Combined evaluation of sexually transmitted infections in HIV-infected pregnant women and infant HIV transmission

Background Sexually transmitted infections (STIs) including Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Treponema pallidum (TP), and cytomegalovirus (CMV) may lead to adverse pregnancy and infant outcomes. The role of combined maternal STIs in HIV mother-to-child transmission (MTCT) was evaluated in mother-infant pairs from NICHD HPTN 040. Methodology Urine samples from HIV-infected pregnant women during labor were tested by polymerase chain reaction (PCR) for CT, NG, and CMV. Infant HIV infection was determined by serial HIV DNA PCR testing. Maternal syphilis was tested by VDRL and confirmatory treponemal antibodies. Results A total of 899 mother-infant pairs were evaluated. Over 30% had at least one of the following infections (TP, CT, NG, and/or CMV) detected at the time of delivery. High rates of TP (8.7%), CT (17.8%), NG (4%), and CMV (6.3%) were observed. HIV MTCT was 9.1% (n = 82 infants). HIV MTCT was 12.5%, 10.3%, 11.1%, and 26.3% among infants born to women with CT, TP, NG or CMV respectively. Forty-two percent of HIV-infected infants were born to women with at least one of these 4 infections. Women with these infections were nearly twice as likely to have an HIV-infected infant (aOR 1.9, 95% CI 1.1–3.0), particularly those with 2 STIs (aOR 3.4, 95% CI 1.5–7.7). Individually, maternal CMV (aOR 4.4 1.5–13.0) and infant congenital CMV (OR 4.1, 95% CI 2.2–7.8) but not other STIs (TP, CT, or NG) were associated with an increased risk of HIV MTCT. Conclusion HIV-infected pregnant women identified during labor are at high risk for STIs. Co-infection with STIs including CMV nearly doubles HIV MTCT risk. CMV infection appears to confer the largest risk of HIV MTCT. Trial registration NCT00099359.

Introduction Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Treponema pallidum (TP) contribute to formidable global burden of treatable bacterial sexually transmitted infections (STIs) with nearly a quarter of a billion of new cases reported annually. [1] These treatable STIs disproportionately impact the health of pregnant women, particularly adolescents and young women in low and middle-income countries. [1][2][3] Untreated CT, NG, and TP are associated with adverse pregnancy outcomes including spontaneous abortion, stillbirth, preterm labor and delivery. Maternal infection with these conditions has also been associated with neonatal infections such as conjunctivitis (CT, NG), pneumonia (CT), and disseminated infection (TP, NG); TP may be particularly devastating given its associations with multi-organ involvement, failure to thrive, and neonatal death. [4][5][6][7][8][9][10][11] Apart from these bacterial STIs, other sexually transmitted infections including viruses such as cytomegalovirus (CMV) [12,13] are often overlooked but can also have profound infant sequelae including neurodevelopmental anomalies and sensorineural hearing loss when acquired in-utero. [14,15] CMV, which can be transmitted by close or sexual contact, is the major infectious etiology of sensorineural hearing loss and developmental delay, and congenital infection globally may be as high as 1-5%. [16][17][18] Routine antenatal screening for these conditions is not routine practice in many regions of the world, particularly in low and middle-income countries. These practices may have particular importance for high-risk groups such as HIV-infected pregnant women and their infants.
Given earlier findings of our HPTN 040 sub-studies evaluating the individual effects of STIs on HIV MTCT (CT and NG and HIV MTCT; TP and HIV MTCT; and CMV and HIV MTCT), [19][20][21][22] the following analysis provides a more comprehensive evaluation of the risk factors associated with these STIs (CT, NG, TP, and CMV) and the impact of these combined

Baseline characteristics of mother-infant pairs
Among 1684 mother-infant pairs enrolled in the parent study, 899 pairs (53.4%) had specimens available for maternal CT, NG, TP, and infant CMV testing. Specimen availability for testing of all four potential co-infections determined inclusion in the present analysis. As such, 785 mother-infant pairs (approximately 47% of the original HPTN 040 cohort) were excluded. We evaluated potential differences in both populations and determined that both groups were very similar in most parameters (S1 Table). Most women in this analysis (86.2%) were from the Americas (Brazil, Argentina, and the U.S.) compared to South Africa (13.8%). (Table 1) The mean maternal age was 26.5 (SD 6.3) years, and the majority received some prenatal care during pregnancy (69.4%). High rates of alcohol (36.5%), tobacco (37%), and illegal substance (9.8%) usage were noted during pregnancy. There were also high rates of prior poor pregnancy outcomes reported such as stillbirth (4.8%). Median log 10 HIV plasma viral load at the time of delivery was 4.2 (interquartile range = IQR 1.7-6.5) copies/mL, with 58% having HIV viral load >10,000 copies/mL; the median CD4 count was 465 (IQR 12-2160) cells/mm 3 . Preterm delivery (<37 weeks) occurred in 9.2% of infants, and 15.8% were of low birth weight (<2500 grams). Approximately 38.9% of infants experienced a serious adverse event (SAE) during the study period.
Rates of co-infections were high in this sub-cohort; over 30% of women had at least one of the infections of interest (TP, CT, NG, and/or CMV). High rates of TP (8.7%), CT (17.8%), and NG (4%) were found. Based on a positive infant urine CMV PCR at birth, which revealed the presence of congenital CMV (cCMV) infection and was used as a surrogate for active maternal CMV infection, 6.3% of women had CMV infection during pregnancy. (Table 1)

