Yaqiong Guo

Concurrent Infections of Giardia duodenalis, Enterocytozoon bieneusi, and Clostridium difficile in Children during a Cryptosporidiosis Outbreak in a Pediatric Hospital in China

Background Over 200 cryptosporidiosis outbreaks have been reported, but little is known if other enteric pathogens were also involved in some of these outbreaks. Recently, an outbreak of cryptosporidiosis linked to poor hygiene by two Cryptosporidium hominis subtypes occurred in a pediatric hospital ward (Ward A) in China, lasting for more than 14 months. In this study, the concurrence during the outbreak of three other enteric pathogens with a similar transmission route, Giardia duodenalis, Enterocytozoon bieneusi, and Clostridium difficile, was assessed. Methods/Principal Findings The occurrence of G. duodenalis, E. bieneusi, and C. difficile in 78 inpatients from Ward A and 283 and 216 inpatients from two control wards (Wards C and D) in the same hospital was examined using molecular diagnostic tools. Significantly higher infection rates were found in children in Ward A for all study pathogens than in Wards C and D (P<0.01): 9.5% versus 1.4% and 0% for G. duodenalis, 10.8% versus 2.8% and 3.7% for E. bieneusi, and 60.8% versus 37.8% and 27.8% for C. difficile, respectively. These differences were mostly seen in children ≤12 months. Enteric pathogen-positive children in Ward A (31/58 or 53.4%) were more likely to have mixed infections than those in Ward C (4/119 or 3.4%) or D (5/68, 7.4%; P<0.01). Having cryptosporidiosis was a risk factor for G. duodenalis (ORu200a=u200a4.3; Pu200a=u200a0.08), E. bieneusi (ORu200a=u200a3.1; Pu200a=u200a0.04), and C. difficile (ORu200a=u200a4.7; P<0.01) infection. In addition, a lower diversity of G. duodenalis, E. bieneusi, and C. difficile genotypes/subtypes was observed in Ward A. Conclusions/Significance Data from this study suggest that multiple pathogens were concurrently present during the previous cryptosporidiosis outbreak. Examination of multiple enteric pathogens should be conducted when poor hygiene is the likely cause of outbreaks of diarrhea.

Subtyping Cryptosporidium ubiquitum, a Zoonotic Pathogen Emerging in Humans

Cryptosporidium ubiquitum is an emerging zoonotic pathogen. In the past, it was not possible to identify an association between cases of human and animal infection. We conducted a genomic survey of the species, developed a subtyping tool targeting the 60-kDa glycoprotein (gp60) gene, and identified 6 subtype families (XIIa-XIIf) of C. ubiquitum. Host adaptation was apparent at the gp60 locus; subtype XIIa was found in ruminants worldwide, subtype families XIIb-XIId were found in rodents in the United States, and XIIe and XIIf were found in rodents in the Slovak Republic. Humans in the United States were infected with isolates of subtypes XIIb-XIId, whereas those in other areas were infected primarily with subtype XIIa isolates. In addition, subtype families XIIb and XIId were detected in drinking source water in the United States. Contact with C. ubiquitum-infected sheep and drinking water contaminated by infected wildlife could be sources of human infections.

Host Specificity and Source of Enterocytozoon bieneusi Genotypes in a Drinking Source Watershed

