Functional characterization of helminth-associated Clostridiales reveals covariates of Treg differentiation.

BACKGROUND: Parasitic helminths influence the composition of the gut microbiome. However, the microbiomes of individuals living in helminth-endemic regions are understudied. The Orang Asli, an indigenous population in Malaysia with high burdens of the helminth Trichuris trichiura, display microbiotas enriched in Clostridiales, an order of spore-forming obligate anaerobes with immunogenic properties. We previously isolated novel Clostridiales that were enriched in these individuals and found that a subset promoted the Trichuris life cycle. In this study, we aimed to further characterize the functional properties of these bacteria. RESULTS: Clostridiales isolates were profiled for their ability to perform 57 enzymatic reactions and produce short-chain fatty acids (SCFAs) and hydrogen sulfide, revealing that these bacteria were capable of a range of activities associated with metabolism and host response. Consistent with this finding, monocolonization of mice with individual isolates identified bacteria that were potent inducers of regulatory T-cell (Treg) differentiation in the colon. Comparisons between variables revealed by these studies identified enzymatic properties correlated with Treg induction and Trichuris egg hatching. CONCLUSION: We identified Clostridiales species that are sufficient to induce high levels of Tregs. We also identified a set of metabolic activities linked with Treg differentiation and Trichuris egg hatching mediated by these newly isolated bacteria. Altogether, this study provides functional insights into the microbiotas of individuals residing in a helminth-endemic region. Video Abstract.

Applying an evolutionary mismatch framework to understand disease susceptibility.

Noncommunicable diseases (NCDs) are on the rise worldwide. Obesity, cardiovascular disease, and type 2 diabetes are among a long list of "lifestyle" diseases that were rare throughout human history but are now common. The evolutionary mismatch hypothesis posits that humans evolved in environments that radically differ from those we currently experience; consequently, traits that were once advantageous may now be "mismatched" and disease causing. At the genetic level, this hypothesis predicts that loci with a history of selection will exhibit "genotype by environment" (GxE) interactions, with different health effects in "ancestral" versus "modern" environments. To identify such loci, we advocate for combining genomic tools in partnership with subsistence-level groups experiencing rapid lifestyle change. In these populations, comparisons of individuals falling on opposite extremes of the "matched" to "mismatched" spectrum are uniquely possible. More broadly, the work we propose will inform our understanding of environmental and genetic risk factors for NCDs across diverse ancestries and cultures.

Functional characterization of helminth-associated Clostridiales reveals covariates of Treg differentiation.

Parasitic helminths influence the composition of the gut microbiome. However, the microbiomes of individuals living in helminth-endemic regions are understudied. The Orang Asli, an indigenous population in Malaysia with high burdens of the helminth Trichuris trichiura, displayed microbiotas enriched in Clostridiales, an order of spore-forming obligate anaerobes previously shown to have immunogenic properties. We previously isolated novel Clostridiales that were enriched in these individuals and found that a subset promoted the Trichuris life cycle. Here, we further characterized the functional properties of these bacteria. Enzymatic and metabolomic profiling revealed a range of activities associated with metabolism and host response. Consistent with this finding, monocolonization of mice with individual isolates identified bacteria that were potent inducers of regulatory T cell (Treg) differentiation in the colon. Comparisons between variables revealed by these studies identified enzymatic properties correlated with Treg induction and Trichuris egg hatching. These results provide functional insights into the microbiotas of an understudied population.

Psychometric evaluation of Oral Mucositis Daily Questionnaire: A cross-cultural adaptation of the Malay version in multiethnic adult autologous stem cell transplant.

