Molecular characteristics and zoonotic potential of enteric protists in domestic dogs and cats in Egypt.

Introduction: Domestic dogs and cats can be a source of human infection by a wide diversity of zoonotic pathogens including parasites. Genotyping and subtyping tools are useful in assessing the true public health relevance of canine and feline infections by these pathogens. This study investigated the occurrence, genetic diversity, and zoonotic potential of common diarrhea-causing enteric protist parasites in household dogs and cats in Egypt, a country where this information is particularly scarce. Methods: In this prospective, cross-sectional study a total of 352 individual fecal samples were collected from dogs (n = 218) and cats (n = 134) in three Egyptian governorates (Dakahlia, Gharbeya, and Giza) during July-December 2021. Detection and identification of Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. were carried out by PCR and Sanger sequencing. Basic epidemiological variables (geographical origin, sex, age, and breed) were examined for association with occurrence of infection by enteric protists. Results and discussion: The overall prevalence rates of Cryptosporidium spp. and G. duodenalis were 1.8% (95% CI: 0.5-4.6) and 38.5% (95% CI: 32.0-45.3), respectively, in dogs, and 6.0% (95% CI: 2.6-11.4) and 32.1% (95% CI: 24.3-40.7), respectively, in cats. All canine and feline fecal samples analyzed tested negative for E. bieneusi and Blastocystis sp. Dogs from Giza governorate and cats from Dakahlia governorate were at higher risk of infection by Cryptosporidium spp. (p = 0.0006) and G. duodenalis (p = 0.00001), respectively. Sequence analyses identified host-adapted Cryptosporidium canis (n = 4, one of them belonging to novel subtype XXe2) and G. duodenalis assemblages C (n = 1) and D (n = 3) in dogs. In cats the zoonotic C. parvum (n = 5) was more prevalent than host-adapted C. felis (n = 1). Household dogs had a limited (but not negligible) role as source of human giardiasis and cryptosporidiosis, but the unexpected high frequency of zoonotic C. parvum in domestic cats might be a public health concern. This is the first molecular-based description of Cryptosporidium spp. infections in cats in the African continent to date. Molecular epidemiological data provided here can assist health authorities and policy makers in designing and implementing effective campaigns to minimize the transmission of enteric protists in Egypt.

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.

Prevalence Patterns for Enteric Parasites of Chickens Managed in Open Environments of the Western United States.

Growing demand for poultry meat and eggs labeled as organic, cage free, or pasture raised has increased the number of producers that manage chickens outdoors. In these open environments, there are likely diverse enteric parasites sustained by fecal-oral transmission or passage through intermediate invertebrate hosts (e.g., worms and insects) that chickens consume. Enteric parasites can reduce chicken health and productivity, but there are few published data describing the identities or prevalence of these parasites on farms that use open environments in the United States. We surveyed 27 poultry farms with open environments that were situated across a wide geographic range, including California, Oregon, Idaho, and Washington. These farms did not use anticoccidial drugs, coccidia vaccines, or parasiticides. Flock size, enclosure area, flock density, flock rotation frequency, and average flock age were highly correlated for all the farms in this study. We analyzed how enclosure size and flock rotations per year (which represented two axes of variation in management) correlated with prevalence of five observed parasite taxa at the farm level. Across all flocks, we detected by fecal flotation Eimeria spp. (95% flocks), Ascaridia galli (69%), Heterakis gallinarum (52%), Capillaria spp. (39%), Strongyloides avium (13%), tapeworm species (29%), Cryptosporidium spp. (3%), and Dispharynx nasuta (1%). Eighty-five percent of samples were coinfected with two or more parasite taxa. Sixty-seven percent of farms raised only layer chicken breeds, 4% raised only broiler breeds, and 30% raised both layer and broiler breeds. The average age of the broiler flocks was 11.0 wk (±1.1 SE), and flocks were moved 54.7 (±17.9) times annually to new locations in pastures (hereafter, "rotation"). Layer flocks averaged 84.9 (±7.67) wk of age and were moved less often on farms being rotated 20.0 (±6.05) times per year. Generalized linear mixed models indicated that for every 1 m2 increase in enclosure size, the odds of detecting Eimeria spp. increased by 0.03%. Furthermore, for every additional rotation per year, the odds of detecting A. galli decreased by 1.3%. For every additional rotation per year, the odds of detecting tapeworm species increased by 2.2%. We found no evidence that flock spatial management affected prevalence of the other parasites observed on the farms. Farming practices and parasite responses in these systems are highly varied, which makes it difficult to identify potential management interventions for reducing these infections.

