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Background Opportunistic infections represent a serious health problem for HIV-infected people. Among enteric infections, cryptosporidiosis, a severe and life-threatening diarrheal disease, is of particular importance in low economic settings where access to anti-retroviral therapy is limited. Understanding transmission routes is crucial in establishing preventive measures, and requires the use of informative genotyping methods. In this study, we performed a retrospective analysis of Cryptosporidium species in 166 stool samples collected from 155 HIV-infected patients during 1999–2004 at the Siriraj Hospital in Bangkok, Thailand. Results Microscopic examination of stools identified 104 of the 155 patients as positive for Cryptosporidium . Other common pathogens identified were microsporidia, Isospora , Giardia , Strongyloides and Opisthorchis. All samples were tested by amplification of a fragment of the 18S rDNA locus, and sequencing showed the presence of Cryptosporidium hominis ( n  = 42), C. meleagridis ( n  = 20), C. canis ( n  = 12), C. felis ( n  = 7), C. suis ( n  = 6) and C. parvum ( n  = 5). Genotyping at the glycoprotein 60 ( gp60 ) locus revealed substantial variability in isolates of C. hominis and C. meleagridis . Among C. hominis isolates, subtype IeA11G3T3 was the most prevalent, but allelic family Id was the more diverse with four subtypes described, two of which were identified for the first time. Among C. meleagridis isolates, seven subtypes, two of which were new, were found in the allelic family IIIb, along with new subtypes in allelic families IIIe and IIIg. In the four C. parvum isolates, subtype IIoA16G1, a rare subtype previously reported in a Swedish patient who had traveled to Thailand, was identified. Conclusions This study confirms the high susceptibility of HIV-infected individuals to infection with different Cryptosporidium species and subtypes, and further stresses the importance of surveillance for opportunistic intestinal protozoans.

Sexual recombination is a hallmark of eukaryotic evolution. Without recombination, asexual eukaryotes should succumb to deleterious mutations and more rapidly evolving pathogens. Giardia duodenalis , a parasitic protist, sits within one of the earliest-branching eukaryotic lineages and has no known sexual stage. Whether Giardia are ‘ancient asexuals’ has been long explored but is unresolved. Here, we find clear evidence of sex in Giardia and also discover an asexual sublineage that has a broader host range than its sexual ancestor. This asexual lineage is not ancient, and is accumulating deleterious mutations. Unlike its sexual counterparts, its genetic variation lacks the signatures of selection and Red Queen coevolution. We propose a new hypothesis that explains how a mutational meltdown during Muller’s Ratchet might enable asexual pathogens to expand their host ranges transiently. Fittingly, our results suggest that Giardia is not the last exception to, but rather further evidence of, the essentiality of eukaryotic sex. Sexual reproduction is thought to be essential for long-term survival of eukaryotes. This study shows that Giardia, once suspected to be anciently asexual, retains evidence of sex while a newly derived asexual lineage is accumulating mutations and expanding its host range.

Background The genetic variability of a large collection of European samples of the zoonotic pathogen Cryptosporidium parvum has been recently explored on the basis of a novel multi-locus sequence typing (MLST) scheme. In this work, we assessed the usefulness of this scheme to type C. parvum samples from Italy, a country where this pathogen is widespread and associated with human infections. Methods Polymerase chain reaction (PCR) and sequencing for the eight markers of the MLST scheme were performed on 31 human- and 21 animal-derived C. parvum samples. MLST data from 27 samples of animal origin previously sequenced at the genome level were also included. Sequence data for the glycoprotein 60 ( gp60 ) gene were also generated. Phylogenetic and cluster analyses were conducted. Results Full genotyping data were obtained for 72 of 79 samples, and 39 different profiles were categorized, 28 of which were found in individual samples (singletons). A new allele was found at the marker on chromosome 2 in a human-derived sample. When compared with the 154 profiles previously described in Europe, 30 of the 39 profiles (76%) were found to be restricted to Italy, a result compatible with a model of isolation by distance, with geographically structured populations. Analysis of the gp60 sequences identified 19 different subtypes among the 55 samples belonging to family IIa, and 7 different subtypes among the 16 samples belonging to family IId. Phylogenetic and haplotype analyses did not identify clusters related to the host, the geographic origin (i.e., the Italian regions), or the time of collection of the samples but did identify two different populations, mirroring data obtained from whole genome comparative analyses. Conclusions The MLST scheme appears to be a promising method for genotyping C. parvum samples, as it provided higher discrimination compared with gp60 and enabled the recognition of the two major populations circulating in Europe and in Italy. Graphical Abstract

