© The Children's Mercy Hospital, 2014. 03/14 Clinical Laboratory Testing for Detection of...

© The Children's Mercy Hospital, 2014. 03/14

Clinical Laboratory Testing for Detection of Influenza.

Rangaraj Selvarangan. BVSc, PhD, D(ABMM).Associate Professor, UMKC-SOM

Director, Microbiology LaboratoryDirector, Laboratory Medicine Research Affairs

Children's Mercy Hospitals and ClinicsKansas City, MO 64108

International Conference on Flu, June 8-10, 2015Chicago, USA


Objectives• Describe laboratory tests available for detection

of influenza infection

• Identify strengths and weaknesses of traditional and molecular laboratory tests for Influenza

• Discuss FDA ruling on Rapid antigen tests

• Describe the potential clinical impact of rapid molecular testing for influenza


Introduction• Respiratory viral illness is one of the most common infection in

children and adults

• Influenza and RSV cause seasonal outbreaks; early diagnosis improves care.

• Non-influenza respiratory illness are also common; estimated at 500 million/year, economic impact of $40 billion/year1

• Several studies show that a substantial percentage of both out patient (>70%) 2 and in-patient antibiotic use (up to 50%) is either unnecessary or inappropriate.

• In 2008, there were 142,000 visits to emergency departments for adverse events attributed to antibiotics.3

1Fendrick et al Arch Intern Med 2003, 2 Hersch et al Ped 2011, 3Shehab et al Clin Inf Dis 2008


Influenza Epidemics• Member of the Orthomyxovirus, Influenza type A and B

cause seasonal epidemics in humans.

• Transmission via large-particle droplets.

• Incubation 1-4 days.

Every year in the United States, on average:

• 5% to 20% of the population gets the flu

• more than 200,000 people are hospitalized from flu-related complications

• about 36,000 people die from flu-related causes

• Shedding: Adults 5-10 days, Children >10 days, immunocompromised weeks or months.


Influenza in Children• Children aged 0–4 years

– 100/100,000 children for those without high-risk medical conditions.

– 500/100,000 children for those with high-risk medical conditions

– Highest among children aged 0–1 years and are comparable to rates reported among persons aged > 65 years.

• Signs: Abrupt onset of constitutional and respiratory signs (fever, myalgia, headache, sore throat, cough). Children- otitis media, nausea and vomiting.

• Hospitalization: 4%-11% ICU, 3% mechanical ventilation.

• Complications: Otitis media, Bacterial Pneumonia, Encephalitis, encephalopathy, Myocarditis, Myositis

• PPV for clinical definition in children 79% to 88%.


Influenza Epidemics

Prophylaxis Diagnosis Treatment


Influenza Culture

• Tube culture- PMK, MDCK (trypsin in medium), ML

• Co-cultured cells: R-Mix (DHI)- A549+ML, R-Mix Too- A549+MDCK. Rapid results >80% of flu specimens are positive on Day 1.

• R-Mix Too- may be better in FluB recovery and it does not support SARS-CoV.

• Dunn et al JCM 2004. Total 3803 respiratory specimens; compared tube culture to R-Mix SV. Flu A 238/241 (99%), Flu B 36/38 (95%) and RSV 52/60 (87%).

• Cryopreserved R-Mix Ready cells: Kim et al JCV 2008. Higher and early recovery of viruses in R- Mix ready cells Vs tube culture.

• Option for small volume laboratories and surplus inventory during peak winter season.

http://www.google.com/url?url=http://helatermproject.blogspot.com/2012/01/main-obstacles-gey-and-his-assistants.html&rct=j&frm=1&q=&esrc=s&sa=U&ei=0PR1VaPaEIq8ggSmjYHoBw&ved=0CBoQ9QEwAg&sig2=pOYIa16OUq2uGrqzhwIHng&usg=AFQjCNGq0qtnSkq1saEXumVbbugnIaZTpg


Influenza DFA

• DFA for influenza is a highly sensitive and specific test.

• Technical expertise needed, TAT few hours• Sensitivity is affected by sample quality- flocked

swabs provide good cell recovery for DFA. • Screening antibodies for 7 viruses are available

from several vendors. Identification is by mAb.• Cytospin improves DFA performance on slide

preparations.• Combination antibodies FluA/respiratory virus

help for quick screening during peak influenza season. D3 Duet (DHI) SimulFluor (Millipore).


Influenza Rapid Antigen Test

• Rapid antigen test for influenza have moderate sensitivity for seasonal Flu A/B (50-80%) and poor sensitivity (10% to 40%) for 2009 H1N1 virus.

