Rangaraj Selvarangan BVSc PhD DABMM Associate Professor UMKCSOM Director Microbiology Laboratory Director Laboratory Medicine Research Affairs Childrens Mercy Hospitals and Clinics ID: 594132
Download Presentation The PPT/PDF document "Clinical Laboratory Testing for Detectio..." is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
Slide1
Clinical Laboratory Testing for Detection of Influenza.
Rangaraj Selvarangan. BVSc, PhD, D(ABMM).Associate Professor, UMKC-SOMDirector, Microbiology LaboratoryDirector, Laboratory Medicine Research AffairsChildren's Mercy Hospitals and ClinicsKansas City, MO 64108
International Conference on Flu, June 8-10, 2015Chicago, USASlide2
ObjectivesDescribe laboratory tests available for detection of influenza infection
Identify strengths and weaknesses of traditional and molecular laboratory tests for InfluenzaDiscuss FDA ruling on Rapid antigen tests Describe the potential clinical impact of rapid molecular testing for influenzaSlide3
IntroductionRespiratory 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/year1Several 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.31Fendrick et al Arch Intern Med 2003,
2 Hersch et al Ped
2011,
3
Shehab et al
Clin
Inf
Dis
2008
Slide4
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 flumore 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.Slide5
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%.Slide6
Influenza Epidemics
ProphylaxisDiagnosisTreatmentSlide7
Influenza Culture
Tube culture- PMK, MDCK (trypsin in medium), MLCo-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.Slide8
Influenza DFA
DFA for influenza is a highly sensitive and specific test.Technical expertise needed, TAT few hoursSensitivity 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).Slide9
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.Slide10Slide11
Rapid Antigen Test-Flu A/BHost Factors:
AgeDuration of illness Specimen typeSpecimen transportSlide12
Host factorsAge: 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 yieldSpecimen 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., 2010Slide13
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.Slide14
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 Slide15
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.Slide16
Rapid Antigen Test-Flu A/BViral Factors:
Influenza activityViral subtype Slide17
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.orgSlide18
1% Flu
Infected
Not-infected
Positive
TP = 19
FP = 80
PPV
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 = 64
PPV
TP / (TP + FP)
380 / 444 = 86%
Negative
FN = 20
TN = 1536
NPV
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 Result
Test ResultSlide19
RADT
RVPRADT,RVP
ProFlu
PCR
LDT-PCR
FluA
/B
Cx
Influneza
pandemic 2009Slide20
Rapid Antigen
PCR
Pre pH1N1
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%)*
Survey of Lab Methods
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)Slide21
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%).Slide22
Rapid Antigen Test-Flu A/BAnalytical Factors
Test characteristicsResult interpretationPost Analytical FactorsReporting TimeClinical interpretationSlide23
Next –Generation Influenza RADT
3M Influenza
BD Veritor
Quidel SofiaSlide24
Rapid Antigen Tests- Instrument Reader
Hassan et al JCM 2014Slide25
Rapid Antigen Tests- Instrument Reader
Dunn et al DMID 2014Slide26Slide27
Manufacturer 1: ref method Culture; 79% sensitivity (75-83%)Slide28
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
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.Slide29
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 formatsMonitoring Performance:Conduct annual analytical testing of their device with contemporary strainsStandardized 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.Slide30
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 patientsImpact: Improper use of antivirals, overuse of antibiotics, additional diagnostic workup, improper infection control measures, over all increase in health care cost. Slide31
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 Slide32
Molecular Nucleic Acid Amplification Methods for Respiratory Virus DetectionSlide33
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 )Slide34
Alere i Influenza Vs Culture
Specimen DetectionTPFPTN
FN
Total
% Sensitivity (95% CI)
% Specificity (95% CI)
PPV (95% CI)
NPV (95% CI)
Flu A
Children
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 B
Children
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)
Adults13062075100100100100(71.6-100)(92.7-100)(71.6-100)(92.7-100)Bell et al; Journal of Clinical Virology 2014 61, 81-86Slide35
Alere i Influneza Vs
ProFlu PCR Alere™ i Influenza A&B vs. real-time RT-PCRTPFP
TN
FN
Total
% Sensitivity (95% CI)
% Specificity (95% CI)
PPV (95% CI)
NPV (95% CI)
Influenza A
MTS
76
1
70
9
156
89.4
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
77
87.1
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
233
88.8
98.3
98.1
89.8
(81.3-93.7)
(93.3-99.7)
(92.6-99.7)
(82.9-94.3)
Influenza BMTS31−124−155100100100100(86.3-100)(96.3-100)(86.3-100)(96.3-100)NPW/A27−50−
77100100100100(84.5-100)(91.1-100)(84.5-100)(91.1-100)Total58−174−232100100100100(92.3-100)(97.3-100)(92.3-100)(97.3-100)Bell et al; J Clin Microbiol. 2014 Nov;52(11):3992-5. Slide36
Alere i Influenza Vs ProFlu PCRSlide37
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.Slide38
Multiplex RP-NAAT: Extended Panel
Luminex 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)Slide39
CAP-ID Resp Panel-FLu 2014
2014 IDR-A summarySlide40
Flu Antiviral Resistance
Influenza viruses
Antiviral
2009 H1N1
Seasonal
H1N1
Seasonal
H3N2
Flu B
Adamantanes
Resistant
Susceptible
Resistant
N/A
Oseltamivir
Susceptible*
Resistant
Susceptible
Susceptible
Zanamivir
Susceptible
Susceptible
Susceptible
Susceptible
* Few oseltamivir-resistant 2009 H1N1 strains reported
CDCSlide41
Cost of the testReimbursement issuesLoss of detection due to mutations*Competitive inhibition of
analytes in a multiplex assayLimited clinical experience for certain infections: coronavirus, rhinovirus and co-infections. *Hawkinson et al DMID 2013Multiplex RP NAAT DisadvantagesSlide42
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 followedMonitor QC controls and review data periodicallyReflex 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.