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Although Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. are prevalent causes of community-acquired pneumonia, rapid and sensitive diagnosis is difficult. Real-time PCR provides rapid and sensitive diagnosis, however, DNA extraction is still required, which is time-consuming, costly and includes a risk of contamination. Therefore, we aimed to develop triplex real-time PCR without DNA extraction. AmpDirect(R) Plus which inhibits PCR inhibitors was used as the PCR buffer. Melting temperatures of the PCR products for the three bacteria were analyzed by SYBR green triplex real-time PCR and were found to be significantly different. Detection limits of bacteria cells diluted in nasopharyngeal aspirates (NPAs) were comparable with the detection limits of previously reported real-time PCR. Our PCR without DNA extraction and probe real-time PCR with DNA extraction showed identical results for the detection of the three bacteria from 38 respiratory specimens (sputum, endotracheal aspirates, and NPAs) collected from patients with pneumonia. No cross-reaction with other bacteria was observed. Our triplex real-time PCR successfully detected and differentiated the three bacteria. Although further field tests are required, our assay is a promising method for the rapid and cost-effective detection of the three bacteria.

Background: Mycoplasma pneumoniae is one of the most common causes of childhood community-acquired pneumonia (CAP), particularly in school-age children. Information regarding this infection in pre-school age children is lacking., Objective: To determine the prevalence of M. pneumoniae in young children aged under 5 years with CAP., Material and Method: This prospective study was conducted at Queen Sirikit National Institute of Child Health (QSNICH), Bangkok, Thailand between December 2001 and November 2002. We enrolled children aged 2 to 5 years with a clinical and radiological diagnosis of CAP. Acute and convalescent sera were collected and measured by using a particle agglutination test. Polymerase chain reaction (PCR) assay for M. pneumoniae was detected from nasopharyngeal secretions. Criteria for diagnosis were defined as > or = 4-found rising of mycoplasma antibody or titer > or = 1:160 with positive PCR., Results: Thirteen out of 113 CAP patients were diagnosed as mycoplasma pneumonia. Three of them were diagnosed by > or = 4-fold rising of mycoplasma antibody while another 10 patients were diagnosed by mycoplasma titer > or = 1:160 with positive PCR for M. pneumoniae. Clinical symptoms and signs of these 13 mycoplasma pneumonia in young patients were fever (85%), cough (92%), dyspnea (85%), diarrhea (15%), rales (85%), wheezing or rhonchi (46%), and skin rash (15%). Leucocytosis (wbc > 15,000/cumm) was found in 46%. Chest x-rays revealed interstitial infiltration (71%), patchy infiltration (29%) and no pleural effusion was detected. Choices of antibiotic were erythromycin (31%), beta lactam antibiotics (61%), and antibiotic was not prescribed in one patient (8%). Sixty-nine percent of the patients improved, while 31% did not, possibly due to the use of beta lactam antibiotics, or non use of antibiotics., Conclusion: Mycopalsma pneumonia is not uncommon in children aged 2-5 years with CAP. Clinical signs, symptoms and radiological findings are non-specific and cannot be differentiated from other causes of CAP.

Background: The prevalence of community-acquired pneumonia (CAP) caused by atypical pathogens in Thai children is unknown., Objective: To examine the prevalence of Mycoplasma pneumoniae and Chlamydophila pneumoniae infections in paediatric patients (aged 2-15 years) with CAP in three academic hospitals using standardised laboratory techniques. The characteristics of atypical pneumonia were also compared with other causes of CAP., Methods: Diagnosis of current infection was based on a four-fold or more rise in antibody serum samples or persistently high antibody titres together with the presence of mycoplasmal or chlamydial DNA in secretions., Results: Of 245 patients with CAP, 17.5% of cases were caused by atypical pathogens (M. pneumoniae 14.3%, C. pneumoniae 2.8% and co-infection 0.4%). We also found atypical pathogens in young children aged 2-5 years. The clinical and laboratory findings did not distinguish atypical pneumonia from other CAPs. Segmental or lobar consolidation on chest X-rays was more common in atypical pneumonia, while dyspnoea was more prominent in other CAPs., Conclusion: Our data show a high prevalence of M. pneumoniae and C. pneumoniae in Thai children with CAP, including in children aged 2-5 years.

Objective: To determine the prevalence and clinical features of mycoplasma pneumoniae in Thai children with community acquired pneumonia (CAP)., Material and Method: Diagnosis of current infection was based on > or = 4 fold rise in antibody sera or persistently high antibody titers together with the presence of mycoplasma DNA in respiratory secretion. The clinical features were compared between children who tested positive for M pneumoniae, and those whose results were negative., Results: Current infection due to M. pneumoniae was diagnosed in 36 (15%) of 245 children with paired sera. The sensitivity and specificity of polymerase chain reaction (PCR) in diagnosing current infection in the present study were 78% and 98% respectively. The mean age of children with mycoplasma pneumoniae was higher than CAP with unspecified etiology. The presenting manifestations and initial laboratory finding were insufficient to predict mycoplasma pneumoniae precisely, the presence of chest pain and lobar consolidation on chest X-ray, however, were significant findings in children with mycoplasma pneumoniae., Conclusion: The present study confirms that M. pneumoniae plays a significant role in CAP in children of all ages. Children with this infection should be identified in order to administer the appropriate antibiotic treatment.