Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Cryptosporidium hominis infection diagnosed by real-time PCR-RFLP.

ABSTRACT: There are approximately 20 known species of the genus Cryptosporidium, and among these, 8 infect immunocompetent or immunocompromised humans. C. hominis and C. parvum most commonly infect humans. Differentiating between them is important for evaluating potential sources of infection. We report here the development of a simple and accurate real-time PCR-based restriction fragment length polymorphism (RFLP) method to distinguish between C. parvum and C. hominis. Using the CP2 gene as the target, we found that both Cryptosporidium species yielded 224 bp products. In the subsequent RFLP method using TaqI, 2 bands (99 and 125 bp) specific to C. hominis were detected. Using this method, we detected C. hominis infection in 1 of 21 patients with diarrhea, suggesting that this method could facilitate the detection of C. hominis infections.

SUBMITTER: Cheun HI

PROVIDER: S-EPMC3712111 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Cryptosporidium hominis infection diagnosed by real-time PCR-RFLP.

Cheun Hyeng-Il HI Kim Kyungjin K Yoon Sejoung S Lee Won-Ja WJ Park Woo-Yoon WY Sim Seobo S Yu Jae-Ran JR

The Korean journal of parasitology 20130630 3

There are approximately 20 known species of the genus Cryptosporidium, and among these, 8 infect immunocompetent or immunocompromised humans. C. hominis and C. parvum most commonly infect humans. Differentiating between them is important for evaluating potential sources of infection. We report here the development of a simple and accurate real-time PCR-based restriction fragment length polymorphism (RFLP) method to distinguish between C. parvum and C. hominis. Using the CP2 gene as the target, w ...[more]

PMID: 23864748

Similar Datasets

Multicentric evaluation of a new real-time PCR assay for quantification of Cryptosporidium spp. and identification of Cryptosporidium parvum and Cryptosporidium hominis.

Project description:Cryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocompromised patients. Laboratory diagnosis of cryptosporidiosis relies on microscopy, antigen detection, and nucleic acid detection and analysis. Among the numerous molecular targets available, the 18S rRNA gene displays the best sensitivity and sequence variations between species and can be used for molecular typing assays. This paper presents a new real-time PCR assay for the detection and quantification of all Cryptosporidium species associated with the identification of Cryptosporidium hominis and Cryptosporidium parvum. The sensitivity and specificity of this new PCR assay were assessed on a multicentric basis, using well-characterized Cryptosporidium-positive and -negative human stool samples, and the efficiencies of nine extraction methods were comparatively assessed using Cryptosporidium-seeded stool samples and phosphate-buffered saline samples. A comparison of extraction yields showed that the most efficient extraction method was the Boom technique in association with mechanical grinding, and column extraction showed higher binding capacity than extraction methods based on magnetic silica. Our PCR assay was able to quantify at least 300 oocysts per gram of stool. Satisfactory reproducibility between laboratories was observed. The two main species causing human disease, Cryptosporidium hominis and Cryptosporidium parvum, were identified using a duplex real-time PCR assay with specific TaqMan minor-groove-binding ligand (MGB) probes for the same amplicon. To conclude, this one-step quantitative PCR is well suited to the routine diagnosis of cryptosporidiosis since practical conditions, including DNA extraction, quantification using well-defined standards, and identification of the two main species infecting humans, have been positively assessed.

| S-EPMC3719639 | biostudies-literature

Development of real-time PCR for detection of Mycoplasma hominis.

Project description:BACKGROUND: Mycoplasma hominis is associated with pelvic inflammatory disease, bacterial vaginosis, post partum fever, sepsis and infections of the central nervous system often leading to serious conditions. Association with development of female infertility has also been suggested, but different publications present different results. We developed a sensitive and fast diagnostic real-time PCR to test clinical samples from women undergoing laparoscopic examination before fertility treatment. To develop a test for the detection and quantification of M. hominis we selected a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (gap), as a target. RESULTS: Real-time PCR was optimized to detect 10 copies of M. hominis PG21 genomic DNA. A fluorescence signal was measured for all 20 other M. hominis isolates, and melting curves analysis showed variations in the melting temperature in agreement with sequence variation in the region of the probes. There was no amplification of other mycoplasmal DNA and human DNA. Eighty-three patient cervical swab samples from infertile women were cultured for M. hominis in the BEa medium. Two of the samples (2.4%) were positive after 48 hours of incubation. The real-time PCR detected the same two samples positive, and the DNA concentrations in the clinical specimens were calculated to 37.000 copies/ml and 88.500 copies/ml, respectively. CONCLUSION: The results demonstrate that real-time PCR may prove to be a rapid alternative to the traditional cultivation method. Information on bacterial load in genital swabs can be obtained. The assay allowed detection of M. hominis in a closed system reducing the risk of contamination by amplicon carry-over.

| S-EPMC518963 | biostudies-literature

New Diagnostic Real-Time PCR for Specific Detection of Mycoplasma hominis DNA.

