Gastrointestinal manifestations of dengue infection in adults.

This is a retrospective study of the gastrointestinal symptoms, signs and laboratory parameters in adult dengue patients admitted to Kuala Lumpur Hospital from 1st December 2004 to 31st December 2004. Clinical and laboratory parameters that may predict the need for intensive care were investigated. Six hundred sixty-six patients with clinical and biochemical features consistent with dengue infection were identified. Patients were stratified into those who required intensive care and those who were managed in non high dependency wards. Serum alanine aminotransaminase (ALT) levels were normal in 22.8% of patients and 5.9% of patients had acute fulminant hepatitis. More patients with dengue haemorrhagic fever (DHF) had elevated ALT levels as compared to patients with classic dengue fever (DF) (p = 0.012). Patients with DF had a statistically significant lower mean ALT level as compared to patients with DHF. Abdominal pain (p = 0.01) and tenderness (p<0.001), gastrointestinal bleed (p<0.001), jaundice (p<0.001), hepatomegaly (p<0.001) and ascites (p<0.001) were predictors of need for intensive care. We conclude that gastrointestinal manifestations are very common in dengue patients. Presence of abdominal pain and tenderness, gastrointestinal bleed, jaundice, hepatomegaly and ascites can be used to triage patients requiring intensive care.

Deaths in children during an outbreak of hand, foot and mouth disease in Peninsular Malaysia--clinical and pathological characteristics.

From July through December 1997, 11 previously healthy children in Peninsular Malaysia succumbed to an illness clinically characterised by an acute severe refractory left-ventricular failure, following a brief prodromal illness, in the midst of an outbreak of hand, foot and mouth disease (HFMD), similar to the reported experience in Sarawak and Taiwan. Retrospective reviews of the clinical features and results of laboratory, pathological and virological investigations of cases were conducted. The median age of the 11 case-patients was 31 months (range, 13 to 49 months); 6 were males. A brief prodromal illness of 3 days (range, 2 to 5 days) was characterised by fever (axillary temperature > 38 degrees C) (100%), oral ulcers (72%), extremity rashes (45%) and significant vomiting (55%). Upon hospitalisation, 7 of 11 case-patients had features suggestive of cardiogenic shock, while 4 of 11 case-patients developed shock during hospitalisation as evidenced by marked sustained tachycardia (heart rate > or = 180 beats per minute), poor peripheral pulses and peripheral perfusion, mottled extremities, pulmonary oedema (haemorrhagic pulmonary secretions in 8 of 11 cases during tracheal intubation, often precipitated by conservative crystalloid boluses, and radiographic evidence of acute pulmonary oedema in 5 of 7 cases) and markedly impaired left ventricular function on echocardiographic examination (7 of 7 cases). Three of 4 case-patients had aseptic meningitis while one case-patient also had an acute flaccid paraparesis. Despite supportive therapy, death occurred within a median of 13.4 hours following hospitalization. Post-mortem findings (all 8 specimens examined) consistently demonstrated brain-stem encephalitis with foci of neuronal necrosis and micro-abscesses. None of the 11 specimens examined revealed histological evidence of myocarditis. Enterovirus 71 (EV71) was detected in 10 of 11 case-patients, many (7) from various sterile tissue sites (5 from central nervous tissues). No other viruses were isolated or identified. Clinical features and pathological studies closely paralleled the reported experience in Sarawak and Taiwan. The uniform necropsy findings of necrotizing brain-stem encephalitis coupled with essentially normal myocardial histology, in concert with the concurrent and consistent detection of EV71 points to a primary EV71 encephalitis; as yet unclear neurogenic mechanisms may account for the cardiovascular manifestations.

Five-year surveillance of acute flaccid paralysis in Malaysia.

