Lopinavir/ritonavir-based antiretroviral treatment (ART) versus efavirenz-based ART for the prevention of malaria among HIV-infected pregnant women

Background: Human immunodeficiency virus (HIV)-infected pregnant women are at increased risk of malaria and its complications. In vitro and in vivo data suggest that the HIV protease inhibitors lopinavir/ritonavir may have potent antimalarial activity. We sought to evaluate whether lopinavir/ritonavir-based antiretroviral therapy (ART) reduced the risk of placental malaria. Methods: HIV-infected, ART-naive pregnant women were enrolled between gestational weeks 12 and 28 and randomly assigned to receive lopinavir/ritonavir-based or efavirenz-based ART. Women received daily trimethoprim-sulfamethoxazole prophylaxis and insecticide-treated bed nets at enrollment and were followed up to 1 year after delivery. The primary outcome was placental malaria, defined by the detection of malaria parasites, using microscopy or polymerase chain reaction (PCR) analysis of placental blood specimens. Secondary outcomes included placental malaria, defined by histopathologic results; adverse birth outcomes; incidence of malaria; and prevalence of asymptomatic parasitemia. Analyses were done using an intention-to-treat approach. Results: Of 389 subjects randomly assigned to a treatment group, 377 were followed through to delivery. There was no significant difference in the risk of placental malaria, as defined by thick smear or PCR findings, between the lopinavir/ritonavir-based and efavirenz-based ART arms (7.4% vs 9.8%; P =.45). Similarly, there were no differences in secondary outcomes between the 2 treatment arms. Conclusions: Lopinavir/ritonavir-based ART did not reduce the risk of placental or maternal malaria or improve birth outcomes, compared with efavirenz-based ART. The study was conducted from December 2009 to March 2013 in Tororo, Uganda, an area of high-intensity malaria transmission [14]. Women were recruited from the Tororo District Hospital (TDH) antenatal clinic, The AIDS Support Organization (TASO), and other health centers in the district. Eligible women were ≥16 years of age, infected with HIV-1 confirmed by two assays, lived within 30 km of the study site, and had a pregnancy between 12–28 weeks gestation by last menstrual period with confirmation by ultrasound. Women were eligible for enrollment at any CD4 cell count. Women were excluded if they had ever received highly active combination ART or single dose nevirapine or other abbreviated monotherapy or dual therapy in the last 24 months. Women were also excluded if they had prior dose-limited toxicity to TMP-SMX within 14 days, active tuberculosis or other WHO stage 4 diseases, cardiac disease, or abnormal screening laboratory values including, hemoglobin <7.5 g/dL, absolute neutrophil count <750/mm3, platelet count <50 000/mm3, ALT >225 U/L, AST >225 U/L, total bilirubin ≥2.5 times the upper limit of normal, and creatinine ≥1.8 times the upper limit of normal. All participants provided written informed consent in their preferred language. The study protocol was approved by the Makerere University School of Medicine Research and Ethics Committee, the Uganda National Council for Science and Technology, and the University of California–San Francisco Committee on Human Research. This was an open-label, single-site, randomized controlled trial of LPV/r-based ART versus EFV-based ART among HIV-infected, ART-naive pregnant Ugandan women. Participants were randomized in a 1:1 ratio after stratification by gravidity (gravida 1 vs gravida ≥2) and gestational age at enrollment (<24 weeks vs ≥24 weeks). Randomization was performed in permuted blocks of 2 or 4. The dosing of study drugs was as follows: EFV 600 mg once daily; or LPV/r (Aluvia; AbbVie Pharmaceuticals, North Chicago, IL) 200 mg/50 mg, 2 tablets twice daily, with an increase to 3 tablets twice daily from gestation week 30 until delivery, after which 2 tablets were administered twice daily. Women in both arms received lamivudine/zidovudine 150 mg/300 mg twice daily. Tenofovir was used in cases of zidovudine intolerance. At enrollment, all women had their medical history recorded, underwent physical examination (including assessment of gestational age, calculated by last menstrual period and ultrasonographic biometry), and underwent baseline laboratory studies. All women received daily TMP-SMX prophylaxis (160 mg/800 mg), a long-lasting ITN, and a basic care package that included multivitamins and condoms. Throughout the study, women received standard antenatal care that accorded with Uganda Ministry of Health guidelines [15]. Women returned to the study clinic every 4 weeks for scheduled visits, including collection of blood specimens for routine blood smears, as well as for any health conditions requiring evaluation. Transportation costs were reimbursed for all clinic visits. Women who presented with a documented fever (tympanic temperature, ≥38.0°C) or history of fever in the previous 24 hours had a blood specimen obtained by finger prick for a thick blood smear. If the smear was positive for malaria parasites, malaria was diagnosed. Episodes of uncomplicated malaria were treated with AL, and complicated malaria was treated with quinine in accordance with Ugandan guidelines. Women were encouraged to deliver at TDH. Women delivering at home were visited by study staff at the time of delivery or as soon as possible afterward. At delivery, a standardized assessment was completed, including