> Infectious Diseases Research Centre
Virology Unit| Introduction | Research | Diagnostic Services |
The Institute for Medical Research (IMR) was founded in 1901 by the First British Resident-General of the Federated Malay States , Sir Frank Swettenham. It took 23 years later, in 1924 during the First World War, for a Virology Laboratory to be created in the IMR in the form of Pasteur Institute branch; its sole purpose was for diagnosing rabies and for preparation of anti-rabies vaccine. Thus the history of virus research in the IMR is synonymous with the history of rabies in Malaysia .
In the last two decades, many communicable diseases due to bacterial microbes have come under control and diseases caused by viruses have emerged to become major health problems in Malaysia . Also, at different times during the past 90 years different viruses were responsible for causing public health problems. In the past, public health problems encountered were due to rabies, smallpox, influenza, enterovirus and arboviruses, while more recently, other viral infections have been added such as the hepatitis viruses, Human Immunodeficiency Viruses, Chikungunya , Nipah and SARS corona virus.
Research at the Virology Unit since the early days was aimed at elucidating the importance of human viruses causing disease in the local setting, determining the epidemiology, initiating and maintaining surveillance, and developing of new technology for the provision of referral diagnostics for these diseases. There was also the provision of diagnostic support to both clinical and public health departments, with such services often necessitating the in-house production of reagents and viral antigens, leading to the development of rapid tests.
1. Hepatitis
Since the early 1980s, the Virology Unit has played a major role in establishing baseline epidemiological data on Hepatitis Viruses such as Hepatitis A, B, C and D.
In the 1980s a sero prevalence study showed that Hepatitis Virus A was endemic in Malaysia ; it occurred sporadically throughout the year and most adults by the age of forty years were immune to the Hepatitis A Virus, the susceptible age group were children under 10 years of age.
In studies carried out over three years from 1987 to 1989, among acute hepatitis cases that come to medical attention and/or which occur as community outbreaks, the Hepatitis Virus A was found to be the predominant aetiological agent (48%), followed by HNANB (28%), HBV (18%), with HDV associated in 5% of HBV cases.
The risk of acute Hepatitis A was found to be highest (80%) in children aged 6 years to 15 years among one thousand one hundred children hospitalised for acute viral hepatitis, followed by children (66%) aged 1to 5 years of age, followed by young adults (29%) aged 20 years and more.
Between 1994 - 1997 in a retrospective analysis of acute hepatitis A cases in Kelantan and Trengganu , the highest attack rate was also in school-going children aged 6 to 15 years, and the occurrence of HAV was recorded throughout the year in those two states.
The studies on Hepatitis B in the early 1980s onwards recorded baseline data for various population groups, this led to the initiation of the National Hepatitis B Immunisation Programme for newborn and other risk populations in 1988. Our studies also led towards developing strategies for the reduction of transmission of Hepatitis B and C Viruses in the form of a nationwide screening programme for transfused blood and blood products. In the early 1990's, molecular diagnostic techniques such as the HBV and HCV nucleic acid detection techniques were set up to complement the existing serological assays in order to boost laboratory diagnostic support for medical and gastro-enterology clinicians within the Ministry of Health, and also as research tools to further propel research activities.
Studies since 1987 to determine the viral aetiology of chronic liver disease (chronic active, chronic persistent hepatitis, cirrhosis, hepatocellular carcinoma) indicated that a large proportion of cases (85%)in this study were associated with HBV; in 75% of cases HBV was the sole viral agent detected while another 10% of cases showed both HBV and HCV. HCV alone accounted for less than 3% of the total chronic cases investigated (Sinniah et al, 1993).
Current data further confirms this observation about HBV, while HCV now accounts for about 11% of the total chronic liver cases investigated.
Currently, the Virology Unit has undertaken an IRPA funded project –“Characterization of virological markers associated with HCC in patients chronically infected with HBVand/or HCV”. One of the aims of this study is to construct a sequence database of Malaysian HBV and HCV viruses detected from patients that have different sequelae in order to analyse HBV and HCV variants associated with HCC. This will facilitate the development of a mutational sequence programme for analysis and identification of unique viral mutations.
Future research would include charting the local epidemiology of the new hepatitis viruses using improved and newer diagnostic technologies, and the evaluation of commercial hepatitis diagnostic kits, intended for use in the Ministry of Health screening programmes .
