Initial Data on the Molecular Epidemiology of Cryptosporidiosis in Lebanon

Cryptosporidium spp. represent a major public health problem worldwide and infect the gastrointestinal tract of both immunocompetent and immunocompromised persons. The prevalence of these parasites varies by geographic region, and no data are currently available in Lebanon. To promote an understanding of the epidemiology of cryptosporidiosisin this country, the main aim of this study was to determine the prevalence Cryptosporidium in symptomatic hospitalized patients, and to analyze the genetic diversity of the corresponding isolates. Fecal specimens were collected in four hospitals in North Lebanon from 163 patients (77 males and 86 females, ranging in age from 1 to 88 years, with a mean age of 22 years) presenting gastrointestinal disorders during the period July to December 2013. The overall prevalence of Cryptosporidium spp. infection obtained by modified Ziehl-Neelsen staining and/or nested PCR was 11%, and children <5 years old showed a higher rate of Cryptosporidium spp. The PCR products of the 15 positive samples were successfully sequenced. Among them, 10 isolates (66.7%) were identified as C. hominis, while the remaining 5 (33.3%) were identified as C. parvum. After analysis of the gp60 locus, C. hominis IdA19, a rare subtype, was found to be predominant. Two C. parvum subtypes were found: IIaA15G1R1 and IIaA15G2R1. The molecular characterization of Cryptosporidium isolates is an important step in improving our understanding of the epidemiology and transmission of the infection.     

SHP-2 Mediates Cryptosporidium parvum Infectivity in Human Intestinal Epithelial Cells

The parasite, Cryptosporidium parvum, induces human gastroenteritis through infection of host epithelial cells in the small intestine. During the initial stage of infection, C. parvum is reported to engage host mechanisms at the host cell-parasite interface to form a parasitophorous vacuole. We determined that upon infection, the larger molecular weight proteins in human small intestinal epithelial host cells (FHs 74 Int) appeared to globally undergo tyrosine dephosphorylation. In parallel, expression of the cytoplasmic protein tyrosine phosphatase Src homology-2 domain-containing phosphatase 2 (SHP-2) increased in a time-dependent manner. SHP-2 co-localized with the C. parvum sporozoite and this interaction increased the rate of C. parvum infectivity through SH2-mediated SHP-2 activity. Furthermore, we show that one potential target that SHP-2 acts upon is the focal adhesion protein, paxillin, which undergoes moderate dephosphorylation following infection, with inhibition of SHP-2 rescuing paxillin phosphorylation. Importantly, treatment with an inhibitor to SHP-2 and with an inhibitor to paxillin and Src family kinases, effectively decreased the multiplicity of C. parvum infection in a dose-dependent manner. Thus, our study reveals an important role for SHP-2 in the pathogenesis of C. parvum. Furthermore, while host proteins can be recruited to participate in the development of the electron dense band at the host cell-parasite interface, our study implies for the first time that SHP-2 appears to be recruited by the C. parvum sporozoite to regulate infectivity. Taken together, these findings suggest that SHP-2 and its down-stream target paxillin could serve as targets for intervention.     

Identification of Cryptosporidium felis in a Cow by Morphologic and Molecular Methods

Apicomplexan Cryptosporidium parasites infect a wide range of vertebrate hosts. While some species are limited to a single host group, such as Cryptosporidium baileyi, which infects chickens, other species of this genus, such as C. parvum, infect a wide range of mammalian species from mice to humans. During an investigation of Cryptosporidium infection in cattle on a farm in northern Poland, we identified an infection caused by C. felis, in addition to known infections with C. muris and C. parvum. This new infection was identified based on the size of the oocysts (mean size, 4.3 ± 0.4 μm; range, 3.5 to 5.0 μm), as well as by analysis of the molecular sequence of the variable region of the small-subunit rRNA. This finding demonstrates the complex host specificity and circulation in the environment of Cryptosporidium species.     

