Acute HIV infection (AHI) diagnosis, defined as the period between the appearance of HIV RNA and detectable HIV antibodies, as measured by western blot, continues to be a global challenge. Cohort studies in Europe and the Americas suggest that during AHI, the rate of HIV transmission is 26 times higher compared to chronic infection in sero-discordant couples , and account for up to 50% of new infections in MSM [2–5]. Mathematical modeling predicts that during AHI in a high-risk MSM cohort prior to initiation of antiretroviral therapy (ART), a patient on ART could cumulatively infect 65% less individuals over a 180-day period compared to one who is treatment-naïve . Another study with HIV-serodiscordant couples reported a reduction in HIV transmission if prevention interventions were performed during early HIV infection . An ongoing AHI study in Thailand demonstrated that patients treated with ART during AHI had a 14-fold reduction in HIV DNA in peripheral blood CD4+ T cells, implying a reduction in the HIV reservoir . A recent study in the simian immunodeficiency virus/rhesus macaque model is consistent with that seen in humans . Therefore, it has been argued that treatment should be initiated early following HIV infection, for clinical benefit and transmission prevention [10,11].
Fourth-generation (4thG) combination HIV antigen/antibody immunoassays have been used in Europe since 1997, and are increasingly used in Asia . The main goal of their development was to permit earlier detection of viral infection. The US Food and Drug Administration (FDA) approved the first 4thG immunoassay in June 2010. These assays, unlike the more routinely used second (2ndG) and third-generation (3rdG) immunoassays, detect not only antibodies but also HIV p24 antigen. It has been estimated that the use of 4thG immunoassay in the diagnosis of HIV infection has shortened the ‘window’ to diagnosis relative to 3rdG immunoassay by 4–15 days, depending on the test kit used [12–14].
Nucleic acid testing (NAT) has been proposed for even earlier detection of HIV infection. The US FDA has approved the Aptima NAT assay (Gen-Probe Inc., San Diego, California, USA) for the diagnosis of HIV infection in conjunction with other diagnostic tests, or independently, if repeat specimens are reactive. The Association of Public Health Laboratories and the US Centers for Disease Control have proposed the use of NAT (either with single or pooled specimens) for detecting AHI . Whereas the Aptima assay is currently not approved for pooled sample testing, studies have shown that pooling can detect HIV RNA in samples nonreactive with 4thG immunoassay [14,16,17]. The use of NAT on 4thG immunoassay nonreactive samples showed increased AHI case-detection rates, ranging from 12.7 to 46.6%, but was dependent on the 4thG immunoassay test kit used .
Thailand is currently experiencing an explosive HIV epidemic in young MSM . Our aim was to identify AHI patients as early as possible to initiate ART for improved disease prognosis. We incorporated NAT as an additional platform to assess whether it would cost effectively increase the number of AHI cases detected relative to 4thG immunoassay. We describe the impact of NAT in combination with 4thG immunoassay on the diagnosis of AHI.
The Thai Red Cross AIDS Research Centre (TRCARC) is an anonymous HIV voluntary counseling and testing (VCT) center in Bangkok. Clients are provided a unique identifier at their first testing visit, which is retained for subsequent visits, and an optional self-administered demographic and risk-behavior questionnaire. Data and samples are stored without any personal identifiers. We previously reported on the demographics of the clientele at the TRCARC from 2006 to 2007. Clients were predominantly heterosexual (60%), with MSM representing 15% of the population, although they had the highest prevalence of HIV infection (25%) .
RV254/SEARCH 010 is a study conducted at the TRCARC, identifying AHI and offering immediate ART to patients. The study was approved by the Institutional Review Boards of Chulalongkorn University in Thailand and the Walter Reed Army Institute of Research in the United States. Samples from HIV-testing clients were screened (S01) to identify and enroll (V01) patients with AHI (clinicaltrials.gov identification numbers: NCT00796146 and NCT00796263) [8,21]. The current study was performed from June 2009 to February 2014.
