Evaluation of five diagnostic methods for Strongyloides stercoralis infection in Amhara National Regional State, northwest Ethiopia.

BACKGROUND: Strongyloides stercoralis is an intestinal parasite that can cause chronic infection, hyperinfection and/or a dissemination syndrome in humans. The use of techniques targeting ova fails to detect S. stercoralis, as only larvae of the parasite are excreted in faeces. Due to the absence of "Gold" standard diagnostic method for S. stercoralis, there is a paucity of reported data worldwide. OBJECTIVE: This study aimed to evaluate the performance of diagnostic methods of S. stercoralis infection by taking the composite reference as a "Gold" standard. METHODS: A cross-sectional study was conducted among 844 schoolchildren in Amhara Region, Ethiopia, from April to December 2019. Stool samples were collected and processed with formol-ether concentration technique (FECT), spontaneous tube sedimentation technique (STST), Baermann concentration technique (BCT), agar plate culture (APC) and real-time polymerase chain reaction (RT-PCR). Sensitivity, specificity, positive predictive value, and negative predictive value of each diagnostic method were computed against the composite reference. The agreements of diagnostic methods were evaluated by Kappa value at 95% CI. RESULTS: The composite detection rate of S. stercoralis by the five diagnostic methods was 39.0% (329/844). The detection rate of the parasite from stool samples by FECT, STST, BCT, APC and RT-PCR was 2.0% (17/844), 4.0% (34/844), 10.2% (86/844), 10.9% (92/844) and 28.8% (243/844), respectively. The highest detection rate (37.8%; 319/844) of S. stercoralis was recorded by a combination of BCT, APC, and RT-PCR followed by a combination of STST, BCT, APC and RT-PCR (37.3%; 315/844). The sensitivity of FECT, STST, BCT, APC and RT-PCR against the composite reference was 5.2%, 10.3%, 26.4%, 28.0% and 73.9%, respectively. The diagnostic agreements of RT-PCR, APC, BCT, STST and FECT with the composite reference in detection of S. stercoralis were substantial (0.775), fair (0.321), fair (0.305), slight (0.123), and slight (0.062), respectively. CONCLUSION: RT-PCR detected the highest number of S. stercoralis infections. A combination of RT-PCR with APC and/or BCT better detected S. stercoralis from stool samples compared to other combinations or single diagnostic methods. Therefore, RT-PCR and combination of RT-PCR with APC and/or BCT diagnostic methods should be advocated for detection of S. stercoralis infection.

Prevalence and public health relevance of enteric parasites in domestic dogs and cats in the region of Madrid (Spain) with an emphasis on Giardia duodenalis and Cryptosporidium sp.

BACKGROUND: Pet dogs and cats exert an unquestionable beneficial effect in the well-being of their owners, but can also act as a source of zoonotic infections if improperly cared. OBJECTIVES: We investigated the occurrence, risk factors, genetic variability and zoonotic potential of intestinal parasites in dogs and cats attended in a clinical veterinary setting in Spain. METHODS: Canine (n = 252) and feline (n = 35) faecal samples were collected during 2017-2019 and analysed by coproparasitological methods. A rapid lateral immunochromatographic test (ICT) was used for detecting Giardia duodenalis and Cryptosporidium sp. Samples positive at microscopy examination and/or ICT were reassessed by molecular methods. RESULTS: Overall, 48.8% (123/252) of dogs and 48.6% (17/35) of cats were infected by enteric parasites. In dogs, G. duodenalis was the most prevalent species (40.9%), followed by Cystoisospora sp. (7.1%), and Toxocara canis (5.2%). In cats, Joyeuxiella sp. and Toxocara cati were the dominant species (20.0% each), followed by G. duodenalis (14.3%), D. caninum (5.7%) and Cystoisospora felis and Toxascaris leonina (2.9% each). Pups and kittens were more likely to harbour intestinal parasites and develop clinical signs. Sequence analyses of dog isolates revealed the presence of assemblages A (n = 1), C (n = 4), D (n = 4) and C+D (n = 1) within G. duodenalis; C. parvum (n = 1) and C. canis (n = 4) within Cryptosporidium and PtEb IX (n = 1) in Enterocytozoon bieneusi. A novel C. canis subtype family, named XXi, is reported. CONCLUSIONS: Our results highlight that (i) well-cared dogs carry zoonotic enteric protozoan parasites of public health relevance, (ii) proper hygiene practices and routine veterinary treatment are essential to prevent zoonotic infections, (iii) vulnerable populations should avoid contact with pups/kittens with diarrhoea and (iv) infected dogs might be major contributors to the environmental contamination with soil-transmitted helminths (STHs) eggs.