2. HIV
In 1985, the Virus Research Division undertook a sero-survey in the country in collaboration with the Epidemiologists from the Ministry of Health with the aim of determining the sero-epidemiology of HIV in Malaysia . The sero-survey involved screening a total of 7000 individuals from selected population groups. Although none of these were positive for HIV-1 antibodies, the National Screening Programme for donated blood was launched in 1985 by MOH. Following this the National AIDS Reference Laboratory (NARL) was set up at IMR in 1986, to assist the MOH in addressing the problem of HIV infection in Malaysia through various activities listed below:
2.1 Screening and confirmation of HIV infection for government and private hospitals.
2.2 Providing technical support and training to government peripheral screening centres which carry out anti-HIV testing. In line with WHO recommendations, the anti-HIV two test screening strategy for high risk individuals was introduced into these peripheral centres in 1995, with retention of technically demanding assays such as Western Blot, Polymerase Chain Reaction, etc., in the National HIV Reference Centre.
2.3 Evaluating new commercial anti-HIV Ministry of Health screening programs.
2.4 Representation in various committees such as the Ministry of Health's AIDS Technical Committee and AIDS Task Force.
2.5 Contributing to National data on HIV prevalence through sero-surveillance studies.
2.6 Research, the Virology Division initiated a sero surveillance study of HIV-2 incidence in Malaysia in 1990. This study is being continued.
Facilities for PCR was established in 1991 as a research tool and since 1992 has been extended as a diagnostic service for paediatric cases. In 1995, studies were initiated on Malaysian HIV-1 variants/ sub-types; this study utilizing analysis of the V3 loop sequences of the HIV-1 envelope region showed that HIV-1 subtypes B, C and E are prevalent among infected Malaysian IVDUs with majority having sub-type B.
The B sub-types showed a greater homology to B genotype strains reported in Thailand than to the North American or European consensus sequences.
3. Dengue
The first dengue outbreak in Malaysia was recorded in 1902 and the prevalence has been increasing ever since. A nationwide study to determine the risk factors of dengue fever was carried out by the IMR. These research activities and continuous surveillance has provided vital information to the health authorities to initiate and maintain relevant control and preventive measures.
A large cohort study in urban children was carried out by the Division in 1986 and 1989 to determine the risk factors of Dengue/DHF. This study showed that by age 12, almost 100% of urban children had been exposed to the dengue virus. It also showed that independant of other factors, children whose families carry out sufficient measures to prevent mosquito larval breeding have a 50% lower risk of dengue infection than those who did not. These research activities and continuous surveillance has provided vital information to the health authorities to initiate relevant control and preventive measures.
Currently, the in-house modified IgM-ELISA is utilised as the first-line screening test for dengue in the Division. It has also increased the efficiency of diagnosis of acute dengue. However, in some cases, definitive diagnosis cannot be obtained in acute samples.
During the early and mid 1990s, molecular techniques were established, such as, PCR for rapid diagnosis of dengue and Japanese Encephalitis. More recently, the rapid RT-PCR was evaluated and found to be more sensitive than viral isolation. Rapid RT-PCR performed directly on serum samples, that is, without the RNA extraction step, gave 100% sensitivity; and when compared to the in-house ELISA-IgM, it is a more sensitive diagnostic tool for samples taken within the first four days of disease. Although the rapid RT-PCR is available as a diagnostic test in the Division, but because of the cost and specialised nature of the technique, it is concluded that rapid RT-PCR should only be performed as a supplement to the existing rapid ELISA-IgM or dengue blot, or in special situations for certain sero-negative cases. This direct RT-PCR technique also enables sero-typing of the dengue virus.
