Bertagnolio Silvia, Soto-Ramirez Luis, Pilon Richard, Rodriguez Roberto, Viveros Monica, Fuentes Luis, Harrigan P Richard, Mo Theresa, Sutherland Don, Sandstrom Paul
World Health Organization, HIV Department, Geneva, Switzerland.
Antivir Ther. 2007;12(1):107-13.
Field-friendly methods for HIV drug resistance (HIVDR) surveillance in resource-limited regions are urgently needed. Despite evidence that dried blood spots (DBS) are suitable for HIV serology, viral load and CD4+ T-cell enumeration, no study has evaluated DBS for HIVDR genotyping. We assessed the feasibility of genotyping HIV-1 from field-collected DBS stored under challenging environmental conditions.
We prospectively collected specimens from newly diagnosed, treatment-naive HIV-positive subjects in Mexico. Whole blood was spotted onto filter cards, air dried at ambient temperature and stored with desiccant at 37 degrees C and 85% humidity for 3 months. Genotypes obtained from DBS-extracted nucleic acids using an in-house nested reverse transcription-PCR method were compared to genotypes derived from matched plasma.
Genotypes from 103 phylogenetically matched plasma and DBS were compared. In total, 90.1% of all DBS specimens could be amplified in either the region of HIV protease or the region of reverse transcriptase. Failure to amplify from DBS did not correlate with low plasma viral loads. Between paired specimens, the median nucleotide similarity was 99.95%. In the nine specimens with drug resistance mutations, all differences between pairs were partial discordances. Mutations identified in plasma were found in the majority of replicate DBS amplifications.
The results suggest that genotypes obtained from DBS are equivalent to those from plasma. DBS are a promising public health tool for HIVDR surveillance of treatment-naive subjects, especially in regions where specimens might be exposed to severe environmental conditions and where logistical difficulties could prevent timely specimen processing. More studies are needed to validate DBS for patient monitoring.
资源有限地区急需便于实地操作的HIV耐药性（HIVDR）监测方法。尽管有证据表明干血斑（DBS）适用于HIV血清学、病毒载量及CD4+T细胞计数，但尚无研究评估DBS用于HIVDR基因分型的情况。我们评估了在具有挑战性的环境条件下储存的实地采集的DBS样本进行HIV-1基因分型的可行性。
我们前瞻性地收集了墨西哥新诊断的、未接受过治疗的HIV阳性受试者的样本。将全血滴在滤纸上，在室温下空气干燥，并与干燥剂一起在37摄氏度和85%湿度下储存3个月。使用内部巢式逆转录-PCR方法从DBS提取的核酸中获得的基因型与匹配血浆中获得的基因型进行比较。
比较了103对系统发育匹配的血浆和DBS的基因型。总体而言，所有DBS样本中有90.1%在HIV蛋白酶区域或逆转录酶区域能够扩增。无法从DBS中扩增与血浆病毒载量低无关。在配对样本之间，核苷酸相似性中位数为99.95%。在9个具有耐药性突变的样本中，配对样本之间的所有差异均为部分不一致。血浆中鉴定出的突变在大多数重复的DBS扩增中都能发现。
结果表明，从DBS获得的基因型与从血浆中获得的基因型相当。DBS是一种很有前景的公共卫生工具，可用于对未接受过治疗的受试者进行HIVDR监测，特别是在样本可能暴露于恶劣环境条件且后勤困难可能妨碍及时样本处理的地区。需要更多研究来验证DBS用于患者监测的情况。
