Abstract
The aim of this study was to improve detection of Mycoplasma pneumoniae and Chlamydia pneumoniae in clinical specimens by developing a multiplex real-time PCR assay that includes identification of macrolide-resistant M. pneumoniae. Novel assays targeting a M. pneumoniae conserved hypothetical protein gene, M. pneumoniae 23S rRNA gene mutations associated with macrolide resistance and human β-globin gene (an endogenous internal control) were designed and combined with a previously published C. pneumoniae PCR targeting ompA gene. The resulting quadraplex PCR was validated with a panel of clinical specimens supplemented with external quality assessment specimens, simulated specimens and various bacterial and viral strains. The obtained results were compared to those obtained by reference PCRs or confirmed by sequencing (typing of macrolide resistance). The novel multiplex PCR assay was in 100 % agreement with reference PCRs. Four M. pneumoniae strains with macrolide resistance-associated mutations were identified among 42 strains, which comprises 9.5 % of the study material. Amplification of an internal control excluded sample-derived inhibition possibly leading to false-negative reporting. In conclusion, we have developed a resources conserving multiplex real-time PCR assay for simultaneous detection of M. pneumoniae, C. pneumoniae and the most common mutations leading to macrolide resistance in M. pneumoniae. The assay is a widely useful tool for detection of these respiratory pathogens and will also shed light on the occurrence of macrolide resistance in M. pneumoniae.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1457-x) contains supplementary material, which is available to authorized users.
Highlights
Mycoplasma pneumoniae and Chlamydia pneumoniae are common respiratory pathogens causing approximately 10–30 % and 5–10 %, respectively, of community acquired pneumonias (Atkinson et al 2008; Kuo et al 1995)
The aim of this study was to improve detection of M. pneumoniae and C. pneumoniae in clinical specimens by developing a time-saving and inexpensive multiplex real-time PCR assay for detection of C. pneumoniae, M. pneumoniae and the most common mutations in the M. pneumoniae 23S rRNA gene leading to resistance for macrolide antibiotics
Sensitivity of co‐amplification To assess the sensitivity of co-amplification of C. pneumoniae, M. pneumoniae and human beta-globin by multiplex real-time PCR, a range of concentrations of M. pneumoniae M129 and C. pneumoniae K-6 DNA were co-amplified
Summary
Mycoplasma pneumoniae and Chlamydia pneumoniae are common respiratory pathogens causing approximately 10–30 % and 5–10 %, respectively, of community acquired pneumonias (Atkinson et al 2008; Kuo et al 1995). Mycoplasma pneumoniae infection can occur in all age groups, but the severity of the disease tends to increase with the age of the patient. M. pneumoniae infection can lead to a severe pneumonia (Waites and Talkington 2004). Resistance to macrolides is caused by point mutations in the bacterial 23S rRNA gene which reduces the affinity of the antibiotic for the ribosomes (Wolff et al 2008). The most common point mutations leading to the resistance are located in the 23S rRNA gene at positions 2063 and 2064, and include transition A to G or C (Waites and Talkington 2004; Lucier et al 1995)
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