Development of a Genomics-Based PCR Assay for Detection of Mycoplasma pneumoniae in a Large Outbreak in New York State

Abstract

A genomics-based PCR method was developed and used to test specimens from patients involved in a large outbreak of Mycoplasma pneumoniae in a closed religious community in New York State. New P1 adhesin gene primers were designed to bind to 9 of 10 target sequences in the repetitive-element sequences obtained from the whole genome sequence of M. pneumoniae. This PCR method had a sensitivity of 0.006 CFU and a specificity of 100% for M. pneumoniae. The PCR was validated by testing a subset of patient samples by culture and comparing the results to those obtained by PCR. Of the initial 280 samples tested, 73 were positive by PCR and 22 were positive by culture. All samples positive by culture were also positive by PCR. Follow-up testing of selected patients 3 to 6 weeks after antibiotic treatment revealed that eight samples remained positive by PCR and that three samples remained positive by culture. Additionally, no nonspecific PCR inhibition was detected as a result of the specimen type, transport medium, or sample preparation methodology. The study demonstrates that the PCR described here is a rapid, sensitive, and specific method for the identification of M. pneumoniae and was helpful for the detection and monitoring of the outbreak.Mycoplasma pneumoniae is primarily a respiratory pathogen that is responsible for approximately 15 to 20% of all community-acquired pneumonias (8). The clinical presentation of patients with M. pneumoniae infection is not significantly different from that of patients with infections caused by other respiratory pathogens such as Chlamydia pneumoniae (22), so diagnosis of infection relies primarily on laboratory testing. M. pneumoniae is, however, a fastidious organism, requiring a laborious effort and 21 days or more for growth in culture. Likewise, serology is lacking in sensitivity, has questionable specificity (22), and is dependent on specific collection times relative to the onset of illness (5). DNA probes have also been used, but cross-reactivity between M. pneumoniae and Mycoplasma genitalium has been observed, and methods that use these probes also lack sensitivity (4).Numerous PCR approaches have been developed to provide a rapid, sensitive method for the detection of M. pneumoniae. PCR targets have included the P1 adhesin gene (3,4,5,12,17,21), the ATPase operon (11), genomic clones (2, 7), or a combination of these (1). The P1 adhesin gene is responsible for cytadherence, which is necessary for colonization and infection of host cells by M. pneumoniae (22). This gene is also an intriguing target for PCR because of its repetitive nature within the M. pneumoniae genome. Approximately 8% of this genome is composed of repetitive DNA elements with regions homologous to the P1 adhesin gene (10). Use of the entire genomic DNA sequence enabled us to design primers that would theoretically amplify the maximum number of target molecules, resulting in an improved sensitivity of the PCR for M. pneumoniae.A large outbreak of respiratory illness occurred in a closed religious communit...

Molecular Diagnosis of Mycoplasma pneumoniae Respiratory Tract Infections

“…Specimens suitable for the detection of M. pneumoniae include sputum (74) and bronchoalveolar lavage (BAL) specimens (40,66), nasopharyngeal and throat swabs (25,78), nasopharyngeal aspirates (20,28,36), tracheal aspirates (1,26), pleural fluid specimens (52), and transthoracic needle aspirations (17). More unusual specimens, such as nasal polyps (29), open-lung biopsies, and autopsy specimens (65), have also been tested.…”

“…Waring et al studied a large outbreak of M. pneumoniae in a closed religious community in New York State (78). Throat swab specimens were collected and processed for culture and PCR.…”

Portación faríngea de Mycoplasma pneumoniae en niños chilenos

“…También se ha demostrado portación de M. pneumoniae en adultos; Gnarpe y cols la demostraron en 4,6% de adultos sanos durante un período de baja incidencia del microorganismo y 13,5% de portación durante un período epidémi-co 15 . Waring y cols comunicaron 15% de portación en un brote de infecciones causadas por M.pneumoniae que afectó a una comunidad religiosa cerrada en Nueva York 16 . La portación faríngea de M. pneumoniae puede representar la persistencia de la bacteria luego de una infección sintomática.…”

Comparison of P1 and 16S rRNA genes for detection of Mycoplasma pneumoniae by nested PCR in adults in Zhejiang, China

“…The P1 adhesin gene is an attractive target for PCR because it repeats up to 10 times within the M. pneumoniae genome, which increases the sensitivity of PCR assay [16]. Another important target is 16S rRNA, or rather rDNA.…”

“…The P1 adhesin gene is an intriguing target gene for PCR because of its repetitive nature within the genome [14]. About 8% of the M. pneumoniae genome consists of repetitive DNA elements with regions homologous to the P1 adhesin gene, thus allowing an increase in the sensitivity of a PCR assay [16]. The 16S rRNA gene is also an attractive candidate as a target due to its unique organization and the presence of conserved and variable regions on its abundant high-copy number [14,17].…”