Thelaziosis in humans, a zoonotic infection, Spain, 2011.

After Thelazia callipaeda infection in dogs and cats were reported in Spain, a human case of thelaziosis in this country was reported, suggesting zoonotic transmission. The active reproductive status of this nematode in situ indicates that humans are competent hosts for this parasite.

Prevalence of Strongyloides stercoralis infection and associated clinical symptoms among schoolchildren living in different altitudes of Amhara National Regional State, northwest Ethiopia.

BACKGROUND: Strongyloides stercoralis is a parasite that causes strongyloidiasis in humans. It is prevalent in the tropics and sub-tropics where poor sanitation is a common problem. The true prevalence of S. stercoralis in Ethiopia is underestimated due to the lack of a "Gold" standard diagnostic method. Moreover, its prevalence across altitudinal gradient in Amhara Region has not been studied. METHODS: A cross-sectional study was conducted among 844 schoolchildren in Amhara Region from April to December 2019. A stool sample was collected from each study participant and processed using formol ether concentration technique (FECT), spontaneous tube sedimentation technique (STST), Baermann concentration technique (BCT), agar plate culture (APC) and real-time polymerase chain reaction (RT-PCR). Data were entered using EpiData and analyzed by SPSS version 23 statistical software. Prevalence of S. stercoralis infection was determined using a single diagnostic technique and combination of techniques. Association of clinical variables with S. stercoralis infection was assessed by logistic regression and independent variables with p<0.05 were considered statistically significant. RESULTS: Prevalence of soil-transmitted helminths (STHs) and S. mansoni infections was 38.0% and 20.4%, respectively. Among STHs, the prevalence of hookworm infection was 32.8%. Prevalence of S. stercoralis infection was 39.0%, 28.8%, 10.9%, 10.3%, 4.0% and 2.0% by the respective, combinations of the five methods, RT-PCR, APC, BCT, STST and FECT. The highest prevalence rates, 48.2%, 45.0% and 41.1% of S. stercoralis were recorded in the age group of 12-14 years, males and rural dwellers, respectively. Prevalence rates of S. stercoralis infection in highland, semi-highland and lowland areas were 40.4%, 41.8% and 25.9%, respectively. Having abdominal pain (AOR = 2.48; 95% CI:1.65-3.72), cough (AOR = 1.63;95%CI:1.09-2.42), urticaria (AOR = 2.49;95%CI:1.50-4.01) and being malnourished (AOR = 1.44;95%:1.10-2.01) were significantly associated with strongyloidiasis. CONCLUSION: Prevalence of S. stercoralis infection was high and varied across different altitudes in Amhara Region. Some clinical syndromes were found to be significantly associated with S. stercoralis infection. Therefore, proper diagnosis and preventive strategies against S. stercoralis infection are highly recommended to be devised and implemented in Amhara Region.

Epidemiology of intestinal helminthiases in a rural community of Ethiopia: Is it time to expand control programs to include Strongyloides stercoralis and the entire community?

