A New Multiplex PCR Assay Reveals the Occurrence of E. bangladeshi alongside E. histolytica and E. moshkovskii in Eastern India.

PURPOSE: Epidemiological studies on amoebic infections are complicated due to morphologically identical and clinically important Entamoeba species. Therefore, newer, simpler, and more economical diagnostic techniques are required for differentiating clinically important Entamoeba species. METHODS: We developed a single-round multiplex PCR assay to identify E. histolytica, E. moshkovskii, E. dispar, E. bangladeshi, and E. coli. Primers were designed based on variations in 18 S rRNA sequences. Sensitivity and specificity were assessed using known positive and negative samples. Furthermore, we screened 472 diarrheal samples using this technique alongside the reference PCR method to evaluate its suitability for epidemiological studies and clinical diagnosis. DNA sequencing and phylogenetic analysis of the isolates were conducted. All statistical analyses of the data were performed using GraphPad Prism. RESULTS: The designed primers successfully yielded species-specific PCR products of different sizes as expected. We did not observe any non-specific amplifications of the primer set. The diagnostic performance was also convincing. After screening clinical samples using the method, we observed that 2.33% (n = 11) tested positive for E. moshkovskii, 1.06% (n = 5) tested positive for E. histolytica, and 0.85% (n = 4) tested positive for E. bangladeshi in the studied area. DNA sequencing further confirmed the identified species. The constructed phylogenetic tree also demonstrated clear separation of the detected species lineages. CONCLUSION: The study suggests the multiplex PCR assay could be a reliable diagnostic tool for amoebic infections. This study is particularly significant as it marks the first reported occurrence of E. bangladeshi since its documentation in South Africa and its native Bangladesh.

A rapid economical multiplex PCR-RFLP method for molecular detection and genotyping of Giardia duodenalis clinical isolates.

Giardia duodenalis is a common cause of diarrheal illness in regions with limited resources. The demand for rapid and cost-effective detection and genotyping methods in large-scale epidemiological studies and clinical diagnostics is imperative. Hence, we developed a multiplex PCR-RFLP technique targeting the tpi gene of G. duodenalis. The assay successfully screened G. duodenalis positive clinical samples (6.33 %; 36/565). It was also able to categorize the isolates into assemblages A (41.66 %; 13/36) and B (58.33 %; 23/36), as well as into subassemblages: AI (13.8 %; 5/36), AII (27.77 %; 10/36), BIII (36.11 %; 15/36) and BIV (22.22 %; 8/36). High diagnostic sensitivity (94.2 %), specificity (100 %) and accuracy (97.1 %) of the PCR assay were obtained, indicating its reliability for diagnosing giardiasis. Notably, the assay demonstrated close concordance with microscopy (κ=0.85) and reference PCR (κ=0.98) results. The optimized method offers a cost-effective and rapid approach for G. duodenalis detection and genotyping, convenient for epidemiological studies and clinical diagnostics.

Andrographolide induced cytotoxicity and cell cycle arrest in Giardia trophozoites.

Giardiasis is a prevalent parasitic diarrheal disease caused by Giardia lamblia, affecting people worldwide. Recently, the availability of several drugs for its treatment has highlighted issues such as multidrug resistance, limited effectiveness and undesirable side effects. Therefore, it is necessary to develop alternative new drugs and treatment strategies that can enhance therapeutic outcomes and effectively treat giardiasis. Natural compounds show promise in the search for more potent anti-giardial agents. Our investigation focused on the effect of Andrographolide (ADG), an active compound of the Andrographis paniculata plant, on Giardia lamblia, assessing trophozoite growth, morphological changes, cell cycle arrest, DNA damage and inhibition of gene expression associated with pathogenic factors. ADG demonstrated anti-Giardia activity almost equivalent to the reference drug metronidazole, with an IC50 value of 4.99 µM after 24 h of incubation. In cytotoxicity assessments and morphological examinations, it showed significant alterations in trophozoite shape and size and effectively hindered the adhesion of trophozoites. It also caused excessive ROS generation, DNA damage, cell cycle arrest and inhibited the gene expression related to pathogenesis. Our findings have revealed the anti-giardial efficacy of ADG, suggesting its potential as an agent against Giardia infections. This could offer a natural and low-risk treatment option for giardiasis, reducing the risk of side effects and drug resistance.

