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Near-Care Molecular Testing for Dengue, Zika, and Related Pathogens
Jesse Waggoner, MDAssistant Professor
Department of Medicine, Division of Infectious DiseasesEmory University School of Medicine
Department of Global HealthRollins School of Public Health
Sirohi, et al. Science, 2016; 352:467Brent Swails, CNN
Buenos días
A todos tengo que dar algo
cada semana y cada día,
un regalo de color azul,
un pétalo frío del bosque,
y ya de mañana estoy vivo
mientras los otros se sumergen
en la pereza, en el amor,
yo estoy limpiando mi campana,
mi corazón, mis herramientas.
Tengo rocío para todos.
- Pablo Neruda, “A mis obligaciones”
Disclosures
• Disclosures• DENV multiplex and pan-DENV assays have been licensed by
Globavir, Inc.
• “ZCD” assay has been licensed by Bio-Rad
• Our group has received funding from Cepheid to evaluate primers and probes in the GeneXpert device
Acknowledgements
• Much of the work described here was performed in collaboration with
• Benjamin A. Pinsky, Stanford University
• Eva Harris, University of California, Berkeley
• Both of whom will be speaking in Session 4 this afternoon
The ZCD Assay
The ZCD Assay
• Dengue Virus (DENV)– Targets 5’ UTR-capsid gene– Detects, but does not distinguish,
the four DENV serotypes
• Chikungunya Virus (CHIKV)– Targets a region of nsp2– Can be performed to quantitate
CHIKV viral load
• Zika Virus (ZIKV)– Targets a region of NS4B gene– Can be performed to quantitate
ZIKV viral load
• Single reaction, real-time RT-PCR for the detection and differentiation of the following:
ZCD Assay Evaluation
• ZCD assay was compared to a published rRT-PCR for ZIKV using 133 consecutive samples– Comparator was designed for 2007 Yap
Island strain– Performed on an ABI 7500 instrument
according to published methods
• Additional testing of 346 patients in Nicaragua demonstrated the utility of a sensitive, multiplex assay– Mono-infection: 192; Coinfection: 71– Viral load was lower in ZIKV than CHIKV or
DENV– Clinically, patients were difficult to
differentiate from one another
ZIKV rRT-PCR
Pos Neg Total
ZCD
Ass
ay Pos 25 31 56
Neg 1 76 77
Total 26 107 133
Comparator interpreted as publishedComparator interpretation similar to
ZCD assay
Waggoner, et al. Emerg Infect Dis, 2016; 22: 1295
Lanciotti, et al. Emerg Infect Dis, 2008; 14: 1232
ZIKV rRT-PCR
Pos Neg Total
ZCD
Ass
ay Pos 47 9 56
Neg 3 74 77
Total 50 83 133
Waggoner, et al, CID, 2016; 63: 1584
Near-Care Molecular Test for ZIKV and DENV
• On-demand assay performance
• Random access platform
• Turn-around time of 1-2 hours
• Decreased complexity
• Limits lab requirementsPatient Interaction
Near-careDiagnostics
Clinical LaboratoryMolecular Diagnostics
Cepheid GeneXpert®
• Simple, random access platform
– Available instruments with 4, 8, 16, 48 or 80 chambers
• Accomplishes nucleic acid extraction, amplification and detection in a single-use, disposable sample cartridge
• Can be used for a growing number of diagnostic tests
• Xpert MTB/RIF
• HIV
• Chlamydia & Gonorrhea
GeneXpert® Device – Closed System
Add 500 μL of Sample into Xpert Cartridge
(Pipette provided)
Add binding reagent to cartridge
(included in kit)
Sample Purification
Result Interpretation and Reporting
One-Step RT-PCR
Nucleic Acid Extraction
Master Mix Reconstitution
(w/ Internal Control)
Answer Out: ~ 90 min<5 min Hands-On
GeneXpert® Device – Open Cartridge
Add 500 μL of Sample into Xpert Cartridge
(Pipette provided)
Add binding reagent to cartridge
(included in kit)
Sample Purification
Result Interpretation and Reporting
One-Step RT-PCR
Nucleic Acid Extraction
Master Mix Reconstitution
(w/ Internal Control)
Answer Out: ~ 90 minAdd Lyophilized Reagent Beads to Open Cartridge
Advantages
• Medium complexity test (closed test)– Limited sample manipulation– Cartridges individually wrapped
• Random access platform– Each compartment can perform separate tests on individual samples
