Decreased Seroreactivity in Individuals Initiating AntiretroviralTherapy during Acute HIV Infection
Mark M. Manak,a Linda L. Jagodzinski,b Ashley Shutt,a Jennifer A. Malia,b Mike Leos,c Jason Ouellette,a Siriwat Akapirat,d
Donn L. Colby,e Nittaya Phanuphak,e Leigh Anne Eller,a Merlin L. Robb,a Mark de Souza,e Jintanat Ananworanich,a,e
Sheila A. Peel,b on behalf of the RV254/SEARCH010 and the RV217 Study Teams
aU.S. Military HIV Research Program, Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., Bethesda, Maryland, USAbU.S. Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, Maryland, USAcBio-Rad Laboratories, Inc., Benicia, California, USAdDepartment of Retrovirology, Armed Forces Research Institute of Medical Sciences, Bangkok, ThailandeSEARCH, Thai Red Cross AIDS Research Centre, Bangkok, Thailand
ABSTRACT Antiretroviral therapy (ART) during acute HIV infection (AHI) interruptsviral dynamics and may delay the emergence of serological markers targeted by cur-rent HIV screening and confirmatory assays, thus creating challenges for correctlyclassifying HIV infection status. The performance of three HIV antigen/antibody com-bination (HIV Ag/Ab Combo) assays (the Bio-Rad GS, Abbott Architect, and Bio-RadBioPlex 2200 assays) was evaluated with samples collected from RV254/South EastAsia Research Collaboration in HIV 010 (RV254/SEARCH010) study (Bangkok, Thai-land) participants at weeks 12 and 24 following the initiation of ART at Fiebig stageI (FI) (n � 23), FII (n � 39), or FIII/IV (n � 22). Supplemental, confirmatory testing wasperformed by the Geenius HIV 1/2 and HIV-1 Western blot assays (Bio-Rad). Samplesfrom 30 untreated, HIV-1-infected individuals demonstrated robust HIV Ag/Ab Comboassay reactivity with well-developed HIV-1 Western blotting profiles by 24 weeks afterinfection. In contrast, 52.2% of samples from individuals initiating ART at FI, 7.7% ofsamples from individuals initiating ART at FII, and 4.5% of samples from individualsinitiating ART at FIII/IV were nonreactive by the HIV Ag/Ab Combo assays, with 36.4to 39.1% of samples having low signal-to-cutoff (S/CO) results by the Architect andBioPlex assays (S/CO � 10). Seroreversion from a reactive to a nonreactive statuswas observed in 10 individuals initiating ART at FII and 3 individuals initiating ART atFIII/IV. The Geenius and HIV-1 Western blot assay results were negative or indetermi-nate for 73.9% and 69.6% of individuals, respectively, treated at FI; 50.0% and 26.3%of individuals, respectively, treated at FII; and 54.5% and 40.9% of individuals, re-spectively, treated at FIII/IV. Virologic suppression of HIV-1 by ART during AHI im-pedes seroconversion to biomarkers of infection, limiting the utility of HIV Ag/AbCombo and supplemental, confirmatory assays for infection status determination.
KEYWORDS HIV Ag/Ab Combo assay, HIV Geenius assay, acute HIV infection,antiretroviral therapy, human immunodeficiency virus, immunodiagnostics,seroreversion
Current guidelines recommend the initiation of antiretroviral therapy (ART) as soonas possible after the diagnosis of HIV infection to minimize the risk of forward
transmission, interrupt the seeding of latent reservoirs, delay disease progression, andultimately, enhance overall health outcomes (1–7). However, the reduction or elimina-tion of viral expansion by ART in acute HIV infection (AHI) to below the thresholdrequired for evolution of an anti-HIV immune response may block or delay theemergence of HIV-specific antigen (Ag) and/or antibody (Ab) targets for detection of
Citation Manak MM, Jagodzinski LL, Shutt A,Malia JA, Leos M, Ouellette J, Akapirat S, ColbyDL, Phanuphak N, Eller LA, Robb ML, de SouzaM, Ananworanich J, Peel SA, on behalf of theRV254/SEARCH010 and the RV217 StudyTeams. 2019. Decreased seroreactivity inindividuals initiating antiretroviral therapyduring acute HIV infection. J Clin Microbiol57:e00757-19. https://doi.org/10.1128/JCM.00757-19.