The TRCARC standard HIV diagnostic algorithm incorporates 4thG immunoassay, but not NAT. The RV254/SEARCH 010 study expanded the algorithm to include NAT and additional immunoassays at S01 to detect AHI (Fig. 1). During the course of the study, three 4thG immunoassays (AxSYM HIV antigen/antibody Combo, Abbott Laboratories, Wiesbaden, Germany from June 2009 to December 2010; HIV Combi Assay, Roche Diagnostics, London, UK from January 2011 to June 2011; and the ARCHITECT HIV antigen/antibody Combo, Abbott Diagnostics, Delkenheim, Germany from July 2011 to February 2014) were used due to reagent supply issues or the manufacturer phasing out a test kit. These 4thG immunoassays use recombinant transmembrane protein and/or peptides to gp41 of HIV group M and O and detect HIV antigen by anti-p24 monoclonal antibody . Two of the three 4thG immunoassays used are reported to have a similar time delay in comparison to HIV NAT detection: AxSYM – 5.8 days, HIV ARCHITECT – 5.2 days, with the HIV Combi having a longer time interval of 7.8 days . Nonreactive 4thG immunoassay samples were pooled and screened by qualitative NAT (Fig. 1). Patients with reactive/nonreactive 4thG immunoassays or reactive 4thG immunoassays/nonreactive 3rdG enzyme immunoassay (EIA) (Genscreen HIV 1/2; Bio-Rad, Marnes la Coquette, France) or 2ndG EIA (Genetic Systems rLAV EIA; Bio-Rad Laboratories, Redmond, Washington, USA), replaced in 2013 with Avioq HIV-1 Microelisa (Avioq, Inc., Research Triangle Park, North Carolina, USA) and western blot (Genetic Systems HIV-1; Bio-Rad Laboratories) negative or indeterminate tests were contacted and requested to return to the TRCARC for a repeat blood draw within 10 days to confirm HIV infection and V01 with the identical assay platforms used at S01 (Figs 1 and 2). HIV p24 antigen assay (ABL Inc., Kensington, Maryland, USA) with a lower limit of detection of 3.1 pg/ml was performed on S01 and V01 samples, without immune-complex dissociation. Samples below the limit of detection were scored as 0 pg/ml p24 antigen for data analysis. Patients were further categorized using the Fiebig (F): FI, NAT+, p24antigen−, HIV IgM EIA−, western blot−; FII, NAT+, p24antigen+, HIV IgM EIA−, western blot−; FIII, NAT+, p24antigen+, HIV IgM EIA+, western blot−; FIV, NAT+, p24antigen+/−, HIV IgM+, western blot indeterminate; FV, NAT+, p24antigen+/−, HIV IgM+, western blot+ (without p31 band); FVI, NAT+, p24antigen+/−, HIV IgM+, western blot+ ; and 4thG – 4thG 1, NAT+/4thGimmunoassay−/3rdG immunoassay−; 4thG 2, NAT+/4thG immunoassay+/3rdG immunoassay−; 4thG 3, NAT+/4thG immunoassay+/3rdG immunoassay+/western blot− or indeterminate  staging systems for AHI. Patients were predominantly infected with CRF01_AE [8,20].
HIV detection by nucleic acid testing
The Aptima HIV qualitative assay has probes specific for two regions of the HIV genome and a 98% detection frequency of 30 copies/ml, correlating with approved quantitative HIV RNA tests [23,24]. An analyte signal/cut-off (S/CO) ratio of at least 1 indicates that the sample is reactive for HIV RNA. Samples submitted for NAT were first pooled in a single step into a total volume of 600 μl, although the pool size could vary from 3 to 30 individuals. The number of individuals per pool was a function of the number of 4thG immunoassay nonreactive patients visiting the TRCARC in a single day and costs. All samples were tested within 24 h of collection, as the goal was to identify AHI as quickly as possible at screening for enrollment into RV254/SEARCH 010. A prior study at the TRCARC had used 40 patients per pool , but the maximum pool size was reduced to 30 patients based on technical issues and costs, as deconvoluting the pool would allow testing of up to 90 (three pools) versus 80 patients (two pools) in a single assay run . In the event that the pool size was of three samples, 200 μl of each was pooled, and if the pool size was of 30 samples, then 20 μl of each specimen was pooled. Pooled samples (500 μl volume) were then assayed according to the manufacturer's instructions. Reactive pools were deconstructed and individual samples were re-tested by the Aptima to identify the HIV RNA-positive patient(s), and a quantitative viral load assay was also performed (sample volume permitting) on all patients at V01 (Fig. 2). The quantitative viral load assay used was the Amplicor 1.5 ultrasensitive assay with a lower detection limit of 50 HIV RNA copies/ml, which was later replaced by the COBAS TaqMan HIV-1 version 1.0, with a lower limit of detection of 48 copies/ml (both from Roche Diagnostics, Branchburg, New Jersey, USA). Viral load quantitative tests were performed at the College of American Pathologists accredited facilities in Bangkok.
