Search Results (26)

Background: The microscopic examination of stool samples remains the reference method for the diagnosis of intestinal protozoal infections; however, this technique is time consuming and requires experienced and well-trained operators. Therefore, there is a growing interest in molecular diagnostic techniques, including commercial PCR assays. The aim of this multicentric study was to evaluate a commercial real-time PCR for the detection of intestinal protozoa in fecal samples. Methods: The samples were routinely examined using conventional techniques, such as macro- and microscopic examination after concentration, Giemsa or Trichromic stain, Giardia duodenalis, Entamoeba histolytica/dispar or Cryptosporidium spp. antigens research, and amoebae culture. The samples were frozen by the participating laboratories, retrospectively extracted and examined with one-step real-time PCR multiplex using the Allplex™ GI-Parasite Assay (Seegene Inc., Seoul, Korea). Results: A total of 368 samples were analyzed from 12 Italian laboratories. Compared to traditional techniques, the sensibility and specificity of the real-time PCR kit were as follows: 100% and 100% for Entamoeba histolytica, 100% and 99.2% for Giardia duodenalis, 97.2% and 100% for Dientamoeba fragilis, and 100% and 99.7% for Cryptosporidium spp., respectively. Conclusions: The Allplex™ GI-Parasite Assay exhibited excellent performance in the detection of the most common enteric protozoa. Full article

Although Dientamoeba fragilis is a common protozoan in humans, its pathogenicity and clinical significance in human diseases remain poorly understood. This study aimed to determine the frequency of D. fragilis in adult ulcerative colitis patients and to assess its relationship with clinical findings, disease characteristics, and biochemical parameters. Patient data were analysed in a prospective, single-centre, cross-sectional design. Faecal samples were consecutively collected from June to December 2024 and screened for D. fragilis positivity using polymerase chain reaction. Of the 110 patients, 33 (30%) were in the active stage of the disease, while 77 (70%) were in remission. The overall frequency of D. fragilis was 10.9% (n = 12), with all isolates classified as genotype 1 according to SSU rRNA sequence analysis. Other protozoa identified were Blastocystis sp. (n = 5, 4.5%), Entamoeba coli (n = 1, 0.9%), and Iodamoeba bütschlii (n = 1, 0.9%). Two patients were co-infected with D. fragilis and Blastocystis. No significant associations were found between D. fragilis positivity and the disease stage, gastrointestinal symptoms, treatment response, or biochemical findings. In conclusion, despite the relatively small sample size, these findings highlight a limited clinical role of D. fragilis in adult ulcerative colitis patients. Full article

Background: Microscopy is the conventional method for the identification of gastrointestinal parasitic pathogens in fecal specimens; however, it presents numerous challenges, including high technical expertise burden, multiple staining procedures, and prolonged turnaround time. Molecular methods provide higher throughput and potentially higher sensitivity and specificity. Methods: We validated a commercial, automated DNA extraction platform and multiplex parasitic real-time PCR panel (Seegene Allplex^TM GI-Parasite Assay) detecting six protozoal pathogens: Blastocystis hominis (Bh), Cryptosporidium spp., Cyclospora cayetanensis (Cc), Dientamoeba fragilis (Df), Entamoeba histolytica (Eh), and Giardia lamblia (Gl) in unpreserved fecal specimens submitted for diagnostic parasitology. Microscopy was the reference standard for all organisms, with stool ELISA as an additional reference assay for Eh. Results: Among 461 unpreserved fecal specimens, sensitivity, specificity, positive predictive and negative predictive values of the enteric multiplex for fresh specimens were as follows: 93%, 98.3%, 85.1%, 99.3% for Bh; 100% for all measures in Cryptosporidium and Cc; 100%, 99.3%, 88.5%, 100% for Df; 33.3%, 100%, 100%, 99.6% for Eh; and 100%, 98.9%, 68.8%, 100% for Gl, respectively. With the addition of 17 frozen specimens, the sensitivity for Eh increased to 75%. On a per-batch basis, the molecular platform reduced pre-analytical and analytical testing turnaround time by 7 h. Conclusions: The enteric multiplex platform provides a useful diagnostic tool for clinically relevant enteric protozoa, including Cryptosporidium spp., Cyclospora cayetanensis, Dientamoeba fragilis, and Giardia lamblia. Further evaluation of the assay is required for Entamoeba histolytica prior to clinical use; however, given the widespread availability of confirmatory serology and stool antigen testing for E. histolytica, such performance limitations are of lesser concern. Full article

