Occurrence and genetic diversity of Enterocytozoon bieneusi (Microsporidia) in owned and sheltered dogs and cats in Northern Spain.

Enterocytozoon bieneusi is an obligate intracellular protist-like fungi parasite that infects numerous mammal hosts including humans, raising concerns of zoonotic transmission. There is little information available on the presence and diversity of E. bieneusi genotypes in companion animals. Here, we determined the occurrence and genetic diversity of E. bieneusi in domestic dogs and cats from Northern Spain. A total of 336 genomic DNA samples extracted from canine (n = 237) and feline (n = 99) faecal specimens were retrospectively investigated. The presence of E. bieneusi was assessed by PCR of the rRNA internal transcribed spacer (ITS) gene. The parasite was detected in 3.0% (3/99) and 0.8% (2/237) of the cats and dogs examined, respectively. All three feline positive samples were from stray cats living in an urban setting, whereas the two canine samples were from owned dogs living in rural areas. Sequence analysis revealed the presence of two genotypes in dogs, BEB6 and PtEb IX, and two genotypes in cats, D and Peru11. The identification of Peru11 in a cat and BEB6 in a dog constitutes the first report of those genotypes in such hosts as well as first report in Spain. This is also the first evidence of genotype D in cats and PtEb IX in dogs in Spain. Three out of the four genotypes, BEB6, D and Peru11, have been previously reported as human pathogens and are potentially zoonotic indicating that dogs and cats need to be considered potential sources of human infection and environmental contamination.

Comparison of three diagnostic methods (microscopy, RDT, and PCR) for the detection of malaria parasites in representative samples from Equatorial Guinea.

BACKGROUND: Malaria in Equatorial Guinea remains a major public health problem. The country is a holo-endemic area with a year-round transmission pattern. In 2016, the prevalence of malaria was 12.09% and malaria caused 15% of deaths among children under 5 years. In the Continental Region, 95.2% of malaria infections were Plasmodium falciparum, 9.5% Plasmodium vivax, and eight cases mixed infection in 2011. The main strategy for malaria control is quick and accurate diagnosis followed by effective treatment. Early and accurate diagnosis of malaria is essential for both effective disease management and malaria surveillance. The quality of malaria diagnosis is important in all settings, as misdiagnosis can result in significant morbidity and mortality. Microscopy and RDTs are the primary choices for diagnosing malaria in the field. However, false-negative results may delay treatment and increase the number of persons capable of infecting mosquitoes in the community. The present study analysed the performance of microscopy and RDTs, the two main techniques used in Equatorial Guinea for the diagnosis of malaria, compared to semi-nested multiplex PCR (SnM-PCR). RESULTS: A total of 1724 samples tested by microscopy, RDT, and SnM-PCR were analysed. Among the negative samples detected by microscopy, 335 (19.4%) were false negatives. On the other hand, the negative samples detected by RDT, 128 (13.3%) were false negatives based on PCR. This finding is important, especially since it is a group of patients who did not receive antimalarial treatment. CONCLUSIONS: Owing to the high number of false negatives in microscopy, it is necessary to reinforce training in microscopy, the "Gold Standard" in endemic areas. A network of reference centres could potentially support ongoing diagnostic and control efforts made by malaria control programmes in the long term, as the National Centre of Tropical Medicine currently supports the National Programme against Malaria of Equatorial Guinea to perform all of the molecular studies necessary for disease control. Taking into account the results obtained with the RDTs, an exhaustive study of the deletion of the hrp2 gene must be done in EG to help choose the correct RDT for this area.

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

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 ß-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.

Detection and molecular characterisation of Giardia duodenalis, Cryptosporidium spp. and Entamoeba spp. among patients with gastrointestinal symptoms in Gambo Hospital, Oromia Region, southern Ethiopia.

