Center for Emerging Infectious Diseases Univ. of Iowa College of Public Health

1. BD ProbeTec™ ET System for the Detection of Mycoplasma pneumoniae from Clinical Respiratory Specimens Center for Emerging Infectious Diseases Univ. of Iowa College of Public Health 200 Hawkins Dr., C21K GH Iowa City, IA 52245 Tel (319) 384-5008 Fax (319) 384-5004 www.public-health.uiowa.edu/CEID Gregory C. Gray1, Lynn B. Duffy2, Charlotte A. Gaydos3, Christine C. Ginocchio4, William LeBar5, Judy Lovchik6, Margaret R . Hammerschlag7, Tony Mazzulli8, Richard Rothman3 1Center for Emerging Infectious Diseases, University of Iowa College of Public Health, Iowa City, IA; 2University of Alabama at Birmingham, Birmingham, AL; 3Johns Hopkins University, Baltimore, MD; 4North Shore Long Island Jewish Health System Laboratories, Lake Success, NY; 5Hospital Consolidated Laboratories – Providence Hospital, Southfield, MI; 6University of Maryland Medical Center, Baltimore, MD; 7SUNY Downstate Medical Center, Brooklyn, NY; 8Mount Sinai Hospital Department of Microbiology, Toronto, Ontario Revised Abstract Table 2. BD ProbeTec ET lower respiratory MP assay vs. culture • Study purpose: To evaluate the BD ProbeTec ET Mycoplasma pneumoniae Amplified DNA Assay*, which is part of a group of assays for atypical pneumonia including Legionella pneumophila, and Chlamydiaceae family* (*Not for sale in the U.S., these assays are under FDA review). Background:Mycoplasma pneumoniae (MP) is a frequent cause of acute respiratory infection, especially pneumonia, and may lead to severe morbidity and death. In some populations, MP may cause as much as 20% of community acquired pneumonia (CAP). MP infections are difficult to detect; culture and serology lack sensitivity, while molecular techniques are often complex. We evaluated a new Strand Displacement Amplification (SDA) assay on the BD ProbeTec™ ET System (BD Diagnostics) for the detection of MPamong patients with CAP. Methods: Patients one year old and greater with radiographic evidence of pneumonia were enrolled at 7 medical sites between October 2002 and July 2003. Lower respiratory specimens (LRS) and throat swabs (TS) were collected from patients who granted informed consent. Specimens were tested for evidence of MPby culture, PCR, and the BD ProbeTec ET MP Assay. The University of Alabama at Birmingham’s mycoplasma laboratory performed MP culture of throat swabs and PCR procedures using previously published techniques. The MP Assay employs simultaneous amplification and real-time detection of target DNA and an internal control using amplification primers and fluorescently labeled detector probes. LRS and TS panels spiked with high and low levels of MP were tested in the MP Assay for reproducibility at 3 sites. Results: During the nine-month study, 5 (1.6%) of 316 enrolled patients had some evidence of MPinfection. The specificity of the MP Assay compared to culture was 99% for both LRS and TS. Specimens that were discrepant (MP Assay vs. culture) were further studied with PCR. All 3 MP Assay positive but culture negative specimens (one LRS and 2 TS) were positive for MP by PCR. Two specimens (LRS and TS from the same patient) that were MP Assay negative but culture positive were both found to be PCR negative. The LRS reproducibility with spiked samples was 92% for the low level and 100% for the high level and negatives. The TS reproducibility with spiked samples was 100% for all three levels. Conclusions: These data suggest that the MP Assay has excellent specificity, reproducibility, and good correlation with PCR for detection of MP. Materials & Methods *=PCR negative (patient H1013) **PCR positive (patient T021) • Subjects: Patients ≥ 1 year of age with signs and radiographic evidence of pneumonia were enrolled from 7 medical sites; patients with antibiotic therapy <14 days • Specimens: After informed consent, patients permitted the collection of: • Lower respiratory specimen (LRS) - sputum (expectorated, induced, endotracheal suction or collected during bronchoscopy) • Throat swabs (TS) • Sera (patients 5-13 years only) – acute sera only • Laboratory methods: • All patients: LRS andTS evaluated with BD ProbeTec™ ET MP assay. TS collected and expressed in SP4 medium for MP culture and MP PCR (University of Alabama) • Children 5-13 years: Serological IgM test for MP Table 3. BD ProbeTec ET throat swab MP assay vs. culture *=PCR negative (H1013) **PCR positive (patients I022 and N106) Table 4. BD ProbeTec ET MP lower respiratory assay - reproducibility Results • A total of 5 (1.6%) of the 316 enrolled pneumonia patients had evidence of MP infection by BD ProbeTec™ ET MP assay • The specificity of BD ProbeTec™ET MP assay was 99% for LRS and TS • Discrepant (BD ProbeTec™ET vs. culture) specimens were MP PCR positive. • Two specimens (LRS and TS from the same patient) that were BD ProbeTec™ET negative but culture positive were all found to be PCR negative. • The BD ProbeTec™ET LRS overall reproducibility was 92% for the low level, 100% for the high level and negatives. • The BD ProbeTec™ET TS overall reproducibility was 100% for all levels. Introduction *One sample excluded from analysis due to an operator pipetting error. • The BD ProbeTec™ ET System uses a proprietary real time molecular amplification technology, Strand Displacement Amplification (SDA), in a closed reagent system that does not require unidirectional workflow or separate work areas. • Using simple, dried down, ready-to-use reagents and smooth workflow, the system assay time is 1 hour for up to 94 specimens. • In the United States, this system has three FDA cleared assays: • Chlamydia trachomatis (CT) assay • Chlamydia trachomatis/ Neisseria gonorrhoeae (CT/GC) assay • Legionella pneumophila (LP) assay Table 5. BD ProbeTec ET MP throat swab - reproducibility Table 1. Data from specimens that had at least some laboratory evidence of MP *One sample excluded from analysis due to an operator pipetting error. • Mycoplasma pneumonia (MP): • Frequent cause of community acquired pneumonia • Treatable but difficult to detect through currently available rapid tests, culture, and serology Conclusions These data suggest that the BD ProbeTec ET MP Assay has excellent specificity, excellent reproducibility, and may be as sensitive as PCR in detecting MP. MP: Mycoplasma pneumoniae TS: Throat swab LRS: Lower respiratory specimen