Charlotte A. Gaydos, MS, MPH, Dr PH Professor Division of Infectious Diseases Johns Hopkins University Baltimore, Maryland, USA

How epidemiology and diagnostics drive public policy--

lessons from influenza

Public Health Practice Grand Rounds Johns Hopkins Bloomberg School of Public Health

Objectives

1. Requirements for rapid point-of-care (POC) tests for influenza in real time

2. Limitations of some Rapid POC tests

3. Overview of some new POC tests

4. Overview of a new rapid PLEX-ID technology which can identify and genotype influenza samples for just- in- time surveillance needs

Requirements for rapid point-of-care (POC) tests for influenza in real time

1. Sensitive

2. Specific

3. Fast

4. Simple

5. Inexpensive

ASSURED: WHO recommendations:

a. Affordable b. Sensitive c. Specific d. User Friendly e. Rapid and Robust f. Equipment-Free g. Deliverable to End-Users

Current Types of Tests for Influenza and Respiratory Viruses

Rapid Tests insensitive, non-specific DFA rapid, less sensitive, specific Shell Vial culture sensitive Roller Tube culture sensitive, slow Real time qPCR/RT-PCR sensitive, slow Pyrosequencing expensive Flow through microsphere array rapid, expensive

Rapid Flu Dx Test Influenza A Type % Positive (#+/# tested) Binax NOW Influenza A&B Novel H1N1 40% (18/45) Seasonal H1N1 60 % (3/5) H3N2 80% (12/15) Directigen EZ Flu A+B Novel H1N1 49% (21/43) Seasonal H1N1 75% (3/4) H3N2 83% (10/12) QuickVue A+B Novel H1N1 69% (31/45) Seasonal H1N1 80% (4/5) H3N2 80% (12/15)

Positivity Rates for 3 Rapid Influenza Diagnostic Tests

CDC, MMWR August 17, 2009

Performance of the QuickVue Rapid Test for Influenza

Faix D et al. N Engl J Med 2009;10.1056/NEJMc0904264

NHRC Influenza Testing: 4/20/01 – 5/30/09

3066 Total Specimens Processed

Confirmed S-OIV 273 (8.9%)

Seasonal H1N1 18 (0.61%)

Seasonal H3N2 31 (2.0%)

QuickVue Rapid Test (Quidel) results available for 767 patients

Influenza Type #Pos by QuickVue #Pos by PCR

Sensitivity 95% CI

S-OIV 20/39 52% 35% - 67%

Seasonal H1N1 12/19 63% 39% - 82%

Seasonal H2N3 6/19 31% 14% - 57%

Specificity of the test, compared to RT-PCR, was 99% in all cases

Rapid Influenza Diagnostic Test Sensitivity Specificity Binax NOW Influenza A&B 38.3% 100% BD Directigen EZ Flu A+B 46.7% 100% QuickVue Influenza A+B 53.3% 100%

Rapid Antigen Test for Diagnosis of Pandemic (Swine) Influenza A/H1N1 (N=84)

Vasoo et al. CID 2009;49:1090-1093

Influenza Diagnostic Test Sensitivity Specificity Rapid Ag Test 21.2% 99.5% (BinexNOW or 3M Rapid Detection) DFA 47.2% 99.6% R-Mix 98.49% 100%

Evaluation of Multiple test methods for Detection of Novel 2009 H1N1 in NYC

(N=1831)

Ginocchio et al. J Clin Vir 2009;45:191-195

H1N1pdm 2009 H3N2v 2011-12

Influenza: Always a challenge; always changing

•Influenza A (H3N2) variant (H3N2v) virus infection was were detected in the United States during July 2011–July 2012

H3N2 influenza A with H1N1pdm09 M (matrix) gene • In 2012, 306 cases of H3N2v infections were reported to CDC from 13 states (12 in 2011 in 5)

IN (138 cases), OH (106), WS (20), MD (12), PA (11), MI (6), IL & MN (4), WV (3), HI & UT (1) ~93% occurred in persons aged <18 years, 16 hospitalized, 1 death

New influenza H3N2v

•All reported direct or indirect exposure to swine at fairs

New variant: Minnesota Health: reports that the number of H1N2v cases has increased to 4

• H3N2v viruses can be detected by qualified U.S. public health laboratories using the CDC Flu rRT-PCR Dx Panel. Initially, if specimens tested positive for influenza A, H3, and pandemic influenza A markers and negative for H1 and pandemic H1 markers, they were reported as inconclusive.