Objetive: The aim of this study was to describe the impact of non-pharmaceutical interventions (NPIs) against SARS-CoV-2 in patients with symptoms of enteric protozoa (EP), including Blastocystis spp., Dientamoeba fragilis, Giardia lamblia, Cryptosporidium spp., Entamoeba histolytica, and Cyclospora cayetanensis, in the overall population and in patients who were consulted at a National Referral Center for Imported Tropical Diseases (NRCITD patients) from a healthcare area in Madrid (Spain). Method: Data on patients with positive RT-PCR results for EP were collected. The periods analyzed were prepandemic (P0, 1 April 2019–31 March 2020), and the first (P1, 1 April 2020–31 March 2021), second (P2, 1 April 2021–31 March 2022), and third (P3, 1 April 2022–31 March 2023) pandemic years. We compared the prevalence, median age, absolute incidence (EP per 100,000 population of each period), and patient profile (NRCITD vs. non-NRCITD) during the study periods using Fisher’s test (p < 0.05) and the T-test (p < 0.001). Results: During P0, 24.8%, [95% CI: 23.9–25.6] of patients tested for EP RT-PCR were positive, 22.6% [95% CI: 21.5–23.7] were positive in P1, 20.4%, [95% CI: 19.5–21.3] were positive during P2, and 20% [95% CI: 19.2–20.9] of patients tested during P3 were positive. During the study, there was no difference in the median ages. The prevalence and absolute incidence of EP showed a decreasing trend during the pandemic for the NRCITD and non-NRCITD patients (p < 0.05). Conclusion: Blastocystis spp. and D. fragilis showed a lower decrease in prevalence during P1 (p > 0.05) due to the higher detection of colonized patients during the SARS-CoV-2 pandemic. However, G. lamblia and Cryptosporidium spp. showed the highest decrease in prevalence and absolute incidence during P2 (p < 0.05) because of the NPIs implemented during the SARS-CoV-2 pandemic. The NTRCID patients showed a higher prevalence of Blastocystis spp. than the non-NTRCID patients during every period studied (p < 0.001). E. histolytica and C. cayetanensis showed a homogeneous trend. Full article

Recently a number of broad-range stool parasite PCR assays have been developed. However, there is ongoing disagreement regarding their diagnostic performance, as various studies have produced contradictory results. In this study, we compared the diagnostic accuracy of the Seegene Allplex GI-Parasite and Allplex GI-Helminth assays (SA) with the conventional methods used at the travel clinic of the Institute of Tropical Medicine (ITM) including microscopy, antigen testing, and molecular detection in order to provide insights into the strengths and limitations of this diagnostic tool which may be crucial to select the most appropriate diagnostic tools for the suspected pathogen. A total of 97 native stool samples from 95 patients with suspected gastrointestinal illness were analyzed, including 26 from a frozen collection and 71 prospectively collected samples. The diagnostic performance of SA was notably superior to the conventional workflow in detecting Dientamoeba fragilis (sensitivity 100% vs. 47.4%) and Blastocystis hominis (sensitivity 95% vs. 77.5%). SA had a comparable performance with the conventional workflow in detecting pathogenic protozoa (sensitivity 90% vs. 95%). In contrast, SA had a much lower diagnostic performance in detecting helminths (59.1%) compared to the conventional workflow (100%). We conclude that the Seegene Allplex GI-Parasite assay may be useful for protozoa screening in low-endemic industrialized countries. However, the Allplex GI-Helminth assay is not recommended due to its suboptimal performance compared to microscopy. Full article

The study was conducted to identify cluster patterns of enteric microorganisms with potential etiological relevance for infectious gastroenteritis in stool samples of individuals from Ghana, which is a known high-endemicity setting for infectious gastroenteritis. These patterns were compared to previous observations with specimens from Colombian indigenous people in order to assess potentially stable clustering for temporally and spatially distinct populations from high-endemicity regions. By doing so, the study aimed to identify stable clusters as markers of microbial interaction with potential importance for etiological relevance assignment in cases of multiple enteric pathogen detections. Stool samples from 1569 Ghanaian individuals (875 from HIV patients, 30 from HIV-negative control adult patients, and 644 from children < 2 years of age) were assessed for enteric microorganisms by applying real-time PCR. As a result, nucleic acids of bacterial microorganisms were most frequently detected, followed by protozoa, microsporidia, and helminths. Interestingly, the cluster assessment confirmed interaction patterns known from the previous analysis with Colombian indigenous people, demonstrating a high likelihood of Blastocystis hominis for clustering with other microorganisms and a prominent, potentially mediating role of Dientamoeba fragilis for microbial interactions within the clusters. In conclusion, the assessment confirmed conserved clustering of enteric microorganisms with potential etiological relevance for human infectious gastroenteritis over geographically distinct high-endemicity settings. Furthermore, the composition of abundant microorganisms is more important than regional factors for the determination of the interplay of enteric microorganisms in the human gut. Thereby, some microbial pathogens and commensals seem more susceptible to a changing microbial composition in the human gut than others. Full article