OBJECTIVES: To assess the prevalence and genetic diversity of the enteric protozoa species G. duodenalis, Cryptosporidium spp. and Entamoeba histolytica in individuals with gastrointestinal symptoms compatible with infections by these pathogens seeking medical attention in a rural area in southern Ethiopia. METHODS: A total of 92 stool samples were initially screened by direct microscopy and immunochromatography and further confirmed by molecular methods. G. duodenalis-positive samples were molecularly characterised by multilocus genotyping of the glutamate dehydrogenase and ß-giardin genes of the parasite. PCR and DNA sequence analysis of the gene encoding the 60-kDa glycoprotein was used for the subtyping of Cryptosporidium isolates. Detection and differential diagnosis of E. histolytica/dispar were conducted by real-time PCR. RESULTS: PCR-based prevalences were 10.9% for G. duodenalis, 1.1% for Cryptosporidium spp. and 3.3% for Entamoeba spp. Seven (four novel and three known) subtypes of G. duodenalis assemblage B were identified at the GDH locus and 5 (one novel and four known) at the BG locus. A novel variant of C. hominis subtype IbA9G3 was also identified. Two Entamoeba isolates were assigned to E. dispar and an additional one to E. histolytica. CONCLUSION: Although preliminary, our results strongly suggest that giardiasis, cryptosporidiosis and amoebiasis represent a significant burden in Ethiopian rural population.

Identification and Molecular Characterization of Four New Blastocystis Subtypes Designated ST35-ST38.

Three recent studies of Blastocystis epidemiology in mammalian hosts identified four novel sequences that appeared to share B. lapemi as the most similar sequence. However, full-length ssu rRNA gene sequences were not available to confirm the validity of these new subtypes. In the present study, Nanopore MinION sequencing was used to obtain full-length reference sequences for each of the new subtypes. Additionally, phylogenetic analyses and pairwise distance comparisons were performed to confirm the validity of each of these new subtypes. We propose that the novel sequences described in this study should be assigned the subtype designations ST35-ST38. The full-length reference sequences of ST35-ST38 will assist in accurate sequence descriptions in future studies of Blastocystis epidemiology and subtype diversity.

Detection of high levels of mutations involved in anti-malarial drug resistance in Plasmodium falciparum and Plasmodium vivax at a rural hospital in southern Ethiopia.

BACKGROUND: In Ethiopia, malaria is caused by Plasmodium falciparum and Plasmodium vivax, and anti-malarial drug resistance is the most pressing problem confronting control of the disease. Since co-infection by both species of parasite is common and sulphadoxine-pyrimethamine (SP) has been intensively used, resistance to these drugs has appeared in both P. falciparum and P. vivax populations. This study was conducted to assess the prevalence of anti-malarial drug resistance in P. falciparum and P. vivax isolates collected at a rural hospital in southern Ethiopia. METHODS: A total of 1,147 patients with suspected malaria were studied in different months across the period 2007-2009. Plasmodium falciparum dhfr and dhps mutations and P. vivax dhfr polymorphisms associated with resistance to SP, as well as P. falciparum pfcrt and pfmdr1 mutations conferring chloroquine resistance, were assessed. RESULTS: PCR-based diagnosis showed that 125 of the 1147 patients had malaria. Of these, 52.8% and 37.6% of cases were due to P. falciparum and P. vivax respectively. A total of 10 cases (8%) showed co-infection by both species and two cases (1.6%) were infected by Plasmodium ovale. Pfdhfr triple mutation and pfdhfr/pfdhps quintuple mutation occurred in 90.8% (95% confidence interval [CI]: 82.2%-95.5%) and 82.9% (95% CI: 72.9%-89.7%) of P. falciparum isolates, respectively. Pfcrt T76 was observed in all cases and pfmdr1 Y86 and pfmdr1 Y1246 in 32.9% (95% CI: 23.4%-44.15%) and 17.1% (95% CI: 10.3-27.1%), respectively. The P. vivax dhfr core mutations, N117 and R58, were present in 98.2% (95% CI: 89.4-99.9%) and 91.2% (95% CI: 80.0-96.7%), respectively. CONCLUSION: Current molecular data show an extraordinarily high frequency of drug-resistance mutations in both P. falciparum and P. vivax in southern Ethiopia. Urgent surveillance of the emergence and spread of resistance is thus called for. The level of resistance indicates the need for implementation of entire population access to the new first-line treatment with artemether-lumefantrine, accompanied by government monitoring to prevent the emergence of resistance to this treatment.

[Detection of antimalarial activity for new compounds by isotopic microtest].

