Genetic and environmental interactions contribute to immune variation in rewilded mice.

The relative and synergistic contributions of genetics and environment to interindividual immune response variation remain unclear, despite implications in evolutionary biology and medicine. Here we quantify interactive effects of genotype and environment on immune traits by investigating C57BL/6, 129S1 and PWK/PhJ inbred mice, rewilded in an outdoor enclosure and infected with the parasite Trichuris muris. Whereas cellular composition was shaped by interactions between genotype and environment, cytokine response heterogeneity including IFNγ concentrations was primarily driven by genotype with consequence on worm burden. In addition, we show that other traits, such as expression of CD44, were explained mostly by genetics on T cells, whereas expression of CD44 on B cells was explained more by environment across all strains. Notably, genetic differences under laboratory conditions were decreased following rewilding. These results indicate that nonheritable influences interact with genetic factors to shape immune variation and parasite burden.

Supplementation of red palm olein-enriched biscuits improves levels of provitamin A carotenes, iron, and erythropoiesis in vitamin A-deficient primary schoolchildren: a double-blinded randomised controlled trial.

PURPOSE: Vitamin A deficiency (VAD) remains a significant contributor to childhood morbidity and mortality in developing countries; therefore, the implementation of sustainable and cost-effective approaches to control VAD is of utmost pertinence. This study aims to investigate the efficacy of red palm olein (RPO)-enriched biscuit supplementation in improving vitamin A, haematological, iron, and inflammatory status among vitamin A-deficient schoolchildren. METHODS: We conducted a double-blinded, randomised controlled trial involving 651 rural primary schoolchildren (8-12 years) with VAD in Malaysia. The schoolchildren were randomised to receive either RPO-enriched biscuits (experimental group, n = 334) or palm olein-enriched biscuits (control group, n = 317) for 6-month duration. RESULTS: Significant improvements in retinol and retinol-binding protein 4 levels were observed in both groups after supplementation (P < 0.001). The improvement in retinol levels were similar across groups among subjects with confirmed VAD (P = 0.40). Among those with marginal VAD, greater improvement in retinol levels was recorded in the control group (P < 0.001) but lacked clinical significance. The levels of α- and ß-carotenes, haematological parameters (haemoglobin, packed cell volume, mean corpuscular volume and mean corpuscular haemoglobin) and iron enhanced more significantly in the experimental group (P < 0.05). The significant reduction in the prevalence of microcytic anaemia (- 21.8%) and high inflammation (- 8.1%) was only observed in the experimental group. CONCLUSION: The supplementation of RPO-enriched biscuits enhanced levels of provitamin A carotenes, iron, and erythropoiesis, and exhibited anti-inflammatory effects. Therefore, the incorporation of RPO into National Nutritional Intervention Programs may be a potential measure to improve the health status of vitamin A-deficient children, among various other interventions. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov (NCT03256123).

High prevalence of malnutrition and vitamin A deficiency among schoolchildren of rural areas in Malaysia using a multi-school assessment approach.

Childhood malnutrition is known as a public health concern globally. The present study aims to assess the anthropometry and blood biochemical status of rural primary schoolchildren in Malaysia. A total of 776 children (7-11 years old) from ten rural primary schools from five states were included in this study. Nutritional outcomes were assessed based on sex, age group and school categories among the children (median age: 9 years (P25:8, P75:10)). The overall prevalence of malnutrition was 53·4 %. Vitamin A deficiency (VAD) was recorded at 20·6 and 39·8 % based on retinol and retinol-binding protein (RBP) levels, respectively. Anaemia, iron deficiency (ID), iron-deficiency anaemia (IDA) and elevated inflammation were found at 14·9, 17·9, 9·1 and 11·5 %, respectively. Malnutrition, VAD, anaemia, ID, IDA and elevated inflammation were more prevalent among Orang Asli (OA) schoolchildren compared with Non-Orang Asli schoolchildren. Higher occurrences of VAD and anaemia were also found among children aged <10 years. Retinol, RBP, α-carotene, ferritin and haemoglobin levels were lower among undernourished children. Besides, overweight/obese children exhibited a higher level of high-sensitivity C-reactive protein. Multivariate analysis demonstrated that OA school children (adjusted OR (AOR): 6·1; 95 % CI 4·1, 9·0) and IDA (AOR: 3·6; 95 % CI 1·9, 6·6) were associated with stunting among this population. The present study revealed that malnutrition, micronutrient deficiencies and anaemia are prevalent among rural primary schoolchildren in Malaysia, especially those from OA schools and younger age children (<10 years). Hence, more appropriate and targeted measures are needed to improve the nutritional status of these children.

