Mother-to-child transmission of HIV-1 occurs in 6% to 47% of children born to untreated HIV-1-seropositive mothers.1-4 Several prophylactic regimens using different drugs and varying durations of treatment have proven to be effective in reducing perinatal HIV transmission in clinical trials. Zidovudine (ZDV or AZT) was the first drug shown to decrease mother-to-child transmission of HIV-1 and is a recommended component of multidrug regimens for prevention of neonate HIV-1.5-7 This drug blocks HIV replication by directly interfering with viral DNA synthesis through incorporation of the active triphosphate analogue into the elongating viral DNA chain. The analog drug then acts as a chain terminator because these drugs lack a 3′ hydroxyl group, and the necessary 3′ to 5′ phosphodiester bond cannot be formed with another nucleotide to continue replication of the DNA chain.8
Antiretroviral treatment with ZDV from the 14th week until the end of pregnancy has markedly reduced the vertical transmission rate in Europe and North America from 22% to only 6%, as shown in the AIDS Clinical Trials Group (ACTG) 076/ANRS 024 trial.6,9 The nature and duration of that particular ZDV regimen make it complex and expensive, and thus impractical for less-developed areas of the world such as Southeast Asia. In addition, it requires prolonged antenatal and neonatal administration of antiretroviral drugs, which could be associated with an increased risk for drug toxicity.10 For these reasons, shorter and less expensive antiretroviral regimens have been tested with successful results, albeit to a lesser degree.5,11,12 The optimal duration of therapy that would minimize HIV transmission and toxic side effects to the fetus and neonate has not been well established, however. There have been several studies on mother-to-child transmission of HIV-1,13-20 but few have dealt with subtype E, the most common subtype in Southeast Asia.21 Whether the same regimens are applicable to all subtypes is unknown.
There is evidence that subtypes B and E do not behave identically. Subtypes B and E are present in Thailand; however, subtype B is mainly found among intravenous drug users, whereas subtype E is predominantly found in prostitutes and heterosexuals.22,23 It has been proposed that there are significantly different degrees of contagiousness between the 2 subtypes.24-26 Estimates of the heterosexual transmission rate of subtype E are 10 to 50 times higher than those of subtype B.27,28 Cellular reservoirs differ as well, with subtype E often residing in Langerhans type histiocytes,29 whereas subtype B is more often found in follicular dendritic cells. Subtype E is genetically distinct from subtype B. There are genetic variations within type B that contribute to drug resistance.30 Hence, it is reasonable to postulate that subtype E might respond differently to drugs used in the treatment of subtype B.
Although the concept of direct maternal-fetal transmission is now accepted, the exact timing of this remains unclear. It is generally believed that most pediatric HIV type 1 infections occur at or near birth.3,31-33 Using a mathematic model, it could be shown that 95% of cases of transmission in utero likely occur less than 8 weeks before delivery.34 On this basis, we conducted a prospective study among pregnant women infected with HIV-1 subtype E, who received ZDV for different durations during the prenatal period but not earlier than the 28th week of gestation. In this way, we were able to analyze to what extent duration of therapy influenced HIV-1 infection of the neonate and placenta.
MATERIALS AND METHODS
The study group consisted of 50 HIV-seropositive pregnant women on ZDV prophylaxis who were patients at the Maharaj Nakorn Chiang Mai University Hospital or Health Promotion Center Region 10 at Nakornping Hospital. Seropositivity was determined by enzyme-linked immunosorbent assay (ELISA) and confirmed by Western blot analysis. Pre-entry evaluations included hemoglobin, white blood cell count and differential, CD4 and CD8 count, HIV RNA viral load, creatinine, serum glutamic-pyruvic transaminase (SGPT), hepatitis B surface antigen, and hepatitis C serology. Women were accepted into the study and randomized provided that they had received ZDV for at least 2 weeks, agreed not to breast-feed, and had the following laboratory values within 21 days before randomization: hemoglobin >8.0 g/dL, absolute neutrophils count >750 cells/mm3, SGPT <5 times the upper limit of normal, and creatinine <1.5 mg/dL. Exclusion criteria included ZDV prophylaxis for less than 2 weeks, laboratory values not in the acceptable range, maternal or fetal condition or concomitant treatment contraindicating ZDV, oligohydramnios, unexplained polyhydramnios or in utero anemia, or medical need for immediate highly active antiretroviral therapy. Women were monitored every 2 weeks until delivery and then at 10 days, 6 weeks, and 4 months postpartum. Adherence to ZDV therapy was assessed by pill count. ZDV prophylaxis consisted of 300 mg administered twice daily, switching to 300 mg administered every 3 hours from the onset of labor until delivery. Twenty-seven women were randomized to “short-term” ZDV lasting from 14 to 35 days before delivery (median = 28 days), whereas the other 23 women received “long-term” ZDV lasting from 62 to 92 days (median = 76 days). Written consent was obtained from all patients.
