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Abstract Background Blastocystis has been described as the most common intestinal parasite in humans and has an increased impact on public health. However, the transmission of this parasite has not been conclusively determined. Methods To contribute to a better comprehension of the epidemiology of this infection, a cross-sectional survey aimed at providing the first documented data on the prevalence and risk factors associated with Blastocystis infection was carried out among three Orang Asli tribes (Proto-Malay, Negrito and Senoi) in selected villages at Negeri Sembilan, Perak and Pahang, Peninsular Malaysia. Faecal samples were examined by formalin-ether sedimentation and trichrome staining techniques. Results Of 500 individuals, 20.4% (102) were detected positive for Blastocystis; 13.3% (20/150) of Proto-Malays, 21.6% (30/139) of Negritos and 24.7% (52/211) of Senois were positive for Blastocystis, respectively. The positive cases showed a decrease with increasing age and most of the positive cases were observed in individuals less than 15 years old. Multivariate analysis confirmed that drinking untreated water and the presence of other family members infected with Blastocystis were significant risk factors of infection among the three tribes and overall population studied. Conclusion Essentially, the findings highlighted that Blastocystis infection is prevalent among Orang Asli communities in Malaysia. Further studies using molecular approaches to distinguish the subtype of Blastocystis is needed. The present study also revealed that this infection may be transmitted through waterborne and human-to-human contact. Therefore, interventions with the provision of clean water supply for the communities and health education especially to the parents are urgently required.

Objective: The aim of this study is to determine the prevalence of malnutrition in children living in endemic areas of soil-transmitted helminthes. Methods: An observational study was carried out on 281 Orang Asli children aged 2-15 years in eight villages in Selangor, Malaysia. Assessment was carried out using anthropometric measurements and examination of blood and faecal samples.The Z-score for weight-for-height was used to denote underweight as an overall indicator of malnutrition. Height-for-age Z-score was used as an indicator for stunting while weight-for-height Z-score for wasting. Faecal samples were collected and screened for soil-transmitted helminthiasis using Kato-Katz technique. Albumin estimation was carried out on blood samples using standard technique. Results: The overall prevalence of mild and significant underweight was 32.1% and 56.5% respectively. The prevalence of mild stunting was 25.6% while another 61.3% had significant stunting. The overall prevalence of mild and significant wasting was 39.0% and 19.5% respectively. The mean albumin level was 44.81 ± 5.75 g/L and 28.0% of the children with albumin level below 35 g/L. The overall prevalence of ascariasis, trichuriasis and hookworm infection were 61.9%, 98.2% and 37.0% respectively and of these 19.0%, 26.0% and 3.0% of the children have severe infection of the respective worms. Conclusion: Thus, the high prevalence of malnutrition in these children due to severe infection of ascariasis, trichuriasis and hookworm infection could not be ruled out. [ABSTRACT FROM AUTHOR]

Background: This study evaluated the performance of routine permanent stain and cultivation method in comparison with polymerase chain reaction assay as the reference technique to detect Blastocystis sp., Methods: A cross-sectional study was conducted among aboriginal populations that reside in Pahang, Peninsular Malaysia in Feb to Mar 2015. A total of 359 stool samples were examined using Wheatley's trichrome stain, in-vitro cultivation in Jones' medium and PCR assay. Positive amplicons were subjected to sequencing and phylogenetic analysis., Results: Fifty-six (15.6%) samples were detected positive with Blastocystis sp. by Wheatley's trichrome stain and 73 (20.3%) by in-vitro culture, while PCR assay detected 71 (19.8%) positive samples. Detection rate of Blastocystis sp. was highest in combination of microscopic techniques (27.9%). The sensitivity and specificity of Wheatley's trichrome staining and in-vitro culture techniques compared to PCR assay were 49.3% (95% CI: 37.2-61.4) and 92.7% (95% CI: 89.1-95.4) and 39.4% (95% CI: 28.0-51.8) and 84.4% (95% CI: 79.7-88.4), respectively. However, the sensitivity [60.6% (95% CI: 48.3-71.9)] of the method increased when both microscopic techniques were performed together. False negative results produced by microscopic techniques were associated with subtype 3. The agreement between Wheatley's trichrome stain, in-vitro culture and combination of microscopic techniques with PCR assay were statistically significant by Kappa statistics (Wheatley's trichrome stain: K = 0.456, P <0.001; in-vitro culture: K = 0.236, P <0.001 and combination techniques: K = 0.353, P <0.001)., Conclusion: The combination of microscopic technique is highly recommended to be used as a screening method for the diagnosis of Blastocystis infection either for clinical or epidemiological study to ensure better and accurate diagnosis., Competing Interests: Conflict of interest The authors declare that there is no conflict of interest.