Patrones de prevalencia de parásitos entéricos de pollos manejados en ambientes abiertos en el oeste de los Estados Unidos. La creciente demanda de carne de pollo y huevos etiquetados como orgánicos, sin jaula o criados en pastoreo ha aumentado el número de productores que manejan pollos al aire libre. En estos entornos abiertos, es probable que existan diversos parásitos entéricos que permanecen debido a la transmisión fecal-oral o por su paso a través de huéspedes invertebrados intermedios (por ejemplo, gusanos e insectos) que son consumidos por los pollos. Los parásitos entéricos pueden reducir la salud y la productividad de los pollos, pero existe poca información publicada que describa las identidades o la prevalencia de estos parásitos en granjas que utilizan entornos abiertos en los Estados Unidos. Se realizó una encuesta incluyendo 27 granjas avícolas con entornos abiertos que estaban situadas en un amplio rango geográfico, incluyendo California, Oregón, Idaho y Washington. Estas granjas no usaban medicamentos anticoccidiales, vacunas contra coccidias ni parasiticidas. El tamaño de la parvada, el área de pastoreo, la densidad de la parvada, la frecuencia de rotación de la parvada y la edad promedio de la parvada estuvieron altamente correlacionados para todas las granjas en este estudio. Se analizó cómo el tamaño del recinto y las rotaciones de parvadas por año (que representaban dos ejes de variación en el manejo) se correlacionaban con la prevalencia de cinco taxones de parásitos observados a nivel de granja. En todas las parvadas, se detectó por flotación fecal Eimeria spp. (95% de las parvadas), Ascaridia galli (69%), Heterakis gallinarum (52%), Capillaria spp. (39%), Strongyloides avium (13%), especies de nemátodos planos (29%), Cryptosporidium spp. (3%) y Dispharynx nasuta (1%). El ochenta y cinco por ciento de las muestras estaban coinfectadas con dos o más taxones de parásitos. El sesenta y siete por ciento de las granjas criaban solo razas de gallinas de postura, el 4% solo criaban razas de pollos de engorde y el 30% criaban razas de gallinas de postura y de pollos de engorde. La edad promedio de las parvadas de pollos de engorde fue de 11.0 semanas (±1.1 SE) y las parvadas se trasladaron 54.7 (±17.9) veces al año a nuevas ubicaciones en los pastos (en adelante, "rotación"). Las parvadas ponedoras promediaron 84.9 (± 7.67) semanas de edad y se trasladaron con menos frecuencia en granjas que se rotaron 20.0 (± 6.05) veces al año. Los modelos lineales mixtos generalizados indicaron que por cada aumento de un metro cuadrado en el tamaño del área de pastoreo, las probabilidades de detectar Eimeria spp. se incrementaron en un 0.03%. Además, por cada rotación adicional por año, las probabilidades de detectar A. galli disminuyeron en un 1.3%. Por cada rotación adicional por año, las probabilidades de detectar especies de tenia aumentaron en un 2.2%. No encontramos evidencia de que el manejo del espacio de la parvada afectara la prevalencia de los otros parásitos observados en las granjas. Las prácticas agrícolas y las respuestas de los parásitos en estos sistemas son muy variadas, lo que dificulta la identificación de posibles intervenciones de manejo para reducir estas infecciones.

Prevalence of endoparasitic and viral infections in client-owned cats in metropolitan Bangkok, Thailand, and the risk factors associated with feline hookworm infections.

The prevalence of enteric parasites in cats in metropolitan Bangkok has not been updated in over 13 years. The main objectives of this study include updating the prevalence of endoparasitism in client-owned cats, status of retroviral infections and determining the association between feline hookworm infection and possible risk factors. A total of 509 fecal samples were collected from client-owned cats in 2014-2015 and examined by a wet fecal mount technique. If additional sample remained, a PBS-ethyl acetate sedimentation was done (n = 229), and ZnSO4 centrifugal flotation was also performed if there was sufficient remaining sample (n = 105). At least one parasite was observed in 32.0% (163/509) of cats, with Ancylostoma being the most common intestinal parasite detected in 21.6% (110/509) of cats. Other parasitic infections detected by fecal examinations included Toxocara (6.9%; 35/509), Platynosomum (3.7%; 19/509), Cystoisospora (3.5%; 18/509), Taenia (2.9%; 15/509), Spirometra (1.6%; 8/509), Dipylidium (0.4%; 2/509), and Opisthorchis-like trematode (0.2%; 1/509). Examination for Giardia infection was conducted with the SNAP® Giardia Test, a coproantigen test, on a subset of the fecal samples (233/509) and revealed a positive result on 3.9% (9/233) of samples. Plasma samples were analyzed using the SNAP® Triple Test detecting antigens of Feline Leukemia Virus (FeLV) and Dirofilaria immitis while also detecting antibodies to Feline Immunodeficiency Virus (FIV). Antigens of FeLV and antibodies to FIV were found in 7.1% (19/269) and 5.2% (14/269) of cats, respectively. None of the cats were found to have circulating antigen of Dirofilaria immitis using this test. No association between retroviral and endoparasitic infections was found. From multivariable logistic regression examining associated factors, the ability of cats to access the outdoors (adjusted OR = 3.22, 95% CI; 1.42-7.87) and having tapeworm segments or adult helminths in feces (adjusted OR = 3.31, 95% CI; 1.34-8.21) were significantly associated with the finding of hookworm eggs in feces. This work presents the most up-to-date data on enteric feline parasite prevalence in the metropolitan Bangkok area from which fecal samples were directly collected from cats. Consequently, this study emphasizes that diagnosis of parasitic infections and the routine use of antiparasitic medications should be encouraged by veterinarians and to owners in order to reduce the reservoir of potentially zoonotic parasites.