Characteristics of STI positive and STI negative HIV-infected pregnant women
Characteristics of mother-infant pairs were compared for women with and without STIs (TP, CT, NG, and/or CMV). (Table 2) Most women with STIs were under 30 years of age (77.3%). Rates of STIs (35.6%) were highest among young women (13-24 years). The results from the adjusted multivariable model indicated that younger women were 1.6 to nearly two times more likely to have an STI (aOR 1.9, 95% CI 1.3-2.8 for those 13-24 years of age and aOR 1.6,  (Table 3) Infants born to women with one of these STIs (CT, NG, TP, and/or CMV) were nearly twice as likely to be HIV-infected (OR 1.8, 95% CI 1.1-2.9) compared to those born to women without these infections; these findings (aOR 1.9, 95% CI 1.1-3.0) persisted after adjusting for risk factors including maternal HIV viral load, maternal HIV log 10 viral load, maternal CD4 count, prolonged rupture of Detectable CMV from the urine of women and infants were important risk factors for infant HIV acquisition. Women with CMV detected at the time of labor and delivery were over four times more likely to have an HIV-infected infant than those without CMV viruria in adjusted analyses (aOR 4.4, 95% CI 1.5-13.0). Similarly, women whose infants had CMV detected in urine after birth (i.e. congenital CMV; cCMV) were more likely to also have infants with HIV-infected than those without CMV viruria (i.e. without congenital CMV) in the unadjusted analysis (OR 4.1, 95% CI 2.2-7.8), but this did not remain statistically significant in adjusted analysis. (Table 3) Other factors associated with infant HIV acquisition included log 10 HIV maternal viral load (aOR 1.9, 95% CI 1.4-2.5), illegal substance use during pregnancy   (Table 3) Further evaluation was done to compare the proportion of maternal STI infections (0 vs 1 vs 2 vs 3 STIs) between HIV-infected and HIV-uninfected infants. (Table 4) Of the 35 HIVinfected infants born to women with STIs (CT, NG, TP, and/or CMV), the majority were those born to women with one STI (n = 24, 68.6%) as opposed to two (n = 10, 28.6%) or three STIs (n = 1, 2.9%). However, significant differences were noted in the proportion of maternal STI infections (0 vs 1, 2 or 3 STIs) and infant HIV-infection, which ranged from 7.5% (47/626) for 0 STIs to 23.8% (10/42) for 2 STIs. Additional regression analyses were done to elucidate these relationships between the number of STIs and HIV MTCT. Women with two STIs were 3.4 times more likely to transmit HIV to their infants than those without STIs (aOR 3.4, 95% CI 1.5-7.7). (Table 3)