BACKGROUND: Soil transmitted helminths are highly prevalent worldwide. Globally, approximately 1.5 billion people are infected with Ascaris lumbricoides, Trichuris trichiura or hookworm. Endemic countries carry out periodic mass treatment of at-risk populations with albendazole or mebendazole as a control measure. Most prevalence studies have focused on school aged children and therefore control programs are implemented at school level, not at community level. In this study, the prevalence of intestinal helminths, including Strongyloides stercoralis, was examined using a comprehensive laboratory approach in a community in north-western Ethiopia. METHODS: A cross-sectional survey was conducted on 792 individuals ≥5 years old in randomly selected houses in a rural district. Stools were examined using three techniques: a formol-ether concentration, the Baermann technique and a real time polymerase chain reaction test (these last two specific for S. stercoralis). Statistical analyses were performed between two large age groups, children (≤14 years old) and adults (≥15 years old). RESULTS: The prevalence of helminths was 91.3%; (95% CI: 89.3-93.3%). Hookworm was the most prevalent, 78.7% (95% CI 75.6-81.4%), followed by S. stercoralis 55.7% (95% CI 52.2-59.1%). Co-infection with both was detected in 45.4% (95% CI 42.0-49.0%) of the participants. The mean age of hookworm-infected individuals was significantly higher than non-infected ones (p = 0.003). Also, S. stercoralis infection was significantly associated with age, being more prevalent in adults (p = 0.002). CONCLUSIONS: This is the highest prevalence of S. stercoralis detected in Ethiopia so far. Our results highlight the need of searching specifically for infection by this parasite since it usually goes unnoticed if helminth studies rely only on conventional diagnostic techniques, i.e. Kato-Katz. Moreover, the focus of these programs on children undermines the actual prevalence of hookworm. The adult population acts as a reservoir for both hookworm and S. stercoralis and this fact may negatively impact the current control programs in Ethiopia which only target treatment of school aged children. This reservoir, together with a lack of adequate water, sanitation and hygiene, increases the probability of re-infection in children. Finally, the high prevalence of S. stercoralis found calls for a comprehensive diagnostic approach in endemic areas in addition to a revision of control measures that is, adding ivermectin to current albendazole/mebendazole, since it is the drug of choice for S. stercoralis.

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.

Human antimicrobial peptide histatin 5 is a cell-penetrating peptide targeting mitochondrial ATP synthesis in Leishmania.

Histatin 5 (Hst5) is a human salivary antimicrobial peptide that targets fungal mitochondria. In the human parasitic protozoa Leishmania, the mitochondrial ATP production is essential, as it lacks the bioenergetic switch between glycolysis and oxidative phosphorylation described in some yeasts. On these premises, Hst5 activity was assayed on both stages of its life cycle, promastigotes and amastigotes (LC(50)=7.3 and 14.4 microM, respectively). In a further step, its lethal mechanism was studied. The main conclusions drawn were as follows: 1) Hst5 causes limited and temporary damage to the plasma membrane of the parasites, as assessed by electron microscopy, depolarization, and entrance of the vital dye SYTOX Green; 2) Hst5 translocates into the cytoplasm of Leishmania in an achiral receptor-independent manner with accumulation into the mitochondrion, as shown by confocal microscopy; and 3) Hst5 produces a bioenergetic collapse of the parasite, caused essentially by the decrease of mitochondrial ATP synthesis through inhibition of F(1)F(0)-ATPase, with subsequent fast ATP exhaustion. By using the Hst5 enantiomer, it was found that the key steps of its lethal mechanism involved no chiral recognition. Hst5 thus constitutes the first leishmanicidal peptide with a defined nonstereospecific intracellular target. The prospects of its development, by its own or as a carrier molecule for other leishmanicidal molecules, into a novel anti-Leishmania drug with a preferential subcellular accumulation are discussed.

Exploring interactions between Blastocystis sp., Strongyloides spp. and the gut microbiomes of wild chimpanzees in Senegal.

BACKGROUND: Gut parasites exert an important influence on the gut microbiome, with many studies focusing on the human gut microbiome. It has, however, undergone severe richness depletion. Hygienic lifestyle, antimicrobial treatments and altered gut homeostasis (e.g., chronic inflammation) reduce gut microbiome richness and also parasite prevalence; which may confound results. Studying species closely related to humans could help overcome this problem by providing insights into the ancestral relationship between humans, their gut microbiome and their gut parasites. Chimpanzees are a particularly promising model as they have similar gut microbiomes to humans and many parasites infect both species. AIMS: We study the interaction between gut microbiome and enteric parasites in chimpanzees. Investigating what novel insights a closely related species can reveal when compared to studies on humans. METHODS: Using eighty-seven faecal samples from wild western chimpanzees (Pan troglodytes verus) in Senegal, we combine 16S rRNA gene amplicon sequencing for gut microbiome characterization with PCR detection of parasite taxa (Blastocystis sp., Strongyloides spp., Giardia duodenalis, Cryptosporidium spp., Plasmodium spp., Filariae and Trypanosomatidae). We test for differences in gut microbiota ecosystem traits and taxonomical composition between Blastocystis and Strongyloides bearing and non-bearing samples. RESULTS: For Blastocystis, twelve differentially abundant taxa (e.g., Methanobrevibacter), including Prevotella and Ruminococcus-Methanobrevibacter enterotype markers, replicate findings in humans. However, several richness indices are lower in Blastocystis carriers, contradicting human studies. This indicates Blastocystis, unlike Strongyloides, is associated to a "poor health" gut microbiome, as does the fact that Faecalibacterium, a bacterium with gut protective traits, is absent in Blastocystis-positive samples. Strongyloides was associated to Alloprevotella and five other taxonomic groups. Each parasite had its unique impact on the gut microbiota indicating parasite-specific niches. Our results suggest that studying the gut microbiomes of wild chimpanzees could help disentangle biological from artefactual associations between gut microbiomes and parasites.

