Dual infection with HIV and malaria adversely affects pregnancy outcome beyond the effects of each single infection.1 When pregnant women are HIV infected, malaria is more frequent and leads to an increased risk of maternal anemia and low birth weight,2–5 which in turn contribute to excess mortality in mothers and infants.6,7 Moreover, malaria during pregnancy can increase the maternal HIV viral load, with possible increased transplacental transmission of the virus.8,9
To prevent malaria in HIV-infected pregnant women, the World Health Organization (WHO) currently recommends the use of insecticide-treated bed nets plus preventive drug treatment.10 To prevent opportunistic infections, immunocompromised women (CD4 cell count <350/mm3; WHO HIV clinical stage, >1) receive cotrimoxazole (CTX) prophylaxis that also has antimalarial activity.11–14 In less immunocompromised women (CD4 count >350/mm3), WHO recommends an intermittent preventive treatment (IPTp) during pregnancy involving at least 3 supervised intakes of an antimalarial drug starting in the second trimester.15,16 Of the commonly used IPTp regimens, sulfadoxine-pyrimethamine (SP-IPTp) has been most extensively evaluated both in HIV-negative women17–20 and more recently in HIV-infected women.21,22 As resistance to sulfa drugs increases rapidly, there is a need to test alternative drugs.23 A recent trial showed that MQ-IPTp was effective in HIV-negative pregnant women.24
However, there is still an important evidence gap in terms of defining optimal malaria prophylaxis for HIV-infected pregnant women, because neither CTX prophylaxis nor alternative drugs to SP in IPTp have ever been evaluated by clinical trials in this specific population. To help fill this gap, we conducted the first randomized controlled trials evaluating CTX for malaria prevention in HIV-infected pregnant women, with MQ-IPTp, alone and in combination, for comparison.
We designed 2 parallel open-label, noninferiority controlled trials based on the participants' immunodeficiency levels. Those with either CD4 counts of <350 per cubic millimeter or advanced HIV disease, for whom CTX prophylaxis was mandatory, received either daily CTX or daily CTX with MQ-IPTp. Less immunocompromised women or those with partially recovered CD4 counts since initiating antiretroviral treatment (ART), for whom CTX was no longer mandatory, received either daily CTX or MQ-IPTp alone.
Study Setting and Participants
In southern Benin, Plasmodium falciparum malaria transmission is intense and perennial, with peaks during rainy seasons.25 Overall HIV prevalence in Benin is around 2%26 and reaches 5% among pregnant women in Cotonou. The 2 trials were conducted in 5 urban hospitals in Cotonou, and Porto-Novo implementing a program for prevention of mother-to-child transmission of HIV (PMTCT).
HIV-infected pregnant women aged >18 years and living permanently in the study area were enrolled between 16 and 28 weeks of gestation after giving written informed consent. Exclusion criteria included age <18 years, history of a neuropsychiatric disorder, severe kidney or liver disease, and serious adverse reaction with MQ, sulfa drugs, or quinine. Receiving IPTp before enrollment was not an exclusion criterion, although to be eligible, the last dosage must have been taken at least 1 month before enrollment. Additionally, women requiring antimalarial treatment at baseline were enrolled at least 2 weeks after the completion of their treatment. Based on their HIV treatment history, WHO stage, and CD4 counts, women were allocated to the “CTX mandatory” (CM) trial or the “CTX not mandatory” (CNM) trial.
Randomization and Masking
In each trial, randomization was stratified according to the study site and the number of previous pregnancies (primigravid vs. multigravid). The study coordination center retained the master list and assigned treatments by phone. This trial was blinded only to the microscopists who evaluated blood smears.
Interventions and Follow-up
At enrollment, sociodemographic characteristics, obstetric and HIV history, and data on malaria prevention were recorded. A venous blood sample was collected for HIV viral load, CD4 count, complete blood count (CBC), and malaria diagnosis. Women received insecticide-treated bed nets and daily supplementation with 100 mg ferrous sulfate and 5 mg folic acid.
