Association of recent HIV infection and in-utero HIV-1 transmission
Taha, Taha E.a; James, Maria M.b; Hoover, Donald R.c; Sun, Jina; Laeyendecker, Oliverd,e; Mullis, Caroline E.e; Kumwenda, Johnstone J.f; Lingappa, Jairam R.g; Auvert, Bertranh,i,j; Morrison, Charles S.k; Mofensen, Lynne M.l; Taylor, Allanm; Fowler, Mary G.b; Kumenda, Newton I.a; Eshleman, Susan H.b
aDepartment of Epidemiology, Johns Hopkins University Bloomberg School of Public Health
bDepartment of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland
cDepartment of Statistics and Biostatistics and Institute for Health, Healthcare Policy and Aging Research, Rutgers University, Piscataway, New Jersey
dLaboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
eDepartment of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
fDepartment of Medicine, College of Medicine, University of Malawi, Blantyre, Malawi
gDepartments of Global Health, Medicine, and Pediatrics, University of Washington, Seattle, Washington, USA
hInstitut National de la Santé et de la Recherche Médicale, INSERM U1018, CESP, Villejuif
iHôpital Ambroise-Paré, Assistance Publique- Hôpitaux de Paris, Boulogne
jUniversity of Versailles, Guyancourt, France
kClinical Sciences, Family Health International, Durham, North Carolina
lPediatric, Adolescent, and Maternal AIDS Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Rockville, Maryland
mEpidemiology Branch, Division of HIV/AIDS Prevention, Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA.
Correspondence to Susan H. Eshleman, MD, PhD, Professor, Department of Pathology, The Johns Hopkins University School of Medicine, Ross Bldg., Room 646, 720 Rutland Ave., Baltimore, MD 21205, USA. Tel: +1 410 614 4734; fax: +1 410 502 9244; e-mail: email@example.com
Received 25 February, 2011
Revised 21 April, 2011
Accepted 9 May, 2011
Objective: We previously developed a multiassay algorithm (MAA) to identify recent HIV infection that includes the BED-capture enzyme immunoassay, an avidity assay based on the Genetic Systems HIV-1/HIV-2 + O enzyme immunoassay, CD4 cell count, and HIV viral load. We used this MAA to evaluate the association between recent maternal HIV infection and in-utero transmission of HIV.
Methods: Plasma samples were collected at delivery from 2561 HIV-infected women in the postexposure prophylaxis of infants-Malawi trial. The MAA described above was used to identify women with recent HIV infection. Logistic regression models assessed association between recent HIV infection and in-utero HIV transmission (defined as a positive infant HIV DNA test at birth).
Results: Seventy-three women were identified as recently infected using the MAA. Those women were younger and had lower parity than women who were identified as not recently infected using the MAA (P < 0.0001 for age and parity). The frequency of in-utero HIV transmission was 17.8% among women identified as recently infected, compared with 6.7% among women identified as not recently infected (13/73 vs. 166/2488, P = 0.001). In a multivariate model, three factors were independently associated with in-utero HIV transmission: recent infection [adjusted odds ratio (AOR): 2.49, 95% confidence interval (CI): 1.30–4.78, P = 0.006], log10 HIV viral load at delivery (AOR: 2.01, 95% CI: 1.60–2.51, P < 0.0001), and younger age (per 10 year increase, AOR: 0.66, 95% CI: 0.43–0.93, P = 0.02).
Conclusion: Results obtained using a MAA suggest that recent maternal HIV acquisition is strongly associated with in-utero HIV transmission, independent of HIV viral load at delivery.
In sub-Saharan Africa, prevalence of HIV infection among young women attending antenatal clinics often exceeds 20% . Studies have shown that women who acquire HIV infection in the postpartum period are at increased risk for postnatal mother-to-child transmission (MTCT) of HIV [2–4]. Women are also at risk of acquiring HIV infection during pregnancy. In a South African study, incident HIV infection was four times more frequent in pregnant women than in nonpregnant women . In that study, 3% of women acquired HIV infection during pregnancy, testing positive approximately 24 weeks after an initial negative test . Because HIV viral loads are often very high near the time of HIV infection [6,7], and because high HIV viral load is one of the factors most strongly associated with MTCT of HIV [8–11], acquisition of HIV infection by women shortly before the onset of pregnancy or during pregnancy is likely to be associated with an increased risk of in-utero HIV transmission.
