Development of a gp60-subtyping method for Cryptosporidium felis

Background: Feline cryptosporidiosis is an increasing problem, especially in catteries. In humans, close contact with cats could be a potential source of infection although the risk of contracting cryptosporidiosis caused by Cryptosporidium felis is considered to be relatively low. Sequencing of the 60-kDa glycoprotein gene is a commonly used tool for investigation of the genetic diversity and transmission dynamics of Cryptosporidium species. However, until now the sequence of gp60 from C. felis has not been available and genotyping has been limited to less discriminatory markers, such as 18S rRNA, COWP and HSP70. Methods: We have identified the gp60 orthologue within the genome sequence of C. felis, and used the sequence to design a nested PCR for subtyping purposes. A total of 128 clinical isolates of both feline and human origin, were used to evaluate the marker. Results: Sequence analysis revealed large variations between the different samples. The C. felis gp60 lack the characteristic serine-tract found in many other cryptosporidian orthologues, instead it has an insertion of variable length (361-742 nt). Also, two cases of suspected zoonotic transmission of C. felis between cats and humans were successfully confirmed. Conclusions: We have identified the gp60 gene in C. felis and show how this highly variable marker can be used in epidemiological investigations

Differentiation of Blastocystis and parasitic archamoebids encountered in untreated wastewater samples by amplicon-based next-generation sequencing.

BACKGROUND: Application of next-generation sequencing (NGS) to genomic DNA extracted from sewage offers a unique and cost-effective opportunity to study the genetic diversity of intestinal parasites. In this study, we used amplicon-based NGS to reveal and differentiate several common luminal intestinal parasitic protists, specifically Entamoeba, Endolimax, Iodamoeba, and Blastocystis, in sewage samples from Swedish treatment plants. MATERIALS AND METHODS: Influent sewage samples were subject to gradient centrifugation, DNA extraction and PCR-based amplification using three primer pairs designed for amplification of eukaryotic nuclear 18S ribosomal DNA. PCR products were sequenced using ILLUMINA® technology, and resulting sequences were annotated to species and subtype level using the in-house BION software, sequence clustering, and phylogenetic analysis. RESULTS: A total of 26 samples from eight treatment plants in central/southern Sweden were analysed. Blastocystis sp. and Entamoeba moshkovskii were detected in all samples, and most samples (n?=?20) were positive for Entamoeba coli. Moreover, we detected Entamoeba histolytica, Entamoeba dispar, Entamoeba hartmanni, Endolimax nana, and Iodamoeba bütschlii in 1, 11, 4, 10, and 7 samples, respectively. The level of genetic divergence observed within E. nana and E. moshkovskii was 20.2% and 7.7%, respectively, across the ~400-bp region studied, and two clades of E. moshkovskii were found. As expected, Blastocystis sp. subtypes 1-4 were present in almost all samples; however, ST8 was present in 10 samples and was the only subtype not commonly found in humans that was present in multiple samples. CONCLUSIONS: Entamoeba and Blastocystis were identified as universal members of the "sewage microbiome". Blastocystis sp. ST8, which has been rarely reported in humans, was a very common finding, indicating that a hitherto unidentified but common host of ST8 contributed to the sewage influent. The study also provided substantial new insight into the intra-generic diversity of Entamoeba and Endolimax

Frequent In Vitro Recombination in Internal Transcribed Spacers 1 and 2 during Genotyping of Pneumocystis jirovecii