Three visits were scheduled (between 16 and 28 weeks, 24 and 32 weeks, and 28 and 36 weeks) with at least 1-month interval between them. In the MQ-IPTp treatment groups, the investigator administered 15-mg/kg MQ (Cipla, Mumbai, India) with a rich-fat collation under direct observation. In case of vomiting within 30 minutes, an extra dose was administered. Where required, women received CTX at the daily dose of 800 mg sulfamethoxazole and 160 mg trimethoprim. Additionally, clinical and adherence information were recorded, and a venous blood sample was collected for CBC, CD4 count, and parasitology.
Febrile women were referred for malaria screening. Intravenous quinine was administered in case of severe malaria. On scheduled visits, participants who were asymptomatic or had low parasitemia (<1000 parasites/μL) were treated by the MQ-IPTp dose (in the MQ group). Otherwise, women received arthemeter–lumefantrine (Coartem; Novartis, Morris Plains, NJ) or oral quinine.
At delivery, thick and thin blood smears were made from the placenta and umbilical cord. Infants were evaluated at delivery, at 6 weeks, 4 months, and 2 months after weaning. After pediatric clinical examination, a blood sample was tested for malaria, hemoglobin, and HIV polymerase chain reaction (PCR). Additionally, PMTCT was provided according to national guidelines, as described elsewhere.27
Venous samples were collected in EDTA tubes on each visit. Hemoglobin level was determined as part of CBC, or if not available, by the colorimetric method (Hemocue, Angelhorn, Sweden). Absolute CD4 count and percentage were assessed by Cyflow (Partec, Münster, Germany) or Facscount (Beckton Dickinson, Franklin Lakes, NJ) cytometer.
HIV viral load was assessed by real-time quantitative PCR using 2000 Abbott thermocyclers (Abbott Laboratories, Abbott Park, IL) provided by the national AIDS control program, with a 40 copies per milliliter detection threshold. In infants, qualitative PCR was performed using the same automats.
For malaria diagnosis, thick and thin blood smears were stained with Giemsa and read by 2 independent microscopists, with conciliation of discrepancies by a third reader. The slide was considered negative after 200 high-power fields were read. For positive slides, parasite density was determined per 500 leukocytes under the assumption of an average leukocyte count of 8000 cells per milliliter.
To increase P. falciparum detection after the follow-up period, we retrospectively performed a real-time (quantitative) duplex PCR assay on all filter paper DNA extracts collected at enrollment and delivery, as described elsewhere.28
The primary end point was the prevalence of placental parasitemia assessed by microscopy. Secondary end points were incident parasitemia during follow-up, the prevalence of peripheral parasitemia at delivery, the mean birth weight, and proportion of low birth weight infants (<2500 g), the mean hemoglobin level, and proportion of anemic women (<9.5 g/dL)29 at delivery. Malarial infections detected by PCR in placental and peripheral blood at delivery were added as secondary end points after a protocol amendment.
Women were sensitized to self-report all adverse events (AEs) whenever they occurred and come to the hospital for adequate treatment, if necessary. Clinical and laboratory AEs were also systematically recorded on each visit. In addition, the investigator observed early adverse reactions to MQ within 30 minutes after supervised intake and collected later reactions by phone the same evening or the next day. A medical examination was performed 2 weeks after each CTX initiation to search for cutaneous reactions. The severity of AEs was graded using standard scale ranging from 1 (minor) to 4 [serious AE (SAE)].29 Spontaneous abortions, stillbirths, and congenital abnormalities were considered SAEs. An independent data and safety monitoring board (DSMB) reviewed all SAEs.
In each trial, we expected placental parasitemia prevalence at 2% in the MQ-IPTp groups, close to 1.7% found in Beninese HIV-negative women.24 With a noninferiority margin of 5%, type 1 error of 2.5% (1-sided) and power of 80%, 123 women were required in each treatment group. To account for 15%–20% losses to follow-up, we planned to enroll 284–296 women in each trial. As this sample size could not be achieved after a 2-year recruitment period, we conducted conditional power and error analyses (see SDC 1, http://links.lww.com/QAI/A485).
For data management and analysis, Access 2003 (Microsoft Corporation, Redmond, WA), Stata 11 (StatCorp LP, College Station, TX), and StatXact 9 (Cytel, Cambridge, MA) were used. An interim analysis of the first 140 deliveries was planned in each trial and finally performed in the CM trial only (sample size not reached in the other trial because of slow recruitment). Because there were neither relevant treatment differences regarding placental parasitemia nor unacceptable AEs, no modifications were requested by the DSMB.
