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Pattern and Frequency of Seroreactivity to Routinely Used Serologic Tests in Early-Treated Infants With HIV

Puthanakit, Thanyawee MDa,b; Ananworanich, Jintanat MDc,d,e; Akapirat, Siriwat PhDf; Pattanachaiwit, Supanit MScg; Ubolyam, Sasiwimol PhDb; Assawadarachai, Vatcharain MScf; Sawangsinth, Panadda BSce; Jupimai, Thidarat MSca; Anugulruengkitt, Suvaporn MDa; Tawan, Monta RNa; Kosalaraksa, Pope MDh; Borkird, Thitiporn MDi; Suntarattiwong, Piyarat MDj; Kanjanavanit, Suparat MDk; de Souza, Mark S. PhDe, on behalf of the RV475/HIV-NAT209 Study Group

Author Information
JAIDS Journal of Acquired Immune Deficiency Syndromes: March 1, 2020 - Volume 83 - Issue 3 - p 260-266
doi: 10.1097/QAI.0000000000002254



Infants acquire HIV infection either in utero, at delivery, or during breastfeeding, with antibody development appearing in most infants at age 6 months in the absence of antiretroviral therapy (ART).1 Immediate ART initiation is life-saving in infants and is standard of care. However, the viral suppressive effects of early therapy may inhibit or delay seroconversion, a phenomenon also described in adults treated during acute HIV infection (AHI).2–7 Continued HIV seronegativity or seroreversion in early-treated children poses a diagnostic challenge for Thailand and other low- and middle-income countries (LMIC), where HIV serologic testing at greater than age 9 months, coinciding with waning maternal anti-HIV antibody, may be used to confirm or exclude HIV infection.8,9 Knowledge of which serologic tests perform best in the context of early treatment could inform accurate HIV diagnosis in children and adolescents infected at birth.

Most studies of diagnostic serology in HIV-infected infants have included immunoassay(s) (IA) using limited HIV antigens, such as Env, to detect antibody. These IA almost always contain at a minimum the highly conserved immunodominant epitope of gp41.2,4,7,10–15 Despite earlier detection of AHI using antigen/antibody combination IA, the fourth-generation (4thG) IA has shown decreased sensitivity in detecting HIV infection in early-treated infants and adults treated with ART during AHI.2,3,5 Low sensitivity of several rapid diagnostic test(s) (RDT) used in national health systems before ART for early infant diagnosis of HIV infection has been reported.10,11 There have also been findings of lower HIV-specific antibody avidity in both infants and adults treated with ART for prolonged periods of time.16 The evolution of de novo HIV-specific antibodies seems to differ between adults and children with the former initially developing antibodies to Env gp41 and the latter to Env gp160 following infection.17

Many LMIC have adopted HIV-1 testing algorithms in lower-tiered health facilities using RDT-based HIV testing algorithms based on World Health Organization guidelines for both adults and infants.18 In 2016, approximately 74 million HIV RDT were performed at President Emergency Plan for AIDS Relief-supported sites.19 Given the increased use of RDT globally, their problems in diagnosing HIV infection in both treated and untreated infants and the reduced sensitivity of the 4thG IA in early treated infants and adults, we assessed the impact of early ART (within the first 6 months of life) with widely used HIV diagnostic assays in LMIC.


Study Participants

The study is a prospective cohort of HIV-infected infants from 6 hospitals in Thailand: King Chulalongkorn Memorial, Srinagarind, Hat Yai, Queen Sirikit National Institute of Child Health, Nakornping, and Prachomklao. All infants had 2 positive HIV DNA polymerase chain reaction results and initiated ART within the first 6 months of life.

From July 2015 to October 2018, blood samples were collected from 58 HIV-infected infants, 25 and 12 of whom had longitudinal annual sample collections for up to 3 years and 4 years of treatment, respectively (see Table 1, Supplemental Digital Content, provides details on the longitudinal samples for each infant). These 25 infants were assessed for longitudinal reactivity to HIV diagnostic test kits.

