Dataset Information

Detection of Cryptosporidium in stool samples of immunocompromised patients.

ABSTRACT: INTRODUCTION:Cryptosporidium species is the most common opportunistic enteric parasite encountered in the immunocompromised patients. Considering the need to diagnose them early relies mostly on rapid tests such as antigen detection by immunochromatographic test (ICT), ELISA, and microscopy. However, the sensitivity and specificity varies with different methods and different kits used. This study was conducted to determine the intestinal parasitic profile in immunocompromised patients and to assess the diagnostic accuracy of the ICT using ImmunoCard STAT kit in detecting Cryptosporidium spp. MATERIALS AND METHODS:The patients in this study were divided into two groups: one group was immunocompromised patients (n = 73) and the other was nonimmunocompromised individuals (n = 73). Stool microscopy, ICT, and polymerase chain reaction (PCR) were carried out for all stool samples. RESULTS:Totally, 4 (5.4%) of 73 patients of the study group were positive for Cryptosporidium. The species detected were Cryptosporidium parvum and Cryptosporidium hominis. PCR was taken as gold standard in the current study. PCR detected Cryptosporidium in four samples while ICT in two samples and microscopy in one sample. CONCLUSION:Cryptosporidium was found to be the most common enteric parasite in the immunocompromised patients studied, followed by Cystoisospora, Entamoeba histolytica, and Strongyloides stercoralis. Although the ICT is a rapid test, it was less sensitive and more expensive in comparison to the PCR; hence, its utility appears to be limited in our setting.

SUBMITTER: Vanathy K

PROVIDER: S-EPMC5369273 | biostudies-literature | 2017 Jan-Jun

REPOSITORIES: biostudies-literature

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Detection of <i>Cryptosporidium</i> in stool samples of immunocompromised patients.

Vanathy K K Parija Subhash Chandra SC Mandal Jharna J Hamide Abdoul A Krishnamurthy Sriram S

Tropical parasitology 20170101 1

<h4>Introduction</h4><i>Cryptosporidium</i> species is the most common opportunistic enteric parasite encountered in the immunocompromised patients. Considering the need to diagnose them early relies mostly on rapid tests such as antigen detection by immunochromatographic test (ICT), ELISA, and microscopy. However, the sensitivity and specificity varies with different methods and different kits used. This study was conducted to determine the intestinal parasitic profile in immunocompromised pati ...[more]

PMID: 28459014

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Project description:The protozoan parasite Cryptosporidium is an important cause of diarrheal disease (cryptosporidiosis) in humans and animals, with significant morbidity and mortality especially in severely immunocompromised people and in young children in low-resource settings. Due to the sexual life cycle of the parasite, transmission is complex. There are no restrictions on sexual recombination between sub-populations, meaning that large-scale genetic recombination may occur within a host, potentially confounding epidemiological analysis. To clarify the relationships between infections in different hosts, it is first necessary to correctly identify species and genotypes, but these differentiations are not made by standard diagnostic tests and more sophisticated molecular methods have been developed. For instance, multilocus genotyping has been utilized to differentiate isolates within the major human pathogens, Cryptosporidium parvum and Cryptosporidium hominis. This has allowed mixed populations with multiple alleles to be identified: recombination events are considered to be the driving force of increased variation and the emergence of new subtypes. As yet, whole genome sequencing (WGS) is having limited impact on public health investigations, due in part to insufficient numbers of oocysts and purity of DNA derived from clinical samples. Moreover, because public health agencies have not prioritized parasites, validation has not been performed on user-friendly data analysis pipelines suitable for public health practitioners. Nonetheless, since the first whole genome assembly in 2004 there are now numerous genomes of human and animal-derived cryptosporidia publically available, spanning nine species. It has also been demonstrated that WGS from very low numbers of oocysts is possible, through the use of amplification procedures. These data and approaches are providing new insights into host-adapted infectivity, the presence and frequency of multiple sub-populations of Cryptosporidium spp. within single clinical samples, and transmission of infection. Analyses show that although whole genome sequences do indeed contain many alleles, they are invariably dominated by a single highly abundant allele. These insights are helping to better understand population structures within hosts, which will be important to develop novel prevention strategies in the fight against cryptosporidiosis.

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

Dataset Information

Detection of Cryptosporidium in stool samples of immunocompromised patients.

Publications

Detection of <i>Cryptosporidium</i> in stool samples of immunocompromised patients.

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