Predominant Virulent IbA10G2 Subtype of Cryptosporidium hominis in Human Isolates in Barcelona: A Five-Year Study

Background

Cryptosporidium infection is a worldwide cause of diarrheal disease. To gain insight into the epidemiology of the infection in a certain geographic area, molecular methods are needed to determine the species/genotypes and subtypes.

Methodology/Principal Findings

From 2004 to 2009, 161 cryptosporidiosis cases were detected in two hospitals in Barcelona. Diagnosis was performed by microscopic observation of oocysts in stool specimens following modified Ziehl-Neelsen staining. Most cases (82%) occurred in children. The number of cases increased in summer and autumn. Molecular characterization of Cryptosporidium was performed in 69 specimens, and C. hominis and C. parvum were identified in 88.4% and 10.1% of the cases, respectively. C. meleagridis was detected in one specimen. Subtyping based on the gp60 polymorphism showed six subtypes, four C. hominis and two C. parvum. Subtype IbA10G2 was the most prevalent subtype corresponding to 90% of all C. hominis isolates. This is the first report on the distribution of specific Cryptosporidium subtypes from humans in Spain.

Conclusions/Significance

In our geographic area, the anthroponotic behavior of C. hominis, the lower infective dose, and the higher virulence of certain subtypes may contribute to the high incidence of human cryptosporidiosis caused by the IbA10G2 subtype. Further studies should include populations with asymptomatic shedding of the parasite.     

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.     

Generation of whole genome sequences of new Cryptosporidium hominis and Cryptosporidium parvum isolates directly from stool samples

Background

Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA.

Results

The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585).The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing.

Conclusion

This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.     

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.     

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.     

Molecular characterisation of Cryptosporidium outbreaks in Western and South Australia

Molecular typing at the 18S rRNA and Gp60 loci was conducted on Cryptosporidium-positive stool samples from cases collected during 2007 Western Australian and South Australian outbreaks of cryptosporidiosis. Analysis of 48 Western Australian samples identified that all isolates were C. hominis and were from five different Gp60C. hominis subtype families. The IbA10G2 subtype was most common across all age groups (37/48). In South Australia, analysis of 24 outbreak samples, identified 21 C. hominis isolates, two C. parvum isolates and one sample with both C. hominis and C. parvum. All C. hominis isolates were identified as the IbA10G2 subtype.     

Burden of Diarrhea,Hospitalization and Mortality Due to Cryptosporidial Infections in Indian Children

Background

Cryptosporidium spp. is a common, but under-reported cause of childhood diarrhea throughout the world, especially in developing countries. A comprehensive estimate of the burden of cryptosporidiosis in resource-poor settings is not available.

Methodology/Principal Findings

We used published and unpublished studies to estimate the burden of diarrhea, hospitalization and mortality due to cryptosporidial infections in Indian children. Our estimates suggest that annually, one in every 6–11 children <2 years of age will have an episode of cryptosporidial diarrhea, 1 in every 169–633 children will be hospitalized and 1 in every 2890–7247 children will die due to cryptosporidiosis. Since there are approximately 42 million children <2 years of age in India, it is estimated that Cryptosporidium results in 3.9–7.1 million diarrheal episodes, 66.4–249.0 thousand hospitalizations, and 5.8–14.6 thousand deaths each year.

Conclusions/Significance

The findings of this study suggest a high burden of cryptosporidiosis among children <2 years of age in India and makes a compelling case for further research on transmission and prevention modalities of Cryptosporidium spp. in India and other developing countries.     

High diversity of Cryptosporidium subgenotypes identified in Malaysian HIV/AIDS individuals targeting gp60 gene

Background

Currently, there is a lack of vital information in the genetic makeup of Cryptosporidium especially in developing countries. The present study aimed at determining the genotypes and subgenotypes of Cryptosporidium in hospitalized Malaysian human immunodeficiency virus (HIV) positive patients.

Methodology/Principal Findings

In this study, 346 faecal samples collected from Malaysian HIV positive patients were genetically analysed via PCR targeting the 60 kDa glycoprotein (gp60) gene. Eighteen (5.2% of 346) isolates were determined as Cryptosporidium positive with 72.2% (of 18) identified as Cryptosporidium parvum whilst 27.7% as Cryptosporidium hominis. Further gp60 analysis revealed C. parvum belonging to subgenotypes IIaA13G1R1 (2 isolates), IIaA13G2R1 (2 isolates), IIaA14G2R1 (3 isolates), IIaA15G2R1 (5 isolates) and IIdA15G1R1 (1 isolate). C. hominis was represented by subgenotypes IaA14R1 (2 isolates), IaA18R1 (1 isolate) and IbA10G2R2 (2 isolates).

