Near-Care Molecular Testing for Dengue, Zika, and Related Pathogens

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1 Brent Swails, CNN Near-Care Molecular Testing for Dengue, Zika, and Related Pathogens Jesse Waggoner, MD Assistant Professor Department of Medicine, Division of Infectious Diseases Emory University School of Medicine Department of Global Health Rollins School of Public Health Sirohi, et al. Science, 2016; 352:467

2 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

3 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

4 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

5 The ZCD Assay

6 The ZCD Assay Single reaction, real-time RT-PCR for the detection and differentiation of the following: 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

7 ZCD Assay ZCD Assay Evaluation Comparator interpretation similar to Comparator ZCD interpreted assay as published 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 Lanciotti, et al. Emerg Infect Dis, 2008; 14: 1232 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 Pos Neg Total Waggoner, et al. Emerg Infect Dis, 2016; 22: 1295 Waggoner, et al, CID, 2016; 63: 1584

8 Near-Care Molecular Test for ZIKV and DENV Near-care Diagnostics On-demand assay performance Random access platform Turn-around time of 1-2 hours Clinical Laboratory Molecular Diagnostics Decreased complexity Limits lab requirements Patient Interaction

9 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

10 GeneXpert Device Closed System Nucleic Acid Extraction Sample Purification Master Mix Reconstitution (w/ Internal Control) Add binding reagent to cartridge (included in kit) One-Step RT-PCR Add 500 μl of Sample into Xpert Cartridge (Pipette provided) Result Interpretation and Reporting <5 min Hands-On Answer Out: ~ 90 min

11 GeneXpert Device Open Cartridge Nucleic Acid Extraction Sample Purification Master Mix Reconstitution (w/ Internal Control) Add binding reagent to cartridge (included in kit) One-Step RT-PCR Add 500 μl of Sample into Xpert Cartridge (Pipette provided) Result Interpretation and Reporting Add Lyophilized Reagent Beads to Open Cartridge Answer Out: ~ 90 min

12 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

13 Advantage of GeneXpert : Availability WHO Roll-out Data as of the 4 th Quarter of

14 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

15 Flexibility for Detection of Different Pathogens

16 Interchangeable multiplex molecular DENV* pan-denv* DENV Multiplex* pan-denv - revised diagnostics Other Flaviviruses Zika* Yellow Fever* West Nile* Japanese Encephalitis Leptospira* All species* Pathogenic species* Malaria Pan-Plasmodium* P. falciparum* 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

17 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 Sample Purification Add binding reagent to cartridge Add 500 μl of Sample into Xpert Cartridge Nucleic Acid Extraction Add Lyophilized Reagent Beads Master Mix Reconstitution One-Step RT-PCR Result Interpretation and Reporting

18 Validation C t ZIKV 10 y = x R² = Log 10 copies/µl C t DENV y = x R² = Log 10 copies/µl

19 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

20 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?

21 Practical Use of the Platform

22 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

23 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

24 Gracias a todos! Hope Clinic, Emory University Hillview Lab, Stanford University UCB School of Public Health IICS-UNA, Asunción, Paraguay