Enteric parasites and socio-epidemiological variables in na academic community

Studies linking human health to environmental conditions are essential since parasitic diseases are connected to environmental and sanitary aspects. This study identified the prevalence of enteric parasites in an academic community in the municipality of Santo Antônio de Jesus, Bahia, Brazil. The purpose was to determine the existence, or not, of links between infections and socio-epidemiological variables, such as personal hygiene habits, the presence of sewage systems and the environment. Participants answered a questionnaire and received universal collectors for fecal samples. Spontaneous sedimentation methods and Rugai were used for diagnosis. One hundred twenty-one samples were analyzed, in which a 38.8% parasite prevalence was detected as well as a 61.7% rate of monoparasitism, as well as a predominance of protozoa Endolimax nana (78.7%) and Giardia duodenalis (21.3%). Among parasitized individuals, 97.9% lived in the Recôncavo Baiano region. The following statistical significance stands out in the findings, with p<0.05: individuals who had already bathed in the local river were more likely to be parasitized than those who had not (p = 0.034) and individuals who washed their hands more frequently before meals proved to be less prone to intestinal parasitic infections (p = 0.018). Results evidenced the presence of enteric parasites in a number of participants in spite of their being university students. The socio-epidemiological variables analyzed brought to light characteristics that favor the establishment of the epidemiological infection triad, such as improper packaging of household waste on disposal and no records of regular domestic water tank cleaning.

Molecular Diversity of Giardia duodenalis, Cryptosporidium spp., and Blastocystis sp. in Symptomatic and Asymptomatic Schoolchildren in Zambézia Province (Mozambique).

Infections by the protist enteroparasites Giardia duodenalis, Cryptosporidium spp., and, to a much lesser extent, Blastocystis sp. are common causes of childhood diarrhoea in low-income countries. This molecular epidemiological study assesses the frequency and molecular diversity of these pathogens in faecal samples from asymptomatic schoolchildren (n = 807) and symptomatic children seeking medical attention (n = 286) in Zambézia province, Mozambique. Detection and molecular characterisation of pathogens was conducted by polymerase chain reaction (PCR)-based methods coupled with Sanger sequencing. Giardia duodenalis was the most prevalent enteric parasite found [41.7%, 95% confidence interval (CI): 38.8‒44.7%], followed by Blastocystis sp. (14.1%, 95% CI: 12.1‒16.3%), and Cryptosporidium spp. (1.6%, 95% CI: 0.9‒2.5%). Sequence analyses revealed the presence of assemblages A (7.0%, 3/43) and B (88.4%, 38/43) within G. duodenalis-positive children. Four Cryptosporidium species were detected, including C. hominis (30.8%; 4/13), C. parvum (30.8%, 4/13), C. felis (30.8%, 4/13), and C. viatorum (7.6%, 1/13). Four Blastocystis subtypes were also identified including ST1 (22.7%; 35/154), ST2 (22.7%; 35/154), ST3 (45.5%; 70/154), and ST4 (9.1%; 14/154). Most of the genotyped samples were from asymptomatic children. This is the first report of C. viatorum and Blastocystis ST4 in Mozambique. Molecular data indicate that anthropic and zoonotic transmission (the latter at an unknown rate) are important spread pathways of diarrhoea-causing pathogens in Mozambique.

Evaluation of Giardia intestinalis, Entamoeba histolytica and Cryptosporidium hominis/Cryptosporidium parvum in human stool samples by the BD MAXTM Enteric Parasite Panel.