ABSTRACT To assess the host specificity of Enterocytozoon bieneusi and to track the sources of E. bieneusi contamination, we genotyped E. bieneusi in wildlife and stormwater from the watershed of New York Citys source water, using ribosomal internal transcribed spacer (ITS)-based PCR and sequence analyses. A total of 255 specimens from 23 species of wild mammals and 67 samples from stormwater were analyzed. Seventy-four (29.0%) of the wildlife specimens and 39 (58.2%) of the stormwater samples from streams were PCR positive. Altogether, 20 E. bieneusi genotypes were found, including 8 known genotypes and 12 new ones. Sixteen and five of the genotypes were seen in animals and stormwater from the watershed, respectively, with WL4 being the most common genotype in both animals (35 samples) and stormwater (23 samples). The 20 E. bieneusi genotypes belonged to five genogroups (groups 1, 3, 4, and 7 and an outlier), with only 23/113 (20.4%) E. bieneusi-positive samples belonging to zoonotic genogroup 1 and 3/20 genotypes ever being detected in humans. The two genogroups previously considered host specific, groups 3 and 4, were both detected in multiple groups of mammals. Thus, with the exception of the type IV, Peru11, and D genotypes, which were detected in only 7, 5, and 2 animals, respectively, most E. bieneusi strains in most wildlife samples and all stormwater samples in the watershed had no known public health significance, as these types have not previously been detected in humans. The role of different species of wild mammals in the contribution of E. bieneusi contamination in stormwater was supported by determinations of host-adapted Cryptosporidium species/genotypes in the same water samples. Data from this study indicate that the host specificity of E. bieneusi group 3 is broader than originally thought, and wildlife is the main source of E. bieneusi in stormwater in the watershed.

Occurrence of human-pathogenic Enterocytozoon bieneusi, Giardia duodenalis and Cryptosporidium genotypes in laboratory macaques in Guangxi, China.

Captive nonhuman primates have been identified as common hosts of Enterocytozoon bieneusi, Giardia duodenalis, Cryptosporidium hominis, and Cyclospora spp., thus are potential reservoirs of some enteric parasites in humans. However, few studies have examined the source and human-infective potential of enteric parasites in laboratory nonhuman primates. In the present work, 205 fecal specimens were collected from three groups of captive Macaca fascicularis kept in different densities in a laboratory animal facility in Guangxi, China, and examined by PCR for E. bieneusi, G. duodenalis, Cryptosporidium spp., and Cyclospora spp. The infection rates of E. bieneusi and G. duodenalis were 11.3% and 1.2% in Group 1 (young animals kept individually; n=168), 72.2% and 11.1% in Group 2 (young animals kept in groups; n=18), and 31.6% and 5.3% in Group 3 (adults kept in groups; n=19), respectively. Sequence analysis of PCR products showed the presence of five E. bieneusi genotypes, with genotype D (in 16/36 genotyped specimens) and a new genotype (in 15/36 genotyped specimens) as the dominant genotypes. All five E. bieneusi genotypes belonged to the zoonotic group (Group 1). The G. duodenalis genotypes (assemblages AII and B) in five specimens and C. hominis subtype (IdA14) in one specimen were also known human-pathogens, although the Cyclospora seen in one animal appeared to be unique to macaque monkeys. The higher infection rate in younger animals reared in groups and common occurrence of zoonotic genotypes indicated that human-pathogenic E. bieneusi could be transmitted efficiently in captive nonhuman primates, and group-housing was a risk factor for transmission of zoonotic pathogens in young nonhuman primates in research facilities.

Dominance of Giardia duodenalis assemblage A and Enterocytozoon bieneusi genotype BEB6 in sheep in Inner Mongolia, China

To examine the occurrence and genotype distribution of Giardia duodenalis and Enterocytozoon bieneusi in sheep, fecal specimens were collected from 162 lambs and 213 ewes on seven farms in the Hulunbeier Prairie in Inner Mongolia, China. By PCR analysis of the triose-phosphate isomerase gene, 16 of the 375 (4.3%) specimens were positive for G. duodenalis, with 13 sequenced successfully belonging to assemblage A. Lambs had a significantly higher infection rate than ewes (8.6% versus 0.9%, respectively). The dominance of assemblage A in sheep was supported by PCR analysis of the β-giardin and glutamate dehydrogenase genes; 4 of 7 β-giardin-positive specimens and 3 of 5 glutamate dehydrogenase-positive specimens were identified as assemblage A. A much higher occurrence of E. bieneusi was detected by PCR analysis of the ribosomal internal transcribed spacer; 77.8% of lambs and 62.9% of ewes were positive for the pathogen. Two genotypes of E. bieneusi were found: BEB6 and CM7. Genotype BEB6 was seen in 237 animals and on all seven farms, whereas genotype CM7 was detected in 23 animals from six farms. These data indicate that sheep in Inner Mongolia are commonly infected with G. duodenalis assemblage A and E. bieneusi genotype BEB6, two zoonotic pathogens in China.