HIV infection (8–11). Negative results on standard HIV Ab tests are common amongHIV-1-infected children who initiate ART at an early age (12–15). At 2 years of age, abouthalf of all HIV-1-infected infants who initiated ART within 2 weeks of birth wereseronegative by HIV immunoassays and rapid device tests, whereas 11% were serone-gative if ART was initiated at 12 and 24 weeks of age. All children initiating ART laterthan 24 weeks of age were seropositive by HIV immunoassays at 2 years of age (6, 16).In the absence of ongoing antigenic stimulation, seroconversion, as determined bysecond-generation immunoassay Ab tests, also failed to occur among adult participantstreated during AHI (9, 15, 17, 18). Several studies reported seroreversion from reactiveto nonreactive results by HIV Ag/Ab combination (Combo) assays in HIV-infectedparticipants who initiated ART during AHI (8, 14, 15, 17–20). A delay in the appearanceof serological markers and, in some cases, reversion to nonreactive results wereobserved in participants who initiated ART during AHI prior to the development of a fullAb response (RV254/South East Asia Research Collaboration in HIV 010 study [theRV254/SEARCH010 study], Thai Red Cross AIDS Research Centre, Bangkok, Thailand)(18). The present study expands upon the aforementioned RV254/SEARCH010 studyand assesses the ability of current HIV screening and supplemental, confirmatory assaysto detect HIV biomarkers of infection in this early-treated cohort.
MATERIALS AND METHODSClinical specimens. The plasma from HIV-infected participants used in this evaluation was selected
from two studies in which very early HIV infection was identified by screening of individuals at high riskof HIV infection with the Hologic Aptima HIV-1 RNA qualitative assay (the Aptima assay). The evolutionof serological markers in 30 untreated participants was examined by the use of samples obtained fromthe RV217 Early HIV Cohort study conducted in Kenya, Uganda, Tanzania, and Thailand (21, 22) and wascompared to that in participants enrolled in the RV254/SEARCH010 study in Bangkok, Thailand, whoinitiated ART prior to the establishment of fully evolved HIV-1 Ab responses (10, 13, 18, 23). The stage ofHIV infection at the time of ART initiation was assigned on the basis of the Fiebig staging system: Fiebigstage I (FI; HIV RNA positive, p24 Ag negative, HIV Ab negative; n � 23), Fiebig stage II (FII; HIV RNApositive, p24 Ag positive, HIV Ab negative; n � 39), Fiebig stage III (FIII; HIV Ab positive, Western blot [WB]assay negative; n � 13), and Fiebig stage IV (FIV; HIV Ab positive, HIV-1 WB assay indeterminate; n � 9)(18, 24).
Assays. Serial specimens from untreated individuals and from those who initiated therapy at Fiebigstage I to IV of infection were tested at the HIV Diagnostic Reference Laboratory (HDRL) in Silver Spring,MD, by the Bio-Rad GS HIV Combo Ag/Ab Combo enzyme immunoassay (EIA) (the BRC assay), theBio-Rad GS HIV-1/HIV-2 Plus O EIA (the 1/2/O assay), and the Bio-Rad GS HIV-1 Western blot (HIV-1 WB)and the Geenius HIV 1/2 (Geenius) supplemental assays. The Bio-Rad Genscreen HIV-1 Ag assay (the p24Ag assay) was used to determine the concentration of serum HIV-1 p24 antigen. Testing of untreatedindividuals (from the RV217 cohort) was conducted at frequent intervals for up to 4 to 7 weeks after thefirst Aptima reactivity by the 1/2/O and BRC assays. HIV-1 WB testing for 15 of 30 individuals wasconducted on serial samples from the first Aptima-reactive sample to 24 weeks of treatment. Individualstreated at Fiebig stages I to IV (the RV254/SEARCH010 cohort) were tested by the 1/2/O, BRC, WB, andGeenius assays at weeks 1, 2, 12, and 24. Testing by the Abbott Architect HIV Ag/Ab Combo (ARC) assayand assignment of the Fiebig stages of the samples from the RV254/SEARCH010 cohort were performedat the Thai Red Cross AIDS Research Centre, Bangkok, Thailand, as previously described (18). Testing bythe Bio-Rad BioPlex 2200 HIV Ag-Ab Combo (BPX) assay was performed at Bio-Rad, Inc., in Benicia, CA.