Conclusions/Significance

These findings highlighted the presence of high diversity of Cryptosporidium subgenotypes among Malaysian HIV infected individuals. The predominance of the C. parvum subgenotypes signified the possibility of zoonotic as well as anthroponotic transmissions of cryptosporidiosis in HIV infected individuals.     

Longitudinal multi-locus molecular characterisation of sporadic Australian human clinical cases of cryptosporidiosis from 2005 to 2008

Cryptosporidium is a gastrointestinal parasite that is recognised as a significant cause of non-viral diarrhea in both developing and industrialised countries. In the present study, a longitudinal analysis of 248 faecal specimens from Australian humans with gastrointestinal symptoms from 2005 to 2008 was conducted. Sequence analysis of the 18S rRNA gene locus and the 60 kDa glycoprotein (gp60) gene locus revealed that 195 (78.6%) of the cases were due to infection with Cryptosporidium hominis, 49 (19.8%) with Cryptosporidium parvum and four (1.6%) with Cryptosporidium meleagridis. A total of eight gp60 subtype families were identified; five C. hominis subtype families (Ib, Id, Ie, If and Ig), and two C. parvum subtype families (IIa and IId). The Id subtype family was the most common C. hominis subtype family identified in 45.7% of isolates, followed by the Ig subtype family (30.3%) and the Ib subtype family (20%). The most common C. parvum subtype was IIaA18G3R1, identified in 65.3% of isolates. The more rare zoonotic IId A15G1 subtype was identified in one isolate. Statistical analysis showed that the Id subtype was associated with abdominal pain (p < 0.05) and that in sporadic cryptosporidiosis, children aged 5 and below were 1.91 times and 1.88 times more likely to be infected with subtype Id (RR 1.91; 95% CI, 1.7-2.89; p < 0.05) and Ig (RR 1.88; 95% CI, 1.10-3.24; p < 0.05) compared to children aged 5 and above. A subset of isolates were also analysed at the variable CP47 and MSC6-7 gene loci. Findings from this study suggest that anthroponotic transmission of Cryptosporidium plays a major role in the epidemiology of cryptosporidiosis in Western Australian humans.     

Comparative genomic analysis reveals occurrence of genetic recombination in virulent Cryptosporidium hominis subtypes and telomeric gene duplications in Cryptosporidium parvum

Background

Cryptosporidium hominis is a dominant species for human cryptosporidiosis. Within the species, IbA10G2 is the most virulent subtype responsible for all C. hominis–associated outbreaks in Europe and Australia, and is a dominant outbreak subtype in the United States. In recent yearsIaA28R4 is becoming a major new subtype in the United States. In this study, we sequenced the genomes of two field specimens from each of the two subtypes and conducted a comparative genomic analysis of the obtained sequences with those from the only fully sequenced Cryptosporidium parvum genome.

Results

Altogether, 8.59-9.05 Mb of Cryptosporidium sequences in 45–767 assembled contigs were obtained from the four specimens, representing 94.36-99.47% coverage of the expected genome. These genomes had complete synteny in gene organization and 96.86-97.0% and 99.72-99.83% nucleotide sequence similarities to the published genomes of C. parvum and C. hominis, respectively. Several major insertions and deletions were seen between C. hominis and C. parvum genomes, involving mostly members of multicopy gene families near telomeres. The four C. hominis genomes were highly similar to each other and divergent from the reference IaA25R3 genome in some highly polymorphic regions. Major sequence differences among the four specimens sequenced in this study were in the 5′ and 3′ ends of chromosome 6 and the gp60 region, largely the result of genetic recombination.

Conclusions

The sequence similarity among specimens of the two dominant outbreak subtypes and genetic recombination in chromosome 6, especially around the putative virulence determinant gp60 region, suggest that genetic recombination plays a potential role in the emergence of hyper-transmissible C. hominis subtypes. The high sequence conservation between C. parvum and C. hominis genomes and significant differences in copy numbers of MEDLE family secreted proteins and insulinase-like proteases indicate that telomeric gene duplications could potentially contribute to host expansion in C. parvum.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1517-1) contains supplementary material, which is available to authorized users.     