Demographic and HIV disease-related characteristics are described as median (minimum-to-maximum range) or frequencies. The time from history of self-reported HIV exposure within the past 30 days to the S01 visit was estimated for each patient. The median time was used for patients with multiple dates of possible HIV exposure. Incidence of HIV infection combining both the NAT and sequentially reactive/nonreactive immunoassay samples was calculated as previously described using a 28-day period from RNA detection to antibody development per 100 person-years in HIV-seronegative patients diagnosed with the standard TRCARC testing algorithm (Fig. 1) [25–27]. Prevalence of HIV was calculated by dividing the total number of patients, defined as HIV-seropositive with the standard TRCARC algorithm, by the total number of clients tested. Ninety-five percent Cls were calculated using the Clopper–Pearson interval. The Mann–Whitney U test and the chi-square test were used to compare continuous and categorical variables between groups. Spearman's correlation was used to assess the association between p24antigen and HIV RNA levels. Analyses were performed with Stata (Stata Statistical Software: Release 12; StataCorp, College Station, Texas, USA) and Prism version 6.0e (Graphpad Software, La Jolla, California, USA).
Between June 2009 and February 2014, 74 334 clients visited the TRCARC for HIV VCT, of whom 8129 tested positive using the TRCARC diagnostic algorithm. The prevalence (95% CI) of HIV over the 4.7-year study period was 10.9% (10.7–11.2). Using the 4thG immunoassay reactive/3rdG immunoassay nonreactive (or reactive with 2ndG nonreactive) and 4thG immunoassay nonreactive/NAT pooling screening strategy, 112 AHI patients were identified. All patients were enrolled into the RV254/SEARCH 010 study and were confirmed with supplemental tests at V01 and subsequent visits. Eighty-one patients were identified by 4thG immunoassay reactivity and an additional 31 (28%) by NAT screening of 4thG immunoassay nonreactive samples. As different 4thG immunoassay assays were used during the course of the study, we assessed the frequency of detection of each kit relative to NAT reactivity. The currently used ARCHITECT assay detected 66 of 86 (77%) AHI, followed by AxSYM [13/22 (59%)] and HIV Combi [2/4 (50%)]. Estimates of the incidence of AHI in HIV-seronegative patients, using 4thG immunoassay reactive with sequential nonreactive less sensitive EIA only was 1.6% (95% CI 1.2–1.9) per 100 person-years, which increased to 2.2% (95% CI 1.8–2.6) when combined with NAT on 4thG nonreactive specimens (Fig. 1). The majority of AHI patients were FI  and 4thG 2  or higher (Table 1). A total of 6565 pools from 66 205 patients with 4thG nonreactive immunoassays were tested by NAT. The median pool size was 14 (3–39) and results were invalid for 300 pools (5%), due to failure of the kit calibrators. Samples in the invalid pools were re-tested and were NAT nonreactive. Of the 6565 pools, 31 (0.5%) were reactive, with a median pool size of 15 individuals (12–17). Deconstructing the pools for individual NAT yielded single reactive specimens in each pool. The S/CO ratio was similar when testing pooled versus individual samples (median 23.44 versus 23.99, respectively; P = 0.68). AHI infection at S01 detected by NAT on 4thG immunoassay nonreactive specimens increased the number of patients at FI by almost two-fold (Table 1).
Quantitative viral load assays were positive for all 31 patients, but due to sample volume limitations at S01, end-point viral load measurements were available for only 28 patients, with the remaining three having viral load at least 100 000 RNA copies/ml. The median viral load for 4thG immunoassay nonreactive samples was 2482 (82–84 545) compared to 577 900 HIV RNA copies/ml (2590–37 050 000) for 4thG immunoassay reactive specimens (P < 0.001). The Aptima assay detected HIV RNA at a concentration of more than 6 copies RNA/ml.