•On August 7, CDC updated the results interpretation of the CDC Flu rRT-PCR Dx Panel for H3N2v for public health laboratories. Specimens with these findings may now be reported as “presumptive positive for influenza A (H3N2)v virus” and, for the ongoing investigations, cases with presumptive-positive test results at the state or local public health laboratory will now be classified as confirmed, as are those cases confirmed at CDC. •The CDC Flu rRT-PCR Dx Panel is available in public health laboratories but is not a point-of-care test available to clinicians

New influenza H3N2v

Evaluation of seven FDA-cleared RIDTs for the ability to detect H3N2v viral antigens — CDC, 2012

RIDT (manufacturer)

Name Approved Specimens

Analyzer interpret

BinaxNOW Influenza A&B (Alere)

BinaxNOW NP swab Nasal wash/ aspirate/swab

No

Directigen EZ Flu A+B (Becton-Dickinson

Directigen NP wash/Aspirate /swab throat swab

No

SAS FluAlert A&B (SA Scientific)

FluAlert Nasal wash/aspirate No

QuickVue Influenza A+B Test (Quidel)

QuickVue NP swab Nasal wash /aspirate/swab

No

Sofia Influenza A+B (Quidel)

Sofia NP aspirate/swab/wash Nasal wash

Required

BD Veritor System for Rapid Detection of Flu A+B (Becton) Dickinson)

Veritor NP swab/nasal swab Required

Xpect Flu A&B (Remel)

Xpect Nasal wash/swab Throat swab

No

Number of 10-fold virus dilutions (maximum = five) detected by seven FDA-cleared RIDTs, by H3N2v strain designation — CDC, 2012

Strain TCID50/ml

Binax NOW

Direct-ogen

Flu Alert

Quick View

Sofia Veritor Xpect

A/Kansas /12/2009

104.5 1 4 U U

2

4

4

A/Pennslylv 14/2012

104.5 2 4 U 2 2

4

3

A/Minnesota /11/2012

104.5 U 3 U U

3

3

2

A/Indiana /08/2011

106.0 1 3 U U

2

3

2

A/Indiana /10//2011

104.0 U 3 U U

2

4

2

A/West VA /06/2011

106.0 2 3 U 2 4

4

2

A/Iowa /07/2011

104.5 2 4 1 1 3

4

3

•The fact that a negative RIDT result should not be considered as conclusive evidence of lack of infection with influenza A (H3N2)v •Results from RIDTs, both positive and negative, always should be interpreted in the broader context of the circulating influenza strains present in the area, level of clinical suspicion, severity of illness, and risk for complications in a patient with suspected infection • Clinicians should minimize the occurrence of false RIDT results by strictly following the manufacturer’s instructions, collecting specimens within the first 72 hours of illness, and confirming RIDT results by sending a specimen to a public health laboratory, who will send to CDC

Additional CDC guidance available at http://www.cdc.gov/flu/swineflu/h3n2v-testing.htm.

CDC Recommendations

Two new amplified flu POC tests

GeneXpert® Flu, Cepheid

FilmArray, Idaho Technology

GeneXpert® Flu No Requirement for dedicated PCR area

CLIA Moderate Complexity

3

• Results in ~ 1 hr • POC, random access

Flocked swab or Nasal Asp/wash

3. Binding reagent into port 1

Xpert® Flu Assay Result Interpretation

• Xpert Flu provides display of: – Flu A Positive – Flu A Positive with

2009 H1N1 Detected or

– Flu B Positive • Reporting of 2009

H1N1 requires presence of both Flu A and 2009 H1N1 targets

Xpert® Flu Assay Published Data

Denver Children’s Hospital: Xpert® Flu is a highly

accurate. No prior molecular training

required. Clinically actionable

Prodesse ProFlu+

Sensitivity Specificity

Xpert Flu (Flu A)

99.1% 100% Xpert Flu

(Flu B) 100% 100%

Xpert Flu (2009 H1N1)

97.9% 99.5%

University of Virginia:

Xpert® Flu is a rapid, robust, sensitive, highly specific and user-friendly test

Reduced complexity allows for 24hr test availability

Prodesse ProFlu+

Sensitivity Specificity

Xpert Flu (Flu A)

