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Research Letter

Detection of HIV RNA in dried blood spots and oral fluids

Stekler, Joanne D.a,b; Ure, Georgea; Dragavon, Joanc; Chang, Mingc; Coombs, Robert W.a,c

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doi: 10.1097/QAD.0000000000001477
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There has been increasing appreciation for the need to employ HIV-1 tests able to detect acute infection during the antibody-negative ‘window period’ [1]. Beginning in 2001, several health departments created pooled nucleic acid amplification testing (NAAT) programs to increase acute infection case finding [2–5]. However, there are barriers to widespread implementation of HIV-1 NAAT, including the fact that high-risk persons prefer less invasive specimen collection methods over venipuncture [6]. In developing countries, needs for venipuncture, sample storage, and advanced technology are other potential barriers.

In 2010, we began a prospective, cross-sectional study to compare the ability of point-of-care and laboratory-based HIV-1 tests with detect acute and early infection in real time [6–8]. This report describes findings from a pilot substudy to evaluate the performance of NAAT on non-venipuncture specimens that could be collected at home or other settings without immediate access to NAAT.

Men and transgender persons reporting sex with men in the prior year were recruited when seeking HIV testing at the Public Health – Seattle & King County Sexually Transmitted Disease Clinic. The University of Washington Institutional Review Board approved this study, and participants gave verbal consent. An additional $20 was given for substudy participation. Persons testing HIV-negative could participate quarterly.

HIV screening procedures are described elsewhere [6–8]. Oral fluid was collected using a flocked swab (Copan Diagnostics, Murrieta, California, USA) rubbed twice in the buccal sulcus between the top and bottom teeth and gums and inserted into a 2-ml cryovial. Swabs were transported within 60 min of collection to the UW Retrovirology Laboratory and immediately placed on dry ice until they could be stored at −80°C. Based on our preliminary work (data not shown), HIV-1 NAAT had lower yield when performed on saliva compared with oral mucosal surface fluid collected as described here.

Dried blood spots (DBS) were collected through fingerstick whole blood specimens into a 50 μl microcollection pipette and dispensed onto a DBS card (Whatman 903 protein saver card; GE Healthcare, Little Chalfont, UK). DBS were allowed to dry undisturbed for 24–72 h at ambient temperature and were placed into a foil packet with desiccant pouch and humidity indicator card (Humonitor MS20003–2; Multisorb Technologies Inc., Buffalo, New York, USA).

Frozen specimens from all HIV-infected participants, including antibody-positive and antibody-negative persons, were selected for batch testing after enrollment was complete. An equal number of specimens from HIV-negative participants were tested to blind laboratory staff to HIV status.

One DBS was excised using a laser cutter [9] and soaked in 2.5 ml of lysis buffer (bioMerieux, Marcy-l’Étoile, France) with agitation for 2 h. The lysate then was quantified using the Abbott RealTime HIV-1 assay and the result was corrected for a dilution factor of 50 to give an estimated HIV-1 RNA copies/ml of blood [10].

We conducted chart reviews for participants with discordant test results to confirm HIV-1 infection. Participants with reactive enzyme immunoassay and positive western blot or detectable HIV-1 RNA were considered to have confirmed infection. Analyses were descriptive, focusing on mean, median, and range of HIV-1 RNA levels detected in blood, DBS, and oral fluid swabs of persons with acute and established infection.

Between 22 February 2010 and 1 August 2014, there were 3438 study visits through the main study; 3407 visits were by men, 24 visits were by transgender women, and seven visits were by transgender men [8]. Table 1 shows the demographic and HIV test results of the eight (2.6%) participants newly diagnosed with HIV-1 infection among 308 substudy visits. Two had acute HIV-1 infection (enzyme immunoassay negative), two had discordant point-of-care test results, and four tested positive on all HIV tests performed. These study participants had a median blood plasma or serum HIV-1 RNA of 61 500 copies/ml (interquartile range 7500–146 000, range 6000–347 000); HIV-1 RNA was detected in 7 (87.5%, 95% confidence interval 47.3–99.7%) DBS and 3 (37.5%, 95% confidence interval 8.5–75.5%) oral fluid swabs. HIV-1 RNA was not detected in either DBS or oral fluid from one participant with established infection. All HIV-negative specimens tested negative (data not shown).

Table 1
Table 1:
Clinical and research HIV test results for eight HIV-infected study participants.

These data suggest that HIV-1 NAAT performed on DBS may be a relatively accurate substitute for performance on blood specimens but do not support the use of oral fluids. This observation, in conjunction with our prior work showing that HIV antibody testing performed on oral fluids is significantly less accurate than testing on fingerstick whole blood [7,11], suggests that oral fluid specimens should not be prioritized in the development of new testing technologies.

Prior studies of oral fluids and saliva have shown greater yield [12–16]. HIV-1 RNA levels in oral fluids are 10- to 100-fold lower than in blood plasma [12,13,16] and may be less than 10 000 copies/ml, even during acute HIV-1 infection [12]. However, in one study, HIV-1 RNA was detected in the saliva of three untreated study participants who had undetectable blood plasma HIV RNA levels [16].

Our study had several limitations that were primarily because of small numbers. We recruited persons not previously diagnosed with HIV-1 infection to enroll individuals naïve to antiretroviral therapy and identify persons with acute infection. It was particulalrly challenging to recruit persons with established infection who had just received a new HIV-1 diagnosis through the main study.

Our results suggest that research into HIV testing using DBS should be advanced. Home self-testing for HIV has potential to reach HIV-positive persons who would not otherwise test for HIV. However, self-testing will reduce access to sensitive tests and could result in increased HIV prevalence because of window period testing [17] unless NAAT can be tailored to home specimen collection. Of the 116 men randomized to self-testing in our iTest study [18], 34 (29%) accepted DBS back-up testing. During the 15-month study period, we received 41 DBS from 17 participants, and all test results were negative (unpublished data). Pooling DBS eluates could reduce cost and time [19]; however, pooling would have resulted in our failure to detect two participants with DBS HIV-1 RNA levels below the level of quantitation. Future work should continue to expand HIV testing and testing options for high-risk populations.


We would like to thank everyone who participated in this project.

The study was supported by NIH R01 MH-83630, U01 AI-38858, UM1 AI-068618, UM1 AI-106701, and P30 AI-27757. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Alere (Waltham, Massachusetts, USA) provided Determine HIV 1/2 Ag/Ab Combo tests and controls throughout the duration of the project.

Conflicts of interest

There are no conflicts of interest.


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