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Worldwide, amoebic liver abscess (ALA) can be found in individuals in non-endemic areas, especially in foreign-born travelers. We performed a retrospective analysis of ALA in patients admitted to French hospitals between 2002 and 2006. We compared imported ALA cases in European and foreign-born patients and assessed the factors associated with abscess size using a logistic regression model. We investigated 90 ALA cases. Patient median age was 41. The male:female ratio was 3.5:1. We were able to determine the origin for 75 patients: 38 were European-born and 37 foreign-born. With respect to clinical characteristics, no significant difference was observed between European and foreign-born patients except a longer lag time between the return to France after traveling abroad and the onset of symptoms for foreign-born. Factors associated with an abscess size of more than 69 mm were being male (OR = 11.25, p<0.01), aged more than 41 years old (OR = 3.63, p = 0.02) and being an immigrant (OR = 11.56, p = 0.03). Percutaneous aspiration was not based on initial abscess size but was carried out significantly more often on patients who were admitted to surgical units (OR = 10, p<0.01). The median time to abscess disappearance for 24 ALA was 7.5 months. In this study on imported ALA was one of the largest worldwide in terms of the number of cases included males, older patients and foreign-born patients presented with larger abscesses, suggesting that hormonal and immunological factors may be involved in ALA physiopathology. The long lag time before developing ALA after returning to a non-endemic area must be highlighted to clinicians so that they will consider Entamoeba histolytica as a possible pathogen of liver abscesses more often.

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.

Background Although chronic morbidity in humans from soil transmitted helminth (STH) infections can be reduced by anthelmintic treatment, inconsistent diagnostic tools make it difficult to reliably measure the impact of deworming programs and often miss light helminth infections. Methods Cryopreserved stool samples from 796 people (aged 2–81 years) in four villages in Bungoma County, western Kenya, were assessed using multi-parallel qPCR for 8 parasites and compared to point-of-contact assessments of the same stools by the 2-stool 2-slide Kato-Katz (KK) method. All subjects were treated with albendazole and all Ascaris lumbricoides expelled post-treatment were collected. Three months later, samples from 633 of these people were re-assessed by both qPCR and KK, re-treated with albendazole and the expelled worms collected. Results Baseline prevalence by qPCR ( n  = 796) was 17 % for A. lumbricoides , 18 % for Necator americanus , 41 % for Giardia lamblia and 15 % for Entamoeba histolytica . The prevalence was <1 % for Trichuris trichiura, Ancylostoma duodenale, Strongyloides stercoralis and Cryptosporidium parvum . The sensitivity of qPCR was 98 % for A. lumbricoides and N. americanus , whereas KK sensitivity was 70 % and 32 %, respectively. Furthermore, qPCR detected infections with T. trichiura and S. stercoralis that were missed by KK, and infections with G. lamblia and E. histolytica that cannot be detected by KK. Infection intensities measured by qPCR and by KK were correlated for A. lumbricoides (r = 0.83, p  < 0.0001) and N. americanus (r = 0.55, p  < 0.0001). The number of A. lumbricoides worms expelled was correlated ( p  < 0.0001) with both the KK (r = 0.63) and qPCR intensity measurements (r = 0.60). Conclusions KK may be an inadequate tool for stool-based surveillance in areas where hookworm or Strongyloides are common or where intensity of helminth infection is low after repeated rounds of chemotherapy. Because deworming programs need to distinguish between populations where parasitic infection is controlled and those where further treatment is required, multi-parallel qPCR (or similar high throughput molecular diagnostics) may provide new and important diagnostic information.

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.

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.

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.

Background. Diarrhea causes enormous morbidity and mortality in developing countries, yet the relative importance of multiple potential enteropathogens has been difficult to ascertain. Methods. We performed a longitudinal cohort study from birth to 1 year of age in 147 infants in Dhaka, Bangladesh. Using multiplex polymerase chain reaction, we analyzed 420 episodes of diarrhea and 1385 monthly surveillance stool specimens for 32 enteropathogen gene targets. For each infant we examined enteropathogen quantities over time to ascribe each positive target as a probable or less-likely contributor to diarrhea. Results. Multiple enteropathogens were detected by the first month of life. Diarrhea was associated with a state of overall pathogen excess (mean number of enteropathogen gene targets (±SE), 5.6 ± 0.1 vs 4.3 ± 0.1 in surveillance stool specimens; P < .05). After a longitudinal, quantitative approach was applied to filter out less-likely contributors, each diarrheal episode still had an average of 3.3 probable or dominant targets. Enteroaggregative Escherichia coli, Campylobacter, enteropathogenic E. coli, rotavirus, and Entamoeba histolytica were the most frequent probable contributors to diarrhea. Rotavirus was enriched in moderate to severe diarrheal episodes. Conclusions. In this community-based study diarrhea seemed to be a multipathogen event and a state of enteropathogen excess above a high carriage baseline.

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.

Background Pathogenic intestinal protozoa exhibit a global distribution and are significant causes of diarrhea, estimated to affect approximately 3.5 billion individuals annually. These intestinal infections continue to pose formidable diagnostic challenges. Microscopy remains the reference diagnostic method for intestinal protozoa, but is limited in terms of sensitivity, specificity and the ability to differentiate closely related species. Additionally, microscopy requires an experienced microbiologist. Emerging diagnostic methods, such as immunochromatography and enzyme-linked immunosorbent assay (ELISA), are regarded as suitable techniques for rapid screening. Molecular diagnostic technologies, particularly real-time PCR (RT-PCR), are gaining traction in non-endemic areas characterised by low parasitic prevalence owing to their enhanced sensitivity and specificity, although these techniques still face various technical challenges. Methods In this multicentre study involving 18 Italian laboratories, we compared the performance of a commercial RT-PCR test (AusDiagnostics) and an in-house RT-PCR assay against traditional microscopy for identifying infections with Giardia duodenalis, Cryptosporidium spp., Entamoeba histolytica and Dientamoeba fragilis . Results The study analysed 355 stool samples, of which 230 samples were freshly collected and 125 had been stored in preservation media. The data from our analyses show complete agreement between the AusDiagnostics and in-house PCR methods for the detection of G. duodenalis , with both methods demonstrating high sensitivity and specificity, similar to those of conventional microscopy. For Cryptosporidium spp. and D. fragilis detection, both methods showed high specificity but limited sensitivity, likely due to inadequate DNA extraction from the parasite. Molecular assays seem to be critical for the accurate diagnosis of E. histolytica . Overall, PCR results from preserved stool samples were better than those from fresh samples, likely due to better DNA preservation in the former. Conclusions Molecular methods show promise for the diagnosis of intestinal protozoan infections. The molecular assays tested in this investigation performed well for G. duodenalis and Cryptosporidium spp. in fixed faecal specimens, while D. fragilis detection was inconsistent. These results suggest that although PCR techniques are promising in terms of reliable and cost-effective parasite identification, further standardisation of sample collection, storage and DNA extraction procedures is necessary for consistent results. Graphical Abstract

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.