Molecular Surveillance of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi by Genotyping and Subtyping Parasites in Wastewater

Background

Despite their wide occurrence, cryptosporidiosis and giardiasis are considered neglected diseases by the World Health Organization. The epidemiology of these diseases and microsporidiosis in humans in developing countries is poorly understood. The high concentration of pathogens in raw sewage makes the characterization of the transmission of these pathogens simple through the genotype and subtype analysis of a small number of samples.

Methodology/Principal Findings

The distribution of genotypes and subtypes of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in 386 samples of combined sewer systems from Shanghai, Nanjing and Wuhan and the sewer system in Qingdao in China was determined using PCR-sequencing tools. Eimeria spp. were also genotyped to assess the contribution of domestic animals to Cryptosporidium spp., G. duodenalis, and E. bieneusi in wastewater. The high occurrence of Cryptosporidium spp. (56.2%), G. duodenalis (82.6%), E. bieneusi (87.6%), and Eimeria/Cyclospora (80.3%) made the source attribution possible. As expected, several human-pathogenic species/genotypes, including Cryptosporidium hominis, Cryptosporidium meleagridis, G. duodenalis sub-assemblage A-II, and E. bieneusi genotype D, were the dominant parasites in wastewater. In addition to humans, the common presence of Cryptosporidium spp. and Eimeria spp. from rodents indicated that rodents might have contributed to the occurrence of E. bieneusi genotype D in samples. Likewise, the finding of Eimeria spp. and Cryptosporidium baileyi from birds indicated that C. meleagridis might be of both human and bird origins.

Conclusions/Significance

The distribution of Cryptosporidium species, G. duodenalis genotypes and subtypes, and E. bieneusi genotypes in urban wastewater indicates that anthroponotic transmission appeared to be important in epidemiology of cryptosporidiosis, giardiasis, and microsporidiosis in the study areas. The finding of different distributions of subtypes between Shanghai and Wuhan was indicative of possible differences in the source of C. hominis among different areas in China.     

Cryptosporidium cuniculus and Giardia duodenalis in rabbits: genetic diversity and possible zoonotic transmission

Background

Cryptosporidium and Giardia are the two important zoonotic pathogens causing diarrhea of humans and animals worldwide. Considering the human cryptosporidiosis outbreak and sporadic cases caused by C. cuniculus, the important public health significance of G. duodenalis and little obtained information regarding rabbit infected with Cryptosporidium and Giardia in China, the aim of this study is to determine the prevalence and molecularly characterize Cryptosporidium and Giardia in rabbits in Heilongjiang Province, China.

Methodology/Principal Findings

378 fecal samples were obtained from rabbits in Heilongjiang Province. Cryptosporidium oocysts and Giardia cysts were detected using Sheather''s sugar flotation technique and Lugol''s iodine stain method, respectively. The infection rates of Cryptosporidium and Giardia were 2.38% (9/378) and 7.41% (28/378), respectively. Genotyping of Cryptosporidium spp. was done by DNA sequencing of the small subunit rRNA (SSU rRNA) gene and all the nine isolates were identified as Cryptosporidium cuniculus. The nine isolates were further subtyped using the 60-kDa glycoprotein (gp60) gene and two subtypes were detected, including VbA32 (n = 3) and a new subtype VbA21 (n = 6). G. duodenalis genotypes and subtypes were identified by sequence analysis of the triosephosphate isomerase (TPI) gene. The assemblage B (belonging to eight different subtypes B-I to B-VIII) was found in 28 G. duodenalis-positive samples.

Conclusions/Significance

The rabbits have been infected with Cryptosporidium and Giardia in Heilongjiang Province. The results show that the rabbits pose a threat to human health in the studied areas. Genotypes and subgenotypes of C. cuniculus and G. duodenalis in this study might present the endemic genetic characterization of population structure of the two parasites.     

Increased Missense Mutation Burden of Fatty Acid Metabolism Related Genes in Nunavik Inuit Population

Background

Nunavik Inuit (northern Quebec, Canada) reside along the arctic coastline where for generations their daily energy intake has mainly been derived from animal fat. Given this particular diet it has been hypothesized that natural selection would lead to population specific allele frequency differences and unique variants in genes related to fatty acid metabolism. A group of genes, namely CPT1A, CPT1B, CPT1C, CPT2, CRAT and CROT, encode for three carnitine acyltransferases that are important for the oxidation of fatty acids, a critical step in their metabolism.