• Specificity usually >90%.• Influenza antigen negative results need

confirmatory test performed; Culture, DFA, RT-PCR.

• Benefit of Influenza rapid antigen test in the OP setting – patient triage, infection control, reduced

antibiotic usage, timely antiviral treatment, reduce unnecessary diagnostic workups, overall reduction in medical care cost.


Rapid Antigen Test-Flu A/B

• Host Factors:

– Age

– Duration of illness

– Specimen type

– Specimen transport


Host factors

• Age: Children excrete high viral loads during influenza infections

• Duration of illness: Viral load in respiratory secretions decrease with duration of illness. Testing within 3 days of illness onset improves detection

• Specimen Type: Nasopharyngeal aspirates, nasopharyngeal washes, nasopharyngeal swabs, mid turbinate swabs are preferred specimens. Nasal and throat swabs results in sub-optimal yield

• Specimen Transport: Collection in viral transport medium and rapid transport on ice improves yield

Cheng et al., 2009; Esposito et al., 2011; Loeb et al., 2012; Talbot et al., 2010


Specimen collection

• Nasopharyngeal aspirates and nasal washes: High viral load, diluted, mucous can interfere with EIA/DFA performance. Cell quantity may vary between collection. Suitable for culture, PCR and later flow tests.

• Recent experience indicates that a combination of NP swab and throat swab in VTM may improve diagnostic yield.

• Flocked swabs: More cells collected, less mucous, well suited for DFA. Nasopharyngeal and mid-turbinate swabs available.


Tilt patient head 70 degree angle and against wallInsert swab straight back (not upward) until resistance is metRotate the swab 5-10 times to loosen cellsRemove swab and inoculate VTM


Swab studies

• Abu-Diab et al JCM 2007; NPA Vs NP swab. 455 children. Sensitivity of NP swab 98.5%.

• Allen et al PASCV 2008 poster # M28; MTS Vs NP swab. 203 children, PCR gold standard. NPS =87.5%, MTS =79%. 8% of both NPS and MTS insufficient for DFA. Nurse prefer MTS.

• Selvarangan et al PASCV 2009 poster # M51. 200 children. NPA Vs MTS. RSV antigen and SV culture. RSV antigen sensitivity 66% for MTS and 70% for NPA. Nurses prefer MTS over NPA.



• Viral Factors:

– Influenza activity

– Viral subtype


Test PerformanceSensitivity : proportion of actual positives which are correctly

identified as such (i.e. the percentage of sick people who are identified as having the condition)

Specificity : proportion of negatives which are correctly identified (i.e. the percentage of well people who are identified as not having the condition).

The probability of the presence or absence of disease given the results of a test.

Positive predictive value (PPV) : proportion of patients with positive test results who are correctly diagnosed.

Negative predictive value (NPV): proportion of patients with negative test results who are correctly diagnosed.

Wikipedia.org


1% Flu Infected Not-infected

Positive TP = 19 FP = 80PPV

TP / (TP+FP)19 / 99 = 19%

Negative FN = 1 TN = 1900 NPV

TN / (TN + FN)1900 / 1901= 100%

Sensitivity TP / (TP + FN) 19 / 20 = 95%

Specificity TN / (FP +TN)1900 / 1980 = 96%

20% Flu Infected Not-infected

Positive TP = 380 FP = 64PPV

TP / (TP + FP)380 / 444 = 86%

Negative FN = 20 TN = 1536NPV

TN / (TN + FN)1536 / 1556 = 99%

Sensitivity TP / (TP + FN) 380 / 400 = 95%

Specificity TN / (FP + TN)1536 / 1600 =96%

Beginning and end of the season-It is advisable to confirm all positives by culture due to low PPV

Test

Resu

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Influenza 2009-10

0

20

40

60

80

100

120

# de

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RADTRVP RADT,

RVP

ProFluPCR

LDT-PCRFluA/B Cx

Influneza pandemic 2009


Rapid Antigen PCR

Pre pH1N1Post pH1N1

Pre pH1N1

Post pH1N1

NPO (n=22) 19 (86.4%) 14 (63.6%) 0 1 (4.5%)

Hosp Ntwrk (n=31) 24 (77.4%) 19 (61.3%) 4 (12.9%) 8 (25.8%)