Genetic characterization of the Entamoeba moshkovskii population based on different potential genetic markers.

Entamoeba moshkovskii, according to recent studies, appears to exert a more significant impact on diarrhoeal infections than previously believed. The efficient identification and genetic characterization of E. moshkovskii isolates from endemic areas worldwide are crucial for understanding the impact of parasite genomes on amoebic infections. In this study, we employed a multilocus sequence typing system to characterize E. moshkovskii isolates, with the aim of assessing the role of genetic variation in the pathogenic potential of E. moshkovskii. We incorporated 3 potential genetic markers: KERP1, a protein rich in lysine and glutamic acid; amoebapore C (apc) and chitinase. Sequencing was attempted for all target loci in 68 positive E. moshkovskii samples, and successfully sequenced a total of 33 samples for all 3 loci. The analysis revealed 17 distinct genotypes, labelled M1­M17, across the tested samples when combining all loci. Notably, genotype M1 demonstrated a statistically significant association with diarrhoeal incidence within E. moshkovskii infection (P = 0.0394). This suggests that M1 may represent a pathogenic strain with the highest potential for causing diarrhoeal symptoms. Additionally, we have identified a few single-nucleotide polymorphisms in the studied loci that can be utilized as genetic markers for recognizing the most potentially pathogenic E. moshkovskii isolates. In our genetic diversity study, the apc locus demonstrated the highest Hd value and π value, indicating its pivotal role in reflecting the evolutionary history and adaptation of the E. moshkovskii population. Furthermore, analyses of linkage disequilibrium and recombination within the E. moshkovskii population suggested that the apc locus could play a crucial role in determining the virulence of E. moshkovskii.

Multi-locus sequence analysis reveals phylogenetically segregated Entamoeba histolytica population.

Amoebiasis, caused by the enteric parasite, Entamoeba histolytica, is one of the major food- and water-borne parasitic diseases in developing countries with improper sanitation and poor hygiene. Infection with E. histolytica has diverse disease outcomes, which are determined by the genetic diversity of the infecting strains. Comparative genetic analysis of infecting E. histolytica strains associated with differential disease outcomes from different geographical regions of the world is important to identify the specific genetic patterns of the pathogen that trigger certain disease outcomes of Amoebiasis. The strategy is able to elucidate the genealogical relation and population structure of infecting E. histolytica strains from different geographical regions. In the present study, we have performed a comparative genetic analysis of circulating E. histolytica strains identified from different parts of the world, including our study region, based on five tRNA-linked short tandem repeat (STR) loci (i.e., D-A, NK2, R-R, STGA-D and A-L) and evaluated their potential associations with differential disease outcomes of Amoebiasis. A number of regional-specific, emerging haplotypes of E. histolytica, significantly associated with specific disease outcomes have been identified. Haplotypes, which have a significant positive association with asymptomatic and amoebic liver abscess outcomes, showed a significant negative association with diarrheal outcome, or vice versa. Comparative multi-locus analysis revealed that E. histolytica isolates from our study region are phylogenetically segregated from the isolates of other geographical regions. This study provides a crucial overview of the population structure and emerging pattern of the enteric parasite, E. histolytica.

Short tandem repeat (STR) based sequence typing of Entamoeba histolytica identifies STGA-D locus as a genetic marker, associated with disease outcomes.

