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New real-time PCR detects prolonged norovirus excretion in highly immunosuppressed patients and children.

ABSTRACT: Noroviruses (NoV) are a major cause of epidemic nonbacterial gastroenteritis and affect all age groups worldwide. Three of five NoV genogroups, namely, genogroup I (GI), GII, and GIV, are associated with human disease. Unfortunately, these genogroups demonstrate a high degree of sequence diversity, complicating the design of pan-NoV diagnostic PCR tests. To decrease the risk of false-negative test results, we have developed a new one-step real-time TaqMan reverse transcription-PCR protocol. This protocol detects all human NoV genogroups in one reaction with a sensitivity of 400 virus genome equivalents/reaction for both GI and GII. The use of in vitro-transcribed NoV RNA as an external standard allows (semi)quantification of viral loads in samples. In a retrospective analysis of 206 stool samples from 77 patient episodes, the duration of NoV excretion and the amount of virus excreted were determined. Twenty (26.0%) of these episodes lasted longer than 10 days. Univariate risk factor analysis revealed the patient status after organ transplantation (odds ratio [OR], 7.49 [95% confidence interval, 2.06 to 28.32]; P < 0.001), immunosuppression (OR, 9.19 [95% confidence interval, 2.50 to 35.39]; P < 0.001), and age of less than 10 years (OR, 4.58 [95% confidence interval, 1.36 to 15.77]; P = 0.004) as risk factors for a NoV excretion period of more than 10 days. These findings were confirmed by time-dependent Kaplan-Meier analyses, whereas multivariate Cox regression analyses identified immunosuppression as the sole risk factor. Surprisingly, in contrast to the excretion periods, the viral loads in stools did not increase in connection with age or immunosuppressive status. This fact may be one important piece in the pattern of high-level NoV transmissibility and may have an impact on the development of transmission prevention strategies.

SUBMITTER: Henke-Gendo C

PROVIDER: S-EPMC2738087 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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New real-time PCR detects prolonged norovirus excretion in highly immunosuppressed patients and children.

Henke-Gendo C C Harste G G Juergens-Saathoff B B Mattner F F Deppe H H Heim A A

Journal of clinical microbiology 20090722 9

Noroviruses (NoV) are a major cause of epidemic nonbacterial gastroenteritis and affect all age groups worldwide. Three of five NoV genogroups, namely, genogroup I (GI), GII, and GIV, are associated with human disease. Unfortunately, these genogroups demonstrate a high degree of sequence diversity, complicating the design of pan-NoV diagnostic PCR tests. To decrease the risk of false-negative test results, we have developed a new one-step real-time TaqMan reverse transcription-PCR protocol. This ...[more]

PMID: 19625473

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Project description:BACKGROUND: Coxiella burnetii, the bacterium causing Q fever, is an obligate intracellular biosafety level 3 agent. Detection and quantification of these bacteria with conventional methods is time consuming and dangerous. During the last years, several PCR based diagnostic assays were developed to detect C. burnetii DNA in cell cultures and clinical samples. We developed and evaluated TaqMan-based real-time PCR assays that targeted the singular icd (isocitrate dehydrogenase) gene and the transposase of the IS1111a element present in multiple copies in the C. burnetii genome. RESULTS: To evaluate the precision of the icd and IS1111 real-time PCR assays, we performed different PCR runs with independent DNA dilutions of the C. burnetii Nine Mile RSA493 strain. The results showed very low variability, indicating efficient reproducibility of both assays. Using probit analysis, we determined that the minimal number of genome equivalents per reaction that could be detected with a 95% probability was 10 for the icd marker and 6.5 for the IS marker. Plasmid standards with cloned icd and IS1111 fragments were used to establish standard curves which were linear over a range from 10 to 10(7) starting plasmid copy numbers. We were able to quantify cell numbers of a diluted, heat-inactivated Coxiella isolate with a detection limit of 17 C. burnetii particles per reaction. Real-time PCR targeting both markers was performed with DNA of 75 different C. burnetii isolates originating from all over the world. Using this approach, the number of IS1111 elements in the genome of the Nine Mile strain was determined to be 23, close to 20, the number revealed by genome sequencing. In other isolates, the number of IS1111 elements varied widely (between seven and 110) and seemed to be very high in some isolates. CONCLUSION: We validated TaqMan-based real-time PCR assays targeting the icd and IS1111 markers of C. burnetii. The assays were shown to be specific, highly sensitive and efficiently reproducible. Cell numbers in dilutions of a C. burnetii isolate were reliably quantified. PCR quantification suggested a high variability of the number of IS1111 elements in different C. burnetii isolates, which may be useful for further phylogenetic studies.

Dataset Information

New real-time PCR detects prolonged norovirus excretion in highly immunosuppressed patients and children.

Publications

New real-time PCR detects prolonged norovirus excretion in highly immunosuppressed patients and children.

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