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.

Synthesis and biological evaluation of fluorescent leishmanicidal analogues of hexadecylphosphocholine (miltefosine) as probes of antiparasite mechanisms.

The leishmanicidal mechanism of miltefosine (hexadecylphosphocholine, MT) is not clearly understood. Valuable insights into its mode of action could be obtained by fluorescence techniques, given suitably emitting analogues. In this regard, the synthesis and biological characterization of two fully competent MT fluorescent analogues is reported here: all-(E)-13-phenyltrideca-6,8,10,12-tetraenylphosphocholine (PTE-MT) and all-(E)-13-phenyltrideca-8,10,12-trien-6-ynylphosphocholine (PTRI-MT). Both compounds show large absorption coefficients and a modest, but usable, fluorescence yield. Their activities were very similar to that of MT and were recognized by the MT uptake system of Leishmania. Their localization in living L. donovani promastigotes by confocal microscopy show a homogeneous intracellular distribution of the fluorescence. The concentration of PTRI-MT within the parasites (ca. 1.7 mM) showed a 100-fold enrichment relative to its external concentration. These results are consistent with a multiple target leishmanicidal mechanism for MT and validate the application of these analogues for pharmacokinetic and diagnostic studies concerning the chemotherapy of leishmaniasis.

Molecular prevalence of Entamoeba histolytica/dispar infection among patients attending four health centres in north-west Ethiopia.

The prevalence of amoebiasis is often overestimated owing to its epidemiological overlap with the non-pathogenic Entamoeba dispar To provide evidence for this conjecture, a cross-sectional study was conducted from November 2013 to January 2015. A range of 180-200 µg of semi-solid and formed stools and 200 µL of diarrhoeic stool samples were used for DNA extraction from microscopically E. histolytica/dispar positive samples using the QIAamp® DNA Stool Mini Kit according to manufacturers' instructions. Nested PCR targeting 18S ribosomal RNA gene was used. In 422 microscopically positive E. histolytica/dispar stools, molecular prevalence revealed that E. histolytica infestation was present in only 1.7% (95% confidence interval [CI], 0.47-2.93) and E. dispar was found in 42.2% (95% CI, 37.49-46.91), while 56.2% (95% CI, 51.47-60.93) had neither E. histolytica nor E. dispar (P < 0.001). We conclude that infestation with E. histolytica is rarer in our study areas than was previously believed. Hence, accurate differentiation of E. histolytica and E. dispar is crucial.

Evaluation of five commercial methods for the extraction and purification of DNA from human faecal samples for downstream molecular detection of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Entamoeba spp.

High quality, pure DNA is required for ensuring reliable and reproducible results in molecular diagnosis applications. A number of in-house and commercial methods are available for the extraction and purification of genomic DNA from faecal material, each one offering a specific combination of performance, cost-effectiveness, and easiness of use that should be conveniently evaluated in function of the pathogen of interest. In this comparative study the marketed kits QIAamp DNA stool mini (Qiagen), SpeedTools DNA extraction (Biotools), DNAExtract-VK (Vacunek), PowerFecal DNA isolation (MoBio), and Wizard magnetic DNA purification system (Promega Corporation) were assessed for their efficacy in obtaining DNA of the most relevant enteric protozoan parasites associated to gastrointestinal disease globally. A panel of 113 stool specimens of clinically confirmed patients with cryptosporidiosis (n=29), giardiasis (n=47) and amoebiasis by Entamoeba histolytica (n=3) or E. dispar (n=10) and apparently healthy subjects (n=24) were used for this purpose. Stool samples were aliquoted in five sub-samples and individually processed by each extraction method evaluated. Purified DNA samples were subsequently tested in PCR-based assays routinely used in our laboratory. The five compared methods yielded amplifiable amounts of DNA of the pathogens tested, although performance differences were observed among them depending on the parasite and the infection burden. Methods combining chemical, enzymatic and/or mechanical lysis procedures at temperatures of at least 56°C were proven more efficient for the release of DNA from Cryptosporidium oocysts.