OBJECTIVE: The nation-wide surveillance for acute flaccid paralysis (AFP) was implemented in Malaysia in 1995 and further intensified in 1996 as part of the World Health Organization's (WHO) certification process for polio eradication in the Western Pacific Region. Clinical data on AFP cases during a 5-year surveillance period from 1997 to 2001 were compiled and analysed. RESULTS: Based on 517 cases of AFP reported during this 5-year period, the overall rate of AFP was 1.2 per 100 000 children below 15 years old. The major clinical diagnosis associated with AFP were Guillain-Barre syndrome (30.2%), central nervous system infection (16.2%), transverse myelitis (10.6%) non-polio enterovirus infection (6.2%), and hypokalaemic paralysis (5.2%). This unusual pattern with an excess of CNS infection and non-polio enterovirus infection was attributed to the outbreak of enterovirus 71 infection nation-wide in 1997. According to the WHO virological classification, there was no case of poliomyelitis due to wild poliovirus. Three cases were 'polio compatible', there were no cases of vaccine-associated paralytic polio (VAPP), while 62 cases (12.0%) were merely classified as 'non-polio AFP'. CONCLUSION: Overall, these data suggest the absence of circulation of wild poliovirus in Malaysia from 1997 to 2001. The pattern of AFP in this study is different from other published reports.

Laboratory acute flaccid paralysis surveillance in Malaysia: a decade of commitment to the WHO global polio eradication initiative.

The Institute for Medical Research, Malaysia, was designated the National Reference Laboratory for Poliomyelitis Eradication (NRLPE) in 1992. Since then, our Polio Laboratory has collaborated actively with the Disease Control Division, Ministry of Health (MOH), Malaysia and WHO towards achieving polio eradication. Since 1992, the NRLPE has investigated 1,063 stool specimens from 641 acute flaccidparalysis (AFP) cases. One hundred and one enteroviruses were isolated from these specimens. Positive cell cultures were confirmed by microneutralization assay using standard WHO antisera. All enterovirus isolates were sent to the Victorian Infectious Disease Reference Laboratory in Melbourne, Australia, for further identification and poliovirus intratypic differentiation. Thirty-one out of these 101 virus isolates (30%) were polioviruses (PV) and the remaining 70 (70%) were non-polio enteroviruses (NPEV) which included coxsackie B viruses, echoviruses and enterovirus 71. Three of the poliovirus isolates were wild-type polioviruses isolated in 1992 which were the last wild-type polioviruses isolated in Malaysia. The rest were vaccine-related Sabin-like strains. Monthly reports of the virological investigation of AFP cases are sent to WHO and to the MOH, AFP control committee. The NRLPE continues to play an integral role in AFP surveillance and is committed to the WHO's goal of global polio eradication by the year 2005.

Combined detection and genotyping of Chikungunya virus by a specific reverse transcription-polymerase chain reaction.

A reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of Chikungunya virus infection. Based on the nonstructural protein 1 (nsP1) and glycoprotein E1 (E1) genes of Chikungunya, two primer sets were designed. Total RNA were extracted from the cell culture fluid of Aedes albopictus C6/36 cells inoculated with the S27 prototype virus, isolated in Tanzania in 1953, and the Malaysian strains (MALh0198, MALh0298, and MALh0398), isolated in Malaysia in 1998. For both sets of RNA samples, the expected 354- and 294-base pair (bp) cDNA fragments were amplified effectively from the nsP1 and E1 genes, respectively. Phylogenetic analysis was conducted for the Malaysian strain and other virus strains isolated from different regions in the world endemic for Chikungunya, using partial E1 gene sequence data. The Malaysian strains isolated during the epidemics of 1998 fell into a cluster with other members of the Asian genotype.

Human immunodeficiency virus type 1 subtypes among Malaysian intravenous drug users.

The HIV-1 genetic variation in 60 infected Malaysian intravenous drug users (IDU) was studied by comparison of the nucleotide sequences and their predicted amino acid sequences in the V3 loop of the external glycoprotein gp120. In this study, HIV-1 B, C and E subtypes were identified among Malaysian IDU, with HIV-1 B being the predominant subtype (91.7%). HIV-1 C and HIV-1 E were minority subtypes among Malaysian IDU. Analysis of the amino acid alignment of the C2-V3 region of the env gene suggests a genetic relationship between Thai and Malaysian B and E subtype strains. This study serves as a baseline for monitoring HIV-1 genetic diversity and spread in Malaysia.

Type-3 dengue viruses responsible for the dengue epidemic in Malaysia during 1993-1994.