Despite recent successes in counseling and testing in sub-Saharan Africa, some barriers exist to achieving universal coverage. Potential barriers to wider coverage in Malawi include structural limitations of the healthcare system . In Malawi, women are frequently first tested for HIV infection near the time of delivery. Without a previous HIV test result, it is not possible to identify women who are likely to have acquired HIV infection shortly before or during pregnancy. Women with acute (pre-seroconversion) HIV infection can be identified at the time of delivery using methods such as pooled HIV RNA testing  (i.e., by detecting HIV RNA in samples from women who do not have detectable HIV antibody). However, because the duration of acute HIV infection is short (e.g., 1–2 weeks), that testing approach would only identify women who acquired HIV infection very close to the time of testing. Even if a large number of women are tested and the incidence rate in pregnant women is high, relatively few women with acute HIV infection are likely to be identified using that approach. Using a pooled HIV RNA testing strategy, a study in Malawi documented acute HIV infection in five (0.21%) of 2327 women tested in the third trimester , and a study in South Africa documented acute HIV infection in four (0.9%) of 467 women tested at the first antenatal visit .
Another approach to identifying women who acquired HIV infection shortly before or during pregnancy is to test samples collected at the time of delivery using laboratory methods developed for cross-sectional HIV incidence determination. Serologic (antibody based) assays have been developed for cross-sectional analysis of HIV incidence, based on the premise that the anti-HIV antibody response matures during the course of HIV infection [16–18]. Those assays measure different characteristics of the HIV antibody response (e.g., antibody avidity, titer, isotype, specificity, or the proportion of the antibody response that is HIV specific) [16–18]. Unfortunately, the serologic response to HIV infection is quite variable. Some HIV-infected individuals never attain a mature HIV-antibody response; in others, factors such as advanced HIV disease and viral suppression (natural or antiretroviral drug-induced) can weaken the anti-HIV antibody response [19,20]. For these reasons, individuals with long-standing HIV infection may be misclassified as recently infected using a single serologic assay [21–23].
We previously developed a multiassay algorithm (MAA) for determination of HIV incidence using cross-sectional samples, as part of the HIV Prevention Trials Network (HPTN) Network Laboratory's HIV Incidence Initiative. This MAA uses two serologic assays (the BED-capture enzyme immunoassay  and an avidity assay based on the Genetic Systems HIV-1/HIV-2 + O EIA ) as well as nonserologic biomarkers (CD4 cell count and HIV viral load) to assess HIV incidence [18,25]. These two serologic assays use different HIV antigens to detect and characterize antibodies. The BED assay includes antigens from subtypes B and D, as well as CRF01_AE, whereas the avidity assay includes antigens from a broader spectrum of HIV strains. These two assays also measure different characteristics of the anti-HIV antibody response. The BED assay measures the proportion of antibody that is HIV specific, whereas the avidity assay measures the strength with which anti-HIV antibodies bind to target antigens [21,23,24]. Each of these assays when used alone is known to misclassify some individuals with long-standing HIV infection as recently infected [21–23]. Our MAA uses modified cutoffs for the BED and avidity assays (BED result >1 normalized optical density unit, OD-n; avidity result >80%), and combines results from these assays with results from HIV viral load and CD4 cell count testing (see Methods) [19,22,26]; this approach reduces the rate of misclassification of individuals with long-standing infection as recently infected [19,22,26]. In this study, we used the MAA to identify Malawian women who were likely to have been infected with HIV shortly before or during pregnancy, and evaluated the relationship between recent maternal HIV infection and in-utero HIV transmission. We hypothesized that the high HIV viral loads associated with early HIV infection would contribute to higher levels of in-utero HIV transmission.
