García-Lerma J Gerardo, McNulty Amanda, Jennings Cheryl, Huang Diana, Heneine Walid, Bremer James W
Laboratory Branch, Division of HIV/AIDS Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
J Antimicrob Chemother. 2009 Jul;64(1):33-6. doi: 10.1093/jac/dkp150. Epub 2009 Apr 29.
Dried blood spots (DBS) and dried plasma spots (DPS) are considered convenient alternatives to serum and plasma for HIV drug resistance testing in resource-limited settings. We sought to investigate how extreme conditions could affect the short-term ability to amplify and genotype HIV from DBS.
A panel of six matched DPS/DBS was generated using blood collected from HIV-infected donors. Replicate cards were prepared in 903 filter paper using 50 microL of blood and stored at either -20 degrees C or at 37 degrees C/100% humidity. Nucleic acids were extracted at baseline and after 1, 2, 8 and 16 weeks of storage and were amplified and sequenced using an in-house RT-nested PCR method or the ViroSeq assay.
HIV-1 pol was successfully amplified in all DBS/DPS at baseline and in those stored for up to 16 weeks at -20 degrees C by the in-house assay. In contrast, amplification was rapidly lost during storage at 37 degrees C/100% humidity with only 6/6 and 4/6 DBS specimens amplifiable by the in-house assay at weeks 1 and 2, respectively. Similarly, only two DPS stored at 37 degrees C/100% humidity were amplified by the in-house assay at week 1.
We show that resistance testing from DBS and DPS is severely compromised after 2 and 1 weeks of storage at 37 degrees C/100% humidity with desiccant, respectively. These findings underscore the importance of temperature and humidity for the efficient genotyping of HIV-1 from DBS and DPS, and reiterate the need to rapidly transport specimens from collection sites to locations that have appropriate storage conditions such as -20 degrees C.
在资源有限的环境中，干血斑（DBS）和干血浆斑（DPS）被认为是用于HIV耐药性检测的血清和血浆的便捷替代物。我们试图研究极端条件如何影响从DBS中扩增HIV并进行基因分型的短期能力。
使用从HIV感染供体采集的血液制备一组六个匹配的DPS/DBS。在903型滤纸上使用50微升血液制备重复卡片，并储存在-20℃或37℃/100%湿度环境中。在储存的基线以及1、2、8和16周后提取核酸，并使用内部RT巢式PCR方法或ViroSeq检测法进行扩增和测序。
通过内部检测法，HIV-1 pol在基线时以及在-20℃储存长达16周的所有DBS/DPS中均成功扩增。相比之下，在37℃/100%湿度下储存期间扩增迅速丧失，内部检测法在第1周和第2周分别只能扩增6/6和4/6的DBS标本。同样，在第1周时，内部检测法仅扩增了两个储存在37℃/100%湿度下的DPS。
我们表明，分别在37℃/湿度100%且有干燥剂的条件下储存2周和1周后，从DBS和DPS进行的耐药性检测会受到严重影响。这些发现强调了温度和湿度对于从DBS和DPS高效进行HIV-1基因分型的重要性，并重申了将标本从采集地点迅速转运至具有适当储存条件（如-20℃）地点的必要性。