Amoebiasis, caused by the enteric parasite Entamoeba histolytica has differential disease outcomes. The association of parasite genotypes with outcomes of amoebic infection is still a paradox and requires to be explored. The genetic information of infecting strains from endemic settings of different geographical regions is essential to evaluate the relation. Comparative genetics of E. histolytica clinical isolates from different disease outcomes have been explored based on two tRNA-linked STR loci (STGA-D and A-L). All of the repeat patterns in the A-L locus were newly identified and unique to Indian isolates. The majority of newly identified repeat patterns in STGA-D locus have outcome-specific distributions, predicting the emergence of disease-specific mutations in this target locus. Statistical analysis further reinforces this observation, as identified repeat patterns only from STGA-D but not A-L locus were significantly associated with disease outcomes. Phylogenetic analysis indicates independent segregation and divergence of tRNA-linked STR arrays for each STR locus.

Phylogenetic analyses of Chilomastix and Retortamonas species using in vitro excysted flagellates.

In vitro excystation of cysts of microscopically identified Chilomastix mesnili and Retortamonas sp. isolated from Japanese macaques and Retortamonas sp. isolated from small Indian mongooses could be induced using an established protocol for Giardia intestinalis and subsequently by culturing with H2S-rich Robinson's medium supplemented with Desulfovibrio desulfuricans. Excystation usually began 2 h after incubation in Robinson's medium. DNA was isolated from excysted flagellates after 4 h of incubation or from cultured excysted flagellates. Phylogenetic analysis based on their 18S rRNA genes revealed that two isolates of C. mesnili from Japanese macaques belonged to the same cluster as a C. mesnili isolate from humans, whereas a mammalian Retortamonas sp. isolate from a small Indian mongoose belonged to the same cluster as that of an amphibian Retortamonas spp. isolate from a 'poison arrow frog' [sequence identity to AF439347 (94.9%)]. These results suggest that the sequence homology of the 18S rRNA gene of the two C. mesnili isolates from Japanese macaques was similar to that of humans, in addition to the morphological similarity, and Retortamonas sp. infection of the amphibian type in the small Indian mongoose highlighted the possibility of the effect of host feeding habitats.

Genotyping and epidemiological distribution of diarrhea-causing isolates of Giardia duodenalis in southeastern part of West Bengal, India.

The prevalence and genetic diversity of the protozoan pathogen Giardia duodenalis have been extensively studied worldwide. There is currently a lack of data regarding the genetic variability of the organism in eastern India. Understanding the circulating genotypes and associated risk factors is crucial for effective planning and implementing control measures. Therefore, the objective of the study was to conduct an epidemiological study to determine the prevalence and identify the various genotypes present. This survey adds to our knowledge on the occurrence and distribution of Giardia genotypes in the studied region. The overall prevalence was found to be 6.8%. This parasitic infection was significantly associated with two age groups, i.e., >0-5 years and >5-12 years. Using a multilocus genotyping method, we genotyped 52 human Giardia isolates that were obtained from diarrheal patients. Two distinct assemblages were found in the population-30.8% belonged to assemblage A; 63.5% belonged to assemblage B, prevalent in the population; and 5.7% belonged to a combined assemblage A+B. Sub-assemblage AII was found in 17.3% of the cases, followed by sub-assemblage AI (13.5%). High levels of genetic diversity were found within the population of assemblage B undergoing balancing selection. Overall, the high prevalence of the parasite observed, particularly among children, raises a major concern and necessitates implementation of robust control measures. Furthermore, we report the presence of numerous unique genotypes, circulating in this limited geographical boundary, which can be useful dataset for future studies.

Low Prevalence of Soil-Transmitted Helminth Infections among Children in the States of Chhattisgarh and Himachal Pradesh, India, 2018.