Patients with AHI were predominantly men (96%) and MSM/bisexual (92%), with a median age and CD4+ cell count of 26 years and 353 cells/μl, respectively. The reported median time from HIV exposure to S01 was 17 days (1–53). Eighty-nine patients (74%) had CD4+ cell counts less than 500 cells/μl [median 320 (7–497) at enrollment] (Table 2). Comparing AHI cases detected by the two methods, those detected by NAT had significantly higher median CD4+ cell counts (+109 cells/μl), lower median viral load (—1.4 log10 RNA copies/ml), and a shorter time from HIV exposure (6 days) compared to those detected by 4thG immunoassay (Table 2). The median viral load for patients at screening was 86 503 RNA copies/ml (82–37 050 700). The median time from S01 to V01 was 2 days (1–5), during which time the median viral load increased by 6.6-fold (Table 2) and the highest frequencies of AHI were at FIII and 4thG 3 (Table 1). Of the 31 patients who were 4thG nonreactive at S01, 23 (74%) remained nonreactive at V01. The frequency of p24 antigen detection at S01 was 75% (83/111), which increased to 82% (92/112) at V01. The median HIV p24 antigen plasma concentrations at S01 and V01 were 43 (0–46 040) and 73 pg/ml (0–41 763), respectively. The median viral load in patients with detectable p24 antigen (median 120.0 pg/ml, range 3.4–41 763.0) was 947 747 (26 580–47 887 300) compared to 22 314 RNA copies/ml (1044–322 660) (P < 0.0001) in p24 antigen-negative samples. There was a robust association between quantitative p24 antigen and viral load (r = 0.874, P < 0.0001). Of the 112 patients identified, 111 initiated ART at V01, and one patient was lost to follow-up after V01. CD4+ T-cell counts were available for 84 of the 89 patients, with CD4+ cell counts of less than 500 cells/μl at 24 weeks post-ART initiation, of whom 80 (95%) showed a median increase in CD4+ T-cell count of 233 cells/μl (160–350). Six months following treatment initiation, viral load was suppressed (<50 copies RNA/ml) in 97 of 106 individuals. The number of patients with more than two sexual partners in 107 questionnaire respondents declined from 77 (81%) to 32 (34%; P < 0.001) following AHI diagnosis and protocol enrollment.
The present study represents, to our knowledge, the first large-scale prospective screening comparison between 4thG immunoassay and pooled NAT measured by the Aptima test for the detection of AHI. Previous studies have assessed 4thG immunoassay performance on 3rdG immunoassay prescreened samples confirmed for HIV infection, or 4thG immunoassay screened samples with a smaller sample size [28–30]. A limitation of our study is that we used three different 4thG immunoassay test kits, of which one had a longer detection time compared to the other two relative to NAT detection. This kit was used for 6 of 57 months of the project and may have introduced some bias . The HIV-prevalence estimate was similar to that previously reported (14.1%; 95% CI 13.0–15.0) from 2006 to 2007, for the TRCARC .
The use of NAT increased the number of AHI cases from 12 with 4thG immunoassay to 17 per 10 000 samples screened. One study showed an overall improved AHI detection of 15% relative to the 4thG immunoassay, and a trend for increased detection with increased HIV prevalence , which is in agreement with other studies of AHI [30,31]. Another US study in high-risk clients reported that NAT testing improved AHI detection by 31%, as only 45 of the 64 Aptima reactive samples were 4thG immunoassay reactive, similar to the findings in the current study . One study on the performance of the 4thG HIV ARCHITECT immunoassay using diverse HIV Group M isolates defined the cut-off for p24 antigen detection as 58 500 RNA copies/ml for assay reactivity . Using this criterion, 87 and 65% of the 4thG immunoassay nonreactive study participants at S01 and V01, respectively, were below the cut-off. Our study used human plasma so the p24 antigen may have been complexed to antibody in patients with a longer time interval between virus exposure and screening.
The 4thG immunoassay lessened the time period for HIV diagnosis compared to the 3rdG immunoassay [12–14,31,33]. Current trends for early treatment following exposure to limit the HIV reservoir size, ameliorate disease and prevent transmission favor the use of NAT as a supplemental diagnostic test [5,13,33]. The use of pooled NAT in this and other studies shows that it is a feasible strategy as a supplemental test for detecting AHI, with a relatively rapid turn-around time (2 working days), using the Aptima platform [8,14,25,34,35]. The use of 3rdG, rather than 4thG, immunoassay in combination with NAT may also be a feasible testing strategy, but it is likely to increase costs due to the higher numbers of samples that would be submitted for NAT. Our finding in the current study has been that 4thG is detecting almost twice the number of AHI compared to 3rdG (data not shown), but this would vary with the kits used.
The Aptima platform has previously been used with 16, 32, and 128 specimen pool sizes [16,17], with HIV RNA detected in all runs using a standard diluted to 67 RNA copies/ml . Our study using human plasma detected HIV down to 82 RNA copies/ml in a single sample from a pool of 13, similar to our previous finding using a different NAT platform .