HIV Serology Assays

Infant blood samples were processed at HIV-NAT, Thai Red Cross AIDS Research Centre and stored at −80°C until testing. Each sample was tested with 4 HIV diagnostic assays: 4thG ARCHITECT HIV Ag/Ab Combo (Architect; Abbott Laboratories, Wiesbaden, Germany), 2 qualitative HIV RDT: Abbott Determine HIV1/2 (Determine; Abbott Diagnostics Medical Co., Matsudo, Japan) and SD Bioline HIV-1/2 3.0 (Bioline; Standard Diagnostics Inc., Yongin, Republic of Korea) and the second generation (2ndG) IA: Avioq HIV-1 Microelisa System (Avioq; Avioq Inc., Rockville, MD). The 4thG assay detects Gag p24 antigen and HIV-specific IgM and IgG, whereas all other assays used detect antibody only. The RDT are based on third-generation (3rdG) IA principles—HIV-specific antibody detection by antigen “sandwich” allowing detection of both IgM and IgG. The 2ndG assay is an IgG-sensitive indirect IA, where HIV-specific antibodies are detected following solid-phase binding to antigen via enzyme conjugated anti-human immunoglobulin and substrate. The 4thG IA and the RDT are approved for HIV-1 testing by the Thai Food and Drugs Administration. The RDT were also prequalified by WHO ( for use in diagnostic algorithms. The 4thG and 2ndG IA are approved by the US FDA for use in HIV screening. Table 1 shows the HIV antigens present in each of the IA used.

Point Frequency of Reactivity to 4 Routinely Used HIV Serologic Tests for Infants Initiating ART Within the First 6 Months of Life Assessed Annually for Up to 4 Years on ART

Data were expressed as both reactive (R)/nonreactive (NR) and the sample: cut-off ratio (S/CO) for diagnostic assays providing a quantitative read-out (Architect and Avioq). A S/CO ratio of ≥1 is considered reactive for both assays. S/CO ratios of low values (<10) have been associated with false-positivity on the 4thG HIV IA20 and have also been proposed as a marker for primary versus established HIV-1 infection.21

The frequency of seroreversion was assessed in 25 infants with longitudinal samples greater than age 24 months, because this is the time that maternal antibody would have waned in ART-treated infants as measured by 2ndG, 3rdG and 4thG IA.8,22,23 Seroreversion for this study is defined as test kit results moving from R to NR at any time point in infants with at least one follow-up sample at up to 3 years of ART.

In addition, longitudinal samples from the 25 infants with samples collected at one and 3 years of treatment were assessed by HIV Western blot (WB, Genetic Systems HIV-1 Western blot kit; Bio-Rad Laboratories, Redmond, WA) and interpreted according to the manufacturers' instructions, with the exception of using only a single intensity score to define positive protein reactivity. Samples for each child were parallel-tested on each assay.


Comparisons between and within test kits were assessed using the McNemar test. Continuous variables were described as median (range) or frequencies and compared using the Mann–Whitney or Wilcoxon nonparametric tests. Fisher exact test was used for comparisons between groups. A P value of <0.050 was considered significant.

Human Subject Protection

Parents provided informed consent, and the study was approved by all ethics committees.


The median age at ART initiation was about 2 months with 26% (15/58) of infants initiating ART later than 3 months. The median CD4% at ART initiation was 36 (14–62) for 39 infants for whom data were available. Fifty-five (95%) infants were treated with zidovudine/lamivudine/LPV/r and 3 infants received zidovudine/lamivudine/nevirapine. Viral suppression (<50 copies/mL) was observed in greater than 80% of children following 1 year of ART (Table 2). Serologic testing of specimens by the 4 test kits was performed on 58, 27, 25, and 12 infants at 1, 2, 3, and 4 years after treatment, respectively, and showed wide variation in reactivity between assays (Table 1).