To characterize the dengue epidemic that recently occurred in Malaysia, we sequenced cDNAs from nine 1993-1994 dengue virus type-3 (DEN-3) isolates in Malaysia (DEN-3 was the most common type in Malaysia during this period). Nucleic acid sequences (720 nucleotides in length) from the nine isolates, encompassing the precursor of membrane protein (preM) and membrane (M) protein genes and part of the envelope (E) protein gene were aligned with various reference DEN-3 sequences to generate a neighbor-joining phylogenetic tree. According to the constructed tree, the nine Malaysian isolates were grouped into subtype II, which comprises Thai isolates from 1962 to 1987. Five earlier DEN-3 virus Malaysian isolates from 1974 to 1981 belonged to subtype I. The present data indicate that the recent dengue epidemic in Malaysia was due to the introduction of DEN-3 viruses previously endemic to Thailand.

Enterovirus 71 from fatal and nonfatal cases of hand, foot and mouth disease epidemics in Malaysia, Japan and Taiwan in 1997-1998.

Enterovirus 71 (EV71), one of the major causative agents for hand, foot and mouth disease (HFMD), is sometimes associated with severe central nervous system diseases. In 1997, in Malaysia and Japan, and in 1998 in Taiwan, there were HFMD epidemics involving sudden deaths among young children, and EV71 was isolated from the HFMD patients, including the fatal cases. The nucleotide sequences of each EV71 isolate were determined and compared by phylogenetical analysis. EV71 strains from previously reported epidemics belonged to genotype A-1, while those from recent epidemics could be divided into two genotypes, A-2 and B. In Malaysia, genotype A-2 was more prevalent, while in Japan and Taiwan, B genotype was more prevalent. Two isolates from fatal cases in Malaysia and one isolate from a fatal case in Japan were genotype A-2. However, all isolates from three fatal cases in Taiwan belonged to genotype B. The severity of the HFMD did not link directly to certain genotypes of EV71.

A four year review of acute viral hepatitis cases in the east coast of peninsular Malaysia (1994-1997).

A total of 1,157 sera from jaundiced patients with clinical and biochemical evidence of liver disease received from government hospital in Kelantan and Terengganu, during the period from 1994 to 1997, were investigated to determine the cause. Hepatitis A virus was found to be the main cause in 26.1% (24/92) of symptomatic clinical hepatitis cases in 1994, 47.8% (63/132) in 1995, 66.4% (613/923) in 1996 and 20% (2/10) in 1997. Sera received in 1996 were also tested for hepatitis B, hepatitis C, hepatitis D and hepatitis E. 1.4% (13/923) anti-bodies were found to be positive for HBc IgM indicating recent HBV infection, 5.4% (50/923) for total HCV Ab, 0.9% (8/923) for total HDV Ab and 0.4% (4/923) for anti-HEV IgM. This study shows that HAV is still a major problem in Kelantan and Terengganu, and there is a need to identify effective strategies for prevention and control in these two states.

Comparison of sequences of E/NS1 gene junction of dengue type 3 virus following culture subpassage in C6/36 cells to study the possible occurrence of mutations.

The aim of this study was to determine whether mutations could occur in the dengue virus genome following three subpassages of the virus in a mosquito cell line. This was done because sources of virus isolates used for sequencing studies are usually maintained in cell lines rather than in patients' sera. Therefore it must be assured that no mutation occurred during the passaging. For this purpose, sequencing was carried out using the polymerase chain reaction (PCR) products of the envelope/non-structural protein 1 junction region (280 nucleotides) of dengue type 3 virus. Sequence data were compared between the virus from a patient's serum against the virus subpassaged three times in the C6/36 cell line. We found that the sequence data of the virus from serum was identical to the virus that was subpassaged three times in C6/36 cell line.

Abundance, parity, and Japanese encephalitis virus infection of mosquitoes (Diptera:Culicidae) in Sepang District, Malaysia.

