Phaik Hooi Tio

Antibodies against prM protein distinguish between previous infection with dengue and Japanese encephalitis viruses.

BackgroundIn Southeast Asia, dengue viruses often co-circulate with other flaviviruses such as Japanese encephalitis virus, and due to the presence of shared antigenic epitopes it is often difficult to use serological methods to distinguish between previous infections by these flaviviruses.ResultsConvalescent sera from 69 individuals who were known to have had dengue or Japanese encephalitis virus infection were tested by western blotting against dengue, Japanese encephalitis and West Nile virus antigens. We determined that individuals who had been infected with dengue viruses had IgG responses against the premembrane protein of dengue viruses but not Japanese encephalitis, whereas individuals who had been infected with Japanese encephalitis had IgG specific for the premembrane protein of Japanese encephalitis virus but not the dengue viruses. None reacted with the premembrane protein of West Nile virus. Using the Pearson Chi Square test, it was determined that the difference between the two groups was highly significant with a p value of <0.001.ConclusionThe use of flavivirus premembrane protein in seroepidemiological studies will be useful in determining what flaviviruses have circulated in a community.

Isolation of subgenus B adenovirus during a fatal outbreak of enterovirus 71-associated hand, foot, and mouth disease in Sibu, Sarawak

BACKGROUND In mid-1997, several children died in Sarawak, Malaysia, during an epidemic of enterovirus-71 (EV71) hand, foot, and mouth disease. The children who died had a febrile illness that rapidly progressed to cardiopulmonary failure and the cause was not satisfactorily resolved. We describe the isolation and identification of a subgenus B adenovirus from the children who died. METHODS We studied two groups of children presenting to Sibu Hospital from April 14 to Sept 30, 1997. For children who died, the inclusion criterion was death after febrile illness, and for those who did not die it was acute flaccid paralysis (AFP). Serum and cerebrospinal fluid samples were tested for IgM antibodies to Japanese encephalitis and dengue viruses. Viruses isolated were identified by immunofluorescence, reverse-transcriptase PCR, or PCR and DNA sequencing. FINDINGS Enterovirus was isolated in three (19%) of 16 children who died and in none of the eight surviving children with AFP. However, an agent that was initially difficult to identify was found in ten (63%) children who died and five (63%) surviving children who had AFP. The agents isolated from ten (66.7%) of these 15 children were eventually identified as adenoviruses and were isolated mainly from clinically important sterile sites or tissues. All the enterovirus-positive children who died had this second agent. INTERPRETATION Our data raises doubts that EV71 was the only aetiological agent in these deaths.

Identification and validation of clinical predictors for the risk of neurological involvement in children with hand, foot, and mouth disease in Sarawak

BackgroundHuman enterovirus 71 (HEV71) can cause Hand, foot, and mouth disease (HFMD) with neurological complications, which may rapidly progress to fulminant cardiorespiratory failure, and death. Early recognition of children at risk is the key to reduce acute mortality and morbidity.MethodsWe examined data collected through a prospective clinical study of HFMD conducted between 2000 and 2006 that included 3 distinct outbreaks of HEV71 to identify risk factors associated with neurological involvement in children with HFMD.ResultsTotal duration of fever ≥ 3 days, peak temperature ≥ 38.5°C and history of lethargy were identified as independent risk factors for neurological involvement (evident by CSF pleocytosis) in the analysis of 725 children admitted during the first phase of the study. When they were validated in the second phase of the study, two or more (≥ 2) risk factors were present in 162 (65%) of 250 children with CSF pleocytosis compared with 56 (30%) of 186 children with no CSF pleocytosis (OR 4.27, 95% CI2.79–6.56, p < 0.0001). The usefulness of the three risk factors in identifying children with CSF pleocytosis on hospital admission during the second phase of the study was also tested. Peak temperature ≥ 38.5°C and history of lethargy had the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 28%(48/174), 89%(125/140), 76%(48/63) and 50%(125/251), respectively in predicting CSF pleocytosis in children that were seen within the first 2 days of febrile illness. For those presented on the 3rd or later day of febrile illness, the sensitivity, specificity, PPV and NPV of ≥ 2 risk factors predictive of CSF pleocytosis were 75%(57/76), 59%(27/46), 75%(57/76) and 59%(27/46), respectively.ConclusionThree readily elicited clinical risk factors were identified to help detect children at risk of neurological involvement. These risk factors may serve as a guide to clinicians to decide the need for hospitalization and further investigation, including cerebrospinal fluid examination, and close monitoring for disease progression in children with HFMD.