Evaluation of Neonates
Fifty neonates were evaluated at birth and at 10 days; 6 weeks; and 4, 6, 9, and 12 months after birth. Evaluation at birth included physical examination, hemoglobin, neutrophil count, and creatinine, SGPT, and bilirubin measurements. For HIV diagnosis, peripheral blood drawn at birth and at 6 weeks and 4 and 6 months after birth was spotted onto filter papers, dried, and stored at −20°C until tested by HIV DNA polymerase chain reaction (PCR) using the AMPLICOR HIV-1 DNA version 1.5 assay (Roche Molecular Systems, Alameda, CA). Infants were considered infected if 2 samples obtained on separate occasions were HIV-positive by PCR and uninfected based on 2 negative DNA PCR tests obtained after 1 month of age. For HIV-infected neonates, transmission was labeled “in utero” if the DNA PCR test obtained within 3 days of birth was positive.35
HIV subtyping was carried out using the PCR on DNA extracted from peripheral blood samples. The portion of the genome amplified included the dimerization initiation site (DIS) loop sequence present in infectious recombinant provirus. The DIS loop and flanking sequences were amplified from cell lysates by using a 27-base pair (bp) upstream (sense) primer DIS1 (5′-AAATCTCTAGCAGTGGCGCCCGAACAG-3′) and a 24-bp downstream (antisense) primer DIS2 (5′-CTCTCCTTCTAGCCTCCGCTAGTC-3′) based on published sequences.36 A 165-bp PCR fragment was generated after 30 cycles of PCR amplification according to published conditions.36 A 10-μL sample of the PCR mixture was subjected to digestion with BssHII, which recognizes the sequence GCGCGC found in HIV-1 subtype B but not subtype E, and with ApaLI, which recognizes the sequence GTGCAC found in HIV-1 subtype E but not in subtype B.36 Positive controls were known infected peripheral blood mononuclear cells of both subtypes.
Placentas were collected from all 50 seropositive patients at the time of delivery. Placentas from 25 HIV-seronegative pregnant women were collected at the same hospitals as negative controls. Multiple tissue samples were taken from all placentas, fixed in neutral-buffered formalin, and embedded in paraffin. Sections (3 μm) were mounted on poly-l-lysine-coated slides. Sections were examined by routine light microscopy after staining with Harris hematoxylin-eosin, Gram (Brown-Hopps), mucicarmine, periodic acid-Schiff, Warthin-Starry, Gomori methenamine silver, and Ziehl-Neelsen stains. Microscopic features were evaluated separately by 3 pathologists, without knowledge of which drug regimen had been used.