A 2-yr study of Japanese encephalitis (JE) virus in Sepang District, Selangor, Malaysia, was carried out to identify the mosquito vectors and to determine their seasonal abundance, parity, and infection rates. In total, 81,889 mosquitoes belonging to 9 genera and > 50 species were identified from CDC trap collections augmented with dry ice during 1992 and 1993. Culex tritaeniorhynchus Giles and Culex gelidus Giles were the most abundant species, and both increased in numbers with increases in rainfall. Overall, 45 JE virus isolations were made from 7 species-Cx. tritaeniorhynchus (24), Cx. gelidus (12), Culex fuscocephala Theobald (2), Aedes butleri Theobald (4), Culex quinquefasciatus Say (1), Aedes lineatopennis Ludlow (1), and Aedes (Cancraedes) sp. (1). Based on elevated abundance and JE infection rates, Cx. tritaeniorhynchus appears to be the most important vector of JE virus in Sepang.

Genotypes of Japanese encephalitis virus isolated in three states in Malaysia.

Two hundred forty nucleotides from the pre-membrane gene region of 12 Japanese encephalitis virus (JEV) strains isolated from three different regions of Malaysia from 1993 to 1994 were sequenced and compared with each other and with the JEV strains from different geographic areas in Asia. These 12 Malaysian isolates were classified into two genotypes. The four JEV strains isolated from Sarawak in 1994 and the four JEV strains isolated from Sepang, Selangor in 1993 were classified into one genotype that included earlier isolated strains from Malaysia (JE-827 from Sarawak in 1968 and WTP/70/22 from Kuala Lumpur in 1970). The four JEV strains from Ipoh, Perak in 1994 were classified into another genotype that included JEV strains isolated from northern Thailand and Cambodia. In an earlier report, 10 JEV strains from Sabak Bernam, Selangor in 1992 were classified into the largest genotype that included strains isolated in temperate regions such as Japan, China, and Taiwan. The data indicate that at least three genotypes of JEV have been circulating in Malaysia.

Application of the Serodia-HCV particle agglutination for the detection of antibodies to hepatitis C virus.

The Serodia-HCV Particle Agglutination (HCV-PA) for the detection of HCV antibodies was compared with the Enzyme Immunoassay Test (UBI HCV EIA) for possible in-house use. A total of 150 specimens were analysed using UBI HCV EIA and Serodia-HCV PA. Of these, 80 (53.3%) were both PA and EIA positive and 59 (39.3%) were negative by both techniques. Eleven sera (7.4%) were found to be EIA-positive but PA-negative. These 11 discordant sera were further tested by the LiaTek-HCV III Immunoassay (Organon Teknika). Ten were found to be line immunoassay negative and one was line immunoassay positive. Failure of the PA to detect the HCV positive serum meant that a small proportion of HCV antibody positives may be missed by the PA test. We conclude that (i) EIA should continue to be the first line screening test in our laboratory, (ii) PA with its 100% specificity could be a useful supplementary screen for all EIA-positive sera and finally (iii) line immunoassay could be used on sera to resolve discordant results in the EIA and PA assays.

HIV type 1 subtypes in Malaysia include B, C, and E.

PIP: Peripheral blood mononuclear cell specimens were collected from 13 HIV-1-infected IV drug users in Kuala Lumpur, Malaysia, as well as one HIV-infected baby, between 1992 and 1993. DNA was then amplified by nested polymerase chain reaction and a 345-bp fragment of the C2V3 region of the env gene was sequenced. 11 of the 14 Malaysian sequences clustered with the B' subtype, one different from the typical subtype B US strains HIVMN and HIVSF2. Two sequences grouped in the C subtype and had sister taxa closer to the Indian C subtype sequences than those from Zambia. The sequence from the infant was identified as a subtype E virus, grouped more closely with subtype E strains from Thailand than subtype E viruses from the Central African Republic.^ieng

Cross-sectional serosurvey for Japanese encephalitis specific antibody from animal sera in Malaysia 1993.

Serum specimens were collected from 6 species of animals living in 9 states of Malaysia including Sabah, North Borneo in 1993. Antibodies against Japanese encephalitis (JE) virus in these sera were detected by means of hemagglutination-inhibition (HI) and neutralization (NT) tests. By HI test, 702 of 2,152 (32.6%) sera showed positive results. Higher positive rates were obtained by the NT test, in which 1,787 of 1,927 (92.7%) sera had antibodies against JE virus. All serum specimens with positive HI were confirmed as positive by the NT. Swine sera showed especially higher rates of antibody positive and higher antibody titers compared with other animals. These results suggest that JE infections are widely distributed among many animals of Malaysia, and pig is the most susceptible amplifier host for JE virus.