Background: Alternating wet and dry seasons may play an important role in the acquisition and distribution of Blastocystis subtype infection in the tropics. This cross-sectional study was therefore conducted to provide the prevalence of Blastocystis and to determine the potential risk factors associated with each subtype during the wet and dry seasons in the Aboriginal community, Pahang, Malaysia., Methods: A total of 473 faecal samples were collected: 256 (54.1%) and 217 (45.9%) samples were obtained during the wet (October-November 2014) and the dry season (June 2015), respectively. All fresh faecal samples were subjected to molecular analysis for subtype and allele identification., Results: Of the 473 samples, 42.6% and 37.8% were positive for Blastocystis ST1, ST2, ST3 and ST4 during wet and dry seasons, respectively. Prevalence of Blastocystis ST1 was significantly higher during the wet season compared to the dry season (Z = 2.146, P < 0.05). Analysis of the association of each Blastocystis subtype with socioeconomic characteristics showed the presence of other family members infected with Blastocystis ST3 and the use of stored river water for domestic activities were the significant risk factors for Blastocystis ST3 infections during both seasons. Untreated water supply and low monthly household income (less or equal to RM 500) were the other significant risk factors for Blastocystis ST3 infections during wet and dry season, respectively. The presence of other family members with Blastocystis ST1 and ST2 was the only significant risk factor associated with ST1 and ST2 infections during both seasons. We hypothesise that transmission of Blastocystis ST1, ST2 and ST3 occurred from person to person during both seasons. The waterborne transmission was also identified as a mode of transmission of Blastocystis ST3., Conclusion: The significant risk factors identified in this study were important in the dynamic transmission of Blastocystis infections during both seasons. Provision of treated water supply and health education are affirmative actions to be taken to control Blastocystis infections in this community.

Objectives: To determine the prevalence and risk factors of Blastocystis among underprivileged communities living in rural Malaysia., Methods: This cross-sectional study was conducted among 253 participants aged between 1 and 85 years. Stool samples were examined using Wheatley's trichrome stain after in-vitro cultivation in Jones' medium to detect the presence of Blastocystis. Information pertaining to the demography, socioeconomic and environment were collected using pre-validated questionnaires., Results: The total prevalence of Blastocystis infection was 40.7%. The multiple logistic regression analysis revealed that age ≥15 years (OR = 2.72; 95% CI = 1.47-5.04) and presence of infected family members (OR = 8.56; 95% CI = 4.47-16.38) were the significant risk factors associated with blastocystosis in these communities., Conclusions: Blastocystosis is revealed through this study to be still prevalent among Orang Asli communities in rural Malaysia. The two main approaches that should be implemented by the public health authority in battling this infection would be the screening of other family members and giving treatment to the infected individuals. Moreover, it is imperative for health education on good personal and food hygiene practices are provided in order to reduce the morbidity and transmission of Blastocystis infection among the Orang Asli in their communities meaningfully., (Copyright © 2017 Hainan Medical University. Production and hosting by Elsevier B.V. All rights reserved.)

Most studies of opportunistic infections focus on immunocompromised patients. However, there is a lack of information on microsporidiosis in healthy people (immunocompetent) worldwide. This study aimed to detect and identify microsporidia species in immunocompetent Orang Asli living in Pahang, Malaysia. Orang Asli is a collective term for a group of indigenous people that usually reside in the interior regions of Peninsular Malaysia. They comprise about 0.7% of the total population in Malaysia and 76% of them lived below the poverty line i.e., poor housing conditions with the lack of access to safe drinking water and adequate sanitation, contaminated environment, high illiteracy rate and unhygienic practices by these people. Stool samples were collected from 209 Orang Asli and analyzed for detecting the presence of Enterocytozoon bieneusi and Encephalitozoon intestinalis by polymerase chain reaction assay targeting small subunit ribosomal RNA gene. E. bieneusi was detected in 8 individuals (3.83%). This infection was commonly found in males than females (5.2% vs. 2.7%). All infected Orang Asli were adults, with a mean age of 44years. Diarrhea and other gastrointestinal symptoms were reported in one case (12.5%) among individuals infected with this species. These findings clearly show that exposure to E. bieneusi may actually be common than reported. The accurate detection and identification of microsporidian species by molecular technique will improve therapy, clinical manifestations and prognosis of this infection, as no antiparasitic therapy has been approved for E. bieneusi. It is hoped that these findings will allow the formulation of better health management and disease prevention advisories, and improvement in the standards of health in similar communities., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the tropics, there are too few studies on isolation of Blastocystis sp. subtypes from water sources; in addition, there is also an absence of reported studies on the occurrence of Blastocystis sp. subtypes in water during different seasons. Therefore, this study was aimed to determine the occurrence of Blastocystis sp. subtypes in river water and other water sources that drained aboriginal vicinity of highly endemic intestinal parasitic infections during wet and dry seasons. Water samples were collected from six sampling points of Sungai Krau (K1-K6) and a point at Sungai Lompat (K7) and other water sources around the aboriginal villages. The water samples were collected during both seasons, wet and dry seasons. Filtration of the water samples were carried out using a flatbed membrane filtration system. The extracted DNA from concentrated water sediment was subjected to single round polymerase chain reaction and positive PCR products were subjected to sequencing. All samples were also subjected to filtration and cultured on membrane lactose glucuronide agar for the detection of faecal coliforms. During wet season, Blastocystis sp. ST1, ST2 and ST3 were detected in river water samples. Blastocystis sp. ST3 occurrence was sustained in the river water samples during dry season. However Blastocystis sp. ST1 and ST2 were absent during dry season. Water samples collected from various water sources showed contaminations of Blastocystis sp. ST1, ST2, ST3 and ST4, during wet season and Blastocystis sp. ST1, ST3, ST8 and ST10 during dry season. Water collected from all river sampling points during both seasons showed growth of Escherichia coli and Enterobacter aerogenes , indicating faecal contamination. In this study, Blastocystis sp. ST3 is suggested as the most robust and resistant subtype able to survive in any adverse environmental condition. Restriction and control of human and animal faecal contaminations to the river and other water sources shall prevent the transmission of Blastocystis sp. to humans and animals in this aboriginal community., Competing Interests: The authors declare there are no competing interests.