Two clone lines (Dd2 and 3D7) of Plasmodium falciparum were cultivated continuously in human erythrocytes at 37 degrees C in RPMI 1640 medium with human serum and subjected to 6% sorbitol treatment 2 times in order to obtain highly synchronized cultures. The second generation parasites after the treatment were diluted with human RBC to be a suspension of P. falciparum-human RBC at 2.5% hematocrit and 0.5% parasitemia, and 2 microCi/ml of 8-3H-hypoxanthine was added. Isotopic microtest was employed to detect the antimalarial activity for 20 new compounds. Results revealed that the 20 compounds showed no anti-malarial activity, while the control drugs, chloroquine and quinine, exhibited high efficacy, indicating that the isotopic microtest is a stable and reproducible assay for screening new antimalarials.

Comparative performance evaluation of four commercial multiplex real-time PCR assays for the detection of the diarrhoea-causing protozoa Cryptosporidium hominis/parvum, Giardia duodenalis and Entamoeba histolytica.

BACKGROUND: Multiplex molecular panels are relentlessly replacing conventional methods for the detection of enteric pathogens from stool samples in clinical and research laboratories. Here we evaluated four commercial multiplex real-time PCR assays for the detection of Cryptosporidium hominis/parvum, Giardia duodenalis and Entamoeba histolytica. METHODS: The diagnostic performance of the Gastroenteritis/Parasite Panel I (Diagenode), the RIDAGENE Parasitic Stool Panel (R-Biopharm), the Allplex Gastrointestinal Parasite Panel 4 (Seegene) and the FTD Stool Parasites (Fast Track) real-time PCR methods was assessed against a reference panel of 126 well-characterized DNA samples including Cryptosporidium hominis (n = 29), Cryptosporidium parvum (n = 3), Giardia duodenalis (n = 47), Entamoeba histolytica (n = 3), other parasite species (n = 20), and apparently healthy subjects (n = 24). PRINCIPAL FINDINGS: Obtained diagnostic sensitivities ranged from 53-88% for Cryptosporidium hominis/parvum, and from 68-100% for G. duodenalis. The R-Biopharm method achieved the best performance for the detection of Cryptosporidium hominis/parvum both in terms of diagnostic sensitivity (87.5%) and detection limit (a 100-fold increase compared to other tests). The Fast Track method was particularly suited for the detection of G. duodenalis, achieving a 100% sensitivity and a detection limit at least 10-fold superior. Detection of E. histolytica was similarly achieved by all compared methods except Diagenode. CONCLUSIONS: Diagnostic performance varied largely depending on the method used and the targeted pathogen species. Factors including test sensitivity/specificity, cost, patient population surveyed, laboratory workflow, and diagnostic algorithm should be carefully considered when choosing the most appropriate multiplex PCR platform.

Cryptosporidium-associated diarrhoea in neonatal calves in Algeria.

Neonatal calf diarrhoea triggered by the enteric protozoan parasite Cryptosporidium is a leading cause of morbidity and mortality in calves aged 1-month-old or younger globally. Infected cattle in general and calves in particular have also been demonstrated as major contributors of zoonotic C. parvum oocysts in the environment and have been linked to a number of waterborne outbreaks of human cryptosporidiosis. Little is known on the occurrence, geographical distribution, and molecular diversity of Cryptosporidium infections affecting bovine populations in Algeria. In this study faecal specimens were randomly collected from 460 cattle aged between two days and 18 months on 10 farms located in the provinces of Aïn Defla, Blida, Sétif, and Tizi Ouzou between the autumn of 2015 and the spring of 2016. Faecal samples were microscopically examined using the modified Ziehl-Neelsen acid-fast technique as screening method. Microscopy-positive samples were confirmed by a commercial coproantigen enzyme-linked immunosorbent assay (Bio-X Diagnostics). The identification of Cryptosporidium species and sub-genotypes in confirmed samples was conducted by PCR and sequence analyses of the small subunit ribosomal RNA (ssu rRNA) and the 60 kDa glycoprotein (gp60) genes of the parasite. Overall, 52.2% (240/460) of the investigated cattle tested positive to Cryptosporidium by microscopy. The infection was widespread in all 10 farms surveyed, but was significantly more prevalent in those from Blida in the central part of the country. Bovine cryptosporidiosis affected cattle of all age groups but with different outcomes. Pre-weaned (up to one month old) calves typically presented with diarrhoea, whereas older animals mostly harboured sub-clinical infections. The commercial ELISA used only detected 15.8% (38/240) of the samples that previously tested positive by microscopy, demonstrating a poor performance in field epidemiological surveys. Sequence analysis of the 29 isolates generated at the ssu rRNA loci confirmed the presence of four Cryptosporidium species including C. parvum (72.4%), C. bovis (13.8%), C. andersoni, (3.4%), and C. ryanae (3.4%). Two additional isolates (7.0%) could only be identified at the genus level. Eight out of the 21 isolates assigned to C. parvum were identified as sub-genotype IIaA16G2R1 at the gp60 locus. C. parvum was almost exclusively found infecting pre-weaned calves, whereas C. ryanae and C. andersoni were only detected in asymptomatic animals. Bovine cryptosporidiosis is highly endemic in the surveyed area and represents a veterinary public health concern that should be adequately tackled by Algerian veterinary health authorities and policy makers.