Statistical analysis. Calculations of correlations, linear regression, and paired t test parametricanalysis were conducted in Prism (version 7) software (GraphPad Software, La Jolla, CA).
Regulatory approval. The clinical specimens used in this study were collected between 2009 and2014 under human subjects research protocols approved by the institutional review boards at therespective collecting institutions and at the Walter Reed Army Institute of Research (WRAIR). Specimenswere provided to the laboratory with no accompanying private health or personal identifying informa-tion. The investigator(s) adhered to the policies regarding the protection of human subjects prescribedby the Code of Federal Regulations (CFR), title 45, vol 1, part 46 (25); CFR, title 32, chapter 1, part 219 (26);CFR, title 21, chapter 1, part 50 (protection of human subjects) (27), and Army Regulation 70-25 (28).
RESULTS
The time course of evolution of serological markers of HIV-1 infection in untreatedindividuals was evaluated by the use of serial samples collected from participants in theRV217 study who were identified during AHI. Figure 1A shows the results of the BRCassay for 30 individuals whose first Aptima-reactive sample was detected within 3 to4 days of the last nonreactive result. The BRC assay yielded a reactive result, on average,at 6.5 � 2.8 days (range, 2 to 11 days) after the first detection of RNA, rose rapidly to
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a saturated signal-to-cutoff (S/CO) value of the assay (S/CO, 12 to 16) by 3 to 5 weeks,and remained high thereafter. The evolution of serological reactivity from the time offirst RNA detection is shown in Fig. 1B. The timing of the earliest BRC assay reactivitycorresponds to that of positive results by the p24 Ag assay and represents reactivity tothe HIV-1 p24 Ag component of the BRC assay. The 1/2/O assay, which detects onlyIgM/IgG Ab, was reactive, on average, by day 15.0 � 4.8 (range, days 6 to 22) after theday of detection of the first Aptima RNA-reactive sample. Both the 1/2/O and BRCassays reached maximum assay S/CO values of 10 to 13 and 12 to 16, respectively, byweeks 2 to 3, and the values remained high throughout subsequent testing. HIV-1 WBassay positivity was shown by the percentage of participants with a positive result. Allparticipants were HIV-1 WB assay positive by 4 to 5 weeks and remained positive atsubsequent time points, as expected.
The evolution of p24 antigenicity and HIV-1 seroreactivity after ART initiation foreach individual (RV254/SEARCH010 cohort) was evaluated by the p24 Ag, BPX, and ARCassays. All samples from individuals at FII to IV prior to ART initiation containedquantifiable p24 Ag by the Bio-Rad Ag assay and detectable p24 Ag by the BPX assay,but p24 Ag was nondetectable within 1 week of therapy and remained nondetectableat weeks 12 and 24 on ART (not shown). Figure 2 shows the evolution of Ag/Ab Comboresults by the BPX (solid lines) or by the ARC (dotted lines) assay in cases in whichinsufficient sample was available to be tested by the BPX assay prior to treatmentinitiation. For those cases where both assays were performed on the same sample, theBPX and ARC assays yielded similar results. Samples from all 23 FI individuals wereinitially nonreactive by Ag/Ab Combo assays at week 0; samples from 5 (21.7%) wereweakly reactive (S/CO � 10) and samples from 2 (8.8%) were strongly reactive (S/CO�10) by the BPX or ARC assay by week 1. Samples from 12 FI individuals (52.2%)remained nonreactive throughout the 24-week period. All FII individuals were Ag/AbCombo reactive at ART initiation (week 0); these included 15 of 39 (38.5%) individualswith strong BPX assay S/CO values which declined after 1 week of therapy. Samplesfrom several individuals showed a transient increase in S/CO at week 1, followed by asubsequent decline. Seroreversion from Ag/Ab Combo reactive to nonreactive wasobserved in 10 of 39 (25.6%) individuals who initiated therapy at Fiebig stage II and 3of 22 (15.6%) individuals who initiated therapy at FIII/IV. By week 24, samples from 3(7.7%) FII individuals remained nonreactive, those from 15 (38.5%) were weakly reac-
FIG 1 Time course of evolution of serological markers in untreated HIV-1-infected individuals (RV217 cohort). (A)Reactivity of 30 untreated HIV-1-infected individuals by the Bio-Rad GS Ag/Ab Combo EIA (BRC assay), whichmeasures both p24 antigen and anti-HIV antibody. (B) Average S/CO values for the BRC and 1/2/O assays, theconcentration of p24 Ag measured by the Bio-Rad p24 Ag assay, and the percentage of individuals with HIV-1Western blot (WB) assay-positive samples at each time point.
