Abstract：
Despite increasing scale-up of antiretroviral therapy (ART) coverage, challenges related to adherence and HIV drug resistance (HIVDR) remain. The high cost of HIVDR surveillance is a persistent challenge with implementation in resource-constrained settings. Dried blood spot (DBS) specimens have been demonstrated to be a feasible alternative to plasma or serum for HIVDR genotyping and are more suitable for lower resource settings. There is a need for affordable HIVDR genotyping assays which can amplify HIV-1 sequences from DBS specimens, particularly those with low viral loads, at a low cost. Here, we present an in-house assay capable of reliably amplifying HIV-1 protease and partial reverse transcriptase genes from DBS specimens, which covers the complete World Health Organization 2009 list of drug resistance mutations under surveillance. DBS specimens were prepared using whole blood spiked with HIV-1 at concentrations of 10,000, 5000, 1000, and 500 copies/mL (n=30 for each concentration). Specimens were tested in triplicate. A two-step approach was used consisting of cDNA synthesis followed by nested PCR. The limit of detection of the assay was calculated to be approximately 5000 (95% CI: 3200-10,700) copies/mL for the protease gene and 3600 (95% CI: 2200-10,000) copies/mL for reverse transcriptase. The assay was observed to be most sensitive with higher viral load specimens (97.8% [95% CI: 92.2-99.7]) for both protease and reverse transcriptase at 10,000 copies/mL with performance decreasing with the use of specimens with lower viral loads (46.7% [36.1-57.5] and 60.0% [49.1-70.2] at 500 copies/mL for protease and reverse transcriptase, respectively). Ultimately, this assay presents a promising opportunity for use in resource-constrained settings. Future work should involve validation under field conditions including sub-optimal storage conditions and preparation of DBS with fingerprick blood in order to accurately reflect real-world collection scenarios.
摘要：
尽管抗逆转录病毒疗法（ART）的覆盖面不断扩大，与依从性和HIV耐药性(HIVDR)相关的挑战依然存在.HIVDR监测的高成本是在资源有限的环境中实施的持续挑战。已证明干血斑（DBS）标本是用于HIVDR基因分型的血浆或血清的可行替代方法，并且更适合较低的资源设置。需要负担得起的HIVDR基因分型测定，可以从DBS样本中扩增HIV-1序列，特别是那些病毒载量低的人，以低成本。这里,我们提出了一种能够可靠地从DBS标本中扩增HIV-1蛋白酶和部分逆转录酶基因的内部检测方法，涵盖了世界卫生组织2009年监测的耐药突变的完整清单。使用以10,000、5,000、1,000和500拷贝/mL的浓度掺入HIV-1的全血制备DBS样本(对于每个浓度n=30)。样品一式三份进行测试。使用两步方法，包括cDNA合成，然后进行巢式PCR。对于蛋白酶基因,测定的检测极限计算为约5,000(95%CI:3,200-10,700)拷贝/mL,对于逆转录酶为3,600(95%CI:2,200-10,000)拷贝/mL。对于10,000拷贝/mL的蛋白酶和逆转录酶，病毒载量较高的标本（97.8％[95％CI：92.2-99.7]），观察到该测定最敏感，随着使用具有较低病毒载量的标本（蛋白酶和逆转录酶在500拷贝/mL时为46.7％[36.1-57.5]和60.0％[49.1-70.2]，分别)。最终,该分析为在资源受限的环境中使用提供了有希望的机会.未来的工作应该包括在现场条件下的验证，包括次优的储存条件和用手指刺血制备DBS，以准确反映现实世界的收集场景。