Objective: Mucositis is one of the most feared side effects of cancer treatment. Psychometric analysis of a patient self-assessment score, the oral mucositis daily questionnaire in Malay (OMDQ-Mal) and its construct validity by means of confirmatory factor analysis (CFA) is lacking. This research aimed to test the validity and reliability of OMDQ-Mal. Methods: A total of 114 autologous stem-cell transplantation patients aged ≥ 18 years old at a national hematology center in Malaysia from April 2019 to December 2020 completed OMDQ-Mal concurrently with physician scores. Internal consistency and reproducibility were determined by Cronbach alpha and intraclass correlation coefficient, respectively. Correlations with physician scores were determined by Spearman correlation. Discriminative validity and construct validity were determined by Mann-Whitney U and CFA, respectively. Results: OMDQ-Mal demonstrated high internal consistency (α â€‹= â€‹0.874). Test-retest reliability between paired days were moderate to excellent (95% CI â€‹= â€‹0.676-0.953). Items in OMDQ-Mal had moderate to strong correlations with physician scores (ρ â€‹= â€‹0.503-0.721). Discriminative validity indicated that the scores of scales were significantly different between participants with severe and mild conditions. Construct validity results of loading factors 0.708-0.952; composite reliability 0.879-0.974; average variant extracted 0.710-0.841; and heterotrait-monotrait ratio 0.528 established the convergent and divergent validity. Conclusions: In conclusion, the OMDQ-Mal, which captured important quality of life responses, demonstrated adequate validity and reliability. This was supported by a two-component model CFA. The strong correlation of OMDQ-Mal with both physician scores indicated its potential as a comprehensive patient-reported outcome measure of mucositis of the entire alimentary tract.

Evolutionary mismatch and the role of GxE interactions in human disease.

Globally, we are witnessing the rise of complex, non-communicable diseases (NCDs) related to changes in our daily environments. Obesity, asthma, cardiovascular disease, and type 2 diabetes are part of a long list of "lifestyle" diseases that were rare throughout human history but are now common. A key idea from anthropology and evolutionary biology-the evolutionary mismatch hypothesis-seeks to explain this phenomenon. It posits that humans evolved in environments that radically differ from the ones experienced by most people today, and thus traits that were advantageous in past environments may now be "mismatched" and disease-causing. This hypothesis is, at its core, a genetic one: it predicts that loci with a history of selection will exhibit "genotype by environment" (GxE) interactions and have differential health effects in ancestral versus modern environments. Here, we discuss how this concept could be leveraged to uncover the genetic architecture of NCDs in a principled way. Specifically, we advocate for partnering with small-scale, subsistence-level groups that are currently transitioning from environments that are arguably more "matched" with their recent evolutionary history to those that are more "mismatched". These populations provide diverse genetic backgrounds as well as the needed levels and types of environmental variation necessary for mapping GxE interactions in an explicit mismatch framework. Such work would make important contributions to our understanding of environmental and genetic risk factors for NCDs across diverse ancestries and sociocultural contexts.

Clostridia isolated from helminth-colonized humans promote the life cycle of Trichuris species.

Soil-transmitted intestinal worms known as helminths colonize over 1.5 billion people worldwide. Although helminth colonization has been associated with altered composition of the gut microbiota, such as increases in Clostridia, individual species have not been isolated and characterized. Here, we isolate and sequence the genome of 13 Clostridia from the Orang Asli, an indigenous population in Malaysia with a high prevalence of helminth infections. Metagenomic analysis of 650 fecal samples from urban and rural Malaysians confirm the prevalence of species corresponding to these isolates and reveal a specific association between Peptostreptococcaceae family members and helminth colonization. Remarkably, Peptostreptococcaceae isolated from the Orang Asli display superior capacity to promote the life cycle of whipworm species, including hatching of eggs from Trichuris muris and Trichuris trichiura. These findings support a model in which helminths select for gut colonization of microbes that support their life cycle.

Orang Asli Health and Lifeways Project (OA HeLP): a cross-sectional cohort study protocol.