Evaluation of cold chain monitoring in Kelantan, Malaysia.

An analysis was carried out on a total of 883 cold chain monitor (CCM) cards, which had been attached to batches of poliomyelitis, measles, DPT (diphtheria, pertussis, tetanus) and hepatitis B vaccines, during their transport and storage from the central store in Kuala Lumpur to Kelantan, a state in north-eastern Malaysia; 234 freeze watches attached to hepatitis B vaccines were also analysed. The monitor cards and freeze watches were observed at six levels between the central store and the periphery during distribution of the vaccines, and a colour change in any of the four windows (A, B, C, D) on the CCM cards or the freeze watches was recorded. In addition, 33 unopened vials of oral poliovirus vaccine (OPV), collected from refrigerators in 29 health facilities in Kelantan, were tested for potency using the tissue culture infective dose 50 (TCID50) method; 14 of them (42%) did not meet the WHO criteria for potent vaccines. The results showed that at the final destination 13.4% of all cards remained white while a colour change to blue was observed in 65% in window A, 16.6% in window B, and 4.4% in window C; none had turned blue in window D indicating that the vaccine had not been subjected to temperatures > or = 34 degrees C for 2 hours. All but 2 of the 234 freeze watches had turned purple, which indicates exposure of the hepatitis B vaccines to temperatures below 0 degree C. These results will assist health planners to correct the weaknesses identified in the cold chain system.

The use of polymerase chain reaction (PCR) as a diagnostic tool for dengue virus.

This study describes the use of polymerase chain reaction as a diagnostic tool for detecting and typing of dengue virus. PCR was compared against virus isolation. First RT-PCR was done using dengue consensus primers after which positive samples were subjected to RT-PCR using type-specific primers. This study shows that the local strains of the dengue virus could be detected using the chosen primers. Furthermore, RT-PCR was found to be more sensitive than virus isolation in identifying the dengue positive samples.

Augmented inflammatory cytokines in primary dengue infection progressing to shock.

Dengue fever (DF) which is caused by four serotypes of dengue virus may in some cases progress into a life threatening situation of dengue haemorrhage fever (DHF) and dengue shock syndrome (DSS). It has been suggested that sequential infection with different dengue virus serotypes predisposes the patient towards DHF/DSS. We report here a primary dengue infection in a 10-year-old boy progressing from DF to DSS while under clinical observation. The report provides unequivocal evidence for the development of DSS in primary dengue infection caused by virus serotype 4. The close relationship between sequential changes in the levels of tumour necrosis factor (TNF), Interleukin 1 and 6 (IL-1 and IL-6) in the serum, to the clinical progression of the disease from DF to DHF/DSS and then to full recovery implicates a pathogenetic role for the inflammatory cytokines. The child also manifested clinical features consistent with Reye's syndrome and this suggests a common pathogenetic origin for DSS and the Reye-like syndrome induced by dengue virus.

HIV-1 variants in South and South-East Asia.

HIV spread in South and South-East Asia is most alarming, and genetic variability of HIV-1 is an important consideration in vaccine development. In this study, we examined the third variable (V3) region of env gene of HIV-1 variants prevalent in Thailand, Malaysia, India, and the Philippines. By phylogenetic tree analyses, an HIV-1 variant from an injecting drug user (IDU) in Thailand belonged to subtype B, and HIV-1 variants from 2 IDUs in Malaysia were classified into 2 subtypes, B and E. One HIV-1 variant from a male homosexual in the Philippines belonged to subtype B. Out of 8 HIV-1 variants from sexually transmitted disease patients in India, 7 belonged to subtype C, and one to subtype A. Although the total number of individuals examined in this study was limited, 4 HIV-1 subtypes were found in South and South-East Asia and large international movements of HIV-1-infected individuals in this region could induce global dissemination of these HIV-1 variants.