Characteristics of Infants Tested

The 2ndG IA showed the highest frequency of reactivity after one year of ART, followed by the 4thG IA and RDT (Table 1). This hierarchy continued through the fourth year of treatment, with a trend for decreased reactivity for the 4thG IA and 2 RDT. None of the 12 infants tested by the Determine were reactive after 4 years of ART. Infants initiating ART within 3 months of life (median age: 1.7; range: 0.2–3.0) demonstrated a significantly lower frequency of reactivity compared with those initiating ART during age 3–6 months (4.2; 3.3–5.4) for all 4 test kits after 1 year of ART, with the 4thG IA showing the greatest difference between the 2 groups (P = 0.005) and the 2ndG IA the least (P = 0.023) (Fig. 1). In addition, the breadth of reactivity for the 4 test kits was lower in the earlier versus late-treated group: median 1 (0–4) versus 3 (1–4), respectively. The median HIV viral loads for infants before ART at less than 3 months (N = 37) versus between ages 3 and 6 months (N = 11) were log10 3.59 (1.40–6.85) and log10 4.93 (1.70–6.32) copies/mL, respectively (P = 0.720). There was no significant difference between the frequency of viremic infants after 1 year of ART between the 2 groups: 10/43 and 1/15 initiating ART at less than and greater than age 3 months, respectively (P = 0.257), although the sample size in the later treated group is small.

Frequency of HIV seroreactivity to 4 clinical IA in 43 infants initiating ART within 3 months (A) and 15 infants (B) initiating ART between age 3 and 6 months. Abbreviations: Architect, ARCHITECT HIV Ag/Ab combo; Determine RDT, Abbott Determine HIV1/2 rapid diagnostic test; Bioline RDT, standard diagnostics Bioline HIV-1/2 3.0 rapid diagnostic test; Avioq, Avioq HIV-1 Microelisa System.

Median S/CO ratios for the 4thG and 2ndG IA did not show any significant change between the first through fourth years of ART. The median S/CO ratio for the 4thG IA was consistently below, whereas that for the 2ndG IA was above the ratio for reactivity at all time-points (Table 2). The median (range) S/CO ratios for the 4thG and 2ndG IA in children showing seropositivity after 3 years of treatment were 5.76 (1.64–256.80) and 2.40 (1.21–8.57), respectively. There was no significant change in the S/CO ratio for children with reactive 4thG IA through 4 years of treatment, with median values of 3.88, 9.91, 5.76, and 2.47 after 1, 2, 3, and 4 years of treatment, respectively (P = 0.693). The frequency of children with 4thG IA S/CO ratios <10 was 93% (54/58), 82% (22/27), 84% (21/25), and 100% (12/12) after 1, 2, 3, and 4 years of ART, respectively. Testing by the 2ndG IA, which saturates at a maximum S/CO ratio range of approximately 8–10, whereas the 4thG IA S/CO ratio saturates at >500, showed a relatively stable S/CO ratio over 4 years of treatment—median S/CO ratios of 2.99, 2.97, 2.40, and 1.65 after 1, 2, 3 and 4 years of treatment, respectively (P = 0.620).

For the 25 infants who were followed longitudinally for 3 years, 96% (24/25) and 88% (22/25) were virologically suppressed at one and 3 years of treatment, respectively. The 3 viremic children on ART for 3 years had HIV viral loads of 2.10, 4.97, and 4.98 log10 copies/mL and all were virologically suppressed at one year after treatment initiation. Comparing the reactivity between the 4thG IA and the other test kits demonstrated a significant difference in reactivity to the 2ndG only at both one and 3 years after treatment: P = 0.016 and P = 0.004, respectively. The Determine and SD RDT showed no significant difference in performance at one and 3 years after treatment (P > 0.370). Comparing each test kit between 1 and 3 years of treatment demonstrated no significant difference in performance for any of the test kits (range: P = 0.182–1.000).

HIV-specific antibodies analyzed by WB at one year of ART were predominantly to the Gag p24 protein (84%), with 20% reactivity to Env protein gp160 and no reactivity to Env gp120 and gp41 proteins. After 3 years of ART, there was a general decline in HIV-specific reactivity for all proteins with the exception of gp160 which increased to 28% and reactivity to gp120 and gp41 for a single child with a viral load of 4.98 log10 copies of HIV RNA/mL (Fig. 2). Seven of 23 children with antibodies to at least one HIV protein were exclusively reactive to Gag p24. The Bioline assay was reactive for only 5/19 (26%) WB p24 positive samples at 3 years of treatment.

Frequency of positive HIV antigens measured by WB assay for 25 infants at 1 and 3 years after ART initiation. *P = 0.045.

