Improved limit of detection for zoonotic Plasmodium knowlesi and P. cynomolgi surveillance using reverse transcription for total nucleic acid preserved samples or dried blood spots.

Background: Zoonotic P. knowlesi and P. cynomolgi symptomatic and asymptomatic infections occur across endemic areas of Southeast Asia. Most infections are low-parasitemia, with an unknown proportion below routine microscopy detection thresholds. Molecular surveillance tools optimizing the limit of detection (LOD) would allow more accurate estimates of zoonotic malaria prevalence. Methods: An established ultra-sensitive Plasmodium genus quantitative-PCR (qPCR) assay targeting the 18S rRNA gene underwent LOD evaluation with and without reverse transcription (RT) for P. knowlesi, P. cynomolgi and P. vivax using total nucleic acid preserved (DNA/RNA Shield™) isolates and archived dried blood spots (DBS). LODs for selected P. knowlesi-specific assays, and reference P. vivax- and P. cynomolgi-specific assays were determined with RT. Assay specificities were assessed using clinical malaria samples and malaria-negative controls. Results: The use of reverse transcription improved Plasmodium species detection by up to 10,000-fold (Plasmodium genus), 2759-fold (P. knowlesi), 1000-fold (P. vivax) and 10-fold (P. cynomolgi). The median LOD with RT for the Kamau et al. Plasmodium genus RT-qPCR assay was ≤0.0002 parasites/µL for P. knowlesi and 0.002 parasites/µL for both P. cynomolgi and P. vivax. The LODs with RT for P. knowlesi-specific PCRs were: Imwong et al. 18S rRNA (0.0007 parasites/µL); Divis et al. real-time 18S rRNA (0.0002 parasites/µL); Lubis et al. hemi-nested SICAvar (1.1 parasites/µL) and Lee et al. nested 18S rRNA (11 parasites/µL). The LOD for P. vivax- and P. cynomolgi-specific assays with RT were 0.02 and 0.20 parasites/µL respectively. For DBS P. knowlesi samples the median LOD for the Plasmodium genus qPCR with RT was 0.08, and without RT was 19.89 parasites/uL (249-fold change); no LOD improvement was demonstrated in DBS archived beyond 6 years. The Plasmodium genus and P. knowlesi-assays were 100% specific for Plasmodium species and P. knowlesi detection, respectively, from 190 clinical infections and 48 healthy controls. Reference P. vivax-specific primers demonstrated known cross-reactivity with P. cynomolgi. Conclusion: Our findings support the use of an 18S rRNA Plasmodium genus qPCR and species-specific nested PCR protocol with RT for highly-sensitive surveillance of zoonotic and human Plasmodium species infections.

No Association between the Plasmodium vivax crt-o MS334 or In9pvcrt Polymorphisms and Chloroquine Failure in a Pre-Elimination Clinical Cohort from Malaysia with a Large Clonal Expansion.

Increasing reports of resistance to a frontline malaria blood-stage treatment, chloroquine (CQ), raises concerns for the elimination of Plasmodium vivax. The absence of an effective molecular marker of CQ resistance in P. vivax greatly constrains surveillance of this emerging threat. A recent genetic cross between CQ sensitive (CQS) and CQ resistant (CQR) NIH-1993 strains of P. vivax linked a moderate CQR phenotype with two candidate markers in P. vivax CQ resistance transporter gene (pvcrt-o): MS334 and In9pvcrt. Longer TGAAGH motif lengths at MS334 were associated with CQ resistance, as were shorter motifs at the In9pvcrt locus. In this study, high-grade CQR clinical isolates of P. vivax from a low endemic setting in Malaysia were used to investigate the association between the MS334 and In9pvcrt variants and treatment efficacy. Among a total of 49 independent monoclonal P. vivax isolates assessed, high-quality MS334 and In9pvcrt sequences could be derived from 30 (61%) and 23 (47%), respectively. Five MS334 and six In9pvcrt alleles were observed, with allele frequencies ranging from 2 to 76% and 3 to 71%, respectively. None of the clinical isolates had the same variant as the NIH-1993 CQR strain, and none of the variants were associated with CQ treatment failure (all P > 0.05). Multi-locus genotypes (MLGs) at 9 neutral microsatellites revealed a predominant P. vivax strain (MLG6) accounting for 52% of Day 0 infections. The MLG6 strain comprised equal proportions of CQS and CQR infections. Our study reveals complexity in the genetic basis of CQ resistance in the Malaysian P. vivax pre-elimination setting and suggests that the proposed pvcrt-o MS334 and In9pvcrt markers are not reliable markers of CQ treatment efficacy in this setting. Further studies are needed in other endemic settings, applying hypothesis-free genome-wide approaches, and functional approaches to understand the biological impact of the TGAAGH repeats linked to CQ response in a cross are warranted to comprehend and track CQR P. vivax.

