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Detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella spp. in clinical specimens using a single-tube multiplex real-time PCR assay.

ABSTRACT: A multiplex real-time PCR assay for the detection of Mycoplasma pneumoniae (MP181), Chlamydia (Chlamydophila) pneumoniae (CP-Arg), Legionella spp. (Pan-Leg), and the human RNase P (RNase P) gene was developed for rapid testing of atypical bacterial respiratory pathogens in clinical specimens. This method uses 4 distinct hydrolysis probes to detect 3 leading causes of community-acquired pneumonia. The assay was evaluated for specificity and sensitivity by testing against 35 related organisms, a dilution series of each specific target and 197 clinical specimens. Specificity testing demonstrated no cross-reactivity. A comparison to previously validated singleplex real-time PCR assays for each agent was also performed. The analytical sensitivity for specific pathogen targets in both the singleplex and multiplex was identical (50 fg), while efficiencies ranged from 82% to 97% for the singleplex assays and from 90% to 100% for the multiplex assay. The clinical sensitivity of the multiplex assay was improved for the Pan-Leg and CP-Arg targets when compared to the singleplex. The MP181 assay displayed equivalent performance. This multiplex assay provides an overall improvement in the diagnostic capability for these agents by demonstrating a sensitive, high-throughput and rapid method. This procedure may allow for a practical and efficient means to test respiratory clinical specimens for atypical pneumonia agents in health care settings and facilitate an appropriate public health response to outbreaks.

SUBMITTER: Thurman KA

PROVIDER: S-EPMC7126442 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella spp. in clinical specimens using a single-tube multiplex real-time PCR assay.

Thurman Kathleen A KA Warner Agnes K AK Cowart Kelley C KC Benitez Alvaro J AJ Winchell Jonas M JM

Diagnostic microbiology and infectious disease 20110311 1

A multiplex real-time PCR assay for the detection of Mycoplasma pneumoniae (MP181), Chlamydia (Chlamydophila) pneumoniae (CP-Arg), Legionella spp. (Pan-Leg), and the human RNase P (RNase P) gene was developed for rapid testing of atypical bacterial respiratory pathogens in clinical specimens. This method uses 4 distinct hydrolysis probes to detect 3 leading causes of community-acquired pneumonia. The assay was evaluated for specificity and sensitivity by testing against 35 related organisms, a d ...[more]

PMID: 21397428

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Project description:BackgroundDiagnosis of community-acquired pneumonia (CAP) caused by Mycoplasma pneumoniae (Mp) in children has been hampered by difficulty in obtaining convalescent serum and time constraints. In this study, the two diagnostic assays that targeted respectively on Mp-antibody and Mp-DNA were retrospectively investigated.MethodsA total of 3146 children were clinically diagnosed to have CAP and were confirmed by chest X-ray during March 2015 to February 2016 in Children's hospital of Hebei Province (China). Both of the sera and sputum samples were collected in 24 h after their admission. The Mp-antibody was examined by the passive particle agglutination assay and a fourfold or greater increase of antibody titers of paired sera or≧1:160 titer of single serum was set as the serology positive. Mp-DNA in the sputum samples was tested by a multiplex-PCR method named GeXP assay (multiplex PCR combined with automated capillary electrophoresis). In order to eliminate the false positive results caused by the asymptomatic carriage after infected by M. pneumoniae, the inconsistent samples were tested by the real-time isothermal transcription-mediated RNA amplification assay (SAT).ResultsThe inter-rated agreement test was performed in 3146 CAP patients, with a highest kappa value in the school-age children as 0.783. There were 6.29% (198/3146) cases showed inconsistent results determined by GeXP and serology assay. All of the 19 GeXP(+)/Serology (-) samples and a randomly chosen 27 from 179 GeXP(-)/Serology (+) samples were tested by SAT assay, and a 97.8% diagnosis agreement was observed between SAT and GeXP assay, but not with the serology assay. In addition, patients who were detected only by serology or only by multiplex-PCR were significantly younger than those with both methods positive (3.0 and 1.5 years vs. 5.0 years, p < 0.01). The Viral-Mp coinfection accounted for 37.0% (97/262), which was more common in winter and spring (p < 0.05) and in the infantile group (p < 0.01), compared to the pure Mp positive ones.ConclusionIn some children CAP cases, the Mp laboratory diagnosis was inconsistent between serology and multiplex-PCR assay. Verified by the SAT assay, the GeXP showed a more sensitive and reliable performance compared with the serology assay. Furthermore, employing the multiplex-PCR could provide more information on the associated pathogens for clinical assessment of CAP.

Dataset Information

Detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella spp. in clinical specimens using a single-tube multiplex real-time PCR assay.

Publications

Detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella spp. in clinical specimens using a single-tube multiplex real-time PCR assay.

Similar Datasets

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