Source of samples that were used for analysis of mother-to-child transmission of HIV
Samples were obtained from HIV-infected women enrolled at the time of delivery in the PostExposure Prophylaxis of Infants in Malawi (PEPI-Malawi) trial (conducted in 2004–2009, NCT00115648) . Approximately 70% of the women enrolled in the PEPI-Malawi trial presented more than 4 h before anticipated delivery; those women were described as early presenters and received a single intrapartum dose of nevirapine (sdNVP) for prevention of MTCT of HIV. In contrast, women with unknown HIV status who presented less than 4 h before anticipated delivery were described as late presenters and did not receive intrapartum sdNVP. Infants of the enrolled women were randomized at birth to receive one of three regimens to prevent postnatal HIV transmission, as described previously . Maternal blood samples used to measure CD4 cell count and HIV viral load were collected at delivery. In-utero HIV transmission was defined as a positive infant HIV DNA test at birth . This study included 2561 (76.8%) of the 3335 women enrolled in the PEPI-Malawi trial; 774 women were excluded from analysis for the following reasons: 470 had no HIV viral load or no CD4 cell count result from the delivery visit, 33 had infants whose HIV infection status was undetermined, 271 did not have an available sample collected at delivery. Nine (0.35%) of the 2561 women started antiretroviral therapy (ART) prior to delivery.
Source of samples that were used to evaluate the performance of the multiassay algorithm
The performance of the MAA was assessed by analyzing samples from adults with known duration of HIV infection who were likely to be infected with subtype C HIV [individuals enrolled in three clinical cohorts: the Hormonal Contraception and HIV Acquisition (HC-HIV) Study , the Orange Farm Male Circumcision Trial (ANRS-1265) [29,30], and the Partners in Prevention HSV/HIV Transmission Study] . This sample set included samples collected from 87 individuals (58 men from South Africa [29,30] and 29 women from Zimbabwe ) less than 180 days after a negative HIV test. Data for CD4 cell count and HIV viral load were not available for those individuals but were assumed to be more than 200 cells/μl and more than 400 copies/ml in these recently infected individuals; none of these individuals were receiving antiretroviral drugs at the time of sample collection. The sample set also included samples from 529 men and women in South Africa and Botswana who were known to have been HIV infected for at least 2 years .
Laboratory analysis of recent HIV infection
The BED assay was performed according to the manufacturer's instructions (Calypte Biomedical Corporation; Lake Oswego, Oregon, USA) , except that an assay cutoff of 1.0 OD-n was used to screen for recent HIV infection (rather than the standard cutoff of 0.8 OD-n). The avidity assay is a modified version of the genetic systems HIV-1/HIV-2 + O EIA (enzyme linked immunoassay, Bio-Rad Laboratories; Redmond, Washington, USA). The avidity assay was performed as previously described [19,24], except that an assay cutoff of 80% was used to screen for recent infection (rather than the standard cutoff of 40%). Women were identified as recently infected using the MAA described previously : BED less than 1.0 OD-n + avidity less than 80% + CD4 more than 200 cells/μl + HIV viral load more than 400 copies/ml (Fig. 1).
For unadjusted comparisons between groups, Fishers exact tests compared categorical variables and Wilcoxon tests compared continuous variables. Univariate and multivariate logistic regression models were used to obtain unadjusted and adjusted odds ratios (AORs) for the association of recent infection with in-utero HIV transmission. Statistical significance was considered for two-sided P = 0.05 or less. All analyses were performed using SAS Software Version 9.1 (Cary, North Carolina, USA).
Written informed consent was obtained from all women for participation in the PEPI-Malawi trial. The PEPI-Malawi trial was approved by Institutional Review Boards in Malawi and the United States as described , including the U.S. Centers for Disease Control and Prevention.
This substudy included 2561 women enrolled in the PEPI-Malawi trial. There was no significant difference in the frequency of in-utero HIV transmission among the 2561 women included in the study and the 774 women who were not included in the study (7.0 vs. 6.2%, respectively, P = 0.48, Fisher's exact test). Fifty-four of the 2561 women included in the study were previously diagnosed with HIV infection in the Nevirapine and Zidovudine (NVAZ) trial, which was conducted in 2000–2004 at the same study sites as the PEPI-Malawi trial [32,33]. Therefore, those 54 women were known not to have recent HIV infection when they subsequently enrolled in the PEPI-Malawi trial. The median duration of time between their enrollment in the NVAZ trial and their subsequent enrollment in the PEPI-Malawi trial was 4.3 years (range: 2.3–6.1 years). Overall, 179 (7.0%) of the 2561 women had infants who were diagnosed with HIV infection at the time of birth (in-utero HIV infection).
