CCQM-P199: Interlaboratory comparability study of HIV-1 RNA copy number quantification
Authors: Devonshire Alison S. , Busby Eloise J. , Jones Gerwyn M. , O’Sullivan Denise M. , Fernandez-Gonzalez Ana , Shibayama Sachie , Fujii Shin-ichiro , Kato Megumi , Emerson Leguizamon Guerrero John , Patricia Tere Peña Claudia , Mercedes Arias Cortes María , Becht Flatschart Roberto , Neves de Medeiros Marcelo , Marcos Saraiva Antonio , Bae Young-Kyung , Yang Inchul , Yoo Hee-Bong , Bogožalec Košir Alexandra , Milavec Mojca , Dong Lianhua , Niu Chunyan , Wang Xia , Morris Phattaraporn , Temisak Sasithon , Cleveland Megan H. , Vallone Peter M. , Burke Daniel , Forbes-Smith Michael , McLaughlin Jacob , Falak Samreen , Hussels Martin , Macdonald Rainer , Kummrow Andreas , Yalçinkaya Burhanettin , Akyurek Sema , Akgoz Muslum , Vonsky Maxim , Runov Andrey , Morris Clare , Almond Neil , Huggett Jim F. 
Published: April 2024

Infection with human immunodeficiency virus (HIV)-1 leads to acquired immunodeficiency syndrome (AIDS) if left untreated. According to UN figures, approximately 39 million people globally were living with HIV in 2022, with 76% of those individuals accessing antiretroviral therapy. Measurement of plasma viral RNA load using calibrated nucleic acid amplification tests (like reverse transcription quantitative PCR, RT-qPCR) is routinely performed to monitor response to treatment and ultimately prevent viral transmission. RNA quantities measured by commercial tests can vary over many orders of magnitude, from trace single copy levels to, in cases, over 109/mL of plasma, presenting an analytical challenge for calibrating across a broad measurement range. Interlaboratory study CCQM-P199 “HIV-1 RNA copy number quantification” (April to September 2019) was conducted under the auspices of the Consultative Committee for Amount of Substance (CCQM) Nucleic Acid analysis Working Group (NAWG), with the aims of supporting national metrology institutes (NMIs) and designated institutes (DIs) development of the capacity and evaluating candidate reference measurement procedures for applied viral nucleic acid measurements.

Thirteen laboratories participated in CCQM-P199 and were requested to report the RNA copy number concentration, expressed in copies per microliter, of the HIV-1 group specific antigen (gag) gene ofin vitrotranscribed RNA molecules at low (≈ 103/μL) and high concentration (≈ 109/μL) (Study Materials 1 and 2, linked by gravimetric dilution) and purified genomic RNA from cultured virus (Study Material 3). Study Materials 1 and 3 were measured by participants using one-step reverse transcription digital PCR (RT-dPCR) (Bio-Rad reagents) and/or two-step RT-dPCR with alternative cDNA synthesis reagents. Study Material 2 was measured by both RT-dPCR (one-step) (n= 4) and orthogonal methods: single molecule flow cytometric counting (n= 2), high performance liquid chromatograph (HPLC) (n= 1) and isotype dilution-mass spectrometry (ID-MS) (n= 1).

Interlaboratory reproducibilities (expressed as %CV) were 21.4 %, 15.3 % and 22.0 % for Study Materials 1, 2 and 3 respectively. Analysis of overdispersion showed that the interlaboratory variation for all three Study Materials was not accounted for in their reported uncertainties, indicating uncharacterized sources of variation remain. Although the mean values of RT-dPCR and orthogonal method results were not statistically significantly different (p= 0.46), the extrapolated mean Study Material 2 results were higher than mean Study Material 1 results (1196vs. 808 /μL;p< 0.05). Follow-up analysis of Study Material 2 purity by ultra-performance liquid chromatography (UPLC) indicated higher molecular weight (MW) impurities constituted 16.6 % of the molecules, which are hypothesised to be the cause of the HPLC and ID-MS results being higher than the majority of Study Material 1 and 2 results.

This study demonstrates that reproducible measurement of RNA templates was achieved by metrology laboratories, illustrating the potential of RT-dPCR combined with complimentary orthogonal approaches to support traceability and precision of contemporary methods for RNA quantification. This study also highlighted that detailed characterization of RNA materials and sources of bias affecting measurements such as RT efficiency is needed to further establish RT-dPCR as a primary reference measurement procedure for RNA copy number quantification.