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tive, and those from 21 (53.8%) were strongly reactive. Samples from individuals whoinitiated ART at FIII/IV also showed an initial decline (BPX assay), with samples from 1(4.5%) individual remaining nonreactive, those from 8 (36.4%) remaining weakly reac-tive, and those from 13 (59.1%) remaining strongly reactive after 24 weeks of treatment.
The percentage of samples from individuals grouped as nonreactive (S/CO � 1.0),weakly reactive (S/CO � 10), or strongly reactive (S/CO � 10) by the BPX or ARC assayby week 12 and week 24 on ART is summarized in Table 1. After 12 weeks of therapyinitiated in early infection, 69.6% of samples from individuals who initiated ART atFiebig stage I, 12.8% of samples from individuals who initiated ART at Fiebig stage II,and 9.1% of samples from individuals who initiated ART at Fiebig stage III/IV werenonreactive. Of those samples that were reactive in the assays, 17.4% (FI), 56.4% (FII),and 40.9% (FIII/FIV) exhibited low S/CO (�10) responses in the BPX and ARC assays. Byweek 24, samples from 52.2%, 7.7%, and 4.5% of individuals initiating therapy at FI, FII,and FIII/IV, respectively, remained nonreactive. Samples from 10 of the 84 (11.9%)individuals initiating ART at FI to FIV in our study achieved the very strong reactiveresponse (S/CO � 100), as is observed in individuals with untreated HIV-1 infection byweek 24.
The results of the BRC, ARC, and BPX Ag/Ab Combo assays with samples fromRV254/SEARCH010 participants who initiated ART at FI to FIV and who were tested after24 weeks on therapy are shown in Fig. 3. The three assays demonstrated overall goodagreement at an S/CO of �10, while at higher reactivities with S/CO values up to �200,
FIG 2 Evolution of BioPlex (BPX) or Architect (ARC) assay reactivity of individuals initiating ART during acute HIVinfection. Solid lines represent BPX assay results, and trends very similar to those seen by the BPX assay were seenby the ARC assay (dotted lines). ARC assay results are shown for individuals that did not have sufficient samplevolume for BPX testing at week 0. Individuals for whom the results are depicted in green were nonreactive by theARC or BPX assay by week 24, while those for whom the results are depicted in blue demonstrated low reactivity(S/CO � 10) and those for whom the results are depicted in red showed strong reactivity (S/CO � 10). Very fewsamples demonstrated the very strong reactive response (S/CO � 100) seen in established infections in untreatedindividuals by 24 weeks of infection.
TABLE 1 Percentage of samples from individuals with nonreactive, weakly reactive, orstrongly reactive results by the BPX or ARC assay at weeks 12 and 24 after initiation oftherapy at FI to FIV
the chemiluminescent ARC assay and the fluorometric BPX assay yielded a broaderdynamic range than the colorimetry-based BRC, which saturates at a maximum S/CO of12 to 16. The ARC and BPX assay results were highly correlated to each other, with anR2 value of 0.8404. The BPX assay, which, in addition to HIV-1/2 reactivity, also providesindependent measures of p24 Ag and anti-HIV Ab detection for each sample, showedthat reactivity, as expected, is due to the anti-HIV Ab component alone, with no p24 Agbeing detected in any samples at week 12 or 24.
