Assessing intra-lab precision and inter-lab repeatability of outgrowth assays of HIV-1 latent reservoir size

Quantitative viral outgrowth assays (QVOA) use limiting dilutions of CD4+ T cells to measure the size of the latent HIV-1 reservoir, a major obstacle to curing HIV-1. Efforts to reduce the reservoir require assays that can reliably quantify its size in blood and tissues. Although QVOA is regarded as a “gold standard” for reservoir measurement, little is known about its accuracy and precision or about how cell storage conditions or laboratory-specific practices affect results. Owing to this lack of knowledge, confidence intervals around reservoir size estimates—as well as judgments of the ability of therapeutic interventions to alter the size of the replication-competent but transcriptionally inactive latent reservoir—rely on theoretical statistical assumptions about dilution assays. To address this gap, we have carried out a Bayesian statistical analysis of QVOA reliability on 75 split samples of peripheral blood mononuclear cells (PBMC) from 5 antiretroviral therapy (ART)-suppressed participants, measured using four different QVOAs at separate labs, estimating assay precision and the effect of frozen cell storage on estimated reservoir size. We found that typical assay results are expected to differ from the true value by a factor of 1.6 to 1.9 up or down. Systematic assay differences comprised a 24-fold range between the assays with highest and lowest scales, likely reflecting differences in viral outgrowth readout and input cell stimulation protocols. We also found that controlled-rate freezing and storage of samples did not cause substantial differences in QVOA compared to use of fresh cells (95% probability of < 2-fold change), supporting continued use of frozen storage to allow transport and batched analysis of samples. Finally, we simulated an early-phase clinical trial to demonstrate that batched analysis of pre- and post-therapy samples may increase power to detect a three-fold reservoir reduction by 15 to 24 percentage points.

定量病毒生长测定法（Quantitative viral outgrowth assays, QVOA）通过对CD4阳性T细胞（CD4+ T cells）进行有限稀释，以检测潜伏HIV-1储存库的规模——该储存库正是攻克HIV-1感染的主要障碍。针对缩小该储存库规模的研究，亟需能够可靠量化其在血液与组织中丰度的检测手段。尽管QVOA被公认为储存库检测的“金标准”，但学界对其准确性与精密度，以及细胞储存条件、实验室特异性操作流程如何影响检测结果，仍缺乏深入认知。由于这一研究空白，当前围绕储存库规模估算的置信区间，以及对治疗干预手段能否改变具有复制能力但转录沉默的潜伏储存库规模的判断，均依赖于针对稀释测定法的理论统计假设。为填补这一研究缺口，我们对来自5名接受抗逆转录病毒治疗（antiretroviral therapy, ART）且病毒得到抑制的受试者的75份外周血单个核细胞（peripheral blood mononuclear cells, PBMC）分装样本，开展了QVOA可靠性的贝叶斯统计分析；上述样本在不同实验室中通过4种不同的QVOA方案完成检测，借此估算了检测精密度以及冷冻细胞储存对预估储存库规模的影响。我们发现，典型的QVOA检测结果与真实值的偏差幅度预计在1.6至1.9倍之间。不同检测方案间存在系统性差异：检测尺度的最高值与最低值之间相差达24倍，这大概率反映了病毒生长读数方法与输入细胞刺激方案的差异。我们还发现，与使用新鲜细胞相比，对样本进行控制性速率冷冻并储存，并不会导致QVOA检测结果出现显著差异（95%概率下变化幅度小于2倍），这支持了继续采用冷冻储存手段以实现样本运输与批量检测的可行性。最后，我们模拟了一项早期临床试验，结果表明，对治疗前后的样本进行批量分析，可将检测到3倍储存库规模缩减的统计效力提升15至24个百分点。