Background: Intestinal parasitic infections (IPIs) are still major health problems in many developing countries including Malaysia, particularly in the poor and socioeconomically deprived rural and remote communities in Peninsular Malaysia. This study was conducted to determine the prevalence of IPIs and to identify the key factors associated with intestinal polyparasitism as well as to evaluate the knowledge, attitude and practices (KAP) on IPIs among rural Orang Asli and Malay communities in Terengganu, Malaysia., Methods: A cross-sectional study was conducted among 340 participants (165 Orang Asli and 175 Malay) aged ≤ 15 years from the Hulu Terengganu and Kemaman districts of Terengganu. Faecal samples were examined for the presence of intestinal parasites by using direct smear, formalin-ether sedimentation, trichrome stain, modified Ziehl Neelsen stain, in vitro cultivation in Jones' medium, Kato Katz and Harada Mori techniques. Demographic, socioeconomic, environmental and behavioural information of the participants and their KAP for IPIs were collected by using a pre-tested questionnaire., Results: Overall, 149 (90.3 %) Orang Asli and 43 (24.6 %) Malay children were infected by at least one parasite species. The overall prevalences of intestinal polyparasitism among the Orang Asli and Malay were 68.5 % (113/165) and 14.3 % (25/175), respectively. Multiple logistic regression analysis showed that using unsafe water supply as a source for drinking water, the presence of domestic animals, not wearing shoes when outside, not washing vegetables before consumption, not washing hands after playing with soil, indiscriminate defecation and the low level of mother's education were the key risk factors for intestinal polyparasitism among the Orang Asli, while working mothers and the presence of domestic animals were the risk factors among the Malay children. Almost all the Malays were well aware about the IPIs while Orang Asli respondents had a poor level of related awareness., Conclusions: This study demonstrates that IPIs are highly prevalent in rural Terengganu, Malaysia. Community awareness about IPIs was found to be imperative in protecting Malay children from these infections. An integrated control programme for the prevention and control of IPIs is highly recommended for these communities, with a special emphasis on the Orang Asli population.

The epidemiology and environmental factors affecting transmission of human microsporidiosis are poorly understood. We conducted the present study to determine the prevalence and risk factors associated with asymptomatic intestinal microsporidiosis among aboriginal school children in the Kuala Krau District, Pahang State, Malaysia. We collected stool samples from 255 school children and examined the samples using Gram-chromotrope Kinyoun stain. We also collected demographic, socioeconomic, environmental and personal hygiene information using a pre-tested questionnaire. Sixty-nine of the children was positive for microsporidia: 72.5% and 27.5% were low (1+) and moderate (2+) excretions of microsporidia spores, respectively. Univariate and multivariate analyses showed being aged 10 years (p = 0.026), using an unsafe water supply as a source for drinking water (p = 0.044) and having close contact with domestic animals (p = 0.031) were all significantly associated with microsporidial infection among study subjects. Our findings suggest asymptomatic intestinal microsporidiosis is common in the study population, more than previously reported. In the study population, control measures need to be implemented, such as good personal hygiene, proper sanitation and safe drinking water supply.

Dientamoeba fragilis is a trichomonad parasite that can infect the gastrointestinal tract of humans causing gastrointestinal disease. Little is known about its epidemiology. We evaluated the prevalence of D. fragilis by conducting a cross sectional study of an Orang Asli population in rural Malaysia. We examined stool samples from 150 participants for D. fragilis using Wheatley's trichrome stain and collected demographic data from each participant using a structured questionnaire. Five participants (3.3%) had D. fragilis in their stool; four of these were aged < 15 years; 3 were male and 2 were female. All participants with positive stool sample for D. fragilis were symptomatic; 3 had diarrhea and 2 had other gastrointestinal symptoms. D. fragilis is present in the study population. Further studies are needed to determine the virulence, pathogenicity and mode of transmission of D. fragilis in the study population.