Large-scale impact assessments of soil-transmitted helminth (STH) programs are essential for determining the frequency of mass drug administration (MDA). In baseline surveys, the prevalence of STHs in the Indian States of Chhattisgarh and Himachal Pradesh was 80.2% in 2015 and 29.0% in 2016, respectively. In 2018, we estimated the prevalence and intensity of STHs after six rounds of biannual MDA in Chhattisgarh and annual MDA in Himachal Pradesh. We conducted multistage cluster sampling surveys in preschool-age children (PSAC), school-age children (SAC), and adolescent cohorts. Stool samples from 3,033 respondents (PSAC, n = 625; SAC, n = 1,363; adolescents, n = 1,045) in Chhattisgarh and 942 respondents (PSAC, n = 192; SAC, n = 388; adolescents, n = 362) in Himachal Pradesh were examined for presence of STH infection using the Kato-Katz method. The overall cluster-adjusted prevalence in Chhattisgarh was 11.6% among all age groups (95% CI, 5.6-22.4)-an 85.5% reduction in the prevalence since 2015. Prevalence was not significantly different across cohorts (PSAC, 11.0% [95% CI, 5.0-22.6]; SAC, 10.9% [95% CI, 5.2-21.6]; adolescents, 12.8% [95% CI, 6.2-24.5]). Ascaris lumbricoides was the most common helminth, with most infections of light intensity. In Himachal Pradesh, only three STH infections were detected in 2018, resulting in a cluster-adjusted prevalence of 0.3% (95% CI, 0.1-1.7)-a 99.0% reduction in prevalence since 2016. All infections were of light intensity. Both states showed substantial improvements in socioeconomic and water, sanitation, and hygiene (WASH) indicators since the baseline surveys. Extensive reductions in prevalence and intensity are linked to sustained, high deworming coverage, as well as socioeconomic WASH indicators.

Functional characterization of phospholipase B enzyme from Giardia lamblia.

The microaerotolarent amitochondriate protozoan Giardia lamblia causes Giardiasis and produces a unique enzyme called Phospholipase B (PLB) in contrast to higher eukaryotes. The enzyme is produced upon induction with oxidative (H2O2) stress, thus leading to prostaglandin E2 (PGE2) production. It exists in dimeric form, and its molecular weight is 56 kDa. This PLB was extracellularly cloned in the pET21d vector. The ORF is 1620 bp (Genbank accession no. -OM939681) long and codes for a protein 539 amino acid long, with a 15 amino acid long amino-terminal signal peptide. The highest enzyme activity of PLB was identified at pH 7.5 and 35 °C. This specific enzyme was also active at 50 °C pH 10, but activity was low. We also analyzed the expression of PLB protein in G. lamblia, which was significantly induced under increased oxidative stress.

Investigating genetic polymorphism in E. histolytica isolates with distinct clinical phenotypes.

Amoebiasis is an infection caused by enteric protozoa, most commonly Entamoeba histolytica, and is globally considered a potentially severe and life-threatening condition. To understand the impact of the parasite genome on disease outcomes, it is important to study the genomes of infecting strains in areas with high disease prevalence. These studies aim to establish correlations between parasite genotypes and the clinical presentation of amoebiasis. We employ a strain typing approach that utilizes multiple loci, including SREHP and three polymorphic non-coding loci (tRNA-linked array N-K2 and loci 1-2 and 5-6), for high-resolution analysis. Distinct clinical phenotype isolates underwent amplification and sequencing of studied loci. The nucleotide sequences were analysed using Tandem Repeats Finder to detect short tandem repeats (STRs). These patterns were combined to assign a genotype, and the correlation between clinical phenotypes and repetitive patterns was statistically evaluated. This study found significant polymorphism in the size and number of PCR fragments at SREHP and 5-6 locus, while the 1-2 locus and NK2 locus showed variations in PCR product sizes. Out of 41 genotypes, two (I6 and I41) were significantly associated with their respective disease outcomes and were found in multiple isolates. We observed that I6 was linked with a symptomatic outcome, with a statistically significant p-value of 0.0183. Additionally, we found that I41 was associated with ALA disease outcome, with a p-value of 0.0089. Our study revealed new repeat units not previously reported, unveiling the genetic composition of E. histolytica strains in India, associated with distinct disease manifestations.