Infection with anthroponotic Cryptosporidium parvum does not fully protect the host against a subsequent challenge with C. hominis

Cryptosporidium hominis and Cryptosporidium parvum are the major Cryptosporidium species that infect humans. Earlier studies in gnotobiotic piglets, model susceptible to both, showed that piglets recovered from infection with C. hominis were fully protected against challenge with same species but incompletely protected against C. parvum challenge. In the present study, piglets were infected with C. parvum first, and after recovery were re-challenged with C. parvum or C. hominis. Again, full protection was only observed when piglets were challenged with the homologous parasite strain. Although the two species are genetically/antigenically almost identical, they do not confer complete protection against each other.     

Prevalence of Cryptosporidium parvum/hominis,Entamoeba histolytica and Giardia lamblia among Young Children with and without Diarrhea in Dar es Salaam,Tanzania

Background

Although enteroparasites are common causes of diarrheal illness, few studies have been performed among children in Tanzania. This study aimed to investigate the prevalence of Cryptosporidium parvum/hominis, Entamoeba histolytica and Giardia lamblia among young children in Dar es Salaam, Tanzania, and identify risk factors for infection.

Methodology/Principal Findings

We performed an unmatched case-control study among children < 2 years of age in Dar es Salaam, recruited from August 2010 to July 2011. Detection and identification of protozoans were done by PCR techniques on DNA from stool specimens from 701 cases of children admitted due to diarrhea at the three study hospitals, and 558 controls of children with no history of diarrhea during the last month prior to enrollment. The prevalence of C. parvum/hominis was 10.4% (84.7% C. hominis), and that of G. lamblia 4.6%. E. histolytica was not detected. The prevalence of Cryptosporidium was significantly higher in cases (16.3%) than in controls (3.1%; P < 0.001; OR = 6.2; 95% CI: 3.7–10.4). G. lamblia was significantly more prevalent in controls (6.1%) than in cases (3.4%; P = 0.027; OR = 1.8; 95% CI: 1.1–3.1). Cryptosporidium infection was found more often in HIV-positive (24.2%) than in HIV-negative children (3.9%; P < 0.001; OR = 7.9; 95% CI: 3.1–20.5), and was also associated with rainfall (P < 0.001; OR = 2.41; 95% CI: 1.5–3.8). Among cases, stunted children had significantly higher risk of being infected with Cryptosporidium (P = 0.011; OR = 2.12; 95% CI: 1.2–3.8). G. lamblia infection was more prevalent in the cool season (P = 0.004; OR = 2.2; 95% CI: 1.3–3.8), and more frequent among cases aged > 12 months (P = 0.003; OR = 3.5; 95% CI: 1.5–7.8). Among children aged 7–12 months, those who were breastfed had lower prevalence of G. lamblia infection than those who had been weaned (P = 0.012).

Conclusions

Cryptosporidium infection is common among young Tanzanian children with diarrhea, particularly those living with HIV, and infection is more frequent during the rainy season. G. lamblia is frequently implicated in asymptomatic infections, but rarely causes overt diarrheal illness, and its prevalence increases with age.     

Genetic Diversity of Cryptosporidium in Children in an Urban Informal Settlement of Nairobi,Kenya

Introduction

Globally Cryptosporidium and Giardia species are the most common non-bacterial causes of diarrhoea in children and HIV infected individuals, yet data on their role in paediatric diarrhoea in Kenya remains scant. This study investigated the occurrence of Cryptosporidium species, genotypes and subtypes in children, both hospitalized and living in an informal settlement in Nairobi.

Methods

This was a prospective cross-sectional study in which faecal specimen positive for Cryptosporidium spp. by microscopy from HIV infected and uninfected children aged five years and below presenting with diarrhoea at selected outpatient clinics in Mukuru informal settlements, or admitted to the paediatric ward at the Mbagathi District Hospital were characterized. The analysis was done by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) of the 18srRNA gene for species identification and PCR-sequencing of the 60 kDa glycoprotein (GP60) gene for subtyping.

Results

C. hominis was the most common species of Cryptosporidium identified in125/151(82.8%) of the children. Other species identified were C. parvum 18/151(11.9%), while C. felis and C. meleagridis were identified in 4 and 2 children, respectively. Wide genetic variation was observed within C. hominis, with identification of 5 subtype families; Ia, Ib, Id, Ie and If and 21 subtypes. Only subtype family IIc was identified within C. parvum. There was no association between species and HIV status or patient type.

