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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 comprehensive meta-analysis study was performed to estimate the reliable national prevalence and molecular epidemiology of amoebiasis in Iran. Nine English and Persian databases were searched to achieve the relevant studies. Pooled estimates were generated and meta-regression was performed. We identified 71 eligible articles involving 330 930 subjects from 25 provinces to be included in the final analysis. Moreover, 17 studies compromising 462 polymerase chain reaction (PCR)-positive isolates performed molecular analysis to inter-species differentiation. The pooled prevalence of Entamoeba infection among Iranian population was about 1% (95% CI 0.8–2.0%). Moreover, regarding Human Development Index (HDI), a higher prevalence was observed in undeveloped provinces. Out of 462 PCR-positive isolates, 83% (95% CI 69–94%) and 12% (95% CI 3–24%) were Entamoeba dispar, Entamoeba histolytica, respectively. In subgroup analysis based on molecular results, in general, population prevalence of Entamoeba dispar and E. histolytica were 91% (95% CI 80–99%) and 7%, (95% CI 0–19%), respectively, while prevalence of these species in patients with gastrointestinal disorders were 75% (95% CI 45–96%) and 18% (95% CI 1–43%), respectively. Our findings indicate the low burden of amoebiasis in Iran. E. dispar, that is mostly non-pathogenic, was identified as most prevalent species. Nevertheless, we suggest more public health interventions in areas with lower HDI.

Amoebae are single-celled parasites frequently colonizing human gut. However, few molecular tools are available for accurate identification. Here, we evaluated a panel of polymerase chain reactions (PCRs) targeting Entamoeba histolytica, Entamoeba dispar, Entamoeba coli, Entamoeba hartmanni, Entamoeba polecki, Endolimax nana and Iodamoeba bütschlii. Thirty-six faecal samples (18 containing at least one amoeba species by microscopy and 18 microscopy negative for amoebae) were tested. Real-time PCRs were used for detection and differentiation of E. histolytica and E. dispar. Conventional PCR with Sanger sequencing were applied for detection and differentiation of E. coli, E. hartmanni, E. polecki, E. nana and I. bütschlii. All microscopy results were confirmed by DNA-based methods. However, more samples were positive for single and mixed amoebic species by DNA-based assays than by microscopy (22 vs 18 and 7 vs 1, respectively). DNA sequencing allowed identification of E. coli subtypes (ST1 and ST2), showed low intra-specific variation within E. hartmanni, identified two phylogenetically distinct groups within E. nana, and identified Iodamoeba at the ribosomal lineage level. Taking into account the high intra-genetic diversity within some of the species at the small subunit (SSU) rRNA gene level, amplification of SSU rRNA genes with subsequent sequencing represents a useful method for detecting, differentiating and subtyping intestinal amoebae.

Epidemiological studies on species-specific Entamoeba infections are scarce due to the morphological similarity of pathogenic Entamoeba histolytica and nonpathogenic E. dispar and E. moshkovskii. The diagnosis of E. histolytica is frequently based on coproantigen (E. histolytica-Gal/GalNAc lectin specific) detection by immunoassays. However, specific E. histolytica-lectin is not expressed in cysts, which are eliminated by asymptomatic individuals leading to false-negative results and an underestimation of amebiasis prevalence. Molecular techniques based on the amplification of parasite DNA have been shown to be a highly sensitive and specific method that allows the detection of different Entamoeba species. This study aimed to assess the frequency of the species from E. histolytica/dispar/moshkovskii complex by molecular and immunological techniques in individuals attended at a public health system in Salvador-Bahia, Brazil. A cross-sectional study involving 55,218 individuals was carried out. The diagnosis was based on microscopy revealing E. histolytica/dispar/moshkovskii complex. The species differentiation was performed by E. histolytica-specific antigen, serological evaluation and by molecular technique. The overall prevalence of E. histolytica/dispar/moshkovskii complex determined by microscopy was approximately 0.49% (273/55,218). E. histolytica-specific antigen detection and molecular characterization returned 100% negativity for E. histolytica. However, serological evaluation returned an 8.9% positivity (8/90). In the stool samples analysed by PCR, it was not possible to identify E. histolytica and E. moshkovskii, although circulating IgG anti-E. histolytica has been detected.

