Transcript respiratory virus validation

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