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Prevalence and molecular characterization of Entamoeba moshkovskii in diarrheal patients from Eastern India [1]

['Sanjib K. Sardar', 'Division Of Parasitology', 'Icmr-National Institute Of Cholera', 'Enteric Diseases', 'Icmr-Niced', 'Kolkata', 'Ajanta Ghosal', 'Tapas Haldar', 'Maimoon Maruf', 'Koushik Das']

Date: 2023-05

Abstract 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 F ST 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.

Author summary Although Entamoeba genus consists of many different species, not all are pathogenic. Entamoeba histolytica is the only species recognized as a definite pathogen associated with intestinal and extraintestinal infections. Nowadays, the importance of other morphologically identical Entamoeba species, like amphizoic E. moshkovskii is increasing, as it has been reported in human patients over the years from different countries. Investigations into the pathogenic potential of E. moshkovskii are ongoing, and only limited studies have been conducted on genetic characterization from India. This study reveals the epidemiology and population structure of E. moshkovskii in diarrhoeal patients from eastern India. Our study showed that the prevalence of E. moshkovskii in diarrhoeal patients is higher than E. histolytica in the studied region. In addition, there was a unique seasonal pattern, found for the infection. The high prevalence rate of sole infection in patients suggests that it is one of the definite etiological agents for diarrhoeal disease in eastern India. The further study identified two SNPs in the 18S rRNA locus, significantly associated with the sole infection cases and hence they can be regarded as a marker for identifying pathogenic isolates of E. moshkovskii. Many novel genotypes were obtained in the study indicating high genetic diversity in E. moshkovskii population conferring adaptability in a changing environment. Further research is needed to ensure proper control measures for this infection for better health care.

Citation: Sardar SK, Ghosal A, Haldar T, Maruf M, Das K, Saito-Nakano Y, et al. (2023) Prevalence and molecular characterization of Entamoeba moshkovskii in diarrheal patients from Eastern India. PLoS Negl Trop Dis 17(5): e0011287. https://doi.org/10.1371/journal.pntd.0011287 Editor: Maria Fantinatti, Fundacao Oswaldo Cruz, BRAZIL Received: October 12, 2022; Accepted: April 5, 2023; Published: May 11, 2023 Copyright: © 2023 Sardar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: Representative sequences obtained in this study were deposited in GenBank under the accession numbers ON965383 - ON965450. Funding: The National Institute of Infectious Diseases (NIID), Tokyo, Japan, and the Indian Council of Medical Research (ICMR), Government of India, provided grant funding to Dr. Sandipan Ganguly. Sanjib K. Sardar received financial support through the ICMR Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Amoebiasis, an infection by enteric protozoa, most commonly Entamoeba histolytica is globally considered a potentially severe and life-threatening infection [1]. The motile form of the parasite, the trophozoite inhabits the lumen of the large intestine, where it multiplies and differentiates into a resistant form. These cysts are released into the environment [1,2]. Consecutively, this resistant form is responsible for the transmission of the infection to another host via the faecal-oral route. Together with the pathogenic species Entamoeba histolytica, the genus Entamoeba includes many other species that reside in the human intestinal lumen, namely E. dispar, E. moshkovskii, E. bangladeshi, E. polecki, E. coli, and E. hartmanni [3]. In many cases, infections with the trophozoites of Entamoeba spp. can result in harmless colonization in the intestinal lumen of asymptomatic carriers, passing cysts in their stool (non-invasive infection). In other cases, the trophozoites invade the intestinal mucosa (intestinal disease) and, through the bloodstream, reach extraintestinal sites in other tissues such as the liver, brain, and lungs (extraintestinal disease) with consequential pathologic manifestations [4]. Abdominal pain, diarrhoea, nausea, vomiting and flatulence are the acute symptoms of amoebiasis caused by the eukaryotic parasite Entamoeba histolytica [4,5,6]. Other Entamoeba species are mostly commensals or are said to rarely infect humans [7]. Amoebic infection is one of the most prevalent parasitic diseases in India and worldwide [8]. It is significantly associated with poor sanitation and socioeconomic status than the geographical location’s climate [9]. In most cases, amoebiasis is routinely diagnosed by light microscopy of a wet smear or stained stool samples [10]. This technique is inexpensive and simple, but it has limited sensitivity and specificity, such as being unable to differentiate between the cyst and trophozoites of the pathogenic species Entamoeba histolytica and the rest commensal species like Entamoeba dispar and the newly identified Entamoeba bangladeshi and the amphizoic amoeba Entamoeba moshkovskii which sporadically