers
°C of the specimen is very crucial

35. SURVEILLANCE : VIRUS ISOLATION TECHNIQUE
SPECIMEN CRITERIA
TEST ABLE TO DETECT
LAB TURN AROUND TIME
SURVEILLANCE
*Suitable specimen
▪ Enterovirus 70
▪ Enterovirus 71
▪ Coxsackie A16 virus
▪ Coxsackie B virus
▪ Echo virus Type 4
▪ Echo virus Type 6
▪ Echo virus Type 9
▪ Echo virus Type 11
▪ Echo virus Type 30
▪ Polio virus Type 1
▪ Polio virus Type 2
▪ Polio virus Type 3
▪ Herpes simplex virus Type 1
▪ Herpes simplex virus type 2
14 DAYS

36. OUTBREAK/CLUSTER : MOLECULAR TECHNIQUE
SPECIMEN CRITERIA
TEST ABLE TO DETECT
LAB TURN AROUND TIME
OUTBREAK
CLUSTER
*SUITABLE SPECIMEN
Enterovirus (Not Enterovirus 71)
Enterovirus 71
3 DAYS

37. TESTING ALGORITHM FOR ENTEROVIRUSES (HFMD)
Sample
Outbreak investigation
<5 samples for each cluster / outbreak
surveillance/diagnostic – Virus isolation
Inoculate in cell line (Vero & RD)
PCR –Pan Enterovirus
CPE
No CPE
Positive
Negative*
Harvest
No virus isolated
PCR – EV71
Report as:
PCR Enterovirus Not Detected
IFA Staining
(Pan Enterovirus Screening Using MAb)
Final Report
Enterovirus Isolation :
No Virus Isolated
Positive
Negative
Positive
Negative*
IFA Staining
(Typing using MAb )
IFA Staining
(Typing HSV 1 & 2 )
Report as:
PCR Enterovirus Detected (Not EV71)
Report as:
PCR Enterovirus Detected (EV71)
Positive
Negative
Untypeable for Pan-Enterovirus panel
Positive
Final Report
Enterovirus Isolation :
Herpes Simplex Virus 1/ Herpes Simplex Virus 2 Isolated
Further Investigation
PCR-Enterovirus
Final Report
Enterovirus Isolation :
Enterovirus 71 Isolated/
Enterovirus 70 Isolated/
Coxsackie A16/
Coxsackie A24 Isolated/
Poliovirus Type 1,2,3 Isolated/
Echovirus Type 4,6,9,11,30 Isolated/
Coxsackie B Isolated 38

40. ONLINE RESULT Username & Password
Request by using moh.gov.my

41. THANK YOU Acknowledgement: Zoonosis Sector Disease Control Division
National Public Health Laboratory