Molecular epidemiology of blastocystosis in Malaysia: does seasonal variation play an important role in determining the distribution and risk factors of Blastocystis subtype infections in the Aboriginal community?

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.

Occurrence of Blastocystis sp. in water catchments at Malay villages and Aboriginal settlement during wet and dry seasons in Peninsular Malaysia.

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.

PREVALENCE OF DIENTAMOEBA FRAGILIS AMONG AN ORANG ASLI POPULATION IN RURAL MALAYSIA.

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.

HUMAN GIARDIASIS IN MALAYSIA: CORRELATION BETWEEN THE PRESENCE OF CLINICAL MANIFESTATION AND GIARDIA INTESTINALIS ASSEMBLAGE.

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.

An improved stool concentration procedure for the detection of Cryptosporidium oocysts in Orang Asli stool samples.

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.

Molecular epidemiology of giardiasis among Orang Asli in Malaysia: application of the triosephosphate isomerase gene.

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.

Soil-transmitted helminth infections and associated risk factors in three Orang Asli tribes in Peninsular Malaysia.

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 (

Different clinical outcomes of Entamoeba histolytica in Malaysia: does genetic diversity exist?

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.

Blastocystis infection in Malaysia: evidence of waterborne and human-to-human transmissions among the Proto-Malay, Negrito and Senoi tribes of Orang Asli.

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.

Evaluation of formalin-ether sedimentation and trichrome staining techniques: its effectiveness in detecting Entamoeba histolytica/dispar/moshkovskii in stool samples.

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.

Molecular epidemiology of amoebiasis in Malaysia: highlighting the different risk factors of Entamoeba histolytica and Entamoeba dispar infections among Orang Asli communities.

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.

First molecular identification of Entamoeba moshkovskii in Malaysia.

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.

Wintergreen oil: a novel method in Wheatley's trichrome staining technique.

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.

Giardiasis among different tribes of Orang Asli in Malaysia: highlighting the presence of other family members infected with Giardia intestinalis as a main risk factor.

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.

Severity of Malaria cases reported in urban and rural hospitals in Malaysia.

The age distribution, types of infection and clinical patterns of malaria were compared in patients admitted to an urban and a rural hospital. Analysis of the cases seen in urban setting characteristically indicated a relatively low transmission rate of the disease, whereas the mean inoculation rate in patients from the rural hospital was found to be at least twenty folds higher. Plasmodium vivax was the predominant causative species in the urban hospital (p = 0.01), infecting mostly adult (p = 0.001) males (p = 0.01). The geometric mean parasite count at 3432/microl among the urban patients was significantly higher than that in the rural patients at 1422/microl (p = 0.04). Coma and death were more common among the cases seen in the urban hospital (p = 0.003), while severe anemia was the significant complication in the rural setting. Overall, the provisional diagnosis of malaria was relatively low in the urban hospital (p = 0.02). The results from this study highlighted the need to define the extent of malaria in urban areas. This report attempts to identify the non-climatic determinants of the infection and, furthermore, to provide a more informed basis to describe the burden of the disease.