Clinical manifestations of giardiasis vary from asymptomatic infection to chronic diarrhea. A total of 611 stool samples from Aboriginal participants residing in Jelebu, Gerik and Temerloh States, Malaysia, ages 2 to 74 years were screened for Giardia intestinalis using microscopic examination and sequence analysis of a fragment of nested-PCR amplified triosephosphate isomerase (tpi) gene. Demographic data was collected through a structured questionnaire. tpi was successfully amplified from 98/110 samples microscopically positive for G. intestinalis, with 62 and 36 belonging to assemblage A and B, respectively. There is a significant correlation between assemblage A and symptomatic infection only in participants of < 15 years of age. In the other age group, host factors may have more effects on the presence of clinical signs and symptoms than G. intestinalis assemblage types.

To improve the stool concentration procedure, we modified different steps of the standard formalin-ether concentration technique and evaluated these modifications by examining stool samples collected in the field. Seven samples were found positive by the modified formalin-ether concentration technique (M-FECT). Therefore, the M-FECT procedure provides enhanced detection of Cryptosporidium oocysts., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: This cross-sectional study aimed to investigate the current prevalence and risk factors associated with intestinal polyparasitism (the concurrent infection with multiple intestinal parasite species) among Orang Asli school children in the Lipis district of Pahang state, Malaysia., Methods/principal Findings: Fecal samples were collected from 498 school children (50.6% boys and 49.4% girls), and examined by using direct smear, formalin-ether sedimentation, trichrome stain, modified Ziehl Neelsen stain, Kato-Katz, and Harada Mori techniques. Demographic, socioeconomic, environmental, and personal hygiene information were collected by using a pre-tested questionnaire. Overall, 98.4% of the children were found to be infected by at least one parasite species. Of these, 71.4% had polyparasitism. The overall prevalence of Trichuris trichiura, Ascaris lumbricoides, hookworm, Giardia duodenalis, Entamoeba spp., and Cryptosporidium spp. infections were 95.6%, 47.8%, 28.3%, 28.3%, 14.1% and 5.2%, respectively. Univariate and multivariate analyses showed that using an unsafe water supply as a source for drinking water, presence of other family members infected with intestinal parasitic infections (IPI), not washing vegetables before consumption, absence of a toilet in the house, not wearing shoes when outside, not cutting nails periodically, and not washing hands before eating were significant risk factors associated with intestinal polyparasitism among these children., Conclusions/significance: Intestinal polyparasitism is highly prevalent among children in the peninsular Malaysian Aboriginal communities. Hence, effective and sustainable control measures, including school-based periodic chemotherapy, providing adequate health education focused on good personal hygiene practices and proper sanitation, as well as safe drinking water supply should be implemented to reduce the prevalence and consequences of these infections in this population.

Background: Despite the intensive global efforts to control intestinal parasitic infections, the prevalence of soil-transmitted helminth (STH) infections is still very high in many developing countries particularly among children in rural areas., Methods: A randomized, double-blind, placebo-controlled trial was conducted on 250 Aboriginal schoolchildren in Malaysia to investigate the effects of a single high-dose of vitamin A supplementation (200,000 IU) on STH reinfection. The effect of the supplement was assessed at 3 and 6 months after receiving interventions; after a complete 3-day deworming course of 400 mg/daily of albendazole tablets., Results: Almost all children (98.6%) were infected with at least one STH species. The overall prevalence of ascariasis, trichuriasis and hookworm infection was 67.8%, 95.5% and 13.4%, respectively. Reinfection rates of Ascaris, Trichuris and hookworm were high; at 6 months, assessment reached 80% of the prevalence reported before treatment. There were no significant differences in the reinfection rates and intensities of STH between vitamin A supplemented-children and those who received placebo at 3 and 6 months (p > 0.05)., Conclusions: Vitamin A supplementation showed no protective effect against STH reinfection and this could be due to the high endemicity of STH in this community. Long-term interventions to reduce poverty will help significantly in reducing this continuing problem and there is no doubt that reducing intestinal parasitic infection would have a positive impact on the health, nutrition and education of these children., Trial Registration: This trial was registered at clinicaltrials.gov as NCT00936091.

Currently, information on prevalence of soil-transmitted helminth (STH) infections among different tribes of Orang Asli (aboriginal) is scarce in Malaysia. The present study is a cross-sectional study aimed at determining the factors associated with the prevalence of STH infections among the Proto-Malay, Negrito and Senoi tribes. Faecal samples were collected from 500 participants and socioeconomic data was collected via pre-tested questionnaire. All samples were processed using formalin-ether sedimentation and Wheatley's trichrome staining. Trichuris trichiura (57%) was the most common STH seen among the participants, followed by Ascaris lumbricoides (23.8%) and hookworm (7.4%). Trichuriasis and ascariasis showed an age-dependency relationship; significantly higher rates were observed among Senois who aged <15 years. Likewise, Negritos also showed an age-dependency association with ascariasis affecting mainly the under 15 years old individuals. Multivariate logistic regression model indicated the following predictors of trichuriasis among these communities; being aged <15 years, consuming raw vegetables, belonging to a large household members (≥8) and earning low household income (