Methods

Exome sequencing and SNP array genotyping were used to examine the genetic variations in the six genes encoding for the carnitine acyltransferases in 113 Nunavik Inuit individuals.

Results

Altogether ten missense variants were found in genes CPT1A, CPT1B, CPT1C, CPT2 and CRAT, including three novel variants and one Inuit specific variant CPT1A p.P479L (rs80356779). The latter has the highest frequency (0.955) compared to other Inuit populations. We found that by comparison to Asians or Europeans, the Nunavik Inuit have an increased mutation burden in CPT1A, CPT2 and CRAT; there is also a high level of population differentiation based on carnitine acyltransferase gene variations between Nunavik Inuit and Asians.

Conclusion

The increased number and frequency of deleterious variants in these fatty acid metabolism genes in Nunavik Inuit may be the result of genetic adaptation to their diet and/or the extremely cold climate. In addition, the identification of these variants may help to understand some of the specific health risks of Nunavik Inuit.     

The first reported cases of human cryptosporidiosis caused by Cryptosporidium hominis in Slovak Republic

Cryptosporidiosis belongs to the important parasitic infections with zoonotic potential and the occurrence in European countries is rare. The first cases of cryptosporidiosis caused by Cryptosporidium hominis detected in the Slovak republic were described here. Collection of examined humans consisted of five family members. Faecal specimens were examined by formalin sedimentation, by the Sheather??s sugar flotation and by immunochromatography and visualised by the Ziehl?CNeelsen acid fast stain. A fragment of the Cryptosporidium small subunit ribosomal RNA gene was amplified by nested polymerase chain reaction and species was determined by restriction fragment length polymorphism analysis with the endonucleases SspI and VspI. C. hominis was found in faeces of two immunocompetent siblings (a 7-year-old boy and a 2-year-old girl). The symptoms occurred only in the boy as gastrointestinal disorders lasting 5?days, and manifested by abdominal pain, an elevated body temperature (37.2?°C), mild diarrhoea, accompanied by lassitude, depression and anorexia. Ultrasonic scan revealed enlarged spleen and mezenteric lymph nodes. Microscopic examination of the stool sample revealed numerous Cryptosporidium oocysts. The DNA typing identified C. hominis subtype IbA10G2. Cryptosporidium was also detected in the boy??s sister without any complications and symptoms. Their father, mother and grandmother were parasitologically negative. The source of infection remained unknown. Human cases in present study reflect necessity of systematic attention on intestinal parasites diagnostic inclusive of cryptosporidia.     

Molecular Characterization of Cryptosporidium spp. in Children from Mexico

Cryptosporidiosis is a parasitic disease caused by Cryptosporidium spp. In immunocompetent individuals, it usually causes an acute and self-limited diarrhea; in infants, infection with Cryptosporidium spp. can cause malnutrition and growth retardation, and declined cognitive ability. In this study, we described for the first time the distribution of C. parvum and C. hominis subtypes in 12 children in Mexico by sequence characterization of the 60-kDa glycoprotein (GP60) gene of Cryptosporidium. Altogether, 7 subtypes belonging to 4 subtype families of C. hominis (Ia, Ib, Id and Ie) and 1 subtype family of C. parvum (IIa) were detected, including IaA14R3, IaA15R3, IbA10G2, IdA17, IeA11G3T3, IIaA15G2R1 and IIaA16G1R1. The frequency of the subtype families and subtypes in the samples analyzed in this study differed from what was observed in other countries.     

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.     

Epidemiology and molecular relationships of Cryptosporidium spp. in people, primates, and livestock from Western Uganda

Background

Cryptosporidium is one of the most common parasitic diarrheal agents in the world and is a known zoonosis. We studied Cryptosporidium in people, livestock, and non-human primates in the region of Kibale National Park, Uganda. Land use change near the park has resulted in fragmented forest patches containing small, remnant populations of wild primates that interact intensively with local people and livestock. Our goal was to investigate risk factors for Cryptosporidium infection and to assess cross-species transmission using molecular methods.