infects humans [3,10,11]. These four species are genetically related and morphologically indistinguishable with different biochemical features [3]. Amoebiasis affects approximately 50 million people in tropical regions and nearly 100,000 deaths are reported annually [12]. After malaria and schistosomiasis, it is the third foremost parasitic cause of death in humans [13]. Although all deaths could be due to invasive E. histolytica infestation, the prevalence is overestimated due to its epidemiological overlap with other morphologically indistinguishable species, specifically Entamoeba dispar and Entamoeba moshkovskii [14]. Moreover, cysts of another nonpathogenic amoeba, Entamoeba hartmanni can be mystified with E. histolytica under the microscope which is even smaller in size (> 10 μm) [15–16]. In many geographical areas, the actual prevalence of each species is not well characterized particularly for E. moshkovskii, as some reports suggest that it has a potential role in provoking human disease [17]. So, it is important to understand the molecular epidemiology of E. moshkovskii in endemic countries in the study of amoebiasis and it has become crucial in the last decade. Entamoeba moshkovskii was first described as a distinct species from Moscow by Tshalaia in 1941. It was primarily considered to be a free-living environmental strain and still regarded as a common protozoan species found in anoxic sediments to brackish coastal pools. It is osmotolerant in nature and can be cultured in various media suitable for intestinal protozoa, in which it grows easily at temperatures of 10–15° C and 37° C [18–19]. Although all the characteristics differentiate E. moshkovskii from E. histolytica and E. dispar, the entire life-cycle, including excystment and metacystic development, closely resemble E. histolytica and E. dispar. The size of the amoeba trophozoites varies from 10 to 120μ, with an average of 25μ and cysts vary from 5 to 16 μ, with an average of 10μ [19]. In 1961, an E. histolytica-like strain was obtained from a resident of Laredo, Texas, who suffered from diarrhoea, weight loss, and epigastric pain and the strain was named E. histolytica Laredo strain which shared many biological characteristics with E. moshkovskii. Both the Laredo strain and E. moshkovskii grew easily at room temperature, were osmotolerant, and resistant to drugs used in the chemotherapy of amoebiasis, for instance, emetine [20]. Subsequent molecular studies revealed that the E. histolytica Laredo strain is E. moshkovskii, the first human isolate of E. moshkovskii [20–21]. Nowadays importance of E. moshkovskii is increasing in the study of amoebiasis, and it is reported as a common Entamoeba infection in humans in some settings. Colonization of E. moshkovskii in human hosts has been reported in countries such as the United States, Italy, Iran, Turkey, Bangladesh, India (Pondicherry), Kenya, Australia, Indonesia, Colombia, Malaysia, Tunisia, Tanzania and Brazil [22–34]. Most of the stool samples in these studies were submitted to clinical microbiology laboratories from patients with gastrointestinal complications, indicating that E. moshkovskii might be associated with pathogenicity. In India (Pondicherry), it is reported that E. moshkovskii cannot invade intestinal mucosa and does not have any ingested erythrocytes, unlike that E histolytica. [29]. In HIV-1-infected persons in northern Tanzania, E. moshkovskii is also not associated with clinical indicators. But in Bangladesh, this species has been identified as the only likely pathogen in individuals with gastrointestinal clinical manifestations, including dysentery [26,27]. However, in these patients, no studies of viral or bacterial agents were conducted to rule out other pathogens or potential pathogens. Another study in Bangladesh by Shimokawa et al, 2012 also pointed out a possible cause of diarrhoea in infants, which was due to E. moshkovskii infection [27]. While in Malaysia, it was isolated from both symptomatic and asymptomatic cases [28]. In the murine model of intestinal amebiasis, E. moshkovskii also caused diarrhoea, weight loss, and colitis [27]. Thus, the pathogenicity of E. moshkovskii in humans remains unclear. For parasite identification, phylogeny and genetic characterization, small subunits of nuclear ribosomal RNA (18S rRNA) loci are recognized as potential targets [35–38]. Therefore, amplification and sequencing of this gene is being extensively used for decades [2,12,26,31–32]. Moreover, genetic variations based on 18S rRNA can be significant in pathogenicity for parasites. To date, limited studies have been conducted on the genetic and phylogenetic characterization of E. moshkovskii in India [29] and remain largely unknown. In this study, we aimed to determine the epidemiology and molecular characterization based on 18S rRNA of amphizoic amoeba Entamoeba moshkovskii in human stool samples from an active surveillance study on enteric pathogens in and around Kolkata, India. We have also investigated the level of genetic diversity and established the genetic structure among the obtained local isolates using a molecular analysis tool.

