Evaluation of a Novel Commercial Real-Time PCR Assay for the Simultaneous Detection of Cryptosporidium spp., Giardia duodenalis, and Entamoeba histolytica

Thorough independent assessment of the diagnostic performance of novel diagnostic assays is essential to ascertain their true usefulness and applicability in routine clinical practice. This is particularly true for commercially available kits based on multiplex real-time PCR aimed to detect and differentiate multiple pathogens in a single biological sample. Cryptosporidium spp., Giardia duodenalis, and Entamoeba histolytica are the most common diarrhea-causing protozoan species globally. Misdiagnosis is a concern for asymptomatic and chronic infections. Multiplexing, i.e., the detection of more than one parasite in a single test by real-time PCR, allows high diagnostic performance with favorable cost-effectiveness. We conducted a clinical evaluation of the VIASURE Cryptosporidium, Giardia, & E. histolytica real-time PCR assay (CerTest Biotec, San Mateo de Gallego, Spain) against a large panel (n = 358) of well-characterized DNA samples positive for Cryptosporidium spp. (n = 96), G. duodenalis (n = 115), E. histolytica (n = 25), and other parasitic species of the phyla Amoebozoa (n = 11), Apicomplexa (n = 14), Euglenozoa (n = 8), Heterokonta (n = 42), Metamonada (n = 37), Microsporidia (n = 4), and Nematoda (n = 6). DNA samples were obtained from clinical stool specimens or cultured isolates in a national reference center. Estimated sensitivity and specificity were 0.96 and 0.99 for Cryptosporidium spp., 0.94 and 1 for G. duodenalis, and 0.96 and 1 for E. histolytica, respectively. Positive and negative predictive values were calculated as 1 and 0.98 for Cryptosporidium spp., 0.99 and 0.98 for G. duodenalis, and 1 and 0.99 for E. histolytica, respectively. The assay identified six Cryptosporidium species (Cryptosporidium hominis, Cryptosporidium parvum, Cryptosporidium canis, Cryptosporidium felis, Cryptosporidium scrofarum, and Cryptosporidium ryanae) and four G. duodenalis assemblages (A, B, C, and F). The VIASURE assay provides rapid and accurate simultaneous detection and identification of the most commonly occurring species and genetic variants of diarrhea-causing parasitic protozoa in humans. IMPORTANCE Thorough independent assessment of the diagnostic performance of novel diagnostic assays is essential to ascertain their true usefulness and applicability in routine clinical practice. This is particularly true for commercially available kits based on multiplex real-time PCR aimed to detect and differentiate multiple pathogens in a single biological sample. In this study, we conducted a clinical evaluation of the VIASURE Cryptosporidium, Giardia, & E. histolytica real-time PCR assay (CerTest Biotec) for the detection and identification of the diarrhea-causing enteric protozoan parasites Cryptosporidium spp., G. duodenalis, and E. histolytica. A large panel of well-characterized DNA samples from clinical stool specimens or cultured isolates from a reference center was used for this purpose. The VIASURE assay demonstrated good performance for the routine testing of these pathogens in clinical microbiological laboratories

Co-Infection with Cryptosporidium meleagridis and Enterocytozoon bieneusi in an HIV+ Colombian Patient

A 44-year-old human immunodeficiency virus-infected (HIV+) female with severe immunodeficiency Category 3 (C3) diagnosed in 2010 was admitted to hospital with acute diarrhoea. She was non-adherent to antiretroviral therapy (ART) and had a previous suspicion of respiratory symptoms with a cough that had been persisting for 15 days. Clinical examination revealed severe immune deterioration (viral load: 109,655 copies/mL; CD4+ count: 14 cells/mm3), respiratory symptoms (negative sputum Gram stain and tuberculosis culture), and neurological deterioration (serological assays negative for Cryptococcus spp. and Toxoplasma gondii). A coproculture was negative for Campylobacter spp., Salmonella spp., and Shigella spp. Ziehl–Neelsen staining of faecal smears revealed the presence of Cryptosporidium spp. oocysts. PCR testing and sequencing confirmed a concomitant infection with C. meleagridis and Enterocytozoon bieneusi. The patient was treated with metronidazole (500 mg every 8 h for 5 days) and nitazoxanide (500 mg every 12 h for 14 days). After requesting voluntary discharge and abandoning ART and parasiticidal treatments, she experienced a dramatic deterioration of her state of health and contact with her was lost. Our results have demonstrated that molecular-based testing improves the detection of opportunistic pathogens that are difficult to detect by routine microscopy, allows for transmission dynamics investigations, and assists in choosing the best chemotherapeutical option.This research was funded by the Health Institute Carlos III (ISCIII) and the Ministry of Economy and Competitiveness (Spain) under project PI16CIII/00024.S