97.3% 100% Xpert Flu

(Flu B) 94.4% 100%

Xpert Flu (2009 H1N1)

100% 100%

Influenza A Influenza A Influenza B

Influenza B

Sensitivity Specificity Sensitivity Specificity Xpert Flu 97.3% 100% 100% 100% Direct Fluorescent Antibody

95.9%

99.2%

100%

100%

Binax NOW 62.2% 100% 54.5% 100% BD Directogen

71.6% 100% 48.5% 100%

Gen Xpert Fu amplified assay compared to rapid influenza tests

*Sensitivity and specificity were calculated using rRT-PCR as the comparator DiMaio et al. J of Vir Meth, 2012

Film Array

Virus No. of specimens Sens % Spec % PRO-/FA+ PRO+/FA+ P RO+/FA- PRO-/FA+ Adenovirus 181 6 5 0 54.5 100 Influenza A (H1-2009) 191 1 0 0 100 100 Influenza B 191 1 0 0 100 100 Metapneumo 180 12 0 0 100 100 Parainflu 1 187 3 0 2 100 98.9 Parainflu 3 179 10 0 3 100 98.4 RSV 181 10 0 1 100 99.5 Prodesse assays were considered the gold standard for comparison. Viruses targeted by both FilmArray RP and Prodesse assays, but not detected during this study: influenza A H1 seasonal virus, influenza A H3 seasonal virus, and parainfluenza virus 2 PRO, Prodesse; FA, FilmArray Respiratory Panel

Loeffelholtz et al. JCM 49:4083-4088, 2012

Comparison of positive and negative results by FilmArray RP and Prodesse assays for viruses

detectable by both assays

vSensitivity and specificity of the FilmArray RP (FA) and xTAG RVP (xTAG) versus PCR-

confirmed results Virus No. detecte Film Assay Sens % Spec % FA+, xTAG+ FA+, xTAG- FA-, xTAG+ FA-, xTAG- Influ A 32 0 1 167 97 100 Influ B 7 0 0 193 100 100 RSV 37 8 0 155 100 100 Parainflu 15 1 0 184 100 100 Rhino/Entero 39 4 2 155 95.6 100 Adenovirus 9 0 1 190 90 99.5 Metapneumo 6 1 0 193 100 100

Rand et al. JCM 49:2449-2453, 2011

Comparison of the FilmArray System to Real-Time PCR for Detection of Respiratory Pathogens in Children •215 frozen archived pediatric respiratory specimens previously characterized as either negative or positive for one or more pathogens by real-time PCR were examined using the FilmArray RP •Agreement between FilmArray RP and real-time PCR assays for shared analytes was 98.6% (kappa0.92 ) The combined positive percent agreement was 89.4%; the negative percent agreement was 99.6% Positive % agreement Negative % Agreement •Influenza A virus H1 100 100 Influenza A virus H3 88.2 100 Influenza A virus 2009 H1 94.1 100 Influenza B virus 88.2 100

Pierce et al. JCM 50:364-371, 2012

Recent Reviews

Chartrand et al. Annals Inter Med 156:500-511, 2012

Vijayan et al. Emerg Infect Dis 18:1414-1421, 2012

Kumar at al. Clin Microbiol Rev 25:344-361, 2012

Evaluation of Sofia fluorescent immunoassay for

Influenza A/B virus

Lee et al. J Clin Vir 2012 (in press)

Sofia FIA displayed sensitivities of 82.2% and 77.8% for strains A and B respectively, compared to the RT-PCR

Sensitivities of BinaxNOW A/B antigen kit, and Directigen Flu A and B were 54.8%, and 68.5% for influenza A

Sensitivities of BinaxNOW A/B antigen kit, and Directigen Flu A and B were 62.5%, and 52.8% for influenza B

How Do We Do Surveillance?