Material and methods Ethical statement The ethical clearance of this study was reviewed and approved by the Institutional Human Ethics Committee of the Indian Council of Medical Research-National Institute of Cholera and Enteric Diseases (IRB Number: A-1/2015-IEC). Written informed consent statements were obtained from every participant. In the case of children, voluntary written informed consent statements were taken from their caregivers (parent/guardian). Study area and population This is a hospital-based systemic surveillance study conducted from March 2017 to February 2020. The target population is patients from different parts of Kolkata and adjacent areas admitted to Infectious Disease & Beliaghata General Hospital and Dr. B C Roy Post Graduate Institute of Paediatric Sciences Hospital, Kolkata, with diarrheal complaints. These two hospitals are referral centres for the treatment of diarrheal diseases along with government health care facilities. Non-diarrhoeal cases have not been included in this study. Sample collection and microscopy This is a hospital-based systemic surveillance study conducted among the patients admitted to Infectious Disease & Beliaghata General Hospital and Dr B C Roy Post Graduate Institute of Paediatric Sciences Hospital, Kolkata, India, with diarrheal complaints from March 2017 to February 2020. These two hospitals are referral centres for the treatment of diarrheal diseases along with government health care facilities. Non-diarrhoeal cases have not been included in this study. This study collected and screened a total of 3258 samples from ID Hospital and 2793 from B C Roy Hospital. The faecal samples were collected from the patients admitted to the hospital by trained medical professionals in the presence of attending physicians on the first day of hospitalisation and before antibiotic therapy. Samples were collected in a sterile container with a unique identification number. Once the samples were collected, they were immediately sent to laboratories for testing after written informed consent was obtained from patients. All stool samples were microscopically examined in triplicate in saline and iodine wet mounts. Trichome staining also has been used for Entamoeba moshkovskii identification. The Uninuclear, binuclear, trinuclear, or tetranuclear cysts or trophozoites of Entamoeba spp observed were recorded. DNA extraction DNA was isolated directly from clinical samples using STOOL DNA Minikit (QIAGEN, USA) as per the manufacturer’s protocol, followed by genetic identification through PCR amplification via species-specific primers. For molecular detection of parasites, species-specific primers have been generated using Primer3 Software. Primer design We have designed a nested PCR system targeting the 18S rRNA gene to detect E. moshkovskii from stool samples. The first set of primer (outer primer set) was used to amplify the 18S rRNA locus of the Entamoeba genus (Genus specific PCR assay), and the second set of primer (Nested primer set) was used for the identification of E. moshkovskii species only. The forward and reverse primers of the Entamoeba genus were designed from highly conserved regions of 18S rRNA locus of five phylogenetically close Entamoeba species viz. E. histolytica, E. dispar, E. moshkovskii, E. bangladeshi and E. nuttalli. The following sequences were analysed: E. histolytica, GenBank accession no. X56991 (1947 bp); Entamoeba dispar, GenBank accession no. Z49256 (1949 bp); E. moshkovskii, GenBank accession no. AF149906 (1944 bp); Entamoeba bangladeshi, GenBank accession no. KR025411 (1927 bp); Entamoeba nuttalli, GenBank accession no. AB485592.1 (2431 bp); Entamoeba coli, GenBank accession no. AF149914 (2101 bp); Entamoeba chattoni, GenBank accession no. AF149912 (1963 bp); Entamoeba polecki, GenBank accession no. AF149913 (1858 bp); E. invadens, GenBank accession no. AF149905 (1965 bp). The sequences were aligned using the ClustalW program (https://www.genome.jp/tools-bin/clustalw). Primer 3 online software was used to design the primers. Primer length and melting temperature were considered. Primer sequences specific for Entamoeba spp. were as follows: EntaS_F: CTGCCAGTATTATATGCTGATGTT and EntaS_R: TCTCCTTCCTCTAAATAAGGAGATTTA. The ability of genus-specific primer to amplify the 18S rRNA of the five Entamoeba species was verified using genomic DNA preparation. In the second round, a nested primer set was designed to amplify the E. moshkovskii species DNA fragment of the same gene. The nested E. moshkovskii-specific primer sequences were EM_779bpNF: AACTAACGAAGGAGATGAAGTGAG and EM_779bpNR: GCCAGAGACATCGATTAAAATG. PCR amplification The genus-specific PCR assay was adjusted to ensure the amplification of each target. We performed PCR amplification in a final volume of 50 mL containing 1X PCR buffer and 1U of Biotaq DNA polymerase (Bioline, UK) to obtain the primary PCR product. After MgCl 2 concentrations ranging from 1.0–4.0 mM were checked, the optimal concentration of MgCl 2 was found to be 2.5 mM. 0.2 μM of each forward and reverse primer was found to be the optimal concentration for best results. After running a gradient PCR, the optimal annealing temperature was fixed to 55°C. The amplification was done in a thermal cycler as follows: 5 min at 94°C, followed by 35 cycles, each of 94°C for 45 sec, 55°C for 50 seconds and then 72°C for 50 seconds with a final extension at 72°C for 7 mins. Amplified PCR products were separated by agarose gel electrophoresis and visualised in a UV transilluminator after 0.5 μm/ml of ethidium bromide staining. The genus-targeted PCR products of Entamoeba spp showed 1803 bp—2046 bp amplicon on the agarose gel, depending on the species. For the detection of E. moshkovskii the primary products went through the second round of PCR amplification using species-specific primer pair. 1.0 μL of primary PCR product was used as a template for the nested PCR reaction. To obtain the nested PCR product, we performed PCR amplification in a final volume of 50 μL reaction mixture containing 1X PCR buffer, 4.0 mM MgCl 2 , 0.2 mM of each dNTP, 0.2 μM of each forward and reverse primer (EM_779bpNF & EM_779bpNR) and 1U of Biotaq DNA polymerase (Bioline, UK). Reactions were performed in a thermal cycler PCR system (Applied Biosystem). The PCR reaction was started with an initial denaturation step at 94°C for 3 minutes and then subjected to 35 cycles of 94°C for 40 seconds, 57°C for 35 seconds and 72°C for 45 Seconds, followed by a final extension at 72°C for 7 minutes. The nested PCR was performed in 2.0 mM MgCl 2 . Nested PCR generates a 779 bp fragment in the presence of E. moshkovskii. Amplified PCR products were separated by electrophoresis in 1.5% agarose gel (Lonza SeaKem® LE Agarose)s in 1X Tris boric Acid EDTA buffer and visualised in a UV transilluminator after 0.5 μm/ml of ethidium bromide staining. The specificity of Genus specific PCR was tested using genomic DNA preparation of five Entamoeba species viz. E. histolytica, E. moshkovskii, E. dispar, E. bangladeshi and E. nuttali. The specificity of E. moshkovskii species-specific primer set was also assessed against DNA extracted from faecal samples of other pathogens, namely E. histolytica, E. dispar, E. bangladeshi, E. nuttali, E. coli, Giardia lamblia, Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium viatorum and mixed bacterial infections. All the tested DNA samples were subjected to the abovementioned amplification protocol. The sensitivity of the nested PCR system was also evaluated using