Conclusion

C. hominis is the most common species associated with diarrhoea in the study population. There was high genetic variability in the C. hominis isolates with 22 different subtypes identified, whereas genetic diversity was low within C. parvum with only one subtype family IIc identified.     

Outbreak of cryptosporidiosis due to Cryptosporidium parvum subtype IIdA19G1 in neonatal calves on a dairy farm in China

Neonatal diarrhea is one of the most important syndromes in dairy cattle. Among enteropathogens, Cryptosporidium spp. are primary causes of diarrhea, but outbreaks due to cryptosporidiosis are rarely reported in cattle. From January to April in 2016, severe diarrhea was observed in over 400 neonatal dairy calves on a large dairy farm in Jiangsu Province of East China. Approximately 360 calves died due to watery diarrhea despite antibiotic therapy. In this study, 18 fecal specimens were collected from seriously ill calves on this farm during the diarrhea outbreak, and analysed for common enteropathogens by enzymatic immunoassay (EIA). In a post-outbreak investigation, 418 and 1372 specimens collected from animals of various age groups were further analysed for rotavirus and Cryptosporidium spp. by EIA and PCR, respectively, to assess their roles in the occurrence of diarrhea on the farm. Cryptosporidium spp. were genotyped using established techniques. Initial EIA tests showed that 15/18 seriously ill calves during the outbreak were positive for Cryptosporidium parvum, while 8/18 were positive for rotavirus. The overall infection rate of Cryptosporidium in pre-weaned calves on the farm was 22.7%, with odds of the Cryptosporidium infection during the outbreak 4.4–23.5 times higher than after the outbreak. Four Cryptosporidium spp. were identified after the outbreak including C. parvum (n = 79), Cryptosporidium ryanae (n = 48), Cryptosporidium bovis (n = 31), and Cryptosporidium andersoni (n = 3), with co-infections of multiple species being detected in 34 animals. Infection with C. parvum (73/79) was found in the majority of calves aged ≤3 weeks, consistent with the age of ill calves during the outbreak. All C. parvum isolates were identified as subtype IIdA19G1. In the post-outbreak investigation, C. parvum infection was associated with the occurrence of watery diarrhea in pre-weaned calves, C. ryanae infection was associated with moderate diarrhea in both pre- and post-weaned calves, while no association was identified between rotavirus infection and the occurrence of diarrhea. Results of logistic regression analysis further suggested that C. bovis infection might also be a risk factor for moderate diarrhea in calves. Thus, we believe this is the first report of a major outbreak of severe diarrhea caused by C. parvum IIdA19G1 in dairy calves. More attention should be directed toward preventing the dissemination of this virulent subtype in China.     

Aged HCT-8 Cell Monolayers Support Cryptosporidium parvum Infection

Cell culture assays in various formats have been used to study the infectivity of Cryptosporidium spp. as well as to determine the infectivity of naturally occurring oocysts in water. Currently, cell culture assays for infectious Cryptosporidium spp. in water have largely been limited to practice in research laboratories. One obstacle to the routine use of Cryptosporidium cell culture assays for the analysis of water samples is the coordination of water sample collection and processing with readiness of cell culture monolayers. For most Cryptosporidium cell culture assays, monolayers are allowed to develop for 24 to 48 h to reach 80 to 100% confluence prior to inoculation. In this study, we used immunofluorescent assay microscopy to evaluate freshly confluent (2-day-old) and aged (8- to 67-day-old) HCT-8 cell monolayers for their ability to support Cryptosporidium parvum infection. HCT-8 monolayers as old as 67 days were clearly shown to support infection. In two of three experiments, aged monolayers (8- to 11-day-old and 11- to 22-day-old, respectively) developed the same number of C. parvum clusters of infection as freshly confluent monolayers. Results suggest that it may be possible to use cell monolayers from freshly confluent to 3 weeks old on hand for infectivity assays without having to schedule sample processing to coincide with development of freshly confluent monolayers. This would make Cryptosporidium cell culture assays much more feasible for water quality and utility laboratories.     

