1. Xpert HIV-1 Viral Load: Beyond Plasma
Lara Noble, Wendy Stevens, Lesley Scott
Department of Molecular Medicine and Haematology, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand and The National Health Laboratory Service, National Priority Programmes, Johannesburg, South Africa

2. VL testing guidelines
WHO: VL monitoring 6 and 12 months after initiating ART, thereafter every 12 months
If patient displays virological failure: >1000c/ml
Intensive adherence counselling
Repeat VL after 3 months
47/56 LMIC recommend VL monitoring, but it is frequently unavailable*
*Medicins Sans Fronteirs, 2015, Putting HIV and HCV to the Test: A Product Guide for Point-of-Care CD4 and Laboratory-Based and Point-of-Care Virological HIV and HCV Tests

3. Addressing the gaps Existing VL services scaled
Red pin (existing centralized VL labs)
16 (m2000, COBAS up to 8800)
Extending VL services through POC?
Green pin (POC community Hubs)
11 identified by CD4
Yellow pin (anticipated true POC)
9 identified by CD4 modeling
*13 sites enrolling in feasibility pilot study – plasma
Maximizing platform integration
Small circles (current GeneXpert TB)
207
Blue: 54 Gx4
Green: 144 Gx 16
Red: 9 (n=2 Gx48, n=7 Gx80)

4. Xpert® HIV-1 VL Assay: what do we know?
GeneXpert
Fully automated
Real-time molecular
Modular (cartridge based)  polyvalent
Xpert HIV-1 Viral Load
Requires 1ml plasma
TAT <95mins
Preference for RNA extraction
Targets 3’ end 5’ LTR
Detects HIV Group M,O,N and recombinants.
Two internal quantification standards.
Linear range: 40 – 10million c/ml
LOD ~20c/ml
Xpert HIV-1 VL is interchangeable with existing technologies in South Africa.

5. Xpert HIV-1 VL: Excellent concordance
Roche TaqMan v2, Abbott RT HIV, bioMerieux NucliSens EasyQ HIV-1(v2) & Hologics Aptima HIV-1 Quant Dx

6. Programme integration: exploiting instrument polyvalency
Slide courtesy Alaine Nyaruhirira, Management Science for Health, Pretoria, South Africa

7. Low Volume Plasma Specimens
Repeat testing
Paediatric patients
Centrifugation still required
PBS buffer required (not supplied)
Dilution factor must be applied to results!
Program into assay or LIS
Supply a conversion chart?

8. Decentralisation: whole blood
OFF-LABEL!!!
Decentralisation: whole blood
DBS or whole blood
Challenges
Decreased volume (reduced sensitivity)
Cell-associated DNA (reduced specificity)
False positive = unnecessary repeat testing
False positive(post-adherence) = unnecessary regimen switch
False negative = undetected virological failure with potential for drug resistance
DBS testing
Extend services
Easy to collect
Easy to transport at room temperature (1-2 weeks)
Uses existing infrastructure (courier and lab)
High throughput
Challenges:
Still centralised: TAT
POC testing
Expand services
Increase access
Time to reportable result = hours not days (35mins-90mins)
Low throughput
Challenges:
Cost, availability, Clinic workflow, task shifting
Requires quality framework aligned with central laboratories
Conclusions
Xpert HIV-1 VL can be performed with 200ul and 500ul plasma specimens diluted in PBS, provided that a conversion factor is applied to minimize clinical misclassification.
The dilution factor can be applied to absolute copies/ml and linear regression or bias is applicable to log copies/ml.
The supplier is encouraged to automate the conversion and reporting to limit calculation and transcription error.
Further clinical evaluation is being performed to assess the utility of these methods in a real-world setting using clinical specimens.

9. DBS: 70µl
Range of buffers and elution conditions tested: Xpert HIV-1 Qual buffer; 56oC, 500rpm, 15 minutes
DBS read higher in the low range (<3log) and lower in the high range (>3log) If we apply dilution factor to all specimens, cell associated DNA causes upward misclassification  Use a conversion factor only when the absolute VL>1000c/ml

10. What about POC? Whole blood R&D
100µl whole blood (VENOUS) mixed with 1mL TRIS buffer directly into cartridge
Can’t apply the dilution factor to all specimens because of cell-associated DNA
Applying dilution factor to specimens with absolute WB VL>1000 does bring results closer to the plasma, but 11 patients are still downward misclassified.
~4.7% upwards misclassification
~12.8% downwards misclassification
Data: Natasha Gous, Lara Noble

11. POC R&D Ongoing laboratory and POC study at CMJAH, Johannesburg
Few errors
1 volume error
1 probe-check error
Easy to perform
Venous and capillary whole blood results are very similar.
Application of dilution factor as for venous whole blood.

12. Considerations
Polyvalent instrumentation cost saving, same supplier, same servicing
One instrument can be used with different ADF for each test type.
Connectivity C360 available and useful for monitoring
Random access and workflow in lab can accommodate different test types
HR requirements: Same staff can be trained for both assays as processing not difficult
Options for whole blood VL testing, small plasma volume testing with conversion factor and finger stick option (but all currently off label)
TB and HIV programs supportive since HIV VL centralised model can now expand and remain plasma based. Business manager regions allows for test/lab consolidation
Cartridges are the same colour
Separate specimen processing areas required
HIV processing requires centrifugation
Centrifugation location away from UPS and Gx instrument
Health professions council registration for technicians performing TB vs HIV testing requires policy change
Instrument utility demand more on HIV to ensure specimen integrity (RNA) not compromised (PPT tubes off label)

13. Conclusions Off-label testing is possible
Dilute plasma (200ul or 500ul)
DBS (requires a thermomixer)
Whole Blood – venous or finger stick (100ul)
Buffer standardisation is vital.
A conversion factor should be applied to minimize clinical misclassification.
The supplier is encouraged to automate the conversion and reporting to limit calculation and transcription error.
Leverage existing GeneXpert platform (207 TB testing sites)
Further clinical evaluation is being performed in collaboration with Cepheid to assess the utility of these methods in a real-world settings.

14. Dept. Molecular Medicine and Haematology
Acknowledgements
Department of Molecular Medicine and Haematology, Wits University and the NHLS, National Priority Program (Dr Sergio Carmona, Dr Leigh Berrie, Dr Pedro da Silva), R&D team (Violet Molopa)
CLS (Linda Bethlehem, Valencia Moswane)
Clinical partners WRHI, Dr Sarah Stacey, Right to care, CMJAH ARV treat clinic, NHLS phlebotomy staff
Patients enrolled in the study
Cepheid R&D team
Funders (specifically ACTG, Bill and Melinda Gates Foundation, CDC, Global Fund, SA/UK-MRC Newton Fund)