High prevalence of Strongyloides stercoralis in school-aged children in a rural highland of north-western Ethiopia: the role of intensive diagnostic work-up.

BACKGROUND: Soil-transmitted helminthiases (hookworms, Ascaris lumbricoides and Trichuris trichiura) are extremely prevalent in school-aged children living in poor sanitary conditions. Recent epidemiological data suggest that Strongyloides stercoralis is highly unreported. However, accurate data are essential for conducting interventions aimed at introducing control and elimination programmes. METHODS: We conducted a cross-sectional survey of 396 randomly selected school-aged children in Amhara region in rural area in north-western Ethiopia, to assess the prevalence of S. stercoralis and other intestinal helminths. We examined stools using three techniques: conventional stool concentration; and two S. stercoralis-specific methods, i.e. the Baermann technique and polymerase chain reaction. The diagnostic accuracy of these three methods was then compared. RESULTS: There was an overall prevalence of helminths of 77.5%, with distribution differing according to school setting. Soil-transmitted helminths were recorded in 69.2%. Prevalence of S. stercoralis and hookworm infection was 20.7 and 54.5%, respectively, and co-infection was detected in 16.3% of cases. Schistosoma mansoni had a prevalence of 15.7%. Prevalence of S. stercoralis was shown 3.5% by the conventional method, 12.1% by the Baermann method, and 13.4% by PCR, which thus proved to be the most sensitive. CONCLUSIONS: Our results suggest that S. stercoralis could be overlooked and neglected in Ethiopia, if studies of soil-transmitted helminths rely on conventional diagnostic techniques alone. A combination of molecular and stool microscopy techniques yields a significantly higher prevalence. In view of the fact that current control policies for triggering drug administration are based on parasite prevalence levels, a comprehensive diagnostic approach should instead be applied to ensure comprehensive control of helminth infections.

Prevalence and Genetic Diversity of Giardia duodenalis and Cryptosporidium spp. among School Children in a Rural Area of the Amhara Region, North-West Ethiopia.

BACKGROUD: Giardia duodenalis and Cryptosporidium spp. are enteric protozoan causing gastrointestinal illness in humans and animals. Giardiasis and cryptosporidiosis are not formally considered as neglected tropical diseases, but belong to the group of poverty-related infectious diseases that impair the development and socio-economic potential of infected individuals in developing countries. METHODS: We report here the prevalence and genetic diversity of G. duodenalis and Cryptosporidium spp. in children attending rural primary schools in the Bahir Dar district of the Amhara Region, Ethiopia. Stool samples were collected from 393 children and analysed by molecular methods. G. duodenalis was detected by real-time PCR, and the 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 species was carried out by sequencing of a partial fragment of the small-subunit ribosomal RNA gene. PRINCIPAL FINDINGS: The PCR-based prevalences of G. duodenalis and Cryptosporidium spp. were 55.0% (216/393) and 4.6% (18/393), respectively. A total of 78 G. duodenalis isolates were successfully characterized, revealing the presence of sub-assemblages AII (10.3%), BIII (28.2%), and BIV (32.0%). Discordant typing results AII/AIII and BIII/BIV were identified in 7.7% and 15.4% of the isolates, respectively. An additional five (6.4%) isolates were assigned to assemblage B. No mixed infections of assemblages A+B were found. Extensive genetic variation at the nucleotide level was observed within assemblage B (but no within assemblage A), resulting in the identification of a large number of sub-types. Cryptosporidium diversity was demonstrated by the occurrence of C. hominis, C. parvum, and C. viatorum in the population under study. CONCLUSIONS: Our data suggest an epidemiological scenario with an elevated transmission intensity of a wide range of G. duodenalis genetic variants. Importantly, the elevated degree of genetic diversity observed within assemblage B is consistent with the occurrence of intra-assemblage recombination in G. duodenalis.