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

BackgroundCryptosporidium 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.ResultsAltogether, 8.59-9.05xa0Mb 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.ConclusionsThe 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.

Periparturient transmission of Cryptosporidium xiaoi from ewes to lambs.

The mechanism for the maintenance of Cryptosporidium infection in sheep between yearly lambing periods is not clear. Previously, periparturient shedding of oocysts as the result of stress from lambing was suspected to be a mechanism for the initiation of Cryptosporidium infection in lambs, but this has never been verified by genotyping studies. In this study, fecal specimens from four age groups of sheep in Inner Mongolia, China were examined for Cryptosporidium spp. by PCR-restricted fragment length polymorphism and sequence analyses of the small subunit (SSU) rRNA gene, including 59 ewes 1 week before parturition, 154 ewes at parturition, 87 lambs of 3-4 weeks, and 75 lambs of 15-16 weeks. The Cryptosporidium infection rate in ewes at parturition (7.8%) was significantly higher than at 1 week before parturition (1.7%). Higher infection rates were found in lambs (18.4% and 26.7% for 3-4-week-old and 15-16-week-old lambs, respectively). Most (10/13) Cryptosporidium-positive ewes were shedding Cryptosporidium xiaoi, which was also the dominant species (15/16) in neonatal lambs of 3-4 weeks in age (15/16). The less common species in ewes, Cryptosporidium ubiquitum, was not found in lambs of 3-4 weeks but was the dominant species (14/20) in lambs of 15-16 weeks. The major zoonotic Cryptosporidium species, C. parvum (of the IIaA15G2R1 subtype), was only found in one lamb. These data support the occurrence of periparturient transmission of Cryptosporidium spp., especially C. xiaoi, from ewes to lambs.

Subtyping Novel Zoonotic Pathogen Cryptosporidium Chipmunk Genotype I

ABSTRACT Cryptosporidium chipmunk genotype I is an emerging zoonotic pathogen in humans. The lack of subtyping tools makes it impossible to determine the role of zoonotic transmission in epidemiology. To identify potential subtyping markers, we sequenced the genome of a human chipmunk genotype I isolate. Altogether, 9,509,783 bp of assembled sequences in 853 contigs were obtained, with an N50 of 117,886 bp and >200-fold coverage. Based on the whole-genome sequence data, two genetic markers encoding the 60-kDa glycoprotein (gp60) and a mucin protein (ortholog of cgd1_470) were selected for the development of a subtyping tool. The tool was used for characterizing chipmunk genotype I in 25 human specimens from four U.S. states and Sweden, one specimen each from an eastern gray squirrel, a chipmunk, and a deer mouse, and 4 water samples from New York. At the gp60 locus, although different subtypes were seen among the animals, water, and humans, the 15 subtypes identified differed mostly in the numbers of trinucleotide repeats (TCA, TCG, or TCT) in the serine repeat region, with only two single nucleotide polymorphisms in the nonrepeat region. Some geographic differences were found in the subtype distribution of chipmunk genotype I from humans. In contrast, only two subtypes were found at the mucin locus, which differed from each other in the numbers of a 30-bp minisatellite repeat. Thus, Cryptosporidium chipmunk genotype I isolates from humans and wildlife are genetically similar, and zoonotic transmission might play a potential role in human infections.

