Jorge L. Muñoz-Jordán, Ph.D. Chief, Molecular Diagnostics and Research Laboratory Centers for...

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:− denguePCRordering@CDC.GOV− denguePCRsupport@CDC.GOV− 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