In the study comparing two sources of reagents for Dengue IgM-ELISA test , it was found that the sensitivity of suckling mouse brain (SMB)-derived dengue type 2 (D2) antigen and D2 monoclonal antibody (MAb) supplied by Dr N Karabatsos from United States is 50% and tissue culture derived antigen (TCA) and dengue cross-reactive monoclonal anitbody MAb MF4/5/A5/C3-3 supplied by Dr Jane Cardosa is 75%, compared to the gold standard of Haemagglutination Inhibition test (HI). While the specificity was 95.2% by the SMB/anti D2 set and 90.3% by the TCA/MAbMF4/5/A5/C3-3 set. The results showed that the second set of reagents can be effectively used in dengue IgM-ELISA and is significantly more sensitive than the first set of reagents (Chi SQR=12.26, P<0.01) at less than 1% risk. However there is no significant difference between the two reagents (Chi SQR=2.45, 0.5>P>0.1) at 10% risk. Also, in our effort to be self-supporting in the production of diagnostic reagents we have elucidated the optimal temperature that will increase the dengue viral antigen yield. It was found that a temperature of 37 0 C resulted in better antigen production followed by 32 0 C and 28 0 C. Dengue 2 (D2) and Dengue 3 (D3) antigens reached a high titre earlier (3 days) than Dengue 1 (D1) and Dengue 4 (D4) (5days). However D2 favours a higher temperature of 37 0 C and D1 favours a temperature of 32 0 C. These findings could also be utilised for optimising dengue antigen yield in tissue culture.
Work has been done to determine the best purification step for PCR products as there has been an attempt to directly sequence PCR products. For this purpose, two different methods of purification were used, namely, Millipore's Microfilter Spin Unit System and Boerhinger Mannheim Biochemica's Quick Spin Column. The results indicate that the sequencing pattern from the first purification method is superior to the second purification method. Sequencing was also done to determine whether mutation occurs during passaging of the virus in C6/36 tissue cell. Sequencing data of dengue-3 virus amplified directly from human serum were compared against virus derived after three passages in C6/36 cell line and no differences were observed. Subsequent work resulted in characterization of the envelope/non-structural 1(E/NS1) gene junction of five local dengue 3 strains and these sequences were submitted to the Genbank. Characterization of the dengue-3 viruses responsible for the dengue epidemic in Malaysia , during 1993-1994, indicate that these virus strains genetically resembled the dengue-3 strains reported to be endemic in Thailand . In seeking to determine nucleotide variations in relations to pathogenicity and virulence of the virus, sequencing of the E/NS1 region of 8 local dengue -3 strains were carried out and found them to be similar to those isolates from other parts of South-East Asia .
4. Japanese Encephalitis
Japanese Encephalitis (JE) has been shown to occur in Malaysia since 1951 and it was the IMR that first demonstrated the presence of JE in Malaysia . Small-scale sero-surveys in the past and laboratory surveillance carried out in the IMR have shown that JE was a common infecting agent for not only in man and horses in Malaysia , but for a wide variety of other animals and mosquitoes. The clinical features of JE in Malaysia were documented by the IMR during an outbreak of JE in Pulau Langkawi in 1979. More recent sero-survey in animals confirmed that almost all animals (viz. pigs, buffaloes, cattle, sheep, goats, birds, and especially swine) could act as vectors in Malaysia .
Although viral encephalitis is a notifiable disease, specific diagnosis for an aetiological agent has been lacking. Therefore cases of JE cannot be quantified accurately. At present, JE is neither clasified as an entity in the Malaysian medical record systems, nor it is a notifiable disease. Based on our research findings, there are now efforts to centralise JE surveillance and control. Presently, the Virology Division is involved in the national encephalitis viral survey that will actually quantify the JE problem in the paediatric age group and to help formalise a national vaccination policy.
The JE IgM-ELISA is a method of choice to demonstrate antibody to JE virus, in both blood and CSF samples. However, the incidental detection of serum antibodies to JE does not necessarily point to the cause of neurological illness. Therefore, the rapid RT-PCR was evaluated and optimised for use in diagnosis of JEV in CSF. The Virology Division is in the process of producing reagents for these tests.
The Virus Division has also sequenced the pre-M gene of JE virus strains isolated in Malaysia in 1992 and have found that they belong to the largest genotypic group that includes strains isolated in temperate regions ( Japan , China and Taiwan ). Subsequently, isolates from 1993 to 1994 were classified into two different genotypes.
5. TORCHES (Rubella)
The TORCHES Study was initiated primarily in the 1980's to aid the Ministry of Health to formulate a vaccination policy for rubella. The TORCHES sero-survey established that 45% of women of childbearing age (14-44 years) were susceptible to rubella infection and that among the 5 TORCHES diseases, rubella was the commonest cause of congenital infection in a newborn. These findings influenced the implementation of the National Rubella Immunisation Programme in 1986. The TORCHES study has been continued as a surveillance programme to determine the impact that the National Rubella Immunisation Programme has on congenital disease incidence .