Deformability Assessment of Waterborne Protozoa Using a Microfluidic-Enabled Force Microscopy Probe

Many modern filtration technologies are incapable of the complete removal of Cryptosporidium oocysts from drinking-water. Consequently, Cryptosporidium-contaminated drinking-water supplies can severely implicate both water utilities and consumers. Existing methods for the detection of Cryptosporidium in drinking-water do not discern between non-pathogenic and pathogenic species, nor between viable and non-viable oocysts. Using FluidFM, a novel force spectroscopy method employing microchannelled cantilevers for single-cell level manipulation, we assessed the size and deformability properties of two species of Cryptosporidium that pose varying levels of risk to human health. A comparison of such characteristics demonstrated the ability of FluidFM to discern between Cryptosporidium muris and Cryptosporidium parvum with 86% efficiency, whilst using a measurement throughput which exceeded 50 discrete oocysts per hour. In addition, we measured the deformability properties for untreated and temperature-inactivated oocysts of the highly infective, human pathogenic C. parvum to assess whether deformability may be a marker of viability. Our results indicate that untreated and temperature-inactivated C. parvum oocysts had overlapping but significantly different deformability distributions.     

Prevalence and Molecular Characterization of Cryptosporidium in Goats across Four Provincial Level Areas in China

This study assessed the prevalence, species and subtypes of Cryptosporidium in goats from Guangdong Province, Hubei Province, Shandong Province, and Shanghai City of China. Six hundred and four fecal samples were collected from twelve goat farms, and the overall infection rate was 11.4% (69/604). Goats infected with Cryptosporidium were found in eleven farms across four provincial areas, and the infection rate ranged from 2.9% (1/35) to 25.0% (9/36). Three Cryptosporidium species were identified. Cryptosporidium xiaoi (45/69, 65.2%) was the dominant species, followed by C. parvum (14/69, 20.3%) and C. ubiquitum (10/69, 14.5%). The infection rate of Cryptosporidium spp. was varied with host age and goat kids were more susceptible to be infected than adult goats. Subtyping C. parvum and C. ubiquitum positive samples revealed C. parvum subtype IIdA19G1 and C. ubiquitum subtype XIIa were the most common subtypes. Other C. parvum subtypes were detected as well, such as IIaA14G2R1, IIaA15G1R1, IIaA15G2R1 and IIaA17G2R1. All of these subtypes have also been detected in humans, suggesting goats may be a potential source of zoonotic cryptosporidiosis. This was the first report of C. parvum subtypes IIaA14G2R1, IIaA15G1R1 and IIaA17G2R1 infecting in goats and the first molecular identification of C. parvum and its subtypes in Chinese goats.     

A cell culture platform for Cryptosporidium that enables long-term cultivation and new tools for the systematic investigation of its biology

Cryptosporidium parasites are a major cause of diarrhoea that pose a particular threat to children in developing areas and immunocompromised individuals. Curative therapies and vaccines are lacking, mainly due to lack of a long-term culturing system of this parasite. Here, we show that COLO-680N cells infected with two different Cryptosporidium parvum strains produce sufficient infectious oocysts to infect subsequent cultures, showing a substantial fold increase in production, depending on the experiment, over the most optimistic HCT-8 models. Oocyst identity was confirmed using a variety of microscopic- and molecular-based methods. This culturing system will accelerate research on Cryptosporidium and the development of anti-Cryptosporidium drugs.     

Detection of Cryptosporidium parvum Oocysts on Fresh Produce Using DNA Aptamers

There are currently no standard methods for the detection of Cryptosporidium spp., or other protozoan parasites, in foods, and existing methods are often inadequate, with low and variable recovery efficiencies. Food testing is difficult due to the low concentrations of parasites, the difficulty in eluting parasites from some foods, the lack of enrichment methods, and the presence of PCR inhibitors. The main objectives of the present study were to obtain DNA aptamers binding to the oocyst wall of C. parvum, and to use the aptamers to detect the presence of this parasite in foods. DNA aptamers were selected against C. parvum oocysts using SELEX (Systematic Evolution of Ligands by EXponential enrichment). Ten rounds of selection led to the discovery of 14 aptamer clones with high affinities for C. parvum oocysts. For detecting parasite-bound aptamers, a simple electrochemical sensor was employed, which used a gold nanoparticle-modified screen-printed carbon electrode. This aptasensor was fabricated by self-assembling a hybrid of a thiolated ssDNA primer and the anti- C. parvum aptamer. Square wave voltammetry was employed to quantitate C. parvum in the range of 150 to 800 oocysts, with a detection limit of approximately 100 oocysts. The high sensitivity and specificity of the developed aptasensor suggests that this novel method is very promising for the detection and identification of C. parvum oocysts on spiked fresh fruits, as compared to conventional methods such as microscopy and PCR.     