In Situ Polymerase Chain Reaction
In situ PCR was performed on sections of placenta using 12.5 μM of primers: SK38 (sense; 5′ ATA ATC CAC CTA TCC CAG TAG GAG AAA T), SK39 (antisense; 5′ TTT GGT CCT TGT CTT ATG TCC AGA ATG C), TN01 (sense; 5′ GGA AGT GAC ATA GCA GG), and TN02 (antisense; 5′ CTA CAT AGT CTC TGA AGG G); 0.2 mM of each deoxynucleoside triphosphate (dNTP), 0.15 U of Taq DNA polymerase, and 1 × PCR buffer. Primers SK38 and SK39 contain sequences shared by subtypes B and E, whereas primers TN01 and TN02 are specific for subtype E.37 The mixture was contained in 15 μL, which allowed the entire tissue section to be covered, after which a glass coverslip was added and sealed with clear nail polish. The amplification process was achieved using a Hybaid in situ PCR thermocycler (Franklin, MD) under the following conditions: 94°C for 30 seconds, 61°C for 1 minute, and 72°C for 1 minute for 30 cycles. The cover slip was removed, and the slide was washed in 2 × standard saline citrate (SSC) for 2 minutes and PBS for a further 2 minutes. Subsequently, the sections were postfixed in freshly made 4% paraformaldehyde for 5 minutes, dehydrated in graded alcohol, and air-dried. Sections were then incubated overnight at room temperature with the biotinylated probe, TN03 (5′ ATC CTG GGA TTA AAT AAA ATA GTA AGA ATG TAT AGC CC), which hybridizes to the PCR reaction product. The TN03 probe was then visualized by tyramide amplification using the TSA-Indirect ISH kit (NEN Life Science Products, UK) according to the manufacturer's protocol. Briefly, sections are blocked for 30 minutes in TNB buffer at room temperature and are then incubated for 30 minutes in horseradish peroxidase-conjugated streptavidin, followed by 5 minutes in biotin tyramide solution. For visualization of the signal, sections were incubated for 30 minutes in horseradish peroxidase-conjugated streptavidin. Diaminobenzidine was used as the chromogen and hematoxylin as a counterstain.
Cells expressing HIV proviral DNA by in situ PCR were quantitated as the number of positive cells per 100 decidual glandular epithelial cells. The degree of infectivity was graded as grade 0 (no HIV expression), grade 1 (≤5% cells positive), grade 2 (>5%-10% cells positive), grade 3 (>10%-30% cells positive), and grade 4 (>30% cells positive).
Statistical comparisons between short-term and long-term therapy groups were carried out using the standard Student t test and Pearson χ2 test. The 2-tailed Fisher exact test was used to evaluate differences in placental pathologic findings between short-term and long-term therapy groups. For all tests, P values of 0.05 or less were considered to be significant.
Evaluation of HIV-1 Subtype E-Seropositive Pregnant Women
Risk factors for HIV were determined for the 50 women: 44 were housewives whose husbands were HIV-positive and had contact with a commercial sex worker; 4 others had no such contact but had received blood transfusions; and of the remaining 2, 1 was a commercial sex worker and 1 was an intravenous drug user. Seven (14%) of 50 women delivered before 37 weeks of gestation. Ten (20%) of 50 women delivered through cesarean section, and 3 of these procedures were performed before the onset of labor. Laboratory findings of women in the study were similar between the short-term and long-term ZDV prophylaxis groups. Selected parameters are shown in Table 1. CD8-positive cells outnumbered CD4-positive cells in both groups. There was no significant difference between the 2 groups in terms of CD4-positive and CD8-positive cell counts. Whereas the median viral load in the short-term ZDV group was higher than in the long-term group, the difference was not significant. All 50 HIV-seropositive mothers in this study were infected with subtype E of the HIV-1 virus.
There were no significant differences on clinical examination or on routine laboratory testing in neonates born to mothers on short-term ZDV prophylaxis compared with mothers on long-term prophylaxis (results not shown). Four neonates were positive for HIV-1 as detected by PCR on peripheral blood samples. All these neonates were from the short-term prophylaxis group; all were born full term: 2 by vaginal delivery and 2 by cesarean section. One of 4 neonates had positive PCR results at 10 days after birth in keeping with in utero transmission.35 The other 3 neonates had negative DNA PCR results at 10 days but positive results at 6 weeks and 4 months of age in keeping with peripartum transmission.