Wild micromammal host spectrum of zoonotic eukaryotic parasites in Spain. Occurrence and genetic characterisation

Micromammals have historically been recognized as highly contentious species in terms of the maintenance and transmission of zoonotic pathogens to humans. Limited information is currently available on the epidemiology and potential public health significance of intestinal eukaryotes in wild micromammals. We examined 490 faecal samples, grouped into 155 pools, obtained from 11 micromammal species captured in 11 Spanish provinces for the presence of DNA from Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi and Blastocystis sp. The presence of Leishmania spp. was investigated in individual spleen samples. All micromammal species investigated harboured infections by at least one eukaryotic parasite, except Apodemus flavicollis, Myodes glareolus, Sorex coronatus and Sciurus vulgaris, but the sample size for these host species was very low. Cryptosporidium spp. was the most prevalent species found (3.7%, 95% confidence interval [CI]: 2.2–5.7), followed by G. duodenalis (2.8%, 95% CI: 1.6–4.6) and E. bieneusi (2.6%, 95% CI: 1.4–4.3). All pooled faecal samples tested negative for Blastocystis sp. Leishmania infantum was identified in 0.41% (95% CI: 0.05–1.46) of the 490 individual spleen samples analysed. Sequence analyses allowed the identification of Cryptosporidium andersoni (5.9%), C. ditrichi (11.7%), C. muris (5.9%), C. parvum (5.9%), C. tyzzeri (5.9%), rat genotypes CR97 (5.9%) and W19 (5.9%), vole genotypes V (11.7%) and VII (5.9%) and Cryptosproridium spp. (35.3%) within Cryptosporidium (n = 17). Known genotypes C (66.7%) and Peru11 (25.0%) and a novel genotype (named MouseSpEb1, 8.3%) were detected within E. bieneusi (n = 12). None of the G. duodenalis-positive samples could be genotyped at the assemblage level. Molecular data indicate that wild micromammals were primarily infected by rodent-adapted species/genotypes of eukaryotic pathogens and thereby have a limited role as a source of human infections. The presence of ruminant-adapted species C. andersoni along with finding C. parvum is indicative of an overlap between domestic/peri-domestic and sylvatic transmission cycles of these agents.This work was supported by the Spanish Ministry for Science and Innovation under projects CGL2011-30274 and CGL2015-71255-P and by the BBVA Foundation under project TOPIGEPLA (2014 call). Additional funding was obtained from the Spanish Ministry for Science and Innovation under projects CGL2017-89866-R and E-RTA-2015-0002-C02-02 and by the Health Institute Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness under project PI19CIII/00029. David González-Barrio is the recipient of a Sara Borrell Research Contract (CD19CIII/00011) funded by the Spanish Ministry of Science, Innovation and Universities. Alejandro Dashti is the recipient of a PFIS contract (FI20CIII/00002) funded by the Spanish Ministry of Science and Innovation and Universities. The ‘Grupo de Rehabilitación de la Fauna Autóctona y su Hábitat’ (GREFA) provided partial funding and invaluable logistic and workforce support for samplings in NW Spain, along with many students and staff from the Autonomous University of Madrid (UAM).Peer reviewe

Molecular detection and characterization of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi infections in dromedary camels (Camelus dromedaries) in Egypt