Background: Giardia duodenalis is a flagellate parasite which has been considered the most common protozoa infecting human worldwide. Molecular characterization of G. duodenalis isolates have revealed the existence of eight groups (Assemblage A to H) which differ in their host distribution. Assemblages A and B are found in humans and in many other mammals., Methods: This cross-sectional study was conducted to identify assemblage's related risk factors of G. duodenalis among Orang Asli in Malaysia. Stool samples were collected from 611 individuals aged between 2 and 74 years old of whom 266 were males and 345 were females. Socioeconomic data were collected through a pre-tested questionnaire. All stool samples were processed with formalin-ether sedimentation and Wheatley's trichrome staining techniques for the primary identification of G. duodenalis. Molecular identification was carried out by the amplification of a triosephosphate isomerase gene using nested-PCR assay., Results: Sixty-two samples (10.2%) were identified as assemblage A and 36 (5.9%) were assemblage B. Risk analysis based on the detected assemblages using univariate and logistic regression analyses identified subjects who have close contact with household pets i.e. dogs and cats (OR = 2.60; 95% CI = 1.42, 4.78; P = 0.002) was found to be significant predictor for assemblage A. On the other hand, there were three significant risk factors caused by assemblage B: (i) children ≤15 years old (OR = 2.33; 95% CI = 1.11, 4.87; P = 0.025), (ii) consuming raw vegetables (OR = 2.82; 95% CI = 1.27, 6.26; P = 0.011) and (iii) the presence of other family members infected with giardiasis (OR = 6.31; 95% CI = 2.99, 13.31; P < 0.001)., Conclusions: The present study highlighted that G. duodenalis infection among Orang Asli was caused by both assemblages with significant high prevalence of assemblage A. Therefore, taking precaution after having contact with household pets and their stool, screening and treating infected individuals, awareness on the importance of good health practices and washing vegetables are the practical intervention ways in preventing giardiasis in Orang Asli community.

Iron deficiency anaemia (IDA) is the most common nutritional deficiency in the world including developed and developing countries. Despite intensive efforts to improve the quality of life of rural and aboriginal communities in Malaysia, anaemia and IDA are still major public health problems in these communities particularly among children. A randomized, double-blind, placebo-controlled trial was conducted on 250 Orang Asli (aboriginal) schoolchildren in Malaysia to investigate the effects of a single high-dose of vitamin A supplementation (200,000 IU) on iron status indices, anaemia and IDA status. The effect of the supplement was assessed after 3 months of receiving the supplements; after a complete 3-day deworming course of 400 mg/day of albendazole tablets. The prevalence of anaemia was found to be high: 48.5% (95% CI=42.3, 54.8). Moreover, 34% (95% CI=28.3, 40.2) of the children had IDA, which accounted for 70.1% of the anaemic cases. The findings showed that the reduction in serum ferritin level and the increments in haemoglobin, serum iron and transferrin saturation were found to be significant among children allocated to the vitamin A group compared to those allocated to the placebo group (p<0.01). Moreover, a significant reduction in the prevalence of IDA by almost 22% than prevalence at baseline was reported among children in the vitamin A group compared with only 2.3% reduction among children in the placebo group. In conclusion, vitamin A supplementation showed a significant impact on iron status indices and IDA among Orang Asli children. Hence, providing vitamin A supplementation and imparting the knowledge related to nutritious food should be considered in the efforts to improve the nutritional and health status of these children as a part of efforts to improve the quality of life in rural and aboriginal communities.

Background: Giardia duodenalis infection and malnutrition are still considered as public health problems in many developing countries especially among children in rural communities. This study was carried out among Aboriginal (Orang Asli) primary schoolchildren in rural peninsular Malaysia to investigate the burden and the effects of Giardia infection on growth (weight and height) of the children., Methods/findings: Weight and height of 374 children aged 7-12 years were assessed before and after treatment of Giardia infection. The children were screened for Giardia parasite using trichrome staining technique. Demographic and socioeconomic data were collected via face-to-face interviews using a pre-tested questionnaire. Overall, 22.2% (83/374) of the children were found to be infected with Giardia. Nutritional status of children was assessed and the results showed that the mean weight and height were 23.9 kg (95% CI = 23.3, 24.5) and 126.6 cm (95% CI = 125.6, 127.5), respectively. Overall, the prevalence of severe underweight, stunting and wasting were 28.3%, 23.8% and 21.0%, respectively. Multiple linear regression analyses showed sex, Giardia infection and household monthly income as the significant determinants of weight while sex and level of mother's education were the significant determinants of height. Weight and height were assessed at 3 and 6 months after treatment of Giardia infection. It was found that Giardia infection has a significant association with the weight of children but not with height., Conclusions/significance: This study reveals high prevalence of Giardia infection and malnutrition among Aboriginal children in rural Malaysia and clearly highlights an urgent need to identify integrated measures to control these health problems in the rural communities. Essentially, proper attention should be given to the control of Giardia infection in Aboriginal communities as this constitutes one of the strategies to improve the nutritional status of Aboriginal children.

