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Amoebiasis, an enteric protozoan disease caused by Entamoeba histolytica, is a public health problem in many developing countries, causing up to 100,000 fatal cases annually. Detection of the pathogenic E. histolytica and its differentiation from the non-pathogenic Entamoeba spp. play a crucial role in the clinical management of patients. Laboratory diagnosis of intestinal amoebiasis in developing countries still relies on labour-intensive and insensitive methods involving staining of stool sample and microscopy. Newer and more sensitive methods include a variety of antigen detection ELISAs and rapid tests; however, their diagnostic sensitivity and specificity seem to vary between studies, and some tests do not distinguish among the Entamoeba species. Molecular detection techniques are highly sensitive and specific and isothermal amplification approaches may be developed into field-applicable tests; however, cost is still a barrier for their use as a routine laboratory test method in most endemic areas. Laboratory diagnosis of extraintestinal amoebiasis faces challenges of lack of definitive detection of current infection and commercially available point-of-care tests. For both types of amoebiasis, there is still a need for highly sensitive and specific tests that are rapid and cost-effective for use in developing countries where the disease is prevalent. In recent years, new molecules of diagnostic value are being discovered and new tests developed. The advances in ‘omics’ technologies are enabling discoveries of new biomarkers that may help distinguish between different infection stages.

Pathogenic intestinal protozoa infections are responsible for substantial mortality and morbidity, particularly in settings where people lack improved sanitation and safe drinking water. We assessed the relation between access to, and use of, sanitation facilities and water treatment and infection with intestinal protozoa. We did a systematic review and searched PubMed, ISI Web of Science, and Embase from inception to June 30, 2014, without restrictions on language. All publications were examined by two independent reviewers and were included if they presented data at the individual level about access or use of sanitation facilities or water treatment, in combination with individual-level data on human intestinal protozoa infections. Meta-analyses using random effects models were used to calculate overall estimates. 54 studies were included and odds ratios (ORs) extracted or calculated from 2 × 2 contingency tables. The availability or use of sanitation facilities was associated with significantly lower odds of infection with Entamoeba histolytica or Entamoeba dispar (OR 0·56, 95% CI 0·42–0·74) and Giardia intestinalis (0·64, 0·51–0·81), but not for Blastocystis hominis (1·03, 0·87–1·23), and Cryptosporidium spp (0·68, 0·17–2·68). Water treatment was associated with significantly lower odds of B hominis (0·52, 0·34–0·78), E histolytica or E dispar (0·61, 0·38–0·99), G intestinalis (0·63, 0·50–0·80), and Cryptosporidium spp infections (0·83, 0·70–0·98). Availability and use of sanitation facilities and water treatment is associated with lower odds of intestinal protozoa infections. Interventions that focus on water and sanitation, coupled with hygiene behaviour, should be emphasised to sustain the control of intestinal protozoa infections. Swiss National Science Foundation (project numbers PBBSP3-146869 and P300P3-154634), Medicor Foundation, European Research Council (614739-A_HERO).

In this study, a single-step duplex polymerase chain reaction procedure was developed for rapid, specific and sensitive identification of Entamoeba histolytica and for its diagnostic differentiation from E. dispar. Specific oligonucleotide primers were combined for the amplification of a cysteine proteinase 5 gene target sequence of 242 bp, present only in E. histolytica. Additionally, another oligonucleotide primer pair for both the E. histolytica and E. dispar actin gene target of 300 bp was designed to amplify only from amoebae DNA. The PCR developed was specific and efficiently identified and differentiated these parasites from each other in either cultured parasites or from stool material.

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.

Intestinal protozoa infections present a major public health challenge, particularly in areas with poor sanitation and limited access to clean water. Effective diagnostic methods are critical, yet traditional microscopy, though widely used for its simplicity, lacks the sensitivity and specificity of modern techniques like real-time Polymerase Chain Reaction (qPCR), making the latter a more effective tool for monitoring and assessing the burden of intestinal protozoa diseases. In this study, we implemented two duplex qPCR assays to detect Entamoeba dispar  +  Entamoeba histolytica and Cryptosporidium spp. +  Chilomastix mesnili , along with singleplex assays for Giardia duodenalis and Blastocystis spp., using a 10 µL reaction volume. This marks the first molecular detection of Chilomastix mesnili by qPCR, enhancing diagnostic precision. Using these, we analyzed stool samples from 70 patients on Pemba Island, Tanzania, before and 54 samples after treatment with 20, 25, or 30 mg of emodepside or placebo, aiming to assess protozoa prevalence for this region and emodepside’s potential antiprotozoal effects. Our qPCR reliably detected protozoa in 74.4% of samples, with Entamoeba histolytica and Entamoeba dispar in 31.4% of cases. Notably, one-third of these infections were caused by Entamoeba histolytica . No significant reduction in protozoa was observed after emodepside treatment compared to placebo. The study highlights the utility of qPCR in providing species-level differentiation and improving the speed and cost-effectiveness of testing. The high prevalence of protozoa in this region underscores the need for continued monitoring and control efforts, though emodepside was not effective against protozoa infections.