For analysis of the primary end point, we computed exact tests to determine the upper limit of the 2-sided 95% confidence interval (CI) of the difference between CTX and MQ treatment groups. Noninferiority was assessed, if the upper limit of the 2-sided 95% CI was inferior to 5% (noninferiority margin) in modified intent-to-treat (mITT) analysis (including all women for whom the primary end point was collected) and per protocol (PP) analysis (restricted to women who had correctly received the allocated treatments). Secondary end points and AEs were compared using Fisher exact test for proportions or Wilcoxon nonparametric test for means in both the mITT and the PP populations.
The protocol received ethical clearance by French (Colité Consultatif de Déontologie et d'Ethique de l'Institut de Recherche pour le Développement) and Beninese (Ministry of Health) Institutional Review Boards.
Trials Profiles and Baseline Characteristics
During the 2-year enrollment period (December 2009 to December 2011), 533 women were screened, among whom the majority was eligible for the CNM trial, that is, with CD4 count >350. Therefore, two-thirds of the 432 women randomized (N = 292) were allocated to the CM trial, and one-third (N = 140) was allocated to the CNM trial (Fig. 1).
The Supplemental Digital Content (see Table, SDC 2, http://links.lww.com/QAI/A485) shows baseline characteristics of the randomized population. HIV-related characteristics differed between trials, but within each trial, populations were similar in the 2 treatment groups. In both trials, 6% of women had positive P. falciparum microscopy and 38% were positive by PCR detection. Sixty-three percent of women reported having slept under bed net the 3 previous nights.
After randomization, 1 woman in the CNM trial refused to continue before receiving the allocated MQ treatment. There were 2 protocol violations at enrollment (contraindication not reported) in the MQ group of the CM trial. The first woman experienced seizures 8 days after MQ intake and recovered after anticonvulsant treatment. She later reported history of epilepsy. The other reported history of depression at the second visit while she was well tolerating MQ. Both women were asked to discontinue MQ.
Seven (5.0%) and 19 (6.5%) women were lost to follow-up in the CNM and CM trials, respectively, 2 (1.4%) and 7 (2.4%) experienced spontaneous abortion, and 1 died of HIV-related disease (in the CM trial). Finally, 130/140 (92%) and 263/292 (90%) of women in the CNM and CM trials, respectively, were followed until delivery of a live or dead infant after 28 weeks of pregnancy (usual definition for stillbirth as opposed to spontaneous abortion),30 and 108 (77%) and 233 (80%), respectively, had the primary end point assessed.
The proportions of women not included in the mITT population were similar in different trials and treatment groups (Fig. 1). Overall, women with missing primary end point had been more frequently diagnosed for HIV during the ongoing pregnancy (56% vs. 38%, P = 0.003), and their HIV diagnosis was more recent (2.4 vs. 19.0 months ago in median, P = 0.005). Other characteristics did not differ.
In both trials, the mean gestational age was 21.8 (SD: 3.8) weeks at the first visit, 27.9 weeks (SD: 2.9) at the second visit, and 33.3 weeks (SD: 2.2) at the third visit. For patients allocated to MQ, the mean dose administered at the first visit was 14.2 mg/kg (SD: 1.1 mg/kg). Accounting for physiologic weight gain during pregnancy, it was 13.7 mg/kg (SD: 1.0) at the second visit and 13.4 mg/kg (SD: 1.2) at the third visit. For 3 women, MQ intake was followed by early vomiting justifying second administration. Adherence to MQ-IPTp was total, except for 1 woman who left the clinic before the supervised intake. Ninety percent and 95% of women self-reported total adherence to CTX prophylaxis and ART during their whole follow-up, respectively.
Deviations in the administration of the allocated treatment concerned 12 women (11% of the mITT population) and 8 women (3%) in the CNM and CM trials, respectively (Fig. 1). Adverse drug reactions (ADRs) caused MQ discontinuation in 1 and 3 women of the CNM and CM trials, respectively. CTX was discontinued in 1 woman of the CM trial.
Primary End Point
Two women had a positive placental blood smear, 1 in the CTX-alone group of each trial. The noninferiority results are shown in Table 1.