Genetic Diversity within Cryptosporidium parvum and Related Cryptosporidium Species

To assess the genetic diversity in Cryptosporidium parvum, we have sequenced the small subunit (SSU) rRNA gene of seven Cryptosporidium spp., various isolates of C. parvum from eight hosts, and a Cryptosporidium isolate from a desert monitor. Phylogenetic analysis of the SSU rRNA sequences confirmed the multispecies nature of the genus Cryptosporidium, with at least four distinct species (C. parvum, C. baileyi, C. muris, and C. serpentis). Other species previously defined by biologic characteristics, including C. wrairi, C. meleagridis, and C. felis, and the desert monitor isolate, clustered together or within C. parvum. Extensive genetic diversities were present among C. parvum isolates from humans, calves, pigs, dogs, mice, ferrets, marsupials, and a monkey. In general, specific genotypes were associated with specific host species. A PCR-restriction fragment length polymorphism technique previously developed by us could differentiate most Cryptosporidium spp. and C. parvum genotypes, but sequence analysis of the PCR product was needed to differentiate C. wrairi and C. meleagridis from some of the C. parvum genotypes. These results indicate a need for revision in the taxonomy and assessment of the zoonotic potential of some animal C. parvum isolates.     

Comparison of Method 1623 and Cell Culture-PCR for Detection of Cryptosporidium spp. in Source Waters

Analysis of Cryptosporidium occurrence in six watersheds by method 1623 and the integrated cell culture-PCR (CC-PCR) technique provided an opportunity to evaluate these two methods. The average recovery efficiencies were 58.5% for the CC-PCR technique and 72% for method 1623, but the values were not significantly different (P = 0.06). Cryptosporidium oocysts were detected in 60 of 593 samples (10.1%) by method 1623. Infectious oocysts were detected in 22 of 560 samples (3.9%) by the CC-PCR technique. There was 87% agreement between the total numbers of samples positive as determined by method 1623 and CC-PCR for four of the sites. The other two sites had 16.3 and 24% correspondence between the methods. Infectious oocysts were detected in all of the watersheds. Overall, approximately 37% of the Cryptosporidium oocysts detected by the immunofluorescence method were viable and infectious. DNA sequence analysis of the Cryptosporidium parvum isolates detected by CC-PCR showed the presence of both the bovine and human genotypes. More than 90% of the C. parvum isolates were identified as having the bovine or bovine-like genotype. The estimates of the concentrations of infectious Cryptosporidium and the resulting daily and annual risks of infection compared well for the two methods. The results suggest that most surface water systems would require, on average, a 3-log reduction in source water Cryptosporidium levels to meet potable water goals.     

Molecular Characterization of Cryptosporidium spp. among Children in Rural Ghana

BackgroundThe relevance of Cryptosporidium infections for the burden of childhood diarrhoea in endemic settings has been shown in recent years. This study describes Cryptosporidium subtypes among symptomatic and asymptomatic children in rural Ghana to analyse subtype-specific demographic, geographical, seasonal and clinical differences in order to inform appropriate control measures in endemic areas.Conclusions/SignificanceCryptosporidiosis is characterized by seasonal anthroponotic transmission of strains typically found in Sub-Saharan Africa. The infection mainly affects young infants, with vomiting and diarrhoea being one of the leading symptoms in C. parvum infection. Combining molecular typing and clinical data provides valuable information for physicians and is able to track sources of infections.     

Distribution and Clinical Manifestations of Cryptosporidium Species and Subtypes in HIV/AIDS Patients in Ethiopia

Background

Cryptosporidiosis is an important cause for chronic diarrhea and death in HIV/AIDS patients. Among common Cryptosporidium species in humans, C. parvum is responsible for most zoonotic infections in industrialized nations. Nevertheless, the clinical significance of C. parvum and role of zoonotic transmission in cryptosporidiosis epidemiology in developing countries remain unclear.