IntroductionFew studies have investigated the occurrence of microeukaryotic gut parasites in dromedary camels in Egypt, and the majority of these investigations are based on microscopic analysis of fecal material.MethodsHerein, we assessed the occurrence, molecular diversity, and zoonotic potential of protozoan (Cryptosporidium spp. and Giardia duodenalis) and microsporidian (Enterocytozoon bieneusi) pathogens in individual fecal samples (n = 102) of dromedary camels with (n = 26) and without (n = 76) diarrhea from Aswan Governorate, Upper Egypt. Other factors possibly associated with an increased risk of infection (geographical origin, sex, age, and physical condition) were also analyzed. The SSU rRNA or ITS genes were targeted by molecular (PCR and Sanger sequencing) techniques for pathogen detection and species identification.Results and discussionThe most abundant species detected was G. duodenalis (3.9%, 4/102; 95% CI: 1.1–9.7), followed by Cryptosporidium spp. (2.9%, 3/102; 95% CI: 0.6–8.4). All samples tested negative for the presence of E. bieneusi. Sequence analysis data confirmed the presence of zoonotic C. parvum (66.7%, 2/3) and cattle-adapted C. bovis (33.3%, 1/3). These Cryptosporidium isolates, as well as the four Giardia-positive isolates, were unable to be amplified at adequate genotyping markers (Cryptosporidium: gp60; Giardia: gdh, bg, and tpi). Camels younger than 2 years old were significantly more likely to harbor Cryptosporidium infections. This connection was not statistically significant, although two of the three cryptosporidiosis cases were detected in camels with diarrhea. The spread of G. duodenalis infections was unaffected by any risk variables studied. This is the first report of C. parvum and C. bovis in Egyptian camels. The finding of zoonotic C. parvum has public health implications since camels may function as sources of oocyst pollution in the environment and potentially infect livestock and humans. Although preliminary, this study provides useful baseline data on the epidemiology of diarrhea-causing microeukaryotic parasites in Egypt. Further research is required to confirm and expand our findings in other animal populations and geographical regions of the country

Zoonotic "Enterocytozoon bieneusi" genotypes in free-ranging and farmed wild ungulates in Spain

Microsporidia comprises a diverse group of obligate, intracellular, and spore-forming parasites that infect a wide range of animals. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and other mammals and birds. Data on the epidemiology of E. bieneusi in wildlife are limited. Hence, E. bieneusi was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were collected from free-ranging (n = 1058) and farmed (n = 324) wild ungulates from five Spanish bioregions. The parasite was detected only in red deer (10.4%, 68/653) and wild boar (0.8%, 3/359). Enterocytozoon bieneusi infections were more common in farmed (19.4%, 63/324) than in wild (1.5%, 5/329) red deer. A total of 11 genotypes were identified in red deer, eight known (BEB6, BEB17, EbCar2, HLJD-V, MWC_d1, S5, Type IV, and Wildboar3) and three novel (DeerSpEb1, DeerSpEb2, and DeerSpEb3) genotypes. Mixed genotype infections were detected in 15.9% of farmed red deer. Two genotypes were identified in wild boar, a known (Wildboar3) and a novel (WildboarSpEb1) genotypes. All genotypes identified belonged to E. bieneusi zoonotic Groups 1 and 2. This study provides the most comprehensive epidemiological study of E. bieneusi in Spanish ungulates to date, representing the first evidence of the parasite in wild red deer populations worldwide. Spanish wild boars and red deer are reservoir of zoonotic genotypes of E. bieneusi and might play an underestimated role in the transmission of this microsporidian species to humans and other animal

Human-Borne Pathogens: Are They Threatening Wild Great Ape Populations?

Climate change and anthropic activities are the two main factors explaining wild great ape habitat reduction and population decline. The extent to which human-borne infectious diseases are contributing to this trend is still poorly understood. This is due to insufficient or fragmented knowledge on the abundance and distribution of current wild great ape populations, the difficulty obtaining optimal biological samples for diagnostic testing, and the scarcity of pathogen typing data of sufficient quality. This review summarises current information on the most clinically relevant pathogens of viral, bacterial, parasitic, and fungal nature for which transmission from humans to wild great apes is suspected. After appraising the robustness of available epidemiological and/or molecular typing evidence, we attempt to categorise each pathogen according to its likelihood of truly being of human origin. We further discuss those agents for which anthroponotic transmission is more likely. These include two viral (Human Metapneumovirus and Respiratory Syncytial Virus), one bacterial (diarrhoeagenic Escherichia coli), and two parasitic (Cryptosporidium spp. and Giardia duodenalis) pathogens. Finally, we identify the main drawbacks impairing research on anthroponotic pathogen transmission in wild great apes and propose research lines that may contribute to bridging current knowledge gaps

Occurrence and subtype distribution of Blastocystis sp. in humans, dogs and cats sharing household in northern Spain and assessment of zoonotic transmission risk.