This narrative review was aimed at collecting updated knowledge on the risk factors, illnesses caused, and measures for the prevention of protozoan infections transmitted by food and drinking water. Reports screened dated from 2019 to the present and regarded global prevalence in food handlers, occurrence in food and drinking water, impact on human health, and recently reported outbreaks and cases of severe infections attributable to the dietary route. Cryptosporidium spp., Cyclospora cayetanensis, Entamoeba histolytica, and Cystoisospora belli were the protozoans most frequently involved in recently reported waterborne and foodborne outbreaks and cases. Blastocystis hominis was reported to be the most widespread intestinal protozoan in humans, and two case reports indicated its pathogenic potential. Dientamoeba fragilis, Endolimax nana, and Pentatrichomonas hominis are also frequent but still require further investigation on their ability to cause illness. A progressive improvement in surveillance of protozoan infections and infection sources took place in developed countries where the implementation of reporting systems and the application of molecular diagnostic methods led to an enhanced capacity to identify epidemiological links and improve the prevention of foodborne and waterborne protozoan infections. Full article

Multiple microbial detections in stool samples of indigenous individuals suffering from chronic gastroenteric disorder of a likely infectious origin, characterized by recurring diarrhea of variable intensity, in the rural north-east of Colombia are common findings, making the assignment of etiological relevance to individual pathogens challenging. In a population of 773 indigenous people from either the tribe Wiwa or Kogui, collider bias analysis was conducted comprising 32 assessed microorganisms including 10 bacteria (Aeromonas spp., Campylobacter spp., enteroaggregative Escherichia coli (EAEC), enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), Salmonella spp., Shiga toxin-producing Escherichia coli (STEC), Shigella spp./enteroinvasive Escherichia coli (EIEC), Tropheryma whipplei and Yersinia spp.), 11 protozoa (Blastocystis spp., Cryptosporidium spp., Cyclospora spp., Dientamoeba fragilis, Entamoeba coli, Entamoeba bangladeshi/dispar/histolytica/moshkovskii complex, Entamoeba histolytica, Endolimax nana, Giardia duodenalis, Iodamoeba buetschlii and Pentatrichomonas hominis), 8 helminths (Ascaris spp., Enterobius vermicularis, Hymenolepis spp., Necator americanus, Schistosoma spp., Strongyloides spp., Taenia spp. and Trichuris spp.), microsporidia (Encephalocytozoon spp.) and fungal elements (microscopically observed conidia and pseudoconidia). The main results indicated that negative associations potentially pointing towards collider bias were infrequent events (n = 14), while positive associations indicating increased likelihood of co-occurrence of microorganisms quantitatively dominated (n = 88). Microorganisms showing the most frequent negative associations were EPEC (n = 6) and Blastocystis spp. (n = 3), while positive associations were most common for Trichuris spp. (n = 16), Dientamoeba fragilis (n = 15), Shigella spp./EIEC (n = 12), Ascaris spp. (n = 11) and Blastocystis spp. (n = 10). Of note, positive associations quantitively dominated for Blastocystis spp. In conclusion, collider bias assessment did not allow clear-cut assignment of etiological relevance for detected enteric microorganisms within the assessed Colombian indigenous population. Instead, the results suggested complex microbial interactions with potential summative effects. Future studies applying alternative biostatistical approaches should be considered to further delineate respective interactions. Full article

