Introduction
In 1999, more than 1600 children were infected with HIV every day worldwide, most of them in developing countries acquiring the virus through mother-to-child transmission (MTCT) [1]. Up to 25–40% of women attending antenatal clinics in some urban areas in sub-Saharan Africa are infected with HIV, and at least a quarter are likely to pass on the infection to their babies [1]. MTCT occurs either in utero, during or close to birth, or postnatally through breast milk. It is estimated that at least 40%, and possibly up to 80% of transmission occurs around the time of birth [2]. The exact mechanisms of MTCT remain unclear, but exposure to maternal infected secretions or blood, either during labour or delivery, has been suggested to increase the risk of MTCT. First-born twins are at increased risk of infection compared with the second twin [3]. A duration of rupture of membranes (ROM) longer than 4 h is associated with an increased risk of MTCT [4]. Elective caesarean section has been found to reduce the risk of MTCT in a non-breastfeeding population [5].
Zidovudine given antenatally, intrapartum and postnatally according to a regimen known as ACTG 076 has been proved to reduce MTCT by 67%[6]. In association with antiretroviral therapy during pregnancy and delivery, elective caesarean section further reduces the risk of transmission to 2% in the absence of breastfeeding [5]. Simpler regimens have also proved effective in breastfeeding populations in developing countries [7,8]. Recently, the prescription of a single dose of nevirapine to the mother at the onset of labour and to the infant within the first 3 days of life showed a reduction of MTCT by half [9]. However, the implementation of such strategies in resource-poor settings will require important additional funding. Besides the drug supply, HIV voluntary counselling and testing must be provided on a large scale in antenatal clinics. Therefore, cheap, safe, and easy interventions, not requiring antenatal HIV testing, to be used alone or in association with antiretroviral treatment, are still needed.
Disinfection of the vagina with aqueous chlorhexidine during labour was shown to be a safe intervention, reducing the neonatal morbidity and mortality caused by group B streptococci [10]. In vitro, chlorhexidine gluconate inactivates HIV at a concentration of 0.2%[11,12]. Consequently, vaginal lavages with chlorhexidine during labour have been suggested as a method to reduce MTCT, either by inactivating the virus or by decreasing the viral concentration in the birth canal. In Malawi, vaginal cleansing with 0.25% chlorhexidine solution every 4 h during labour, and washing of the baby with the same solution at birth, did not show an overall reduction of global MTCT. However MTCT was significantly lower in the sub-group of women with ROM longer than 4 h [13]. It was unclear, however, whether the lack of impact in the Malawi trial was due to wrong assumptions on the potential effect of the lavage on the virus, or to technical constraints. In Malawi, vaginal cleansing was carried out using chlorhexidine-soaked cotton wool, whereas a vaginal douching technique had been used in the Scandinavian studies. Therefore, we repeated the Malawi trial in a prospective study examining the effect on perinatal HIV transmission of vaginal lavage with chlorhexidine, using a plastic catheter for vaginal douching instead of genital cleansing with cotton wool.
Methods
The study was conducted between 1996 and 1999 in Coast Provincial General Hospital, Mombasa, Kenya. Ethical approval was obtained from the Kenyatta National Hospital Ethical and Research Committee. The intervention used diluted chlorhexidine acetate (Hibitane) at a concentration of 0.2% during the first 17 months. After the publication of the Malawi trial, showing no overall reduction of MTCT with chlorhexidine 0.25%[13], and after careful discussion with all stakeholders, the concentration of chlorhexidine was increased to 0.4% for the last 11 months of recruitment. Chlorhexidine has already proved to be safe [10], and is routinely used in many maternity hospitals all over the world.