6. Enteroviruses
Clinical poliomyelitis was recognised as a clinical entity in the country (when it was Malaya then) many years before laboratory diagnosis became available. In 1959, a Poliomyelitis Unit was set up at the IMR. Surveys were planned in collaboration with the Ministry of Health to aid the implementation of control measures. The Division of Virology has been accredited as the WHO Poliovirus National Laboratory for Polio Eradication since 1992.
The cases of polio reported are dated back to 1967 when 39 cases were reported; the numbers increased to as high as 768 in 1973. Due to that, the government launched a National Immunisation Programme for Poliomyelitis in the same year. Consequently, the number of cases has dropped tremendously; the last major outbreak occurred in 1977 with 121 cases. As part of the poliomyelitis surveillance, all cases of acute flaccid paralysis (AFP) are reported and investigated at this laboratory.
Other than poliovirus, the division is also actively involved in the surveillance and research of other enteroviruses such as Enterovirus 71 associated with hand, foot and mouth (HFMD) syndrome and meningo-encephalitis. Several EV71 and enteroviral isolates were obtained from paralytic or encephalitic cases during the 1997 outbreak and thereafter, which coincided with large outbreaks of HFMD in young children. These isolates were submitted for sequencing to our collaboraters at the National Institute of Health, Japan and the Centre for Diseases Control, Atlanta , USA . The viral surveillance of HFMD and acute flaccid paralysis is continuing.
The Virology Division serves as the main Public Health Laboratory in the Ministry of Health providing diagnostic services for prevalent medical viral infections. Its services extends to most government hospitals, clinics and veterinary departments in the country. It also serves as the following:-
i) National AIDS Reference Laboratory (NARL)
With regards to HIV/AIDS, the NARL was involved in several studies in selected high-risk groups. This was in addition to the confirmatory tests carried out on all referred samples and cases. The NARL conducted the National Quality Assurance Anti-HIV Screening Programme for 53 government HIV screening laboratories.
ii) National Reference Laboratory for Poliomyelitis Eradication which since 1992 has provided laboratory support for the National Polio Eradication Programme.
iii) WHO National Influenza Centre
iv) National Laboratory for potency testing of Viral Vaccines (specifically for measles and polio)
v) National SARS CoV Reference Centre
vi) The Division is one of eight YFV designated vaccination centres in Malaysia .
The range of diagnostic services provided at the Virology Division, IMR include:
1. Reference diagnostic service (serology) for various viral infections
2. Specific viral cultures using tissue culture and animal innoculation
3. Specific viral diagnosis by antigen detection
4. Reference services for HIV
5. Reference services for Hepatitis A, B, C & D viruses and other hepatotrophic viruses
6. Reference diagnostic service for certain Sexually Transmitted Diseases (STD), for example, Chlamydia
7. Potency testing of viral vaccines
8. Consultancy services (for the evaluation of commercial kits)
9. Yellow fever immunisation and certification for travellers
The Unit provides consultancies to the following:-
1. Hospital laboratories and various divisions under the Ministry of Health Malaysia, such as the Vector-Borne Disease Control and AIDS Units and the Communicable Disease Centre
2. Ministry of Health's Drug (Disinfectant) review sub-committee
3. Ministry of Health Microbiology subcommittee of the National Quality Assurance Programme for laboratory services
4. Ministry of Health HIV/AIDS Clinical Management subcommittee
5. Ministry of Health committee for animal care and use in research.
The Unit conducts the (1) virology module of the SEAMEO-TROPMED Diploma in Medical Microbiology course and (2) participates in the Diploma in Applied Parasitology and Entomology Course held at the IMR.
The Division is also involved in the training of Medical Laboratory Technologists attending the Diploma and Advanced Courses in Medical Laboratory Technology.
Post-graduate students from local universities and visiting scientists (foreign as well as local) also attend the above courses.
1. Centre for Disease Control (CDC), Atlanta , USA
2. Nagasaki University , Japan
3. Osaka University , Japan
Cffice Telephone Number: 03 - 26162671
Office Email: virus2@imr.gov.my
| Introduction | Research | Diagnostic Services |