Challenges for Influenza Typing and Surveillance for Respiratory Viruses

•*Challenge to have a surveillance tool that gives a rapid diagnosis, as well as a genotype, that can provide public health guidance for “just-in-time” diagnostics

•Epidemics happen quickly: 4wk H1N1pdm

•Called Type A influenza: “not H3” and “not H1”, thus “un-subtypeable”

*Metzgar, D. et al. J Clin Microbiol. 48:4228-4234., 2010

PLEX-ID as a New Surveillance Tool

• PLEX-ID Technology combines broad amplification with PCR, and electrospray ionization mass spectrometry

• Designed to provide broad identification, detailed genotyping and characterization, and recognition of known and emerging strains

• Designed to track a potential pandemic in real time

•Comprehensive sample tracking •Analysis of 300+ samples in 24 hours

•Simplified, easy to interpret results

Respiratory Virus Surveillance I Assay

• Influenza virus validation at Ibis • >650 blinded samples

–Nasal aspirates, nasal swabs, nasal washes, throat swabs, bronchial washes, tracheal aspirates

–Samples from 1999-2006, across United States

–Correctly identified all Influenza A types • 149 H3N2 • 34 H1N1

–67 Influenza B InfluenzaSensitivity 96.8%Specificity 97.5%

PPV 96.0%NPV 98.0%

Sampath. R., K.L. Russell, C. Massire, M.W. Eshoo, V. Harpin, L.B. Blyn, et al. Rapid Surveillance of the Global Spread of Emerging Influenza Virus Genotypes by Mass Spectrometry. PLoS ONE. May 2007, issue 5, e489.

Novel Influenza Virus: 2009 H1N1

Human Avian Swine

Before the H1N1 pandemic strain was added to the PLEX database, it appeared as a hybrid of several strains from different hosts – human, avian, and swine

CDC ID T5000 Inferred Subtype PB1 NP M1 PA NS1 NS2 Comparison to CDC ID

Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 35 26 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 - Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 35 26 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 - 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 - Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 - Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 36 25 27 24 36 35 20 28 34 29 16 26 Matched

Pandemic Influenza Samples from April-June 2009; Deyde et al. Plos One Oct 2010 5:e13293

CDC ID T5000 Inferred Subtype

PB1 NP M1 PA NS1 NS2 Comparison to CDC ID

Swine-H1N1 A/California/04/2009 38 33 25 32 35 21 20 25 24 28 24 29 35 26 28 23 35 35 21 28 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 38 33 25 32 35 21 20 25 23 29 25 28 35 26 28 23 35 35 21 28 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 38 33 25 32 35 21 20 25 24 28 24 29 35 26 28 23 35 36 21 27 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 23 29 25 28 35 26 28 23 35 36 21 27 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 38 33 25 32 35 21 20 25 22 30 25 28 35 26 28 23 35 35 21 28 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 38 33 25 32 35 21 20 25 23 29 25 28 35 26 28 23 35 36 21 27 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 38 33 25 32 35 21 20 25 23 29 25 28 35 26 28 23 35 36 21 27 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 38 33 25 32 34 22 21 25 24 28 24 29 35 26 28 23 35 35 21 28 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 37 34 25 32 35 21 20 25 23 29 25 28 - 35 36 21 27 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 37 34 25 32 35 21 20 25 24 28 24 29 - 35 36 21 27 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 37 34 25 32 35 21 20 25 24 28 24 29 - 35 36 21 27 34 29 16 26 Matched Swine-H1N1 A/California/04/2009 37 34 25 32 35 21 20 25 23 29 25 28 - 35 36 21 27 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 38 33 25 32 35 21 20 25 23 29 25 28 35 26 28 23 35 35 21 28 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 38 33 25 32 35 21 20 25 23 29 25 28 35 26 28 23 35 36 21 27 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 - 35 21 20 25 23 29 25 28 35 26 28 23 35 35 21 28 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 38 33 25 32 35 21 20 25 23 29 25 28 35 26 28 23 35 35 21 28 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 23 29 25 28 38 25 29 20 35 36 21 27 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 38 33 25 32 35 21 20 25 24 28 24 29 35 26 28 23 35 35 21 28 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 23 29 25 28 35 26 28 23 35 35 21 28 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 39 33 24 32 35 21 20 25 24 28 24 29 35 26 28 23 35 35 21 28 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 37 34 25 32 35 21 20 25 23 29 25 28 - 35 36 21 27 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 38 33 25 32 35 21 20 25 24 28 24 29 35 26 28 23 35 36 21 27 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 - 35 21 20 25 24 28 24 29 35 26 28 23 35 36 21 27 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 38 33 25 32 35 21 20 25 24 28 24 29 35 26 28 23 35 35 21 28 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 38 33 25 32 35 21 20 25 23 29 25 28 35 26 28 23 35 36 21 27 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 38 33 25 32 35 21 20 25 23 29 25 28 35 26 28 23 35 36 21 27 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 37 34 25 32 35 21 20 25 24 28 24 29 35 26 28 23 35 36 21 27 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 39 32 24 33 35 21 20 25 24 28 24 29 35 26 28 23 35 36 21 27 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 38 33 25 32 35 21 20 25 22 30 25 28 35 26 28 23 35 36 21 27 33 30 17 25 Matched Swine-H1N1 A/California/04/2009 38 33 25 32 35 21 20 25 24 28 24 29 35 26 28 23 35 35 21 28 33 30 17 25 Matched