INTRODUCTION: Non-communicable disease (NCD) risk is influenced by environmental factors that are highly variable worldwide, yet prior research has focused mainly on high-income countries where most people are exposed to relatively homogeneous and static environments. Understanding the scope and complexity of environmental influences on NCD risk around the globe requires more data from people living in diverse and changing environments. Our project will investigate the prevalence and environmental causes of NCDs among the indigenous peoples of Peninsular Malaysia, known collectively as the Orang Asli, who are currently undergoing varying degrees of lifestyle and sociocultural changes that are predicted to increase vulnerability to NCDs, particularly metabolic disorders and musculoskeletal degenerative diseases. METHODS AND ANALYSIS: Biospecimen sampling and screening for a suite of NCDs (eg, cardiovascular disease, type II diabetes, osteoarthritis and osteoporosis), combined with detailed ethnographic work to assess key lifestyle and sociocultural variables (eg, diet, physical activity and wealth), will take place in Orang Asli communities spanning a gradient from remote, traditional villages to acculturated, market-integrated urban areas. Analyses will first test for relationships between environmental variables, NCD risk factors and NCD occurrence to investigate how environmental changes are affecting NCD susceptibility among the Orang Asli. Second, we will examine potential molecular and physiological mechanisms (eg, epigenetics and systemic inflammation) that mediate environmental effects on health. Third, we will identify intrinsic (eg, age and sex) and extrinsic (eg, early-life experiences) factors that predispose certain people to NCDs in the face of environmental change to better understand which Orang Asli are at greatest risk of NCDs. ETHICS AND DISSEMINATION: Approval was obtained from multiple ethical review boards including the Malaysian Ministry of Health. This study follows established principles for ethical biomedical research among vulnerable indigenous communities, including fostering collaboration, building cultural competency, enhancing transparency, supporting capacity building and disseminating research findings.

Host immune response against DENV and ZIKV infections.

Dengue is a major public health concern, affecting almost 400 million people worldwide, with about 70% of the global burden of disease in Asia. Despite revised clinical classifications of dengue infections by the World Health Organization, the wide spectrum of the manifestations of dengue illness continues to pose challenges in diagnosis and patient management for clinicians. When the Zika epidemic spread through the American continent and then later to Africa and Asia in 2015, researchers compared the characteristics of the Zika infection to Dengue, considering both these viruses were transmitted primarily through the same vector, the Aedes aegypti female mosquitoes. An important difference to note, however, was that the Zika epidemic diffused in a shorter time span compared to the persisting feature of Dengue infections, which is endemic in many Asian countries. As the pathogenesis of viral illnesses is affected by host immune responses, various immune modulators have been proposed as biomarkers to predict the risk of the disease progression to a severe form, at a much earlier stage of the illness. However, the findings for most biomarkers are highly discrepant between studies. Meanwhile, the cross-reactivity of CD8+ and CD4+ T cells response to Dengue and Zika viruses provide important clues for further development of potential treatments. This review discusses similarities between Dengue and Zika infections, comparing their disease transmissions and vectors involved, and both the innate and adaptive immune responses in these infections. Consideration of the genetic identity of both the Dengue and Zika flaviviruses as well as the cross-reactivity of relevant T cells along with the actions of CD4+ cytotoxic cells in these infections are also presented. Finally, a summary of the immune biomarkers that have been reported for dengue and Zika viral infections are discussed which may be useful indicators for future anti-viral targets or predictors for disease severity. Together, this information appraises the current understanding of both Zika and Dengue infections, providing insights for future vaccine design approaches against both viruses.

Diversity and natural selection of Merozoite surface Protein-1 in three species of human malaria parasites: Contribution from South-East Asian isolates.

The present study aimed to examine the genetic diversity of human malaria parasites (i.e., P. falciparum, P. vivax and P. knowlesi) in Malaysia and southern Thailand targeting the 19-kDa C-terminal region of Merozoite Surface Protein-1 (MSP-119). This region is essential for the recognition and invasion of erythrocytes and it is considered one of the leading candidates for asexual blood stage vaccines. However, the genetic data of MSP-119 among human malaria parasites in Malaysia is limited and there is also a need to update the current sequence diversity of this gene region among the Thailand isolates. In this study, genomic DNA was extracted from 384 microscopy-positive blood samples collected from patients who attended the hospitals or clinics in Malaysia and malaria clinics in Thailand from the year 2008 to 2016. The MSP-119 was amplified using PCR followed by bidirectional sequencing. DNA sequences identified in the present study were subjected to Median-joining network analysis with sequences of MSP-119 obtained from GenBank. DNA sequence analysis revealed that PfMSP-119 of Malaysian and Thailand isolates was not genetically conserved as high number of haplotypes were detected and positive selection was prevalent in PfMSP-119, hence questioning its suitability to be used as a vaccine candidate. A novel haplotype (Q/TNG/L) was also detected in Thailand P. falciparum isolate. In contrast, PvMSP-119 was highly conserved, however for the first time, a non-synonymous substitution (A1657S) was reported among Malaysian isolates. As for PkMSP-119, the presence of purifying selection and low nucleotide diversity indicated that it might be a potential vaccine target for P. knowlesi.

