Culture-Independent Detection and Genotyping of Mycoplasma pneumoniae in Clinical Specimens from Beijing, China.

Fei Zhao,Xiaoxia Tao,Jianzhong Zhang,Lihua He,Fanliang Meng,Liyong Liu,Qijing Zhang

doi:10.1371/journal.pone.0141702

Fei Zhao, Xiaoxia Tao + Show 5 more

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https://doi.org/10.1371/journal.pone.0141702

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Abstract

A duplex real-time PCR assay was designed for simultaneous detection and genotyping of Mycoplasma pneumoniae (M. pneumoniae). The detection/typing performance of this duplex PCR method, targeting specific genes for M. pneumoniae type 1 (mpn 459) and type 2 (mpna 5864), was compared to that of the previously published MpP1 real-time PCR assay and the genotyping method for the adhesin P1 gene (mpn 141). A total of 1,344 throat swab specimens collected from patients in Beijing, China were tested for M. pneumoniae by bacterial culture, MpP1 real-time PCR assay, and our duplex PCR assay, and positive detection rates of 26.9%, 34.4%, and 33.7%, respectively, were obtained. The duplex PCR method demonstrated high sensitivity and accuracy for detecting and genotyping M. pneumoniae, and significant differences in genotyping ability were observed when compared to the conventional P1 gene-based method. M. pneumoniae type 1 was the predominate genotype from 2008 to 2012 in Beijing, and a shift from type 1 to type 2 began to occur in 2013. To our knowledge, this is the first reported incidence of a type shift phenomenon of M. pneumoniae clinical isolates in China. These genotyping results provide important information for understanding recent changes in epidemiological characteristics of M. pneumoniae in Beijing.

Highlights

Mycoplasma pneumoniae (M. pneumoniae), a small prokaryote devoid of a cell wall, is one of the most common etiological agents of human respiratory tract infections
Based on the crossing threshold (CT) values for the serial 10-fold dilutions of DNA, the lowest detectable limit (LDL) of M. pneumoniae type 1 and type 2 were approximately 8.1 fg and 9.3 fg DNA, respectively (Table 2). Both these amounts are equivalent to approximately 3 colony forming units (CFU)
We demonstrated that our duplex real-time PCR assay can simultaneously detect and type M. pneumoniae directly from extracted DNA in clinical specimens, without cultivation of the pathogen

Summary

Introduction

Mycoplasma pneumoniae (M. pneumoniae), a small prokaryote devoid of a cell wall, is one of the most common etiological agents of human respiratory tract infections. About 10–40% of all cases of community acquired pneumonia can be attributed to M. pneumoniae, which occurs in epidemic peaks at intervals of 3–7 years [1,2,3]. Severe cases with extrapulmonary involvement can result in hospitalization and death due to neurological disease, such as encephalitis [4, 5]. Culture-Independent Typing of M. pneumoniae in China for timely diagnosis. While the culturing of M. pneumoniae is time-consuming (2–8 weeks) and unreliable, serological detections require the availability of paired sera exhibiting IgG antibodies, which is age- and time-dependent [6]. Especially real-time PCR methods, are currently used for rapid, sensitive, and specific detection of M. pneumoniae in clinical specimens [7,8,9,10]

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