Upload
betty-turner
View
213
Download
0
Embed Size (px)
Citation preview
Jorge L. Muñoz-Jordán, Ph.D.Chief, Molecular Diagnostics and Research Laboratory
Centers for Disease Control and Prevention (CDC)Division of Vector-Borne Infectious Diseases
Dengue BranchPuerto Rico
Real-Time RT-PCRY su generalización en las
Américas
Illness
Kinetics of Diagnostic Targets During Dengue Illness
0 5
IgM
Virus
Days Post-Onset of Fever
90
100 -
% D
ete
ctio
n
Molecular Diagnostic Tests Many Laboratory-Developed Tests (LDT)
published Few-to-none accredited by regulatory
agencies Few-to-none extensively validated for their
performance in the diagnosis of human disease (not just analytical sensitivity)
Many commercial primer sets(self-denominated “tests”)
How do we compare them fairly?
Lab Developed Tests (LDT) Vs. In-vitro Diagnostic Devices (IDV)
LDT Restricted for their intended use:
diagnosis of symptomatic cases Unchangeable elements
established and validated by developer
Sensitivity and performance precisely established or ensured by developer
Requirements for large, external performance and reproducibility studies for accreditation
Requirement for quality assurance in the production of test elements
IDV
YesNo
YesNo
YesNo
YesNo
YesNo
Usefulness of RT-PCR during dengue acute illness
75% of patients reported ≤ 5 days of symptoms High viremias and late IgM response Detectable vRNA levels
− Viremias can be approx. 107 virus particles/ml− RT-PCR sensitivity: 103 virus particles/ml
High specificity Positive result is a definite proof of infection All-around good reference test
Vision
A widely available molecular assay that diagnosis dengue disease and identifies DENV serotype in a high proportion of cases globally
% pick up of DENV strains before and after reformulation
RT-PCR Assay
DENV OldAssay
New Assay
DENV-1
20 % 100%
DENV-2
15% 90%
DENV-3
25 % 100%
DENV-4
60% 100%Phylogenetic analysis of current strains
DENV-1
N=16
DENV-2
N=16
DENV-3
N=15
DENV-4
N=7
Santiago et al., 2013
RT-PCR Results (Yap outbrek investigation)
New Primers and Probe(Santiago et al, 2013)
Old Primers and Probe(Johnson et al, 2015)
15 % Questionable positives(poor shape of amplificationplots call results into question)
= cutoff sensitivity
75 % Positives
3 of 5 Yap sequence mismatches are resolved by
new CDCprimers/probes
CTC TCC GAG AAC GGG TCT CGA CTT TAA
CAG GCT ATG GCA CTG TCA CGA T
CCA TTT GCA GCA ACA CCA TCT C
D2-F
D2-R
D2-P
Mismatches with original primers/probes
Mismatches with new primers/probes
Dengue Real Time RT-PCR
R² = 0.9995R² = 0.9969
2
3
4
5
6
7
8
9
10
0 1 2 3 4 5 6 7DILUTION
Singleplex
LO
G
GC
E
DENV-3
Multiplex
R² = 0.9885R² = 0.9974
2
3
4
5
6
7
8
9
10
0 1 2 3 4 5 6 7DILUTION
LO
G
GC
E
DENV-3
Adapted to Pandemic Flu Platform
CDC-Dengue Branch standard platform
Santiago et al., 2013
Clinical PerformanceMultiplex CDC DENV-1-4 Real-Time RT-PCR Assay Comparison Results
Reference Method (IgM Conversion)
Positive Negative Total
DENV-1-4 RT-PCR
Positive 100 4 104
Negative 2 265 267
Total 102 269 371
Value 95% Confidence Interval
Positive percent agreement 98.03% 78.12 – 92.34
Negative percent agreement 98.51% 96.20 – 99.10