Correction: Helminth Colonization Is Associated with Increased Diversity of the Gut Microbiota.

[This corrects the article DOI: 10.1371/journal.pntd.0002880.].

Characterization of benzimidazole resistance in Haemonchus contortus: integration of phenotypic, genotypic and proteomic approaches.

The field strain of Haemonchus contortus has a long history of anthelmintic resistance. To understand this phenomenon, the benzimidazole resistance profile was characterized from the Malaysian field-resistant strain by integrating phenotypic, genotypic and proteomic approaches. The faecal egg count reduction test (FECRT) demonstrated that benzimidazole resistance was at a critical level in the studied strain. The primary resistance mechanism was attributed to F200Y mutation in the isotype 1 ß-tubulin gene as revealed by AS-PCR and direct sequencing. Furthermore, the protein response of the resistant strain towards benzimidazole (i.e., albendazole) treatment was investigated via two-dimensional difference gel electrophoresis (2D-DIGE) and tandem liquid chromatography-mass spectrometry (LC-MS/MS). These investigations illustrated an up-regulation of antioxidant (i.e., ATP-binding region and heat-shock protein 90, superoxide dismutase) and metabolic (i.e., glutamate dehydrogenase) enzymes and down-regulation of glutathione S-transferase, malate dehydrogenase, and other structural and cytoskeletal proteins (i.e., actin, troponin T). Findings from this study are pivotal in updating the current knowledge on anthelmintic resistance and providing new insights into the defence mechanisms of resistant nematodes towards drug treatment.

A holistic approach is needed to control the perpetual burden of soil-transmitted helminth infections among indigenous schoolchildren in Malaysia.

A cross-sectional survey was conducted among 1,142 Orang Ali schoolchildren in six states of Peninsular Malaysia to investigate the current prevalence and risk factors of STH infections. Faecal samples were examined using direct smear, formalin-ether sedimentation, Kato-Katz, and Harada-Mori methods. A pre-tested questionnaire was used to collect information on the demographic, socioeconomic, personal hygiene, and health status of the participants. Overall, 70.1% (95% CI = 67.4, 72.7) of the participants were infected with at least one of the STH species. The prevalence of Ascaris lumbricoides, Trichuris trichiura, and hookworm infections was 63.1%, 61.8% and 11.5%, respectively. Moderate-to-heavy STH infections accounted for 61.3% of the total infections. Univariate and logistic regression analyses revealed different sets of risk factors, with age (> 10 years) being the significant risk factor of all three STH species. Moreover, other species-specific risk factors were identified including being a member of the Senoi tribe, family size (≥ 7 members), school size (150-250 pupils), maternal unemployment, unimproved source of drinking water, lacking improved toilet in the house, inadequate WASH facilities at school, not washing hands before eating, and not washing fruits before eating; presence of domestic animals, and not wearing shoes when outside. The high prevalence of STH infections found in the study population exceeds the WHO policy intervention threshold (20% prevalence). Thus, an innovative holistic approach should be adopted to control STH infections among these children as part of the efforts to improve the quality of life of the entire Orang Asli population. .

Identification of Plasmodium knowlesi Merozoite Surface Protein-119 (PkMSP-119 ) novel binding peptides from a phage display library.

