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Abstract Body

Background and aims

Robust Cryptosporidium oocysts shed in faeces can be transmitted through water, food or objects and directly between animals and people. Molecular tests may identify species but there is no internationally harmonised multilocus genotyping (MLG) scheme for public health microbiology. This paper will update on the epidemiology of Cryptosporidium in the COVID-19 pandemic, and the use of genotyping to monitor changes, detect and inform outbreak investigations.

Methods

C. parvum and C. hominis cases in England and Wales, 2015–2021, were analysed in an interrupted time-series analysis. To improve outbreak investigations a MLG scheme for C. parvum, based on multilocus variable number of tandem repeats analysis (MLVA), was validated and applied.

Results

During the COVID-19 pandemic C. hominis fell by 94% and C. parvum by 26%. There was a discernible shift in seasonality for C. parvum, peaking later into summer-autumn. MLVA typability of 259 C. parvum validation samples was 85%, discriminatory power for unrelated samples was 0.99 and 79% MLGs were unique (Robinson et al., 2022). In an outbreak linked to milk from an on-farm vending machine, patient and cattle samples were indistinguishable (Gopfert et al., submitted).

Conclusions

Molecular surveillance is essential to monitor and understand changes and their drivers. Stronger evidence can be obtained in outbreak investigations by improved genotyping. Genetic clusters of C. parvum might indicate unrecognised outbreaks.

Introduction

Human cryptosporidiosis is a major global health problem, a primary cause of diarrhoea, and is largely driven by zoonotic (Cryptosporidium parvum) or anthroponotic (C. parvum and C. hominis) transmission. The epidemiology of cryptosporidiosis varies globally with geography and socioeconomics. Understanding this is critical to control and yet heavily depends on studies driven by single-locus genotyping. Here, we undertook a global population genomic study of the predominant human-infectious Cryptosporidium, C. hominis, and explored the implications of these data for the current understanding of its epidemiology and virulence.

Methods

Global genomic dataset was used to determine population structure, genetic diversity, and introgression.

Results

We detected two lineages with distinct biology and demography that diverged ~500 years ago. Lineage 1 is almost exclusively represented by isolates from Africa and Asia and appears to have undergone recent population contraction. Lineage 2 was found in Europe, North America, and Oceania, and appears to be expanding. We detected genomic regions of introgression following secondary contact between the lineages, likely associated with increased human migration. This resulted in genomic islands of putative virulence genes undergoing diversifying selection and adaptive evolution, suggesting a coevolutionary arms race with the host and increasing virulence.

Conclusions

We consider these lineages two subspecies and propose the names C. hominis hominis and C. hominis aquapotentis for Lineages 1 and 2 respectively.

Introduction

Understanding of Cryptosporidium sp. epidemiology is crucial to control the spread of this parasitic disease. Nowadays, most of the C. parvum and C. hominis epidemiological studies are based on gp60 gene subtyping using Sanger (Sg) sequencing method. Unfortunately, Sg method present the limit of being unable to detect DNA mixtures and so mixed infections. Among the new sequencing methods, Next Generation Sequencing (NGS) seems to be an interesting solution to overcome Sg limit’s, while working on many samples simultaneously. Thus, the aim of our study was to (i) evaluate the added value of NGS as a molecular typing tool for cryptosporidiosis, (ii) investigate the genetic diversity of the parasite and the frequency of mixed infections in human cases and (iii) assess NGS usefulness in Cryptosporidium sp. outbreaks investigations.

Methods

111 DNA extracts from C. hominis and C. parvum positive samples were used in order to (i) assess the suitability of NGS protocol to subtype Cryptosporidium sp. at gp60 gene locus, (ii) evaluate NGS ability to explore parasitic genetic diversity and (iii) explore NGS capacities on epidemic situation.

Results

NGS proved suitable to C. parvum and C. hominis subtyping at gp60 gene locus and to detect DNA mixtures. Applied to epidemic samples, NGS reported more genetic informations compared to Sg method, especially when working on many samples simultaneously, and by detecting more genetic diversity.

Conclusions

This study confirms the usefulness of NGS applied to C. hominis and C. parvum epidemiological studies, especially aimed at detecting minority variants.

Introduction

Cryptosporidium is a leading cause of moderate-to-severe diarrhea in children. Nitazoxanide is the only FDA-approved medication available for cryptosporidiosis treatment, but it has limited efficacy in malnourished children and is ineffective in immunocompromised individuals. Therefore, more effective treatment options are urgently needed.

Methods

We have optimized a novel strategy to silence essential genes in Cryptosporidium using next generation Antisense oligonucleotides ASOs. These molecules consist of a short segment of DNA flanked by two segments of RNA, linked with a phosphorothioate backbone improved by the incorporation of a modified sugar in the RNA backbone. This ASOs molecule is called Fluoroarabinonucleic acid, or 2’ FANA. In this work we tested ASOs-FANA to silence multiple Cryptosporidium genes in cells infected with this parasite.