Knowledge, Attitude and Practices Towards Cryptosporidium Among Public Swimming Pool Patrons and Staff in Western Australia.

PURPOSE: There is a dearth of research conducted on the Knowledge, Attitude and Practices (KAP) of swimming pool patrons and staff to determine their understanding of the importance of Cryptosporidium and its transmission in swimming pools. METHODS: We conducted a KAP survey of public swimming pool patrons (n = 380) and staff (n = 40) attending five public swimming pools in Western Australia (WA). RESULTS: Knowledge, attitudes and practices (KAP) of Cryptosporidium varied between patrons and staff but were generally limited. Only 26.1% and 25.0% of patrons and staff had heard of Cryptosporidium, while 17.4% and 10.0% knew that it causes diarrhoea, respectively. Thirty-one percent of patrons were aware of their pool policy concerning gastroenteritis and Cryptosporidium, compared to 62.5% of staff. Less than 50% of patrons demonstrated awareness of how features within the pool environment were relevant to the control of Cryptosporidium. Only about a third of patrons (35%) and staff (37.5%) were aware that showering before swimming reduced the risk of gastroenteritis. CONCLUSION: Raising awareness about hygiene-related practices through the delivery of targeted health education messages to the general public is essential to reduce the burden of Cryptosporidium infections in aquatic environments.

Molecular analysis of cryptosporidiosis cases in Western Australia in 2019 and 2020 supports the occurrence of two swimming pool associated outbreaks and reveals the emergence of a rare C. hominis IbA12G3 subtype.

Cryptosporidium is an important protozoan parasite and due to its resistance to chlorine is a major cause of swimming pool-associated gastroenteritis outbreaks. The present study combined contact tracing and molecular techniques to analyse cryptosporidiosis cases and outbreaks in Western Australia in 2019 and 2020. In the 2019 outbreak, subtyping at the 60 kDa glycoprotein (gp60) gene identified 89.0% (16/18) of samples were caused by the C. hominis IdA15G1 subtype. Amplicon next generation sequencing (NGS) at the gp60 locus identified five C. hominis IdA15G1 subtype samples that also had C. hominis IdA14 subtype DNA, while multi locus sequence typing (MLST) analysis on a subset (n = 14) of C. hominis samples identified three IdA15G1 samples with a 6 bp insertion at the end of the trinucleotide repeat region of the cp47 gene. In 2020, 88.0% (73/83) of samples typed were caused by the relatively rare C. hominis subtype IbA12G3. Four mixed infections were observed by NGS with three IdA15G1/ IdA14 mixtures and one C. parvum IIaA18G3R1 sample mixed with IIaA16G3R1. No genetic diversity using MLST was detected. Epidemiological and molecular data indicates that the outbreaks in 2019 and 2020 were each potentially from swimming pool point sources and a new C. hominis subtype IbA12G3 is emerging in Australia. The findings of the present study are important for understanding the introduction and transmission of rare Cryptosporidium subtypes to vulnerable populations.

Zoonotic infection by Cryptosporidium fayeri IVgA10G1T1R1 in a Western Australian human.