Identification of new leishmanicidal peptide lead structures by automated real-time monitoring of changes in intracellular ATP.

Leishmanicidal drugs interacting stoichiometrically with parasite plasma membrane lipids, thus promoting permeability, have raised significant expectations for Leishmania chemotherapy due to their nil or very low induction of resistance. Inherent in this process is a decrease in intracellular ATP, either wasted by ionic pumps to restore membrane potential or directly leaked through larger membrane lesions caused by the drug. We have adapted a luminescence method for fast automated real-time monitoring of this process, using Leishmania donovani promastigotes transfected with a cytoplasmic luciferase form, previously tested for anti-mitochondrial drugs. The system was first assayed against a set of well-known membrane-active drugs [amphotericin B, nystatin, cecropin A-melittin peptide CA(1-8)M(1-18)], plus two ionophoric polyethers (narasin and salinomycin) not previously tested on Leishmania, then used to screen seven new cecropin A-melittin hybrid peptides. All membrane-active compounds showed a good correlation between inhibition of luminescence and leishmanicidal activity. Induction of membrane permeability was demonstrated by dissipation of membrane potential, SYTOX trade mark Green influx and membrane damage assessed by electron microscopy, except for the polyethers, where ATP decrease was due to inhibition of its mitochondrial synthesis. Five of the test peptides showed an ED50 around 1 microM on promastigotes. These peptides, with equal or better activity than 26-residue-long CA(1-8)M(1-18), are the shortest leishmanicidal peptides described so far, and validate our luminescence assay as a fast and cheap screening tool for membrane-active compounds.

Application of real-time PCR for the detection of Strongyloides spp. in clinical samples in a reference center in Spain.

Strongyloidiasis is one of the major intestinal helminthic infections in humans with a worldwide distribution, affecting especially tropical and subtropical regions. This disease can occur without any symptoms or as a potentially fatal hyperinfection or disseminated infection. Definitive diagnosis of Strongyloides stercoralis infection relies mainly on demonstration of larvae in stool, but at present there is no gold standard for this diagnosis. Our main objective was to evaluate a real-time PCR targeting the 18S rRNA gene of Strongyloides spp. and to compare it with routine parasitological methods. DNA from Strongyloides venezuelensis was used to optimize PCR protocols obtaining an analytical sensitivity of 0.1 pg of parasite DNA per sample. Sensitivity and specificity of real-time PCR on fecal samples from 231 patients screened for suspected strongyloidiasis attending two hospitals in Madrid were 93.8% and 86.5%, respectively. No significant differences were found when comparing Ct-values of positive PCR between parasitological positive and negative samples. This study showed that real-time PCR is an effective tool for diagnosing strongyloidiasis and could be applied in association with parasitological methods in epidemiological studies in endemic areas. It would be also important to assess its performance in immunocompromised populations who are at risk of fatal disease.

Prevalence and genotype identification of Toxoplasma gondii in wild animals from southwestern Spain.

We used PCR to detect Toxoplasma gondii in the principal game species in southwestern Spain. We detected T. gondii in 32.2% of animals tested. Prevalences varied from 14.7% in wild boar (Sus scrofa) to 51.2% in red fox (Vulpes vulpes). The most prevalent genotype was type II (50.0%), followed by type III (20.6%) and type I (5.9%). Mixed infections (11.8%) were detected in wild boar (types I+III) and red fox (types II+III). Polymorphic strains (11.8%) were detected in several species. The high prevalence and the genetic variability shown could have implications for infection of farm animals and humans.

Serological study of Trypanosoma cruzi, Strongyloides stercoralis, HIV, human T cell lymphotropic virus (HTLV) and syphilis infections in asymptomatic Latin-American immigrants in Spain.