A more detailed picture of the relative performance of the BRC, ARC, and BPX assaysat 24 weeks after ART initiation stratified by those who initiated therapy at FI, FII, orFIII/IV is shown in Fig. 4. By 2 to 4 weeks of infection, samples from untreatedHIV-infected individuals consistently showed S/CO values near the maximum range ofthe BRC assay (S/CO � 12 to 16) and 1/2/O assay (S/CO � 10 to 13) and remained highthereafter (Fig. 1). In contrast, more than half (12 of 23) of the samples from individualsinitiating ART at Fiebig stage I remained nonreactive (S/CO � 1) by all three Ag/AbCombo assays at week 24. An additional 8 samples demonstrated very low S/COreactivity (S/CO � 10) by the ARC and BPX assays, with those from only two individualsdemonstrating an S/CO of �100. Samples from all individuals treated at Fiebig stagesII and III/IV were reactive by all three assays prior to treatment initiation (at week 0), butby 24 weeks on ART, samples from 3 of the 39 FII individuals and 1 of the 22 FIII/IVindividuals were nonreactive by all three assays.
The ability of the supplemental, confirmatory assays to confirm HIV reactivity insamples from individuals who initiated ART during AHI was evaluated using theGeenius and HIV-1 WB assays. The HIV-1 WB assay profiles of all 15 of 30 untreatedindividuals tested demonstrated typical HIV-1 WB assay seroconversion profiles. Anexample for an untreated individual is shown in Fig. 5. The reactivity to each antigenincreased with time, yielding a mature, florid HIV-1 WB assay profile at weeks 11 and 21.
FIG 3 Comparison of reactivity of the Architect (ARC), Bio-Rad (BRC), and BioPlex (BPX) Ag/Ab Comboassays in individuals who initiated ART at FI to FIV and who were tested after 24 weeks on ART.
FIG 4 Distribution of S/CO values at week 24 after initiation of ART at Fiebig stage I, II or III/IV by theBio-Rad (BRC), Architect (ARC), or BioPlex (BPX) HIV Ag/Ab Combo assays. The horizontal bars show thegeometric mean and standard deviation for each set.
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In contrast to untreated HIV-1-infected individuals, who seroconverted to HIV-1 WBassay positivity by 7 weeks after the first Aptima RNA reactivity, individuals whoinitiated ART at FI to FIV showed a much slower evolution of HIV-1 WB assay antigenreactivity. The HIV-1 WB assay demonstrated only faint antigen reactivity by 24 weekson ART, which in many cases was insufficient for interpretation as a positive result. Asshown by the examples in Fig. 5, a number of individuals initiating ART during earlystages of AHI were either negative or indeterminate by an HIV-1 WB assay after 12 and24 weeks on ART. Although specific HIV-1 WB assay patterns were variable amongindividuals, even those who scored HIV-1 WB assay positive failed to develop theexpected florid antigen reactivity typically observed in untreated individuals.
The reactivity to the HIV p24, p31, gp41, and gp160 antigens, as detected by theGeenius and WB assays at 24 weeks after ART initiation in early infection, is shown inFig. 6. The reactivity of all antigens was significantly inhibited when ART was initiatedat Fiebig stage I. The reactivity to gp41 in samples from individuals initiating therapy
FIG 5 (Left) Evolution of HIV-1 Western blot (WB) assay profiles of an untreated HIV-1-infected individual with timeafter the first Aptima-reactive RNA sample. (Right) HIV-1 WB assay profiles for selected individuals at weeks 12 and24 following initiation of ART at Fiebig stages I to IV of HIV infection. MW, molecular weight marker.
FIG 6 Reactivity of HIV antigens by the Geenius (GN) and HIV-1 Western blot (WB) assays at 24 weeksafter initiation of therapy at FI (green), F2 (yellow), and FIII/IV (blue).
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at FI to FIV was comparable by the Geenius and HIV-1 WB assays, while detection ofgp160 (P � 0.005) and p24 (P � 0.0001) at each Fiebig stage was significantly lower bythe Geenius assay than by the HIV-1 WB assay. Reactivity to p31, a late-appearingantigen, was either low or negative by both the Geenius and HIV-1 WB assays at24 weeks after early ART initiation.