In the present study, a 37-year-old immunosuppressed female in Western Australia (WA) was identified as positive for Cryptosporidium by microscopy and treated with nitazoxanide. Molecular analyses at the 18S ribosomal RNA (18S) and 60 kDa glycoprotein (gp60) loci identified C. fayeri subtype IVgA10G1T1R1, which had previously been identified in western grey kangaroos (Macropus fuliginosus) in WA. Next generation sequencing (NGS) of the gp60 locus confirmed the absence of mixed infections with other Cryptosporidium species. This is only the second report of C. fayeri in a human host highlighting the zoonotic potential of this wildlife-associated species. Routine diagnosis using molecular methods in laboratories is required to better understand the diversity and epidemiology of Cryptosporidium parasite.

Retrospective analysis of Cryptosporidium species in Western Australian human populations (2015-2018), and emergence of the C. hominis IfA12G1R5 subtype.

Cryptosporidium species are a major cause of diarrhoea worldwide. In the present study, a retrospective analysis of 109 microscopically Cryptosporidium-positive faecal specimens from Western Australian patients, collected between 2015 and 2018 was conducted. Sequence analysis of the 18S rRNA and the 60 kDa glycoprotein (gp60) gene loci identified four Cryptosporidium species: C. hominis (86.2%, 94/109), C. parvum (11.0%, 12/109), C. meleagridis (1.8%, 2/109) and C. viatorum (0.9%, 1/109). Subtyping at the gp60 locus identified a total of 11 subtypes including the emergence of the previously rare C. hominis IfA12G1R5 subtype in 2017 as the dominant subtype (46.7%, 21/45). This subtype has also recently emerged as the dominant subtype in the United States but the reasons for its emergence are unknown. This is also the first report of C. viatorum in humans in Australia and a novel subtype (XVaA3g) was identified in the one positive patient.

Feeding behavior of Mimomyia (Etorleptiomyia) luzonensis (Ludlow, 1905) (Diptera, Culicidae) in Peninsular Malaysia.

Mosquitoes are vectors of various human diseases in the tropics including yellow fever, dengue, malaria and West Nile virus. Mosquitoes can act as vectors between wildlife and humans, which is particularly important for diseases where wild animals serve as reservoirs of parasites in the absence of human infections. Research has mainly focused on the medical impacts of Anopheles, Aedes, Mansonia and Culex, however, very little attention has been directed towards other mosquito genera, especially those which act as vectors of diseases of wildlife. We have observed adults of Mimomyia (Etorleptiomyia) luzonensis (Ludlow, 1905) feeding on a toad, Ingerophrynus parvus, near an oil palm plantation settlement in Setia Alam, Selangor state, Peninsular Malaysia. Mimomyia is known to feed on reptiles and amphibians, and is a documented vector of several arboviruses, including West Nile virus. The observation of Mimomyia feeding on a common toad near a human settlement highlights a need to understand the relationships between mosquitoes, toads and humans from an ecological perspective. We report on-site observations of the feeding habit of Mimomyia; the first records from Malaysia.

Intestinal Myiasis in a Malaysian Patient Caused by Larvae of Clogmia albipunctatus (Diptera: Psychodidae).

We report a case of human intestinal myiasis in a 41-yr-old female patient presented at a clinic in Seri Kembangan, Selangor, Malaysia. Larvae passed out in the patient's feces were sent to the Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. DNA barcoding confirmed the second case of intestinal myiasis in Malaysia involving the larvae of Clogmia albipunctatus (Duckhouse) (Diptera: Psychodidae). We review reported cases of myiasis and discuss the present case of intestinal myiasis in an urban patient.

First report of brown widow spider sightings in Peninsular Malaysia and notes on its global distribution.