Intestinal parasitic infections remain among the most common infectious diseases worldwide. This study aimed to estimate their prevalence and provide a detailed analysis of geographical distribution of intestinal parasites in the metropolitan region of Rio de Janeiro, considering demographic, socio-economic, and epidemiological contextual factors. The cross-section survey was conducted among individuals attending the Evandro Chagas National Institute of Infectious Diseases (FIOCRUZ, RJ) during the period from April 2012 to February 2015. Stool samples were collected and processed by sedimentation, flotation, Kato-Katz, Baermann-Moraes and Graham methods, iron haematoxylin staining and safranin staining. Of the 3245 individuals analysed, 569 (17.5%) were infected with at least one parasite. The most common protozoa were Endolimax nana (28.8%), Entamoeba coli (14.8%), Complex Entamoeba histolytica/Entamoeba dispar (13.5%), Blastocystis hominis (12.7%), and Giardia lamblia (8.1%). Strongyloides stercoralis (4.3%), Schistosoma mansoni (3.3%), Ascaris lumbricoides (1.6%), and hookworms (1.5%) were the most frequent helminths. There was a high frequency of contamination by protozoa (87%), and multiple infections were observed in 141 participants (24.8%). A positive association between age (young children) and gender (male) with intestinal parasites was observed. Geospatial distribution of the detected intestinal parasitic infections was not random or homogeneous, but was influenced by socioeconomic conditions (through the material deprivation index (MDI)). Participants classified in the highest levels of deprivation had higher risk of having intestinal parasites. This study provides the first epidemiological information on the prevalence and distribution of intestinal parasitic infections in the Rio de Janeiro metropolitan area. Intestinal parasites, especially protozoa, are highly prevalent, indicating that parasitic infections are still a serious public health problem. MDI showed that intestinal parasites were strongly associated with the socioeconomic status of the population, thus making it possible to identify social vulnerable areas.

Detection of Entamoeba histolytica and its differentiation from Entamoeba dispar is an important goal of the clinical parasitology laboratory. The aim of this study was the identification and differentiation of E. histolytica and E. dispar by MALDI-TOF MS, in order to evaluate the application of this technique in routine diagnostic practice. MALDI-TOF MS was applied to 3 amebic reference strains and to 14 strains isolated from feces that had been differentiated by molecular methods in our laboratory. Protein extracts from cultures of these strains (axenic cultures for the 3 reference strains and monoxenic cultures for the 14 field isolates) were analyzed by MALDI-TOF MS and the spectra obtained were analyzed by statistical software. Five peaks discriminating between E. histolytica and E. dispar reference strains were found by protein profile analysis: 2 peaks (8,246 and 8,303 Da) specific for E. histolytica and 3 (4,714; 5,541; 8,207 Da) for E. dispar. All clinical isolates except one showed the discriminating peaks expected for the appropriate species. For 2 fecal samples from which 2 strains (1 E. histolytica and 1 E. dispar) out of the 14 included in this study were isolated, the same discriminating peaks found in the corresponding isolated amebic strains were detected after only 12h (E. histolytica) and 24h (E. dispar) of incubation of the fecal samples in Robinson's medium without serum. Our study shows that MALDI-TOF MS can be used to discriminate between E. histolytica and E. dispar using in vitro xenic cultures and it also could have potential for the detection of these species in clinical samples.

Amoebiasis is the second leading cause of death from parasitic disease worldwide. The causative protozoan parasite, Entamoeba histolytica, is a potent pathogen. Secreting proteinases that dissolve host tissues, killing host cells on contact, and engulfing red blood cells, E histolytica trophozoites invade the intestinal mucosa, causing amoebic colitis. In some cases amoebas breach the mucosal barrier and travel through the portal circulation to the liver, where they cause abscesses consisting of a few E histolytica trophozoites surrounding dead and dying hepatocytes and liquefied cellular debris. Amoebic liver abscesses grow inexorably and, at one time, were almost always fatal, but now even large abscesses can be cured by one dose of antibiotic. Evidence that what we thought was a single species based on morphology is, in fact, two genetically distinct species—now termed Entamoeba histolytica (the pathogen) and Entamoeba dispar (a commensal)—has turned conventional wisdom about the epidemiology and diagnosis of amoebiasis upside down. New models of disease have linked E histolytica induction of intestinal inflammation and hepatocyte programmed cell death to the pathogenesis of amoebic colitis and amoebic liver abscess.

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.