Although the microscopic examination of stool samples remains the reference method of choice for the diagnosis of intestinal protistan infections, this method is time-consuming and requires experienced and well-trained operators. The purpose of this study was to evaluate the level of agreement between the BD MAX TM Enteric Parasite Panel (EPP) and microscopy for the detection of Giardia intestinalis (Lambl, 1859), Cryptosporidium spp. and Entamoeba histolytica Schaudinn, 1903 in stool samples. The study included faecal samples of 362 patients who were admitted to our hospital due to gastrointestinal complaints. In the microscopic examination, which was made with the native-lugol method on the stool samples that were taken from the patients, cysts, trophozoites and eggs of the parasite were examined. The diagnosis of G. intestinalis, Cryptosporidium parvum Tyzzer, 1912 and Cryptosporidium hominis Morgan-Ryan, Fall, Ward, Hijjawi, Sulaiman, Fayer, Thompson, Olson, Lal et Xiao, 2002, and E. histolytica was made in the faecal samples using the EPP assay. In the microscopic examination, Cryptosporidium spp. positive stool samples were stained with kinyoun's acid-fast. In the microscopic examination, parasites were detected in 41 (11%) of the 362 stool samples. In contrast, EPP assay identified parasites in 23 (6.3%) of the samples. In the microscopic examination, E. histolytica and Entamoeba dispar Brumpt, 1925 were detected in 22 (6.1%) of the samples, G. intestinalis was seen in 15 (4.1%), and C. parvum or C. hominis were detected in three (0.8%); these values were five (1.4%), 16 (4.4%) and two (0.5%) positive with the EPP assay. Although C. parvum or C. hominis were detected as positive in the microscopic examination of three samples, only two of the samples were positive in both EPP assay and kinyoun's acid-fast method. The EPP assay is a relatively simple test that can distinguish E. histolytica and E. dispar, but it cannot replace microscopy in the diagnosis of amoebiasis. Diagnosis for G. intestinalis and C. parvum/C. hominis with the BD MAXTM enteric parasite panel was equivalent to that with microscopy. We believe that E. histolytica must be diagnosed with nucleic acid amplification tests that have a high sensitivity and specificity like EPP assay in certain patient groups.

Protist enteroparasites in wild boar (Sus scrofa ferus) and black Iberian pig (Sus scrofa domesticus) in southern Spain: a protective effect on hepatitis E acquisition?

BACKGROUND: Several studies have independently evaluated the occurrence of hepatitis E virus (HEV) and enteroparasites in swine, but no surveys have been conducted to jointly assess the prevalence and genetic diversity of enteroparasites in pigs and wild boars, their sympatric transmission between hosts, and their potential interaction with HEV. METHODS: We prospectively collected serum and faecal samples from black Iberian domestic pigs and wild boars from southern Spain between 2015‒2016. We evaluated for HEV in serum and faeces, and for the presence of enteroparasites (Giardia duodenalis, Cryptosporidium spp., Blastocystis sp., Neobalantidium coli and Strongyloides spp.) in the same faecal samples. The prevalence of each intestinal parasite species was calculated. RESULTS: A total of 328 animals (56.7% black Iberian pigs and 43.3% wild boars) were included in the study. The overall global prevalence of HEV in serum was 16.8%. The overall global prevalence of each enteroparasite species was 19.5% for G. duodenalis, 8.2% for Cryptosporidium spp., 41.8% for Blastocystis sp., 31.4% for N. coli, and 8.8% for Strongyloides spp. HEV-infected animals showed a significantly lower prevalence of G. duodenalis (3.2 vs 20%; P = 0.002) and Blastocystis sp. (38.7 vs 80%; P < 0.001) than those uninfected by HEV. Animals carrying G. duodenalis and Blastocystis sp. infections showed a significantly lower rate of HEV infection than those not harbouring these enteroparasites (P < 0.001). CONCLUSIONS: Our study found a high prevalence of enteroparasites in black Iberian pigs and wild boars in southern Spain, suggesting a sympatric co-transmission of some of the species investigated. It is suggested that extracellular G. duodenalis and Blastocystis sp. might have a protective effect on HEV acquisition in swine.

Detection of enteric parasite DNA in household and bed dust samples: potential for infection transmission.

BACKGROUND: Enteric parasites are transmitted in households but few studies have sampled inside households for parasites and none have used sensitive molecular methods. METHODS: We collected bed and living room dust samples from households of children participating in a clinical trial of anthelmintic treatment in rural coastal Ecuador. Dust was examined for presence of DNA specific for 11 enteric parasites (Ascaris lumbricoides, Trichuris trichiura, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Toxocara canis and T. cati, Giardia lamblia, Blastocystis hominis, Cryptosporidium spp., and Entamoeba histolytica) by quantitative PCR (qPCR). RESULTS: Of the 38 households sampled, 37 had positive dust for at least one parasite and up to 8 parasites were detected in single samples. Positivity was greatest for B. hominis (79% of household samples) indicating a high level of environmental fecal contamination. Dust positivity rates for individual pathogens were: S. stercoralis (52%), A. lumbricoides (39%), G. lamblia (39%), Toxocara spp. (42%), hookworm (18%) and T. trichiura (8%). DNA for Cryptosporidium spp. and E. histolytica was not detected. Bed dust was more frequently positive than floor samples for all parasites detected. Positivity for A. lumbricoides DNA in bed (adjusted OR: 10.0, 95% CI: 2.0-50.1) but not floor dust (adjusted OR: 3.6, 95% CI: 0.3-37.9) was significantly associated with active infections in children. CONCLUSIONS: To our knowledge, this is the first use of qPCR on environmental samples to detect a wide range of enteric pathogen DNA. Our results indicate widespread contamination of households with parasite DNA and raise the possibility that beds, under conditions of overcrowding in a humid tropical setting, may be a source of transmission.

Diagnóstico molecular de parasitosis intestinales.