Women were admitted in the delivery room at a cervical dilation of at least 4 cm. Obstetric data were collected as well as data on the timing of lavage. Mothers in late second stage at the time of admission were not included. Planned or emergency caesarean section, suspected congenital abnormality, obstetric contraindication to vaginal manipulation (e.g. placenta praevia), known allergy to chlorhexidine, serious maternal illness, complicated labour or delivery were all excluded, as well as women living too far from the hospital to allow follow-up. Selected patients were allocated to intervention by alternating blocks of time. For logistical reasons, a regime of 9 days of lavage followed by 5 days of non-lavage was introduced early on in the trial. This was then balanced by a regime of 4 days of lavage and 10 days of non-lavage at the end of the recruitment period.
During the first stage of labour, the procedure was explained and verbal consent was sought to perform the lavage, as this procedure was not part of routine Kenyan obstetric care. During the vaginal examination, a plastic catheter attached to a 60 ml syringe was gently introduced up to the cervix. The cervical area, where a greater shedding of the HIV has been described [14], was flushed with 60 ml of the solution. The syringe was then refilled without removing the catheter, and a second flushing with the same amount was performed, while withdrawing the catheter slowly. The lavage was repeated every 3 h until delivery. After a short training and supervision period, the obstetric staff carried out the procedure without any problems. During the non-intervention time, chlorhexidine was removed from the delivery room and patients received the usual hygienic labour care, including cleansing of the vulva and the perineal area with savlon. To allow the intervention to work, mothers who delivered within one hour from admission were excluded from both arms of the trial. An intention-to-treat analysis was performed on the remaining mothers. The intervention was further considered adequate if there was at least one hour between the first lavage and delivery, and not more than 4 h between the last lavage and delivery. Subsequent analysis excluded women who did not fulfil these criteria, but the number of women excluded for this reason was small (Fig. 1).
Before discharge and within 48 h after delivery, information about the study was provided in a group session to women who delivered a liveborn baby. Mothers who delivered a stillborn baby or whose infant died in the early neonatal period were excluded from further follow-up. A written consent to participate was obtained individually. For mothers willing to participate, a venous blood sample was taken for HIV and syphilis testing and measurement of the haemoglobin level. At the same time, 250 μl of venous blood were collected in ethylenediamine tetraacetic acid-coated tubes from all babies (day 1 sample) and stored at −20°C until analysis. Data on maternal sociodemographic status and obstetric history, signs of adverse effects of chlorhexidine lavage and baby's health were recorded, as well as all the information necessary for home visiting.
Infants were seen for a follow-up visit at 6 weeks (between 4 and 9 weeks) and 14 weeks (between 10 and 20 weeks), which is the usual time to return for immunization. During the 6 week visit, mothers were offered their HIV test result. HIV-positive women received post-test counselling by a project counsellor. Blood samples were collected from the babies born to HIV-positive mothers at 6 and 14 weeks. HIV-positive women who did not show up for a follow-up visit were visited at home by the project counsellors and were encouraged to come back to the hospital. HIV-negative mother–infant pairs were discharged from the study after the 6th week of follow-up.
Maternal HIV status was determined by enzyme-linked immunosorbent assay (Detect-HIV; Biochem ImmunoSystems, Montreal, Canada). Positive samples were confirmed by a second enzyme-linked immunosorbent assay (Recombigen; Cambridge Biotech, Worcester, MA, USA). Mothers with discordant results were excluded from the analysis, unless their serostatus was confirmed on a sample taken during the follow-up visit. Maternal syphilis status was tested by rapid plasma reagin (RPR; Becton Dickinson, Cockeysville, MD, USA). HIV infection of infants born to HIV-1-positive mothers was determined by polymerase chain reaction (PCR). DNA was extracted from whole blood using the QIAamp DNA blood kit (QIAGEN, Hilden, Germany). Part of the human leukocyte antigen DQ gene was amplified first to control the quality of the DNA. PCR was performed as described by Vandamme et al. [15] using the pol primers and the env primers. Samples with discordant results for the HIV pol and env PCR were tested again using the gag primer set described by Poss et al.[16]. All PCR analysis was performed in the AIDS Reference Laboratory of the University of Ghent, Belgium.
