Clinical Laboratory Testing for Detection
of Influenza.
Rangaraj Selvarangan. BVSc, PhD, D(ABMM).
Associate Professor, UMKC-SOM
Director, Microbiology Laboratory
Director, Laboratory Medicine Research Affairs
Children's Mercy Hospitals and Clinics
Kansas City, MO 64108
International Conference on Flu, June 8-10, 2015
Chicago, USA
© The Children's Mercy Hospital, 2014. 03/14
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
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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
3
et al Arch Intern Med 2003, 2 Hersch et al Ped 2011, 3Shehab et al Clin Inf Dis 2008
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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.
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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%.
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Influenza Epidemics
Prophylaxis
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Diagnosis
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Treatment
Influenza Culture
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•
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.
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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).
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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.
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Rapid Antigen Test-Flu A/B
• Host Factors:
– Age
– Duration of illness
– Specimen type
– Specimen transport
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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
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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 midturbinate swabs available.
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Tilt patient head 70 degree angle and against wall
Insert swab straight back (not upward) until resistance is met
Rotate the swab 5-10 times to loosen cells
Remove swab and inoculate VTM
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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.
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Rapid Antigen Test-Flu A/B
• Viral Factors:
– Influenza activity
– Viral subtype
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Test Performance
Sensitivity : 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
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1% Flu
Test Result
Positive
Negative
Infected
Not-infected
TP = 19
FN = 1
Sensitivity TP / (TP + FN)
19 / 20 = 95%
FP = 80
PPV
TP / (TP+FP)
19 / 99 = 19%
TN = 1900
NPV
TN / (TN + FN)
1900 / 1901= 100%
Specificity TN / (FP +TN)
1900 / 1980 = 96%
Beginning and end of the seasonIt is advisable to confirm all positives by culture due to low PPV
Test Result
20% Flu
Positive
Negative
Infected
Not-infected
TP = 380
FN = 20
Sensitivity TP / (TP + FN)
380 / 400 = 95%
FP = 64
PPV
TP / (TP + FP)
380 / 444 = 86%
TN = 1536
NPV
TN / (TN + FN)
1536 / 1556 = 99%
Specificity TN / (FP + TN)
1536 / 1600 =96%
© The Children's Mercy Hospital, 2014. 03/14
60
40
2F
16 eb
-F
eb
2M
16 ar
-M
30 ar
-M
13 ar
-A
p
27 r
-A
11 pr
-M
25 ay
-M
a
8- y
Ju
22 n
-Ju
n
6Ju
20 l
-J
3- u l
Au
17 g
-A
u
31 g
-A
u
14 g
-S
e
28 p
-S
e
12 p
-O
26 ct
-O
c
9- t
No
23 v
-N
o
7- v
D
21 ec
-D
ec
4Ja
18 n
-Ja
1- n
F
15 eb
-F
eb
1M
15 ar
-M
29 ar
-M
12 ar
-A
pr
# detected
Influneza pandemic 2009
Influenza 2009-10
120
100
80
RADT
RVP RADT,
RVP
ProFlu
PCR
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LDT-PCR
FluA/B Cx
20
0
Survey of Lab Methods
Rapid Antigen
Pre pH1N1
PCR
Post 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%)*
Selvarangan et al ASM conference 2010
Use of molecular methods for detection of respiratory viruses
2009 = 18% (Selvarangan, ClinMicronet Survey)
2011= 36% (Miller, ClinMicronet Survey)
2013 = 63% (Miller, ClinMicronet Survey)
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Influenza Testing
10000
8000
6000
4000
2000
0
2010-11
2011-12
2012-13
Flu A/B pos
2013-14
2014-15
Tested
Influenza Testing 2014-15
450
400
350
300
250
200
150
100
50
0
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%).
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Rapid Antigen Test-Flu A/B
• Analytical Factors
– Test characteristics
– Result interpretation
• Post Analytical Factors
– Reporting Time
– Clinical interpretation
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Next –Generation Influenza RADT
BD Veritor
Quidel Sofia
3M Influenza
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Rapid Antigen Tests- Instrument Reader
Hassan et al JCM 2014
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Rapid Antigen Tests- Instrument Reader
Dunn et al DMID 2014
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Manufacturer 1: ref method Culture; 79% sensitivity (75-83%)
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FDA Ruling May 2014-RIDT
Reference Method: Viral culture
Sensitivity
Flu A Point estimate of 90% (95% C.I . >80%)
Flu B Point estimate of 80% (95% C.I. >70%)
Specificity
lower bound of the 95% CI exceeding 90% for both, Flu A and Flu B.
