The feasibility of modified HIV and antiretroviral drug testing using self-collected dried blood spots from men who have sex with men

The “Evaluation of HIV Self-Testing Among MSM Project” (eSTAMP) was a CDC funded study that evaluated the effect of providing self-testing kits on sexual risk behaviors, partner testing, and linkage to care after self-tested positive. The study recruited MSM online to participate in a formative study [6] in May–December 2014 and a randomized controlled trial (RCT) [4] in March 2015–December 2016. Eligibility for eSTAMP included male at birth and identified as male, ≥ 18 years old, residing in the US, HIV-negative or unaware of HIV status, able to read in English, anal sex with ≥1 man during the past 12 months, not on antiretroviral medications to prevent HIV, never in an HIV vaccine trial, and not having a bleeding disorder. In the formative study, after completing a baseline survey participants were mailed a study package containing one OraQuick In-Home HIV test (OraSure Technologies, Bethlehem, PA), a self-test version of Sure Check HIV 1/2 Assay (Chembio Diagnostic System Inc., Medford, NY) used under an Investigational Device Exemption from the Food and Drug Administration (FDA), and a DBS collection kit. Participants were asked to submit test results online after self-testing. In the one-year RCT, participants were required to complete not only baseline but also quarterly follow-up online surveys. They were mailed a study package after completing the12-month follow-up survey or reporting an HIV positive test result. All eSTAMP participants who received the study package were instructed to collect their own DBS sample within 48 h of self-testing. The DBS collection kit included written instructions, a web-link to a demonstration video, two Whatman 903 protein saver DBS cards (GE healthcare Bio-sciences, Pittsburgh, PA), two Microtainer contact-activated lancets (Becton Dickinson and Co., Franklin Lakes, NJ), two sterile gauze, two alcohol swabs, two bandages, three desiccants, a humidity indicator card (Poly Lam Products, Corp., Williamsville, NY), two gas-permeable plastic zipper seal bags, and a pre-paid return envelope. All participants provided electronic informed consent. Figure 1 illustrates the flow of DBS from collection to processing at the CDC laboratory.

The American Men’s Internet Survey (AMIS) study is an annual online behavioral surveillance survey of MSM residing in the US [7, 8]. Eligibility criteria included male at birth and male gender identity, ≥15 years old, residing in the US, and reported oral or anal sex with a man at least once at any time in the past. Survey respondents were asked to provide an email address for future study invitations. Participants of the 2017 and 2018 data collection cycles who were at least 18 years of age and self-reported a positive HIV status were emailed an invitation to participate in the DBS VL study. After consenting to participate in the DBS collection and completing an online survey, participants were mailed a DBS collection kit containing written instructions, one Whatman 903 protein saver DBS card, two contact-activated lancets, two sterile gauze, two alcohol pads, two bandages, five desiccants, a humidity indicator card, one plastic zipper bag, and a pre-paid return envelope. In the online survey, participants answered questions about whether they missed any ARV doses in the past 4 and 30 days and how often they took ARV as directed in the past 30 days. Participants were considered ARV adherent if they didn’t report missing any doses in the past 4 days and reported “always” or “almost always” taking ARV as directed in the past 30 days. The electronic informed consent in the AMIS protocol included only VL testing.

Participants from both studies were instructed to write the date they collected their blood on the card and to follow the mailing instructions provided with the DBS collection kits.

DBS from eSTAMP were first tested with the FDA-approved Avioq HIV-1 Microelisa System to detect antibody to HIV (Avioq) (Avioq, Inc., Rockville, MD) and, if repeatedly reactive, followed with GS HIV-1 Western Blot (WB) (Bio-Rad laboratories, Redmond, WA) for confirmation. Each test requires one 6-mm punch of DBS and were performed following package insert instructions [9, 10]. For DBS cards with sufficient spots, a modified GS HIV Combo Ag/Ab EIA (Ag/Ab) (Bio-Rad laboratories) DBS protocol was performed to detect HIV p24 antigen and antibody, followed by a modified Geenius HIV-1/2 Supplemental Assay (Geenius) (Bio-Rad laboratories) DBS protocol if repeatedly reactive. These two modified protocols were validated and published previously [11]. Due to the limited blood spots per card, we modified the Abbott RealTime HIV-1 RNA DBS protocol [12] with a limit of detection (LOD) of 2.92 log₁₀ (copies/mL) using a 12 mm perforated circle (~ 70 μL whole blood) to use four 6 mm punches of DBS (~ 50 μL whole blood) [13]. The validated 4-punch protocol showed a linearity agreement of R² = 0.924 when comparing the DBS and 0.8 mL plasma protocols (unpublished data).

Concentrations of Tenofovir (TFV), Emtricitabine (FTC), Abacavir (ABC), Lamudivine (3TC), Efavirenz (EFV), and Raltegravir (RAL), in DBS were measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) (Sciex, Foster City, CA). TFV/FTC as well as ABC/3TC capture the nucleoside analog backbones of commonly used antiretroviral regimens. The non-nucleoside reverse transcriptase inhibitor EFV was a component in some regimens at the time of sampling. The integrase inhibitor RAL is commonly added to nucleoside analog backbones in antiretroviral therapy. The procedure was described in our previous publication [13].