Background. Transmission of Entamoeba histolytica generally occurs by fecal excretion of cysts followed by oral ingestion of contaminated food or water. However, fecal-oral transmission may occur within households and long-term care institutions, and sexual transmission occurs among men who have sex with men. Epidemiologically linked clusters of E. histolytica infection are rare in industrialized countries. We report such a sexually linked cluster in Canada. Methods. An index case involving a young female with an amebic liver abscess led to an epidemiological investigation of sexual contacts. Anti-amebic serological analysis, stool specimen examinations, and abdominal ultrasounds were done for the contacts. Enzyme-linked immunosorbent assay was done for stool antigen specific to E. histolytica. Genotyping and phylogenetic analysis was performed on 1 stool isolate. Results. By tracing sexual contacts related to the index case, we uncovered a cluster of 7 cases of amebiasis (3 with liver abscesses). Oral-anal sex was common in the group; the 5 female individuals were bisexual (4) or homosexual (1). The outbreak strain was genotyped, and cluster analysis indicated that this virulent strain differed substantially from asymptomatic or diarrheal E. histolytica isolates. Conclusions. E. histolytica can be transmitted by heterosexual activity as well as male and female homosexual activity. Patients with amebiasis should be counselled about possible sexual transmission.

Background The outcome of an Entamoeba histolytica infection is variable and can result in either asymptomatic carriage, immediate or latent disease (diarrhea/dysentery/amebic liver abscess). An E. histolytica multilocus genotyping system based on tRNA gene-linked arrays has shown that genetic differences exist among parasites isolated from patients with different symptoms however, the tRNA gene-linked arrays cannot be located in the current assembly of the E. histolytica Reference genome (strain HM-1:IMSS) and are highly variable. Results To probe the population structure of E. histolytica and identify genetic markers associated with clinical outcome we identified in E. histolytica positive samples selected single nucleotide polymorphisms (SNPs) by multiplexed massive parallel sequencing. Profile SNPs were selected which, compared to the reference strain HM-1:IMSS sequence, changed an encoded amino acid at the SNP position, and were present in independent E. histolytica isolates from different geographical origins. The samples used in this study contained DNA isolated from either xenic strains of E. histolytica trophozoites established in culture or E. histolytica positive clinical specimens (stool and amebic liver abscess aspirates). A record of the SNPs present at 16 loci out of the original 21 candidate targets was obtained for 63 of the initial 84 samples (63% of asymptomatically colonized stool samples, 80% of diarrheal stool, 73% of xenic cultures and 84% of amebic liver aspirates). The sequences in all the 63 samples both passed sequence quality control metrics and also had the required greater than 8X sequence coverage for all 16 SNPs in order to confidently identify variants. Conclusions Our work is in agreement with previous findings of extensive diversity among E. histolytica isolates from the same geographic origin. In phylogenetic trees, only four of the 63 samples were able to group in two sets of two with greater than 50% confidence. Two SNPs in the cylicin-2 gene (EHI_080100/XM_001914351) were associated with disease (asymptomatic/diarrhea p = 0.0162 or dysentery/amebic liver abscess p = 0.0003). This study demonstrated that there are genetic differences between virulent and avirulent E. histolytica strains and that this approach has the potential to define genetic changes that influence infection outcomes.