In the CM trial, CTX efficacy for the prevention of placental parasitemia was not more than 5% inferior to the association of CTX + MQ-IPTp. In the CNM trial, because of the small sample size obtained, noninferiority could not be conclusively assessed. However, high-calculated conditional power (95%, see SDC 1, http://links.lww.com/QAI/A485) indicated that the probability of detecting noninferiority was high, if we had continued enrollments to reach the target sample size.
Sensitivity analyses successively excluded, then counted as failures, the 9 women who had received quinine or Coartem during follow-up, all in the CM trial (see Table, SDC 3, http://links.lww.com/QAI/A485). These analyses corroborated results of the main analysis, although the second approach lacked sufficient power to reach statistical significance.
Secondary End Points
In the CM trial, using PCR for P. falciparum detection in the placental blood, we detected no infection (0/105) in women receiving CTX + MQ versus 5/103 infections with CTX alone (P = 0.03 and 0.06 in mITT and PP analyses, respectively; Table 2). The low incidence rate of malaria parasitemia did not differ between treatment groups. No statistically significant differences were either observed regarding peripheral parasitemia at delivery, maternal hemoglobin, birth weight, or prematurity in any trial.
Adverse Events in the Mother
Vomiting, nausea, dizziness, and fatigue were more frequently reported with MQ (36% vs. 0% in both trials for dizziness, 37% vs. 3% in the CNM trial, and 34% vs. 0% in the CM trial for vomiting, P < 0.0001; Table 3). The time of occurrence of these AEs ranged from few minutes (for early vomiting) to one day after intake and 90% were resolved within 3 days. Overall, 57% of women receiving MQ-experienced ADRs (122/213), mostly graded minor (62%) or moderate (33%). ADRs were more frequent after the first intake (see Figure, SDC 4, see http://links.lww.com/QAI/A485). Adverse neuropsychiatric reactions to MQ were not reported except for seizures in an erroneously enrolled epileptic woman.
Four cutaneous reactions were linked to CTX, among which 1 exanthema graded 3, caused treatment discontinuation. No SAE was linked to MQ or CTX by the DSMB except the seizures already mentioned.
Adverse Events in the Offspring
Although not significantly, spontaneous abortions tended to be more frequent in the CTX + MQ treatment group of the CM trial (4.1% vs. 0.7%, P = 0.12) (see Table, SDC 5, http://links.lww.com/QAI/A485).
One polymalformative syndrome was associated with intrauterine fetal death, and one hydrocephaly required surgical drainage. Other congenital abnormalities were benign. Finally, 7 early neonatal deaths and 11 later infant deaths were recorded (1.8% and 2.8%, respectively), mostly because of neonatal infection and acute diarrhea. After reviewing all SAEs, the DSMB did not see strong evidence for a link with the study drugs.
Mother-to-Child Transmission of HIV
Implementation of PMTCT in this study has been detailed elsewhere.27 Only 4/393 women delivered without receiving ART. The end-of-pregnancy viral load was undetectable for 159 of the 221 women tested (71.9%). Of the 388 live infants born, 378 (97.4%) received ART during the first weeks of life; 2/3 were breastfed for a median duration of 6 months and 1/3 received formula milk; only 1 received mixed feeding.
HIV status could be determined in 229 of the 329 infants followed for more than 6 weeks (70%). Two infants were HIV infected, both in the CM trial (1 in each treatment group). The MTCT rate was 0.9% (95% exact CI: 0.1% to 3.1%).
We present here the first randomized controlled trials providing evidence of the protection conferred by CTX in HIV-infected pregnant women. In women with low CD4 count, we compared CTX with the combination of CTX + MQ-IPTp. In less immunocompromised women, we compared CTX directly with MQ-IPTp. At delivery, the prevalence of placental parasitemia was <2% in CTX-alone groups and 0% in MQ-IPTp groups of both trials. In the CM trial, CTX prophylaxis alone proved not inferior to the association CTX + MQ-IPTp. Additionally, the proportion of P. falciparum infections detected by PCR decreased from 36% at enrollment to 5%–7% at delivery (placental and peripheral blood) in the women treated with CTX alone and from 41% to 0%–5% in the women taking CTX + MQ, indicating that both strategies decreased dramatically parasitemias, even submicroscopic.