Blastocystis sp. is probably the most common enteric parasite in humans globally. Although the role of Blastocystis in human disease is still controversial, epidemiological and experimental evidence suggests that pathogenicity may be associated with certain subtypes of the protist. Since the life cycle of Blastocystis is maintained through still elusive pathways, companion animals have attracted the attention of researchers as potential reservoirs of human infections. In order to evaluate the risk of zoonotic transmission of Blastocystis, we investigated the occurrence and molecular diversity of this microorganism in human, canine and feline populations sharing temporal and spatial settings in the province of Álava, northern Spain. A total of 268 (including 179 human, 55 canine and 34 feline) faecal specimens were obtained from 63 family households during February-December 2014. Detection of Blastocystis was achieved by PCR amplification and sequencing of small subunit rRNA genes. Blastocystis was found in 35.2% (95% CI: 0.29%-0.42%) of the human stool samples analysed, but not in any of the canine or feline faecal specimens investigated. Out of the 63 PCR-positive human samples, 84.1% (53/63) were successfully subtyped, allowing the identification of the subtypes ST2 (62.3%), ST3 (17.0%), ST1 (13.2%) and ST4 (7.5%). No mixed subtype infections were identified. Blastocystis carriage was independent of the gender and region of origin of the affected individuals, but children in the age groups of >5-10 years and >10-15 years were significantly more affected by the protist. None of the risk factors considered (water-use practices, contact with livestock, contact with individual undergoing diarrhoeal episodes) were associated with increased prevalence of Blastocystis. Our data demonstrate that pet dogs and cats play a negligible role as natural reservoirs of human Blastocystis infection in this geographic region, although the applicability of these results should be corroborated in future molecular epidemiological studies.Funding Information: Health Institute Carlos III, Ministry of Economy and Competitiveness. Grant Number: CP12/03081S

Prevalence and associated risk factors of intestinal parasites among schoolchildren in Ecuador, with emphasis on the molecular diversity of Giardia duodenalis, Blastocystis sp. and Enterocytozoon bieneusi.

BackgroundIntestinal helminths, including Soil-Transmitted Helminth (STH), and Gastrointestinal Protist (GP) infections are major contributors to the global burden of disease, particularly in low-income countries such Ecuador. Their epidemiology in these settings is largely unknown.MethodologyThis prospective cross-sectional study investigates the carriage of intestinal helminths, including STH, and GP in asymptomatic schoolchildren (3-11 years) in the Chimborazo and Guayas provinces, Ecuador. Single stool samples (n = 372) and epidemiological questionnaires on demographics and potential risk factors were collected from participating schoolchildren. Conventional microscopy examination was used as screening method, and molecular (PCR and Sanger sequencing) assays were used to further investigate the epidemiology of some GP. A multivariate logistic regression analysis was used to evaluate the strength of the association of suspected risk factors with the presence of helminths and GP.Principal findingsAt least one intestinal parasite species was observed by microscopy in 63.2% (235/372) of the participating schoolchildren. Enterobius vermicularis (16.7%, 62/372; 95% CI: 13.0-20.9) and Blastocystis sp. (39.2%, 146/372; 95% CI: 34.2-44.2) were the most prevalent among helminths and GP, respectively. Assemblages A (50.0%), B (37.5%) and A+B (12.5%) were detected within Giardia duodenalis and ST3 (28.6%), ST1 and ST2 (26.2% each), and ST4 (14.3%) within Blastocystis sp. Three genotypes, two known (A: 66.7%; KB-1: 16.7%) and a novel (HhEcEb1, 16.7%) were identified within Enterocytozoon bieneusi. Municipality of origin, household overcrowding, and poor sanitation and personal hygiene habits were risk factors for childhood intestinal parasites colonization.Conclusions/significanceDespite massive government drug administration programs, STH and GP infection remain a public health concern in paediatric populations living in poor-resource settings. Molecular analytical methods are required to better understand the epidemiology of these intestinal parasites. This study provides novel information on the occurrence of Blastocystis sp. and E. bieneusi genetic variants circulating in Ecuadorian human populations

