Laboratory diagnosis of Mycoplasma pneumoniae respiratory tract infections in children

Abstract

Mycoplasma pneumoniae is a common respiratory tractpathogen that causes up to 40% of cases of community-acquired pneumonia in children [1]. A specific diagnosis isessential because treatment of M. pneumoniae infection withβ-lactam antibiotics is ineffective. In routine laboratories,serology remains an important diagnostic tool [2, 3].However, it can only provide a retrospective diagnosis andpaired samples are required. Recently developed PCRtechniques show high levels of specificity and sensitivity forthe rapid detection of M. pneumoniae in clinical specimens[4, 5]; however, PCR alone is not always sufficient for adiagnosis [3]. In lieu of a gold-standard diagnostic method,we aimed to evaluate the laboratory methods currently usedto diagnose M. pneumoniae infection in order to find the onemost suitable for rapidly diagnosing the illness, especially inthe early phase of disease.During a 15-month period, a throat swab (viscose swab)andfirstserumspecimenweretakenonadmissionfromatotalof 75 children (42 males, 33 females; mean age 6.2 years;range 4 months–14 years) who were hospitalized fortreatment of a respiratory tract infection (RTI). According tothe guidelines of the British Thoracic Society, 53 of thesechildren had pneumonia, 16 pharyngitis and 6 tracheobron-chitis. A second serum specimen was obtained 9–24 dayslater.ThethroatspecimenswereexaminedforthepresenceofM. pneumoniae using culture in methylene blue–glucosediphasic medium (Oxoid, Basingstoke, UK), a sandwichenzyme-immunoassay for antigen detection (EIA-Ag Virion/Serion, Germany) and a PCR technique for DNA detection(extracted DNA was amplified by the primer pair P1-1 andP1-3 for the P1 adhesin gene [6]). Antibodies against M.pneumoniae were measured using the immunofluorescenceassay (IFA) for IgG and IgM (Vircell, Granada, Spain),enzyme-linked immunosorbent assay (ELISA) for IgG(Platelia BioRad, Marnes-la-Coquette, France) and IgA(Virotech, Russelsheim, Germany), and capture-ELISA forIgM (Platelia BioRad). Toconfirm thespecificityofIgMandIgAantibodydetection,theWesternblottechnique(Virotech)wasadditionallyperformed.Thismethodiscurrentlythemostspecific for detecting anti-M. pneumoniae antibodies [3]. ForIgM, a positive result requires ≥9 bands including P1protein; for IgA, >24 bands are required including P1protein. In the present study a current or recent infectionwas considered to have occurred definitely if at least two ofthe following criteria were met: (1) positive PCR and/orculture, (2) positive IgM antibodies (in the first and/or thesecond sample), (3) seroconversion or significant increase ofIgG antibodies (fourfold increase for IFA, twofold increasefor ELISA), or high IgG titers >40 AU/ml for ELISA.M. pneumoniae was detected by PCR in the throat-swabspecimens of 11 of 75 patients, by culture in three and bythe antigen detection test in only one. All culture-positivesamples were also positive by PCR. The complete resultsobtained using the various methods for the samples of eachof the 75 children studied are shown in Table 1.According to the diagnostic criteria, a definite diagnosisof current or recent M. pneumoniae infection was obtainedfor a total of 12 of the 75 pediatric patients (Table 1; patients1–12; ten with pneumonia and two with pharyngitis). Four