Project description:Mycoplasma hominis is a fastidious micro-organism causing genital and extragenital infections. We developed a specific real-time PCR that exhibits high sensitivity and low intrarun and interrun variabilities. When applied to clinical samples, this quantitative PCR allowed to confirm the role of M. hominis in three patients with severe extragenital infections.

| S-EPMC2913506 | biostudies-literature

Cryptosporidium hominis infection of the human respiratory tract.

Project description:Cryptosporidium oocysts, observed in a natural sputum sample of a patient with HIV, were further studied by using DNA markers to determine the species of the parasite. C. hominis was identified as the species infecting the patient's respiratory tract, a finding that strengthens evidence regarding this pathogen's role in human disease.

| S-EPMC2725888 | biostudies-literature

Comparative performance evaluation of four commercial multiplex real-time PCR assays for the detection of the diarrhoea-causing protozoa Cryptosporidium hominis/parvum, Giardia duodenalis and Entamoeba histolytica.

Project description:BackgroundMultiplex molecular panels are relentlessly replacing conventional methods for the detection of enteric pathogens from stool samples in clinical and research laboratories. Here we evaluated four commercial multiplex real-time PCR assays for the detection of Cryptosporidium hominis/parvum, Giardia duodenalis and Entamoeba histolytica.MethodsThe diagnostic performance of the Gastroenteritis/Parasite Panel I (Diagenode), the RIDAGENE Parasitic Stool Panel (R-Biopharm), the Allplex Gastrointestinal Parasite Panel 4 (Seegene) and the FTD Stool Parasites (Fast Track) real-time PCR methods was assessed against a reference panel of 126 well-characterized DNA samples including Cryptosporidium hominis (n = 29), Cryptosporidium parvum (n = 3), Giardia duodenalis (n = 47), Entamoeba histolytica (n = 3), other parasite species (n = 20), and apparently healthy subjects (n = 24).Principal findingsObtained diagnostic sensitivities ranged from 53-88% for Cryptosporidium hominis/parvum, and from 68-100% for G. duodenalis. The R-Biopharm method achieved the best performance for the detection of Cryptosporidium hominis/parvum both in terms of diagnostic sensitivity (87.5%) and detection limit (a 100-fold increase compared to other tests). The Fast Track method was particularly suited for the detection of G. duodenalis, achieving a 100% sensitivity and a detection limit at least 10-fold superior. Detection of E. histolytica was similarly achieved by all compared methods except Diagenode.ConclusionsDiagnostic performance varied largely depending on the method used and the targeted pathogen species. Factors including test sensitivity/specificity, cost, patient population surveyed, laboratory workflow, and diagnostic algorithm should be carefully considered when choosing the most appropriate multiplex PCR platform.

| S-EPMC6453453 | biostudies-literature

Dual probe DNA capture for sensitive real-time PCR detection of Cryptosporidium and Giardia.

Project description:Nucleic acid amplification for the enteropathogens Cryptosporidium and Giardia is complicated by low target template concentrations and PCR inhibitors. In this work we designed dual capture oligonucleotides for both Cryptosporidium and Giardia 18S rRNA targets which when utilized during DNA extraction from stool improved the limit of detection of our multiplex PCR assay by 1-2 logs, to as little as 10 cysts. When applied to clinical specimens, the method improved the real-time PCR C(T) by an average of 10.7 ± 9.7 cycles. This work provides a highly sensitive protocol for Cryptosporidium and Giardia when limit of detection is of utmost importance.

| S-EPMC3319247 | biostudies-literature

First case of type E wound botulism diagnosed using real-time PCR.

Project description:Wound botulism is a growing problem among injecting drug users. The condition is often difficult to diagnose, with laboratory confirmation in only 50% of the cases. Here we present a real-time PCR-based method for the diagnosis of wound botulism caused by Clostridium botulinum. The assay includes an internal amplification control which is amplified simultaneously with the genes encoding neurotoxin types A, B, and E. This method was used to detect the first case of wound botulism in an injecting drug user in Sweden. In addition, to the best of our knowledge, this is the first reported case of wound botulism caused by C. botulinum type E.

| S-EPMC2168498 | biostudies-literature

Intraspecies polymorphism of Cryptosporidium parvum revealed by PCR-restriction fragment length polymorphism (RFLP) and RFLP-single-strand conformational polymorphism analyses.