Maternal samples collected at delivery from the 2561 women were analyzed using the BED and avidity assays. Results from those assays were combined with results from CD4 cell count and HIV viral load testing to identify women with recent HIV infection (Fig. 1). The performance of the MAA in individuals likely to be infected with subtype C HIV was evaluated by analyzing samples from individuals from subtype C endemic areas who had known duration of infection (see Methods). In those individuals, the positive predictive value of the MAA for identifying individuals with recent HIV infection (defined as <180 days) was 93.9%; that result can be compared with the result obtained by testing the same sample set with the avidity assay alone using a standard assay cutoff of 40% (positive predictive value: 90.9%), and to the result obtained by testing the same sample set with the BED assay alone using a standard assay cutoff of 0.8 OD-n (positive predictive value: 71.7%).
Using the MAA described above, 73 (2.9%) of the 2561 women were identified as recently infected; the remaining 2488 women were identified as not recently infected. All but nine of the 2488 women were identified as not recently infected based on results of serologic testing (BED >1.0 OD-n and/or avidity >80%, Fig. 1). Those nine women had low BED and/or low avidity test results. Four of those nine women had CD4 cell counts less than 200 cells/μl and five of those women had viral loads less than 400 copies/ml (Fig. 1). Individuals who have low CD4 cell counts and low HIV viral loads are unlikely to be recently infected; furthermore, those factors have been associated with misclassification of individuals with nonrecent HIV infection as recently infected using serologic HIV incidence assays [20,34,35].
None of the 54 women who had known nonrecent HIV infection when they enrolled in the PEPI-Malawi trial were misclassified as recently infected using the MAA (none out of 54 vs. 73/2507, P = 0.40, Fishers exact test). In addition, nine women (0.4% of the 2561 women included in this study) were receiving ART at the time of enrollment into the PEPI-Malawi trial (a coformulated regimen of stavudine, lamivudine, and nevirapine; Triommune, Cipla, India); none of those women were identified as recently infected using the MAA and none had an infant who was HIV-infected in utero. The median time between antiretroviral treatment initiation and enrollment in the PEPI-Malawi trial for those nine women was 56 days (range: 3–263, interquartile range: 25–97 days). They had a median CD4 cell count of 180 (range: 33–701 cells/μl, five had a CD4 cell count <200 cells/μl) and a median log10 HIV viral load of 2.73 (range: 2.30–4.36 copies/ml) at enrollment.
Table 1 shows demographic, clinical, and laboratory characteristics of the 73 women who were identified as recently infected using the MAA and the 2488 women identified as not recently infected by the MAA. Women who were identified as recently infected using the MAA were younger and had lower parity than the women who were identified as not recently infected (median age: 23 vs. 26 years, P < 0.0001, Wilcoxon test; median parity: two vs. three pregnancies, P < 0.0001, Wilcoxon test). Women identified as recently infected by the MAA also had significantly higher CD4 cell counts at delivery; this is partly explained by the fact that the MAA excludes women with CD4 cell counts below 200 cells/μl from the recently infected group. There was a significant association between recent maternal HIV infection (defined using the MAA) and in-utero HIV transmission. The frequency of in-utero HIV transmission was 17.8% in the recently infected group, compared with only 6.7% in the not recently infected group (13/73 vs. 166/2488, P = 0.001, Table 1). There was no significant association between identification of women as recently infected using the MAA and other demographic, clinical, and laboratory variables (Table 1).
We considered the possibility that some women who were identified as not recently infected based on CD4 cell count or HIV viral load data alone might have been misclassified; among the 2488 women identified as not recently infected, only nine had a BED result less than 1.0 OD-n and an avidity result less than 80% (four with a CD4 cell count <200 cells/μl and five with a viral load <400 copies/ml). Even if all nine of those women were included in the recently infected group, recent infection would still have been significantly associated with an increased risk of in-utero HIV infection (P = 0.0062).