BACKGROUND: The brown widow spider (Latrodectus geometricus Koch, 1841) has colonised many parts of the world from its continent of origin, Africa. By at least 1841, the species had successfully established populations in South America and has more recently expanded its range to the southern states of North America. This highly adaptable spider has been far more successful in finding its niche around the world than its famous cousins, the black widow, Latrodectus mactans, found in the south-eastern states of North America, and the red-back, Latrodectus hasselti, found mostly in Australia, New Zealand and Japan. METHODS: We performed an extensive web search of brown widow sightings and mapped the location of each sighting using ArcGIS. Specimens reputedly of the species L. geometricus were collected at three localities in Peninsular Malaysia. The spiders were identified and documented based on an examination of morphological characteristics and DNA barcoding. RESULTS: The spiders found in Peninsular Malaysia were confirmed to be Latrodectus geometricus based on their morphological characteristics and DNA barcodes. We recorded 354 sightings of the brown widow in 58 countries, including Peninsular Malaysia. CONCLUSION: Reports from the Americas and the Far East suggest a global-wide invasion of the brown widow spider. Herein we report the arrival of the brown widow spider in Peninsular Malaysia and provide notes on the identification of the species and its recently expanded range.

Ectoparasites of murids in peninsular Malaysia and their associated diseases.

A considerable number of rat-borne ectoparasite studies have been conducted since the early 1930s in the Malayan Peninsula (now known as peninsular Malaysia). The majority of studies were field surveys and collections of specimens across the region, and were conducted primarily to catalogue the ectoparasite host distribution and discover novel species. This has generated a signification amount of information, particularly on the diversity and host distribution; other aspects such as morphology, host distribution and medical significance have also been investigated. Amongst the four main groups (mites, fleas, ticks, lice), rat-borne mites have received the most attention with a particular emphasis on chiggers, due to their medical importance. More recent studies have examined the distribution of ectoparasites in rats from different habitat type simplicating a high prevalence of zoonotic species infesting rat populations. Despite being capable of transmitting dangerous pathogens to human, the health risks of rat-borne ectoparasites appear to be small with no serious outbreaks of diseases recorded. Although an extensive number of works have been published, there remain gaps in knowledge that need to be addressed, such as, the distribution of under studied ectoparasite groups (listrophorids and myobiids), determining factors influencing infestation, and understanding changes to the population distribution over time.

First report of brown widow spider sightings in Peninsular Malaysia and notes on its global distribution

Background:The brown widow spider (Latrodectus geometricus Koch, 1841) has colonised many parts of the world from its continent of origin, Africa. By at least 1841, the species had successfully established populations in South America and has more recently expanded its range to the southern states of North America. This highly adaptable spider has been far more successful in finding its niche around the world than its famous cousins, the black widow, Latrodectus mactans, found in the south-eastern states of North America, and the red-back, Latrodectus hasselti, found mostly in Australia, New Zealand and Japan.Methods:We performed an extensive web search of brown widow sightings and mapped the location of each sighting using ArcGIS. Specimens reputedly of the species L. geometricus were collected at three localities in Peninsular Malaysia. The spiders were identified and documented based on an examination of morphological characteristics and DNA barcoding.Results:The spiders found in Peninsular Malaysia were confirmed to be Latrodectus geometricus based on their morphological characteristics and DNA barcodes. We recorded 354 sightings of the brown widow in 58 countries, including Peninsular Malaysia.Conclusion:Reports from the Americas and the Far East suggest a global-wide invasion of the brown widow spider. Herein we report the arrival of the brown widow spider in Peninsular Malaysia and provide notes on the identification of the species and its recently expanded range.(AU)

First report of brown widow spider sightings in Peninsular Malaysia and notes on its global distribution

Background: The brown widow spider (Latrodectus geometricus Koch, 1841) has colonised many parts of the world from its continent of origin, Africa. By at least 1841, the species had successfully established populations in South America and has more recently expanded its range to the southern states of North America. This highly adaptable spider has been far more successful in finding its niche around the world than its famous cousins, the black widow, Latrodectus mactans, found in the south-eastern states of North America, and the red-back, Latrodectus hasselti, found mostly in Australia, New Zealand and Japan. Methods: We performed an extensive web search of brown widow sightings and mapped the location of each sighting using ArcGIS. Specimens reputedly of the species L. geometricus were collected at three localities in Peninsular Malaysia. The spiders were identified and documented based on an examination of morphological characteristics and DNA barcoding. Results: The spiders found in Peninsular Malaysia were confirmed to be Latrodectus geometricus based on their morphological characteristics and DNA barcodes. We recorded 354 sightings of the brown widow in 58 countries, including Peninsular Malaysia. Conclusion: Reports from the Americas and the Far East suggest a global-wide invasion of the brown widow spider. Herein we report the arrival of the brown widow spider in Peninsular Malaysia and provide notes on the identification of the species and its recently expanded range.