The p24 antigen assay detected 17% of 4thG nonreactive samples, but was negative in 4% of 4thG immunoassay reactive samples at the screening visit (data not shown). The window for p24 antigen detection is very short and would not identify patients at FI and could miss patients at FIV and above , which could have been up to 47% (53/112) of patients detected by NAT and sequential immunoassay at screening in this study. The p24 antigen assay detected AHI at an equivalent frequency as the 4thG immunoassay at both S01 (76 versus 72%, respectively) and V01 (82 versus 81%, respectively). One study, which used similar technology to that used in the current study, reported that the sensitivity of p24 antigen tests in detecting AHI was 79% compared to 100% for NAT . However, there is currently no US FDA-licensed or CE-marked p24 antigen test kit for HIV diagnostics, and the assay has lower throughput and a longer performance time compared to the 4thG immunoassay platform used in this study.
On the basis of the epidemiologic studies in MSM residing in Bangkok showing HIV prevalence and incidence ranging from 21.7 to 25.3% [19,20] and 2.7 to 5.9 per 100 person-years, respectively [19,25], those presenting to the TRCARC are generally at high risk. A limitation of this study was that risk factors were unavailable for all TRCARC clients, so we could not assess whether MSM were more likely to have AHI relative to other groups. A previous study at the TRCARC reported that AHI is biased towards MSM .
The cost of a 4thG immunoassay at the TRCARC (excluding labor and controls) is approximately $8 and screening by the Aptima platform run as a full kit including pooling is $27. Minimum costs for screening of the study cohort by 4thG only would be $616 972. A NAT pooling strategy of 14 samples per pool would incur a minimal cost of $144 473, although given our experience, 15 individuals per pool are preferred, further reducing NAT screening costs to $135 486. The addition of NAT in the screening procedure described here increases per patient testing costs from $8.33 for 4thG immunoassay only to $10.16 for 4thG immunoassay + NAT – an increase of less than 25%. Additionally, alternative NAT assays with high sensitivity and specificity may further reduce costs. Estimates from the United States have proposed that AHI missed by 4thG immunoassay and detected by NAT were from high-risk patients who could potentially result in 0–3.4 new infections over 1 year. These new infections could incur a lifetime US healthcare cost in excess of $1 million, which could be avoided provided the participants changed their risk behavior . Identifying AHI in the current study resulted in the group demonstrating a marked reduction from prediagnosis in the number of sexual partners at 6 months following protocol entry.
Our experience with the use of pooled NAT over the past 8 years has shown that it is feasible for detecting AHI, but the decision to incorporate this strategy should depend on the HIV infection risk of the testing clientele. One US study tested over 18 000 low-risk patients using pooled NAT and reported no AHI, suggesting that pooled NAT be reserved for high-risk clients . Studies in sub-Saharan Africa have proposed implementation of a risk-score algorithm using clinical, laboratory, and behavioral data obtained from a verbally administered questionnaire to reduce AHI testing costs [38,39]. Such an algorithm would be beneficial, but implementation at a large VCT center might require additional staff to administer the questionnaire due to the large number of clients.
Currently, the TRCARC performs AHI intervention studies in which early detection of infection is critical; thus pooled NAT testing and the additional costs are warranted.
The study team is grateful to the individuals who volunteered to participate in this study and the staff at the Thai Red Cross AIDS Research Centre and the Department of Retrovirology, US Army Medical Component, Armed Forces Research Institute of Medical Sciences (AFRIMS).
The RV254/SEARCH 010 Study Group includes from SEARCH/TRCARC/HIV-NAT: Nipat Teeratakulpisarn, Donn Colby, Duanghathai Sutthichom, Somprartthana Rattanamanee, Peeriya Mangyu, Sasiwimol Ubolyam, Pacharin Eamyoung, Suwanna Puttamaswin, Somporn Tipsuk and Putthachard Sangtawan; from AFRIMS: Viseth Ngauy, Robert O’ Connell, Siriwat Akapirat, Nantana Tantibul, Hathairat Savadsuk and Vatcharain Assawadarachai; from the US Military HIV Research Program: Nelson Michael, Merlin Robb and Sodsai Tovanabutra.
J.A., N.P. and M.DeS. conceived, designed, and drafted the article. P.P., J.F., E.K., N.C., N.L.M. and J.K. participated in the study design, coordination, and data interpretation and clinical care of the patients. S.Pi. and M.DeS. compiled and analyzed the data. S.Pu. and R.T. participated in the laboratory testing, data compilation, and interpretation. All authors read, provided input in the article, and approved the final article.
Funding source: The study was funded by the US Military HIV Research Program, Walter Reed Army Institute of Research, Rockville, Maryland, under a cooperative agreement (W81XWH-07-2-0067) between the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., and the US Department of Defense.
Conflicts of interest
Disclaimer: The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of any of the institutions mentioned above, the US Department of the Army, or the US Department of Defense, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government or The Thai Red Cross AIDS Research Centre.
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