Logistic regression analysis was performed to assess the association between in-utero HIV infection and identification of women as recently infected using the MAA, controlling for other factors (Table 2). In a multivariate model (which excluded parity because it was correlated with age), increased risk of in-utero transmission was significantly associated with recent HIV infection [as defined by the MAA, (AOR): 2.49, 95% confidence intervals (CI): 1.30–4.78, P = 0.006], HIV viral load at delivery (per log10 increase, AOR: 2.01, 95% CI: 1.60–2.51, P < 0.0001), and younger age (per 10 year increase, AOR: 0.66, 95% CI: 0.43–0.93, P = 0.02), but not with CD4 cell count at delivery, or other factors (Table 2).
We used a MAA to identify women in the PEPI-Malawi trial who were likely to have had recent HIV infection at the time of delivery. The frequency of in-utero HIV transmission was significantly higher among women who were identified as recently infected using the MAA compared with women who were identified as not recently infected (17.8 vs. 6.7%, P = 0.001). The high frequency of in-utero HIV transmission in women with recent HIV infection is consistent with the dynamics of HIV infection, because those women were likely to have been acutely infected during pregnancy. HIV viral load is typically very high during the acute stage of HIV infection [6,7], and high HIV viral load is one of the factors most strongly associated with an increased risk of MTCT [8–11]. In this study, identification of women as recently infected using the MAA and maternal HIV viral load at delivery were independently associated with in-utero HIV transmission.
The independence of HIV viral load at delivery and recent HIV infection (as defined by the MAA) suggests that other factors associated with recent HIV infection may increase the risk of in-utero HIV transmission. This hypothesis is consistent with data from studies of heterosexual HIV transmission. In heterosexual transmission, adults with recent HIV infection are more likely to transmit HIV than adults with chronic (nonrecent) HIV infection . This is not explained by differences in HIV viral load alone  and suggests that some viral strains present early in infection may be more fit for transmission [38,39]. Adults with recent HIV infection are also more likely to have immature anti-HIV antibody responses (e.g. low anti-HIV antibody titer or avidity, low proportion of immunoglobulin G anti-HIV antibody). During pregnancy, women who have immature anti-HIV antibody responses might have lower proportions of antibody-associated or neutralized HIV. Previous studies have found that women who transmit HIV to their infants in utero are less likely to have neutralizing antibodies than are women who transmit HIV to their infants at or after delivery [40,41]. Further studies are needed to evaluate the relative contributions of high HIV viral load, viral transmission phenotype, and the quality or level of the HIV antibody response to in-utero HIV transmission in women with recent HIV infection.
There are some limitations of this study. Because we cannot determine the actual time of infection for women in this study, we do not know what proportion of women who were identified as recently infected using the MAA were infected during pregnancy, rather than in the months preceding pregnancy. We also recognize that some women who acquired HIV infection during pregnancy may not have been identified using the MAA, and that the MAA may also have classified some women as recently infected who had chronic HIV infection. Several observations from this report provide indirect evidence supporting use of the MAA. First, the MAA identified 2.9% of the women as recently infected; that result is consistent with findings from a South African study, where 3% of women acquired HIV infection during pregnancy based on longitudinal HIV serological testing . Second, none of 54 women with known long-standing HIV infection and none of the nine women on ART were misclassified as recently infected using the MAA. Third, women identified as recently infected using the MAA were younger than other women in the cohort; younger age has been associated with HIV acquisition in sub-Saharan Africa [42,43]. We also note that BED and avidity test results are not significantly affected by pregnancy; therefore, pregnancy is not likely to impact the performance of the MAA .
This study suggests that women who acquire HIV near the onset of pregnancy or during pregnancy have significantly higher risk of transmitting HIV to their infants in utero than do women with chronic HIV infection. Unfortunately, identification of women with recent HIV infection is difficult unless there is a prior history of a recent negative HIV antibody test, because use of methods such as the MAA to detect recent HIV infection is not feasible in large scale in resource-limited countries. In high-prevalence settings, retesting during the third trimester of women who tested HIV-negative shortly before pregnancy or earlier in pregnancy would allow identification of women with recent HIV infection . In that subset of women, triple antiretroviral drug prophylaxis for prevention of MTCT of HIV  may be preferable given the increased risk of HIV transmission associated with recent HIV infection. However, it is important to recognize that in-utero infection might have already occurred by the time that maternal infection would have been diagnosed using this strategy. For these reasons, the results of our study emphasize that prevention of HIV acquisition in women during pregnancy is a critical step in preventing in-utero HIV transmission and reducing the global epidemic of pediatric HIV/AIDS.