Seroconversion and seroreversion were observed with all generations of test kits over 3 years of therapy (Table 3). Seroconversion for the 4thG IA and RDT over 3 years of ART was only observed in up to 12% of infants (Table 3), with one infant showing seroconversion across all 3 assays. Two infants who were reactive to all test kits after 3 years of therapy were also viremic with HIV viral loads of 4.97 and 4.98 log10 copies/mL. Comparing the pattern of reactivity at year one versus year 3 of ART showed concordant serology profiles for 17/25 (68%), 6/25 (24%), and 2/25 (8%) for the 2ndG IA, 4thG IA, and 2 RDT, respectively. The Determine RDT showed seroreversion for 8/25 (32%) infants, 3 of whom also seroreverted by the 4thG IA and Bioline RDT. Three of 4 seroreversion events observed for the 2ndG IA were unique to that assay. Five infants who were seroreactive to all tests at year one of ART, seroreverted to at least one test kit. The most common pattern of nonreactivity over 3 years of ART across the diagnostic assays was to Architect, Determine, and Bioline which was observed in 12/25 infants. Three children were seronegative to all assays used over 3 years of ART and the one child for whom samples were available for 4 years of ART remained seronegative. All 3 children initiated ART within 3 months of life. The WB assay did not show frank seroconversion from negative to positive and seroreversion from positive to negative between one and 3 years of ART, with 4 infants seroconverting from indeterminate to positive and 3 infants regressing from positive to indeterminate.

Cumulative Frequency of Serology Profiles to 4 Routinely Used HIV Serologic Tests in 25 Infants Initiating ART Within the First 6 Months of Life Assessed for Up to 3 Years on Treatment


HIV-specific antibody measurement in infants is confounded by the presence of maternal antibody,24 which clears in HIV-uninfected children at a median of 13.9 months, although seroreactivity has been reported at up to age 24 months.22 The current longitudinal study in HIV-infected infants treated with early ART demonstrated low levels of 4thG IA reactivity through 4 years of treatment, which has been previously reported for the Architect in cross-sectional studies.2,7,12

The lack of association between viral load at baseline and increased HIV-specific seroreactivity is in contrast to other studies, but is likely due to the smaller number of viral load measurements in the group treated between ages 3 and 6 months, compared with other reports.4,12,25 However, the lower frequency of seroreactivity to HIV clinical assays in the less than 3 versus 3–6 month treated groups is in agreement with published findings, despite the narrower time range for the latter group in the current study.4,7,12

The 2ndG IA showed the highest frequency of reactivity relative to the other test kits despite its being least sensitive than the other 3 assays used in diagnosing HIV infection in adults.26 However, it was the only assay used that measured antibody responses to potentially all HIV antigens. Although it may be argued that children aged 12–24 months may have had residual maternal antibody, this is unlikely following more than 2 years of treatment although one report using the relatively insensitive first-generation IA projected 1.2% seropositivity in HIV-exposed uninfected children aged 24 months.22 A study in Vietnam in HIV-uninfected infants born to infected mothers reported seroreversion to 3rdG IA in all infants by age 24 months.23 A more recent report from Thailand of HIV-exposed, uninfected infants receiving early ART and tested at 12, 18, and 24 months by 3rdG and 4thG IA reported that all infants seroreverted by age 24 months, although 4thG IA testing was associated with delayed seroreversion at age 18 months relative to the 3rdG IA.8

The 4thG IA and RDT used in this study detect antibody to HIV Env (gp41) with the Bioline also detecting antibody to HIV Gag p24 (Table 1). The 4thG IA is the only assay tested capable of detecting HIV antigen, but does not discriminate between HIV antigen and/or antibody reactivity. However, given that most infants were virologically suppressed, the assay was primarily detecting antibody. All infants with a viral load equivalent to or greater than the 4thG IA limit of detection (4.70 log10 HIV RNA copies/mL)27 were reactive for this assay (data not shown).

The finding that most infants throughout the study demonstrated low S/CO ratios in the 4thG IA similar to the level often classified as false-positive results20,28 could confound diagnosis particularly when used with RDT only in diagnostic algorithms.