First case report of Cyclosporiasis from eastern India: Incidence of Cyclospora cayetanensis in a patient with unusual diarrheal symptoms.

Cyclospora cayetanensis, a recently described coccidian parasite causes severe gastroenteric disease worldwide. Limited studies are found on the incidence of C. cayetanensis infection from India; hence remains largely unknown. To date, no case of cyclosporiasis from eastern India has been reported. In this study, we described an incidental case of C. cayetanensis in a 30 years old Bengali female patient with no travel history from eastern India. In June 2022, the patient presented with a history of diarrhoea persisting for more than two months with continuous passage foul smelling stools for which she took multiple antibiotics that were ineffective. There were no Salmonella, Shigella, or Vibrio-like organisms in the patient's faecal sample, and Toxin A/B of Clostridium difficile was also not detected by ELISA. The patient was HIV-negative. Finally, UV autofluorescence and DNA-based diagnosis confirmed the presence of C. cayetanensis, and the treatment with a combination of appropriate antibiotics was successful. This case report could raise awareness about C. cayetanensis associated diarrhoeal cases in India.

Prevalence and molecular characterization of Entamoeba moshkovskii in diarrheal patients from Eastern India.

BACKGROUND: Importance of the amphizoic amoeba Entamoeba moshkovskii is increasing in the study of amoebiasis as a common human pathogen in some settings. Limited studies are found on the genetic and phylogenetic characterization of E. moshkovskii from India; hence remain largely unknown. In this study, we determined the prevalence and characterized the E. moshkovskii isolates in eastern India. METHODS: A three-year systemic surveillance study among a total of 6051 diarrhoeal patients from ID Hospital and BC Roy Hospital, Kolkata was conducted for E. moshkovskii detection via a nested PCR system targeting 18S rRNA locus. The outer primer set detected the genus Entamoeba and the inner primer pair identified the E. moshkovskii species. The 18S rRNA locus of the positive samples was sequenced. Genetic and phylogenetic structures were determined using DnaSP.v5 and MEGA-X. GraphPad Prism (v.8.4.2), CA, USA was used to analyze the statistical data. RESULT: 4.84% (95%CI = 0.0433-0.0541) samples were positive for Entamoeba spp and 3.12% (95%CI = 0.027-0.036) were infected with E. moshkovskii. E. moshkovskii infection was significantly associated with age groups (X2 = 26.01, P<0.0001) but not with gender (Fisher's exact test = 0.2548, P<0.05). A unique seasonal pattern was found for E. moshkovskii infection. Additionally, 46.56% (95%CI = 0.396-0.537) were sole E. moshkovskii infections and significantly associated with diarrheal incidence (X2 = 335.5,df = 9; P<0.0001). Sequencing revealed that the local E. moshkovskii strains were 99.59%-100% identical to the prototype (GenBank: KP722605.1). The study found certain SNPs that showed a correlation with clinical features, but it is not necessarily indicative of direct control over pathogenicity. However, SNPs in the 18S rRNA gene could impact the biology of the amoeba and serve as a useful phylogenetic marker for identifying pathogenic E. moshkovskii isolates. Neutrality tests of different coinfected subgroups indicated deviations from neutrality and implied population expansion after a bottleneck event or a selective sweep and/or purifying selection in co-infected subgroups. The majority of FST values of different coinfected subgroups were <0.25, indicating low to moderate genetic differentiation within the subgroups of this geographical area. CONCLUSION: The findings reveal the epidemiological significance of E. moshkovskii infection in Eastern India as the first report in this geographical area and expose this species as a possible emerging enteric pathogen in India. Our findings provide useful knowledge for further research and the development of future control strategies against E. moshkovskii.

Development of a simple PCR-RFLP technique for detection and differentiation of E. histolytica, E. dispar and E. moshkovskii.