Background: Studies on microsporidial infection mostly focus on immunodeficiency or immunosuppressive individuals. Therefore, this cross-sectional study describes the prevalence and risk factors of microsporidiosis among asymptomatic individuals in Malaysia., Methods/findings: Four hundred and forty seven stool samples were collected and examined for microsporidia after staining with Gram-chromotrope Kinyoun. Demographic, socioeconomic, environmental, and behavioral information were collected by using a pre-tested questionnaire. Overall, 67 (15%) samples were positive for microsporidia. The prevalence of infection was significantly higher among individuals aged more than 15 years compared to those aged <15 years (OR = 1.97, 95% CI = 1.08, 3.62; P = 0.028). Furthermore, logistic regression analysis confirmed that the presence of other family members infected with microsporidia (OR = 8.45; 95% CI = 4.30, 16.62; P<0.001) and being a consumer of raw vegetables (OR = 2.05; 95% CI = 1.15, 3.66; P = 0.016) were the significant risk factors of this infection., Conclusions: These findings clearly show that exposure to microsporidia is common among Aboriginal population. Further studies using molecular approach on microsporidia isolates from asymptomatic individuals is needed to determine species-specific. The risk factors associated with microsporidiosis will help in identifying more clearly the sources of the infection in the environment that pose a risk for transmission so that preventive strategies can be implemented.

The present study was conducted to investigate the clinical outcomes of Entamoeba histolytica infection in symptomatic and asymptomatic Orang Asli (aborigine) communities in Malaysia. Examination was performed on 500 stool samples obtained from Orang Asli communities in 3 different states using formalin-ether concentration, trichrome staining, and single-round PCR techniques. Out of 500 stool samples, single infection of E. histolytica, Entamoeba dispar, and Entamoeba moshkovskii was identified in 3.2%, 13.4%, and 1%, respectively. In addition, 10 samples had mixed infections with E. histolytica and E. dispar. Six samples containing E. dispar were also positive for E. moshkovskii, and only 2 samples had E. histolytica in association with E. dispar and E. moshkovskii. Seventeen E. histolytica-positive samples were from symptomatic subjects, whereas the remaining 11 samples came from asymptomatic subjects. These findings suggest a predominant distribution of pathogenic potential of E. histolytica strains in this community. Therefore, further studies on genotyping of E. histolytica is required, to find out association between E. histolytica genotype and the outcome of the infection.