The authors thank the women and infants who participated in the PEPI-Malawi trial, and the PEPI-Malawi study team in Malawi. The authors also thank the laboratory staff at the College of Medicine, University of Malawi-Johns Hopkins University Research Project in Blantyre, Malawi for their assistance with sample processing and shipping for the PEPI-Malawi study. The authors thank the study teams and participants of the following three studies for providing samples and data used to evaluate the performance of the multiassay algorithm: Hormonal Contraception and HIV Acquisition (HC-HIV) Study, the Orange Farm Male Circumcision Trial (ANRS-1265), and the Partners in Prevention HSV/HIV Transmission Study. The authors thank Shauna Wolf for help with sample and data management, and Kevin Eaton for assistance with sample testing.
The sources of support are as follows: First, International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) Group, American Recovery and Reinvestment Act award (cooperative agreement 3U01AI068632-05S3). Second, The HPTN sponsored by the National Institute of Allergy and Infectious Diseases (NIAID), the National Institute on Drug Abuse, the National Institute of Mental Health, and the Office of AIDS Research, of the National Institutes of Health (NIH), Department of Health and Human Services (DHHS) (U01AI068613). Third, The Centers for Disease Control and Prevention (cooperative agreement U50/CCU022061). Fourth, IMPAACT Group (U01-AI068633). Fifth, The Partners in Prevention HSV/HIV Transmission Study (grant #26469, Bill and Melinda Gates Foundation). Sixth, the Hormonal Contraception and HIV Acquisition (HC-HIV) Study (contract N01-HD-0-3310 through Family Health International, The Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), NIH, DHHS). Seventh, The Division of Intramural Research, NIAID, NIH.
All of the authors reviewed the manuscript and provided input into the manuscript prior to submission. In addition, individual authors had the following contributions:
T.E.T. contributed to study design, data analysis, data interpretation, and manuscript preparation; U.S. Principal Investigator for the PEPI-Malawi trial. M.M.J. coordinated laboratory studies, analyzed laboratory data, contributed to manuscript preparation. D.R.H., senior statistician for this study; statistician for the PEPI-Malawi trial, contributed to manuscript preparation. J.S. was data analyst for this study. O.L. was responsible for overseeing BED and avidity testing and reviewing BED and avidity test results and contributed to manuscript preparation. C.E.M. performed BED and avidity testing. J.J.K. was clinician in charge of clinical care for participants in the PEPI-Malawi trial. J.L. provided samples and data used to analyze the performance of the multiassay algorithm. B.A. provided samples and data used to analyze the performance of the multiassay algorithm. C.S.M. provided samples and data used to analyze the performance of the multiassay algorithm. L.M.M. was the NICHD medical officer for the PEPI-Malawi trial, was involved with the original design of the PEPI-Malawi trial and with monitoring of adverse events during the trial, and contributed to manuscript revisions. A.T. was the CDC medical officer for the PEPI-Malawi trial. M.G.F. was the former CDC medical officer for the PEPI-Malawi trial, was involved with the original design of the PEPI Malawi trial and with monitoring of adverse events during the trial, and contributed to manuscript preparation. N.I.K. was the Malawi principal investigator for the PEPI-Malawi trial, was involved with the original design of the PEPI-Malawi trial and with monitoring of adverse events during the trial. S.H.E. conceived of the study, coordinated the study, was responsible for study design, data interpretation, and manuscript preparation. The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the US Centers for Disease Control and Prevention or the NIH. Use of trade names is for identification purposes only and does not constitute endorsement by the US Centers for Disease Control and Prevention or the NIH. A portion of this work was presented at the 18th Conference on Retroviruses and Opportunistic Infections (Feb 2011; Boston, MA, USA). The authors have no conflicts of interest to declare.
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