The Epidemiology, Clinical Features, and Long-Term Prognosis of Japanese Encephalitis in Central Sarawak, Malaysia, 1997–2005

BACKGROUND Japanese encephalitis is a major public health problem in Asia. However, there is little data on the long-term outcome of Japanese encephalitis survivors. METHODS We prospectively evaluated children with serologically confirmed Japanese encephalitis over an 8.3-year period. The patients were assessed and their outcomes were graded with a functional outcome score at hospital discharge and at follow-up appointments. We examined how patient outcome at hospital discharge compared with that at long-term follow-up visits, when changes in outcome occurred, and the prognostic indicators of the eventual outcome. RESULTS One hundred and eighteen patients were recruited into the study, and 10 (8%) died during the acute phase of illness. At hospital discharge, 44 (41%) of the 108 patients who survived had apparent full recovery; 3 (3%) had mild, 28 (26%) had moderate, and 33 (31%) had severe neurological sequelae. Eighty six of the 108 patients were followed up for a median duration of 52.9 months (range, 0.9-114.9 months). During follow-up, 31 patients experienced improvement, but 15 patients experienced deterioration in their outcome grade. In most cases, assessment during the first 3-6 months after hospital discharge was predictive of the long-term outcome. More than one-half of the patients continued to experience neuropsychological sequelae and behavioral disorders. A combination of poor perfusion, Glasgow coma score < or =8, and > or =2 witnessed seizures predicted a poor long-term outcome with 65% sensitivity and 92% specificity. CONCLUSIONS Neurological assessment of Japanese encephalitis survivors at hospital discharge does not predict long-term outcome. Seizures and shock are treatable risk factors for a poor outcome at hospital discharge and at long-term follow-up visits.

Two dimensional VOPBA reveals laminin receptor (LAMR1) interaction with dengue virus serotypes 1, 2 and 3.

BackgroundThe search for the dengue virus receptor has generated many candidates often identified only by molecular mass. The wide host range of the viruses in vitro combined with multiple approaches to identifying the receptor(s) has led to the notion that many receptors or attachment proteins may be involved and that the different dengue virus serotypes may utilize different receptors on the same cells as well as on different cell types.ResultsIn this study we used sequential extraction of PS Clone D cell monolayers with the detergent β-octylglucopyranoside followed by sodium deoxycholate to prepare a cell membrane-rich fraction. We then used 2 dimensional (2D) gel electrophoresis to separate the membrane proteins and applied a modified virus overlay protein binding assay (VOPBA) to show that dengue virus serotypes 1, 2 and 3 all interact with the 37 kDa/67 kDa laminin receptor (LAMR1), a common non-integrin surface protein on many cell types.ConclusionAt least 3 of the 4 dengue serotypes interact with the 37 kDa/67 kDa laminin receptor, LAMR1, which may be a common player in dengue virus-cell surface interaction.

Dengue virus serotype 2 from a sylvatic lineage isolated from a patient with dengue hemorrhagic fever.

Dengue viruses circulate in both human and sylvatic cycles. Although dengue viruses (DENV) infecting humans can cause major epidemics and severe disease, relatively little is known about the epidemiology and etiology of sylvatic dengue viruses. A 20-year-old male developed dengue hemorrhagic fever (DHF) with thrombocytopenia (12,000/ul) and a raised hematocrit (29.5% above baseline) in January 2008 in Malaysia. Dengue virus serotype 2 was isolated from his blood on day 4 of fever. A phylogenetic analysis of the complete genome sequence revealed that this virus was a member of a sylvatic lineage of DENV-2 and most closely related to a virus isolated from a sentinel monkey in Malaysia in 1970. This is the first identification of a sylvatic DENV circulating in Asia since 1975.

