HIV-infected pregnant women should be offered antiretroviral prophylaxis to prevent perinatal HIV transmission, as well as optimal therapies for their own health . Zidovudine (ZDV) treatment reduces perinatal transmission by nearly 70%  despite the fact that it reduces viral load little, if at all. Because the risk of vertical transmission decreases with lower maternal viral load [3,4], combination regimens (containing ZDV) with a more pronounced antiviral effect may well be more effective than ZDV alone in preventing such transmission. Furthermore, the majority of HIV-infected women meet the current criteria for antiviral treatment with two reverse transcriptase inhibitors (RTI) and a protease inhibitor and ZDV monotherapy is no longer considered optimal therapy . However, toxicity of combination therapies for mothers and children is a major concern.
Anaemia is the major toxicity of ZDV, and carcinogenesis may be a potential long-term complication in neonates, since vaginal tumours in rodents exposed to high doses of ZDV have occurred . To date, no cases of tumours or fetal toxicity have been reported in humans [7,8]. Minimal data are available concerning the safety of other antiretrovirals [9–12]. Tolerance of lamivudine (3TC; with or without ZDV) given to more than 48 pregnant women and for 1 week to the newborn was good [9–11]; didanosine (with ZDV) given to 12 pregnant women was also well tolerated . There are no published data on the safety of stavudine. Ritonavir, saquinavir and nelfinavir are classified as US Food and Drug Administration pregnancy category B and indinavir (IDV) is classified as category C, due to the theoretical risk of hyperbilirubinaemia and kidney stones in neonates . Nevertheless, an increasing number of HIV-infected pregnant women will receive combination antiretroviral therapy. The aim of this study is to report any adverse event observed in HIV-infected pregnant women and their newborns exposed to combination antiretroviral therapy in Switzerland.
We performed an observational study in Switzerland. HIV-infected pregnant women on double RTI therapy with or without protease inhibitors and their newborns were identified in the Swiss HIV Cohort Study, the Swiss Collaborative HIV and Pregnancy Study, the Swiss Neonatal HIV Study, and from private practitioners.
Laboratory and clinical data were extracted from the patients'; charts and reflected values/events obtained throughout the pregnancy. Antiviral drugs, HIV RNA levels (Amplicor HIV Monitor, Roche, Basel, Switzerland; limit of detection, 400 copies/ml), CD4 cell count, concomitant treatments, clinical events and abnormal laboratory values (1979 World Health Organization classification) were recorded. Primary caesarean section was defined as an abdominal delivery before the onset of labour and rupture of membranes, performed either at week 38 in order to prevent HIV transmission or for obstetrical reasons. Data extracted from newborn charts included Apgar score, weight, height, head circumference, clinical events, antiviral treatment and detection of p24 antigen, HIV RNA and DNA. Children were classified as HIV-infected if they had two different positive HIV virological tests on separate blood samples. HIV infection was excluded if two or more tests were negative after 1 month of age, with at least one performed after 4 months of age.
Any clinical event was considered an adverse event. Data of newborns were compared with those from the population of the Swiss Neonatal HIV Study . For comparisons of categorical values, χ2 tests were used, and for continuous and ordinal data, Kruskal-Wallis analysis of variance by ranks was used. For these tests and for logistic regression (univariate and multivariate), the software package STATISTICA (StatSoft, Inc., Tulsa, Oklahoma, USA) was used.
From October 1996 to 30 April 1998, 37 HIV-infected pregnant women with combined antiretroviral treatment during pregnancy were identified. Nine (20%) were found to be HIV-positive during pregnancy. Twenty received two RTI, 16 received two RTI plus one or two protease inhibitors, and one received two RTI plus hydroxyurea. Twelve women became pregnant while on treatment; amongst them five were receiving protease inhibitors. ZDV and 3TC were part of the treatment in 33 cases, and stavudine and didanosine in four cases. Protease inhibitors were introduced during pregnancy in 11 women and had to be stopped in one woman. Nine patients received IDV, four received ritonavir, two received nelfinavir, and two received saquinavir. Other treatments during pregnancy were vitamins and iron substitution in five patients, cotrimoxazole in two patients, pentamidine aerosols in one patient, and amoxicilline-clavulanate in two patients; six injecting drug users received benzodiazepines.
During the first trimester, 28 patients were in Centers for Disease Control and Prevention (CDC) stage A, seven were in stage B, and two were in stage C; no major HIV-related disease occurred during pregnancy. Median CD4 cell count was 372 × 106/l (range, 51–813 × 106/l), median viraemia was 7206 copies/ml (range, 33–139 479 copies/ml), and 19% had less than 400 copies/ml. During the third trimester the median viraemia was 200 copies/ml (range, 39–34 927 copies/ml) and 62% of patients had less than 400 copies/ml.
By 30 April 1998, the women had given birth to 30 children, one extrauterine pregnancy occurred, and six women were still pregnant. Delivery was vaginal in 16 cases (54%); amongst these women, labour was induced in three, and two had delivery with forceps. Twelve primary and two secondary caesarean sections were performed. Twenty-two patients (73%) received intravenous ZDV during delivery.