assessment of gestational age and of birth weight, using an electronic scale. Following delivery, participants continued to receive ART and were followed up to 52 weeks after delivery or until study completion, in March 2013. Thick and thin blood smears were stained with 2% Giemsa and read in duplicate by experienced microscopists who were blinded to study arm and to each other's readings. A smear was considered negative if no parasites were seen after review of 100 high-powered fields. Discrepant readings were settled by a third microscopist. Clinical laboratory tests, including complete blood count, determination of CD4+/CD8+ T-lymphocyte subsets, and measurement of HIV-1 RNA load by polymerase chain reaction (Cobas Amplicor, version 1.5, Roche Molecular Diagnostics, Pleasanton, CA) were performed at enrollment and during the follow-up period. Placental specimens were collected within 30 minutes of delivery in the hospital (or as early as possible, if delivery occurred at home) and included placental blood from an incision on the maternal surface and placental tissue. Placental blood was tested for malaria parasites, using a thick blood smear in accordance with the procedures described above, a rapid diagnostic test (RDT; Paracheck-Pf, Orchid, Goa, India) performed immediately after placental blood was collected, and PCR of dried blood spots. For PCR, aliquots of approximately 25 µL of placental blood were placed on filter paper, air dried, and stored in individual bags with desiccant. DNA was extracted from dried blood spots by use of Chelex and was amplified using a nested PCR reaction as previously described [16]. PCR reactions were performed at the University of California–San Francisco by experienced staff and included appropriate controls and quality assurance. For histopathologic analysis, a 2 × 2 full-thickness biopsy specimen was placed in 10% fresh buffered formalin, using a 1:10 ratio of tissue to formalin. After 24 hours in formalin, placental tissue was trimmed into a 1 × 1-cm block of tissue and placed into a fresh jar of 10% buffered formalin (1:10 ratio) and stored out of direct sunlight. Tissue specimens were fixed with ethanol and xylene, embedded in paraffin wax, sectioned into slides, and stained with hematoxylin-eosin and Giemsa. Slides for histopathologic analysis were read by a trained investigator (V. A.) and included examination for malaria parasites and hemozoin pigment in intervillous fibrin and macrophages, using standardized criteria as previously described [17]. Quality control of histopathologic findings was performed on a random sample of 25% of positive slides and 10% of negative slides by an experienced pathologist (Atis Muehlenbachs, Centers for Disease Control and Prevention). The primary end point for this study was placental malaria, defined as the detection of P. falciparum in a thick blood smear or by PCR of placental blood. Secondary outcomes included (1) prevalence of placental malaria, as determined by RDT or histopathologic analysis; (2) adverse birth outcomes, including stillbirth (intrauterine fetal demise ≥20 weeks of gestation) or spontaneous abortion (miscarriage <20 weeks of gestation), preterm delivery (<37 weeks of gestation), low birth weight (<2500 g), neonatal death within 28 days of delivery, or a composite outcome, defined as any of the adverse birth outcomes listed above; and (3) measures of maternal malaria, including the incidence of symptomatic malaria and the prevalence of asymptomatic parasitemia at the time routine blood smears were performed, stratified by the periods during and after pregnancy. For women giving birth to twins, outcomes were classified according to whether either infant met criteria for the outcome of interest. HIV-related outcomes, including maternal and infant safety profile, as well as virologic and immunologic responses, are presented in a separate report [18]. To test the hypothesis that use of LPV/r-based ART will reduce the prevalence of placental malaria, as defined by positive findings of a placental blood smear or PCR, we assumed a prevalence of 20% in the EFV-based ART arm, based on previous data from the study site [19]. We then calculated that a sample size of 500 was needed to show a 48.5% reduction in prevalence of placental malaria in the LPV/r-based arm (2-sided significance level, 0.05; power, 80%), assuming that 15% of women enrolled would not have evaluable results at delivery. Because of the slower than anticipated study accrual, 389 women were enrolled. Data were double entered in Access (Microsoft, Redmond, WA), and analyses were performed using Stata, version 12 (StataCorp, College Station, TX). All analyses were done using an intention-to-treat approach. Comparisons of baseline characteristics and birth outcomes were made using a t test for continuous variables and a χ2 test for categorical variable. Comparisons of malaria incidence between study arms were made using a negative binomial regression model. Comparison of the risk of recurrent malaria with 42 days of treatment with AL was made using a Cox proportional hazard model. with adjustment for repeat observations in the same study participant. Comparison of the prevalence of asymptomatic parasitemia at the time routine blood smears were performed was made using generalized estimating equations, with adjustment for repeated measures in the same patient by using exchangeable correlation and robust standard errors. A P value of <.05 was considered statistically significant.