Epidemiological studies on amoebic infections are complicated by morphological overlap between the pathogenic E. histolytica, the commensal E. dispar and the amphizoic E. moshkovskii, necessitating molecular identification. The present study developed a simple and economical 18S PCR-RFLP method for the simultaneous detection and differentiation of the three species. PCR products were differentiated by Tat1 restriction digestion generating three different RFLP patterns. Validation was conducted by screening 382 faecal samples from human patients from Kolkata, India, hospitalized for diarrhoea. Analysis indicated that the PCR-RFLP could successfully differentiate between the three species and was confirmed by sequence analysis. This method could prove useful for clinical and epidemiological studies of amoebiasis.

Molecular evidence suggests the occurrence of Entamoeba moshkovskii in pigs with zoonotic potential from eastern India.

Entamoeba moshkovskii Tshalaia, 1941 is prevalent in developing countries and it is considered to be primarily a free-living amoeba, which is morphologically indistinguishable, but biochemically and genetically different from the human infecting, pathogenic Entamoeba histolytica Schaudinn, 1903. The pathogenic potential of this organism is still under discussion. Entamoeba moshkovskii in human stool samples has been reported in different countries such as the United States, Italy, Australia, Iran, Turkey, Bangladesh, India (Pondicherry), Indonesia, Colombia, Malaysia, Tunisia, Tanzania and Brazil, but no data are available about the occurrence of E. moshkovskii in farm animals. This study provides data on the occurrence of E. moshkovskii in pigs in a total of 294 fresh faecal samples collected from five different regions in Kolkata, West Bengal, India. Stool samples were tested by nested PCR using primers targeting SSU rDNA of E. moshkovskii. The amplified PCR products were further confirmed by RFLP technique. Purified nested PCR products were also sequenced and identified via BLAST program run on the NCBI website to confirm species along with their genetic characteristics of the E. moshkovskii isolates. Overall 5.4 % samples were identified as E. moshkovskii positive. Results of this study demonstrate that swine can host E. moshkovskii and should be considered as a potential natural reservoir for E. moshkovskii. However, the occurrence of E. moshkovskii infection in pigs was not statistically associated with their faecal consistency, sex and developmental stage.

Epidemiology of Soil-Transmitted Helminth Infections among Primary School Children in the States of Chhattisgarh, Telangana, and Tripura, India, 2015-2016.

Soil-transmitted helminth (STH) infections are highly prevalent in many developing countries, affecting the poorest and most deprived communities. We conducted school-based surveys among children studying in first to fifth standard in government schools in the Indian States of Chhattisgarh, Telangana, and Tripura to estimate the prevalence and intensity of STH infections during November 2015 and January 2016. We adopted a two-stage cluster sampling design, with a random selection of districts within each agro-climatic zone in the first stage. In the second stage, government primary schools were selected by probability proportional to size method from the selected districts. We collected information about demographic details, water, sanitation, and hygiene (WASH) characteristics and stool samples from the school children. Stool samples were tested using Kato-Katz method. Stool samples from 3,313 school children (Chhattisgarh: 1,442, Telangana: 1,443, and Tripura: 428) were examined. The overall prevalence of any STH infection was 80.2% (95% confidence interval [CI]: 73.3-85.7) in Chhattisgarh, 60.7% (95% CI: 53.8-67.2) in Telangana, and 59.8% (95% CI: 49.0-69.7) in Tripura. Ascaris lumbricoides was the most prevalent STH infection in all three states. Most of the STH infections were of light intensity. Our study findings indicate that STH infections were highly prevalent among the school children in Chhattisgarh, Telangana, and Tripura, indicating the need for strengthening STH control program in these states. The prevalence estimates from the survey would serve as a baseline for documenting the impact of the National Deworming Day programs in these states.

Molecular Identification of Cryptosporidium viatorum Infection in a Patient Suffering from Unusual Cryptosporidiosis in West Bengal, India.