Twenty-nine newborns received oral ZDV for 6 weeks; one received didanosine after 2 weeks of ZDV, one continued ZDV because of proven HIV infection, one stopped ZDV after 4 weeks because of anaemia, and two received a combination with 3TC or didanosine.
Adverse events during pregnancy
Anaemia was observed in 15 women, 14 of whom were receiving ZDV and 3TC. Anaemia led to the replacement of ZDV in one patient and to blood transfusions in one patient on didanosine plus stavudine who was known to have sickle cell disease. Two injecting drug users had hepatitis (grade ≥ 3) while on double or triple therapy; liver function test values decreased even though the treatment was continued. One patient receiving didanosine plus stavudine had grade 1 hyper-amylasaemia with elevation of aspartate aminotransferase and hyperbilirubinaemia. Four patients had severe nausea, and IDV had to be stopped because of onset of vomiting in one patient. Two cases of diarrhoea, one case of grade 1 leukopenia and one case of grade 2 thrombocytopenia were present in patients on double or triple therapy. Two patients without protease inhibitors had glucose intolerance. One case of diabetes occurred in a patient on triple therapy, with insulin administered during the last days of pregnancy. Two out of nine women treated with IDV during pregnancy developed echographically proven nephrolithiasis; in these cases IDV was continued. One patient on triple therapy had onset of high blood pressure, which led to the induction of labour for fetal distress.
Adverse events in newborns
There were no still-births. Ten children were premature (born before 37 weeks; median, 35.9 weeks; range, 27.9–36.7 weeks). Amongst these children, three were born through primary caesarean section. Birth weight, height and head circumference were in the normal range (10th-90th percentile) in 22 neonates. Four newborns had weight below 10th percentile, two had height below 10th percentile, one had head circumference below 10th percentile and two above 90th percentile. Eight mild cases of anaemia were observed: three were already present at birth and one worsened and led to withdrawal of ZDV (the baby had also vomiting). Cryptorchidism was present in two children exposed to RTI only. Cutaneous angiomas appeared in two babies exposed to ZDV–3TC–IDV or stavudine–3TC–IDV. One premature baby, exposed to ZDV–3TC had an opiate withdrawal syndrome and transient hepatitis. Two newborns (gestational age, 37.7 and 33.0 weeks) had non-life-threatening intracranial haemorrhages detected by transfontanellar cerebral echography (small left frontoparietal haemorrhage in one, and small infraependymal cysts in the premature baby); they had been exposed in utero to ZDV–3TC–IDV or stavudine–3TC–IDV. Extrahepatic biliary atresia, with vestigial remains of gallbladder and cystic duct, was discovered at 1 month of age in a child who had been exposed to ZDV–3TC–IDV since the second trimester of pregnancy. He benefited from a portoenterostomy procedure at week 6 (Table 1).
Prematurity below 37 weeks was found in 33% (10 out of 30) compared with 17% in newborns from the Swiss Neonatal HIV study exposed to ZDV (19 out of 112) and 14% of those without antiretroviral exposure (63 out of 452). The odds ratio of prematurity after combination therapy versus no antiretrovirals was 2.73 [95% confidence interval (CI), 1.37–6.86], and 2.30 (95% CI, 1.17–7.10) after adjustment for opiate use, clinical stage, and primary caesarean section. No differences were observed concerning intrauterine growth and Apgar score. Intracranial lesions visualized by ultrasound were reported in two premature children (0.4%) out of 537 infants from comparison groups versus two (6.6%) out of 30 in our study (P < 0.0001).
None of the children were breastfed. By 30 April 1998, vertical HIV transmission could be assessed in 15 children, and determinations of p24 antigen, HIV RNA and DNA were negative in all but one. In this case, the mother had been on ZDV since the second trimester, and 3TC was added during the third trimester. However, she admitted poor compliance and the viral load during the third trimester was 3077 copies/ml. A short follow-up (< 4 months) precluded a full assessment of HIV transmission in the remaining 15 cases (Table 1).
All 37 pregnant women in this series received two nucleoside analogues and 16 also received protease inhibitors. The most common adverse event was anaemia, occurring in 42% of women, all of whom were exposed to ZDV and 3TC. There were no serious adverse events in the few patients exposed to didanosine and stavudine. One woman on triple therapy had insulin-requiring diabetes. Diabetes is a known complication of pregnancy; however, because insulin resistance is a side-effect of protease inhibitors , IDV may have played a role in revealing diabetes in this case. Two cases of kidney stones and one case of vomiting were probably related to IDV.