This study was conducted to evaluate two routinely microscopic diagnostic methods in comparison with single-round PCR assay as the reference technique to detect Entamoeba histolytica/dispar/moshkovskii. Examination was performed on 500 stool samples obtained from Orang Asli communities in different states of Malaysia using formalin-ether sedimentation, trichrome staining and single-round PCR techniques. Ninety-three stool samples were detected E. histolytica/dispar/moshkovskii positive by routine microscopy, while single-round PCR detected 106 positive samples. Additional positives detected by PCR assay were eventually confirmed to be negative by both microscopic techniques. Detection rate of E. histolytica/dispar/moshkovskii was highest in combination techniques (18.6%), followed by trichrome staining (13.4%) and formalin-ether sedimentation (11.2%) techniques. Single-round PCR detected 21.2% of the stool samples. The sensitivity and specificity of formalin-ether sedimentation and trichrome staining techniques compared to the reference technique were 31.1% (95% CI: 29.0-36.0) and 94.2% (95% CI: 89.8-98.9), and 53.8% (95% CI: 46.0-76.2) and 97.5% (95% CI: 92.8-99.1), respectively. However, the sensitivity [59.4% (95% CI: 48.9-78.5)] of the method increased when both techniques were performed together, but the specificity decreased to 92.4% (95% CI: 81.0-98.0). The agreement between the reference technique, trichrome staining and combination techniques were statistically significant by Kappa statistics (trichrome staining: K = 0.592, p < 0.05; combination techniques: K = 0.543, p < 0.05). Hence, the combination technique is recommended to be used as a screening method in the diagnosis of E. histolytica/dispar/moshkovskii infections either for clinical or epidemiological study., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Currently, species-specific information on Entamoeba infections is unavailable in Malaysia and is restricted worldwide due to the re-description of pathogenic Entamoeba histolytica and non-pathogenic Entamoeba dispar and Entamoeba moshkovskii. Therefore, this cross-sectional study was conducted to provide the first known documented data on the true prevalence of these three species in western Malaysia using a molecular method. Another aim of this study was to determine the association of potential risk factors associated with each Entamoeba sp. A total of 500 stool samples from three Orang Asli tribes were randomly collected. The overall prevalence of E. histolytica, E. dispar and E. moshkovskii determined by microscopy was 18.6% (93/500). Molecular analysis revealed that while most Entamoeba-positive individuals were infected with E. dispar (13.4%), followed by E. histolytica (3.2%) and E. moshkovskii (1.0%), the present findings show low prevalence rates of mixed infections with E. histolytica and E. dispar (2%), E. dispar and E. moshkovskii (1.2%) and association infections of E. histolytica, E. dispar and E. moshkovskii (0.4%). Logistical regression analysis indicates that the dynamics of the transmission of the three Entamoeba spp. was different. Of six statistically significant variables observed in the univariate analysis, three were retained as significant risk factors for E. histolytica infection in the logistical regression model. These factors were (i) not washing hands after playing with soil or gardening (Odds ratio (OR)=4.7; 95% confidence level (CI)=1.38, 16.14; P=0.013), (ii) indiscriminate defecation in the river or bush (OR=5.7; 95% CI=1.46, 21.95; P=0.012) and (iii) close contact with domestic animals (OR=5.4; 95% CI=1.36, 2.51; P=0.017). However, subjects with family members who were infected with E. histolytica/E. dispar/E. moshkovskii (OR=3.8; 95 CI=2.11, 6.86; P<0.001) and those who consumed raw vegetables (OR=1.8; 95% CI=1.01, 3.23; P=0.047) were more likely to be infected with E. dispar. On the other hand, no associated factor was identified with E. moshkovskii infection. Nevertheless, diarrhoea (P=0.002) and other gastroenteritis symptoms (P<0.001) were only associated with E. histolytica infection. The present study provides new insight into the distribution and risk factors of E. histolytica, E. dispar and E. moshkovskii infections among Orang Asli communities in Malaysia. Identifying the different risk factors of E. histolytica and E. dispar infections will help in the planning specific strategies in the control and prevention of each infection in the communities. Moreover, it emphasises the need for molecular methods to determine the species-specific prevalence of Entamoeba spp., (Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Entamoeba moshkovskii and Entamoeba dispar are microscopically indistinguishable from the pathogenic species Entamoeba histolytica. Although sporadic cases of human infection with E. moshkovskii have been reported, the amoeba is still considered primarily as a free-living amoeba. A cross-sectional study was carried out among Orang Asli communities in 3 different states of Peninsular Malaysia. Fecal samples were examined by formalin-ether sedimentation and trichrome staining techniques and then single-round PCR assay was used to detect E. moshkovskii. Out of 500 fecal samples examined microscopically, 93 (18·6%) samples were positive for E. histolytica/E. dispar/E. moshkovskii complex cysts and/or trophozoites. PCR products were detected in 106 fecal samples. E. moshkovskii isolates were detected in 13 (12·3%) fecal samples. Of the 13 E. moshkovskii-positive samples, 5 were of single isolation of E. moshkovskii, 6 were also positive for E. dispar, and only 2 samples were positive for E. dispar and E. histolytica. Moreover, 3 E. moshkovskii-positive samples were collected from symptomatic individuals while the remaining 10 samples were from asymptomatic subjects. This is the first report on the identification of E. moshkovskii in Malaysia. Further studies are needed to confirm the pathogenicity of E. moshkovskii infection and determine the epidemiology among Orang Asli communities in Malaysia.