Comm Hosp (n=43) 40 (93%) 36 (83.7%) 0 0

Academic Inst (n=33) 18 (55%) 8 (24%)* 5 (15%) 14 (42%)*

Survey of Lab Methods

Selvarangan et al ASM conference 2010

Use of molecular methods for detection of respiratory viruses2009 = 18% (Selvarangan, ClinMicronet Survey)2011= 36% (Miller, ClinMicronet Survey)2013 = 63% (Miller, ClinMicronet Survey)


2010-11 2011-12 2012-13 2013-14 2014-150

100020003000400050006000700080009000

10000

Influenza Testing

Flu A/B pos Tested

11/1

7/20

14

11/2

7/20

14

12/7

/201

4

12/1

7/20

14

12/2

7/20

14

1/6/

2015

1/16

/201

5

1/26

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2/5/

2015

2/15

/201

5

2/25

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3/7/

2015

3/17

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5

3/27

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5

4/6/

2015

4/16

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4/26

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50100150200250300350400450

Influenza Testing 2014-15

Positive A Positive B

The updated VE estimate against influenza A H3N2 viruses was 18% (95% confidence interval (CI): 6%-29%).The VE estimate against influenza B viruses this season was 45% (95% CI: 14% – 65%).



• Analytical Factors

– Test characteristics

– Result interpretation

• Post Analytical Factors

– Reporting Time

– Clinical interpretation


Next –Generation Influenza RADT

3M Influenza

BD Veritor

Quidel Sofia


Rapid Antigen Tests- Instrument Reader

Hassan et al JCM 2014


Rapid Antigen Tests- Instrument Reader

Dunn et al DMID 2014


Manufacturer 1: ref method Culture; 79% sensitivity (75-83%)


FDA Ruling May 2014-RIDT

Reference Method: Viral culture SensitivityFlu A Point estimate of 90% (95% C.I . >80%)Flu B Point estimate of 80% (95% C.I. >70%)Specificitylower bound of the 95% CI exceeding 90% for both, Flu A and Flu B.Reference Method: Molecular methodSensitivityFlu A Point estimate of 80% (95% C.I. >70%)Flu B Point estimate of 80% (95% C.I. >70%)Specificitylower bound of the 95% CI exceeding 90% for both, influenza A and influenza B.


FDA Ruling May 2014-RIDTLaboratory Tests:

Influenza viral antigens or influenza viral gene segments (protein or nucleic acid), either in single unit test formats or multi-test formats

Monitoring Performance:

Conduct annual analytical testing of their device with contemporary strains

Standardized panel of viruses selected in coordination with FDA. Dilutions at 10e2 and 10e5 TCID50/mL in triplicate.

Detection of all replicates at least at one dilution. Standardized panels of well characterized viral stocks could possibly be available from CDC or commercial vendors.

The testing could be conducted in-house or at a contract laboratory. Absence of analytical reactivity would be reflected in labeling as a limitation.

Emerging Influenza strains: Provide analytical reactivity report to FDA within 60 days of emerged virus strain availability.


Diagnostic Challenges• Problem1: Lack of a rapid and highly sensitive

method for detection of Influenza at POC setting.

• Problem 2: Lack of sensitive method for extensive detection of respiratory viruses in hospitalized patients

– Impact: Improper use of antivirals, overuse of antibiotics, additional diagnostic workup, improper infection control measures, over all increase in health care cost.


Diagnostic Challenge-Solution

• Problem 1: Lack of a rapid and highly sensitive method for detection of Influenza at POC setting.

• Solution- RADT-Instrument read, Rapid multiplex RP-NAAT- Alere™ i Influenza A & B, iQuum Liat Influenza A/B, Focus Simplexa™ FluA/B & RSV Direct , Cepheid Xpert® Flu, and GenXpert and BioFire Filmarray™ RP

• Considerations: Clinical performance data limited, Cost, clinical experience limited, Lack of complete understanding of the hierarchy among these tests


Molecular Nucleic Acid Amplification Methods for

Respiratory Virus Detection


Multiplex RP-NAAT: Rapid

BioFire Filmarray™ RP (~60 min)

Cepheid Xpert® Flu(75 min) Focus Simplexa™ FluA/B & RSV Direct (~60 min)

iQuum Liat Influenza A/B (20 min )

Alere™ i Influenza A & B (15 min )


Alere i Influenza Vs Culture

Specimen Detection TP FP TN FN Total

% Sensitivity (95% CI)

% Specificity (95% CI)

PPV (95% CI)

NPV (95% CI)

Flu AChildren 128 5 320 1 454

99.2 98.4 96.2 99.6

(95.1-99.9) (96.2-99.4) (91.0-98.6) (98.0-99.9)