Pneumocystis jirovecii is the causative agent of Pneumocystis pneumonia (PCP) in immunocompromised persons. Knowledge of the transmission and epidemiology of PCP is still incipient, and investigations on these subjects are based exclusively on applications of molecular typing techniques. The polymorphic internal transcribed spacers ITS1 and ITS2 in the ribosomal DNA operon, which in the P. jirovecii genome exist as single-copy DNA, are commonly used as target loci for isolate typing. In the course of genotyping P. jirovecii in respiratory specimens from PCP patients by amplification and cloning of a large number of ITS sequences, we found mixed infections (two or more types) in 50% of the samples. In a majority of the specimens with mixed infections, we detected many ITS haplotypes (combinations of ITS1 and ITS2 types) that appeared to be products of recombination between globally common ITS haplotypes present in the same sample. Here we present results of a series of experiments showing that essentially all ITS recombinants are chimeras formed during the genotyping process. Under standard conditions, as many as 37% of the amplified sequences could be hybrid DNA artifacts. We show that by modifying PCR amplification conditions, ITS chimera formation could be largely abolished and the erroneous establishment of artifactual haplotypes avoided. The accurate assessment of genetic diversity is fundamental for a better understanding of the epidemiology and biology of P. jirovecii infections

Protozoan infections are under-recognized in Swedish patients with gastrointestinal symptoms

In acute gastroenteritis (GE), identification of the infectious agent is important for patient management and surveillance. The prevalence of GE caused by protozoa may be underestimated in Swedish patients. The purpose was to compare the prevalence ofE. histolytica,Cryptosporidiumspp.,G. intestinalis, andC. cayetanensisin samples from patients where the clinician had requested testing for gastrointestinal parasites only (n = 758) to where testing for bacterial GE only (n = 803) or where both parasite and bacterial testing (n = 1259) was requested and a healthy control group (n = 197). This prospective cohort study was conducted in Region Jonkoping County, Sweden (October 2018-March 2019). Fecal samples were analyzed with microscopy and real-time PCR.Cryptosporidiumspp. was detected in 16 patients in the bacterial GE group and in 13 in the both bacterial and parasite group; no cases were detected in the group were only parasite infection was suspected.C. cayetanensiswas detected in two patients in the bacterial GE group. One case ofE. histolyticawas detected in the bacterial group and one in the both bacterial and parasite group.G. intestinaliswas detected in 14 patients in the parasite only group, 12 in the both parasite and bacterial group, three in the bacterial GE group, and one in the control group. Diarrhea caused by protozoa, especially Cryptosporidium was under-recognized by clinicians and is likely more common than hitherto estimated in Sweden. A more symptom-based diagnostic algorithm may increase detection and knowledge about protozoan infections.Funding Agencies|Karolinska InstitutetKarolinska Institutet; Futurum, Region Jonkoping County</p

Cryptosporidium species and subtypes identified in human domestic cases through the national microbiological surveillance programme in Sweden from 2018 to 2022

Abstract Background The intestinal protozoan parasite Cryptosporidium is an important cause of diarrheal disease worldwide. A national microbiological surveillance programme was implemented in Sweden in 2018 in order to increase knowledge of the molecular epidemiology of human cryptosporidiosis to better understand transmission patterns and potential zoonotic sources. This article summarises the results of the first five years of the surveillance programme. Methods Cryptosporidium-positive faecal and DNA samples from domestically acquired infections were collected from clinical microbiological laboratories in Sweden. Species and subtype determination was performed using 60 kDa glycoprotein and/or small subunit ribosomal RNA gene analysis. Results Between 2018 and 2022, 1654 samples were analysed and 11 different species were identified: C. parvum (n = 1412), C. mortiferum (n = 59), C. hominis (n = 56), C. erinacei (n = 11), C. cuniculus (n = 5), C. meleagridis (n = 3), C. equi (n = 2), C. ubiquitum (n = 2), and one each of C. canis, C. ditrichi and C. felis. Subtyping revealed seven subtype families of C. parvum (new subtype families IIy and IIz) and 69 different subtypes (11 new subtypes). The most common C. parvum subtypes were IIdA22G1c, IIdA24G1, IIdA15G2R1 and IIaA16G1R1b. For C. hominis, four different subtype families and nine different subtypes (two new subtypes) were identified. For additional species, two new subtype families (IIIk and VId) and nine new subtypes were identified. All successfully subtyped C. mortiferum cases were subtype XIVaA20G2T1, confirming previous findings in Sweden. Several outbreaks were identified of which the majority were foodborne and a few were due to direct contact with infected animals. Conclusion Infection with C. parvum is the leading cause of human cryptosporidiosis acquired in Sweden, where more than 90% of domestic cases are caused by this zoonotic species and only a small proportion of cases are due to infection with other species. The rodent-associated C. mortiferum is considered an emerging zoonotic species in Sweden and the number of domestically acquired human cases has surpassed that of infection with C. hominis. A high diversity of species and subtypes, as well as diversity within the same subtype, was detected. Also, cryptosporidiosis appears to affect adults to a great extent in Sweden

