Kathleen A. Thurman

Evaluation of Three Real-Time PCR Assays for Detection of Mycoplasma pneumoniae in an Outbreak Investigation

ABSTRACT We compared the performances of three recently optimized real-time PCR assays derived from distinct genomic regions of Mycoplasma pneumoniae during an outbreak. Comprehensive evaluation established that a newly described toxin gene represents a superior target for detecting M. pneumoniae DNA in clinical specimens, although use of multiple targets may increase testing confidence.

Comparison of Laboratory Diagnostic Procedures for Detection of Mycoplasma pneumoniae in Community Outbreaks

BACKGROUND Mycoplasma pneumoniae continues to be a significant cause of community-acquired pneumonia (CAP). A more definitive methodology for reliable detection of M. pneumoniae is needed to identify outbreaks and to prevent potentially fatal extrapulmonary complications. METHODS We analyzed 2 outbreaks of CAP due to M. pneumoniae. Nasopharyngeal and/or oropharyngeal swab specimens and serum samples were obtained from persons with clinically defined cases, household contacts, and asymptomatic individuals. Real-time polymerase chain reaction (PCR) for M. pneumoniae was performed on all swab specimens, and the diagnostic utility was compared with that of 2 commercially available serologic test kits. RESULTS For cases, 21% yielded positive results with real-time PCR, whereas 81% and 54% yielded positive results with the immunoglobulin M and immunoglobulin G/immunoglobulin M serologic tests, respectively. For noncases, 1.8% yielded positive results with real-time PCR, whereas 63% and 79% yielded serologically positive results with the immunoglobulin M and immunoglobulin G/immunoglobulin M kits, respectively. The sensitivity of real-time PCR decreased as the duration between symptom onset and sample collection increased, with a peak sensitivity of 48% at 0-21 days. A specificity of 43% for the immunoglobulin M antibody detection assay was observed for persons aged 10-18 years, but the sensitivity increased to 82% for persons aged 19 years. DISCUSSION Thorough data analysis indicated that no single available test was reliable for the identification of an outbreak of CAP due to M. pneumoniae. A combination of testing methodologies proved to be the most reliable approach for identification of outbreaks of CAP due to M. pneumoniae, especially in the absence of other suspected respiratory pathogens.

Detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella spp. in clinical specimens using a single-tube multiplex real-time PCR assay

Abstract A multiplex real-time PCR assay for the detection of Mycoplasma pneumoniae (MP181), Chlamydia (Chlamydophila) pneumoniae (CP-Arg), Legionella spp. (Pan-Leg), and the human RNase P (RNase P) gene was developed for rapid testing of atypical bacterial respiratory pathogens in clinical specimens. This method uses 4 distinct hydrolysis probes to detect 3 leading causes of community-acquired pneumonia. The assay was evaluated for specificity and sensitivity by testing against 35 related organisms, a dilution series of each specific target and 197 clinical specimens. Specificity testing demonstrated no cross-reactivity. A comparison to previously validated singleplex real-time PCR assays for each agent was also performed. The analytical sensitivity for specific pathogen targets in both the singleplex and multiplex was identical (50 fg), while efficiencies ranged from 82% to 97% for the singleplex assays and from 90% to 100% for the multiplex assay. The clinical sensitivity of the multiplex assay was improved for the Pan-Leg and CP-Arg targets when compared to the singleplex. The MP181 assay displayed equivalent performance. This multiplex assay provides an overall improvement in the diagnostic capability for these agents by demonstrating a sensitive, high-throughput and rapid method. This procedure may allow for a practical and efficient means to test respiratory clinical specimens for atypical pneumonia agents in health care settings and facilitate an appropriate public health response to outbreaks.

Community Outbreak of Mycoplasma pneumoniae Infection: School-Based Cluster of Neurologic Disease Associated with Household Transmission of Respiratory Illness

BACKGROUND We investigated an outbreak of severe neurologic disease and pneumonia that occurred among students at 4 schools in Rhode Island. METHODS We identified cases of encephalitis, encephalomyelitis, and pneumonia that occurred among schoolchildren from 1 September 2006 through 9 February 2007, and we performed serologic tests, polymerase chain reaction (PCR) analysis, and culture for the detection of multiple pathogens in oropharyngeal and nasopharyngeal specimens. Students with positive results of M. pneumoniae IgM serologic testing and no alternative diagnosis were considered to be infected with M. pneumoniae. At school A, we used questionnaires to identify students and their household contacts who made visits to physicians for pneumonia and cough. We compared observed and expected rates of pneumonia. RESULTS Rates of pneumonia among elementary students (122 cases/1000 student-years) were > 5-fold higher than expected. Three students had encephalitis or encephalomyelitis, and 76 had pneumonia. Of these 2 groups of students, 2 (66%) and 57 students (75%), respectively, had M. pneumoniae infection. M. pneumoniae was detected by PCR in 10 students with pneumonia; 5 of these specimens were cultured, and M. pneumoniae was isolated in 4. Of 202 households of students attending school A, 20 (10%) accounted for 61% of visits to physicians for pneumonia or cough. Of 19 household contacts of students with pneumonia, 8 (42%) developed pneumonia and 6 (32%) reported visits for cough. CONCLUSIONS M. pneumoniae caused a community-wide outbreak of cough illness and pneumonia and was associated with the development of life-threatening neurologic disease. Although M. pneumoniae was detected in schools, its transmission in households amplified the outbreak. Interrupting household transmission should be a priority during future outbreaks.