Elements for a global In-vitro Diagnostic (IVD)
Assay for dengue
IDD
Global Instrumentatio
n and Software
Common reagents
(RNA extraction, RT-PCR…)
Current Primes &Probes
CDC DENV-1-4 RT-PCR Assay (First FDA-approved dengue molecular IVD)
Kit and manual
Primers, probes and controls made and distributed by CDC
Ancillary Reagents
• Roche and Qiagen RNA extraction
• Invitrogen RT-PCR• Prequalified lots• Purchased by user
Equipment
• Roche and Qiagen • ABI 7500 Dx
Present in most labs
Public Health Labs(183) Research (5) Private/Commercial (10) US Dept. of Defense (16)
Requests for CDC DENV-1-4 Real Time RT-PCR Assay
(2013-2015)
G.A. Santiago
• 190 Laboratories• 95% Proficient
Harmonization of Dengue Diagnostics(New Testing algorithm and Training)
Costa Rica, 2012
El Salvador, 2013 (5 Countries)
Atlanta, 2013 (12 Countries, 20 US State Labs)
Cuba, 2014 (11 Countries)
Atlanta 2014 (19 Countries)
Sponsored by PAHO
Implementation of Best Diagnostic Practices
Informe Epidemiologico, Costa Rica
Weekly Report, Puerto Rico
Sustainability
Strong commitment from CDC and stakeholders (E.g. PAHO)
190 Labs globally (900 kits in 3 years) = 180,000 PCR tests
Resources:− [email protected]− [email protected]− www.cdc.gov/dengue− Santiago et al. 2013
Towards a One Sample Approach:
Simplify and decentralize surveillance
Help Clinicians and Patients
Can we positively diagnose dengue in most cases if we run the necessary tests on any sample at any time during the disease progress?
Demonstrative Study (The Algorithm Study)
• 1200 dengue suspected cases collected in Puerto Rico between 2008 and 2013 with the following characteristics:• Clinical diagnosis of dengue• Paired samples
• 1st sample (0-5 days)• 2nd sample (> 5 days)
• All samples were tested with three tests:• CDC DENV-1-4 RT-PCR Assay• IgM ELISA • NS1 Assay
Examples:
Demonstrative Study (The Algorithm Study)
Days Post-onset
0 2 4 6 8 101 3 5 7 9
1 IgM-, PCR- IgM+, PCR- Dengue Positive2 IgM-, PCR+ IgM+, PCR+ Dengue Positive3 IgM-, PCR- IgM-, PCR- Not dengue
4 IgM-, NS1- IgM+, NS1- Dengue Positive5 IgM-, NS1+ IgM+, NS1+ Dengue Positive6 IgM-, NS1- IgM-, NS1 Not dengue
Demonstrative Study (The Algorithm Study)
Based on CDC study of 1200 paired samples from confirmed dengue cases
Sensitivity of Dengue Diagnostic Tests
Diagnosis in one sample
One serum sample(0-7 days of illness)
IgM +NS1
IgM + RT+PCR
One serum sample(≥ 7 days of illness) IgM
AcknowledgementsCDC-DVBID Dengue BranchMolecular Dx. LabEdgardo Vergne Gilberto SantiagoJesus Vazquez Yashira QuileJoan Cosme Juan F. MedinaFreddy Medina Candimar ColónBetsy Ramos
Luis Manuel Santiago Kay Tomashek and team Elizabeth Hunsperger and team Vera Soltero and team Harold Margolis
CDC-DVBID-ADB Robert Lanciotti Barbara JohnsonINCIENSA (Costa Rica)Laboratorio de Salud Pública de Puerto Rico
CDC Influenza Div.Julie VillanuevaRoy Johnson
CDC NCEZID - Reg. AffairsSusan HollingsworthHye-Joo Kim
CDC NCEZID – DSRKanwar BediOwen HerzeghDennis BagarozziNicky Sulaiman
University of Nuevo Leon– Ana Rivas– Kame Galan
Florida Department of HealthTexas Department of Health
FDA-Div. Diagnostic Devices