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.

Seroprevalence of Nipah Virus Infection in Peninsular Malaysia.

Nipah virus (NiV) outbreak occurred in Malaysia in 1998. The natural host reservoir for NiV is Pteropus bats, which are commonly found throughout Malaysia. Humans become infected when NiV spills over from the reservoir species. In this study, NiV serosurveillance in Peninsular Malaysia, particularly among the indigenous population, was performed. The collected samples were tested for presence of NiV antibodies using a comparative indirect enzyme-linked immunosorbent assay based on the recombinant NiV nucleocapsid (rNiV-N) protein. We found that 10.73% of the participants recruited in this study had antibodies against rNiV-N, suggesting possible exposure to NiV.

Detection of Schistosoma spindale ova and associated risk factors among Malaysian cattle through coprological survey.

The present study was conducted to determine the occurrence of Schistosoma spindale ova and its associated risk factors in Malaysian cattle through a coprological survey. A total of 266 rectal fecal samples were collected from six farms in Peninsular Malaysia. The overall infection rate of S. spindale was 6% (16 of 266). Schistosoma spindale infection was observed in two farms, with a prevalence of 5.4% and 51.9%, respectively. This trematode was more likely to co-occur with other gastro-intestinal parasites (i.e., Dicrocoelium spp., Paramphistomum spp., strongyle, Eimeria spp. and Entamoeba spp.). Chi-square analysis revealed that female cattle are less likely to get S. spindale infection as compared to male cattle (OR = 0.3; 95% CI = 0.08-1.06; p < 0.05), and cattle weighing lower than 200 kg, were significantly at higher risk than those higher than 200 kg (OR = 5; 95% CI = 1.07-24.79; p < 0.05) to the infection. Multivariate analysis confirmed that among the cattle in Malaysia, the age (cattle with two year old and higher: OR = 21; 95% CI = 2.48-179.44; p < 0.05) and weight (weighing 200 kg and lower: OR = 17; 95% CI = 3.38-87.19; p < 0.05) were risk factors for S. spindale infection among Malaysian cattle.

Loop-mediated isothermal amplification assay for detection of generic and verocytotoxin-producing Escherichia coli among indigenous individuals in Malaysia.

We have successfully developed a Loop-mediated isothermal amplification (LAMP) assay that could specifically detect generic Escherichia coli (E. coli). This assay was tested on 85 bacterial strains and successfully identified 54 E. coli strains (average threshold time, Tt = 21.26). The sensitivity of this assay was evaluated on serial dilutions of bacterial cultures and spiked faeces. The assay could detect 10(2) CFU/mL for bacterial culture with Tt = 33.30 while the detection limit for spiked faeces was 10(3) CFU/mL (Tt = 31.12). We have also detected 46 generic E. coli from 50 faecal samples obtained from indigenous individuals with 16% of the positive samples being verocytotoxin-producing E. coli (VTEC) positive. VT1/VT2 allele was present in one faecal sample while the ratio of VT1 to VT2 was 6 : 1. Overall, our study had demonstrated high risk of VTEC infection among the indigenous community and most of the asymptomatic infection occurred among those aged below 15 years. The role of asymptomatic human carriers as a source of dissemination should not be underestimated. Large scale screening of the VTEC infection among indigenous populations and the potential contamination sources will be possible and easy with the aid of this newly developed rapid and simple LAMP assay.

Red palm olein-enriched biscuit supplementation lowers Ascaris lumbricoides reinfection at 6-month after anthelmintic treatment among schoolchildren with vitamin A deficiency (VAD).

Notwithstanding the global efforts made to control intestinal parasitic infections, soil-transmitted helminth (STH) infections are still one of the most prevalent infections globally, especially in developing countries. A double-blinded, randomized controlled trial was conducted on 343 primary schoolchildren (8-12 years old) with vitamin A deficiency (VAD) in rural areas of Malaysia to investigate the effects of red palm olein (RPO)-enriched biscuits on STH reinfection rates and infection intensities. The effects of the RPO-enriched biscuits (experimental group, n = 153) and palm olein (PO)-enriched biscuits (control group, n = 190), were assessed at 3- and 6-month after the administration of complete triple-dose albendazole (one dose of 400 mg for three consecutive days). The overall STH infection rate at baseline was recorded at 65.6%. At 6-month, a significantly lower reinfection rate of A. lumbricoides was observed in the experimental group (35.3%) compared to the control group (60.0%) (P< 0.05), and a significant reduction in fecal egg count (epg) of A. lumbricoides was observed in the experimental group from baseline (P< 0.001), but no significant reduction was observed in the control group. No significant differences in the reduction of infection intensities of T. trichiura and hookworm were observed between experimental and control groups at 3- and 6-month (P>0.05). These findings suggest the potential beneficial effects of RPO-enriched biscuit supplementation on the reinfection of A. lumbricoides, which could be attributed to its high carotenoids content by enhancing host immune response and mucosal epithelium integrity. However, further studies are warranted to confirm whether RPO supplementation could result in similar parasite-specific beneficial effects in other community settings, as well as to explore the underlying mechanisms.