Possible zoonotic transmission of Cryptosporidium felis in a household

In humans, the risk of contracting cryptosporidiosis caused by Cryptosporidium felis is considered to be relatively low, and most of the confirmed cases have been observed in immunocompromised patients. Both anthroponotic and zoonotic transmission routes have been suggested. Here, we report a case of suspected zoonotic transmission of C. felis from a cat to a human. The cat developed diarrhea several months before such symptoms were displayed by its owner, a 37-year-old immunocompetent woman. The presence of identical C. felis SSU rRNA, HSP70, and COWP gene sequences was verified in both hosts. In conclusion, it is highly probable that the cat was the initial source of infection and not the opposite. Our results show that Cryptosporidium infection can be transmitted from pets to humans and that molecular analysis is needed to confirm the identity of the oocysts

Cryptosporidium as a testbed for single cell genome characterization of unicellular eukaryotes

Background: Infectious disease involving multiple genetically distinct populations of pathogens is frequently concurrent, but difficult to detect or describe with current routine methodology. Cryptosporidium sp. is a widespread gastrointestinal protozoan of global significance in both animals and humans. It cannot be easily maintained in culture and infections of multiple strains have been reported. To explore the potential use of single cell genomics methodology for revealing genome-level variation in clinical samples from Cryptosporidium-infected hosts, we sorted individual oocysts for subsequent genome amplification and full-genome sequencing. Results: Cells were identified with fluorescent antibodies with an 80 % success rate for the entire single cell genomics workflow, demonstrating that the methodology can be applied directly to purified fecal samples. Ten amplified genomes from sorted single cells were selected for genome sequencing and compared both to the original population and a reference genome in order to evaluate the accuracy and performance of the method. Single cell genome coverage was on average 81 % even with a moderate sequencing effort and by combining the 10 single cell genomes, the full genome was accounted for. By a comparison to the original sample, biological variation could be distinguished and separated from noise introduced in the amplification. Conclusions: As a proof of principle, we have demonstrated the power of applying single cell genomics to dissect infectious disease caused by closely related parasite species or subtypes. The workflow can easily be expanded and adapted to target other protozoans, and potential applications include mapping genome-encoded traits, virulence, pathogenicity, host specificity and resistance at the level of cells as truly meaningful biological units

Cryptosporidium chipmunk genotype I : An emerging cause of human cryptosporidiosis in Sweden

Most cases of cryptosporidiosis in humans are caused by Cryptosporidium parvum or Cryptosporidium hominis. However, more uncommon species are increasingly being recognised to cause infection in humans. Here we report that Cryptosporidium chipmunk genotype I, which has various rodents as its natural host, is the third most common source of human cryptosporidiosis in Sweden. We also describe the first small outbreak of cryptosporidiosis caused by Cryptosporidium chipmunk genotype I and report the first case of zoonotic transmission of Cryptosporidium chipmunk genotype I from a red squirrel to a human. Cryptosporidium chipmunk genotype I was identified in 20 human cases, including 16 sporadic cases, three outbreak-related cases, and one zoonotic case, as well as in two squirrel samples. Gp60 subtyping which was successful for 19 human cases and two squirrel samples showed that all samples harboured the same subtype, XIVaA20G2T1. The work presented here suggests that red squirrel is a natural host of Cryptosporidium chipmunk genotype I and that infection with Cryptosporidium chipmunk genotype I is an emerging cause of domestic cryptosporidiosis in Sweden and a potential source of outbreaks