Prevalence of intestinal parasites, with emphasis on the molecular epidemiology of Giardia duodenalis and Blastocystis sp., in the Paranaguá Bay, Brazil: a community survey.

BACKGROUND: Intestinal protozoan parasites are major contributors to the global burden of gastrointestinal disease causing significant socioeconomic consequences. Children living in resource-poor settings with restricted access to water and sanitary services are particularly at risk of these infections. METHODS: A prospective, community-based, cross-sectional survey was conducted in Paraná (southern Brazil) between May 2015 and May 2016. A total of 766 stool samples were individually collected from volunteers (male/female ratio: 0.99; age range: 0-76 years) and used for investigating the presence of intestinal helminth and protozoan species by routine microscopic procedures including the Kato-Katz and modified Ritchie concentration methods and the Ziehl-Neelsen stain technique. Quantitative real-time PCR confirmed microscopy-positive samples for Giardia duodenalis and the assemblages and sub-assemblages determined by multilocus sequence-based genotyping of the glutamate dehydrogenase (gdh) and ß-giardin (bg) genes of the parasite. Identification of Blastocystis subtypes was carried out by amplification and sequencing of a partial fragment of the small-subunit ribosomal RNA gene (SSU rDNA) of this heterokont microorganism. RESULTS: Overall, 46.1% (353/766) of the participants were infected/colonised by at least one intestinal parasite/commensal species. Protozoan and helminth species were detected in 42.7% and 10.1% of the surveyed population, respectively. Blastocystis sp. (28.2%), Endolimax nana (14.9%), and Giardia duodenalis (11.0%) were the most prevalent species found among protozoans and Ascaris lumbricoides (5.0%), Trichuris trichiura (4.6%) and hookworms (1.0%) among helminths. A total of 38 G. duodenalis-positive samples were genotyped at gdh and bg markers, revealing the presence of the sub-assemblages AII (47.4%), AII/AIII (2.6%), BIII (5.3%), BIV (26.3%) and BIII/BIV (13.1%). Two samples (5.3%) were only identified as assemblage B. AII was predominantly found in females aged 5-9 years and was associated with a higher likelihood of reporting gastrointestinal symptoms. A total of 102 Blastocystis-positive samples were successfully subtyped at the SSU rRNA gene revealing the presence of ST1 (36.3%), ST2 (15.7%), ST3 (41.2%), ST4 (2.9%), ST6 (1.0%) and ST8 (2.9%). CONCLUSIONS: Data presented here indicate that enteric parasites still represent a pressing health concern in Paraná, Brazil, probably due to sub-optimal water, sanitation and hygiene conditions. A mostly anthroponotic origin is suspected for G. duodenalis and Blastocystis sp. infections.

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.

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.

Transmission of malaria and genotypic variability of Plasmodium falciparum on the island of Annobon (Equatorial Guinea).