Results

After the infection, we treated infected cells up to 3 days with ASOs, we observed a dramatic reduction of infection levels (~90%) in all cells treated with specific ASOs FANA but not with scramble ASOs.

Conclusions

Overall, our results show the potential use of antisense therapy as alternative for Cryptosporidiosis treatment.

Introduction

Cryptosporidiosis is an important diarrhoeal disease that can be transmitted from humans and animals. In Slovenia, the average annual incidence of the disease is less than 1 per 100,000 inhabitants, which is below the European average. At the Institute of Microbiology and Immunology, almost 80 % of all reported Slovenian human cases are diagnosed. The distribution of species and gp60 subtypes is tracked every year.

Methods

Isolates from sporadic symptomatic cases diagnosed between 2015 and 2021 were analysed by PCR and sequencing of 18S rRNA and gp60 genes.

Results

Of 104 isolates, the infecting Cryptosporidium sp. was identified in 98 (94 %) cases; 12 of 98 (12 %) were C. hominis and 86 (88 %) were C. parvum. Of the C. parvum isolates, 78 (91 %) were successfully subtyped and assigned to subtype families IIa (75 isolates), IId (two isolates) and IIl (one isolate) and 14 subtypes. In subtype family IIa, 11 different subtypes were identified, with IIaA15G2R1 (41 isolates) being the predominant subtype, followed by IIaA16G1R1 (10 isolates) and IIaA16G2R1 and IIaA17G1R1 (five isolates each). Other C. parvum subtypes caused four or fewer cases of cryptosporidiosis each. Of the C. hominis isolates, all were successfully subtyped and six different subtypes were identified within four subtype families (Ia, Ib, Id and If). C. hominis IbA10G2 was predominant (five cases), while other subtypes were causative agents of cryptosporidiosis in only one or two cases. More than half of the C. hominis cases were imported.

Conclusions

Similar to the results of our studies conducted between 2000 and 2014, C. parvum IIaA15G2R1 is still the predominant causative agent of human cryptosporidiosis in Slovenia, indicating that zoonotic transmission plays an important role in human cryptosporidiosis.

Introduction

In Denmark, cryptosporidiosis is not under national surveillance. In December 2021, an outbreak of diarrhoea due to Cryptosporidium was identified among 30-40 employees (teachers and kitchen personnel) at a high school in Funen, Denmark. This report highlights the need for an active surveillance system that can facilitate the detection of clusters and prompt investigations of potential outbreaks.

Methods

Samples positive for Cryptosporidium at the local clinical microbiology department (Odense University Hospital) were sent to Statens Serum Institut for species identification and glycoprotein 60-based genotyping.

Results

Cryptosporidium parvum was identified in 13 samples; however, no less than three genotypes were identified (genotypes IIdA22G1c, IIdA25G1, and IIdA26G1b), none of which are commonly observed in Denmark. At least one of the genotypes was highly suspected to be linked to the outbreak (IIdA26G1b).

Over the next month, an outbreak of Cryptosporidium was suspected in the neighbouring administrative region of Zealand. However, the patients involved were geographically scattered. Gp60 data for these patients are pending and will reveal whether these two clusters can be linked.

Conclusions

Investigation into any links between the two outbreaks is ongoing. At present, no vehicle that could be responsible for the outbreak(s) has yet been identified.

In Denmark, all clinical microbiology laboratory data are available in the national Microbiological Database ‘MiBa’. With typing facilities in place at Statens Serum Institut, optimal conditions exist for national real-time monitoring of laboratory-confirmed cases of Cryptosporidium and outbreak investigations in Denmark.

Introduction

Microsporidia and Cryptosporidium species are prominent agents of enteritis, capable of causing severe chronic diarrhea in immunocompromised individuals worldwide. It is not possible to identify the parasites at the species level solely based on microscopy. This study aimed to identify Microsporidia and Cryptosporidium parvum and determine their transmission routes through nested PCR, sequencing, and phylogenetic analysis in immunocompromised humans.

Methods

201 stool samples were examined by microscopic methods. Genomic DNA was extracted from all stool samples and specific fragments of the Cryptosporidium parvum beta-tubulin gene and, the Microsporidia SSU rRNA gene was amplified. Furthermore, positive samples were sequenced for genotype identification and bioinformatics analysis.

Results

Based on the microscopic analysis of stool samples, 3 (1.5%) and 27(13.4%) samples were considered positive for Cryptosporidium oocysts and Microsporidia spores, respectively. Species analysis showed the presence of C. parvum in 15 (7.4%) and E. bieneusi in 10 (5%) of the samples, respectively. DNA sequencing analysis of the E. bieneusi and C.parvum-positive specimens confirmed the PCR results and indicated 96–100% similarity with sequences in GenBank.

Conclusions

This study revealed that PCR, sequencing, and phylogenetic analysis are very powerful tools for species identification. Phylogenetic and haplotype network analysis showed geographic segregation in the human populations of E. bieneusi and C. parvum. Besides, according to comparative phylogenetics, cryptosporidiosis and microsporidiosis may occur in this region through zoonotic transmission. Our findings increase the current understanding of these infection's transmission dynamics.