Yuwana Podin

Sentinel surveillance for human enterovirus 71 in Sarawak, Malaysia: lessons from the first 7 years

BackgroundA major outbreak of human enterovirus 71-associated hand, foot and mouth disease in Sarawak in 1997 marked the beginning of a series of outbreaks in the Asia Pacific region. Some of these outbreaks had unusually high numbers of fatalities and this generated much fear and anxiety in the region.MethodsWe established a sentinel surveillance programme for hand, foot and mouth disease in Sarawak, Malaysia, in March 1998, and the observations of the first 7 years are described here. Virus isolation, serotyping and genotyping were performed on throat, rectal, vesicle and other swabs.ResultsDuring this period Sarawak had two outbreaks of human enterovirus 71, in 2000 and 2003. The predominant strains circulating in the outbreaks of 1997, 2000 and 2003 were all from genogroup B, but the strains isolated during each outbreak were genetically distinct from each other. Human enterovirus 71 outbreaks occurred in a cyclical pattern every three years and Coxsackievirus A16 co-circulated with human enterovirus 71. Although vesicles were most likely to yield an isolate, this sample was not generally available from most cases and obtaining throat swabs was thus found to be the most efficient way to obtain virological information.ConclusionKnowledge of the epidemiology of human enterovirus 71 transmission will allow public health personnel to predict when outbreaks might occur and to plan interventions in an effective manner in order to reduce the burden of disease.

Human Enterovirus 71 Disease in Sarawak, Malaysia: A Prospective Clinical, Virological, and Molecular Epidemiological Study

BACKGROUND Human enterovirus (HEV)-71 causes large outbreaks of hand-foot-and-mouth disease with central nervous system (CNS) complications, but the role of HEV-71 genogroups or dual infection with other viruses in causing severe disease is unclear. METHODS We prospectively studied children with suspected HEV-71 (i.e., hand-foot-and-mouth disease, CNS disease, or both) over 3.5 years, using detailed virological investigation and genogroup analysis of all isolates. RESULTS Seven hundred seventy-three children were recruited, 277 of whom were infected with HEV-71, including 28 who were coinfected with other viruses. Risk factors for CNS disease in HEV-71 included young age, fever, vomiting, mouth ulcers, breathlessness, cold limbs, and poor urine output. Genogroup analysis for the HEV-71-infected patients revealed that 168 were infected with genogroup B4, 68 with C1, and 41 with a newly emerged genogroup, B5. Children with HEV-71 genogroup B4 were less likely to have CNS complications than those with other genogroups (26 [15%] of 168 vs. 30 [28%] of 109; odds ratio [OR], 0.48; 95% confidence interval [CI], 0.26-0.91; P=.0223) and less likely to be part of a family cluster (12 [7%] of 168 vs. 29 [27%] of 109; OR, 0.21; 95% CI, 0.10-0.46; P<.0001); children with HEV-71 genogroup B5 were more likely to be part of a family cluster (OR, 6.26; 95% CI, 2.77-14.18; P<.0001). Children with HEV-71 and coinfected with another enterovirus or adenovirus were no more likely to have CNS disease. CONCLUSIONS Genogroups of HEV-71 may differ with regard to the risk of causing CNS disease and the association with family clusters. Dual infections are common, and all possible causes should be excluded before accepting that the first virus identified is the causal agent.

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.

Evaluation of Different Clinical Sample Types in Diagnosis of Human Enterovirus 71-Associated Hand-Foot-and-Mouth Disease

ABSTRACT Human enterovirus 71 and coxsackievirus A16 are important causes of hand-foot-and-mouth disease (HFMD). Like other enteroviruses, they can be isolated from a range of sterile and nonsterile sites, but which clinical sample, or combination of samples, is the most useful for laboratory diagnosis of HFMD is not clear. We attempted virus culture for 2,916 samples from 628 of 725 children with HFMD studied over a 3 1/2-year period, which included two large outbreaks. Overall, throat swabs were the single most useful specimen, being positive for any enterovirus for 288 (49%) of 592 patients with a full set of samples. Vesicle swabs were positive for 169 (48%) of 333 patients with vesicles, the yield being greater if two or more vesicles were swabbed. The combination of throat plus vesicle swabs enabled the identification of virus for 224 (67%) of the 333 patients with vesicles; for this patient group, just 27 (8%) extra patients were diagnosed when rectal and ulcer swabs were added. Of 259 patients without vesicles, use of the combination of throat plus rectal swab identified virus for 138 (53%). For 60 patients, virus was isolated from both vesicle and rectal swabs, but for 12 (20%) of these, the isolates differed. Such discordance occurred for just 11 (10%) of 112 patients with virus isolated from vesicle and throat swabs. During large HFMD outbreaks, we suggest collecting swabs from the throat plus one other site: vesicles, if these are present (at least two should be swabbed), or the rectum if there are no vesicles. Vesicle swabs give a high diagnostic yield, with the added advantage of being from a sterile site.

Molecular phylogeny of modern coxsackievirus A16

Summary.A phylogenetic analysis of VP1 and VP4 nucleotide sequences of 52 recent CVA16 strains demonstrated two distinct CVA16 genogroups, A and B, with the prototype strain being the only member of genogroup A. CVA16 G-10, the prototype strain, showed a nucleotide difference of 27.7–30.2% and 19.9–25.2% in VP1 and VP4, respectively, in relation to other CVA16 strains, which formed two separate lineages in genogroup B with nucleotide variation of less than 13.4% and less than 16.3% in VP1 and VP4, respectively. Lineage 1 strains circulating before 2000 were later displaced by lineage 2 strains.