• Data can be uploaded into the cloud for real-time surveillance
• Reagent stability > 1 year at room temperature
• Maintains sensitive detection– Preliminary data with a DENV multiplex test demonstrates a limit of
detection around 100 copies/mL
Advantage of GeneXpert®: Availability
http://apps.who.int/tb/laboratory/xpertmap/
WHO Roll-out Data as of the 4th Quarter of 2016
Disadvantages
• Requires a power supply– Has a low footprint– Can be run from battery power
• Cost per test– With concessional pricing, this is as low as 10 USD per test for MTb/Rif
• Relatively low throughput– Turn around can be increased with automatic stop protocols, but
assumes single target detection in each sample
• Use of an entire sample aliquot– Nucleic acid eluate is not available for further testing
Flexibility for Detection of Different Pathogens
Interchangeable multiplex molecular diagnostics
• DENV*– pan-DENV*– DENV Multiplex*– pan-DENV - revised†
• Leptospira*– All species*– Pathogenic species*
• Malaria– Pan-Plasmodium*– P. falciparum*
• Other Flaviviruses– Zika*– Yellow Fever*– West Nile*– Japanese Encephalitis†
• Alphaviruses– Chikungunya*– O’nyong-nyong*/†– Mayaro*/ †– Venezuelan Equine Encephalitis †
• Bunyaviridae– Rift Valley Fever†– La Crosse †
* Clinical and analytical evaluation has been performed† Predominantly analytical data available
GeneXpert®: Assay Adaptation• Individual assays can be
prepared in separate beads
• With a standard cycling protocol, can tailor components of a multiplex for– Regional epidemiology– Emergence/identification
of new pathogens
• Plan to first evaluate ZCD assay– Then evaluate
combinations including additional pathogens
Add 500 μL of Sample into Xpert
Cartridge
Add binding reagent to cartridge
Sample Purification
Result Interpretation and Reporting
One-Step RT-PCR
Nucleic Acid Extraction
Master Mix Reconstitution
Add Lyophilized Reagent Beads
Validation
y = -3.5634x + 42.96
R² = 0.99830
5
10
15
20
25
30
35
40
45
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0
Ct
Log10 copies/µL
ZIKV
y = -3.5509x + 44.989
R² = 0.99890
5
10
15
20
25
30
35
40
45
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0
Ct
Log10 copies/µL
DENV-2
Clinical Evaluation – ZCD Xpert
• Side-by-side evaluation– With the ZCD Assay as originally described– Compare performance using different specimen types
• Include patients with a compatible clinical illness– Confirmed positives and negatives– Patients with other confirmed infections
• Ideally multicenter to evaluate performance differences based on– Patient population– Circulating viral strains
• Each bullet point represents a potential challenge for assay validation
Challenges for Validation
• Clinical Specimens– Necessary sample volumes– Definition of gold-standard or reference diagnostic tests– Different/multiple sample types
• Assay Modifications– If an additional test is added, can a smaller repeat validation of
other targets be performed
• Co-Infections– Do these need to be evaluated?– How should they be evaluated?
Practical Use of the Platform
Intended Use and Level of Care
• Intended use– Diagnosis of an acute or recent infection– Syndrome-based testing at triage of with a change in condition– Research testing for targeted enrollment or specimen banking
• Level of care– Outpatient, acute care clinics– Emergency care units– Clinical laboratories– Unit-based laboratories– Mobile care and/or testing units
Target User
• Institutional level– Health care facilities
• Hospitals or even particular units• Clinics
– Unlikely to be a good option for health care systems or large/national reference laboratories
• Individual level– Trained health care workers
• Nurses in the emergency care unit• Specimen processing technicians
– Research staff – Not necessarily clinical laboratory specialists
Gracias a todos!
IICS
Hillview Lab, Stanford University
UCB School of Public Health IICS-UNA, Asunción, Paraguay
Hope Clinic, Emory University