Diagnostic Utility and Clinical Significance of Naso- and Oropharyngeal Samples Used in a PCR Assay To Diagnose Mycoplasma pneumoniae Infection in Children with Community-Acquired Pneumonia

Abstract

PCR assays of naso-and oropharyngeal samples among hospitalized children appear equally effective for the diagnosis of serologically confirmed community-acquired mycoplasmal pneumonia. However, the combination of results from both sites yields optimal sensitivity (57%), specificity (98%), and positive (92%) and negative (82%) predictive values when compared with Mycoplasma pneumoniae enzyme-linked immunosorbent assay.Controversy exists over the role of PCR assays performed on upper respiratory tract (URT) samples for the diagnosis of mycoplasmal pneumonia. It has been suggested that the most sensitive and prompt method of diagnosis in children is a combination of URT PCR and acute-phase serum immunoglobulin M (IgM) enzyme immunoassay (4, 11). However, concerns exist about the potential inability to distinguish between URT carriage of M. pneumoniae versus active disease in children with PCR-positive URT specimens in the setting of pneumonia (6). Furthermore, the optimal site and method of sampling the URT for the detection of M. pneumoniae by PCR are not established in children.Patients. We report on 21 children with serologically proven M. pneumoniae infection and 42 approximately matched M. pneumoniae seronegative controls selected from 154 consecutively enrolled children hospitalized with clinically and radiologically confirmed lower respiratory infections, as described previously (8, 9). The Institutional Review Board of the University of Texas Southwestern Medical Center at Dallas approved the protocol.Laboratory detection of M. pneumoniae. M. pneumoniae enzyme-linked immunosorbent assays (ELISA) were performed by the Diagnostic Mycoplasma Laboratory at The University of Alabama at Birmingham. The assay was considered positive if the IgM was Ն1:10 or if there was Ն4-fold rise in IgG titer (1). Twenty-one of 154 children (13.6%) had serologically proven acute mycoplasmal pneumonia. Forty-two approximately matched M. pneumoniae seronegative control subjects were selected from the same study population.Both nasopharyngeal (Np) and oropharyngeal (Op) swabs were obtained within 24 h of admission by using dacron-tipped swabs (Fisher Scientific). Swab tips were placed in 200-l PCR buffer and stored at Ϫ70°C. Upon thawing, 25 l of protease solution was added, and samples were lysed in Buffer AL (QIAamp blood kit; QIAGEN GmbH) and sonicated at 37°C. Bacterial DNA was extracted by centrifuge columns (QIAGEN GmbH). Single-step DNA amplification was performed with a programmable thermal cycler (GeneAmp PCR system 9600; Perkin-Elmer Cetus, Norwalk, Conn.), based on a method modified from that of Williamson et al. (7,12 ). The 100-l reaction mixture consisted of 40 l of DNA eluent and 60 l of HotStarTaq DNA polymerase master mix (QIAGEN GmbH). The published primer sequences targeted the highly conserved region of the 16S rDNA gene of M. pneumoniae and amplified a unique 290-bp region (12). A 10-l volume of amplified product was separated by electrophoresis in polyacrylamide gel and visualized under UV light after ethidium...

Treatment of Mycoplasma Pneumonia: A Systematic Review

“…Furthermore, coinfection with a pathogen in addition to M. pneumoniae is common but rarely identified via clinical testing, making it difficult to draw specific conclusions about therapeutic effects. 9,38 Finally, no study included in our analysis took into account the duration of illness before the start of antibiotic therapy. It is possible that the timing of intervention relative to the start of symptoms is related to the outcome.…”

Pneumonia in healthy Canadian children and youth: Practice points for management

“…Haemophilus influenzae type b has almost disappeared because of vaccination. Mycoplasma pneumoniae and Chlamydophila pneumoniae are more common causes of pneumonia among school-age children, but they occasionally cause pneumonia in younger children (10,11).…”

Assessment of the Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of <i>Mycoplasma pneumoniae</i> in Pediatric Community-Acquired Pneumonia

“…At present, several methods are available for the definitive diagnosis of M. pneumoniae infections, including culture, serology, and nucleic acid amplification techniques. Detection of M. pneumoniae by polymerase chain reaction (PCR) as a rapid, sensitive, and specific method has been reported by many authors (3)(4)(5). The loop-mediated isothermal amplification (LAMP) assay, a novel nucleic acid amplification method, for the rapid detection of M. pneumoniae was recently developed, and it has been demonstrated to have nearly the same sensitivity and specificity for detecting M. pneumoniae as PCR (6).…”

“…1) 7,400 ± 2,700 2) 6,200 ± 2,300 3) 10,300 ± 4,600 2), 3) º0.01 Serum C-reactive protein on admission (mg/dL) 1) 2.5 ± 2.4 2.5 ± 1.3 2.5 ± 2.6 0.39…”

“…GAS (8), SP (5), HI (4), RSV (2), flu (1) RSV (3), HI (2), GAS (2), flu (1) -PA titer to M. pneumoniae on admission 1) 2 4.0 ± 2.7 2 3.6 ± 2.8 2 2.6 ± 1.8 0.053 PA titer to M. pneumoniae at discharge 1) 2 8.6 ± 1.9 2) 2 8.0 ± 1.8 3) 2 3.0 ± 1.8 2), 3) º0.01…”