Background The flagellated parasite Giardia duodenalis is a major and global cause of diarrhoeal disease. Eight genetically very distinct groups, known as assemblages A to H, have been recognized in the G. duodenalis species complex, two of which (assemblages A and B) infect humans and other mammalian hosts. Informative typing schemes are essential to understand transmission pathways, characterize outbreaks and trace zoonotic transmission. In this study, we evaluated a published multi-locus sequence typing (MLST) scheme for G. duodenalis assemblage A, which is based on six polymorphic markers. Methods We genotyped 60 human-derived and 11 animal-derived G. duodenalis isolates collected in Europe and on other continents based on the published protocol. After retrieving previously published genotyping data and excluding isolates whose sequences showed allelic sequence heterozygosity, we analysed a dataset comprising 146 isolates. Results We identified novel variants at five of the six markers and identified 78 distinct MLST types in the overall dataset. Phylogenetic interpretation of typing data confirmed that sub-assemblage AII only comprises human-derived isolates, whereas sub-assemblage AI comprises all animal-derived isolates and a few human-derived isolates, suggesting limited zoonotic transmission. Within sub-assemblage AII, isolates from two outbreaks, which occurred in Sweden and Italy, respectively, had unique and distinct MLST types. Population genetic analysis showed a lack of clustering by geographical origin of the isolates. Conclusion The MLST scheme evaluated provides sufficient discriminatory power for epidemiological studies of G. duodenalis assemblage A. Graphical Abstract

Background Giardiasis, caused by the protozoan parasite Giardia intestinalis , often presents a treatment challenge, particularly in terms of resistance to metronidazole. Despite extensive research, markers for metronidazole resistance have not yet been identified. Methods This study analysed 28 clinical samples of G. intestinalis from sub-assemblage AII, characterised by varying responses to metronidazole treatment. We focussed on copy number variation (CNV) of the multi-copy flavohemoprotein gene, analysed using digital polymerase chain reaction (dPCR) and next generation sequencing (NGS). Additionally, chromosomal ploidy was tested in 18 of these samples. Flavohemoprotein CNV was also assessed in 17 samples from other sub-assemblages. Results Analyses revealed variable CNVs of the flavohemoprotein gene among the isolates, with no correlation to clinical metronidazole resistance. Discrepancies in CNVs detected from NGS data were attributed to biases linked to the whole genome amplification. However, dPCR helped to clarify these discrepancies by providing more consistent CNV data. Significant differences in flavohemoprotein CNVs were observed across different G. intestinalis sub-assemblages. Notably, Giardia exhibits a propensity for aneuploidy, contributing to genomic variability within and between sub-assemblages. Conclusions The complexity of the clinical metronidazole resistance in Giardia is influenced by multiple genetic factors, including CNVs and aneuploidy. No significant differences in the CNV of the flavohemoprotein gene between isolates from metronidazole-resistant and metronidazole-sensitive cases of giardiasis were found, underscoring the need for further research to identify reliable genetic markers for resistance. We demonstrate that dPCR and NGS are robust methods for analysing CNVs and provide cross-validating results, highlighting their utility in the genetic analyses of this parasite. Graphical Abstract