For infants found to be HIV positive at 6 or 14 weeks, the day 1 sample was tested retrospectively. An infant was considered to have been infected in utero if the day 1 sample was HIV-1-DNA positive. An infant was considered to have been infected perinatally if negative at day 1, and positive at 6 and 14 weeks. Not all patients came for both the 6 and 14 weeks follow-up visit. We considered the baby's HIV status based on the result of the sample taken at 6 weeks after birth or at 14 weeks if a result at 6 weeks was not available.
Assuming a 30% MTCT rate, of which half takes place during labour and delivery, and a potential reduction of the intrapartum transmission rate by 50%, it was calculated that 425 HIV-positive mothers and infants per group would have to be analysed to show a reduction from 30 to 22.5% with a power of 80% and a significance of 90%. After an interim analysis of 250 mother–infant pairs in each group, the intervention showed no effect and the study was stopped, as it was demonstrated that it was highly unlikely to verify the initial hypothesis. Data were analysed using the SPSS package (SPSS, Chicago, IL, USA). Chi-square, Fisher's exact, Student's and Mann–Whitney tests were used for statistical analysis. Odds ratios (OR) and 95% confidence intervals (CI) were calculated. Factors related to lavage were examined specifically, tested in a multivariate logistic regression model including general risk factors for MTCT, and are presented with adjusted OR, 95% CI and P values.
Results
Recruitment took place between June 1996 and March 1999. Circumstances beyond the control of the investigators (nurses’ strike, displacement of study population in one of the peripheral areas of Mombasa) caused temporary interruptions to recruitment for a total of 7 months. Patients recruited 2 months before these events were excluded from analysis, as they were poorly attended during that time.
Out of 10 144 women who delivered during the non-lavage weeks, 3173 (31.3%) were screened, compared with 3217 (34.6%) out of 9306 women who delivered during the intervention weeks (Fig. 1). The main reasons for not being recruited were: living too far away to allow a follow-up, or intending to travel back to the rural home area after delivery (32.1%); late admissions with a short stay in the delivery room (23.6%); and complicated deliveries or serious maternal illnesses (19.1%). The lavage criteria were not fulfilled for 92 women (14 HIV-positive), 37 with less than one hour between first lavage and delivery, and 55 with more than 4 h between last lavage and delivery, because of logistical reasons (overcrowded labour rooms). The average number of lavages before delivery was 2.1 (range 1–11). Infants’ Apgar scores and maternal complaints the day after delivery have been analysed to detect adverse effects of the intervention. Infants’ Apgar scores at 1, 5 and 10 min after birth were comparable in the non-lavage and lavage group (means 8.6, 9.6 and 9.9 versus 8.5, 9.6 and 9.9). The rates of maternal complaints (any complaint, itching or stinging of the genital tract, other complaint) the day after delivery were also comparable (3.4, 2.7 and 0.5% versus 3.4, 2.4 and 0.8% with P = 0.98, 0.47 and 0.13, respectively).
Thirty-six mothers (0.3%) refused to be tested for HIV. In the non-lavage group, 431 (13.7%) mothers were HIV positive versus 467 (14.6%) in the lavage group (P = 0.30). Eight children born to HIV-positive mothers in the group of non-lavage and five in the group of lavage were reported to be dead before 6 weeks.
Respectively, 297 (68.9%) and 309 (66.2%) mother–infant pairs attended a follow-up visit at 6 or 14 weeks (P = 0.38), or both. The analysis presented here only considered HIV-positive women and their infants who came for a follow-up visit, including four twin deliveries in the group of non-lavage and three in the group of lavage women, for whom only the results for the first-born twin were taken into account. Three mothers did not breastfeed their infants. Patients were comparable for most of the demographic parameters, obstetric history and characteristics of delivery (Table 1). There was a significantly higher proportion of primigravidae during the non-lavage than the lavage period (35.5 versus 27.3%, P = 0.03). Women lost to follow-up were similar to the attenders for all the characteristics presented in Table 1.