A narrow range of HIV antigens used to detect HIV antibody is not the only factor related to inferior performance of the 4thG IA and the RDT, as the Bioline also detects p24 and performed similarly to the Determine, but the p24 component has not been separately evaluated for sensitivity (to our knowledge). The WB data support that the p24 component of the Bioline assay is not as sensitive as the gp41 component at detecting HIV antibody.

Studies on the evolution of antibodies to specific HIV antigens in infected infants following ART initiation have been limited. One study using in vitro stimulation of B cells from HIV-infected untreated infants and a clinical HIV WB reported antibody production to gp160 in 60% of patients aged 0–2 months, whereas gp41 and p24 antibodies were observed in less than 25% of infants.1 However, it should be stressed that the study used a commercial WB and the gp160 antibody may have been tetramers of gp41 rather than gp160.29 An Italian study of HIV-infected infants treated within the first 6 months of life for more than 2 years showed 78% seropositivity to the same 4thG IA used in the current study, with the predominant antibody response being to gp160 and p24, although a viral-lysate-based WB was again used, so reactivity to oligomers of gp41 may also have occurred.12 The higher frequency of 4thG IA reactivity in the Italian cohort is probably because of the later median age of ART initiation compared with the current study, because they noted a significant correlation between age of ART initiation and HIV seroreactivity.12 Other reports from Thailand and South Africa in HIV-infected infants treated within the first 3 months of life and tested at 2 years or more following ART with the Architect (Thailand) and a different 4thG IA (South Africa) which also measured gp41 antibody reported seroreactive frequencies of 53% and 36%, respectively, similar to that observed in the current study.2,4 The South African study also used the Determine and reported 41% reactivity, which is considerably higher than what we observed following 2 years of treatment, despite infants in both studies initiating ART at a similar median age (around 7–8 weeks).4 This may be because of the different definition of viral suppression used in the current study <50 HIV RNA copies/mL, whereas the South African study used <400 HIV RNA copies/mL, and there may have been more instances of low-level viremia in that study, because higher viral load is usually associated with increased seropositivity in infants.4,25

HIV-specific antibody to Env gp41 and gp160 and Gag p24 decline in HIV-infected infants treated at less than age 3 months has been reported to be similar to HIV-exposed uninfected infants born to HIV-infected mothers over a 2-year period.25 Another study using WB scoring in HIV-infected infants treated within 6 months of life and tested after more than 2 years of ART reported the frequency of HIV-specific antibodies to gp41 at about 30% and for p24 of 76% which is similar to the current study.12 Low levels of gp41 antibody may account for the nonreactivity observed with the 4thG IA and RDT used in the current study, whereas the relatively high frequency of reactivity observed for the 2ndG IA is likely because of the high frequency of Gag p24 antibodies combined with reactivity to native gp160.

HIV cure trials in infants are being actively pursued given the difficulty of adherence, requirement for life-long ART, drug-associated toxicities, and social stigma.30 Reduction in the HIV reservoir size is considered a key marker for HIV remission.12,30,31 A recent report proposing HIV antibody profiles as an indication of HIV remission strategies reported that low levels of gp41 and loss of antibodies to p24 were associated with HIV cure in adults.31 The use of gp41 antibody levels and/or activity and cumulative HIV antibody scores measured by WB have been proposed as surrogate markers for HIV total DNA levels.7,12,25

Limitations of our study include the small sample size and the breadth of test kits used, which was limited by sample volume. In addition, the intra-individual longitudinal study lacked some infants at 2 years of treatment which could have affected the seroreversion results. However, these results show that more advanced HIV diagnostic assays result in a high frequency of seronegativity in HIV-infected infants treated early in life which is maintained during therapy.

More recent generations of HIV diagnostic assays may not necessarily be the optimum in the context of early-treated pediatric infection, AHI, and pre-exposure prophylaxis, because there have been reports of NR HIV serology profiles in all these groups.2,7,15,32,33 In addition, selection of HIV diagnostic algorithms should follow the World Health Organization guidelines of using assays based on different antigen preparations, although this is often difficult, because many manufacturers do not provide detailed information on the antigen used.