Infections causes by parasites of the gastrointestinal tract are a global public health problem. In industrialised countries, their particular epidemiological (low general prevalence of enteroparasites), economic (high labour costs) and clinical characteristics (constant increase in the number of samples and diagnostic determinations to be performed) have led molecular techniques to progressively replace conventional microscopy as the first-line diagnostic method of these pathogens in modern clinical laboratories. PCR-based techniques, particularly those developed for the simultaneous detection of the various agents that can cause the same infectious disease (syndromic diagnosis), already represent a cost-effective option that allow process automisation, workflow optimisation, and comparison of results among different laboratories, and facilitate accreditation of diagnostic procedures. This review clearly and concisely discusses the current situation of the molecular diagnosis of the main species of intestinal parasites in humans, particularly the enteric protozoans causing diarrhoea (Cryptosporidium spp., Giardia duodenalis, Entamoeba histolytica), the most important members the Microsporidia phyla (Enterocytozoon bieneusi) and Stramenopiles phyla (Blastocystis sp.), as well as the helminths transmitted by soil (Ancylostoma spp., Ascaris lumbricoides, Necator americanus, Strongyloides stercoralis and Trichuris trichiura) and food (Anisakis spp., Clonorchis sinensis, Fasciola spp., Taenia solium, and Trichinella spiralis). Special attention is paid to the description of available techniques and formats, to their diagnostic benefits and the most widely used genetic markers for their detection, both in clinical laboratories and genotyping in referral and research centres.

A hospital qPCR-based survey of 10 gastrointestinal parasites in routine diagnostic screening, Marseille, France.

There is a scarcity of recent epidemiological data on intestinal parasitic infections in France. We conducted a prospective study aimed at estimating the prevalence of 10 enteric parasites in Marseille, France, using real-time polymerase chain reaction (PCR)-based diagnosis. A total of 643 faeces from 488 patients referred to the Parasitology-Mycology Laboratory of the University Hospital of Marseille over a 6 months period were included. DNA was extracted using a semi-automated method. Parasites of interest were detected using singleplex quantitative PCRs (qPCRs). For positive samples, the Blastocystis subtype was determined by sequence analysis. During the study, the overall prevalence of enteric parasites was 17%. Blastocystis sp. was the most frequent species (10.5%), followed by Dientamoeba fragilis (2.3%) and Giardia intestinalis (2.3%). The prevalence of other parasites was <1% each. The ST3 Blastocystis subtype was predominant (43.6%) and the other subtypes identified were ST1, ST2, ST4 and ST6. This is the first time that a qPCR-based diagnosis has been used to survey the prevalence of 10 enteric parasites in a French University Hospital. This study confirms that fast, specific, sensitive and simultaneous detection in a single stool sample by qPCR clearly outperforms conventional microscopy-based diagnosis. Furthermore, qPCR is particularly well suited to surveying gastroenteritis agents.

Evaluation of two DNA extraction methods for the PCR-based detection of eukaryotic enteric pathogens in fecal samples.

OBJECTIVE: Efficient and easy-to-use DNA extraction and purification methods are critical in implementing PCR-based diagnosis of pathogens. In order to optimize the routine clinical laboratory diagnosis of eukaryotic enteric pathogens, we compare, via quantitative PCR cycle threshold (Ct) values, the efficiency of two DNA extraction kits: the semi-automated EZ1® (Qiagen) and the manual QIAamp® DNA Stool Mini Kit (Qiagen), on six protozoa: Blastocystis spp., Cryptosporidium parvum/hominis, Cyclospora cayetanensis, Dientamoeba fragilis, Giardia intestinalis and Cystoisospora belli and one microsporidia: Enterocytozoon bieneusi. RESULTS: Whereas EZ1® (Qiagen) and QIAamp® DNA Stool Mini Kit (Qiagen) yielded similar performances for the detection of Cryptosporidium spp. and D. fragilis, significant lower Ct values (p < 0.002) pointed out a better performance of EZ1® on the five remaining pathogens. DNA extraction using the semi-automated EZ1® procedure was faster and as efficient as the manual procedure in the seven eukaryotic enteric pathogens tested. This procedure is suitable for DNA extraction from stools in both clinical laboratory diagnosis and epidemiological study settings.

Prevalence and molecular characterization of Strongyloides stercoralis, Giardia duodenalis, Cryptosporidium spp., and Blastocystis spp. isolates in school children in Cubal, Western Angola.

