Title HereEvaluation of the Film Array Respiratory Panel for Detection of Mycoplasma pneumoniae and Respiratory Viruses in Multiple Specimen Types

Christine Robinson1, Kristin Pretty1, Daniel Olson2, and Samuel Dominguez2 Departments of Pathology and Laboratory Medicine1 and Pediatrics2 , Children’s Hospital Colorado1,2 and University of Colorado2,

Anschutz Medical Campus, CO

BACKGROUND

OBJECTIVES

METHODS

RESULTS

CONCLUSIONS

ORGANISM AND SIGNIFCANCE

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.

OUTBREAK

An outbreak of Mp-associated respiratory disease and unusual cluster of Mp- associated 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.

Hospitalized child with severe SJS skin and eye involvement

M. pneumoniae Scanning electron micrograph.

Organisms in FA Respiratory Panel

Viruses Bacteria

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

Rhinovirus/enterovirus

Film Array System

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.

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.

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.

Mp – ANALYTICAL VALIDATION: In progress

FILM ARRAY RESP. PANEL - TESTING OF MULTIPLE SPECIMEN TYPES FOR VIRUS

MYCOPLASMA PNEUMONIAE

SJS Cases & Mp Association: Jan 2012 – Nov 2013

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1

2

3

4Mycoplasma PCR not obtained

negative Mycoplasma PCR

positive Mycoplasma PCR

Admit date

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A. Film Array RP Detects Outbreak 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.

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.

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.

All FA Results; Feb 2013-Feb 2014

FILM ARRAY RESP. PANEL - TESTING OF MULTIPLE SPECIMEN TYPES FOR MP

RESULTS

Film Array RP

Specimen Types Mp Pos Tested Pos Rate

FDA Cleared NPS 9 741 1.2%

Lab-Validated

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%

Total Tested 85 * 4929 1.7%

* 89% of Mp FA positives were in lab-validated specimens

C. Agreement between CDC and Focus Mp PCRs

A. Mp Detected in Many Specimen Types

A. Many Specimen Types Testable B. Validation of FA-RP vs. Luminex RVP B. Validation of FA-RP Mp PCR vs. Other PCRs

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 .

Specimens other than NPS can be tested for viruses by Film Array RP and resulted with confidence

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

* IVD version **RUO version

Evaluation of the Film Array Respiratory Panel for Detection of Mycoplasma pneumoniae and Respiratory Viruses in Multiple Specimen Types

Christine Robinson1, Kristin Pretty1, Daniel Olson2, and Samuel Dominguez2 Departments of Pathology and Laboratory Medicine1 and Pediatrics2 , Children’s Hospital Colorado1,2 and University of Colorado2,

Anschutz Medical Campus, CO