OBJECTIVE: Plasmodium knowlesi, the fifth human malaria parasite, has caused mortality in humans. We aimed to identify P. knowlesi novel binding peptides through a random linear dodecapeptide phage display targeting the 19-kDa fragment of Merozoite Surface Protein-1 protein. METHODS: rPkMSP-119 protein was heterologously expressed using Expresso® Solubility and Expression Screening System and competent E. cloni® 10G cells according to protocol. Three rounds of biopanning were performed on purified rPkMSP-119 to identify binding peptides towards rPkMSP-119 using Ph.D.™-12 random phage display library. Binding sites of the identified peptides to PkMSP-119 were in silico predicted using the CABS-dock web server. RESULTS: Four phage peptide variants that bound to PkMSP-119 were identified after three rounds of biopanning, namely Pkd1, Pkd2, Pkd3 and Pkd4. The sequences of both Pkd1 and Pkd2 consist of a large number of histidine residues. Pkd1 showed positive binding signal with 6.1× vs. BSA control. Docking results showed that Pkd1 and Pkd2 were ideal binding peptides for PkMSP-119 . CONCLUSION: We identified two novel binding peptides of PkMSP-119 , Pkd1 (HFPFHHHKLRAH) and Pkd2 (HPMHMLHKRQHG), through phage display. They provide a valuable starting point for the development of novel therapeutics.

OBJECTIF: Plasmodium knowlesi, le cinquième parasite du paludisme humain, cause la mortalité chez l'homme. Nous avons cherché à identifier les nouveaux peptides de liaison de P. knowlesi par le biais d'une présentation linéaire aléatoire de phages dodécapeptidiques ciblant le fragment de 19 kDa de la protéine-1 de surface du mérozoïte. MÉTHODES: La protéine rPkMSP-119 a été exprimée de façon hétérologue en utilisant le système de criblage de solubilité et d'expression Expresso® et des cellules compétentes E. cloni® 10G conformément au protocole. Trois cycles de biopanning ont été effectués sur rPkMSP-119 purifié pour identifier les peptides de liaison sur rPkMSP-119 en utilisant la banque de présentation aléatoires de phages Ph.D.™-12. Les sites identifiés de liaison des peptides à PkMSP-119 ont été prédits in silico en utilisant le Web serveur CABS-dock. RÉSULTATS: Quatre variantes de peptides phagiques qui se lient à PkMSP-119 ont été identifiées après trois cycles de biopanning, à savoir Pkd1, Pkd2, Pkd3 et Pkd4. Les séquences de Pkd1 et Pkd2 consistent en un grand nombre de résidus histidine. Pkd1 a montré un signal de liaison positif de 6,1 x par rapport au contrôle BSA. Les résultats d'amarrage ont montré que Pkd1 et Pkd2 étaient des peptides de liaison idéaux pour PkMSP-119 . CONCLUSION: Nous avons identifié deux nouveaux peptides de liaison de PkMSP-119 , Pkd1 (HFPFHHHKLRAH) et Pkd2 (HPMHMLHKRQHG), grâce à la présentation de phages. Ils constituent un point de départ précieux pour le développement de nouvelles thérapies.

Linking the effects of helminth infection, diet and the gut microbiota with human whole-blood signatures.

Helminth infection and dietary intake can affect the intestinal microbiota, as well as the immune system. Here we analyzed the relationship between fecal microbiota and blood profiles of indigenous Malaysians, referred to locally as Orang Asli, in comparison to urban participants from the capital city of Malaysia, Kuala Lumpur. We found that helminth infections had a larger effect on gut microbial composition than did dietary intake or blood profiles. Trichuris trichiura infection intensity also had the strongest association with blood transcriptional profiles. By characterizing paired longitudinal samples collected before and after deworming treatment, we determined that changes in serum zinc and iron levels among the Orang Asli were driven by changes in helminth infection status, independent of dietary metal intake. Serum zinc and iron levels were associated with changes in the abundance of several microbial taxa. Hence, there is considerable interplay between helminths, micronutrients and the microbiota on the regulation of immune responses in humans.

Prevalence and risk factors of Strongyloides stercoralis infection among Orang Asli schoolchildren: new insights into the epidemiology, transmission and diagnosis of strongyloidiasis in Malaysia.