BACKGROUND: Human infections by the gastrointestinal helminth Strongyloides stercoralis and the enteric protozoans Giardia duodenalis, Cryptosporidium spp. and Blastocystis spp. are not formally included in the list of 20 neglected tropical diseases prioritised by the World Health Organization. Although largely underdiagnosed and considered of lower public health relevance, these infections have been increasingly demonstrated to cause significant morbidity and even mortality globally, particularly among children living in resource-poor settings. METHODS: In this cross-sectional survey the prevalence, frequency and molecular diversity of S. stercoralis, G. duodenalis, Cryptosporidium spp. and Blastocystis spp. were investigated in a school children population in the province of Benguela (Angola). A total of 351 stool samples were collected during January to June 2015. The presence of S. stercoralis and G. duodenalis was confirmed by qPCR methods. Giardia duodenalis assemblages and sub-assemblages were determined by multilocus sequence-based genotyping of the glutamate dehydrogenase and ß-giardin genes of the parasite. Detection and identification of Cryptosporidium and Blastocystis species and subtypes was carried out by amplification and sequencing of a partial fragment of the small-subunit ribosomal RNA gene of both protozoan. Analyses of risk factors potentially associated with the transmission of these pathogens were also conducted. RESULTS: Prevalences of S. stercoralis, G. duodenalis, Cryptosporidium spp., and Blastocystis spp. were estimated at 21.4% (95% CI: 17.1-25.7%), 37.9% (95% CI: 32.8-43.0%), 2.9% (95% CI: 1.1-4.5%) and 25.6% (95% CI: 21.18-30.2%), respectively. Overall, 64.1% (225/351) of the children were infected by at least one of the pathogens investigated. Sequence analyses of the 28 G. duodenalis isolates that were successfully genotyped allowed the identification of sub-assemblages AI (14.3%), AII (14.3%), BIII (7.1%) and BIV (25.0%). Discordant typing results AII/AIII and BIII/BIV were identified in 7.1% and 14.3% of the isolates, respectively. A total of five additional isolates (17.9%) were identified as assemblage B. Three Cryptosporidium species including C. hominis (70%), C. parvum (20%) and C. canis (10%) were found circulating in the children population under study. A total of 75 Blastocystis isolates were assigned to the subtypes ST1 (30.7%), ST2 (30.7%), ST3 (36.0%), ST5 (1.3%) and ST7 (1.3%), respectively. Children younger than seven years of age had significantly higher risk of infections by protozoan enteropathogens (PRR: 1.35, P < 0.01), whereas being underweight seemed to have a protective effect against these infections (PRR: 0.74, P = 0.005). CONCLUSIONS: The burden of disease attributable to human strongyloidiasis, giardiosis, cryptosporidiosis and blastocystosis in Angola is considerably higher than initially estimated in previous surveys. Surveillance and control of these infections should be jointly tackled with formally considered neglected tropical diseases in order to maximize effort and available resources. Our data also demonstrate the added value of using molecular diagnostic methods in high transmission areas.

A Comparative Study of Four Methods for the Detection of Nematode Eggs and Large Protozoan Cysts in Mandrill Faecal Material.

Coproscopical methods like sedimentation and flotation techniques are widely used in the field for studying simian gastrointestinal parasites. Four parasites of known zoonotic potential were studied in a free-ranging, non-provisioned population of mandrills (Mandrillus sphinx): 2 nematodes (Necatoramericanus/Oesophagostomum sp. complex and Strongyloides sp.) and 2 protozoan species (Balantidium coli and Entamoeba coli). Different coproscopical techniques are available but they are rarely compared to evaluate their efficiency to retrieve parasites. In this study 4 different field-friendly methods were compared. A sedimentation method and 3 different McMaster methods (using sugar, salt, and zinc sulphate solutions) were performed on 47 faecal samples collected from different individuals of both sexes and all ages. First, we show that McMaster flotation methods are appropriate to detect and thus quantify large protozoan cysts. Second, zinc sulphate McMaster flotation allows the retrieval of a higher number of parasite taxa compared to the other 3 methods. This method further shows the highest probability to detect each of the studied parasite taxa. Altogether our results show that zinc sulphate McMaster flotation appears to be the best technique to use when studying nematodes and large protozoa.

Enteric parasitic infection among HIV-infected patients visiting Tribhuvan University Teaching Hospital, Nepal.

BACKGROUND: Enteric parasitic infection among human immunodeficiency virus (HIV) patients has been a significant health problem in developing countries like Nepal. This study was undertaken to access the burden of enteric parasites among HIV patients and its association with their immune status. METHODS: A cross-sectional study, involving 112 HIV sero-positive patients was conducted in Tribhuvan University Teaching Hospital, Public Health Research Laboratory, Kathmandu, Nepal from July 2011 to June 2012. The fecal samples were processed by direct-smear technique, in both normal saline solution and 1% iodine solution as well as modified acid fast staining (Kinyoun's method) after formalin ether concentration and Sheather's sucrose flotation for the identification of enteric parasites. RESULTS: Infection with one or more parasite was seen in 33.9% (n = 38) of the cases enrolled in the study, with the parasite prevalence rate of 41.1% (n = 46). Literacy (OR = 1.9, 95% CI 0.9-4.3) and CD4 T-cell count <200 (OR = 2.5, 95% CI 1.1-5.7) were found to be associated with enteric parasite infection. Similarly, CD4 T-cell count <200 was found to be associated with opportunistic parasitic infection (OR = 3.2, 95% CI 1.2-7.8). Among opportunistic parasites, Giardia duodenalis was the most common (28.3%, n = 13) one. Multi-parasitism was observed in six patients (15.8%). CONCLUSION: Enteric parasitic infections are common in HIV-infected people. The poor immune status as indicated by low CD4 T-cell count may account for higher risk of both opportunistic and non-opportunistic enteric parasitic infection.