BACKGROUND: Several species of protozoa cause acute or chronic gastroenteritis in humans, worldwide. The burden of disease is particularly high among children living in developing areas of the world, where transmission is favored by lower hygienic standards and scarce availability of safe water. However, asymptomatic infection and polyparasitism are also commonly observed in poor settings. Here, we investigated the prevalence of intestinal protozoa in two small fishing villages, Porto Said (PS) and Santa Maria da Serra (SM), situated along the river Tietê in the State of São Paolo, Brazil. The villages lack basic public infrastructure and services, such as roads, public water supply, electricity and public health services. METHODS: Multiple fecal samples were collected from 88 individuals in PS and from 38 individuals in SM, who were asymptomatic at the time of sampling and had no recent history of diarrheal disease. To gain insights into potential transmission routes, 49 dog fecal samples (38 from PS and 11 from SM) and 28 river water samples were also collected. All samples were tested by microscopy and PCR was used to genotype Giardia duodenalis, Blastocystis sp., Dientamoeba fragilis and Cryptosporidium spp. RESULTS: By molecular methods, the most common human parasite was Blastocystis sp. (prevalence, 45% in PS and 71% in SM), followed by D. fragilis (13.6% in PS, and 18.4% in SM) and G. duodenalis (18.2% in PS and 7.9% in SM); Cryptosporidium spp. were not detected. Sequence analysis revealed large genetic variation among Blastocystis samples, with subtypes (STs) 1 and 3 being predominant, and with the notable absence of ST4. Among G. duodenalis samples, assemblages A and B were detected in humans, whereas assemblages A, C and D were found in dogs. Finally, all D. fragilis samples from humans were genotype 1. A single dog was found infected with Cryptosporidium canis. River water samples were negative for the investigated parasites. CONCLUSIONS: This study showed a high carriage of intestinal parasites in asymptomatic individuals from two poor Brazilian villages, and highlighted a large genetic variability of Blastocystis spp. and G. duodenalis.

Limited molecular data exist on zoonotic parasites Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in Angus calves from Guizhou, China. This study constitutes the first molecular epidemiological survey of these pathogens in this region. 817 fecal samples from Angus calves across 7 intensive beef farms (Bijie City). Nested PCR methods targeting SSU rRNA (Cryptosporidium spp.), gp60 (Cryptosporidium bovis subtyping), bg/gdh/tpi (G. duodenalis), and ITS (E. bieneusi) coupled with DNA sequencing were employed. DNA sequences were analyzed against the NCBI. database. Statistical differences were assessed via a generalized linear mixed-effects model. Cryptosporidium spp. prevalence 23.5% (192/817; 95% CI 28.1–34.6%), with C. bovis predominating 89.6% (172/192; 95% CI 84.4–93.5%) and six subtypes (XXVIa-XXVIf). Highest infection in 4–8-week-olds 29.9% (143/479; 95% CI 25.8–34.1%) (p < 0.01). G. duodenalis: 31.3% (256/817; 95% CI 28.1–34.6%) positive, overwhelmingly assemblage E 97.6% (6/256; 95% CI 0.9–5.0%), zoonotic assemblage A was marginal 0.7% (6/817; 95% CI 0.3–1.6%). Farm-level variation exceeded 10-fold (e.g., Gantang: 55.0% (55/100; 95% CI 44.7–65.0%) vs. Tieshi: 4.9% (5/102; 95% CI 1.6–11.1%). E. bieneusi: prevalence 19.7% (161/817; 95% CI 17.0–22.6%), exclusively zoonotic genotypes BEB4: 49.7% (80/161; 95% CI 41.7–57.7%); I: 40.4% (65/161; 95% CI 32.7–48.4%). Strong diarrhea association (p < 0.01) and site-specific patterns (e.g., Guanyindong: 39.2%). While Giardia exhibited the highest prevalence (31.3%) with minimal zoonotic risk, Enterocytozoon—despite lower prevalence (19.7%)—posed the greatest public health threat due to exclusive circulation of human-pathogenic genotypes (BEB4/I) and significant diarrhea association, highlighting divergent control priorities for these enteric parasites in Guizhou calves. Management/Public health impact: Dominant zoonotic E. bieneusi genotypes (BEB4/I) necessitate: 1. Targeted treatment of 4–8-week-old Angus calves. 2. Manure biofermentation (≥55 °C, 3 days), and 3. UV-disinfection (≥1 mJ/cm2) for karst water to disrupt transmission in this high-humidity region.