reference DNA templates by serial dilutions from 10 to 0.000019 ng/μL of DNA. DNA Sequencing The 18S rRNA locus of the positive samples was sequenced to characterize the local isolates of E. moshkovskii. As the same-size amplified products do not unavoidably mean identical DNA sequences, we directly sequenced the PCR products without cloning them into any vector to reduce the chances of any sequence selection. For DNA sequencing of E. moshkovskii positive samples obtained in this study were amplified separately using ExTaq DNA polymerase (Takara, Japan). We used ExTaq because of its higher fidelity than standard Taq with a lower mutation rate. The aforementioned species-specific primer set was employed directly to amplify the 18S rRNA locus of the positive samples by conventional PCR method. PCR was accomplished in a 50 μL reaction mixture containing: 0.50 μL Takara Ex Taq, 5 μL 10X Ex Taq Buffer, dNTP mixture 3 μL, 0.2 μM of each forward and reverse primer (EM_779bpNF & EM_779bpNR), approximately 200 ng stool DNA and nuclease-free water (Ambion™) up to 50 μL. The reaction mixture was subjected to an initial denaturation step at 94°C for 5 mins, followed by 35 cycles of 60 s at 94°C of denaturation, primer annealing for 40 s at 57°C and extension for 55 s at 72°C. A final seven minutes polymerization step at 72°C was also performed. Successfully amplified PCR products were purified using the Roche PCR Gel extraction Kit as per the manufacturer’s protocol. The purified PCR products were sequenced using the standard BigDye terminator V3.1 sequencing kit (Applied Biosystem, USA) following the manufacturer’s instructions. Sequencing was performed with a 5730 DNA analyzer (Applied Biosystem, Foster City, CA, USA). The accuracy of the sequence was verified with sequencing in the 5’ - 3’ direction using both forward and reverse primer separately. Sequence alignment, nucleotide polymorphisms analysis The obtained 18S rDNA gene sequences of E. moshkovskii compared to those available in the GenBank database using the BLAST tool (NCBI - https://blast.ncbi.nlm.nih.gov/Blast.cgi)). All of the obtained sequences were deposited in NCBI GenBank (accession numbers ON965383- ON965450). Multiple alignments of the nucleotide sequence allowed us to analyze nucleotide sequence variations. The obtained sequences were aligned using ClustalW multiple sequence alignment program of GenomeNet Bioinformatics tools and edited manually. This alignment was also performed with MultAlin using identity parameter values of -1 and -0 and the penalty default values to determine sequence variations. All obtained sequences were aligned and adjusted in MEGA X. Substitution matrix (Maximum likelihood/ML) and transition/transversion (ML) bias were estimated using the same software. In the substitution matrix (ML), each entry is the probability of substitution (r) from one base (row) to another base (column). Substitution patterns and rates were estimated under the General Time Reversible model (+G+I) [39]. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories, [+G], parameter = 200.0000). The rate variation model allowed some sites to be evolutionarily invariable ([+I], 0% sites). Rates of different transitional substitutions are shown in bold and those of transversionsal substitutions are shown in italics. Relative values of instantaneous r should be considered when evaluating them. For simplicity, the sum of r values is made equal to 100. For estimating Maximum likelihood values, a tree topology was automatically computed [39]. This analysis involved 68 nucleotide sequences. For transition/transversion (ML) bias determination substitution patterns and rates were estimated under the Tamura-Nei (1993) model (+G+I) [40]. There were a total of 733 positions in