BACKGROUND: Malaria transmission in Equatorial Guinea and its space-time variability has been widely studied, but there is not much information about the transmission of malaria on the small island of Annobon. In 2004, two transversal studies were carried out to establish the malaria transmission pattern on Annobon and analyse the circulating Plasmodium falciparum allelic forms. METHODS: A blood sample was taken from the selected children in order to determine Plasmodium infection by microscopical examination and by semi-nested multiplex PCR. The diversity of P. falciparum circulating alleles was studied on the basis of the genes encoding for the merozoite surface proteins, MSP-1 and MSP-2 of P. falciparum. RESULTS: The crude parasite rate was 17% during the dry season and 60% during the rainy season. The percentage of children sleeping under a bed net was over 80% in the two surveys. During the rainy season, 33.3% of the children surveyed were anaemic at the time of the study. No association was found between the crude parasite rate, the use of bed nets and gender, and anaemia. However, children between five and nine years of age were five times less at risk of being anaemic than those aged less than one year. A total of 28 populations of the three allelic families of the msp-1 gene were identified and 39 of the msp-2 gene. The variability of circulating allelic populations is significantly higher in the rainy than in the dry season, although the multiplicity of infections is similar in both, 2.2 and 1.9 respectively. CONCLUSION: Based on the high degree of geographical isolation of the Annobon population and the apparent marked seasonality of the transmission, it is feasible to believe that malaria can be well controlled from this small African island.

Genetic population structure of Anopheles gambiae in Equatorial Guinea.

BACKGROUND: Patterns of genetic structure among mosquito vector populations in islands have received particular attention as these are considered potentially suitable sites for experimental trials on transgenic-based malaria control strategies. In this study, levels of genetic differentiation have been estimated between populations of Anopheles gambiae s.s. from the islands of Bioko and Annobón, and from continental Equatorial Guinea (EG) and Gabon. METHODS: Genotyping of 11 microsatellite loci located in chromosome 3 was performed in three island samples (two in Bioko and one in Annobón) and three mainland samples (two in EG and one in Gabon). Four samples belonged to the M molecular form and two to the S-form. Microsatellite data was used to estimate genetic diversity parameters, perform demographic equilibrium tests and analyse population differentiation. RESULTS: High levels of genetic differentiation were found between the more geographically remote island of Annobón and the continent, contrasting with the shallow differentiation between Bioko island, closest to mainland, and continental localities. In Bioko, differentiation between M and S forms was higher than that observed between island and mainland samples of the same molecular form. CONCLUSION: The observed patterns of population structure seem to be governed by the presence of both physical (the ocean) and biological (the M-S form discontinuity) barriers to gene flow. The significant degree of genetic isolation between M and S forms detected by microsatellite loci located outside the "genomic islands" of speciation identified in A. gambiae s.s. further supports the hypothesis of on-going incipient speciation within this species. The implications of these findings regarding vector control strategies are discussed.

No molecular epidemiological evidence supporting household transmission of zoonotic Giardia duodenalis and Cryptosporidium spp. from pet dogs and cats in the province of Álava, Northern Spain.

The role of pet dogs and cats as suitable source of human infections by the diarrheagenic protozoan parasites Giardia duodenalis and Cryptosporidium spp. has been a topic of intense debate for long time and still remains a largely unsolved problem. In this cross-sectional molecular epidemiological survey we attempted to investigate whether zoonotic (or zooanthroponotic) disease transmission was occurring among humans and domestic dogs and cats sharing the same spatial and temporal setting in both rural and urban areas of the province of Álava, Northern Spain. A total of 268 (including 179 human, 55 canine, and 34 feline) individual faecal specimens were obtained from 63 family households during February-March and November-December 2014. Detection of G. duodenalis cysts and Cryptosporidium spp. oocysts was achieved by direct fluorescence microscopy (DFAT) and PCR-based methods targeting the small subunit (SSU) ribosomal RNA gene of the parasites. Giardia-positive isolates were subsequently sub-genotyped at the glutamate dehydrogenase (GDH) and ß-giardin (BG) genes. Overall, G. duodenalis infections were identified in 3.4% (6/179) of humans, 29% (16/55) of dogs, and 5.9% (2/34) of cats, respectively. Cryptosporidium spp. infections were detected in 1.1% (2/179) of humans, 5.5% (3/55) of dogs, and 8.8% (3/34) of cats, respectively. Simultaneous infections in human and canine/feline hosts by G. duodenalis or Cryptosporidium spp. were only demonstrated in a single household in which a cat and its owner tested positive for Cryptosporidium by DFAT, but this result could not be confirmed by SSU-PCR. Infections were homogeneously distributed among the studied human or animal populations irrespectively of their sex, age group, or geographical region of origin. Inadequate washing of raw vegetables and fruits was the only risk factor significantly associated to a higher likelihood of having human giardiosis/cryptosporidiosis. Molecular characterization of G. duodenalis isolates revealed the presence of sub-assemblage BIV in a single human isolate. All dog (n=3) and cat (n=2) isolates successfully genotyped were assigned to canine- and feline-specific assemblages C and F, respectively. No mixed assemblage or sub-assemblage infections could be demonstrated. Regarding Cryptosporidium, C. canis was found infecting dogs (n=2), and C. felis a single cat. Attempts to amplify and characterize Cryptosporidium human isolates failed repeatedly. Our results suggest that pet dogs and cats do not seem to play a significant role as suitable reservoirs of human giardiosis or cryptosporidiosis in the province of Álava. We conclude, therefore, that zoonotic transmission of giardiosis or cryptosporidiosis among pet dogs and cats and their owners in this geographical region is very likely a rare event.