Isolation and Enrichment of Cryptosporidium DNA and Verification of DNA Purity for Whole-Genome Sequencing

ABSTRACT Whole-genome sequencing of Cryptosporidium spp. is hampered by difficulties in obtaining sufficient, highly pure genomic DNA from clinical specimens. In this study, we developed procedures for the isolation and enrichment of Cryptosporidium genomic DNA from fecal specimens and verification of DNA purity for whole-genome sequencing. The isolation and enrichment of genomic DNA were achieved by a combination of three oocyst purification steps and whole-genome amplification (WGA) of DNA from purified oocysts. Quantitative PCR (qPCR) analysis of WGA products was used as an initial quality assessment of amplified genomic DNA. The purity of WGA products was assessed by Sanger sequencing of cloned products. Next-generation sequencing tools were used in final evaluations of genome coverage and of the extent of contamination. Altogether, 24 fecal specimens of Cryptosporidium parvum, C. hominis, C. andersoni, C. ubiquitum, C. tyzzeri, and Cryptosporidium chipmunk genotype I were processed with the procedures. As expected, WGA products with low (<16.0) threshold cycle (CT ) values yielded mostly Cryptosporidium sequences in Sanger sequencing. The cloning-sequencing analysis, however, showed significant contamination in 5 WGA products (proportion of positive colonies derived from Cryptosporidium genomic DNA, ≤25%). Following this strategy, 20 WGA products from six Cryptosporidium species or genotypes with low (mostly <14.0) CT values were submitted to whole-genome sequencing, generating sequence data covering 94.5% to 99.7% of Cryptosporidium genomes, with mostly minor contamination from bacterial, fungal, and host DNA. These results suggest that the described strategy can be used effectively for the isolation and enrichment of Cryptosporidium DNA from fecal specimens for whole-genome sequencing.

Genetic similarities between Cyclospora cayetanensis and cecum-infecting avian Eimeria spp. in apicoplast and mitochondrial genomes

BackgroundCyclospora cayetanensis is an important cause for diarrhea in children in developing countries and foodborne outbreaks of cyclosporiasis in industrialized nations. To improve understanding of the basic biology of Cyclospora spp. and development of molecular diagnostic tools and therapeutics, we sequenced the complete apicoplast and mitochondrial genomes of C. cayetanensis.MethodsThe genome of one Chinese C. cayetanensis isolate was sequenced using Roche 454 and Illumina technologies. The assembled genomes of the apicoplast and mitochondrion were retrieved, annotated, and compared with reference genomes for other apicomplexans to infer genome organizations and phylogenetic relationships. Sequence variations in the mitochondrial genome were identified by comparison of two C. cayetanensis nucleotide sequences from this study and a recent publication.ResultsThe apicoplast and mitochondrial genomes of C. cayetanensis are 34,155 and 6,229xa0bp in size and code for 65 and 5 genes, respectively. Comparative genomic analysis showed high similarities between C. cayetanensis and Eimeria tenella in both genomes; they have 85.6xa0% and 90.4xa0% nucleotide sequence similarities, respectively, and complete synteny in gene organization. Phylogenetic analysis of the genomic sequences confirmed the genetic similarities between cecum-infecting avian Eimeria spp. and C. cayetanensis. Like in other coccidia, both genomes of C. cayetanensis are transcribed bi-directionally. The apicoplast genome is circular, codes for the complete machinery for protein biosynthesis, and contains two inverted repeats that differ slightly in LSU rRNA gene sequences. In contrast, the mitochondrial genome has a linear concatemer or circular mapping topology. Eight single-nucleotide and one 7-bp multiple-nucleotide variants were detected between the mitochondrial genomes of C. cayetanensis from this and recent studies.ConclusionsThe apicoplast and mitochondrial genomes of C. cayetanensis are highly similar to those of cecum-infecting avian Eimeria spp. in both genome organization and sequences. The availability of sequence data beyond rRNA and heat shock protein genes could facilitate studies of C. cayetanensis biology and development of genotyping tools for investigations of cyclosporiasis outbreaks.