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Evaluation of the Film Array Respiratory Panel for Detection of Mycoplasma pneumoniae and Respiratory Viruses in Multiple Specimen Types Title Here Christine Robinson1, Kristin Pretty1, Daniel Olson2, and Samuel Dominguez2 1,2 and University of Colorado22, Departments of Pathology and Laboratory Medicine11 and Pediatrics22 , Children’s Hospital Colorado1,2 Anschutz Medical Campus, CO BACKGROUND RESULTS ORGANISM AND SIGNIFCANCE FILM ARRAY RESP. PANEL - TESTING OF MULTIPLE SPECIMEN TYPES FOR VIRUS A. Many Specimen Types Testable B. Validation of FA-RP vs. Luminex RVP 2000 Film Array Pos Film Array Neg Film Array RP 1000 500 0 NPS M. pneumoniae Scanning electron micrograph. Hospitalized child with severe SJS skin and eye involvement NPW FDACleared TA BAL Various Lab-Validated Other A. Mp Detected in Many Specimen Types 20 NPS 45 NPW 40 TA 40 BAL Luminex RVP* Luminex RVP** Luminex RVP** Luminex RVP** Pos Neg 1500 FILM ARRAY RESP. PANEL - TESTING OF MULTIPLE SPECIMEN TYPES FOR MP Total Pos Neg Total Pos Neg Total Pos Neg Total Pos 24 1 25 28 6 34 20 0 20 20 0 20 Neg 1 454 455 1 640 641 0 580 580 0 580 580 Total 25 455 480 29 646 675 20 646 600 20 580 Discrepancy Missed 1 AdV Detected 1 add'l RV FA missed 1 AdV None None Sensitivity 96% 98.70% 100% 100% Specificity 99.80% 99.10% 100% 100% Agreement 99.50% 98.90% 100% 100% 600 Film Array RP Specimen Types FDA Cleared Lab-Validated * IVD version **RUO version OUTBREAK An outbreak of Mp-associated respiratory disease and unusual cluster of Mpassociated SJS was identified in Denver-area children in late 2013, prompting an on-site investigation by CDC. Most of these infections were detected by the Film Array Respiratory Panel (FA RP, Biofire Diagnostics), an FDA-cleared PCR for 17 respiratory viruses and 3 bacteria in nasopharyngeal swabs (NPS). Specimens other than NPS were Mp-positive, although we had not yet validated reporting of bacteria from this test. Other cases were detected by Mp PCR of TS at Focus Diagnostics Laboratory. Total Tested Use of FA for respiratory virus detection begins in early 2013; bacteria to be validated later. If Mp detected, specimen sent to Focus for confirmation before reporting. Mp detection increases significantly in autumn. Mp-Positive Specimens by FA 20 9 741 1.2% NW 64 3421 1.9% BAL 10 441 2.3% TA 2 222 0.1% Other 0 104 0.0% Total 76 * 4188 1.8% 85 * 4929 1.7% Same Specimen as FA Different Specimen as FA Pos Neg Total Pos Neg Total Pos 79 4 83 16 2 18 Neg 0 118 118 0 85 85 Total 79 122 201 16 87 103 Sensitivity 100% 100.00% Specificity 96.70% 97.70% Agreement 98.00% 98% C. Agreement between CDC and Focus Mp PCRs BAL NW Film Array System 5 OBJECTIVES Jan-14 Dec-13 Nov-13 Oct-13 Sep-13 Jul-13 Aug-13 Jun-13 May-13 Apr-13 Mar-13 Jan-13 NPS Feb-13 0 Collection Date B. Association of MP with SJS Link between SJS and Mp observed. CDC investigates on-site; verifies community-wide Mp outbreak and association of Mp with some SJS cases. 9 SJS Cases & Mp Association: Jan 2012 – Nov 2013 Pneumonia due to Mp. ICD9 Defined Cases Oct 2008-Oct 2013 4 8 Mycoplasma PCR not obtained Number of cases 7 6 5 Mp – ANALYTICAL VALIDATION: In progress NPS 15 10 4 3 3 negative Mycoplasma PCR positive Mycoplasma PCR CONCLUSIONS 2 1 Specimens other than NPS can be tested for viruses by Film Array RP and resulted with confidence 2 0 1 OCT13 JUL13 APR13 JAN13 JUL12 OCT12 JAN12 APR12 OCT11 JUL11 APR11 JAN11 OCT10 JUL10 JAN10 APR10 JUL09 OCT09 APR09 JAN09 0 OCT08 Mp – CLINICAL VALIDATION: 288 banked or fresh respiratory specimens positive or negative for Mp by FA were sent to the CDC’s Mycoplasma Laboratory only (19 pos), Focus Reference Laboratory (16 pos, 203 neg), or both laboratories (50 pos) for Mp PCR. Mp strain typing by MLVA and resistance testing was performed at CDC. Pos Rate 88% agreement when the same 50 FA-positive specimens tested by both laboratories 83% positive agreement when different FA-positive specimens from the same patient tested CDC PCR identified 4 Mp-positive specimens resulted as Mp negative at Focus; Focus detected no additional positives . All FA Results; Feb 2013-Feb 2014 Rhinovirus/enterovirus RESPIRATORY VIRUS VALIDATION: Banked or fresh NPW, TA, and BAL positive by Luminex Respiratory Virus Panel (LUM, IUO version), donated LUM-positive NPS, and negative specimens spiked with virus were combined into 2-specimen pools and tested by FA v1.6. The number of expected and observed analytes for each pool was scored. Tested A. Film Array RP Detects Outbreak Bacteria METHODS Mp Pos MYCOPLASMA PNEUMONIAE Influenza A, H1, H1 2009, H3 Mycoplasma pneumoniae Influenza B Chlamydophila pneumoniae RSV Bordetella pertussis Human metapneumovirus Adenovirus Parainfluenza 1-4 Coronaviruses 229E, OC43, HKU1, NL63 Describe a clinical validation of FA for detection of respiratory viruses and Mp in multiple specimen types including NPS, nasopharyngeal washes (NPW), tracheal aspirates (TA), and bronchoalveolar lavage (BAL), using specimens collected during a Mp outbreak. Confirmatory Mp PCR ( CDC and/or Focus) * 89% of Mp FA positives were in lab-validated specimens Organisms in FA Respiratory Panel Viruses B. Validation of FA-RP Mp PCR vs. Other PCRs Film Array RP Mycoplasma pneumoniae (Mp) is a bacterial cause of community-acquired pneumonia and occasional extra-pulmonary disease in school-age children and young adults. Most cases are mild but some are hospitalized. Stevens Johnson Syndrome (SJS), a rare but serious disorder involving skin and mucous membranes usually triggered by medication, can also be induced by Mp. Circulation usually is endemic, but outbreaks can occur. Throat swabs (TS) are the usual specimen sent to detect Mp by PCR. RESULTS Admit date 7 Mp-associated probable SJS cases identified Aug-Nov 2013; 5 considered confirmed. All had severe disease; several had recurrent disease. 3 different strains of Mp were circulating; no single strain was identified in SJS patients All 4 Mp from SJS patients were macrolide sensitive; 7% of 45 Mp from non-SJS patients and contacts were resistant. Detection of bacteria compared to viruses in respiratory tract specimens of children is low An outbreak of Mp tract disease identified by Mp PCR of respiratory tract specimens occurred in late 2013 in Denver area children. Some cases were associated with Stevens-Johnson Disease Clinical detection of M. pneumoniae by Film Array RP was validated in multiple specimen types