Detection and molecular diversity of Giardia duodenalis and Cryptosporidium spp. in sheltered dogs and cats in Northern Spain.

Domestic dogs and cats may act as natural reservoirs of a large number of zoonotic pathogens, including the enteric parasites Giardia duodenalis and Cryptosporidium spp., the most relevant protozoan species causing gastrointestinal disease worldwide. A cross-sectional epidemiological study aiming to assess the prevalence and molecular diversity of G. duodenalis and Cryptosporidium spp. was conducted in an animal rescue centre in the province of Álava (Northern Spain). A total of 194 and 65 faecal dropping samples from individual dogs and cats, respectively, were collected between November 2013 and June 2016. G. duodenalis cysts and Cryptosporidium spp. oocysts were detected by direct fluorescence microscopy and PCR-based methods targeting the small subunit ribosomal RNA gene of these parasites. Overall, G. duodenalis and Cryptosporidium spp. were detected in 33% (63/194) and 4.1% (8/194) of dogs, and 9.2% (6/65) and 4.6% (3/65) of cats, respectively. G. duodenalis and Cryptosporidium co-infections were observed in 1.5% (3/194) of dogs, but not in cats. No significant differences in infection rates could be demonstrated among dogs or cats according to their sex, age group, status, or geographical origin. Multi-locus sequence-based genotyping of the glutamate dehydrogenase and ß-giardin genes of G. duodenalis allowed the characterization of 19 canine isolates that were unambiguously assigned to sub-assemblages AII (n=7), BIII (n=1), and BIV (n=7), and assemblages C (n=3) and D (n=1). Two feline isolates were genotyped as assemblages A and F, respectively. No mixed assemblage or sub-assemblage infections were identified. C. canis (n=5) and C. hominis (n=1) were the Cryptosporidium species found in dogs, whereas C. felis (n=1) was identified in cats. The finding of G. duodenalis sub-assemblages AII, BIII, and BIV circulating in dogs (but not cats) may have zoonotic potential, although most of the AII and BIV isolates sub-genotyped corresponded to genetic variants not previously found in Spanish human populations. Dogs may also act as novel suitable hosts for C. hominis. We recommend to considerer companion animals as sentinel surveillance system for zoonotic giardiasis and cryptosporidiosis in order to minimize the risk of spreading of these parasitic diseases among the human population.

Molecular diversity and frequency of the diarrheagenic enteric protozoan Giardia duodenalis and Cryptosporidium spp. in a hospital setting in Northern Spain.

