Mun Yik Fong

Localization of Dengue Virus in Naturally Infected Human Tissues, by Immunohistochemistry and In Situ Hybridization

Dengue viral antigens have been demonstrated in several types of naturally infected human tissues, but little is known of whether these same tissues have detectable viral RNA. We studied tissue specimens from patients with serologically or virologically confirmed dengue infections by immunohistochemistry (IHC) and in situ hybridization (ISH), to localize viral antigen and RNA, respectively. IHC was performed on specimens obtained from 5 autopsies and 24 biopsies and on 20 blood-clot samples. For ISH, antisense riboprobes to the dengue E gene were applied to tissue specimens in which IHC was positive. Viral antigens were demonstrated in Kupffer and sinusoidal endothelial cells of the liver; macrophages, multinucleated cells, and reactive lymphoid cells in the spleen; macrophages and vascular endothelium in the lung; kidney tubules; and monocytes and lymphocytes in blood-clot samples. Positive-strand viral RNA was detected in the same IHC-positive cells found in the spleen and blood-clot samples. The strong, positive ISH signal in these cells indicated a high copy number of viral RNA, suggesting replication.

High proportion of knowlesi malaria in recent malaria cases in Malaysia

BackgroundPlasmodium knowlesi is a simian parasite that has been recognized as the fifth species causing human malaria. Naturally-acquired P. knowlesi infection is widespread among human populations in Southeast Asia. The aim of this epidemiological study was to determine the incidence and distribution of malaria parasites, with a particular focus on human P. knowlesi infection in Malaysia.MethodsA total of 457 microscopically confirmed, malaria-positive blood samples were collected from 22 state and main district hospitals in Malaysia between September 2012 and December 2013. Nested PCR assay targeting the 18S rRNA gene was used to determine the infecting Plasmodium species.ResultsA total of 453 samples were positive for Plasmodium species by using nested PCR assay. Plasmodium knowlesi was identified in 256 (56.5%) samples, followed by 133 (29.4%) cases of Plasmodium vivax, 49 (10.8%) cases of Plasmodium falciparum, two (0.4%) cases of Plasmodium ovale and one (0.2%) case of Plasmodium malariae. Twelve mixed infections were detected, including P. knowlesi/P. vivax (n = 10), P. knowlesi/P. falciparum (n = 1), and P. falciparum/P. vivax (n = 1). Notably, P. knowlesi (Included mixed infections involving P. knowlesi (P. knowlesi/P. vivax and P. knowlesi /P. falciparum)) showed the highest proportion in Sabah (84/115 cases, prevalence of 73.0%), Sarawak (83/120, 69.2%), Kelantan (42/56, 75.0%), Pahang (24/25, 96.0%), Johor (7/9, 77.8%), and Terengganu (4/5, 80.0%,). In contrast, the rates of P. knowlesi infection in Selangor and Negeri Sembilan were found to be 16.2% (18/111 cases) and 50.0% (5/10 cases), respectively. Sample of P. knowlesi was not obtained from Kuala Lumpur, Melaka, Perak, Pulau Pinang, and Perlis during the study period, while a microscopically-positive sample from Kedah was negative by PCR.ConclusionIn addition to Sabah and Sarawak, which have been known for high prevalence of P. knowlesi infection, the findings from this study highlight the widespread distribution of P. knowlesi in many Peninsular Malaysia states.

Toxoplasmosis: prevalence and risk factors

A total of 200 pregnant women were recruited in this cross-sectional study. The overall seroprevalence of toxoplasmosis in pregnant women was found to be 49%, in which 39%, 4% and 6% for anti-Toxoplasma IgG, IgM and both anti-Toxoplasma IgG and IgM antibodies, respectively. We found the differences in Toxoplasma seroprevalence rates among the races were significant: the highest rate was in the Malays (55.7%), followed by the Indian (55.3%) and the Chinese (19.4%) (P < 0.05) populations. An increase in Toxoplasma seroprevalence with increasing parity was detected (P < 0.05). Women with no children had a prevalence of 39.7%, while women with one or more than two children had a prevalence of 44.2% and 62.9%, respectively. In this study, there was no significant association between Toxoplasma seroprevalence and various possible risk factors in pregnant women (P > 0.05). When multivariate analysis was performed, no significant association between Toxoplasma seroprevalence and history of contact with cats, consumption of undercooked meat and blood transfusion was found (P > 0.05). We did not find any newly diagnosed cases of acute acquired toxoplasmosis in pregnancy during the study period.

