Background: Universal prenatal HIV antibody testing, which does not detect acute HIV, is standard for pregnant women in the United States. Unrecognized HIV acquisition during pregnancy may result in higher rates of perinatal transmission.
Objective: To determine the prevalence of acute (antibody-negative) HIV infection in pregnant women and to assess the potential for prompt initiation of antiretroviral therapy to prevent perinatal transmission.
Methods: From 1 November 2002 to 30 April 2005, all publicly funded HIV testing sites participated in North Carolina's Screening and Tracing Active Transmission (STAT) Program, which retested all specimens that were HIV antibody negative for HIV RNA using specimen pooling. All patients with acute HIV infection were immediately traced for evaluation, confirmatory testing, counseling, and referral services. For this study, all pregnant women with acute HIV were immediately initiated onto antiretroviral therapy and followed prospectively for pregnancy outcomes.
Results: During the study period, 443 women were HIV positive by antibody testing; 15 were HIV antibody negative but positive by RNA assay and of these five were pregnant at the time of testing. The pregnant women received antiretroviral drugs and delivered HIV-uninfected infants. Maternal testing records of all six HIV-infected infants born in North Carolina showed three mothers with chronic HIV infection and three HIV antibody negative at private prenatal testing facilities.
Conclusions: In resource-rich settings, a substantial proportion of residual perinatal transmission may be from HIV acquisition during pregnancy. Standard antibody tests miss acute HIV infection and so algorithms that include pooled HIV RNA testing may improve its detection and represent a further opportunity to prevent perinatal transmission.
From the aSchool of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, USA
bNorth Carolina Department of Health and Human Services HIV/STD Prevention and Care Branch, USA
cNorth Carolina State Laboratory of Public Health, Raleigh, North Carolina, USA
dSchool of Medicine, University of California at San Francisco, San Francisco, California, USA.
Received 19 February, 2007
Revised 15 August, 2007
Accepted 17 August, 2007
Correspondence to Dr. K.B. Patterson, Division of Infectious Diseases, School of Medicine, The University of North Carolina at Chapel Hill, 2104 Bioinformatics Building, Campus Box 7215, Chapel Hill, North Carolina 27599-7215, USA. E-mail: Kristine_patterson@med.unc.edu
Since the onset of the HIV pandemic, tremendous progress has been made in preventing maternal to child transmission (MTCT) of HIV. Between 1992 and 2004, there has been a 95% reduction in the number of perinatally acquired HIV infections in the United States, with only 117 known transmissions in 2003 and 2004 . This reduction in developed countries is the result of a series of measures including routine HIV testing of pregnant women, antiretroviral therapy (ART) for treatment of mothers and prophylaxis of infants, elective cesarean sections when indicated, and the avoidance of breastfeeding .
Antibody testing for HIV is recommended to all pregnant women early in prenatal care to identify chronically infected women. However, this strategy may miss women who have an acute HIV infection (AHI) at the time of testing (HIV antibody negative, HIV-1 RNA positive) . Clinical diagnosis of AHI is uncommon because individuals may be asymptomatic or symptoms may be nonspecific. AHI is associated with a high rate of sexual transmission as there is high viremia . Acquiring HIV while pregnant may increase the risk of perinatal transmission. This report describes the implementation of an enhanced HIV testing strategy in North Carolina for the detection of AHI in pregnancy and the outcome of those pregnancies.
In 2002, the North Carolina Department of Health and Human Services began using an enhanced testing strategy using pooled nucleic acid amplification testing for HIV in addition to standard HIV testing for specimens received from all 171 public testing facilities. Public testing facilities include HIV counseling and testing sites, sexually transmitted disease clinics, family planning clinics, prenatal clinics, correctional facilities, drug-treatment facilities, tuberculosis clinics, community health clinics, hospital clinics, and primary care clinics. The Screening and Tracing Active Transmission (STAT) program identifies individuals who are antibody negative but have detectable HIV RNA. Within 72 h of notification of test results, disease intervention specialists makes contact with all acutely infected individuals for clinical evaluation, follow-up confirmatory testing, partner counseling, and referral to longitudinal clinical care.