This cross-sectional study aimed to determine the prevalence and risk factors of S. stercoralis infection among 1142 Orang Asli primary schoolchildren in six different states of Peninsular Malaysia. Fecal samples were examined using direct smear, formalin-ether sedimentation (FES), agar plate culture (APC) and PCR techniques. Overall, 15.8% of the children were found to be infected with S. stercoralis. The prevalence was 0.2, 1.3, 15.2 and 13.7% by direct smear, FES, APC and PCR, respectively. Multivariate analysis showed that an age of >10 years, being male, belonging to a Proto-Malay tribe, belonging to the Senoi tribe, indiscriminate defecation, using an unimproved water source for drinking water and not wearing shoes when outside were the significant risk factors of infection among these children. In conclusion, we provide new evidence on the occurrence of S. stercoralis in Malaysia to show that there is a relatively high prevalence of infection among Orang Asli schoolchildren. Therefore, the use of specific methods for detecting S. stercoralis should be considered when screening these children for intestinal parasites. Moreover, prevention and control measures specific to S. stercoralis should be integrated into the intestinal parasitic infections control programme in Malaysia.

Molecular Diagnosis of Microsporidia among Immunocompromised Patients in Kuala Lumpur, Malaysia.

Microsporidia are obligate intracellular parasitic fungi causing chronic diarrhea, particularly among immunocompromised patients. The current method used for diagnosis is based on conventional microscopy, which does not differentiate parasites at the species level. The present study was carried out to identify microsporidian species in immunocompromised patients. From March 2016 to March 2017, a total of 289 archived stool samples were examined microscopically for microsporidian spores using Gram-chromotrope Kinyoun (GCK) stain. Positive stool samples by microscopy were subjected to polymerase chain reaction and sequencing for species identification. Based on microscopy examination, the overall prevalence of microsporidian infections was 32.2% (93/289; 95% CI = 27.1-37.8). Of these stool samples, 45 samples were successfully amplified and confirmed as Enterocytozoon bieneusi. No Encephalitozoon intestinalis was detected. Accurate identification of species might help clinicians to decide appropriate management strategies as dissemination risks and treatment response vary for different species, hence improving the management of microsporidian infections.

Understanding Giardia infections among rural communities using the one health approach.

The epidemiology of giardiasis in rural villages in Peninsular Malaysia was examined in the context of the One Health triad that encompasses humans, animals and environment (i.e. river water). A cross-sectional study was carried out among five rural communities in Malaysia to determine the prevalence of Giardia duodenalis in humans, animals and river water. Fecal samples collected from humans and animals were examined by light microscopy. Water was sampled from the rivers adjacent to the target communities and investigated for the occurrence of Giardia cysts. The isolated cysts were further genotyped targeting the glutamate dehydrogenase and triosephosphate isomerase genes. The overall prevalence of G. duodenalis was 6.7% (18/269) and 4.7% (8/169) among humans and animals, respectively. Giardia cysts (mean concentration range: 0.10-5.97 cysts/L) were also found in adjacent rivers at four out of the five villages examined. At Kemensah and Kuala Pangsun, Giardia cysts were isolated from humans [rate: 3.7% each (of 54 each)], animals [rates: 6.3% (of 62) and 11.3% (of 16), respectively] and river water [average concentration of 9 samples each: 0.83±0.81 and 5.97±7.00, respectively]. For both villages at Pos Piah and Paya Lebar, 12.2% (of 98) and 6.1% (of 33) of collected human samples were infected, respectively whilst none of the collected animals samples in these villages were found to be positive. The river water samples of these two villages were also contaminated (average concentration: 0.20±0.35 (of 9) and 0.10±0.19 (of 3), respectively). In conclusion, Giardia cysts were simultaneously observed in the human-animal-environment (i.e., river water) interfaces in at least two of five studied communities highlighting a vital need to improve understanding on the interplay of transmission dynamics, the role of infected humans and animals in contaminating the water sources and the role of water as a vehicle of disease transmission in these communities. Indeed, this study illustrates the One Health approach which is to recognize that the optimal health of humans are interconnected with the well-being of animals and their environment.