Reference Method: Molecular method
Sensitivity
Flu A Point estimate of 80% (95% C.I. >70%)
Flu B Point estimate of 80% (95% C.I. >70%)
Specificity
lower bound of the 95% CI exceeding 90% for both, influenza A and influenza B.
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FDA Ruling May 2014-RIDT
Laboratory 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.
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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.
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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 RPNAAT- 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
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Molecular Nucleic Acid
Amplification Methods for
Respiratory Virus Detection
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Multiplex RP-NAAT: Rapid
Cepheid Xpert® Flu(75 min)
iQuum Liat Influenza A/B (20 min ) Focus Simplexa™ FluA/B &
RSV Direct (~60 min)
Alere™ i Influenza A & B (15 min )
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BioFire Filmarray™ RP (~60 min)
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Alere i Influenza Vs Culture
Specimen
Detection
TP
Children
128
FP
5
TN
320
FN
1
Total
454
% Sensitivity
(95% CI)
% Specificity
(95% CI)
PPV
(95% CI)
NPV
(95% CI)
99.2
98.4
96.2
99.6
(95.1-99.9)
(96.2-99.4)
(91.0-98.6)
(98.0-99.9)
100
96.5
89.4
100
(77.0-100)
(87.0-99.4)
(65.4-98.1)
(92.0-100)
97.2
100
100
99.5
(89.4-99.5)
(98.7-100)
(93.5-100)
(97.9-99.9)
100
100
100
100
(71.6-100)
(92.7-100)
(71.6-100)
(92.7-100)
Flu A
Adults
Flu B
Children
Adults
17
70
13
2
0
0
56
379
62
0
2
0
75
451
75
Bell et al; Journal of Clinical Virology 2014 61, 81-86
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Alere i Influneza Vs ProFlu PCR
Alere™ i Influenza A&B
vs. real-time RT-PCR
MTS
Influenza A
NPW/A
Total
MTS
Influenza B
NPW/A
Total
TP
FP
TN
FN
Total
76
1
70
9
156
27
103
31
27
58
1
2
−
−
−
45
115
124
50
174
4
13
−
−
−
77
233
155
77
232
% Sensitivity % Specificity
(95% CI)
(95% CI)
NPV
(95% CI)
89.4
98.6
98.7
88.6
(80.4-94.7)
(91.3-99.9)
(92.0-99.9)
(79.0-94.3)
87.1
97.8
96.4
91.8
(69.2-95.8)
(87.0-99.9)
(79.8-99.8)
(79.5-97.4)
88.8
98.3
98.1
89.8
(81.3-93.7)
(93.3-99.7)
(92.6-99.7)
(82.9-94.3)
100
100
100
100
(86.3-100)
(96.3-100)
(86.3-100)
(96.3-100)
100
100
100
100
(84.5-100)
(91.1-100)
(84.5-100)
(91.1-100)
100
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.
35
PPV
(95% CI)
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Alere i Influenza Vs ProFlu PCR
Prodesse PCR Ct
35
25
15
Alere i Influenza A Neg
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Alere i Influenza A Pos
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.
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Multiplex RP-NAAT: Extended Panel
Luminex xTAG RVP (n=12) and RVP FAST (n=8)
Nanosphere RV Plus test (n=7)
38
GenMark eSensor RVP (n=14)
BioFire Filmarray RP (n= 20 )
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CAP-ID Resp Panel-FLu 2014
200
180
160
140
120
100
80
60
40
20
0
174
38
44
37
5
2014 IDR-A summary
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Flu Antiviral Resistance
Antiviral
Adamantanes
Oseltamivir
Zanamivir
Influenza viruses
Seasonal
Seasonal
H1N1
H3N2
Resistant
Susceptible
2009 H1N1
Resistant
Susceptible*
Susceptible
Resistant
Susceptible
Flu B
N/A
Susceptible
Susceptible
Susceptible
Susceptible
* Few oseltamivir-resistant 2009 H1N1 strains reported
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CDC
Multiplex RP NAAT Disadvantages
• 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 coinfections.
*Hawkinson et al DMID 2013
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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.
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