The decreased antigen reactivity translates to a high percentage of early-treatedindividuals remaining negative or indeterminate by the Geenius and HIV-1 WB supple-mental, confirmatory tests after 12 and 24 weeks of treatment (Table 2). At 12 weeks ofART, the Geenius assay was positive for only 25% of samples from FI individuals, 44.7%of samples from FII individuals, and 59.1% of samples from FIII/IV individuals, while thesamples from the remaining individuals were negative or indeterminate. Similar resultswere observed at week 24, with samples from 26.1%, 50.0%, and 45.5% of FI, FII, andFIII/IV individuals, respectively, being positive by the Geenius assay. HIV-1 WB assaydetection sensitivity was somewhat higher, with 30.4% (FI), 73.7% (FII), and 59.1%(FIII/IV) samples yielding a positive result at week 24.
DISCUSSION
Previous results from the RV254/SEARCH010 study showed a marked delay in theappearance of or the absence of serological markers of HIV-1 infection in serial samplesfrom individuals who initiated ART during AHI (18). The present study extends theevaluation of samples from this cohort to examine the impact of early ART on thereactivity of newer HIV Ag/Ab Combo assays and the supplemental, confirmatory assaysused to generate laboratory evidence of HIV infection. The detection of serologicalmarkers of HIV-1 infection in samples from ART-treated individuals was contrasted tothe detection of those from untreated individuals from the RV217 Early HIV Cohortstudy (21, 22).
As demonstrated in the RV217 cohort of individuals with early HIV infection, the firstantigen marker appears very early in infection with the emergence of the p24 Ag, whichis detected by Ag/Ab Combo assays. The antigen concentration measured by theBio-Rad p24 Ag assay reaches a maximum concentration at approximately 2 weeks afterthe first RNA detection and then rapidly declines following the emergence of anti-HIV-1Ab. The p24 Ag in all samples from individuals from the RV254/SEARCH010 cohort at FIIto FIV was detectable prior to ART but was nondetectable within 1 week on ART andremained nondetectable thereafter. Since the p24 Ag is no longer detectable inuntreated individuals after 4 to 5 weeks of infection or after 1 week of therapy, the highS/CO values observed by the Combo assays is due to the Ab component only, asexhibited by the 1/2/O assay or the Ab signal in the BPX assay.
Plasma from untreated individuals generated an S/CO at the maximum range forboth the BRC and 1/2/O assays within 3 to 4 weeks of the first Aptima RNA reactivity.HIV-1 WB assay antigen profiles continued to increase in intensity with time, yielding amature, florid HIV-1 WB assay pattern by week 24. Although testing by the BPX or ARC
TABLE 2 Positivity of HIV-1 Western blot and Geenius assays at 12 and 24 weeks afterinitiation of ART at FI, FII, or FIII/IVa
Total 100 100 100 100 100 100 100 100 100 100 100 100aA high percentage of samples tested at 12 and 24 weeks of ART remained negative or indeterminate whentherapy was initiated early. WB, Western blot assay; GN, Geenius assay. POS, positive; IND, indeterminate;NEG, negative.
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assay was not performed on these samples at week 24, results from other studies haveshown that the intensity of the BPX and ARC assay results continues to increase overthis time frame (29–31).
Previous studies examining the ability of HIV Ag/Ab Combo assays to detect AHIshowed comparable performances among BRC, BPX, and ARC assays. The limit ofdetection of the p24 Ag by these assays ranged from 2 to 7 pg, which corresponds to4.4 to 4.9 log copies of RNA per ml. The assays detect HIV infection from 7 to 11 daysearlier than the anti-HIV IgM/IgG immunoassays (32–35). In our study, the results of theBRC, ARC, and BPX assays from samples collected at 24 weeks after the initiation of ARTin AHI also showed generally good agreement at S/CO results below 10. Whereas theBRC assay optical density readout saturates quickly at an S/CO of 12 to 16, the ARC andBPX assays provided a useful measure of the impact of early treatment on the evolutionof Ab reactivity. We show that the high S/CO values typically seen in the ARC (S/CO �
700) and BPX (S/CO � 200) assays by 12 to 24 weeks in untreated individuals (29–31)were greatly suppressed by early ART. Although the most dramatic inhibition ofserologic reactivity at weeks 12 and 24 postinitiation of ART was observed whentreatment was initiated at FI, a significant number of samples from individuals treatedat FII to FIV also demonstrated lower reactivity by the ARC and BPX assays.