Pathologic Findings of Placentas of HIV-Seropositive Women
Placentas were evaluated for presence of the following findings: chorioamnionitis, villitis, villous stromal fibrosis, placental infarction, abnormal villous maturity, acute deciduitis, plasmacellular deciduitis, and decidual necrosis. The most common finding was infarction, which was present in 33% of cases in the short-term therapy group and in 35% of cases in the long-term therapy group. Acute deciduitis was found in 22% of cases in both groups, and chorioamnionitis was found in 19% and 22% of cases in the short-term and long-term therapy groups, respectively. Villitis, villous stromal fibrosis, and abnormal villous maturity were found infrequently in both groups, and plasmacellular deciduitis and decidual necrosis were not found at all. There were no significant differences in any of these pathologic parameters with respect to the 2 treatment groups. With respect to the 4 infected neonates, chorioamnionitis was present in the placenta of the neonate who was HIV-positive by PCR by 10 days of age. Placentas from the other 3 neonates who were HIV-positive by PCR after 10 days of age did not show chorioamnionitis.
HIV Proviral DNA Expression in Placentas of HIV-Seropositive Women
In situ PCR performed on placentas from seropositive women revealed HIV proviral DNA expression only in decidual glandular epithelial cells (Fig. 1). Expression was detected in 30 (60%) of 50 cases, with varying numbers of positive cells as shown in Table 2. For purposes of analysis, grades of 0 and 1 were grouped as “low infectivity” (ie, ≤5% of decidual glandular epithelial cells expressing HIV) and grades 2 through 4 were grouped as “high infectivity” (ie, >5% of decidual glandular epithelial cells expressing HIV). According to this scheme, 67% (18 of 27 cases) of women on short-term ZDV therapy were in the high-infectivity group compared with 22% (5 of 23 cases) on long-term therapy. Alternatively, 33% (9 of 27 cases) of women on short-term ZDV therapy were in the low-infectivity group compared with 78% (18 of 23 cases) on long-term therapy (P < 0.02). No control placental tissues in HIV-uninfected cases revealed proviral DNA signals.
The purpose of our study was to examine the effectiveness of 2 different durations of limited ZDV therapy on transmission of HIV-1 subtype E, beginning no earlier than the 28th week of gestation. This type of regimen is being administered in Southeast Asia, mainly on an empiric basis, because clinically proven regimens are of longer duration and are too expensive to administer in less affluent parts of the world. Moreover, such studies are based on subtype B of HIV-1 rather than on subtype E, which is the predominant type in Southeast Asia. Based on previous experience, the situation with subtype E is far from straightforward. One recent study concluded that there was no significant difference in the transmission rate in women who started ZDV prophylaxis before or after 30 weeks of gestation.20 In contrast, another found that short-term therapy begun at 36 weeks of gestation actually resulted in a worse outcome in neonates than in neonates not exposed to antiretroviral therapy.38 Our study showed that a duration of at least 60 days of prophylaxis was associated with reduced transmission to neonates.
One strength of our study was the availability of placentas in all cases. This allowed an objective measurement of HIV infection as well as providing data about the dynamics of HIV infection in the placenta and the effect of antiretroviral drug therapy on the placenta. Most studies on maternal-neonate transmission of HIV have not included examination of the placenta.13-20
Our study of placentas showed that HIV-1 can be detected in 60% of placentas of seropositive mothers, even when these women are receiving ZDV prophylaxis. Nevertheless, there is evidence of drug efficacy, because only decidual glandular cells were positive for HIV-1 in this group of patients and in none of the fetal contribution to the placenta. This contrasts with the findings of our previous study, in which we examined placentas from seropositive patients not receiving any antiretroviral prophylaxis and found that maternal and fetal cell types were infected by HIV-1, with more than 80% of placentas positive.39 Further evidence in the present study for drug efficacy is that significantly fewer positive cells were noted in women who had received the long-term regimen compared with the short-term regimen (P < 0.02).
Infection of the placenta with HIV-1 did not equate to transmission to the fetus or neonate, implying that other factors are involved in vertical transmission. Nevertheless, in our study of 50 neonates born to seropositive mothers, 4 were found to be infected with HIV-1 subtype E in the prenatal or peripartum period. All 4 were born to mothers who had received the short-term regimen (18, 22, 30, and 40 days, respectively). There was no correlation with maternal HIV viral load or CD4 or CD8 cell counts. There was, however, a correlation with HIV detection in the placenta; all 4 cases were in the high-infectivity group. These results allow us to comment on the optimal regimen that might be considered for ZDV prophylaxis. Because there was fetal or neonate transmission only in mothers receiving <35 days of ZDV prophylaxis prenatally, this duration would not seem to be optimal. The long-term regimen was at least 60 days of therapy, suggesting that this duration might be protective.