Specific, sensitive and rapid detection of human plasmodium knowlesi infection by loop-mediated isothermal amplification (LAMP) in blood samples

BackgroundThe emergence of Plasmodium knowlesi in humans, which is in many cases misdiagnosed by microscopy as Plasmodium malariae due to the morphological similarity has contributed to the needs of detection and differentiation of malaria parasites. At present, nested PCR targeted on Plasmodium ssrRNA genes has been described as the most sensitive and specific method for Plasmodium detection. However, this method is costly and requires trained personnel for its implementation. Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method was developed for the clinical detection of P. knowlesi. The sensitivity and specificity of LAMP was evaluated in comparison to the results obtained via microscopic examination and nested PCR.MethodsLAMP assay was developed based on P. knowlesi genetic material targeting the apical membrane antigen-1 (AMA-1) gene. The method uses six primers that recognize eight regions of the target DNA and it amplifies DNA within an hour under isothermal conditions (65°C) in a water-bath.ResultsLAMP is highly sensitive with the detection limit as low as ten copies for AMA-1. LAMP detected malaria parasites in all confirm cases (n = 13) of P. knowlesi infection (sensitivity, 100%) and none of the negative samples (specificity, 100%) within an hour. LAMP demonstrated higher sensitivity compared to nested PCR by successfully detecting a sample with very low parasitaemia (< 0.01%).ConclusionWith continuous efforts in the optimization of this assay, LAMP may provide a simple and reliable test for detecting P. knowlesi malaria parasites in areas where malaria is prevalent.

Comparative phylogeography reveals a shared impact of pleistocene environmental change in shaping genetic diversity within nine Anopheles mosquito species across the Indo-Burma biodiversity hotspot

South‐East Asia is one of the world’s richest regions in terms of biodiversity. An understanding of the distribution of diversity and the factors shaping it is lacking, yet essential for identifying conservation priorities for the region’s highly threatened biodiversity. Here, we take a large‐scale comparative approach, combining data from nine forest‐associated Anopheles mosquito species and using statistical phylogeographical methods to disentangle the effects of environmental history, species‐specific ecology and random coalescent effects. Spatially explicit modelling of Pleistocene demographic history supports a common influence of environmental events in shaping the genetic diversity of all species examined, despite differences in species’ mtDNA gene trees. Populations were periodically restricted to allopatric northeastern and northwestern refugia, most likely due to Pleistocene forest fragmentation. Subsequent southwards post‐glacial recolonization is supported by a north–south gradient of decreasing genetic diversity. Repeated allopatric fragmentation and recolonization have led to the formation of deeply divergent geographical lineages within four species and a suture zone where these intraspecific lineages meet along the Thai–Myanmar border. A common environmental influence for this divergence was further indicated by strong support for simultaneous divergence within the same four species, dating to approximately 900 thousand years ago (kya). Differences in the geographical structuring of genetic diversity between species are probably the result of varying species’ biology. The findings have important implications for conservation planning; if the refugial regions and suture zone identified here are shared by other forest taxa, the unique and high levels of genetic diversity they house will make these areas conservation priorities.

Entomologic investigation of Plasmodium knowlesi vectors in Kuala Lipis, Pahang, Malaysia

BackgroundThe first natural infection of Plasmodium knowlesi in humans was recorded in 1965 in peninsular Malaysia. Extensive research was then conducted and it was postulated that it was a rare incident and that simian malaria will not be easily transmitted to humans. However, at the turn of the 21st century, knowlesi malaria was prevalent throughout Southeast Asia and is life threatening. Thus, a longitudinal study was initiated to determine the vectors, their seasonal variation and preference to humans and macaques.MethodsMonthly mosquito collections were carried out in Kuala Lipis, Pahang, peninsular Malaysia, using human-landing collection and monkey-baited traps at ground and canopy levels. All mosquitoes were identified and all anopheline mosquitoes were dissected and the gut and gland examined for oocysts and sporozoites. Nested polymerase chain reaction (PCR) was conducted on positive samples, followed by sequencing of the csp gene.Results and discussionAnopheles cracens was the predominant mosquito biting humans as well as the macaques. It comprised 63.2% of the total collection and was the only species positive for sporozoites of P. knowlesi. It was exophagic and did not enter houses. Besides An. cracens, Anopheles kochi was also found in the monkey-bait trap. Both species preferred to bite monkeys at ground level compared to canopy.ConclusionAnopheles cracens, which belongs to the Dirus complex, Leucosphyrus subgroup, Leucosphyrus group of mosquitoes, has been confirmed to be the only vector for this site from Pahang during this study. It was the predominant mosquito at the study sites and with deforestation humans and villages are entering deeper in the forests, and nearer to the mosquitoes and macacques. The close association of humans with macaques and mosquitoes has led to zoonotic transmission of malaria.