Oral and Gut Microbiota Dysbiosis is Associated with Mucositis Severity in Autologous Hematopoietic Stem Cell Transplantation: Evidence from an Asian Population.

Mucositis is a debilitating complication of hematopoietic stem cell transplantation (HSCT). It is unclear how changes in the composition of microbiota, which are modulated by geographical location and ethnicity, may influence immune regulation leading to the development of mucositis, and the study of both oral and gut microbiota in a single population of autologous HSCT in the Asian region is lacking. The present study aimed to characterize the oral and gut microbiota changes, and the impact on both oral and lower gastrointestinal (GI) mucositis, with associated temporal changes in a population of adult recipients of autologous HSCT. Autologous HSCT recipients age ≥18 years were recruited from Hospital Ampang, Malaysia, between April 2019 and December 2020. Mucositis assessments were conducted daily, and blood, saliva, and fecal samples were collected prior to conditioning, on day 0, and at 7 days and 6 months post-transplantation. Longitudinal differences in alpha diversity and beta diversity were determined using the Wilcoxon signed-rank test and permutational multivariate analysis of variance, respectively. Changes in relative abundances of bacteria across time points were assessed using the microbiome multivariate analysis by linear models function. The combined longitudinal effects of clinical, inflammatory, and microbiota variables on mucositis severity were measured using the generalized estimating equation. Among the 96 patients analyzed, oral mucositis and diarrhea (representing lower GI mucositis) occurred in 58.3% and 95.8%, respectively. Alpha and beta diversities were significantly different between sample types (P < .001) and across time points, with alpha diversity reaching statistical significance at day 0 in fecal samples (P < .001) and at day +7 in saliva samples (P < .001). Diversities normalized to baseline by 6 months post-transplantation. Significant microbiota, clinical, and immunologic factors were associated with increasing mucositis grades. Increasing relative abundances of saliva Paludibacter, Leuconostoc, and Proteus were associated with higher oral mucositis grades, whereas increasing relative abundances of fecal Rothia and Parabacteroides were associated with higher GI mucositis grades. Meanwhile, increasing relative abundances of saliva Lactococcus and Acidaminococcus and fecal Bifidobacterium were associated with protective effects against worsening oral and GI mucositis grades, respectively. This study provides real-world evidence and insights into the dysbiosis of the microbiota in patients exposed to conditioning regimen during HSCT. Independent of clinical and immunologic factors, we demonstrated significant associations between relative bacteria abundances with the increasing severity of oral and lower GI mucositis. Our findings offer a potential rationale to consider the inclusion of preventive and restorative measures targeting oral and lower GI dysbiosis as interventional strategies to ameliorate mucositis outcome in HSCT recipients.

Genetic and Environmental interactions contribute to immune variation in rewilded mice.

The relative and synergistic contributions of genetics and environment to inter-individual immune response variation remain unclear, despite its implications for understanding both evolutionary biology and medicine. Here, we quantify interactive effects of genotype and environment on immune traits by investigating three inbred mouse strains rewilded in an outdoor enclosure and infected with the parasite, Trichuris muris. Whereas cytokine response heterogeneity was primarily driven by genotype, cellular composition heterogeneity was shaped by interactions between genotype and environment. Notably, genetic differences under laboratory conditions can be decreased following rewilding, and variation in T cell markers are more driven by genetics, whereas B cell markers are driven more by environment. Importantly, variation in worm burden is associated with measures of immune variation, as well as genetics and environment. These results indicate that nonheritable influences interact with genetic factors to shape immune variation, with synergistic impacts on the deployment and evolution of defense mechanisms.

Effects of helminths on the human immune response and the microbiome.