A significantly higher prematurity rate was found after combination antiretroviral therapy than in newborns not exposed to antiretrovirals. This association persisted after adjusting for several confounding factors. Use of antiretrovirals during organogenesis (first trimester) is of major concern. There are no published clinical data to support or refute the teratogenic risk of antiretroviral drugs, including ZDV [in AIDS Clinical Trials Group (ACTG) trial 076, ZDV was administered beginning in the second trimester]. Animal studies have failed to demonstrate teratogenicity of nucleoside analogues and protease inhibitors . In our study, 43% of women were already on antiretroviral treatment when they became pregnant. Fifteen out of 30 newborns were exposed to antiviral drugs during the first trimester and no teratogenic effects were found. However, adverse events of major concern with origin later in gestation were observed, namely intracerebral haemorrhages in two cases and biliary disease in one. One case of intracerebral haemorrhage occurred in a premature baby and could be considered a complication of prematurity . However, intracerebral haemorrhage in a term neonate in the absence of vascular malformation or a disorder of haemostasis is uncommon . Spontaneous bleeding has occurred in haemophiliacs taking ritonavir or IDV [17,18], indicating that protease inhibitors may interfere with haemostasis. Therefore, the finding of such a complication in a term neonate might be more than just a chance event.
Extrahepatic biliary atresia in a child exposed to ZDV, 3TC and IDV in the second and third trimester was unexpected. Extrahepatic biliary atresia is not a developmental error but rather a progressive sclerosing inflammatory obliteration of the extrahepatic ducts in the perinatal period suggesting a role for environmental factors . Because incidence in the general population is very low (1:14000 live births) , the occurrence of even one case is of concern. No case of biliary atresia was noted in long-term follow-up of the ACTG 076 trial  nor in 664 children in the Swiss Neonatal HIV Study (C. Kind, personal communication, 1998). Minor adverse events observed in newborns were anaemia in 27% of cases: most had been exposed to ZDV-3TC in utero. Because cutaneous angiomas and cryptorchidism (present in two cases each) are frequent abnormalities in newborns, a correlation with exposition to antiviral drugs cannot be assessed. Amongst the six neonates who were exposed in utero to IDV, none had prolonged hyperbilirubinaemia nor renal problems.
The occurrence of unexpected major adverse events among newborns emphasizes the necessity to maintain updated registers concerning pregnancy, newborns and antiretroviral therapy. Observation time has not been sufficient to assess long-term side-effects of highly active antiretroviral therapy, lipodystrophy being the best example . Until further observational studies provide clearer evidence, the present data can assist in the assessment of benefits and risks of combination antiretroviral therapies including protease inhibitors in pregnancy.
The authors are indebted to the following collaborators, mainly private practitioners, for their help in data collection: J-F. Balavoine, J-P. Chaves, D. Genné, D. Oertle, C. Renold, K. Rohling and J. Wintsch.
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Members of the Swiss HIV Cohort Study
M. Battegay (Co-chair of the Scientific Board), E. Bernasconi, Ph. Bürgisser, M. Egger, P. Erb (Chairman of the Laboratories Group), W. Fierz, M. Flepp (Chairman of the Clinics Group), P. Francioli (President of the SHCS, Centre Hospitalier Universitaire Vaudois, Lausanne), H.J. Furrer, P. Grob, B. Hirschel (Chairman of the Scientific Board), L. Kaiser, B. Ledergerber, R. Malinverni, L. Matter, M. Opravil, F. Paccaud, G. Pantaleo, L. Perrin, W. Pichler, J-C. Piffaretti, M. Rickenbach (Head of Data Centre), P. Sudre, J. Schupbach, A. Telenti, P. Vernazza.
Members of the Swiss HIV and Pregnancy Study
Principal investigators: C. Rudin MD (University Children's Hospital, Basel), K. Biedermann (Womens Hospital, Chur); Collaborators: U. Lauper (University Women's Hospital, Zürich), O. Irion, A.P. Brunelli (University Women's Hospital, Geneva), G. Spoletini, A. Schreyer (University Women's Hospital, Lausanne), I. Hösli (University Women's Hospital, Basel), E. Saurenmann (Division of Gynaecology/Obstetrics, Kantonsspital, Luzern), G. Drack (Division of Gynaecology/Obstetrics, Kantonsspital, St Gallen), M. Isenschmid (University Women's Hospital, Berne), C. Kind (Division of Neonatology, Kantonsspital, St Gallen), M. Poorbeik (Central Laboratories of the Swiss Red Cross, Berne). Funding: Swiss Federal Office of Public Health (contract nos 90–7007, 93–7131, 96–7257).
Members of the Swiss Neonatal HIV Study
Principal Investigator: C. Kind (Division of Neonatology, Kantonsspital, St Gallen); Collaborators: D. Nadal (University Children's Hospital, Zürich), C. Rudin (University Children's Hospital, Basel), C-A. Siegrist, C-A. Wyler (University Children's Hospital, Geneva), J-J. Cheseaux (University Children's Hospital, Lausanne), C. Aebi (University Children's Hospital, Berne), H. Gnehm (Children's Hospital, Aarau), G. Schubiger (Children's Hospital, Luzern), J. Klingler (Children's Hospital, Biel), U. Hunziker (Division of Paediatrics, Kantonsspital, Wintherthur), H. Kuchler (Division of Paediatrics, Hopital Cantonal, Sion), M.P. Gianinazzi (Division of Paediatrics, Ospeale Civico, Lugano), U. Bühlmann (Division of Paediatrics, Stadtspital Triemli, Zürich). Funding: Swiss Federal Office of Public Health (contract nos 86–97, 88–244, 90–7046, 93–7158, 96–7261).