Pandemic Influenza Samples Johns Hopkins University October-December 2009

• Know your Rapid POC test, its strengths and limitations

• Some POC tests may not work well with new strains of influenza

• Promising new POC tests are becoming available

• The PLEX-ID Influenza Surveillance Typing Assay can detect newly assorted and shifted or drifted strains (including avian strains such as H5 and other H and N types)

• Be aware of the potential of new influenza strains

Conclusions

Questions?

cgaydos@jhmi.edu 855 N Wolfe St 530 Rangos Bldg. Baltimore, MD 21205 PH 410-614-0932 FAX 410-614-9775

Acknowledgements Kuan-Fu Chen, MD

Faix D et al. N Engl J Med 2009;10.1056/NEJMc0904264

Results of RT-PCR Analyses of 3066 Specimens, According to Influenza A Subtype

vAV Hexon Type 1 300 BoV NP-1 Clinical Sample 4000 B. per Toxin A639 4,000 C. pne ompA TW183 3000 CoV 229E Polymerase VR-740 4 CoV HKU1 Nucleoprotein PCMC 6123 1.96 x 10^6 CoV OC43 Nucleoprotein VR-759 600 CoV NL63 Nucleoprotein NR-470 5 EV 5’ UTR Echovirus 6 30,000 hMPV Nucleoprotein hMPV-16/IA10-2003 Type A1 2 HRV 5’UTR 1A 1 Flu A (H1N1) Matrix, NS1, HA1 A/Brisbane/59/07 200 Flu A (H1N1- 2009) Matrix, NS1, HA1-2009 A/SwineNY/03/2009 100 Flu A (H3N2) Matrix, NS1, HA3 A/Wisconsin/67/2005 5 Flu B Hemagglutinin B/FL/04/06 60 M. pne Toxin M129 – Type 1 30 PIV 1 Hemagglutinin Type 1 500 PIV 2 Fusion Type 2 10 PIV 3 Fusion Type 3 10 PIV 4 Fusion Type 4a 5,000 RSV Matrix RSV Type A 2

FilmArray RP Pouch Pathogens, Gene Targets and LOD95 Organism Gene Target(s) Strain LOD95

Poritz et al. P:os One e26047, 2011

Pandemic 2009 H1N1 strains # Influenza A/New York/15/2009 16

Influenza A/California/05/2009 pandemic-H1N1 7

Influenza A/Santo Domingo/WR1068N/2009(Pandemic-H1N1)-PB1-S4 SNP

3

Influenza A virus A/Singapore/GP2711/2009(Pandemic-H1N1)-NUC-S2 SNP

1

Influenza A/Wisconsin/629-D00698/2009(Pandemic-H1N1)-HA-S1 SNP

1

Influenza A/Hiroshima/220/2009(Pandemic-H1N1) 1

Influenza A/New York/4057/2009(Pandemic-H1N1)-NS1-S1 SNP 2

Influenza A/Japan/PR1070/2009(Pandemic-H1N1)-PA-S3 SNP 1

What circulated in 2010-2111 in Baltimore?

Murillo et al. ICEID 2012

H3N2 strains #

Influenza A/Thailand/CU-B1697/2009 9

Influenza A/ Kentucky/UR07-0148/2008 8

Influenza A/Boston/47/2008(H3N2) 1

Influenza A/New York/3079/2009(H3N2) 1

Influenza A/Texas/AF01/2007(H3N2) 1

Influenza A/California/VRDL334/2009(H3N2)

1

Influenza A/Cheong/2009 1

What circulated in 2010-2111 in Baltimore?

Murillo et al. ICEID 2012