Fish innate immunity against intestinal helminths.

Most individual fish in farmed and wild populations are infected with parasites. Upon dissection of fish, helminths from gut are often easily visible. Enteric helminths include several species of digeneans, cestodes, acanthocephalans and nematodes. Some insights into biology, morphology and histopathological effects of the main fish enteric helminths taxa will be described here. The immune system of fish, as that of other vertebrates, can be subdivided into specific and aspecific types, which in vivo act in concert with each other and indeed are interdependent in many ways. Beyond the small number of well-described models that exist, research focusing on innate immunity in fish against parasitic infections is lacking. Enteric helminths frequently cause inflammation of the digestive tract, resulting in a series of chemical and morphological changes in the affected tissues and inducing leukocyte migration to the site of infection. This review provides an overview on the aspecific defence mechanisms of fish intestine against helminths. Emphasis will be placed on the immune cellular response involving mast cells, neutrophils, macrophages, rodlet cells and mucous cells against enteric helminths. Given the relative importance of innate immunity in fish, and the magnitude of economic loss in aquaculture as a consequence of disease, this area deserves considerable attention and support.

Fast Technology Analysis Enables Identification of Species and Genotypes of Latent Microsporidia Infections in Healthy Native Cameroonians.

Several enteric microsporidia species have been detected in humans and other vertebrates and their identifications at the genotype level are currently being elucidated. As advanced methods, reagents, and disposal kits for detecting and identifying pathogens become commercially available, it is important to test them in settings other than in laboratories with "state-of-the-art" equipment and well-trained staff members. In the present study, we sought to detect microsporidia DNA preserved and extracted from FTA (fast technology analysis) cards spotted with human fecal suspensions obtained from Cameroonian volunteers living in the capital city of Yaoundé to preclude the need for employing spore-concentrating protocols. Further, we tested whether amplicon nucleotide sequencing approaches could be used on small aliquots taken from the cards to elucidate the diversity of microsporidia species and strains infecting native residents. Of 196 samples analyzed, 12 (6.1%) were positive for microsporidia DNA; Enterocytozoon bieneusi (Type IV and KIN-1), Encephalitozoon cuniculi, and Encephalitozoon intestinalis were identified. These data demonstrate the utility of the FTA cards in identifying genotypes of microsporidia DNA in human fecal samples that may be applied to field testing for prevalence studies.

Effect of enteric parasitic infection on serum trace elements and nutritional status in upper Egyptian children.

INTRODUCTION: Enteric parasitic infections still the cause of major health problems among Egyptian children as they have great morbid effect on their physical and cognitive development. Malnutrition makes children more prone to micronutrient deficiency and subsequently more vulnerable to parasitic infection. The present study aimed to identify the effect of intestinal parasitism on micronutrient serum level and children nutritional status. MATERIALS AND METHODS: A case control study was carried out on children from 1 to 6 years old who were attending the Assiut University Children Hospital outpatient clinic, after parasitological stool examination they were divided into Group 1 (G1, n: 60) positive with enteric parasite and Group 2 (G2, n: 60) age and sex matched and free of parasites. Anthropometric measurements were expressed as weight for age (WFA), height for age (HFA), and weight for height (WFH) parameters. Serum zinc (Zn) and copper (Cu) were determined by atomic absorption spectrophotometer. RESULTS: Intestinal parasitic infection rate was 55.7%; more commonly detected parasites were Giardia lamblia 28%, Cryptosporidium sp. 20%, and polyparasitism 18%. All children (G1 and G2) were underweight (WFA) while 63% of G1 were malnourished, either in the form of wasting (WFH) or stunting (HFA) or both aspects. Stunting and wasting were more dominant among children infected with G. lamblia and Cryptosporidium sp. and most of them were below 2 years old. CONCLUSIONS: Coincident decrease in serum Zn level and an increase of serum Cu was more prominent among G. lamblia and Cryptosporidium sp. patients. G. lamblia and Cryptosporidium sp. were found to be more associated with nonstandard children nutritional status beside to an altered micronutrient level.

Zoonotic enteric parasites transmitted from dogs in Egypt with special concern to Toxocara canis infection.