Permanent staining of faecal smears by Wheatley's trichrome technique has been used by many scientists for the detection of parasites in the past and it was found to be highly sensitive. This study was conducted to evaluate the use of Wintergreen oil in comparison with xylene in Wheatley's trichrome staining technique, as the reference technique. In a blind comparison study, 500 collected faecal samples from aboriginal communities were examined. Wintergreen oil was found to be more superior than xylene as a clearing agent in the Wheatley's trichrome staining of polyvinyl alcohol-fixed faecal smears for the identification of intestinal protozoa. Elimination of toxic, carcinogenic, and fire hazards makes Wintergreen oil the preferred choice in routine parasitology examinations., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The flagellate protozoan parasite, Giardia intestinalis, is widely distributed throughout the world with a high prevalence in developing countries in the tropics and subtropics, including Malaysia. Approximately 200 million people are infected with the parasite globally, with 500,000 new cases reported annually. This cross-sectional study was conducted among three tribes of Orang Asli communities in Selangor, Perak and Pahang states of Malaysia. The main objective was to determine the prevalence of and risk factors for giardiasis. Stool samples were collected from 500 individuals aged between 2 and 74 years (males=219, females=281). The samples were examined with formalin-ether sedimentation and trichrome staining techniques. Socioeconomic data were collected through a pre-tested questionnaire. The overall prevalence of giardiasis was 20.0% with the highest prevalence in the Proto-Malays (33.3%) followed by Negritos (20.1%) and Senois (10.4%). The positive cases showed a decrease with increasing age and most of the positive cases were observed in individuals less than 24 years old. Males had significantly higher prevalence than females (χ(2)=5.283, P=0.022). Logistic regression analysis of the overall population studied and the Senoi tribe confirmed that being a child aged less than 15 years, being male, the consumption of raw vegetables and the presence of other family members infected with G. intestinalis were the main risk factors for giardiasis. The presence of other family members infected with G. intestinalis was the only risk factor highlighted in the Proto-Malay and Negrito tribes. Diarrhoea was significantly associated with giardiasis. However, the cause and effect relationship has yet to be determined. Thus, screening family members and treating the infected individuals are the main strategies that should be adopted by the public health authority in combating this infection in Orang Asli communities as well as health education regarding good personal and food hygiene practises., (Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Background: Entamoeba histolytica/Entamoeba dispar/Entamoeba moshkovskii infection is still prevalent in rural Malaysia especially among Orang Asli communities. Currently, information on prevalence of this infection among different ethnic groups of Orang Asli is unavailable in Malaysia. To contribute to a better comprehension of the epidemiology of this infection, a cross-sectional study aimed at providing the first documented data on the prevalence and risk factors associated with E. histolytica/E. dispar/E. moshkovskii infection was carried out among three Orang Asli ethnic groups (Proto-Malay, Negrito, and Senoi) in selected villages in Negeri Sembilan, Perak, and Pahang states, Malaysia., Methods/findings: Faecal samples were examined by formalin-ether sedimentation and trichrome staining techniques. Of 500 individuals, 8.7% (13/150) of Proto-Malay, 29.5% (41/139) of Negrito, and 18.5% (39/211) of Senoi were positive for E. histolytica/E. dispar/E. moshkovskii, respectively. The prevalence of this infection showed an age-dependency relationship, with higher rates observed among those aged less than 15 years in all ethnic groups studied. Multivariate analysis confirmed that not washing hands after playing with soils or gardening and presence of other family members infected with E. histolytica/E. dispar/E. moshkovskii were significant risk factors of infection among all ethnic groups. However, eating with hands, the consumption of raw vegetables, and close contact with domestic animals were identified as significant risk factors in Senoi., Conclusions: Essentially, the findings highlighted that E. histolytica/E. dispar/E. moshkovskii parasites are still prevalent in Malaysia. Further studies using molecular approaches to distinguish the morphologically identical species of pathogenic, E. histolytica from the non-pathogenic, E. dispar and E. moshkovskii are needed. The establishment of such data will be beneficial for the public health authorities in the planning and implementation of specific prevention and control strategies of this infection in different Orang Asli ethnic groups in Malaysia.

This study was conducted to evaluate the modification of the usual Gram-chromotrope staining technique developed in-house known as Gram-chromotrope Kinyoun (GCK) in comparison with the Weber Modified Trichrome (WMT) staining technique; as the reference technique. Two hundred and ninety fecal specimens received by the Microbiology Diagnostic Laboratory of Hospital Universiti Kebangsaan Malaysia were examined for the presence of microsporidial spores. The sensitivity and specificity of GCK compared to the reference technique were 98% and 98.3%, respectively. The positive and negative predictive values were 92.5% and 99.6%, respectively. The agreement between the reference technique and the GCK staining technique was statistically significant by Kappa statistics (K = 0.941, P < 0.001). It is concluded that the GCK staining technique has high sensitivity and specificity in the detection of microsporidial spores in fecal specimens. Hence, it is recommended to be used in the diagnosis of intestinal microsporidiosis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Aims: The mRNA level for interleukin-6 (IL-6) is an important marker of osteoarthritis (OA). The present study aimed to investigate the level of IL-6 mRNA in the cartilage of OA knee while comparing it to the normal cartilage obtained from the same patient., Materials and Methods: A total of 21 patients who underwent total knee replacement were recruited for this study. Sectioning of the destructive cartilage was performed in the medial part of the proximal tibiofemoral cartilage. The unaffected lateral part of the knee in the same patient, served as a control. The mRNA level for IL-6 was assessed using LightCycler 2.0 quantitative real-time polymerase chain reaction (qRT-PCR). actin mRNA was used as an endogenous control., Results: Twelve out of 21 patients (57.1%) exhibited up regulation of IL-6 mRNA in the OA cartilage as compared to the normal cartilage. The rest of the patients (42.9%) showed down regulation of IL-6 mRNA. The statistical analysis showed there was insignificant level of IL-6 mRNA in the OA (1.91 +/- 0.45) as compared to the normal cartilage (1.13 +/- 0.44) (p > 0.05). The inter-individual variation in the level of IL-6 mRNA in the cartilage of idiopathic knee was in accordance with previous findings., Conclusions: These observations suggest IL-6 could also act as a catabolic agent in some patients or its expression might be influenced by other cytokines.

This study determined the prevalence of ocular manifestation of vitamin A deficiency in Orang Asli (Aborigine) children. Night blindness was found in 16.0% of the children, conjunctiva xerosis in 57.3%, Bitot's spot in 2.8%, corneal xerosis in 0.5% and corneal scars in 5.6%. These findings show that history of night blindness had sensitivity, specificity and predictive value (positive) of 47.2, 98.1 and 96.2%, respectively, compared with the standard diagnosis procedure using luxometer readings.