the final dataset. Nucleotide composition, parsimony-informative sites, singleton sites, variable sites (S), the number of haplotypes (h), haplotype diversity (Hd), the average number of nucleotide differences (K), and nucleotide diversity (π) were determined from the aligned sequences using the program DnaSP v5. Genetic structure analysis Partitions of genetic diversity within and among different population subdivisions of local isolates obtained from the present study were calculated using Wright’s F statistics (F ST ). Population subdivision was performed based on the co-infection status of the isolates. F ST is a measure of genetic differentiation among populations. DnaSP v5.0 package was used to estimate the mean pairwise differences between the populations. The significant difference in F ST was from 0 based on 1000 random permutations of the dataset. Four neutrality tests using Tajima’s D, Fu’s FS, Fu and Li’s D and F statistics were also performed through DnaSP v5.0 for assessing the probable population expansion. Phylogenetic analysis The maximum Likelihood method and Tamura-Nei model were applied to infer the evolutionary history [40]. The tree with the highest log likelihood (-6543.31) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites [8 categories (+G, parameter = 200.0000)]. The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 0.42% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 76 nucleotide sequences. There were a total of 822 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [39,41]. Haplotype network construction The relationship between the haplotypes of E. moshkovskii in different coinfected subgroups was inferred by constructing a Median Joining haplotype network in PopART v1.7. The isolates were colour-coded according to specific coinfection groups to derive a relationship with sequenced data. Data collection of other Enteropathogen infections Entamoeba histolytica, Cryptosporidium, and Giardia were identified by PCR method as described elsewhere [42]. Helminth parasites were detected by light microscopy after wet mount. Other enteric pathogens like Vibrio cholera O1/O139, Salmonella spp., Campylobacter jejuni, Rotavirus, astrovirus and adenovirus co-infection information with E. moshkovskii were obtained from the institutional database of ICMR-National Institute of Cholera and Enteric Diseases, Kolkata, India. Cultivation of Entamoeba spp In this study, we used Trophozoites of the E. moshkovskii (An isolate of Thailand), a gift from Dr Seiki Kobayashi, School of Medicine, Keio University. The E. moshkovskii strain was regularly cultivated in our lab using the BIS-33 medium and maintained at a temperature of 25°C. Trophozoites under the log phase of growth were used in the experiments as a positive control. Geographical distribution of E. moshkovskii infection The geographical locations of the patients were obtained from the hospital case record file (CRF). This data was employed to evaluate the spatial distribution of E. moshkovskii in the catchment area. If patients from any area comprised less than 1% of the total sample size, the site was excluded from the dataset. Statistical analysis GraphPad prism v.8.4.2, CA, USA, was used to analyze the data. The relationship between the prevalence of E. moshkovskii with other variables like age, gender, and other co-infections/additional enteropathogens was measured by testing X2. Fisher’s exact test was applied to evaluate the statistical association between gender and the occurrence of infection of E. moshkovskii. Two-way ANOVA was employed in three-year sample comparisons to assess the statistical significance of the seasonal pattern of E. moshkovskii infection. In all cases, a p-value less than 0.05 was considered significant.