AIM: This work aimed to study the role played by dogs in transmitting zoonotic enteric parasites to humans in Egypt and to analyze the risk factors associated with the occurrence of such infection in dogs. Serodiagnosis of anti-Toxocara immunoglobulin G (IgG) antibodies among human beings as well as analyzing risk factors predispose to Toxocara canis infection in human beings are another objectives of this study. MATERIALS AND METHODS: From June to December 2013, a total of 130 fecal samples from 4 dog populations (Military, nomadic and domiciled dogs from rural and high standard districts) and 150 stool samples of 6 occupational groups were examined for the presence of enteric parasitic infection. Moreover, 150 serum samples were collected from humans from whom stool samples were collected and examined for the presence of anti-T. canis antibodies. RESULTS: Enteric parasites were detected in 30% of fecal samples from 4 dog populations in Egypt. High infectivity had been reported in nomadic dogs (63.33%) (Crude odds ratios [COR]=67.36, 95% confidence interval [CI]=8.09-560.8, p<0.000), followed by domiciled dogs from rural areas (40%) (COR=26, 95% CI=3.14-215.54, p=0.003), domiciled dogs from high standard areas (23.33%) (COR=11.87, 95% CI=1.37-102.69, p=0.025) and military dogs (2.5%). Twelve species of enteric parasites were identified, Ancylostomatidae (6.15%), T. canis and Cryptosporidium spp. (5.38%, each), Heterophyes spp. (3.85%), Toxocara leonina and Blastocystis spp. (3.07%), Taenidae eggs (2.31%), Hymenolepis diminuta (1.54%) and Entamoeba canis, Cyclospora cayetanensis, and Paragonimus spp. (0.77%, each). Univariate logestic regression revealed significant association of age (COR=4.73, 95% CI=2.13-10.53, p<0.000), gender (COR=2.63, 95% CI=1.22-5.68, p<0.014), housing system (COR=5.10, 95% CI=2.04-12.75), p<0.000) with enteric parasitic infection in dogs. However, breeds (COR=6.91, 95% CI=0.88-54.52, p=0.067) and type of feeding (COR ranged from 3.5 to 7.62, p>0.05) did not seem to have a significant association among the examined dogs. Enteric parasitic infection was reported in 31/150 human stools (20.67%). Students were the most affected groups (37.14%), followed by nomadic people (24%), house wives (20%), house guarders and military workers (12%, each), and employees (10%). The identified parasites were Cryptosporidium spp. (9.33%), Ascaris lumbercoides (3.33%), Heterophyes spp. and Ancylostoma spp. (2.66%, each) and Paragonimus spp. and Hymenolepis nana (1.33%, each). Toxocara IgG antibodies were detected in 36/150 (24%) serum samples investigated. Toxocara IgG antibodies were more prevalent in males (26.66%) than females (20%). Seroprevalence was highest (17/35, 48.57%) in 7-15 years old (COR=6.93, 95% CI=1.75-27.43, p=0.006). Seroprevalence values for T. canis antibodies were higher in those; raising dogs (29.85%), eating raw vegetables (25.21%) and not washing hands before meals (25.45%). T. canis antibodies were detected in 25% of those contacted with soil compared to 30% of those did not. Students were mostly affected (34.29%), followed by nomadic people (32%), house guarders (28%), housewives (20%), military workers (13%), and employees (10%). CONCLUSION: Detection of enteric parasites in dogs and humans in Egypt substantiates the role posed by dogs in transmitting zoonotic parasites to humans and knock an alarm for common sources of infection for humans and dogs. Common sources may be infected fish or contaminated vegetables that are consumed by dogs or humans or even infected rodents that may contaminate their feed. This pilot study necessitate the need for similar studies and tracing such infection in fish, vegetables, rodent that may be responsible for infecting humans and dogs in order to understand the epidemiology of zoonotic parasitic infection transmitted from dogs to humans.

Enteric Parasitic Infections in Relation to Diarrhoea in HIV Infected Individuals with CD4 T Cell Counts <1000 Cells/µl in Chennai, India.

AIM: This study was conducted to estimate the prevalence of enteric parasites in HIV patients in Chennai and to correlate with CD4 counts and diarrhoeal status. MATERIAL AND METHODS: Faecal specimens from 100 HIV infected individuals with CD4 < 1000/µl were screened for enteric parasites with wet mounts, modified acid-fast stain for coccidian parasites, modified trichrome stain for Microsporidia, before and after the stool concentration. Agar plate culture for Strongyloides was put up. Chi-square and ANOVA tests were used for statistical analysis. RESULTS: Study group comprised of 38 subjects with acute diarrhoea, 30 with chronic diarrhoea (> 2 weeks) and remaining 32 without diarrhoea. Enteric parasites were detected in 33% of subjects; Isoapora belli (21) being the commonest followed by E.histolyt/Entamoeba dispar (5), Entamoeba coli (2), Cryptosporidium spp (2), Hookworms (2), Strongyloides stercoralis (2), Giardia lamblia (1) and Microsporidium spp (1). There was a significant inverse relation between CD4 counts and duration of diarrhoea. Opportunistic parasites were isolated from the subjects with wide range of CD4 counts and different diarrhoeal status but most commonly from chronic diarrhoea patients. CONCLUSION: The prevalence of intestinal parasitic infections in HIV patients is high in Chennai, India, especially at CD4 <1000/µl, I.belli infection being the commonest. Routine screening of all HIV patients with low CD4 counts for coccidian parasitic infections by using simple stool microscopic techniques can help in early diagnosis and treatment.