BACKGROUND: Human giardiosis and cryptosporidiosis are caused by the enteric protozoan parasites Giardia duodenalis and Cryptosporidium spp. Both pathogens are major contributors to the global burden of diarrhoeal disease, affecting primarily children and immunodebilitated individuals in resource-poor settings. Giardiosis and cryptosporidiosis also represent an important, often underestimate, public health threat in developed countries. In Spain only limited information is currently available on the epidemiology of these infections. Molecular data on the diversity, frequency, geographical distribution, and seasonality of G. duodenalis assemblages/sub-assemblages and Cryptosporidium species/sub-genotypes are particularly scarce. METHODS: A longitudinal molecular epidemiological survey was conducted between July 2015 to September 2016 in patients referred to or attended at the Hospital San Pedro (La Rioja, Northern Spain) that tested positive for G. duodenalis (N = 106) or Cryptosporidium spp. (N = 103) by direct microscopy and/or a rapid lateral flow immunochromatographic assay. G. duodenalis infections were subsequently confirmed by real-time PCR and positive isolates assessed by multi-locus sequence genotyping of the glutamate dehydrogenase and ß-giardin genes of the parasite. Cryptosporidium species and sub-genotypes were investigated at the 60 kDa glycoprotein or the small subunit ribosomal RNA genes of the parasite. Sociodemographic and clinical parameters of infected patients were also gathered and analysed. PRINCIPAL FINDINGS: Out of 90 G. duodenalis-positive isolates by real-time PCR a total of 16 isolates were successfully typed. AII (44%, 7/16) was the most prevalent sub-assemblage found, followed by BIV (31%, 5/16) and BIII (19%, 3/16). A discordant genotype result AII/AIII was identified in an additional (6%, 1/16) isolate. No mixed infections A+B were detected. Similarly, a total of 81 Cryptosporidium spp. isolates were successfully typed, revealing the presence of C. hominis (81%, 66/81) and C. parvum (19%, 15/81). Obtained GP60 sequences were assigned to sub-type families Ib (73%, 59/81) within C. hominis, and IIa (7%, 6/81) and IId (2%, 2/81) within C. parvum. A marked inter-annual variation in Cryptosporidium cases was observed. CONCLUSIONS: Human giardiasis and cryptosporidiosis are commonly identified in patients seeking medical care in Northern Spain and represent a more important health concern than initially thought. Assemblage A within G. duodenalis and sub-genotype IbA10G2 within C. hominis were the genetic variants of these parasite species more frequently found circulating in the population under study. Molecular data presented here seem to suggest that G. duodenalis and Cryptosporidium infections arise through anthroponotic rather than zoonotic transmission in this Spanish region.

Association between enteric protozoan parasites and gastrointestinal illness among HIV- and tuberculosis-infected individuals in the Chowke district, southern Mozambique.

Human immune deficiency virus (HIV) and tuberculosis (TB) infections remain major public health issues globally, particularly in sub-Saharan Africa. Impairment of both cell-mediated and humoral immunity by HIV and/or TB infections may limit the host's defences against other pathogens, including the diarrheagenic protozoan Cryptosporidium spp., Giardia intestinalis, and Entamoeba histolytica. During September-December 2015 a cross-sectional study was conducted to assess the prevalence and molecular diversity of these enteric parasites among HIV- and/or TB-infected patients at a medical reference centre in Chowke district, southern Mozambique. A total of 99 stool specimens were initially screened by direct microscopy and further confirmed and characterised by molecular methods. DNA sequence analyses of the genes encoding the small subunit ribosomal RNA and the 60-kDa glycoprotein were used for the typing and sub-typing of Cryptosporidium isolates, respectively. G. intestinalis-positive isolates by real-time PCR were subsequently typed at the glutamate dehydrogenase locus. Differential diagnosis of E. histolytica/dispar was achieved by real-time PCR. G. intestinalis (8.1%) was the enteric protozoan more frequently detected, followed by Cryptosporidium spp. (7.1%), and Entamoeba histolytica/dispar (6.1%). Two HIV-infected (but not TB-infected) patients harbour G. intestinalis and Cryptosporidium spp. co-infections. Two (29%) G. intestinalis isolates were successfully characterised, revealing the presence of known AII and novel BIV genotypes. Four (57%) Cryptosporidium isolates were unmistakeable assigned to C. hominis, identifying two (IbA10G2 and IdA22) sub-types. Cryptosporidium infections were not associated to diarrhoea in HIV-positive patients, probably because improved immune function in the affected individuals due to antiretroviral therapy. G. intestinalis was considered a non-opportunistic pathogen, whereas the presence of E. histolytica could not be confirmed by molecular methods. Based on their common presence in the studied clinical population, we recommend the effective diagnosis and treatment of these enteropathogens for improving the management of HIV and TB patients.