Case Report: A Symptomatic Case of Hymenolepis diminuta Infection in an Urban-Dwelling Adult in Malaysia.

A case of Hymenolepis diminuta infection in a 43-year-old Malaysian male with persistent abdominal colicky pain is reported. Endoscopy revealed whitish worms in the lumen of the small intestine, which were identified as H. diminuta after microscopy. Patient was successfully treated with a single dose of praziquantel (25 mg/kg).

Genetic diversity in the C-terminus of merozoite surface protein 1 among Plasmodium knowlesi isolates from Selangor and Sabah Borneo, Malaysia.

Plasmodium knowlesi, a malaria parasite of macaques, has emerged as an important parasite of humans. Despite the significance of P. knowlesi malaria in parts of Southeast Asia, very little is known about the genetic variation in this parasite. Our aim here was to explore sequence variation in a molecule called the 42kDa merozoite surface protein-1 (MSP-1), which is found on the surface of blood stages of Plasmodium spp. and plays a key role in erythrocyte invasion. Several studies of P. falciparum have reported that the C-terminus (a 42kDa fragment) of merozoite surface protein-1 (MSP-142; consisting of MSP-119 and MSP-133) is a potential candidate for a malaria vaccine. However, to date, no study has yet investigated the sequence diversity of the gene encoding P. knowlesi MSP-142 (comprising Pk-msp-119 and Pk-msp-133) among isolates in Malaysia. The present study explored this aspect. Twelve P. knowlesi isolates were collected from patients from hospitals in Selangor and Sabah Borneo, Malaysia, between 2012 and 2014. The Pk-msp-142 gene was amplified by PCR and directly sequenced. Haplotype diversity (Hd) and nucleotide diversity (л) were studied among the isolates. There was relatively high genetic variation among P. knowlesi isolates; overall Hd and л were 1±0.034 and 0.01132±0.00124, respectively. A total of nine different haplotypes related to amino acid alterations at 13 positions, and the Pk-MSP-119 sequence was found to be more conserved than Pk-msp-133. We have found evidence for negative selection in Pk-msp-42 as well as the 33kDa and 19kDa fragments by comparing the rate of non-synonymous versus synonymous substitutions. Future investigations should study large numbers of samples from disparate geographical locations to critically assess whether this molecule might be a potential vaccine target for P. knowlesi.

Needs of exploring the burden of recent onset seizures due to neurocysticercosis and challenges in southeast Asia focusing on scenario in Malaysia.

Seizures due to neurocysticercosis (NCC) is a neglected human-to-human transmitted disorder and an emerging problem worldwide. A substantial portion of recent onset seizures is known to be attributed to NCC in Taenia solium (T. solium) endemic areas where populations which neither raise pigs nor eat pig meat are also at risk. High prevalence of NCC causing epilepsy has been reported in the underdeveloped areas of Southeast Asia (SEA) however, only fragmentary information on its incidence is available in countries like Malaysia. In Malaysia T. solium infection was previously thought to be infrequent due to Muslim population majority and the religious prohibition of consuming pork, but it is not totally absent. There is an evident lack of knowledge and awareness of the actual burden, routes of transmission, and the impact of NCC in this region. The problem is assumed to be more prevalent particularly in cities because of the frequent inflow of possibly T. solium infected individuals or carriers among those who migrate from neighboring endemic countries to Malaysia. The issue of imported cases that are likely to be emerging in Malaysia is highlighted here. An accurate quantification of regional burdens of epilepsy due to NCC in Malaysia is warranted considering the disease emergence in its neighboring countries. It is suggested that the importance of NCC be recognized through quantification of its burden, and also to collect epidemiological data for its subsequent elimination in line of World Health Organization's mission for control of cysticercosis as a neglected tropical disease. In this review the need as well as a strategy for neuro-care center screening of epilepsy cases, and various issues with possible explanations are discussed. It is also proposed that NCC be declared as a reportable disease which is one of the eradicable public health problems in SEA.