Prenatal testing for HIV is currently not mandatory under North Carolina law. Rather, the law encourages providers to offer testing to all pregnant women on initial presentation into prenatal care. Infants are tested if clinical symptoms suggest HIV, if the mother is newly diagnosed with HIV or has symptoms highly suggestive of HIV, or if the mother is known to be HIV positive.
HIV testing procedures
The North Carolina State Laboratory of Public Health centrally processes all serum samples for routine HIV antibody testing using standard Vironstika HIV-1 enzyme immunoassay (bioMérieux) and Western blot analysis (Bio-Rad, Hercules, California, USA). This is standard HIV testing.
Serum samples that were antibody negative were pooled and tested as previously described  by nucleic acid amplification for HIV-1 RNA using one of the following assays: the Procleix HIV-1 assay (GenProbe, San Diego, California, USA), the NucliSens HIV-1 EasyQ assay, or the NucliSens HIV-1 QL assay (bioMérieux). The NucliSens HIV-1 QL assay is able to detect > 75 copies/ml HIV-1 RNA. Quantitative HIV-1 RNA testing was performed on individual specimens that were antibody negative on routine testing but were positive on the pooled RNA screen, and on samples that were antibody negative but had an indeterminate or negative Western blot. Subjects found to have possible AHI, as defined by a negative/indeterminate antibody test in conjunction with a positive RNA test, were asked to undergo follow-up confirmatory antibody and nucleic acid testing. Subjects were defined as having AHI if the following conditions were met: initially had negative antibody tests and detectable HIV RNA in serum, and seroconverted to HIV positive during follow-up testing. Only subjects meeting these criteria were included in this analysis.
Follow-up of HIV testing and program outcomes
All women with AHI identified through STAT testing were located, counseled, and referred to clinical care via the STAT program . Pregnant women with AHI were initiated onto ART within 14 days of confirmatory diagnosis and were followed prospectively for pregnancy outcomes. After obtaining informed consent, clinical and epidemiological data, including HIV testing outcomes, were collected into a dedicated research database from all clients tested in the STAT program, as approved by the state of North Carolina and the University of North Carolina's Biomedical Institutional Review Board.
All women who consented to routine HIV testing at 171 publicly funded voluntary counseling and testing sites of the North Carolina Department of Health and Human Services from 1 November 2002 to 30 April 2005 were included in this analysis. Maternal HIV testing records were reviewed retrospectively for all HIV-infected infants born in North Carolina whose mothers were tested during this same time period. For the purposes of this analysis, infants were defined as being perinatally infected if they were HIV DNA positive. Data management and descriptive statistics were conducted with SAS software, version 9.1.2 (Cary, North Carolina, USA). Statistical testing for differences between medians was performed using a nonparametric two-sample test and P < 0.05 was considered significant for all analyses.
Between 1 November 2002 and 30 April 2005, 187 135 women were tested for HIV at public sites. The demographics of all women undergoing HIV testing are shown in Table 1. A total of 443 (0.2%) women tested positive for HIV: 428 were HIV antibody positive and 15 were HIV antibody negative but HIV RNA positive (3.4% of all positive women). Of the HIV antibody-positive women, 313 (73%) were black. Heterosexual transmission was reported as the likely mode for 83% of women; risk factors that were commonly reported among antibody-positive women included having a sex partner at risk for HIV, having a sexually transmitted infection, and exchanging sex for noninjecting drugs or money. Five antibody-positive women reported being a victim of sexual assault.
The 15 women with AHI identified through nucleic acid testing of pooled serum samples had a median initial serum HIV-1 RNA load of 55 230 copies/ml (interquartile range, 19 944–811 110) at diagnosis: 12 (80%) were black, 13 (87%) reported heterosexual transmission, and 2 reported the use of injection drugs. Three (20%) women reported symptoms suggestive of acute retroviral syndrome.