Late presentation of Nipah virus encephalitis and kinetics of the humoral immune response

Nipah virus is a newly discovered paramyxovirus transmitted directly from pigs to humans. During a large encephalitis outbreak in Malaysia and Singapore in 1998–9, most patients presented acutely. A 12 year old child is described who developed encephalitis 4 months after exposure to the virus. She was diagnosed by a new indirect IgG enzyme linked immunosorbent assay (ELISA), which is also described. The late presentation and IgG subclass responses had similarities to subacute sclerosing panencephalitis. Nipah virus should be considered in patients with encephalitis even months after their possible exposure.

In Enterovirus 71 Encephalitis With Cardio-Respiratory Compromise, Elevated Interleukin 1β, Interleukin 1 Receptor Antagonist, and Granulocyte Colony-Stimulating Factor Levels Are Markers of Poor Prognosis

BACKGROUND Enterovirus 71 (EV71) causes large outbreaks of hand, foot, and mouth disease (HFMD), with severe neurological complications and cardio-respiratory compromise, but the pathogenesis is poorly understood. METHODS We measured levels of 30 chemokines and cytokines in serum and cerebrospinal fluid (CSF) samples from Malaysian children hospitalized with EV71 infection (n = 88), comprising uncomplicated HFMD (n = 47), meningitis (n = 8), acute flaccid paralysis (n = 1), encephalitis (n = 21), and encephalitis with cardiorespiratory compromise (n = 11). Four of the latter patients died. RESULTS Both pro-inflammatory and anti-inflammatory mediator levels were elevated, with different patterns of mediator abundance in the CSF and vascular compartments. Serum concentrations of interleukin 1β (IL-1β), interleukin 1 receptor antagonist (IL-1Ra), and granulocyte colony-stimulating factor (G-CSF) were raised significantly in patients who developed cardio-respiratory compromise (P = .013, P = .004, and P < .001, respectively). Serum IL-1Ra and G-CSF levels were also significantly elevated in patients who died, with a serum G-CSF to interleukin 5 ratio of >100 at admission being the most accurate prognostic marker for death (P < .001; accuracy, 85.5%; sensitivity, 100%; specificity, 84.7%). CONCLUSIONS Given that IL-1β has a negative inotropic action on the heart, and that both its natural antagonist, IL-1Ra, and G-CSF are being assessed as treatments for acute cardiac impairment, the findings suggest we have identified functional markers of EV71-related cardiac dysfunction and potential treatment options.

A decade of Japanese encephalitis surveillance in Sarawak, Malaysia: 1997–2006

Japanese encephalitis virus (JEV) is an important encephalitis virus in Asia, but there are few data on Malaysia. A hospital‐based surveillance system for Japanese encephalitis (JE) has been in operation in Sarawak, Malaysia, for the last 10 years. JEV is endemic in Sarawak, with cases occurring throughout the year and a seasonal peak in the last quarter (one‐way anova, P < 0.0001). Ninety‐two per cent of 133 cases were children aged 12 years or younger; the introduction of JE vaccination in July 2001 reduced the number of JE cases (84 in the four seasons prior to vs. 49 in the six seasons after, McNemar’s test, P = 0.0001). After implementation of the programme, the mean age of infected children increased from 6.3 to 8.0 years (Student’s t‐test, P = 0.0037), suggesting the need for a catch‐up programme.

Adenovirus type 21-associated acute flaccid paralysis during an outbreak of hand-foot-and-mouth disease in Sarawak, Malaysia

We report the virological and clinical features of 8 children who presented with adenovirus-associated acute flaccid paralysis (AFP) during an epidemic of enterovirus type 71 (EV71)-associated hand-foot-and-mouth disease (HFMD) in Sarawak, Malaysia, in 1997. Neutralization tests and phylogenetic analysis revealed adenovirus type 21 (Ad21), although DNA restriction digests suggested that this virus was different from the prototype Ad21. Four children had upper-limb monoparesis, 2 had lower-limb monoparesis (one of whom had changes in the anterior spinal cord noted on magnetic resonance imaging), and 2 had flaccid paraparesis. At follow-up, 4 children were noted to have made full recoveries and 3 had residual flaccid weakness and wasting. Neurophysiological investigation revealed a mixture of axonal and demyelinating features in motor and sensory nerves, with denervation. These findings suggest that Ad21 might cause AFP by anterior horn cell damage or neuropathy of the brachial or lumbosacral plexus. The occurrence of these unusual adenovirus infections during an outbreak of EV71-associated HFMD suggests that an interaction between the 2 viruses may have occurred.