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Background Leptospirosis diagnoses have increased in Sarawak, Malaysia in recent years. Methods To better understand the burden of disease and associated risk factors, we evaluated 147 patients presenting with clinical leptospirosis to local hospitals in Sarawak, Malaysia for the presence of Leptospira and associated antibodies. Sera and urine specimens collected during the acute illness phase were assessed via a commercially available rapid diagnostic test (Leptorapide, Linnodee Ltd., Antrim, Northern Ireland), an ELISA IgM assay (Leptospira IgM ELISA, PanBio, Queensland, Australia) and a pan-Leptospira real-time PCR (qPCR) assay to estimate disease prevalence and diagnostic accuracy of each method. Microagglutination testing was performed on a subset of samples. Results Overall, 45 out of 147 patients (30.6%) showed evidence of leptospires through qPCR in either one or both sera (20 patients) or urine (33 patients), and an additional ten (6.8%) were considered positive through serological testing, for an overall prevalence of 37.4% within the study population. However, each diagnostic method individually yielded disparate prevalence estimates: rapid test 42.2% for sera and 30.5% for urine, ELISA 15.0% for sera, qPCR 13.8% for sera and 23.4% for urine. Molecular characterization of a subset of positive samples by conventional PCR identified the bacterial species as Leptospira interrogans in 4 specimens. A multivariate risk factor analysis for the outcome of leptospirosis identified having completed primary school (OR = 2.5; 95 CI% 1.0–6.4) and weekly clothes-washing in local rivers (OR = 10.6; 95 CI% 1.4–214.8) with increased likelihood of leptospirosis when compared with those who had not. Conclusion Overall, the data suggest a relatively high prevalence of leptospirosis in the study population. The low sensitivities of the rapid diagnostic test and ELISA assay against qPCR highlight a need for better screening tools.

BackgroundIn a year-long pneumonia etiology study conducted June 2017 to May 2018 in Sarawak, Malaysia, 599 patients’ nasopharyngeal swab specimens were studied with real-time polymerase chain reaction (rPCR)/ reverse-transcription (rRT-PCR) assays for respiratory pathogens known to contribute to the high burden of lower respiratory tract infections. The study team sought to compare real-time assay results with panspecies conventional molecular diagnostics to compare sensitivities and learn if novel viruses had been missed.MethodsSpecimens were studied for evidence of adenovirus (AdV), enterovirus (EV) and coronavirus (CoV) with panspecies gel-based nested PCR/RT-PCR assays. Gene sequences of specimens positive by panspecies assays were sequenced and studied with the NCBI Basic Local Alignment Search Tool software.ResultsThere was considerable discordance between real-time and conventional molecular methods. The real-time AdV assay found a positivity of 10.4%; however, the AdV panspecies assay detected a positivity of 12.4% and the conventional AdV-Hexon assay detected a positivity of 19.6%. The CoV and EV panspecies assays similarly detected more positive specimens than the real-time assays, with a positivity of 7.8% by the CoV panspecies assay versus 4.2% by rRT-PCR, and 8.0% by the EV panspecies assay versus 1.0% by rRT-PCR. We were not able to ascertain virus viability in this setting. While most discordance was likely due to assay sensitivity for previously described human viruses, two novel, possible zoonotic AdV were detected.ConclusionsThe observed differences in the two modes of amplification suggest that where a problem with sensitivity is suspected, real-time assay results might be supplemented with panspecies conventional PCR/RT-PCR assays.

To monitor the incidence of Plasmodium knowlesi infections and determine whether other simian malaria parasites are being transmitted to humans, we examined 1,047 blood samples from patients with malaria at Kapit Hospital in Kapit, Malaysia, during June 24, 2013–December 31, 2017. Using nested PCR assays, we found 845 (80.6%) patients had either P. knowlesi monoinfection (n = 815) or co-infection with other Plasmodium species (n = 30). We noted the annual number of these zoonotic infections increased greatly in 2017 (n = 284). We identified 6 patients, 17–65 years of age, with P. cynomolgi and P. knowlesi co-infections, confirmed by phylogenetic analyses of the Plasmodium cytochrome c oxidase subunit 1 gene sequences. P. knowlesi continues to be a public health concern in the Kapit Division of Sarawak, Malaysian Borneo. In addition, another simian malaria parasite, P. cynomolgi, also is an emerging cause of malaria in humans.

Plasmodium knowlesi, a simian malaria parasite responsible for all recent indigenous cases of malaria in Malaysia, infects humans throughout Southeast Asia. There are two genetically distinct subpopulations of Plasmodium knowlesi in Malaysian Borneo, one associated with long-tailed macaques (termed cluster 1) and the other with pig-tailed macaques (cluster 2). A prospective study was conducted to determine whether there were any between-subpopulation differences in clinical and laboratory features, as well as in epidemiological characteristics. Over 2 years, 420 adults admitted to Kapit Hospital, Malaysian Borneo with knowlesi malaria were studied. Infections with each subpopulation resulted in mostly uncomplicated malaria. Severe disease was observed in 35/298 (11.7%) of single cluster 1 and 8/115 (7.0%) of single cluster 2 infections (p = 0.208). There was no clinically significant difference in outcome between the two subpopulations. Cluster 1 infections were more likely to be associated with peri-domestic activities while cluster 2 were associated with interior forest activities consistent with the preferred habitats of the respective macaque hosts. Infections with both P. knowlesi subpopulations cause a wide spectrum of disease including potentially life-threatening complications, with no implications for differential patient management.