The determination of in-utero MTCT was not possible for four children in the group of non-lavage women (two samples not available and two samples with inconclusive results), and for three children in the group of lavage women (two samples not available and one with inconclusive results). The global MTCT was 21.7% (95% CI 17.4–27.4) in the non-lavage group and 20.5% (95% CI 16.0–25.0) in the lavage group. In-utero MTCT was 5.0%, and was comparable for the two groups (Table 2). Vaginal lavage during delivery can only have an influence on the transmission of HIV occurring during the intra-partum period. Therefore, the efficacy of lavage was calculated in the group of children who were HIV negative at birth. There was no statistical difference between intra-partum MTCT in the group of non-lavage and in the group of lavage, 17.2% (95% CI 12.8–21.6) versus 15.9 % (95% CI 11.7–20.1). There was no evidence of a difference of intra-partum MTCT between the intervention and non-intervention groups, whether the concentration of chlorhexidine used was 0.2 or 0.4%, and whether the duration of ROM was less or equal to 4 h, or longer than 4 h.
In the non-lavage group, none of the factors analysed showed a significant correlation with intrapartum MTCT (Table 3). In the lavage group, a duration of ROM longer than 4 h and a birthweight of less than 2500 g was associated with a higher rate of intrapartum MTCT.
To examine the effect of the intervention under specific conditions, we compared the treatment arms in chosen subsets of the population (Table 4). As the particular timing of performing the intervention was not randomized, we took special care in adjusting for possible confounding factors for MTCT. For example, a patient with a prolonged duration of ROM is more prone to have a lavage after ROM than a woman with ROM just before delivery. All analyses were performed adjusting for maternal age over 27 years (75% percentile), duration of ROM more than 4 h, and birthweight less than 2500 g. We also adjusted for parity, as this variable was not well balanced between groups. For 58 (10.2%) of the 569 mother–infant pairs at risk of intrapartum MTCT the value of one or more outcome variables was missing, therefore, analysis was conducted for 511 mother-infant pairs only. The intrapartum MTCT rate globally was 17.7% for the 249 mother–infant pairs of the non-lavage group and 15.3% for the 262 mother–infant pairs of the lavage group.
The group with 0.4% chlorhexidine lavage administered solely before ROM showed the lowest rate of MTCT (adjusted OR 0.1, 95% CI 0.0–0.9). Neither the total number of lavages nor the duration between the last lavage and the time of delivery appeared to influence intrapartum MTCT.
Discussion
This study examined the effect of vaginal lavage with chlorhexidine on MTCT. The intervention and non-intervention groups were comparable for all baseline characteristics measured, except, for no clear reason, a higher parity in the non-lavage group. The difference was absent at enrolment but emerged only in the group of HIV-positive women who attended a follow-up visit. Women were recruited from the delivery room, and were mostly unaware of their HIV status.
The observed rate of in-utero MTCT was 5.0%, in line with results reported in the literature. Only one-third of all deliveries during the recruitment period were enrolled into the study. However, this did not seem to influence the MTCT rate, which was 22.2% in the non-intervention group, comparable with rates in placebo arms of other intervention studies in breastfeeding populations [7,8,13,17–19]. This adds confidence to the representativeness of the sample.
This intent-to-treat analysis shows no effect of vaginal lavage with chlorhexidine on global MTCT (22.27 versus 21.0%, P = 0.78) nor intrapartum MTCT (17.2 versus 15.9%, P = 0.66). The lack of evidence of an intervention on the effect of intrapartum MTCT can be due to several reasons: (i) breastfeeding might have increased the number of infections reported as intrapartum, as early postpartum infections were not differentiated; (ii) the chlorhexidine concentration, virucidal in vitro, might be too low to be active in vivo; (iii) the secretions and fluids excreted in the birth canal during labour might dilute and inactivate the chlorhexidine solution; (iv) the technique of flushing might be unable to wash away infectious secretions that are too viscous and adhere to the vaginal wall. This might explain why, unlike in our study, birth canal cleansing with cotton wool soaked in a solution of chlorhexidine was found to be associated with a significant reduction of MTCT in mothers with ROM for more than 4 h before delivery [13]. Furthermore, in the latter study, infants were also washed with a chlorhexidine solution. One may debate the choice of an interval of 3 h between consecutive lavages. However, the aim of our study was to find a realistic intervention, feasible in a busy labour ward in a developing country, and this schedule was chosen to fit in with the usual time of review of women in labour in such health services. If infants were infected at the moment of delivery, additional washing at the time of expulsion might be necessary to reduce MTCT.