Using the DBS collected in eSTAMP, we compared the HIV serological results using FDA-approved protocols with laboratory-modified protocols to evaluate the feasibility of HIV testing using DBS. VL and ARV were measured to examine the correlation between VL levels and ARV presence in samples with enough quantity of spots and to fully characterize HIV-positive test results. The VL results from AMIS DBS were used to further assess the feasibility of using the DBS for monitoring viral suppression.

eSTAMP participants were recruited from across the US (38.7% South, 23.5% West, 19.3% Midwest, 18.5% Northeast) and most (69.5%) were between 18 and 34 years old. Participants were mostly non-Hispanic white (67.9%) followed by 19.6% Hispanic, 7.4% black, and 5.1% Asian.

AMIS study participants were from across the US as well and 74.4% were non-Hispanic white, 12.8% Hispanic, 7.7% black, and 5.1% others or multiple races. The majority (56.4%) of AMIS participants were over 50 years old.

From May 2014 to December 2016, 2313 eSTAMP participants were mailed a DBS collection kit and 1456 (62.9%) returned self-collected cards to the CDC laboratory. Twelve cards were rejected for testing; the spots were too small for a single 6 mm punch (n = 6) or the participant’s unique identifier was not written on the card (n = 6). Of 1444 (98.0%) cards eligible for testing, the median time at ambient temperature from collection to arrival at the laboratory was 8 days; 1300 (90.0%) were received within 14 days, 93 (6.5%) from 14 to 21 days, and 51 (3.5%) more than 3 weeks from the collection date written on the card. Regarding the quantity of spots either partially or fully filled with blood, 1286 (89.1%) had five spots, 97 (6.7%) had four spots, 58 (4.0%) had three spots, and three cards (0.2%) had one or two spots filled. Based on the humidity indicator, 637 (44.1%) were exposed to high humidity (≥50%) and 38 (2.6%) did not have a humidity indicator upon arrival at the CDC laboratory. Of the remaining 769 (53.3%) that were within recommended storage condition for Avioq testing (< 50% humidity) [9], 525 (36.4%) and 244 (16.9%) were exposed to 40 and 30% humidity, respectively.

Out of 111 AMIS participants, 78 (70.3%) mailed self-collected DBS back to CDC laboratory for VL testing. Nearly half of them were missing the date of sample collection, therefore we calculated the median days from when the DBS cards were mailed to participants until their arrival at the CDC laboratory. The median number of days until arrival was 11.5 days (range 4 to 70 days) and 67.9% (53/78) of AMIS DBS cards were received within 14 days. All 78 had five spots either partially or fully filled with blood, which was sufficient for VL assay. Six cards (7.7%) were sent without humidity indicators. Ten (12.8%) and 62 (79.5%) were exposed to 40% and ≤ 30% humidity, respectively.

Of 1444 cards tested with Avioq, 32 cards (2.2%) were repeatedly reactive (Fig. 2). Of those, 31 were confirmed as WB HIV-1 positive and one was WB HIV-1 negative. Of 31 Avioq-reactive/WB positive, 27 had sufficient spots for Bio-Rad Ag/Ab testing and all were reactive. The one Avioq-reactive/WB HIV-negative DBS was non-reactive by Bio-Rad Ag/Ab. Among 1400 Avioq-non-reactive DBS, Bio-Rad Ag/Ab was non-reactive in 1397 (99.8%), and three were initially reactive but were not repeated in duplicate due to insufficient quantity. The initial signal/cutoff ratio for the three samples were 1.2, 1.4, and 1.5, respectively, and all three had undetectable VL and ARV levels.

Nine of 27 reactive samples had sufficient quantity of spots for Geenius testing, VL, and ARV measurement by HPLC-MS (Table 1). All were Geenius HIV-1 positive, and two samples without detectable ARV had VL > 5 log₁₀ (copies/mL). In contrast, of seven samples with presence of at least one ARV drug, five had undetectable VL and two had VL values of 3.29 and 3.58 log₁₀ (copies/mL).

To further assess the use of the DBS for VL monitoring, VL results from the AMIS study were compared to self-reported ARV adherence. Of 78 DBS, 4 were excluded due to contradictory responses to questions about taking ARV medications, 63 had undetectable VL, seven had detectable but unquantifiable VL (< 2.92 log₁₀ (copies/mL)), and four had quantifiable VL ranging from 3.04 to 5.30 log₁₀ (copies/mL). Of the four with quantifiable VL, three were not ARV adherent and one was not on ARV treatment. Among the seven with detectable but unquantifiable VL, five participants were considered adherent; one missed doses on 2 of 4 days, and one didn’t record the ARV information in the survey. Table 2 lists the 11 detectable VL results and their self-reported adherence measures. Among 63 participants with undetectable VL, three missed doses on 2 of 4 days, six missed doses on 1 of 4 days, and one was not taking ARV.