We do not have data on why 60 days of therapy would be particularly protective, only that it was the lower limit of duration of therapy in the long-term group. Perhaps shorter courses of therapy lead to emergence of resistant strains. Alternatively, agents that block viral replication, such as ZDV, are not necessarily virucidal and may require the action of other cells or pathways to eliminate the virus. Short courses may not allow sufficient time for this to occur completely. We would also need to obtain data on durations of 35 to 60 days to know if a duration within this range would be equally protective.
Sixty days of prenatal therapy implies beginning around the 31st week of gestation. Previously, the regimen of ZDV prophylaxis recommended by the Thailand Ministry of Public Health started at 34 weeks of gestation (or 42 days of therapy, between our short-term and long-term prophylaxis groups). Based on our finding that 14% of mothers with HIV-1 deliver before 37 weeks of gestation, however, such patients would receive at most 21 days of therapy and would fall into the short-term therapy group, which we found to be inadequate to prevent transmission of HIV to the neonate. To take into account the increased tendency of infected mothers to deliver prematurely, the Thailand Ministry of Public Health now recommends starting ZDV prophylaxis at 28 weeks of gestation until delivery. This would provide 84 days of prophylaxis in cases of full-term births and at least 63 days for gestations of up to 37 weeks. The results of our study would support such a recommendation. This is based on a study of subtype E, however, and whether similar recommendations apply to subtype B remain to be determined.
In our study, short-term ZDV therapy was associated with increased HIV-1 transmission to neonates. There are other aspects of short-term courses of ZDV therapy that may be cause for concern but were not part of this study. As with any antimicrobial agent, shorter courses of therapy have a greater chance of producing drug-resistant forms, and in the cases of mother-to-child transmission, such forms would emerge in infected neonates. Short-term courses have also been associated with early and more rapid disease progression in neonates compared with neonates with no antiretroviral therapy.38 Although it is unclear if these findings, based on subtype E, are applicable to the other subtypes around the world, it does indicate that short-term therapies have potential disadvantages that should be considered relative to the advantages of lower cost and greater simplicity of administration.
The pathologic finding of high infectivity by HIV-1 in the placenta was associated with transmission to the fetus or neonate and might have some predictive value in determining which neonates would benefit from more careful screening. Stated another way, lack of placental infection was never seen in an infected neonate. Nevertheless, this finding could not be used to predict which specific neonates would become infected; clearly, there other factors involved in determining vertical transmission. One issue not addressed in this study is the timing of infection. If infection were to occur before the start of prophylaxis, such neonates might still be born infected. There is evidence from our studies and others that at least some cases of HIV vertical transmission occur as early as 8 weeks of gestation.39-41 If this were a major determinant of vertical transmission, however, one might expect there to be infected neonates in the long-term prophylaxis group as well as in the short-term group.
An interesting observation from the present study is that only cells from the maternal contribution to the placenta were found to be infected. This suggests that HIV infection of the fetus is not simply via infection of the fetal components of the placenta with spread to the fetus itself. It is likely that other pathways are involved, such as physical breaks in the placental barrier or coexisting inflammation that might induce changes in the intrauterine environment via cytokine effects or expression of receptors and/or coreceptors.42 In support of this concept, we and other investigators have reported an increased vertical transmission risk of HIV associated with chorioamnionitis.39,43-45 Moreover, in the present study, chorioamnionitis was found in the placenta of the 1 neonate who was HIV-positive already by 10 days of age. Further studies are needed to delineate the role of these factors and their effects on the placenta and fetus in the setting of vertical transmission of HIV.
The authors thank Lukana Eiangleng for her expert technical assistance.
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Keywords:© 2005 Lippincott Williams & Wilkins, Inc.
HIV-1; placenta; zidovudine; vertical transmission; Thailand