Sarcocystis nesbitti Infection in Human Skeletal Muscle: Possible Transmission from Snakes

Sarcocystis nesbitti is an intracellular protozoan parasite found as sarcocysts within muscle fibers of intermediate hosts (monkey and baboon). The definitive host is suspected to be the snake. We report two cases from a larger cohort of 89 patients who had fever, headache, and generalized myalgia after a trip to Pangkor Island, Malaysia. Sarcocysts were detected in skeletal muscle biopsy specimens by light and electron microscopy from these two patients. DNA sequencing based on the 18S ribosomal DNA region identified the Sarcocystis species as S. nesbitti. We also identified S. nesbitti sequences in the stools of a snake (Naja naja). Phylogenetic analysis showed that these sequences form a cluster with most of the other known Sarcocystis species for which the snake is a definitive host. We believe these two patients were likely to have symptomatic acute muscular sarcocystosis after S. nesbitti infection that may have originated from snakes.

Acute respiratory distress syndrome and acute renal failure from Plasmodium ovale infection with fatal outcome

BackgroundPlasmodium ovale is one of the causative agents of human malaria. Plasmodium ovale infection has long been thought to be non-fatal. Due to its lower morbidity, P. ovale receives little attention in malaria research.MethodsTwo Malaysians went to Nigeria for two weeks. After returning to Malaysia, they fell sick and were admitted to different hospitals. Plasmodium ovale parasites were identified from blood smears of these patients. The species identification was further confirmed with nested PCR. One of them was successfully treated with no incident of relapse within 12-month medical follow-up. The other patient came down with malaria-induced respiratory complication during the course of treatment. Although parasites were cleared off the circulation, the patient’s condition worsened. He succumbed to multiple complications including acute respiratory distress syndrome and acute renal failure.ResultsSequencing of the malaria parasite DNA from both cases, followed by multiple sequence alignment and phylogenetic tree construction suggested that the causative agent for both malaria cases was P. ovale curtisi.DiscussionIn this report, the differences between both cases were discussed, and the potential capability of P. ovale in causing severe complications and death as seen in this case report was highlighted.ConclusionPlasmodium ovale is potentially capable of causing severe complications, if not death. Complete travel and clinical history of malaria patient are vital for successful diagnoses and treatment. Monitoring of respiratory and renal function of malaria patients, regardless of the species of malaria parasites involved is crucial during the course of hospital admission.

Multicentre evaluation of dengue IgM dot enzyme immunoassay

BACKGROUND The traditional methods used in the diagnosis of dengue infection do not lend themselves to field application. As such, clinical specimens have to be sent to a central laboratory for processing which invariably leads to delay. This affects patient management and disease control. The development of the dengue IgM dot enzyme immunoassay has opened up the possibility of carrying out the test in peripheral health settings. OBJECTIVES This multicentre study was conducted to evaluate a new, commercial nitrocellulose membrane based IgM capture enzyme immunoassay. STUDY DESIGN The sensitivity and specificity of the test were compared with in-house dengue IgM enzyme-linked immunoassays routinely performed by each of the selected centres. Known positive and negative dengue specimens, as well as specimens from non-dengue cases, were included in the evaluation. RESULTS Based on 402 specimens tested by the six centres, the sensitivity was 92.1% and specificity 88.1%, with an overall agreement of 92.8% when compared with IgM EIA assays performed on microplates. CONCLUSIONS The results suggest that this commercial kit has a role to play in the diagnosis of dengue infection, especially in peripheral health settings.

Plasmodium knowlesi Reinfection in Human

To the Editor: In 2004, a large number of patients infected with Plasmodium knowlesi (simian malarial species) were reported in Sarawak, Malaysia (1). P. knowlesi infection was also reported in Peninsular Malaysia (2). Here we report a case of human P. knowlesi reinfection. Phylogenetic sequence analysis shows that the first and second infections were caused by different strains of P. knowlesi.