In this study, we have collected and screened a total of 268 stool samples from diarrheal patients admitted to an Infectious disease hospital in Kolkata for the presence of Cryptosporidium spp. The initial diagnosis was carried out by microscopy followed by genus specific polymerase chain reaction assays based on 70 kDa heat shock proteins (HSP70). DNA sequencing of the amplified locus has been employed for determination of genetic diversity of the local isolates. Out of 268 collected samples, 12 (4.48%) were positive for Cryptosporidium spp. Sequences analysis of 70 kDa heat shock proteins locus in 12 Cryptosporidium local isolates revealed that 2.24% and 1.86% of samples were showing 99% to 100% identity with C. parvum and C. hominis. Along with the other 2 major species one recently described globally distributed pathogenic species Cryptosporidium viatorum has been identified. The HSP70 locus sequence of the isolate showed 100% similarity with a previously described isolate of C. viatorum (Accession No. JX978274.1, JX978273.1, and JN846706.1) present in GenBank.

Multilocus sequence typing (MLST) of Entamoeba histolytica identifies kerp2 as a genetic marker associated with disease outcomes.

Amoebiasis caused by protozoan parasite Entamoeba histolytica has diverse infection outcomes. The relationship between parasite genotypes and outcome of amoebic infection is still a paradox and needs to be explored. Genome information of infecting strains from endemic areas throughout the world is essential to explore this relation. Comparative genetics between E. histolytica populations from different disease outcomes have been studied to identify potential genetic markers having single nucleotide polymorphisms (SNPs) significantly associated with specific clinical outcome. Coding and non-coding regions have significantly different rates of polymorphism. Non-synonymous base substitutions were significantly more frequent than synonymous within coding loci. Both synonymous and non-synonymous SNPs within lysine- and glutamic acid rich protein 2 (kerp2) locus were significantly associated with disease outcomes. An incomplete linkage disequilibrium (LD) value with potential recombination events and significant population differentiation (FST) value have also been identified at kerp2 locus within the study population. Presence of disease specific SNPs, potential recombination events, and significant FST value at kerp2 locus indicate that kerp2 gene and its gene product are under constant selection pressure exerted by host on parasite and could also be a potential determinant of disease outcome of E. histolytica infection. Furthermore, E. histolytica isolated from asymptomatic carriers are phylogenetically closer to those causing liver abscess in human and exhibit potential inter-population recombination among them. Individuals with persistent asymptomatic E. histolytica infection may be under high risk of developing amoebic liver abscess formation in future and detailed investigation of asymptomatic individuals from endemic areas should be always required.

Genetic characterization reveals evidence for an association between water contamination and zoonotic transmission of a Cryptosporidium sp. from dairy cattle in West Bengal, India.

Cryptosporidium sp. is an enteric parasite with zoonotic potential, and can infect a wide range of vertebrates, including human. Determining the source of infection and the mode of transmission in a new endemic region is crucial for the control of cryptosporidiosis. In the present study, we have assessed the importance of dairy cattle as a potential source of Cryptosporidium infection for humans in a newly recognized endemic region. Cryptosporidium isolates from dairy calves, humans (farm workers) and nearby water bodies were genetically characterized based on 18SrRNA and hsp70 genes. A high incidence of Cryptosporidium infection was identified in our study region. This finding is of public health concern. Cryptosporidium ryanae rather than Cryptosporidium parvum has been identified as the most prevalent infecting species in the study region. Infections were associated with clinical symptoms of infected animals. An incomplete linkage disequilibrium (LD) value with potential recombination events at 18SrRNA locus were identified for the first time in C. ryanae, which was previously reported as a clonal population. Phylogenetic analysis revealed the presence of identical genotypes of a Cryptosporidium sp. from dairy calves, farm workers and nearby water bodies and indicates an association between water contamination and zoonotic transmission of Cryptosporidiosis in our study region.