Helminths have evolved sophisticated immune regulating mechanisms to prevent rejection by their mammalian host. Our understanding of how the human immune system responds to these parasites remains poor compared to mouse models of infection and this limits our ability to develop vaccines as well as harness their unique properties as therapeutic strategies against inflammatory disorders. Here, we review how recent studies on human challenge infections, self-infected individuals, travelers, and endemic populations have improved our understanding of human type 2 immunity and its effects on the microbiome. The heterogeneity of responses between individuals and the limited access to tissue samples beyond the peripheral blood are challenges that limit human studies on helminths, but also provide opportunities to transform our understanding of human immunology. Organoids and single-cell sequencing are exciting new tools for immunological analysis that may aid this pursuit. Learning about the genetic and immunological basis of resistance, tolerance, and pathogenesis to helminth infections may thus uncover mechanisms that can be utilized for therapeutic purposes.

The Oral, Gut Microbiota and Cardiometabolic Health of Indigenous Orang Asli Communities.

The Orang Asli (OA) of Malaysia have been relatively understudied where little is known about their oral and gut microbiomes. As human health is closely intertwined with the human microbiome, this study first assessed the cardiometabolic health in four OA communities ranging from urban, rural to semi-nomadic hunter-gatherers. The urban Temuan suffered from poorer cardiometabolic health while rural OA communities were undergoing epidemiological transition. The oral microbiota of the OA were characterised by sequencing the V4 region of the 16S rRNA gene. The OA oral microbiota were unexpectedly homogenous, with comparably low alpha diversity across all four communities. The rural Jehai and Temiar PP oral microbiota were enriched for uncharacterised bacteria, exhibiting potential for discoveries. This finding also highlights the importance of including under-represented populations in large cohort studies. The Temuan oral microbiota were also elevated in opportunistic pathogens such as Corynebacterium, Prevotella, and Mogibacterium, suggesting possible oral dysbiosis in these urban settlers. The semi-nomadic Jehai gut microbiota had the highest alpha diversity, while urban Temuan exhibited the lowest. Rural OA gut microbiota were distinct from urban-like microbiota and were elevated in bacteria genera such as Prevotella 2, Prevotella 9, Lachnospiraceae ND3007, and Solobacterium. Urban Temuan microbiota were enriched in Odoribacter, Blautia, Parabacetroides, Bacteroides and Ruminococcacecae UCG-013. This study brings to light the current health trend of these indigenous people who have minimal access to healthcare and lays the groundwork for future, in-depth studies in these populations.

Tinea Imbricata among the Indigenous Communities: Current Global Epidemiology and Research Gaps Associated with Host Genetics and Skin Microbiota.

Tinea imbricata is a unique fungal skin disease that mostly affects indigenous populations in Southeast Asia, Oceania, and Central and South America. The control and management of this disease among these communities are challenging given their remote locations, certain traditional practices, and severe malnutrition status. To date, there are only a handful of reports published globally, which highlights the need for a more holistic approach in addressing this skin disease. Several bodies of evidence and reports have shown that host genetic factors have a profound influence on the pathogenesis of tinea imbricata, while skin microbiota is touted to have a role in the pathogenesis of the disease. However, there are limited studies of how host genetics and skin microbiota impact disease susceptibility in the host. To improve the understanding of this disease and to find possible long-term effective treatment among the affected indigenous communities, a comprehensive literature review is needed. Hence, this review paper aims to present the current status of tinea imbricata among the indigenous communities, together with published findings on the possible underlying reasons for its specific distribution among these communities, particularly on the ways in which host skin microbiota and host genetics affect occurrence and disease patterns. This information provides valuable insights for future research by highlighting the current knowledge gaps in these areas.

Efficacy of triple dose albendazole treatment for soil-transmitted helminth infections.

In Malaysia, soil-transmitted helminth (STH) infections still persist among indigenous communities. In the past, local studies have focused mostly on epidemiologic aspects of STH infections with a scarcity of information on the efficacy of deworming treatment. The present study consisted of 2 phases: a cross-sectional phase on current epidemiological status and risk factors of STH infections and a longitudinal study over 6 weeks on triple dose albendazole efficacy against STH infections. A total of 253 participants were recruited at baseline and a pre-tested questionnaire was administered to obtain information on socio-demographics, environmental and behavioural risk factors. Stool samples were evaluated using a modified Kato-Katz technique. Cure rate (CR) and egg reduction rate (ERR) were assessed at 3 weeks following a 3-day course of 400mg albendazole treatment and infection status were observed again at 6 weeks. Baseline positivity of trichuriasis, ascariasis and hookworm infections were 56.1%, 11.9% and 20.2%, respectively. Multivariate analysis showed age below 18 years old (P = 0.004), without latrine in house (P = 0.042) and indiscriminate defecation (P = 0.032) were associated with STH infections. In the longitudinal study (N = 89), CR for trichuriasis was 64.6%, while CR of 100% was observed for both ascariasis and hookworm. ERR was above 90% for all three STH species. A rapid increased of Trichuris trichiura egg output was observed at 6 weeks. In conclusion, STH infections are highly prevalent among indigenous communities. Children and teenagers, poor sanitation and hygiene behaviour were determinants for STH infections. Triple dose albendazole is found to be efficacious against Ascaris lumbricoides and hookworm infections but has moderate curative effect with high ERR against T. trichiura. Although triple dose albendazole regimen has logistic challenges and may not be a routine option, consideration of this treatment regime may still be necessary in selective communities to reduce high intensity of T. trichiura infection.

