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