Methodology/Principal Findings

In this cross-sectional study, 520 HIV/AIDS patients were examined for Cryptosporidium presence in stool samples using genotyping and subtyping techniques. Altogether, 140 (26.9%) patients were positive for Cryptosporidium spp. by PCR-RFLP analysis of the small subunit rRNA gene, belonging to C. parvum (92 patients), C. hominis (25 patients), C. viatorum (10 patients), C. felis (5 patients), C. meleagridis (3 patients), C. canis (2 patients), C. xiaoi (2 patients), and mixture of C. parvum and C. hominis (1 patient). Sequence analyses of the 60 kDa glycoprotein gene revealed a high genetic diversity within the 82 C. parvum and 19 C. hominis specimens subtyped, including C. parvum zoonotic subtype families IIa (71) and IId (5) and anthroponotic subtype families IIc (2), IIb (1), IIe (1) and If-like (2), and C. hominis subtype families Id (13), Ie (5), and Ib (1). Overall, Cryptosporidium infection was associated with the occurrence of diarrhea and vomiting. Diarrhea was attributable mostly to C. parvum subtype family IIa and C. hominis, whereas vomiting was largely attributable to C. hominis and rare Cryptosporidium species. Calf contact was identified as a significant risk factor for infection with Cryptosporidium spp., especially C. parvum subtype family IIa.

Conclusions/Significance

Results of the study indicate that C. parvum is a major cause of cryptosporidiosis in HIV-positive patients and zoonotic transmission is important in cryptosporidiosis epidemiology in Ethiopia. In addition, they confirm that different Cryptosporidium species and subtypes are linked to different clinical manifestations.     

Comparison of various staining methods for the detection of Cryptosporidium in cell-free culture

The complete development of Cryptosporidium in host cell-free medium first described in 2004, represented a significant advance that can facilitate many aspects of Cryptosporidium research. A current limitation of host cell-free cultivation is the difficulty involved in visualising the life-cycle stages as they are very small in size, morphologically difficult to identify and dispersed throughout the media. This is in contrast to conventional cell culture methods for Cryptosporidium, where it is possible to focus on the host cells and view the foci of infection on the host cells. In the present study, we compared three specific and three non-specific techniques for visualising Cryptosporidium parvum life-cycle stages in cell-free culture; antibody staining using anti-sporozoite and anti-oocyst wall antibodies (Sporo-Glo™ and Crypto Cel), fluorescent in-situ hybridization (FISH) using a Cryptosporidium specific rRNA oligonucleotide probe and the non-specific dyes; Texas Red, carboxyfluorescein diacetate succinimidyl ester (CFSE) and 4,6′ diamino-2-phenylindole dihydrochloride (DAPI). Results revealed that a combination of Sporo-Glo™ and Crypto Cel staining resulted in easy and reliable identification of all life-cycle stages.     

Diversity of Cryptosporidium spp. in Apodemus spp. in Europe

The genetic diversity of Cryptosporidium spp. in Apodemus spp. (striped field mouse, yellow-necked mouse and wood mouse) from 16 European countries was examined by PCR/sequencing of isolates from 437 animals. Overall, 13.7% (60/437) of animals were positive for Cryptosporidium by PCR. Phylogenetic analysis of small-subunit rRNA, Cryptosporidium oocyst wall protein and actin gene sequences showed the presence of Cryptosporidium ditrichi (22/60), Cryptosporidium apodemi (13/60), Cryptosporidium apodemus genotype I (8/60), Cryptosporidium apodemus genotype II (9/60), Cryptosporidium parvum (2/60), Cryptosporidium microti (2/60), Cryptosporidium muris (2/60) and Cryptosporidium tyzzeri (2/60). At the gp60 locus, novel gp60 families XVIIa and XVIIIa were identified in Cryptosporidium apodemus genotype I and II, respectively, subtype IIaA16G1R1b was identified in C. parvum, and subtypes IXaA8 and IXcA6 in C. tyzzeri. Only animals infected with C. ditrichi, C. apodemi, and Cryptosporidium apodemus genotypes shed oocysts that were detectable by microscopy, with the infection intensity ranging from 2000 to 52,000 oocysts per gram of faeces. None of the faecal samples was diarrheic in the time of the sampling.