Adults 17 2 56 0 75

100 96.5 89.4 100

(77.0-100) (87.0-99.4) (65.4-98.1) (92.0-100)

Flu BChildren 70 0 379 2 451

97.2 100 100 99.5

(89.4-99.5) (98.7-100) (93.5-100) (97.9-99.9)

Adults 13 0 62 0 75

100 100 100 100

(71.6-100) (92.7-100) (71.6-100) (92.7-100)

Bell et al; Journal of Clinical Virology 2014 61, 81-86


Alere i Influneza Vs ProFlu PCR

Alere™ i Influenza A&B vs. real-time RT-PCR

TP FP TN FN Total % Sensitivity (95% CI)

% Specificity (95% CI)

PPV (95% CI)

NPV (95% CI)

Influenza A

MTS 76 1 70 9 15689.4 98.6 98.7 88.6

(80.4-94.7) (91.3-99.9) (92.0-99.9) (79.0-94.3)

NPW/A 27 1 45 4 7787.1 97.8 96.4 91.8

(69.2-95.8) (87.0-99.9) (79.8-99.8) (79.5-97.4)

Total 103 2 115 13 23388.8 98.3 98.1 89.8

(81.3-93.7) (93.3-99.7) (92.6-99.7) (82.9-94.3)

Influenza B

MTS 31 − 124 − 155100 100 100 100

(86.3-100) (96.3-100) (86.3-100) (96.3-100)

NPW/A 27 − 50 − 77100 100 100 100

(84.5-100) (91.1-100) (84.5-100) (91.1-100)

Total 58 − 174 − 232100 100 100 100

(92.3-100) (97.3-100) (92.3-100) (97.3-100)

Bell et al; J Clin Microbiol. 2014 Nov;52(11):3992-5.

http://www.ncbi.nlm.nih.gov/pubmed/25210070








Alere i Influenza Vs ProFlu PCR

Alere i Influenza A Neg Alere i Influenza A Pos15

25

35

Pro

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Ct


Cobas Liat Influenza assay

• FDA-cleared, rapid (<20 min) PCR assay (Roche cobas® Liat) to Focus Simplexa™ Flu A/B & RSV Direct using respiratory swabs (n=197).

• The cobas Liat influenza A and B assays demonstrated sensitivities of 99.2% (123/124) and 100% (23/23), respectively, while showing a specificity of 100% for both targets.Binnicker et al: J Clin Microbiol. 2015 Apr 29. pii: JCM.00791-15.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Roche+LIAT+influenza









Multiplex RP-NAAT: Extended PanelLuminex xTAG RVP (n=12) and RVP FAST (n=8)

BioFire Filmarray RP (n= 20 )Nanosphere RV Plus test (n=7)

GenMark eSensor RVP (n=14)


CAP-ID Resp Panel-FLu 2014

BioFire Cepheid GenMark GenProbe Luminex Nanosphere0

20

40

60

80

100

120

140

160

180

200

174

38 44

5

37

11

2014 IDR-A summary


Flu Antiviral Resistance

Influenza viruses

Antiviral 2009 H1N1Seasonal

H1N1Seasonal

H3N2Flu B

Adamantanes Resistant Susceptible

Resistant N/A

Oseltamivir Susceptible

*

Resistant Susceptible

Susceptible

Zanamivir Susceptible Susceptible

Susceptible

Susceptible

* Few oseltamivir-resistant 2009 H1N1 strains reportedCDC


• Cost of the test

• Reimbursement issues

• Loss of detection due to mutations*

• Competitive inhibition of analytes in a multiplex assay

• Limited clinical experience for certain infections: coronavirus, rhinovirus and co-infections.

*Hawkinson et al DMID 2013

Multiplex RP NAAT Disadvantages


Influenza Diagnostics- Summary• Rapid antigen tests are useful for OP management.

• Antigenic drift and shift in Influenza strains influences test performance.

• Specimen collection method and VTM approved for the antigen test need to be followed

• Monitor QC controls and review data periodically

• Reflex antigen negative test result to confirmatory testing

• Rapid molecular tests for Influenza will improve diagnostic yield

• SV culture is a simple assay with Flu isolation mostly by day 1

• Multiplex PCR assays improve Flu detection and other respiratory infections associated with ILI

• Epidemiological data or individual Flu subtype testing may be necessary to determine antiviral resistance.

© The Children's Mercy Hospital, 2014. 03/14 Clinical Laboratory Testing for Detection of...

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