HIV can be detected in the gastric aspirates of neonates after birth [20–22]. In a sub-sample of the study population, we identified HIV in the infant's oropharyngal secretions at birth as an independent factor of MTCT [23]. This supports the suggestion made by Van de Perre [24] that a substantial amount of MTCT might occur through the mucosal route after the oral intake of HIV by the infant. A careful aspiration of the infant's oropharyngal and gastric secretions in association with lavage might reduce infant exposure to HIV. Nevertheless, we cannot exclude the possibility that the source of infection might not be the birth canal, but the amniotic fluid or the placenta. In this case, disinfection of the birth canal during labour or the infant's oral cavity after delivery will not reduce MTCT. An alternative approach with benzalkonium chloride lavage during late gestation, and infant bathing at birth with the same product has proved to be safe and feasible in HIV-positive women in Africa [25]. Its efficacy in reducing MTCT remains to be determined.
Neither the number of lavages nor the duration between the last lavage and delivery had an effect on intrapartum MTCT. However, if we accept that proper adjustment was made for possible confounders, this study generates the hypothesis that administering lavage before ROM is beneficial, especially with a higher concentration of chlorhexidine. This is plausible if we assume that lavage before ROM clears the genital tract of viral particles and infected cells, thereby preventing infectious materials from ascending to the fetus.
Conclusion
A better understanding of the mechanisms of MTCT is still needed to identify targets and design innovative interventions. Given the remaining uncertainty about the possible beneficial effect of performing lavage before ROM with a higher concentration of chlorhexidine, it remains worthwhile to confirm this observation in a specifically designed confirmatory trial. Interventions to reduce MTCT that do not require HIV voluntary counselling and testing are still needed in poor resource settings, where access to comprehensive maternal–child health/voluntary counselling and testing services is scarce outside the context of research. Despite the increasing number of options with respect to the choice of antiretroviral regimens for reducing MTCT of HIV, these regimens remain inaccessible for the majority of HIV-infected mothers. There remains a huge gap and a need for low-cost, safe interventions that do not necessarily require screening for HIV infection, and that are easy and simple to implement in busy healthcare facilities with high HIV prevalence and limited resources.
The vaginal lavage option thus deserves further research as it is simple in use, is low cost and widely available. Vaginal disinfections could be beneficial not only to lower MTCT, but also to reduce neonatal and maternal mortality rates [10,26], and could be used in a broader public health perspective.
Acknowledgements
The authors would like to thank Esther Getambu, Chief Administrator for Coast Provincial General Hospital, Mombasa, Kenya, for facilitating the implementation of the study in the hospital. The authors acknowledge the role and support of Medicus Mundi Belgium, and in particular of Mrs F. Wijckmans. They are grateful for the contribution of Babill Peterson, Department of Obstetrics and Gynaecology, University of Oslo, Norway, in assisting in explaining and supervising the technique of chlorhexidine lavage during the implementation of the study. They acknowledge the contribution in the data analysis of Stijn Vansteelandt, Department of Applied Mathematics and Informatics, Ghent University, Belgium. They also thank Dr Aruna Chudasama, Reinhilde Meilis, Matron Muigai, Isabelle Tilmant, Raymond Caciano, the counsellors, midwives and clinical officers for their daily work for the study. Finally, they are indebted to the women and infants who made this research possible.
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