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This study aimed to determine the prevalence of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii (collectively referred to as Entamoeba complex), using microscopic and molecular methods in Kurdistan Province, northwest of Iran. The relationship between positive Entamoeba species and clinical symptoms was also investigated. Eight positive Entamoeba complex, as well as four Entamoeba complex-like isolates, were detected by microscopic stool examination. DNA was extracted from all positive and from 55 randomly selected negative stool samples. PCR was performed using species-specific 18S rRNA primers for the Entamoeba complex. All positive PCR samples were sequenced. In total, 14 (1.01%) out of 1383 isolates, i.e. 12 microscopy-positive and Entamoeba complex-like isolates and two out of 55 microscopy-negative isolates, were identified via PCR and sequencing. Overall, 0.58% (8/1383) of the isolates were E. dispar, 0.14% (2/1383) E. histolytica, 0.07% (1/1383) E. moshkovskii and 0.22% (3/1383) were mixed of E. histolytica and E. dispar. Based on our findings, the prevalence of E. dispar is greater than that of E. histoltyica. On the other hand, a case of E. moshkovskii was reported for the first time in this region. It seems that some gastrointestinal symptoms may be attributed to Entamoeba species.

Entamoeba moshkovskii is historically considered nonpathogenic. We report a case of severe extraintestinal infection in a patient from eastern India who had abdominal pain, fever, weight loss, anemia, and a splenic abscess. Molecular analysis confirmed E. moshkovskii as the causative agent. This case highlights this parasite's potential to cause severe illness.

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. 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. 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. 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.

Entamoeba histolytica infection (amoebiasis) is the second leading cause of death from parasitic diseases. Epidemiological studies from developed countries have reported an increasing prevalence of amoebiasis and of invasive infections, such as amoebic colitis, among men who have sex with men (MSM) who engage in oral–anal sex. Although most infections with E histolytica are asymptomatic, clinical manifestations of invasive amoebiasis mainly include amoebic colitis and amoebic liver abscess, which are associated with substantial morbidity and medical cost. Laboratory diagnosis of amoebiasis should be based on detection of E histolytica by use of tests with high sensitivity and specificity, such as specific amoebic-antigen or PCR-based assays. Microscopy used in routine clinical laboratories is not sensitive or specific enough for detection of E histolytica. Metronidazole or tinidazole remains the mainstay of treatment for invasive amoebiasis, followed by treatment with luminal agents to prevent relapse and transmission of E histolytica to sexual partners or close contacts.

Infection with Entamoeba histolytica can lead to amebic colitis and to complications including liver abscess. This review summarizes recent research on the pathogenesis and treatment of infection and the prospects for the development of a vaccine. A mucosal IgA response can produce partial, protective immunity to infection with E. histolytica . Diarrheal diseases continue to be major causes of morbidity and mortality in children in developing countries. For example, in Bangladesh 1 in 30 children dies of diarrhea or dysentery by his or her fifth birthday. 1 In developed countries the microorganisms that cause diarrheal disease remain of concern because of their potential use as bioterrorist agents. Bacillary dysentery is most commonly caused by microorganisms belonging to the genus shigella, whereas amebic dysentery is caused by the protozoan parasite Entamoeba histolytica . The annual number of shigella infections throughout the world is believed to be approximately 164 million. 2 Estimates of E. histolytica . . .

TaqMan-probed quantitative PCR (qPCR) is highly valued for diagnosing Entamoeba histolytica infections (amebiasis). However, unclear cycle threshold (Ct) values often yield low-titer positive results, complicating interpretation. This study aimed to optimize qPCR primer-probe sets with logically determined cut-off Ct value using droplet digital PCR (ddPCR). Amplification efficacy was evaluated using ddPCR by measuring absolute positive droplet counts (APD) and mean fluorescence intensity at different PCR cycles and annealing temperatures (AT). A primer-probe specific cut-off Ct value was determined from a standard curve by correlating Ct values with APD. Twenty primer-probe sets targeting small subunit rRNA gene regions (X64142) were designed from previous papers. Amplification efficacy remained consistent at high PCR cycles (50 cycles), but differed at lower PCR cycles (30 cycles), identifying five sets with higher amplification efficiency than other candidates. Of these, only two sets maintained efficiency at higher AT (62°C). Ct value was inversely proportional to the square of APD, defining the specific cut-off Ct value as 36 cycles. Selected primer-probe set with a cut-off effectively differentiated E. histolytica infection in clinical specimens. However, discordant results between Ct value and APD were seen in some cases with high Ct value. Shotgun metagenomic sequencing suggested microbial-independent false positive reactions contributed to these discrepancies, although specific reactants were unidentified. The combination use of ddPCR with qPCR revealed that false positive reactions of qPCR and/or ddPCR commonly happen in stool specimens. Also, this study emphasizes the value of ddPCR for establishing accurate cut-off values with efficient primer-probes.