We determined the prevalence of Entamoeba (E.) histolytica, E. dispar and E. moshkovskii in patients with chronic diarrhoea associated with abdominal pain or discomfort mimicking irritable bowel syndrome. Stool samples were collected from 161 patients with chronic diarrhoea and from 157 healthy controls. Stool microscopy with modified trichrome stain, culture and polymerase chain reaction (PCR) for Entamoeba spp. differentiation was performed. Microscopy demonstrated Entamoeba cysts in 44% (57/129) of patients with diarrhoea compared to 29% (44/151) of controls (P=0·009). In patients with diarrhoea, PCR for E. histolytica was positive in 9% (11/129) (P=0·008), E. dispar in 19% (24/129) (P=0·117) and E. moshkovskii in 19% (24/129) (P<0·001). E. histolytica and E. moshkovskii were significantly associated with diarrhoea while E. dispar was found equally in both groups.

Amoebiasis diagnosis is usually based on microscopy that cannot differentiate pathogenic E. histolytica from morphologically identical non-pathogenic species. 194 fecal samples were collected from diarrheic &/or dysenteric patients and examined for Entamoeba complex microscopically, E. histolytica/E. dispar coproantigen using ICT and E. histolytica coproantigen using Tech lab E. histolytica II ELISA test. Entamoeba complex trophozoites/cysts, E. histolytica/E. dispar coproantigen and E. histolytica coproantigen were detected in 22.2, 14.4 and 3.6 % of samples, respectively. Microscopy and ICT method had limited sensitivity with poor PPV (9.3 and 7.1 %, respectively) and both slightly agree with ELISA test. The prevalence of E. histolytica was low (3.6 %) in studied individuals and was 14 times lower than non-pathogenic amoebae. E. histolytica detection studied individuals was positively associated with mucoid and bloody stool, which makes them disease predictors. E. histolytica fecal ELISA assay for E. histolytica detection surpassed microscopy and E. histolytica/E. dispar ICT assay. This has highlighted the need for practical non-microscopic detection methods that can differentiate between amoeba infections to avoid unnecessary and possibly harmful therapies and to determine the true prevalence and epidemiology of E. histolytica.

A single-tube multiprobe real-time PCR assay for simultaneous detection of Entamoeba histolytica and Entamoeba dispar was developed. One primer pair with 2 species-specific probes was designed based on new SSU RNA regions of the ribosomal DNA-containing episome. The sensitivity is 1 parasite per milliliter of feces and thus superior to the conventional nested PCR and comparable to other published real-time PCR protocols. The applicability for clinical diagnosis was validated with 218 stool specimens from patients. A total of 51 E. histolytica and 39 E. dispar positive samples was detected by the multiprobe real-time PCR compared to 39 and 22 by routine nested PCR diagnosis. The detection rate of Entamoeba species for the multiprobe real-time PCR assays was significantly higher than the nested PCR (40.8% vs. 28.0%, P < 0.01). The test did not show cross reactivity with DNA from Entamoeba moshkovskii, Giardia lamblia, Cryptosporidium sp., Escherichia coli, or other nonpathogenic enteric parasites. The multiprobe real-time PCR assay is simple and rapid and has high specificity and sensitivity. The assay could streamline the laboratory diagnosis procedure and facilitate epidemiological investigation.

Archezoan protists are thought to represent lineages that diverged from other eukaryotes before acquisition of the mitochondrion and other organelles. The parasite Entamoeba histolytica was originally included in this group. Ribosomal RNA based phylogenies, however, place E. histolytica on a comparatively recent branch of the eukaryotic tree, implying that its ancestors had these structures. In this study, direct evidence for secondary loss of mitochondrial function was obtained by isolating two E. histolytica genes encoding proteins that in other eukaryotes are localized in the mitochondrion: the enzyme pyridine nucleotide transhydrogenase and the chaperonin cpn60. Phylogenetic analysis of the E. histolytica homolog of cpn60 confirmed that it is specifically related to the mitochondrial lineage. The data suggest that a mitochondrial relic may persist in this organism. Similar studies are needed in archezoan protists to ascertain which, if any, eukaryotic lineages primitively lack mitochondria.