Our results show that M. pneumoniae is an important pathogen of adolescent and adult CAP patients, consistent with the findings of the CAPNETZ report in 2009 . In the present study, we also demonstrate that, compared with non-mycoplasma infections, CAP patients infected with M. pneumoniae are younger, less likely to have comorbidities, and have longer duration of fever before admission.
After infection by M. pneumoniae, IgM antibodies appear during the first week of the illness, and reach peak titers during the third week. Martínez et al. have found that the sensitivity of IgM assay was 33.3% in diagnosis of acute M. pneumoniae infection in adult CAP patients . In our study, however, the sensitivity of the IgM assay is only 7.4% compared with a fourfold increase of IgG titers. In some adult patients, the antibodies are constantly negative or produced 15 days after onset as a result of multiple previous infections [17, 18]. Therefore, using the IgM assay as the sole test may provide inadequate information.
When compared with PCR, the sensitivity of culture from respiratory specimens has turned out to be as low as 61.5% . Similarly, She et al. reported that culture was unacceptably insensitive for diagnosing M. pneumoniae infection . In our study, however, there was no difference between the positive rates of the two methods, just as Waris et al. found in children . Furthermore, the values of diagnostic parameters, including specificity, sensitivity, positive and negative predictive values and positive likelihood ratio, are the highest among the three methods evaluated here. In particular, a PLR of 10.9 means that there is a reasonable correlation between culture results and true infection. However, the prolonged turnaround time limits the clinical application of culture.
Compared with serology and culture, PCR (especially real-time PCR) is more rapid, practicable and sensitive, and has been suggested to be more suitable for early diagnosis of acute M. pneumoniae infection [21, 22]. Touati et al. have evaluated five commercial kits based on PCR, and found that the sensitivities of the kits were 62–98% . Similarly, Martínez et al. observed the sensitivity, specificity, PPV and NPV of the PCR to be 66.7%, 98.5%, 78.3% and 97.3%, respectively . In the present study, however, the sensitivity, specificity, PPV and NPV of the FQ-PCR kit targeted to 16 S rRNA gene were 40.7%, 88.9%, 50% and 84.6%, respectively. The low sensitivity of PCR testing may be due to the low bacterial load resulting from previous antimicrobial treatment or dilution of the sample in the throat swabs below the limit of detection. The detection limit of our FQ-PCR kit is stated as 10 copies per μl in the manual, which is lower than 0.1–1 copies per μl that other commercial kits reported [23, 24]. In addition, it was found that the efficiency of the PCR assay could be influenced by sample type, and sputum could be superior to other respiratory samples including throat swab .
Possible reasons contributing to discordant results between FQ-PCR or culture, and IgG serology have been explored. Four patients (one positive by FQ-PCR and culture, and three positive by FQ-PCR) might represent asymptomatic carriage of M. pneumoniae as a result of persistence from a previous infection, since these patients’ respective CAP etiologies had been identified with IFVA, hMPV, AdV and HRV. Five patients (three positive by FQ-PCR and culture, and two positive by FQ-PCR or culture and IgM test) showed 2.5–3.8 fold increase of IgG response, which could be explained by an inadequate time interval (mean interval was 17 days). The remaining three patients (only positive by FQ-PCR) were 69, 73 and 74 years old. They failed to develop IgG antibody response in paired sera, which could be interpreted as a deterioration of the immune response due to ageing . These cases highlight the limitations of a serological diagnosis, which could be affected by the timing of specimen collection and the age of the patient.
Recently, in China, Yin et al. demonstrated the good sensitivity and specificity of the JRS scoring system for the early presumptive diagnosis of M. pneumoniae pneumonia . In our cohort, we showed that with a fourfold or greater increase of IgG antibody titers of paired sera as the “gold standard”, the sensitivity and specificity of JRS scoring were 63% and 59.2%, respectively. With positivity either by PCR, culture, IgM, or a fourfold increase of IgG as the diagnosis for M. pneumoniae pneumonia, the sensitivity decreased to 61.9% and specificity increased to 74.7%.
The limitations of the study included: 1) because of cost consideration, we evaluated only one FQ-PCR kit approved by State Food and Drug Administration of China; and 2) in patients who had received antibiotics, including macrolides and fluoroquinolones, the positive rates of culture and FQ-PCR may have been decreased.