Phylogenetic study of six species of Anopheles mosquitoes in Peninsular Malaysia based on inter-transcribed spacer region 2 (ITS2) of ribosomal DNA.

BACKGROUND: Molecular techniques are invaluable for investigation on the biodiversity of Anopheles mosquitoes. This study aimed at investigating the spatial-genetic variations among Anopheles mosquitoes from different areas of Peninsular Malaysia, as well as deciphering evolutionary relationships of the local Anopheles mosquitoes with the mosquitoes from neighbouring countries using the anopheline ITS2 rDNA gene. METHODS: Mosquitoes were collected, identified, dissected to check infection status, and DNA extraction was performed for PCR with primers targeting the ITS2 rDNA region. Sequencing was done and phylogenetic tree was constructed to study the evolutionary relationship among Anopheles mosquitoes within Peninsular Malaysia, as well as across the Asian region. RESULTS: A total of 133 Anopheles mosquitoes consisting of six different species were collected from eight different locations across Peninsular Malaysia. Of these, 65 ITS2 rDNA sequences were obtained. The ITS2 rDNA amplicons of the studied species were of different sizes. One collected species, Anopheles sinensis, shows two distinct pools of population in Peninsular Malaysia, suggesting evolvement of geographic race or allopatric speciation. CONCLUSION: Anopheles mosquitoes from Peninsular Malaysia show close evolutionary relationship with the Asian anophelines. Nevertheless, genetic differences due to geographical segregation can be seen. Meanwhile, some Anopheles mosquitoes in Peninsular Malaysia show vicariance, exemplified by the emergence of distinct cluster of An. sinensis population.

Is there a risk of suburban transmission of malaria in Selangor, Malaysia?

BACKGROUND: The suburban transmission of malaria in Selangor, Malaysia's most developed and populous state still remains a concern for public health in this region. Despite much successful control efforts directed at its reduction, sporadic cases, mostly brought in by foreigners have continued to occur. In addition, cases of simian malaria caused by Plasmodium knowlesi, some with fatal outcome have caused grave concern to health workers. The aim of this study was to investigate the possibility of local malaria transmission in suburban regions of Selangor, which are adjacent to secondary rainforests. FINDINGS: A malaria survey spanning 7 years (2006 - 2012) was conducted in Selangor. A total of 1623 laboratory confirmed malaria cases were reported from Selangor's nine districts. While 72.6% of these cases (1178/1623) were attributed to imported malaria (cases originating from other countries), 25.5% (414/1623) were local cases and 1.9% (31/1623) were considered as relapse and unclassified cases combined. In this study, the most prevalent infection was P. vivax (1239 cases, prevalence 76.3%) followed by P. falciparum (211, 13.0%), P. knowlesi (75, 4.6%), P. malariae (71, 4.4%) and P. ovale (1, 0.06%). Mixed infections comprising of P. vivax and P. falciparum were confirmed (26, 1.6%). Entomological surveys targeting the residences of malaria patients' showed that the most commonly trapped Anopheles species was An. maculatus. No oocysts or sporozoites were found in the An. maculatus collected. Nevertheless, the possibility of An. maculatus being the malaria vector in the investigated locations was high due to its persistent occurrence in these areas. CONCLUSIONS: Malaria cases reported in this study were mostly imported cases. However the co-existence of local cases and potential Plasmodium spp. vectors should be cause for concern. The results of this survey reflect the need of maintaining closely monitored malaria control programs and continuous extensive malaria surveillance in Peninsula Malaysia.