Discussion The study was conducted to determine the prevalence of E. moshkovskii in stool samples of diarrheal patients admitted to Infectious Disease Hospital, Kolkata and B C Roy Hospital, Kolkata. This is the first study done in Eastern India using molecular biological techniques that report the prevalence of E. moshkovskii in clinical stool samples. According to the active surveillance study for the detection of common enteric parasites going on over the past two decades, we have observed a decreasing trend of E. histolytica infection in the last few years around Kolkata [42,44–45]. Also, non-seasonal sporadic infections with E. histolytica have been observed, which is unusual for a tropical area like Kolkata [42,45]. Microscopic investigation of diarrheal stool samples has uncovered the presence of cysts/trophozoites of amoeba that have a similar morphological feature to E. histolytica by a significant proportion throughout the year in Kolkata. After performing PCR-based molecular identification, it was found that the most abundant species of Entamoeba observed in diarrheal stool samples in Kolkata is a morphologically indistinguishable amoeba from E. histolytica and is a related species called E. moshkovskii. A recent study in Egypt showed that 85% of amoebic infections were caused by so-called non-pathogenic Entamoeba spp. such as E. dispar, E. moshkovskii, and E. hartmani [46]. This observation has led to an increased interest in the study of traditionally non-pathogenic Entamoeba species. Humans are a true host for amphizoic amoeba E. moshkovskii [27]. Moreover, recent evidence from different studies supports the pathogenicity of E. moshkovskii [23,27,29–30]. Although E. moshkovskii is identified as a cause of human infection, endemicity has not been appropriately assessed in most epidemiological studies [47]. In this study, we employed microscopic and molecular tools to determine the prevalence and genetic structure of E. moshkovskii in and around Kolkata. The present study reported that Entamoeba spp was prevalent in diarrhoeal stool samples and other enteric pathogens in Kolkata. More than half of the amoebic infection was caused by E. moshkovskii. This finding is alarming as it implies that E. histolytica infections previously decreased and E. moshkovskii has been taking its place. Although E. moshkovskii was highly prevalent in diarrheal patients, we did not find any hematophagous trophozoites of E. moshkovskii during microscopy, indicating its non-invasive nature. These results will boost research for a better understanding of the mechanism of pathogenicity in the parasite. The raw data revealed that a more significant proportion of men were infected with E. moshkovskii than women. Still, the difference was not statistically significant, which indicates that the probability of infection with E. moshkovskii is equal for both genders. Our data revealed that E. moshkovskii infections were most predominant in ages 5–12. The higher prevalence of E. moshkovskii in this age group might be associated with their lack of health education regarding hygiene practices. A recent study in Bangladesh reported that 21% of children aged 2–5 were infected with E. moshkovskii, which was associated with diarrhoea [23]. Many other studies also reported E. moshkovskii as an enteropathogen in patients suffering from diarrhoea or dysentery [7,23,27,29–30]. Our study was conducted among patients with diarrhoeal complaints. A notable percentage of individuals were infected with E. moshkovskii, and the presence of mono-infection/ sole infection of E. moshkovskii was statistically associated with diarrhoeal occurrence. Therefore, the diarrheal incidents associated with E. moshkovskii were not commonly coinfected in Kolkata. These results indicate that E. moshkovskii may not simply be a commensal of the human gut; instead, it acts as a “potential” pathogen causing diarrhoea and other gastrointestinal disorders in the study area. A typical seasonal pattern generally observed in many parasitic infections like E. histolytica and Giardia lamblia usually showed the highest peaks in the wet season. It gradually decreased with the arrival of the dry season [48–52]. Interestingly we observed a unique seasonal pattern of E. moshkovskii infection in Kolkata. We reveal two remarkable peaks of infection in summer and post-fall season. The result of such an unusual study finding has yet to be explained, requiring further detailed investigations. Further, we have performed phylogenetic analysis and multiple alignments of the E. moshkovskii population from Kolkata based on the 18S rRNA locus. Multiple alignments showed that 44.12% (n/N = 30/68) isolates were 100% similar at the sequenced region compared to the Laredo strain of E. moshkovskii, and the rest of the isolates were novel genetic variants. Phylogenetic analysis clarified the relationships among subpopulations clustered together with their respective variants. However, further research is needed using high-resolution molecular markers to conclude whether the subpopulation is a different genotype of E. moshkovskii or a completely new lineage. Since this was a hospital-based surveillance study and patients came from a limited geographical area, the distribution of different genotypes needed to be better understood. But the findings of this study confirmed the distribution of a richly diverse population of E. moshkovskii species in Kolkata and adjacent areas. The findings of this study highlighted the epidemiological significance of E. moshkovskii infection in Eastern