A decrease in Ab detection after early treatment was also observed in other studies,which showed increased frequencies of false-negative results, weakly reactive HIV rapidtest results, and increased numbers of indeterminate HIV-1 WB assay results (11, 36). Ofnote, S/CO values of less than 10 by the ARC and BPX assays have frequently beenassociated with false-positive results (31, 37–39). The lower rate of Ab detection inART-treated individuals also led to increased estimates of recent infection when testedby Ab avidity-based HIV incidence assays, such as the limiting antigen (LAg), Bio-Radavidity, and HIV-1 multiplex assays, which rely on the semiquantitative detection of theintensity of the immune response (29, 40).
Previous studies on the performance of the Geenius assay showed that its perfor-mance was comparable to that of the HIV-1 WB assay for the detection of AHI (41, 42).In contrast, our results showed that the HIV-1 WB assay is more sensitive than theGeenius assay for the confirmation of infection in individuals treated during acuteinfection. Examination of the reactivity to individual antigens detected by the Geeniusassay in ART-treated individuals showed that this assay is less sensitive than the HIV-1WB assay for the detection of p24 and gp160 (Fig. 6), two antigens important forinterpretation of a positive result with this assay. Our results show that antigenreactivity by both the Geenius and WB assays fails to develop after early ART treatment.A high percentage of individuals remain negative or nonreactive by these confirmatoryassays at weeks 12 and 24. The decrease in HIV-1 WB assay reactivity observed at24 weeks after the start of treatment at FII (73.7%) versus FIII/IV (59.1%) (Table 2) maybe due to the small sample size. Statistical analysis showed no significant difference(P � 0.243) between the percentage of HIV-1 WB assay-positive individuals at FII andFIII/IV at week 12 or week 24. The differences in the percentages of Geenius assay-positive individuals were smaller and were also not significant.
These results are similar to those of a previous study which found that in chronic HIVinfection, the p24 and p31 antigens are less frequently detected by the Geenius assaythan by the HIV-1 WB or Inno-Lia assay and that the Geenius assay is less suitable fordistinguishing between acute and chronic infections (43). Our finding that 73.9% ofindividuals who initiated therapy at FI and over 50% of those who initiated therapy atFII or FIII/IV were not confirmed as HIV-1 positive by Geenius assay suggests theseindividuals may be misclassified by current diagnostic algorithms, especially in individ-uals on ART with undetectable HIV-1 RNA.
The impact of a delayed evolution of a serological response, as manifested byweaker reactivity on screening and confirmatory assays, has important implications inevaluating the infection status of individuals who initiate ART in AHI or followingsuspected exposure to HIV. The high rates of false-negative results by rapid device testsfor HIV-1-infected children on long-term ART can lead to misclassification, inadequate
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patient management, and/or the false perception of a lack of infection or cure (44).Frequent false-negative serological results were reported in HIV-1-infected infants whowere breast-fed by mothers on ART or in postexposure prophylaxis (PEP) programs toprevent mother-to-child transmission (45–47). A significant reduction in HIV Ab levels,including a lack of HIV-1 WB assay reactivity and an increase in the rate of false-negativeserological results following ART initiation, was previously reported in other studies (9,48, 49). Individuals from the RV254/SEARCH010 cohort described in this study whoinitiated treatment at FI remained HIV-1 infected, despite the full suppression of HIV-1plasma viremia with no detectable serological markers for over a 2-year period, as HIVRNA rebound occurred when ART was discontinued (50).