Our preliminary findings highlight the importance of careful interpretation of seronegative results from HIV diagnostic tests in the era of early and prolonged ART, to avoid incorrect diagnosis of HIV infection and potential discontinuation of therapy in individuals retested for HIV.


The authors gratefully acknowledge the HIV-NAT 209 study participants and their parents without whom this study would not have been possible. The authors would also like to thank Rapee Trichavaroj, Pornchanok Panjapornsuk and Bhubate Tongchanakarn from the AFRIMS laboratory and Bunruan Sopa and Nuchtida Phongarm from the HIV-NAT laboratory for their invaluable help with the immunoassays.

The RV475/HIV-NAT209 Study Group:

Research Sites; HIV-NAT, TRCARC/Chulalongkorn University: Thanyawee Puthanakit, Mark de Souza, Thidarat Jupimai, Torsak Bunupuradah, Wasana Prasitsuebsai, Watsamon Jantarabenjakul, Suvaporn Anugulruengkitt, Stephen Kerr, Sasiwimol Ubolyam, Apicha Mahanontharit, Napasawan Laopraynak, Preeyarach Klaytong, Tulathip Suwanlerk, Thita Pitimahajanaka, Naruporn Kasipong, Thornthan Noppakaorattanamanee, Kesdao Nanthapisal, Umaporn Methanggool, Sasithorn Bureechai, Panadda Sawangsinth, Chutima Saisaengjan, Monta Intawan; Queen Sirikit National Institute of Child Health: Piyarat Suntarattiwong, Pugpen Sirikutt, Pimsiri Leowsrisook, Yosawadee Na Nakorn, Naruemon Sassungnune; Siriraj Hospital, Mahidol University: Kulkanya Chokephaibulkit, Kanokkarn Wongmayurachat; Srinagarind Hospital, Khon Kaen University: Pope Kosalaraksa, Chanasda Kakkaew, Somjai Rattanamanee; Prachomklao Hospital: Wittaya Petdachai, Manee Yentang, Patcha Panyim, Janyarak Punyim; Nakornping Hospital: Suparat Kanjanavanit, Thida Namwong, Siripim Kamphaengkham; Chiangrai Prachanukroh Hospital: Rawiwan Hansudewechakul, Areerat Khongponoi; Hat Yai Hospital: Thitiporn Borkird, Ratchanee Saksawad,Usa Sukhaphan, Arena Laeyuheem.

Laboratory and Network; PHPT: Gonzague Jourdain, Nicole Ngo-Giang-Huong.

Vaccine and Cellular Immunology laboratory; Chulalongkorn University: Sunee Sirivichayakul; The National Cancer Institute, NIH: Frank Maldarelli; The University of; Sydney: Sarah Palmer; MoPH and ACC network; Thailand MOPH-U.S. CDC Collaboration: Michael Martin, Rangsima Lolekha, Thananda Naiwatanakul, Worawan Faikratok, Benjamas Baipluthong; The Bureau of Health Promotion, MoPH: Danai Teewunda, Sarawut Boonsuk, Chaweewan Tonputsa, Pariwat Tangpong; Clinical Research Center, MoPH: Archawin Rojanawiwat, Hansa Thaisri, Wiroj Puangtubtim, Chaydan Boonrossak; The Bureau of AIDS TB and STIs, MoPH: Sumet Ongwandee, Walairat Chaifoo, Cheewanan Lertpiriyasuwat, Patcharaporn Pawapootarnont, Jiraporn Chucherd, Juthamanee Moonwong; Faculty of Associated Medical Sciences, Chiang Mai University: Tanawan Samleerat; National Health Security Office: Suchada Chaiwut; CRCHUM-Université de Montréal: Louise Leyre, Rémi Fromentin, Amélie Pagliuzza, Marion Pardons, Johann Plantin, Hawley Rigsby, Nicolas Chomont; MHRP; Jintanat Ananworanich, Lydie Trautmann, Suteeraporn Pinyakorn, Oratai Butterworth, Madelaine Ouellette; CRCHUM- Flow Cytometry Platform: Dominique Gauchat.


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HIV fourth-generation immunoassay; HIV rapid diagnostic test; seroreversion; HIV-infected infant; HIV immunoassay; Thailand

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