Project description:A glycoprotein (Cpgp40/15)-encoding gene of Cryptosporidium parvum was analyzed to reveal intraspecies polymorphism within C. parvum isolates. Forty-one isolates were collected from different geographical origins (Japan, Italy, and Nepal) and hosts (humans, calves, and a goat). These isolates were characterized by means of DNA sequencing, PCR-restriction fragment length polymorphism (PCR-RFLP), and RFLP-single-strand conformational polymorphism (RFLP-SSCP) analyses of the gene for Cpgp40/15. The sequence analysis indicated that there was DNA polymorphism between genotype I and II, as well as within genotype I, isolates. The DNA and amino acid sequence identities between genotypes I and II differed, depending on the isolates, ranging from 73.3 to 82.9% and 62.4 to 80.1%, respectively. Those among genotype I isolates differed, depending on the isolates, ranging from 69.0 to 85.4% and 54.8 to 79.2%, respectively. Because of the high resolution generated by PCR-RFLP and RFLP-SSCP, the isolates of genotype I could be subtyped as genotypes Ia1, Ia2, Ib, and Ie. The isolates of genotype II could be subtyped as genotypes IIa, IIb, and IIc. The isolates from calves, a goat, and one Japanese human were identified as genotype II. Within genotype II, the isolates from Japan were identified as genotype IIa, those from calves in Italy were identified as genotype IIb, and the goat isolate was identified as genotype IIc. All of the genotype I isolates were from humans. The Japanese isolate (code no. HJ3) and all of the Nepalese isolates were identified as genotypes Ia1 and Ia2, respectively. The Italian isolates were identified as genotype Ib, and the Japanese isolate (code no. HJ2) was identified as genotype Ie. Thus, the PCR-RFLP-SSCP analysis of this glycoprotein Cpgp40/15 gene generated a high resolution that has not been achieved by previous methods of genotypic differentiation of C. parvum.

| S-EPMC169079 | biostudies-literature

Real-time PCR for quantification of Giardia and Cryptosporidium in environmental water samples and sewage.

Project description:The protozoan pathogens Giardia lamblia and Cryptosporidium parvum are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of Giardia and Cryptosporidium are very low. Primers and TaqMan probes based on the beta-giardin gene of G. lamblia and the COWP gene of C. parvum were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of G. lamblia and one oocyst of C. parvum. A multiplex real-time PCR (qPCR) assay for simultaneous detection of G. lamblia and C. parvum resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify G. lamblia in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of G. lamblia in the sewage. No Cryptosporidium was detected in these samples by either method.

| S-EPMC194944 | biostudies-literature

Detection of viable Cryptosporidium parvum in soil by reverse transcription-real-time PCR targeting hsp70 mRNA.

Project description:Extraction of high-quality mRNA from Cryptosporidium parvum is a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive procedure for Cryptosporidium detection in soil samples. The efficiencies of five RNA extraction methods were compared (mRNA extraction with the Dynabeads mRNA Direct kit after chemical and physical sample treatments, and total RNA extraction methods using the FastRNA Pro Soil-Direct, PowerSoil Total RNA, E.Z.N.A. soil RNA, and Norgen soil RNA purification kits) for the direct detection of Cryptosporidium with oocyst-spiked sandy, loamy, and clay soils by using TaqMan reverse transcription-PCR. The study also evaluated the presence of inhibitors by synthesis and incorporation of an internal positive control (IPC) RNA into reverse transcription amplifications, used different facilitators (bovine serum albumin, yeast RNA, salmon DNA, skim milk powder, casein, polyvinylpyrrolidone, sodium hexametaphosphate, and Salmonella enterica serovar Typhi) to mitigate RNA binding on soil components, and applied various treatments (β-mercaptoethanol and bead beating) to inactivate RNase and ensure the complete lysis of oocysts. The results of spiking studies showed that Salmonella cells most efficiently relieved binding of RNA. With the inclusion of Salmonella during extraction, the most efficient mRNA method was Dynabeads, with a detection limit of 6 × 10(2) oocysts g(-1) of sandy soil. The most efficient total RNA method was PowerSoil, with detection limits of 1.5 × 10(2), 1.5 × 10(3), and 1.5 × 10(4) C. parvum oocysts g(-1) soil for sandy, loamy, and clay samples, respectively.

| S-EPMC3187154 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data