Discussion
The results of this study were notable for the high prevalence of overall STIs (TP, CT, NG, and CMV) in this sub-cohort of HIV-infected pregnant women. The presence of at least one of these STIs represented an increased risk for HIV MTCT, particularly two STIs, with CMV being the most important co-infection contributing to HIV perinatal transmission. In general, limited published studies have focused on evaluating the impact of untreated maternal STIs such as CT, NG, TP, and active CMV infection in pregnancy on HIV motherto-child transmission (MTCT), nor have they comparatively evaluated the potential contribution of individual co-infections within the same cohort on perinatal HIV transmission. The biological plausibility for STIs increasing HIV MTCT risk was suggested in studies of nonpregnant women which demonstrated increased HIV genital shedding in the presence of STIs. [24,25] Chorioamnionitis has also been demonstrated to increase the risk of HIV MTCT. [26][27][28][29][30][31][32][33] Selected studies have demonstrated a link between specific STIs with HIV MTCT, including results from our prior individual HPTN 040 analyses. [19][20][21][22][34][35][36][37] Over 30% of HIV-infected pregnant women in the present analysis had at least one STI (TP, CT, NG, and/or CMV). Our findings re-emphasize that women in our study were at high-risk for multiple STIs apart from HIV, particularly younger women. Predictors of STIs such as younger maternal age has been previously well-documented. [38][39][40][41][42][43] Women enrolled in the parent study were a high-risk group by definition; they were late to present for HIV diagnosis and not on antiretrovirals (ARVs) during pregnancy because they were only diagnosed with HIV at the time of labor and delivery. Nearly 30% did not receive antenatal care and more than a third of women engaged in tobacco, alcohol, and/or illegal substance use during pregnancy.
Collectively, results of this analysis provide additional support that STIs (TP, CT, NG, and/ or CMV) increase the risk of HIV MTCT by nearly two-fold and for those with two STIs, possibly more than three-fold. Forty-three percent of infants with HIV were born to women with at least one of these STIs, while rates of HIV MTCT were most pronounced in women with CMV (>26% but possibly as high as >30% for those with CMV viruria) and CT (12.5%) infection.
Our previous studies of individual STIs also demonstrated that these conditions were associated with an increased risk of HIV MTCT. In our earlier individual analyses, 10% of the original NICHD HPTN 040 maternal cohort had positive syphilis results and were twice as likely to transmit HIV to their infants (adjusted OR 2.1, 95% CI 1.3-1.4). [20] Among 1373 HIVinfected pregnant women with high rates of CT (18.1%) and NG (4.6%), CT appeared to pose a possible 1.5-fold increased risk of HIV MTCT (OR 1.5, 95% CI 0.94-2.3). [19] We believe that the present study did not highlight this MTCT risk as clearly due to the reduction in sample size required to perform a combined analysis, which included testing of subjects for all four co-infections. Apart from our own published results from HPTN 040, literature focusing on the impact of STIs (TP, CT, and NG) and the risk of HIV MTCT has also demonstrated mixed results, particularly for CT and NG. [33,34,36,40] In the present study, maternal CMV (especially CMV viruria) was significantly associated with an increased risk of HIV MTCT (more than four-fold). A prior analysis of the same cohort demonstrated high rates of CMV viruria (9.2%) among HIV-infected pregnant women at the time of delivery, with maternal CMV viruria being associated with a similar but higher risk of HIV MTCT (aOR 5.6, 95% CI 1.9-16.8). [22] The clinical significance of detectable CMV viruria at the time of labor and delivery in HIV-infected pregnant women not on ARVs, however, is not well-understood. Considering that it is very likely that most of the studied women were already CMV seropositive before pregnancy due to the frequent exposure to CMV in these populations, CMV viruria might result from CMV reactivation in the genitourinary tract or reinfection with a new virus strain. [44] The relationship between HIV and CMV perinatal infections is complex, since infection with one may be a risk factor for infection with the other pathogen. Yet, while several studies have shown that HIV-exposure in pregnancy is a risk factor for congenital CMV, fewer have demonstrated that congenital CMV acquisition is also a risk factor for HIV perinatal transmission. [31,37,45,46] To our knowledge, the only other primary study to address this aspect of the relationship was a retrospective case-control study of 293 HIV-infected and HIV-exposed, uninfected infants from Thailand. [37,47] They found that congenital CMV was a risk factor for HIV MTCT, particularly for in utero HIV (OR 8.1, 95% CI 1.5-63.7). [37] In summary, maternal co-infections such as TP, CT, NG, and CMV facilitate HIV perinatal transmission by a variety of means. Genital tract infections from these organisms may lead to cervicitis, which may trigger increased cervico-vaginal HIV viral shedding [24,25,[48][49][50][51][52] or may cause acute or chronic placental inflammation. [26,[30][31][32][33][34] This inflammation at the maternal-fetal interface may induce immune activation and alter cytokine production, increasing viral load, and upregulating the expression of CCR5 T-cell receptors as well as CCR5 HIV co-receptors on Hofbauer cells (macrophages) in the placenta, which contribute towards increased HIV tropism and infectivity. [46,53] It has been suggested that many of these factors are especially important in understanding the role CMV infection plays as a risk factor for HIV MTCT, particularly at the placental interface. These pathogens appear to effectively overcome inherent placental antiviral mechanisms that safeguard against fetal infection. [46,53]

Study limitations
As discussed previously in our results section, specimen availability for testing for potential coinfections determined inclusion in the present analysis. While study inclusion and exclusion populations were similar, some differences were noted in maternal age, mode of delivery, prenatal care, alcohol use, tobacco use, and maternal HIV viral load. In addition, in the parent study, 28% of subjects came from South Africa and 72% from the Americas. This proportion was not maintained in the sub-analysis as specimen availability was limited for South African subjects. Therefore, 13.8% of subjects were from South Africa and the remainder from the Americas in the present analysis, p < 0.0001. This implies that our results might not be as generalizable to the South African site. Additional limitations include inability to test for other STIs and genital tract infections such as bacterial vaginosis, Trichomonas vaginalis, and Herpes simplex virus.

Conclusion
We conducted this analysis to explore the potential synergy between several co-infections and increased risk for HIV MTCT in a large cohort of HIV-infected mothers and HIV-exposed infants. HIV-infected pregnant women in this cohort were at very high risk for STIs (TP, CT, NG, and/or CMV). The presence of one or more infections nearly doubled the odds of HIV MTCT, and the presence of two co-infections tripled the odds. In particular, CMV in HIVinfected pregnant women, especially the presence of maternal CMV viruria at delivery, appeared to confer the largest risk of perinatal HIV transmission. Our study's results emphasize the importance that undiagnosed STIs may play in pregnancy as risk factors for HIV MTCT. Our data underscores the need to augment current existing antenatal care programs to include STI screening, particularly for high-risk women such as young, HIV-infected pregnant women as well as ensuring that HIV-infected women have consistent access to antiretroviral treatment and monitoring, particularly during pregnancy.
Supporting information S1