Rapid real-time PCR assay for detection of A2063G mutation in macrolide-resistant Mycoplasma pneumoniae isolates

Introduction: \nMycoplasma pneumoniae (M. pneumoniae) has been a major cause of community-acquired pneumonia (CAP), accounting for about 10-30% of the cases. Previously, a local study revealed that more than 60% of clinical isolates of M. pneumoniae exerted A2063G mutation, which confers a high level of macrolide drug resistance and results in treatment failure. While A2063G is the only mutation identified locally, a rapid diagnostic assay for detection of this single point mutation is urgently needed for switching the drug of choice. \n \nAims: \nThis study aims to develop a rapid PCR assay for detection of A2063G mutation of M. pneumoniae isolates for our locality, to compare with other commercially available assays and to further confirm the prevalence of A2063G mutation in macrolide-resistance M. pneumoniae (MRMP) in Hong Kong. \n \nMethods: \nA total of 110 respiratory tract samples were collected from 102 patients in Hong Kong Sanatorium and Hospital during April 2013 to April 2014. They were analyzed by an in-house hybridization-probe real-time PCR assay coupled with melting curve analysis to detect the presence of M. pneumoniae and the target A2063G point mutation. Results were compared with a commercial real-time PCR assay and the A2063G point mutation was further confirmed by 23S rRNA gene sequencing. The limit of detection (LOD), mutation threshold determination and cross reactivity of the in-house assay were also evaluated. \n \nResults: \nOver 40% (47/110) of the respiratory tract samples were tested positive for M. pneumoniae by the in-house assay and 36.2% (17/47) of the positive samples exerted A2063G mutation. The limit of detection was 500 copies/ml as evaluated using external quality control samples. Twenty well-characterized clinical isolates of M. pneumoniae were used to evaluate the A2063G mutation threshold. The mutation threshold for A2063G mutant detection was above 60%. This assay did not show any cross-reactivity with common clinical isolates from the respiratory tract samples. \n \nConclusion: \nIn this study, an in-house real-time PCR assay was evaluated and demonstrated its great potential as a rapid clinical diagnostic tool. The assay was highly sensitive and specific in detecting M. pneumoniae and its A2063G mutation from clinical samples in Hong Kong. The results were almost concordant to the current routine testing, with the advantage of lower cost and shorter turnaround time for rapid detection.