Identification of acute HIV infection in pregnant women
Of the 15 women with AHI, five (33%) were pregnant at the time of STAT HIV testing. Initial serum viral loads for pregnant women with AHI (median HIV-1 RNA, 88 787 copies/ml) were similar to nonpregnant women with AHI (P = 0.48). All pregnant women with AHI considered heterosexual transmission as their primary risk factor. The median gestation at HIV diagnosis was 15 weeks (range, 12–16). A summary of the pregnant women with AHI is shown in Table 2. All women were prescribed HAART within 14 days of confirmatory diagnosis. Woman 5 was promptly prescribed HAART after being diagnosed, was noncompliant and subsequently underwent delivery by cesarian section for persistent viremia. The infant of woman 4 was delivered by cesarian section because of obstetrical complications after a prolonged labor. All women received standard ACTG 076 dosing of intravenous zidovudine at the time of delivery and all delivered live singleton infants who received prophylactic zidovudine for 6 weeks. None of these infants was HIV DNA positive at 0–2 days, 4–6 weeks, or 4–6 months of life.
Identification of perinatally infected infants
During the study period, there were six HIV-infected infants reported in North Carolina. Review of maternal HIV testing records for all cases confirmed that three of these infants were born to women whose HIV diagnosis was known prior to their pregnancy. Two of these mothers did not receive routine prenatal care; a third mother received HAART during pregnancy but was only partially compliant and had persistently elevated plasma HIV RNA. She received antepartum intravenous zidovudine and delivered by cesarian section. The remaining three HIV-infected infants were born to women who had undergone routine enzyme immunoassay-based antibody testing through nonpublic testing sites (not participating in the STAT program) between 12 and 18 weeks of gestation. All had negative HIV antibody tests but nucleic acid amplification was not performed. None of the mothers could recall symptoms suggestive of acute retroviral syndrome during or after pregnancy, and none of these infants was breastfed. None of these women could recall being tested for HIV during or immediately following delivery.
None of the three infants born to mothers ‘missed’ by routine nonpublic testing was diagnosed in a timely fashion. Two of the mother–infant pairs were identified only when the mothers underwent repeat antibody testing as a component of routine prenatal testing for a second pregnancy and were found to be HIV antibody positive. Their first infants were subsequently tested and found to be HIV DNA positive. The third mother–infant pair was identified when the infant presented at 3 months of age with Pneumocystis jiroveci pneumonia.
To identify women with AHI in North Carolina, an enhanced testing strategy using pooled HIV RNA testing was utilized for all women who were antibody negative at the time of routine testing. In this study, 3.4% of all newly HIV-positive women had AHI at the time of testing, compared with the 0.5% of HIV-infected individuals who are estimated to be in this window period at any one time in the US population . Looked at another way, antibody testing alone had only 96.6% sensitivity for HIV infection in this group. Additionally, one-third of these women with AHI were pregnant at the time. Identifying AHI in pregnant women using this enhanced testing strategy may have particularly important implications for the prevention of perinatal transmission, especially in populations with an increased incidence of HIV.
During the 29-month study period, the five identified pregnant women with AHI were promptly prescribed HAART, underwent appropriate delivery modalities and no MTCT occurred in this group. We identified three pregnant women who had tested outside the state system and had negative routine antibody testing yet who transmitted HIV to their infants, after most likely acquiring HIV during pregnancy. These three represent 50% of the HIV-infected infants reported in the state over the same time period. We cannot retrospectively assess whether the HIV infection status of these three women would have been detected by an initial screening algorithm providing for HIV RNA reflex testing. However, these cases demonstrate the ongoing need for public health interventions for the prevention and detection of HIV acquisition in pregnant women.
Following current guidelines, pregnant HIV-infected women in care, receiving ART and other appropriate interventions, have a less than 1–2% chance of transmission to their infants [7,8]. This suggests that a proportion of residual transmissions may be a result of acquisition of HIV during pregnancy. Gray et al.  found that women have nearly two times the risk of acquiring HIV while pregnant compared with nonpregnant women irrespective of their sexual behaviors or their partners' plasma/serum HIV RNA. In other populations, the heterosexual transmission of HIV is associated with an increased HIV viremia [10,11], and the rapid dissemination of HIV at high levels into genital secretions [12–14]. These three factors – increased risk of acquisition, increased viremia, and rapid dissemination – could make pregnancy itself a mechanism for efficient, serial transmission of HIV from male sexual partners to pregnant women and subsequently to their infants.