OBJECTIVE: We aimed to perform a serological screening for T. cruzi, Strongyloides stercoralis, HIV, human T cell lymphotropic virus (HTLV) and syphilis in Latin American immigrants admitted to hospital in Spain. METHODS: We have carried out a cross-sectional study of Latin American immigrants admitted to the Hospital General Universitario Alicante (Spain) from June 2012 to May 2014, where screening of Chagas disease, strongyloidiasis, HTLV, HIV and syphilis was performed by serology. RESULTS: A total 180 patients were included in the study. Patients' median age was 38 years old, 123 (68.3%; 123/180) were female and 57 (31.7%; 57/180) male. Five of the 180 (2.5%) patients were positive for Chagas disease; associated with knowledge about Chagas disease (p=0.005), previous contact with patients with Chagas disease (p=0.04) and being Bolivian (p<0.001). Forty-two of the 157 (26.8%) patients were positive for Strongyloides serology; associated positively with being male (p<0.001), eosinophilia (p=0.001), hyper-IgE (p<0.001) and being Ecuadorian (p=0.001), and negatively associated with being Colombian (p=0.03). Positive serology of latent syphilis was found in 1.8% (3/171) of patients. Serology of HTLV was negative in all cases. No new cases of HIV infection were diagnosed. CONCLUSIONS: We propose that Latin American immigrant patients admitted to hospital in Spain be screened for strongyloidiasis, Chagas disease and syphilis by serology.

Detection and molecular characterization of Giardia duodenalis in children attending day care centers in Majadahonda, Madrid, Central Spain.

Infections by the protozoan enteroparasites Giardia duodenalis and Cryptosporidium spp are a major cause of morbidity in children attending day care facilities in developed countries. In this cross-sectional study, we aimed to estimate the occurrence and genotype frequencies of these pathogens in children attending day care centers in Majadahonda, Central Spain. To do so, single stool samples were obtained from 90 children and tested for the presence of G duodenalis and Cryptosporidium spp by conventional microscopy and immunochromatography. Positive results by these techniques were subsequently confirmed by immunofluorescence microscopy. G duodenalis-positive samples were subjected to molecular characterization studies by multilocus sequence-based genotyping of the glutamate dehydrogenase and ß-giardin genes of the parasite. G duodenalis assemblages were confirmed by restriction fragment length polymorphism analyses and sequencing. A socioepidemiological questionnaire was used to identify variables potentially associated with giardiasis/cryptosporidiosis in the population of children under investigation. Overall, G duodenalis and Cryptosporidium spp were detected in 15.5% and 3.3% of stool samples, respectively. Giardiasis and cryptosporidiosis were found in 3/3 and 2/3 day care centers, respectively, affecting mainly infants aged 13 to 24 months. A total of 8 G duodenalis isolates were confirmed as subassemblage BIV, all of them belonging to asymptomatic children. Attempts to genotype Cryptosporidium isolates failed. None of the variables considered could be associated with higher risk of infection with giardiasis or cryptosporidiosis. These results clearly indicate that asymptomatic infections with G duodenalis and Cryptosporidium spp are frequent in <3-year-old children in Central Spain.

Effects of the disruption of the HSP70-II gene on the growth, morphology, and virulence of Leishmania infantum promastigotes.

The 70-kDa heat shock protein (HSP70) is highly conserved among both prokaryotes and eukaryotes and plays essential roles in diverse cellular functions not only under stress but also under normal conditions. In the protozoan Leishmania infantum, the causative agent of visceral leishmaniasis, HSP70 is encoded by two HSP70 genes. Here, we describe the phenotypic alterations of HSP70-II-deficient (Deltahsp70-II) promastigotes. The absence of HSP70-II caused a major alteration in growth as the promastigotes reached stationary phase. In addition, aberrant forms were frequently observed in Deltahsp70-II mutant cultures. An accumulation of cells in the G2/M phase in cultures of the Deltahsp70-II mutant was determined by flow cytometry. Furthermore, Deltahsp70-II promastigotes showed a limited capacity of multiplication within macrophages, even though attachment to and uptake by macrophages did not differ significantly from the wild-type. Moreover, Deltahsp70-II was highly attenuated in BALB/c mouse experimental infections. In mutants re-expressing HSP70-II, the growth rate was restored, the normal morphology was recovered, and interactions with macrophages increased. However, promastigotes re-expressing HSP70-II did not recover their virulence. Overall, these data highlight the essential role played by HSP70-II expression in Leishmania virulence, pointing to this gene as a promising target for therapeutic interventions.