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.

Gut microbiome of helminth-infected indigenous Malaysians is context dependent.

BACKGROUND: While microbiomes in industrialized societies are well characterized, indigenous populations with traditional lifestyles have microbiomes that are more akin to those of ancient humans. However, metagenomic data in these populations remains scarce, and the association with soil-transmitted helminth infection status is unclear. Here, we sequenced 650 metagenomes of indigenous Malaysians from five villages with different prevalence of helminth infections. RESULTS: Individuals from villages with higher prevalences of helminth infections have more unmapped reads and greater microbial diversity. Microbial community diversity and composition were most strongly associated with different villages and the effects of helminth infection status on the microbiome varies by village. Longitudinal changes in the microbiome in response to albendazole anthelmintic treatment were observed in both helminth infected and uninfected individuals. Inference of bacterial population replication rates from origin of replication analysis identified specific replicating taxa associated with helminth infection. CONCLUSIONS: Our results indicate that helminth effects on the microbiota were highly dependent on context, and effects of albendazole on the microbiota can be confounding for the interpretation of deworming studies. Furthermore, a substantial quantity of the microbiome remains unannotated, and this large dataset from an indigenous population associated with helminth infections is a valuable resource for future studies. Video Abstract.

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

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

The study of seroprevalence of hepatitis E virus and an investigation into the lifestyle behaviours of the aborigines in Malaysia.

Malaysia is a non-endemic country for hepatitis E virus (HEV) infection. However, seroprevalence as high as 50% among samples of aboriginal people were reported over two decades ago. A total of 207 samples collected from seven aboriginal villages in rural settlements across two states in Malaysia were analysed for anti-HEV IgG and IgM by an enzyme-linked immunoassay. Following the detection of anti-HEV seroprevalence, we organized health outreach to inform and educate the community. Qualitative interviews were conducted with individuals tested positive for anti-HEV antibodies. Data derived from interviews and observations were used to investigate possible lifestyle behaviours associated with HEV infection. Anti-HEV IgG was detected in six samples (5.9%) from the village of Dusun Kubur. Qualitative inquiry and observation study revealed poor dietary and household hygiene, contaminated food and water, contact with animal faeces, unsanitary and domestic waste disposal, and wildlife reservoirs could be the contributing factors for transmission and acquisition of HEV infection. Investigation during health outreach is important to provide insights for future empirical research and implementation for improvement of lifestyle behaviours among the aborigines. Managing the risk of HEV infection in the aborigines may reduce the risk of HEV transmission to the local communities.

Enrichment of gut-derived Fusobacterium is associated with suboptimal immune recovery in HIV-infected individuals.

We explored the gut microbiota profile among HIV-infected individuals with diverse immune recovery profiles following long-term suppressive ART and investigated the relationship between the altered bacteria with markers of immune dysfunction. The microbiota profile of rectal swabs from 26 HIV-infected individuals and 20 HIV-uninfected controls were examined. Patients were classified as suboptimal responders, sIR (n = 10, CD4 T-cell <350 cells/ul) and optimal responders, oIR (n = 16, CD4 T-cell >500 cells/ul) after a minimum of 2 years on suppressive ART. Canonical correlation analysis(CCA) and multiple regression modelling were used to explore the association between fecal bacterial taxa abundance and immunological profiles in optimal and suboptimal responders. We found Fusobacterium was significantly enriched among the HIV-infected and the sIR group. CCA results showed that Fusobacterium abundance was negatively correlated with CD4 T-cell counts, but positively correlated with CD4 T-cell activation and CD4 Tregs. Multiple linear regression analysis adjusted for age, baseline CD4 T-cell count, antibiotic exposure and MSM status indicated that higher Fusobacterium relative abundance was independently associated with poorer CD4 T-cell recovery following ART. Enrichment of Fusobacterium was associated with reduced immune recovery and persistent immune dysfunction following ART. Modulating the abundance of this bacterial taxa in the gut may be a viable intervention to improve immune reconstitution in our setting.

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.