Background and Objectives: The guidelines for chronic urticaria in children contain recommendations that are often based on adult studies. The diagnostic pathway has not been standardized and the effectiveness of anti-H1, omalizumab, montelukast, and systemic glucocorticoids is rarely reported in the pediatric population. There is a wide variation in the rate of remission of chronic urticaria between studies. The aim of this study is to enhance our understanding of pediatric chronic urticaria. Materials and Methods: This study enrolled 37 children with chronic urticaria aged from 0 to 18 years. Demographic parameters, medical history, clinical features, laboratory data and treatment information were collected. Children were treated with the recommended dosage of second-generation H1-antihistamines, which was increased by up to twofold. Omalizumab was added for refractory anti-H1 patients. A three-day course with systemic glucocorticoids was administered for severe exacerbations. Montelukast was administered to some children. Results: Wheals without angioedema were common. Chronic urticaria was spontaneous in 32 children (86.48%), inducible in 2 (5.41%), induced by a parasite in 1 and vasculitic in 2. Treatment of the potential causes of chronic urticaria was of no benefit, except for eradication of Dientamoeba fragilis. Chronic urticaria was resolved within three years in 45.9% of cases. Allergic diseases were present in nine children (24.32%) and autoimmune diseases were present in three (8.11%). All children were treated with anti-H1 at the licensed dose or at a higher dose. A partial or complete response to anti-H1 was observed in 29 (78.38%) patients. Montelukast showed no benefit. All children treated with omalizumab responded. Systemic glucocorticoids were successfully used to treat exacerbations. Conclusions: Our findings indicate that laboratory tests should not be routinely performed in children with chronic urticaria without clinical suspicion. However, comorbidities such as thyroid autoimmune disease and coeliac disease are suggested to be monitored over the chronic urticaria course. These clinical conditions could be diagnosed from the diagnostic framework of chronic urticaria. Increasing the dosage of anti-H1 and omalizumab was effective in children resistant to standard treatment but we still need further studies to generate a standard patient-centered treatment. Full article

Intestinal parasitic infections are one of the most common infectious diseases worldwide, particularly in developing countries. A distinct group at increased risk of infection is military personnel deployed overseas for extended periods, typically six months at a time. The aim of this study was to determine the prevalence of Blastocystis spp. and other intestinal parasites in Polish military personnel returning from deployments to Lebanon (n = 206) and Iraq (n = 220). In this group of subjects, we found Blastocystis spp. (13.6%), Dientamoeba fragilis (3.3%), Entamoeba coli (0.9%), and Endolimax nana (0.5%). Entamoeba histolytica sensu lato and Chilomastix mesnili infections were detected only in one soldier returning from Lebanon and Iraq, respectively. Blastocystis subtype (ST) 3 was predominant in soldiers returning from Lebanon, followed by ST2 and ST1. ST1 infection was predominant in soldiers returning from Iraq, followed by ST3 and ST2. Our study affirms that, deployment abroad is of no influence of the prevalence of parasitic protozoa. However, it would be worth to monitor parasite infection in military personnel returning from tropical zone even if they have no actual symptoms. In addition, it is very important to determine the subtypes of Blastocystis—this may help to clearly define their pathogenicity, especially considering the scarcity of studies on Blastocystis genotypes in Iraqi and Lebanese residents. Full article

Cryptosporidium spp., Giardia duodenalis and Entamoeba histolytica are species of protozoa- causing diarrhoea that are common worldwide, while Entamoeba dispar, Dientamoeba fragilis and Blastocystis sp. appear to be commensal parasites whose role in pathogenicity remains controversial. We conducted the clinical evaluation of five singleplex and one duplex CerTest VIASURE Real-Time PCR Assays against a large panel of positive DNA samples (n = 358), and specifically to Cryptosporidium spp. (n = 96), G. duodenalis (n = 115), E. histolytica (n = 25) E. dispar (n = 11), Blastocystis sp. (n = 42), D. fragilis (n = 37), and related parasitic phylum species such as Apicomplexa, Euglenozoa, Microsporidia and Nematoda. DNA samples were obtained from clinical stool specimens or cultured isolates in a national reference centre. Estimated diagnostic sensitivity and specificity values were 0.94–1 for Cryptosporidium spp., 0.96–0.99 for G. duodenalis, 0.96–1 for E. histolytica, 1–1 for E. dispar, and 1–0.99 for D. fragilis in the evaluated singleplex assays. In the duplex assay for the simultaneous detection of Blastocystis sp. and D. fragilis these values were 1–0.98 and 1–0.99, respectively. Measures of diagnostic precision for repeatability and reproducibility were found to be under acceptable ranges. The assays identified six Cryptosporidium species (C. hominis, C. parvum, C. canis, C. felis, C. scrofarum, and C. ryanae), four G. duodenalis assemblages (A, B, C, and F), and six Blastocystis subtypes (ST1-ST5, and ST8). The evaluated singleplex and duplex VIASURE Real-Time PCR assays provide sensitive, practical, and cost-effective choices to the molecular diagnosis of the main diarrhoea-causing intestinal protists in clinical microbiology and research laboratories. Full article