Multiplex molecular panels are relentlessly replacing conventional methods for the detection of enteric pathogens from stool samples in clinical and research laboratories. Here we evaluated four commercial multiplex real-time PCR assays for the detection of Cryptosporidium hominis/parvum, Giardia duodenalis and Entamoeba histolytica. The diagnostic performance of the Gastroenteritis/Parasite Panel I (Diagenode), the RIDAGENE Parasitic Stool Panel (R-Biopharm), the Allplex Gastrointestinal Parasite Panel 4 (Seegene) and the FTD Stool Parasites (Fast Track) real-time PCR methods was assessed against a reference panel of 126 well-characterized DNA samples including Cryptosporidium hominis (n = 29), Cryptosporidium parvum (n = 3), Giardia duodenalis (n = 47), Entamoeba histolytica (n = 3), other parasite species (n = 20), and apparently healthy subjects (n = 24). Obtained diagnostic sensitivities ranged from 53-88% for Cryptosporidium hominis/parvum, and from 68-100% for G. duodenalis. The R-Biopharm method achieved the best performance for the detection of Cryptosporidium hominis/parvum both in terms of diagnostic sensitivity (87.5%) and detection limit (a 100-fold increase compared to other tests). The Fast Track method was particularly suited for the detection of G. duodenalis, achieving a 100% sensitivity and a detection limit at least 10-fold superior. Detection of E. histolytica was similarly achieved by all compared methods except Diagenode. Diagnostic performance varied largely depending on the method used and the targeted pathogen species. Factors including test sensitivity/specificity, cost, patient population surveyed, laboratory workflow, and diagnostic algorithm should be carefully considered when choosing the most appropriate multiplex PCR platform.

is a major parasitic cause of acute gastroenteritis. In this study, hematological inflammatory indices were assessed in adhesin-positive amoebiasis cases. Adhesin test results and hemogram parameters were evaluated simultaneously in cases who were referred to Necmettin Erbakan University Faculty of Medicine, Medical Parasitology laboratory with suspicion of amoebiasis and whose specific adhesin enzyme-linked immunosorbent assay test was found to be positive between January 2022 and December 2023. In this study, the indices were calculated using haemogram parameters. Age- and sex-matched groups were formed, consisting of cases with adhesin test-positive acute gastroenteritis (APAG) and those with adhesin test-negative acute gastroenteritis (ANAG). In addition to common statistical analyses, the diagnostic performance of various hematologic inflammatory parameters in predicting adhesin positivity was evaluated by receiver operating characteristic analysis. The results of 340 cases were analyzed, including 136 cases under the age of 18. Blood lymphocyte and monocyte levels were significantly lower in the APAG group compared to the ANAG group (p=0.004 and p=0.048, respectively), while no significant differences were observed in the remaining haemogram parameters. There was also no statistically significant difference in C-reactive protein levels between the groups (p=0.061). Among the calculated indices, only the platelet-to-lymphocyte ratio (PLR) showed a significant difference between groups (p=0.017). In the gender-based subgroup analysis of the APAG group, red blood cell levels were found to be lower in female cases (p=0.026), while no significant differences were observed in the calculated indices. This study evaluated the predictive performance of various hematologic inflammatory parameters in determining adhesin test positivity. Although the PLR showed a statistically significant difference between groups, the positive and negative predictive values of all evaluated parameters remained moderate. These findings suggest that the diagnostic utility of these biomarkers is limited.

Entamoeba moshkovskii , recently known as a possible pathogenic amoeba, and the non-pathogenic Entamoeba dispar are morphologically indistinguishable by microscopy. Although PCR was used for differential diagnosis, gel electrophoresis is labor-intensive, time-consuming, and exposed to hazardous elements. In this study, nucleic acid lateral flow immunoassay (NALFIA) was developed to detect E. moshkovskii and E. dispar by post-PCR amplicon analysis. E. moshkovskii primers were labeled with digoxigenin and biotin whereas primers of E. dispar were lebeled with FITC and digoxigenin. The gold nanoparticles were labeled with antibodies corresponding to particular labeling. Based on the established assay, NALFIA could detect as low as 975 fg of E. moshkovskii target DNA (982 parasites or 196 parasites/microliter), and 487.5 fg of E. dispar target DNA (444 parasites or 89 parasites/microliter) without cross-reactivity to other tested intestinal organisms. After testing 91 stool samples, NALFIA was able to detect seven E. moshkovskii (87.5% sensitivity and 100% specificity) and eight E. dispar samples (66.7% sensitivity and 100% specificity) compared to real-time PCR. Interestingly, it detected three mixed infections as real-time PCR. Therefore, it can be a rapid, safe, and effective method for the detection of the emerging pathogens E. moshkovskii and E. dispar in stool samples.