India as it is the first report in this geographical area and exposes the existence of this species as a possible emerging enteric pathogen in India. Most research on E. moshkovskii infection has mainly focused on the prevalence of this pathogen without considering the co-occurrence of other enteric pathogens [22–33]. In this study, a correlation was observed between 18S rRNA SNPs and clinical features. However, the correlation between SNPs and clinical features does not necessarily indicate a direct control of their impact on pathogenicity. The sequence of the 18S ribosomal RNA can be used as a phylogenetic marker, allowing for the identification of pathogenic organisms in clinical samples. It can also be utilized to detect E. moshkovskii isolates and enables further diagnostic testing. The identified SNPs may exhibit an essential role of E. moshkovskii in adapting to the gut environment or in acquiring other enteric pathogens. The nucleotide diversity (π) value is an important index in molecular genetics to determine the degree of genetic polymorphism within a population [53]. The estimator of nucleotide diversity, π, with a higher value than 0.01, suggests comparatively significant variations in most organisms [54–55]. In this study, nucleotide diversity for all subgroups was lower than 0.01, indicating a lower degree of genetic polymorphism among the haplotypes. The haplotype diversity index was highest in the IB/V subgroup and lowest in the ISTH subgroup. The average haplotype diversity index was 0.677, considering differences among the haplotypes of each subgroup. The obtained average haplotype diversity index suggests that the E. moshkovskii population is highly diverse in this geographical area and influenced by a moderate recombination rate [53]. The high levels of genetic diversity suggested the strong viability and adaptability of the E. moshkovskii population. This may increase the average fitness of E. moshkovskii populations in a changing environment. However, the 18S rRNA data showed that the average nucleotide diversity was reasonably low. The nucleotide diversity was low since it is a highly conserved component with minimal nucleotide substitution rates. Our neutrality test results implied that the Sole D and IOEP subpopulations were not under directional selection pressure. IEH subpopulation was influenced by selection pressure, which resulted in the adaptation of these isolates to coexist with E. histolytica via changes in its genetic constitution. Pairwise genetic differentiation (F ST ) among different coinfected subgroups ranged from low to high. The F ST can range from 0 to 1, where 0 suggests complete sharing of genetic material and 1 suggests no sharing [53,55]. According to the standard F ST scale, the fixation index is F ST less than 0.05 = little genetic difference; F ST of 0.05 0.15 = moderate genetic difference; F ST of 0.15–0.25 = great genetic difference and F ST greater than 0.25 = very great genetic difference [43]. The majority of F ST values of different coinfected subgroups obtained in this study were lower than 0.25, indicating low to moderate genetic differentiation within the different coinfected/sole infected subgroups of the E. moshkovskii population in this geographical area. Whereas F ST values with the highest levels of differentiation were observed for IEH subgroups versus three other subgroups–Sole D, IOEP and ISTH indicating a higher genetic differentiation and higher genetic drift or lower gene flow within IEH coinfected subgroups of the parasite [55]. The higher F ST values also implied that this genome region may have undergone positive selection pressure in IEH coinfected subgroup [56]. Therefore, the obtained IEH subgroup might be genetically isolated and corresponds to the speciation process. More studies should be performed using other genetic markers to validate whether the coinfected subgroup of E. moshkovskii really corresponds to a new lineage or only to a different genotype of E. moshkovskii; it should be remembered that we only considered a fragment of 18S rRNA gene in this study.

Conclusion In conclusion, the present study suggested that E. moshkovskii is one of the causative agents for acute diarrhoea in humans. The study found that many diarrhoeal patients infected with this species were negative for other enteric pathogens such as bacteria and viruses. The study recommends further research to understand the transmission dynamics of E. moshkovskii and proper diagnosis to avoid the development of drug-resistant strains. It also highlights the need for public health authorities to implement prevention and control strategies. The findings of the study raise concerns about the importance of proper diagnosis and control of E. moshkovskii infection.

Supporting information S1 Table. List of clinical isolates of E. moshkovskii in diarrheal patients obtained from the hospital-based surveillance study. https://doi.org/10.1371/journal.pntd.0011287.s001 (DOCX) S2 Table. Substitution matrix based on 18S rRNA sequences of the populations of E. moshkovskii collected in and around Kolkata. https://doi.org/10.1371/journal.pntd.0011287.s002 (DOCX)

Acknowledgments The authors extend their appreciation to all patients who took part in the study. We are also grateful for the assistance provided by the hospital staff and sample collector in the sample collection process

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[1] Url: https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0011287

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