Indigenous people live in remote areas of Colombia. Multiple infections with bacteria, protozoa and/or helminths are common, as well as colonization in various forms. This study focused on the question of whether and to what extent various pathogens interact with each other. Therefore, a mathematical approach was retrospectively applied to PCR-based data of 244 stool samples, collected in two datasets. A stable cluster solution of the pathogens assessed was determined, and a unique configuration between Blastocystis hominis/Campylobacter spp./Giardia lamblia forming cluster 1 and Dientaemoeba fragilis was verified. A pathogen density-dependent interplay appeared between the B. hominis/Campylobacter spp./G. lamblia cluster, D. fragilis and Ascaris lumbricoides. The applied mathematical approach demonstrated that co-infections with parasites of questionable pathological relevance such as B. hominis and D. fragilis can be of diagnostic relevance due to their ability to promote or repress other pathogens. With the increasing availability of highly sensitive multiplexed molecular diagnostic approaches even in resource-limited settings, where multiple colonization of infection events with enteric pathogens in parallel are common, the importance of interpreting whole pathogen patterns rather than just individual pathogen detection may become more and more relevant. Full article

Globally, over 3.5 billion people are infected with intestinal parasites each year, resulting in over 200,000 deaths. Three of the most common protozoan pathogens that affect the gastrointestinal tract of humans are Cryptosporidium spp., Giardia intestinalis, and Entamoeba histolytica. Other protozoan agents that have been implicated in gastroenteritis in humans include Cyclospora cayetanensis, Dientamoeba fragilis, Blastocystis hominis, and the microsporidia Enterocytozoon bieneusi and Encephalitozoon intestinalis. Genetic Signatures previously developed a 3base™ multiplexed Real-Time PCR (mRT-PCR) enteric protozoan kit (EP001) for the detection of Giardia intestinalis/lamblia/duodenalis, Cryptosporidium spp., E. histolytica, D. fragilis, and B. hominis. We now describe improvements to this kit to produce a more comprehensive assay, including C. cayetanensis, E. bieneusi, and E. intestinalis, termed EP005. The clinical performance of EP005 was assessed using a set of 380 clinical samples against a commercially available PCR test and other in-house nucleic acid amplification tests where commercial tests were not available. All methods provided at least 90% agreement. EP005 had no cross-reactivity against 82 organisms commonly found in the gut. The EP005 method streamlines the detection of gastrointestinal parasites and addresses the many challenges of traditional microscopic detection, resulting in cost savings and significant improvements in patient care. Full article

Objectives: We aimed to assess the performance of the Novodiag^® Stool Parasites (NSP) assay in the diagnosis of the most common intestinal protozoan and microsporidia infections. Methods: A panel of 167 selected stool samples was retrospectively analysed with the NSP assay and compared to routine microscopy and qPCR methods for the detection of pathogenic protozoa and microsporidia. Results: Whereas specificity was high for all protozoa and microsporidia, NSP sensitivity was strongly dependent on the comparative method used as reference. When compared to microscopic methods, NSP sensitivity was high (96.7 to 100%) for Blastocystis hominis, Entamoeba histolytica and Cyclospora cayetanensis but was lower for Giardia intestinalis (85.2%) and ≤50% for Cystoisospora belli and Dientamoeba fragilis. In comparison to conventional qPCR, the NSP assay demonstrated lower sensitivity characteristics dependent on parasite loads, reaching 60 to 70% for G. intestinalis, D. fragilis, Cryptosporidium spp. and E. histolytica. Sensitivity was 100% for Enterocytozoon bieneusi, but none of the five samples containing Encephalitozoon spp. were detected. Conclusions: The overall performance of the NSP assay in the diagnosis of gastrointestinal protozoa and microsporidia seems to be better than or equivalent to that observed with microscopic methods but inferior to that obtainable with classical targeted qPCR. Full article

Dientamoeba fragilis is a cosmopolitan intestinal protist colonizing the human gut with varying prevalence depending on the cohort studied and the diagnostic methods used. Its role in human health remains unclear mainly due to the very sporadic number of cross-sectional studies in gut-healthy populations. The main objective of this study was to expand knowledge of the epidemiology of D. fragilis in gut-healthy humans and their animals. A total of 296 stool samples from humans and 135 samples from 18 animal species were analyzed. Using qPCR, a prevalence of 24% was found in humans in contrast to conventional PCR (7%). In humans, several factors were found to influence the prevalence of D. fragilis. A more frequent occurrence of D. fragilis was associated with living in a village, traveling outside Europe and contact with farm animals. In addition, co-infection with Blastocystis spp. was observed in nearly half of the colonized humans. In animals, D. fragilis was detected in 13% of samples from eight species using qPCR. Our molecular phylogenies demonstrate a more frequent occurrence of Genotype 1 in gut-healthy humans and also revealed a likely a new protist species/lineage in rabbits related to D. fragilis and other related organisms. Full article