Genotyping of Mycoplasma pneumoniae isolates using real-time PCR and high-resolution melt analysis

Mycoplasma pneumoniae is an important respiratory pathogen, accounting for up to 25% of community-acquired pneumonia, and is a common cause of hospitalized pneumonia in otherwise healthy adults and children. Mycoplasma pneumoniae isolates can be classified into two main genomic groups (type 1 and type 2) based on sequence variation within the gene encoding the major adhesion molecule P1. Although numerous publications have described real-time PCR assays for the detection of M. pneumoniae, none has been able to discriminate the two genomic types. Here, a real-time PCR assay that can distinguish each type of M. pneumoniae utilizing high-resolution melt-curve analysis is reported. Using this method, 102 isolates obtained from patients from 1965 to the present, including those from recent outbreaks, were typed along with reference strains M129 (type 1) and FH (type 2). The results show that 55 isolates (54%) can be classified as type 1 and 47 isolates (46%) as type 2, and 100% correlation was demonstrated when compared with a standard PCR-restriction fragment length polymorphism typing procedure. Typing of isolates obtained from recent outbreaks in the USA has revealed the presence of both types. This assay provides a rapid, reliable and convenient method for typing M. pneumoniae isolates and may be useful for surveillance purposes and epidemiological investigations, and may provide insight into the biology of M. pneumoniae distribution within populations.

Identification of P1 Variants of Mycoplasma pneumoniae by Use of High-Resolution Melt Analysis

ABSTRACT Mycoplasma pneumoniae is a leading cause of community-acquired pneumonia. Although two genetically distinct types of M. pneumoniae are known, variants of each also exist. We used a real-time PCR high-resolution melt genotyping assay to identify clinical variants which may provide greater insight into the genetic distribution of M. pneumoniae strains.

Evaluation of two real-time PCR chemistries for the detection of Chlamydophila pneumoniae in clinical specimens.

Chlamydophila pneumoniae is an atypical bacterial respiratory pathogen that is responsible for approximately 3-10% of community-acquired pneumonia cases. We report the evaluation of two distinct real-time PCR assays for rapid and specific detection of C. pneumoniae. We tested 401 clinical specimens, finding 5.7% positive, and confirmed a localized outbreak.

Investigation of a Chlamydia pneumoniae Outbreak in a Federal Correctional Facility in Texas

BACKGROUND Chlamydia pneumoniae illness is poorly characterized, particularly as a sole causative pathogen. We investigated a C. pneumoniae outbreak at a federal correctional facility. METHODS We identified inmates with acute respiratory illness (ARI) from 1 November 2009 to 24 February 2010 through clinic self-referral and active case finding. We tested oropharyngeal and/or nasopharyngeal swabs for C. pneumoniae by real-time polymerase chain reaction (qPCR) and serum samples by microimmunofluorescence. Cases were inmates with ARI and radiologically confirmed pneumonia, positive qPCR, or serological evidence of recent infection. Swabs from 7 acutely ill inmates were tested for 18 respiratory pathogens using qPCR TaqMan Array Cards (TACs). Follow-up swabs from case patients were collected for up to 8 weeks. RESULTS Among 33 self-referred and 226 randomly selected inmates, 52 (20.1%) met the case definition; pneumonia was confirmed in 4 by radiology only, in 9 by qPCR only, in 17 by serology only, and in 22 by both qPCR and serology. The prison attack rate was 10.4% (95% confidence interval, 7.0%-13.8%). White inmates and residents of housing unit Y were at highest risk. TAC testing detected C. pneumoniae in 4 (57%) inmates; no other causative pathogens were identified. Among 40 inmates followed prospectively, C. pneumoniae was detected for up to 8 weeks. Thirteen (52%) of 25 inmates treated with azithromycin continued to be qPCR positive >2 weeks after treatment. CONCLUSIONS Chlamydia pneumoniae was the causative pathogen of this outbreak. Higher risk among certain groups suggests that social interaction contributed to transmission. Persistence of C. pneumoniae in the oropharynx creates challenges for outbreak control measures.

Improved Detection of Respiratory Pathogens by Use of High-Quality Sputum with TaqMan Array Card Technology.

ABSTRACT New diagnostic platforms often use nasopharyngeal or oropharyngeal (NP/OP) swabs for pathogen detection for patients hospitalized with community-acquired pneumonia (CAP). We applied multipathogen testing to high-quality sputum specimens to determine if more pathogens can be identified relative to NP/OP swabs. Children (<18 years old) and adults hospitalized with CAP were enrolled over 2.5 years through the Etiology of Pneumonia in the Community (EPIC) study. NP/OP specimens with matching high-quality sputum (defined as ≤10 epithelial cells/low-power field [lpf] and ≥25 white blood cells/lpf or a quality score [q-score] definition of 2+) were tested by TaqMan array card (TAC), a multipathogen real-time PCR detection platform. Among 236 patients with matched specimens, a higher proportion of sputum specimens had ≥1 pathogen detected compared with NP/OP specimens in children (93% versus 68%; P < 0.0001) and adults (88% versus 61%; P < 0.0001); for each pathogen targeted, crossing threshold (CT) values were earlier in sputum. Both bacterial (361 versus 294) and viral detections (245 versus 140) were more common in sputum versus NP/OP specimens, respectively, in both children and adults. When available, high-quality sputum may be useful for testing in hospitalized CAP patients.

Comparison of nucleic acid extraction methods for the detection of Mycoplasma pneumoniae.

Four nucleic acid extraction procedures (2 automated and 2 manual) were compared for their efficiency at isolating Mycoplasma pneumoniae DNA. Oropharyngeal swabs from healthy volunteers were spiked with varying amounts of M. pneumoniae, extracted, and tested using real-time polymerase chain reaction. Our data indicate that both automated extraction methods consistently outperform the manual procedures.