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

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

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)

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.

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

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

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?

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.

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

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

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.

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)

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.

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)