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.

Incorrect identification of recent Asian strains of Coxsackievirus A16 as human enterovirus 71: Improved primers for the specific detection of human enterovirus 71 by RT PCR

BackgroundHuman enterovirus 71 has emerged as an important pathogen in the Asia Pacific region and it is important to be able to make a rapid and specific diagnosis for outbreak control. Recent Asian strains of Coxsackievirus A16 have changes in the VP1 gene which causes mispriming of widely used primers for human enterovirus 71 specific identification.MethodsLocal strains of Coxsackievirus A16 were sequenced in the VP4 and VP1 genes and using sequence alignment tools, an improved set of primers were designed for specific identification of human enterovirus 71. These primers were evaluated against virus isolates as well as primary clinical specimens.ResultsA total of 218 virus strains were tested. All 39 human enterovirus 71 isolates were positive and none of the 38 Coxsackievirus A16, 127 other enteroviruses and 14 prototype flaviviruses and adenoviruses were positive when tested with the new primers. When aliquots of primary specimens known to have yielded human enterovirus 71 were retrospectively tested, we found that within 2 months of collection of the specimens, greater than 90% were positive but that the success rate diminished rapidly to 18% after 2 years storage.ConclusionsOur new primers will be useful in rapid diagnosis of human enterovirus 71 infection, and can also be used as a screening tool in surveillance programmes for early warning of human enterovirus 71 transmission.

Burkholderia pseudomallei Isolates from Sarawak, Malaysian Borneo, Are Predominantly Susceptible to Aminoglycosides and Macrolides

ABSTRACT Melioidosis is a potentially fatal disease caused by the saprophytic bacterium Burkholderia pseudomallei. Resistance to gentamicin is generally a hallmark of B. pseudomallei, and gentamicin is a selective agent in media used for diagnosis of melioidosis. In this study, we determined the prevalence and mechanism of gentamicin susceptibility found in B. pseudomallei isolates from Sarawak, Malaysian Borneo. We performed multilocus sequence typing and antibiotic susceptibility testing on 44 B. pseudomallei clinical isolates from melioidosis patients in Sarawak district hospitals. Whole-genome sequencing was used to identify the mechanism of gentamicin susceptibility. A novel allelic-specific PCR was designed to differentiate gentamicin-sensitive isolates from wild-type B. pseudomallei. A reversion assay was performed to confirm the involvement of this mechanism in gentamicin susceptibility. A substantial proportion (86%) of B. pseudomallei clinical isolates in Sarawak, Malaysian Borneo, were found to be susceptible to the aminoglycoside gentamicin, a rare occurrence in other regions where B. pseudomallei is endemic. Gentamicin sensitivity was restricted to genetically related strains belonging to sequence type 881 or its single-locus variant, sequence type 997. Whole-genome sequencing identified a novel nonsynonymous mutation within amrB, encoding an essential component of the AmrAB-OprA multidrug efflux pump. We confirmed the role of this mutation in conferring aminoglycoside and macrolide sensitivity by reversion of this mutation to the wild-type sequence. Our study demonstrates that alternative B. pseudomallei selective media without gentamicin are needed for accurate melioidosis laboratory diagnosis in Sarawak. This finding may also have implications for environmental sampling of other locations to test for B. pseudomallei endemicity.

Unprecedented Melioidosis Cases in Northern Australia Caused by an Asian Burkholderia pseudomallei Strain Identified by Using Large-Scale Comparative Genomics.

ABSTRACT Melioidosis is a disease of humans and animals that is caused by the saprophytic bacterium Burkholderia pseudomallei. Once thought to be confined to certain locations, the known presence of B. pseudomallei is expanding as more regions of endemicity are uncovered. There is no vaccine for melioidosis, and even with antibiotic administration, the mortality rate is as high as 40% in some regions that are endemic for the infection. Despite high levels of recombination, phylogenetic reconstruction of B. pseudomallei populations using whole-genome sequencing (WGS) has revealed surprisingly robust biogeographic separation between isolates from Australia and Asia. To date, there have been no confirmed autochthonous melioidosis cases in Australia caused by an Asian isolate; likewise, no autochthonous cases in Asia have been identified as Australian in origin. Here, we used comparative genomic analysis of 455 B. pseudomallei genomes to confirm the unprecedented presence of an Asian clone, sequence type 562 (ST-562), in Darwin, northern Australia. First observed in Darwin in 2005, the incidence of melioidosis cases attributable to ST-562 infection has steadily risen, and it is now a common strain in Darwin. Intriguingly, the Australian ST-562 appears to be geographically restricted to a single locale and is genetically less diverse than other common STs from this region, indicating a recent introduction of this clone into northern Australia. Detailed genomic and epidemiological investigations of new clinical and environmental B. pseudomallei isolates in the Darwin region and ST-562 isolates from Asia will be critical for understanding the origin, distribution, and dissemination of this emerging clone in northern Australia.

Reliability of automated biochemical identification of Burkholderia pseudomallei is regionally dependent

ABSTRACT Misidentifications of Burkholderia pseudomallei as Burkholderia cepacia by Vitek 2 have occurred. Multidimensional scaling ordination of biochemical profiles of 217 Malaysian and Australian B. pseudomallei isolates found clustering of misidentified B. pseudomallei isolates from Malaysian Borneo. Specificity of B. pseudomallei identification in Vitek 2 and potentially other automated identification systems is regionally dependent.