Our study is limited by its descriptive nature. We cannot estimate the total number of women who acquired HIV during pregnancy in the study period but did not transmit HIV to their infants; nor can we determine if there were additional transmissions of HIV from women who tested in the public or nonpublic testing, as mandatory HIV testing of newborns is currently not performed in North Carolina. Consequently, all children infected during this period may not have been reported and, as a consequence, were not included in this analysis. Still, we can state that a proportion of infected women will be antibody negative when they present for testing, and that one-third of women with AHI that we identified were pregnant.
Our data suggest that enhanced HIV testing and universal antibody rescreening in the third trimester may be needed to reduce MTCT of HIV to the lowest levels possible. We believe the data are sufficient to offer several concrete recommendations. Universal HIV testing of all pregnant women early in pregnancy – similar to syphilis and hepatitis – should be performed and should include reflex RNA testing for antibody-negative women. Similar repeat testing should be performed in the third trimester. Rapid antibody testing with reflex HIV RNA testing should be performed at the time of delivery for women who did not previously receive testing. Confirmatory testing on women with positive HIV RNA results not identified prior to discharge can be achieved through the collaborative efforts of public health officials, obstetricians, and/or pediatricians. Lastly, infants born to mothers who did not undergo repeat testing during pregnancy should be tested for HIV.
The addition of reflex RNA testing to HIV screening algorithms may entail additional cost. We have demonstrated in North Carolina, however, that specimens can be tested efficiently in a centralized public health laboratory if a pooled serum sampling strategy is utilized. We have previously reported that the pooled specimen process resulted in an additional cost of $3.63 per specimen, a cost that included equipment, kits, labor, and administration . This report included a decision analysis from the public health perspective, based on the number of pregnant and nonpregnant AHI identified in the program's first year. This model conservatively projected the number of HIV-infected infants averted at an average of one infant infection every 5 years, presuming 80% adherence to MTCT prevention guidelines and a reduction in the vertical transmission rate from 25 to 14% with ART among pregnant women with AHI. Nonetheless, the use of enhanced testing specifically in antenatal HIV testing sites contributed significantly to the overall cost-effectiveness of the program, with testing in antenatal sites alone resulting in a lifetime cost savings of $11 298 per year. These cost savings were attributable to the enormous cost of lifetime ART  and the impact of undiagnosed infant HIV infection on discounted, quality-adjusted survival .
North Carolina's enhanced testing strategy allowed for intervention and prevention of HIV transmission in all five identified pregnant women with AHI, suggesting that this strategy has had an impact on the residual transmission of MTCT in our state [17–20]. Despite other current efforts, perinatal transmission of HIV still occurs in the United States and other developed countries . What proportion of these transmissions results from unrecognized AHI is not known, but it may be substantial. Perhaps by incorporating HIV into standard prenatal screening, instituting a repeat testing strategy, and utilizing reflex HIV RNA testing, perinatal transmission of HIV in the United States can finally be eliminated. As testing becomes more readily available and accepted, developing countries may also benefit from an enhanced and repeat testing program. Additional research will be needed to demonstrate the usefulness of the approach in resource-poor settings.
The authors are most grateful for the assistance provided by the North Carolina Department of Health and Human Services STAT Disease Intervention Specialists and to Myra Brinson, Juanita Harris, Regina Lee and Vanessa Campbell at NCSLPH for their dedication to the STAT Program.
A special thanks to BioMerieux and GenProbe, who each provided HIV nucleic acid assays free of charge for the first 2 years of the STAT program.
Sponsorship: This project was supported by Building Interdisciplinary Research in Women's Health (BIRCWH) 5 K12 HD 01441-01 (KBP), North Carolina HIV/STD Prevention and Care Branch (PAL, EF, SIM, DW, LW), North Carolina State Laboratory for Public Health (LW, the US National Institutes of Health RO1 MH068686-01 (CDP), and the University of North Carolina Center for AIDS Research P30AI50410 (SAF, JJE).
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