Despite the high rate of success and continued expansion of ART-based preventionand treatment programs, such as treatment as prevention, preexposure prophylaxis(PrEP), and postexposure prophylaxis (PEP) programs, HIV breakthrough infections canstill occur due to nonadherence or the emergence of drug-resistant strains. Theserological and nucleic acid assays used in current HIV diagnostic algorithms may notbe sufficient to accurately identify true infection in settings where antiviral treatmentis provided very early in HIV infection. Individuals who become infected while on PrEPhave slower Ab avidity maturation, prolonged seroconversion profiles, and an increasein the time to full HIV-1 WB assay positivity (from 49 to 80 days) (51, 52). Only 27/57(47%) of RNA-positive individuals who were infected while on PrEP were found to bereactive by the Bio-Rad GS HIV Combo Ag/Ab EIA, and none were HIV-1 positive byeither the HIV-1 WB or the Geenius assay (53). Alternative approaches, such as testingfor cell-associated HIV total nucleic acid (RNA and DNA), may be required to rule outHIV-1 infections in individuals with inconclusive HIV diagnostic test results and may behelpful to confirm infections in such cases (6, 54).
ACKNOWLEDGMENTSWe are grateful to the participants and study volunteers for their contribution and
commitment to make this HIV research possible.This work was supported in part by the U.S. Army Medical Research and Materiel
Command under contract no. W81-XWH-18-C-0337 and W81-XWH-16-C-0225; by theU.S. Military HIV Research Program, Walter Reed Army Institute of Research, under acooperative agreement (agreements W81-XWH-07-2-0067 and W81-XWH-11-2-0174) withthe Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc.; by NIHgrant R01AI108433 between the Henry M. Jackson Foundation for the Advancement ofMilitary Medicine, Inc., and the U.S. Department of Defense (DoD); by the Division of AIDS,U.S. National Institute of Allergy and Infectious Diseases; and by an intramural grant fromthe Thai Red Cross AIDS Research Centre. The U.S. Army Medical Research AcquisitionActivity (Fort Detrick, MD, USA) is the awarding and administering acquisition office for thecooperative agreement. Antiretroviral therapy was supported by the Thai GovernmentPharmaceutical Organization, Gilead, Merck, and ViiV Healthcare.
The RV254/SEARCH010 Study Team comprises the following: from SEARCH/TRCARC,Praphan Phanuphak, Nipat Teeratakulpisarn, Eugene Kroon, Carlo Sacdalan, PhillipChan, Ponpen Tantivayakul, Nitiya Chomchey, Ratchapong Kanaprach, Jintana Intasan,Duanghathai Sutthichom, Peeriya Prueksakaew, Pacharin Eamyoung, Suwanna Putta-maswin, Khunthalee Benjapornpong, Somporn Tipsuk, Tassanee Luekasemsuk, andKultida Poltavee; from Chulalongkorn University, Supranee Buranapraditkun, SuneeSirivichayakul, and Phandee Wattanaboon-yongcharoen; from AFRIMS, Robert O’Connell,Alexandra Schuetz, Siriwat Akapirat, Rapee Trichavaroj, Bessara Nantapinit, NumpuengChurikanont, Saowanit Getchalarat, and Nongluck Sangnoi; and from MHRP, MerlinRobb, Trevor Crowell, Suteeraporn Pinyakorn, Ellen Turk, Oratai Butterworth, CorinneMcCullough, Sodsai Tovanabutra, Lydie Trautmann, and Mark Milazzo. The RV217 StudyTeams consist of Hannah Kibuuka (Uganda), Lucas Maganga (Tanzania), SorachaiNitayaphan (Thailand), and Fred K. Sawe (Kenya).
The views, opinions and/or findings contained in this report are those of the authors andshould not be construed as an official U.S. Department of the Army position, policy, or
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decision, unless so designated by other documentation. The content of this article is solelythe responsibility of the authors and does not necessarily represent the official views of anyof the institutions mentioned above, the U.S. Department of the Army, the U.S. Departmentof Defense, the National Institutes of Health, the U.S. Department of Health and HumanServices, or the United States government and the Thai Red Cross AIDS Research Centre.
J.A. has received honoraria for participating in advisory meetings for ViiV Healthcare,Gilead, Merck, Roche, and AbbVie. The other authors have no conflict of interest.
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