RESUMO
Background & objectives: Mycoplasma pneumoniae is the most important and common cause of community-acquired pneumonia (CAP). The conventional detection methods (culture and serology) lack sensitivity. PCR offers a better approach for rapid detection but is prone to carry over contamination during manipulation of amplification products. Quantitative real-time PCR (qRT-PCR) method offers an attractive alternative detection method. In the present study, qRT-PCR, PCR and serology methods were used to detect M. pneumoniae infection in cases of pneumonias and findings compared. Methods: A total of 134 samples consisting of blood (for serology) and respiratory secretions (for PCR and qRT-PCR) from 134 patients were collected. The blood samples were tested for IgG, IgM and IgA using commercially available kits. For standardization of PCR of M. pneumoniae P1 gene was cloned in pGEMTEasy vector. Specific primers and reporter sequence were designed and procured for this fragment. The qRT-PCR assay was performed to prepare the standard curve for M. pneumoniae positive control DNA template and detection in patient samples. Results: Of the 134 patients, 26 (19%) were positive for antibodies against M. pneumoniae. IgG was positive in 14.92 per cent (20) cases, IgM in 4.47 per cent (6) and IgA was positive in 5.22 per cent (7) cases. In the qRT-PCR assay 19 per cent (26) samples were positive. Of the 26 qRT-PCR positive samples, nine could be detected by serology. PCR was positive for 25 samples. An extra sample negative by PCR was detected by qRT-PCR. Thus, real-time PCR assay, PCR and serology in combination could detect M. pneumoniae infection in 43 patients. Interpretation & conclusions: The study shows that 17 patients were detected by serology alone, 17 were detected by qRT-PCR only and nine patients were positive by both serology and real-time PCR. Of the 134 samples tested, 25 were positive by conventional PCR, but qRT-PCR could detect one more sample that was negative by PCR and serology. These results suggest that a combination of two or three methods may be required for reliable identification of CAP due to M. pneumoniae.
RESUMO
Background & objectives: Legionella pneumophila has been increasingly recognized as an emerging pathogen responsible for community acquired pneumonia (CAP) worldwide. In India, the actual burden is not known. The present study was thus undertaken to see the presence of Legionella infection in patients with community acquired pneumonia admitted in a tertiary care centre in north India. Methods: Both children and adults (n=113) with symptoms of pneumonia were included in the study. Clinical samples (blood, urine, nasopharyngeal aspirates, bronchoalveolar lavage, sputum, etc.) were collected and subjected to culture and other tests. Enzyme linked immunosorbent assay (ELISA) was done by commercial kits for all the three classes of immunoglobulins (IgG, IgM & IgA). Urinary antigen was also detected using commercial kits. Culture was performed on 51 respiratory tract fluid samples. Serum samples of 44 healthy controls were also screened for the presence of anti-legionella antibodies (IgG, IgM & IgA). Results: Thirty one of the 113 cases (27.43%) were serologically positive. Anti-legionella IgG, IgM and IgA antibodies were positive in 7.96, 15.92 and 11.50 per cent patients respectively. In controls, seropositivity was 9.09 (4/44). IgA was positive in 3 and IgM, IgG combined in one. Antigenuria detection by Microwell ELISA kit showed 17.69 per cent positivity. Four antigenuria positive patients were also serologically positive; of these two patients were positive for IgM, hence considered as confirmed cases of Legionella infection. None of the sample was culture positive. Interpretation & conclusions: Combination of serology and antigenuria detection may be a valuable tool for the diagnosis of Legionella infection in absence of culture positivity. In order to evaluate the actual burden of Legionella in community acquired pneumonia, further studies with larger samples need to be done.