Development, Optimisation and Validation of a Novel Multiplex Real-Time PCR Method for the Simultaneous Detection of Cryptosporidium spp., Giardia duodenalis and Dientamoeba fragilis

The enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis and Dientamoeba fragilis are—to various extents—contributors to the burden of gastrointestinal illness in high-income countries. Detection of these pathogens by microscopy examination is challenging because of the limited sensitivity and need for specific staining procedures. We developed and optimised a new multiplex real-time PCR assay for the simultaneous detection of Cryptosporidium spp., G. duodenalis and D. fragilis in clinical (stool) samples. The diagnostic performance of the assay was evaluated against a large panel of well-characterised DNA samples positive for Cryptosporidium spp. (n = 126), G. duodenalis (n = 132) and D. fragilis (n = 49). The specificity of the test was assessed against a DNA panel from other intestinal or phylogenetically related parasites (n = 105) and faecal DNA from individuals without clinical manifestations (n = 12). The assay exhibited a diagnostic sensitivity of 0.90–0.97 and a diagnostic specificity of 1. The limit of detection was estimated for Cryptosporidium (1 oocyst) and G. duodenalis (5 × 10−4 cysts). The method allowed the detection of four Cryptosporidium species (C. hominis, C. parvum, C. meleagridis and C. cuniculus) and five G. duodenalis assemblages (A–E) without cross-reacting with other parasites belonging to the phyla Amoebozoa, Apicomplexa, Euglenozoa, Microsporidia, Nematoda and Platyhelminthes. This newly developed multiplex real-time PCR assay represents a novel alternative for the rapid and accurate detection of Cryptosporidium, G. duodenalis and D. fragilis in clinical settings

Occurrence and molecular epidemiology of Giardia duodenalis infection in dog populations in eastern Spain

Abstract Background Giardia duodenalis is one of the most common enteric parasites in domestic animals including dogs. Young animals are more prone to the infection, with clinical manifestations ranging from asymptomatic to acute or chronic diarrhoea. Dogs are primarily infected by canine-specific (C-D) assemblages of G. duodenalis. However, zoonotic assemblages A and B have been increasingly documented in canine isolates, raising the question of whether and to which extent dogs can act as natural reservoirs of human giardiosis. Methods In this cross-sectional epidemiological survey we assessed the molecular diversity of G. duodenalis in dogs in the province of Castellón, Eastern Spain. A total of 348 individual faecal samples from sheltered (n = 218), breeding (n = 24), hunting (n = 68), shepherd (n = 24), and pet (n = 14) dogs were collected between 2014 and 2016. Detection of G. duodenalis cysts in faecal material was carried out by direct fluorescence microscopy as a screening test, whereas a qPCR targeting the small subunit ribosomal RNA gene of the parasite was subsequently used as a confirmatory method. Results Giardia duodenalis was detected in 36.5% (95% CI: 31.6–41.7%) of dogs. No significant differences in prevalence rates could be demonstrated among dogs according to their sex and geographical origin, but breeding (45.8%; 95% CI: 27.9–64.9%) and sheltered (40.4%; 95% CI: 34.1–47.0%) dogs harboured significantly higher proportions of G. duodenalis. Multi-locus sequence-based genotyping of the glutamate dehydrogenase and β-giardin genes of G. duodenalis allowed the characterization of 35 canine isolates that were unambiguously assigned to assemblages A (14.3%), B (22.9%), C (5.7%), and D (37.1%). A number of inter-assemblage mixed infections including A + B (11.4%), A + D (2.9%), and A + B + D (5.7%) were also identified. Conclusions Data presented here are strongly indicative of high infection pressures in kennelled animals. Zoonotic sub-assemblages AII, BIII, and BIV were responsible for a considerable proportion of the G. duodenalis infections detected, but very few of the genotypes identified have been previously documented in Spanish human populations. Although possible, zoonotic transmission between dogs and humans seems an infrequent event in this Spanish region