Giardia duodenalis, originally regarded as a commensal organism, is the etiologic agent of giardiasis, a gastrointestinal disease of humans and animals. Giardiasis causes major public and veterinary health concerns worldwide. Transmission is either direct, through the faecal-oral route, or indirect, through ingestion of contaminated water or food. Genetic characterization of G. duodenalis isolates has revealed the existence of seven groups (assemblages A to G) which differ in their host distribution. Assemblages A and B are found in humans and in many other mammals, but the role of animals in the epidemiology of human infection is still unclear, despite the fact that the zoonotic potential of Giardia was recognised by the WHO some 30 years ago. Here, we performed an extensive genetic characterization of 978 human and 1440 animal isolates, which together comprise 3886 sequences from 4 genetic loci. The data were assembled into a molecular epidemiological database developed by a European network of public and veterinary health Institutions. Genotyping was performed at different levels of resolution (single and multiple loci on the same dataset). The zoonotic potential of both assemblages A and B is evident when studied at the level of assemblages, sub-assemblages, and even at each single locus. However, when genotypes are defined using a multi-locus sequence typing scheme, only 2 multi-locus genotypes (MLG) of assemblage A and none of assemblage B appear to have a zoonotic potential. Surprisingly, mixtures of genotypes in individual isolates were repeatedly observed. Possible explanations are the uptake of genetically different Giardia cysts by a host, or subsequent infection of an already infected host, likely without overt symptoms, with a different Giardia species, which may cause disease. Other explanations for mixed genotypes, particularly for assemblage B, are substantial allelic sequence heterogeneity and/or genetic recombination. Although the zoonotic potential of G. duodenalis is evident, evidence on the contribution and frequency is (still) lacking. This newly developed molecular database has the potential to tackle intricate epidemiological questions concerning protozoan diseases.

The influence of unicellular eukaryotic microorganisms on human gut health and disease is still largely unexplored. Blastocystis spp. commonly colonize the gut, but its clinical significance and ecological role are currently unsettled. We have developed a high-sensitivity bioinformatic pipeline to detect Blastocystis subtypes (STs) from shotgun metagenomics, and applied it to 12 large data sets, comprising 1689 subjects of different geographic origin, disease status and lifestyle. We confirmed and extended previous observations on the high prevalence the microrganism in the population (14.9%), its non-random and ST-specific distribution, and its ability to cause persistent (asymptomatic) colonization. These findings, along with the higher prevalence observed in non-westernized individuals, the lack of positive association with any of the disease considered, and decreased presence in individuals with dysbiosis associated with colorectal cancer and Crohn’s disease, strongly suggest that Blastocystis is a component of the healthy gut microbiome. Further, we found an inverse association between body mass index and Blastocystis , and strong co-occurrence with archaeal organisms ( Methanobrevibacter smithii ) and several bacterial species. The association of specific microbial community structures with Blastocystis was confirmed by the high predictability (up to 0.91 area under the curve) of the microorganism colonization based on the species-level composition of the microbiome. Finally, we reconstructed and functionally profiled 43 new draft Blastocystis genomes and discovered a higher intra subtype variability of ST1 and ST2 compared with ST3 and ST4. Altogether, we provide an in-depth epidemiologic, ecological, and genomic analysis of Blastocystis , and show how metagenomics can be crucial to advance population genomics of human parasites.

Abstract Cryptosporidiosis is a major global health problem and a primary cause of diarrhea, particularly in young children in low- and middle-income countries (LMICs). The zoonotic Cryptosporidium parvum and anthroponotic Cryptosporidium hominis cause most human infections. Here, we present a comprehensive whole-genome study of C. hominis, comprising 114 isolates from 16 countries within five continents. We detect two lineages with distinct biology and demography, which diverged circa 500 years ago. We consider these lineages two subspecies and propose the names C. hominis hominis and C. hominis aquapotentis (gp60 subtype IbA10G2). In our study, C. h. hominis is almost exclusively represented by isolates from LMICs in Africa and Asia and appears to have undergone recent population contraction. In contrast, C. h. aquapotentis was found in high-income countries, mainly in Europe, North America, and Oceania, and appears to be expanding. Notably, C. h. aquapotentis is associated with high rates of direct human-to-human transmission, which may explain its success in countries with well-developed environmental sanitation infrastructure. Intriguingly, we detected genomic regions of introgression following secondary contact between the subspecies. This resulted in high diversity and divergence in genomic islands of putative virulence genes, including muc5 (CHUDEA2_430) and a hypothetical protein (CHUDEA6_5270). This diversity is maintained by balancing selection, suggesting a co-evolutionary arms race with the host. Finally, we find that recent gene flow from C. h. aquapotentis to C. h. hominis, likely associated with increased human migration, maybe driving the evolution of more virulent C. hominis variants.