Methodology/Principal Findings

Diagnostic PCR revealed a prevalence of Cryptosporidium of 32.4% in humans, 11.1% in non-human primates, and 2.2% in livestock. In the case of humans, residence in one particular community was associated with increased risk of infection, as was fetching water from an open water source. Although 48.5% of infected people reported gastrointestinal symptoms, this frequency was not significantly different in people who tested negative (44.7%) for Cryptosporidium, nor was co-infection with Giardia duodenalis associated with increased reporting of gastrointestinal symptoms. Fecal consistency was no different in infected versus uninfected people or animals. DNA sequences of the Cryptosporidium oocyst wall protein gene placed all infections within a well-supported C. parvum/C. hominis clade. However, the only two sequences recovered from primates in the core of the park''s protected area fell into a divergent sub-clade and were identical to published sequences from C. parvum, C. hominis, and C. cuniculus, suggesting the possibility of a separate sylvatic transmission cycle.

Conclusions/Significance

Cryptosporidium may be transmitted frequently among species in western Uganda where people, livestock, and wildlife interact intensively as a result of anthropogenic changes to forests, but the parasite may undergo more host-specific transmission where such interactions do not occur. The parasite does not appear to have strong effects on human or animal health, perhaps because of persistent low-level shedding and immunity.     

A Novel Sample Processing Method for Rapid Detection of Tuberculosis in the Stool of Pediatric Patients Using the Xpert MTB/RIF Assay

Background

Tuberculosis (TB) is difficult to diagnose in children using molecular tests, because children have difficulty providing respiratory samples. Stool could replace sputum for diagnostic TB testing if adequate sample processing techniques were available.

Methods

We developed a rapid method to process large volumes of stool for downstream testing by the Xpert MTB/RIF (Xpert) TB-detection assay. The method was tested and optimized on stool samples spiked with known numbers of M. tuberculosis colony forming units (CFU), and stools from M. tuberculosis-infected cynomolgus macaques (Macaca fascicularis). Performance was scored on number of positive Xpert tests, the cycle thresholds (Cts) of the Xpert sample-processing control (SPC), and the Cts of the M. tuberculosis-specific rpoB probes. The method was then validated on 20 confirmed TB cases and 20 controls in Durban, South Africa.

Results

The assay’s analytical limit of detection was 1,000 CFU/g of stool. As much as one gram of spiked stool could be tested without showing increased PCR inhibition. In analytical spiking experiments using human stool, 1g samples provided the best sensitivity compared to smaller amounts of sample. However, in Macaques with TB, 0.6g stool samples performed better than either 0.2g or 1.2g samples. Testing the stool of pediatric TB suspects and controls suggested an assay sensitivity of 85% (95% CI 0.6–0.9) and 84% (95% CI 0.6–0.96) for 0.6g and 1.2g stool samples, respectively, and a specificity of 100% (95% CI 0.77–1) and 94% (95% CI 0.7–0.99), respectively.

Conclusion

This novel approach may permit simple and rapid detection of TB using pediatric stool samples.     

Evaluating the Impact of Breastfeeding on Rotavirus Antigenemia and Disease Severity in Indian Children

Objectives

To evaluate the contribution of breastfeeding to Rotavirus (RV)-induced antigenemia and/or RNAemia and disease severity in Indian children (<2 yrs age).

Methods

Paired stool and serum samples were collected from (a) hospitalized infants with diarrhea (n = 145) and (b) healthy control infants without diarrhea (n = 28). Stool RV-antigen was screened in both groups by commercial rapid-test and enzyme immunoassay. The disease severity was scored and real-time-PCR was used for viral-load estimation. Serum was evaluated for RV-antigenemia by EIA and RV-RNAemia by RT-PCR. Data was stratified by age-group and breastfeeding status and compared.

Results

Presence of RV-antigenemia and RV-RNAemia was positively related with presence of RV in stool. Disease severity and stool viral-load was significantly associated with RV-antigenemia[(r = 0.74; CI:0.66 to 0.84; P<0.0001,R² = 0.59) and (r = -0.55; CI:-0.68 to -0.39; P<0.0001,R² = 0.31) respectively], but not with RV-RNAemia. There was significant reduction in RV-antigenemiarate in the breast-fed group compared to non-breastfed infants, especially in 0–6 month age group (P<0.001). Non-breastfed infants were at risk for RV-antigenemia with severe disease manifestations in form of high Vesikari scores correlating with high fever, more vomiting episodes and dehydration.

Conclusion

RV-antigenemia was common in nonbreastfed children with severe RV-diarrhea and correlated with stool RV-load and disease severity.