AIMS The aim of this study was to assess the pharmacokinetic properties of artemether, lumefantrine and their active metabolites in Plasmodium knowlesi malaria. METHODS Malaysian adults presenting with uncomplicated P. knowlesi infections received six doses of artemether (1.7 mg/kg) plus lumefantrine (10 mg/kg) over 3 days. Venous blood and dried blood spot (DBS) samples were taken at predetermined time-points over 28 days. Plasma and DBS artemether, dihydroartemisinin, lumefantrine and desbutyl-lumefantrine were measured using liquid chromatography-mass spectrometry. Multi-compartmental population pharmacokinetic models were developed using plasma with or without DBS drug concentrations. RESULTS Forty-one participants (mean age 45 years, 66% males) were recruited. Artemether-lumefantrine treatment was well tolerated and parasite clearance was prompt. Plasma and DBS lumefantrine concentrations were in close agreement and were used together in pharmacokinetic modelling, but only plasma concentrations of the other analytes were used because of poor correlation with DBS levels. The areas under the concentration-time curve (AUC0-∞ ) for artemether, dihydroartemisinin and lumefantrine (medians 1626, 1881 and 625 098 μg.h/L, respectively) were similar to those reported in previous pharmacokinetic studies in adults and children. There was evidence of auto-induction of artemether metabolism (mean increase in clearance relative to bioavailability 25.2% for each subsequent dose). The lumefantrine terminal elimination half-life (median 9.5 days) was longer than reported in healthy volunteers and adults with falciparum malaria. CONCLUSION The disposition of artemether, dihydroartemisinin and lumefantrine in knowlesi malaria largely parallels that in other human malarias. DBS lumefantrine concentrations can be used in pharmacokinetic studies but DBS technology is currently unreliable for the other analytes.

Population genetic analysis revealed that Plasmodium knowlesi infections in Malaysian Borneo are caused by 2 divergent parasites associated with long-tailed (cluster 1) and pig-tailed (cluster 2) macaques. Because the transmission ecology is likely to differ for each macaque species, we developed a simple genotyping PCR to efficiently distinguish between and survey the 2 parasite subpopulations. This assay confirmed differences in the relative proportions in areas of Kapit division of Sarawak state, consistent with multilocus microsatellite analyses. Analyses of 1,204 human infections at Kapit Hospital showed that cluster 1 caused approximately two thirds of cases with no significant temporal changes from 2000 to 2018. We observed an apparent increase in overall numbers in the most recent 2 years studied, driven mainly by increased cluster 1 parasite infections. Continued monitoring of the frequency of different parasite subpopulations and correlation with environmental alterations are necessary to determine whether the epidemiology will change substantially.

Burkholderia pseudomallei infections are prevalent in Southeast Asia and northern Australia and often misdiagnosed. Diagnostics are often neither sensitive nor rapid, contributing up to 50% mortality rate. In this 2018 pilot study, we enrolled 100 patients aged 6 months–79 years from Kapit Hospital in Sarawak, Malaysia, with symptoms of B. pseudomallei infection. We used three different methods for the detection of B. pseudomallei: a real-time polymerase chain reaction (PCR) assay, a rapid lateral flow immunoassay, and the standard-of-care bacterial culture—the gold standard. Among the 100 participants, 24 (24%) were positive for B. pseudomallei by one or more of the detection methods. Comparing the two individual diagnostic methods against the gold standard—bacterial culture—of any positive test, there was low sensitivity for each test (25–44%) but high specificity (93–98%). It seems clear that more sensitive diagnostics or a sensitive screening diagnostic followed by specific confirmatory diagnostic is needed for this disease.

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.

Background Melioidosis is a serious, and potentially fatal community-acquired infection endemic to northern Australia and Southeast Asia, including Sarawak, Malaysia. The disease, caused by the usually intrinsically aminoglycoside-resistant Burkholderia pseudomallei, most commonly affects adults with predisposing risk factors. There are limited data on pediatric melioidosis in Sarawak. Methods A part prospective, part retrospective study of children aged, Author summary Melioidosis is a serious, and often fatal community-acquired infection endemic to Southeast Asia and northern Australia. It is caused by the environmental saprophyte Burkholderia pseudomallei, a bacterium that is intrinsically resistant to many commonly used antibiotics. Its presence in Sarawak, Malaysian Borneo, has been documented, and recently, a novel gentamicin-susceptible strain discovered. However, there are limited data on the burden and clinical characteristics of melioidosis in Sarawak, both in adults and in children. In this study, we comprehensively investigated all pediatric melioidosis cases in Central Sarawak over a 6-year period. We found that this region has a very high incidence of pediatric melioidosis, and that children frequently presented with disseminated disease and had high fatality rates, despite the absence of any predisposing risk factor. We confirmed that these infections were caused predominantly by gentamicin-susceptible B. pseudomallei strains. We also highlighted other undescribed epidemiological, clinical and microbiological features, which may help in the overall understanding of B. pseudomallei infections. We emphasized the importance of improving the awareness and recognition of melioidosis in children, both in Sarawak and in other endemic regions.