In the CNM trial, although the same trend was observed, small sample size did not allow us to detect noninferiority. Indeed, recruitment was compromised in this trial because most women had a low CD4 count at screening (median 342 cells/mm3) in relation to late HIV diagnosis, as previously reported in cohorts of pregnant women in Africa.31,32 Though the conditional power indicated, it would have been useful to continue recruitment (see SDC 1, http://links.lww.com/QAI/A485), this was unfortunately not possible because of the additional time and financial resources needed.
Several studies have shown decreases in malaria incidence with increased CD4 count.33,34 Additionally, a clinical trial in Uganda recently reported a drastic reduction of malaria incidence in nonpregnant HIV-infected adults with CD4 counts of >200 per cubic millimeter and on CTX.35 Taken together, these observations suggest that CTX prophylaxis may have benefits in malaria prevention among HIV-infected pregnant women, even when the level of immunodeficiency is partially restored.
Two previous observational studies in HIV-infected pregnant women compared CTX to SP-IPTp, with one showing comparable prevalence of placental infection with CTX,13 and the other showing a decrease.14
Critical mutations in DHFR/DHPS genes can impair sulfa drugs efficacy.36 In West Africa, quadruple mutants represent the preponderant haplotypes37; less than 10% of parasites collected from Beninese pregnant women between 2008 and 2011 harbored quintuple mutations (Ndam NT, unpublished data). This low prevalence of highly mutated parasites probably explains the CTX efficacy we observed. However, an upward trend in parasite resistance, as consistently reported in parts of Africa, where SP and CTX have been widely used, may further compromise this efficacy in the future.12
These trials are also the first to test an alternative to SP for IPTp in HIV-infected pregnant women. We chose MQ as the comparator to CTX prophylaxis because it is currently the best candidate to replace SP in IPTp,38 and because we had already assessed MQ-IPTp in HIV-negative women.24 We show here its antimalarial efficacy in HIV-infected pregnant women on both microscopic and submicroscopic parasitemias. Unlike SP which contains a sulfa drug, MQ can be combined with CTX. The association CTX + MQ-IPTp resulted in the absence of placental infection as assessed by PCR.
CTX was well tolerated with only rare adverse cutaneous reactions. MQ-related moderate reactions were commonly reported (by 57% of women), although less frequently than in HIV-negative women (78%),24 and only 2% of these reactions led to treatment discontinuation. These findings regarding the acceptable tolerability profile of MQ provide grounds for an optimistic perspective of its potential role in malaria prevention in this vulnerable population, as discussed elsewhere.39
The overall rate of spontaneous abortions and stillbirths (25/432, 5.7%) was in the range of that reported in pregnant women receiving ART in other African settings (5.2%–7.1%).32 These outcomes occurred slightly more frequently in women treated with MQ, without reaching statistical significance. However, our statistical power for the comparison of uncommon events was limited. In this study, most of the women who were lost to follow-up belonged to the CTX-alone treatment groups, may be because appointments were more flexible for this preventive strategy. Hence, abortions and stillbirths may have been under-reported in these groups. Moreover, our results are consistent with a previous trial of HIV-negative women in Benin,24 where no significant difference was found between groups. Finally, a recent study involving 2506 pregnant women subjected to weekly MQ prophylaxis in industrialized countries showed that the rates of fetal demises were comparable with background rates.40
Beyond the results generated in this study, some limitations need to be considered. The first, already highlighted and discussed, relates to the inability to achieve sufficient enrollment in the CNM trial. Second, despite extensive effort, placental blood was not collected in 20% of the randomized women. Although high, this proportion was similar with that reported in other trials (18% in Benin,24 29% in Malawi).22 Baseline characteristics did not differ between women included in the primary analysis and those excluded, except for duration since HIV diagnosis. Moreover, the proportion of women lost for measurement of the primary end point was comparable between groups, so we believe this effect did not have a major impact on our results. To account for missing primary end point, we performed an mITT analysis rather than a missing = failure analysis, since this provides a more realistic definition of failure. PP analysis confirmed the results of mITT analysis in both trials.
MQ-IPTp was administered as a directly observed treatment, maximizing women's adherence. Fewer than 10% of the women reported incomplete adherence to CTX; however, we are aware that the validity of these self-reported data is questionable.41 Moreover, we cannot be certain that comparable high adherence would be obtained under standard care, that is, removed from a clinical trial setting.
In our study, the distribution of insecticide treated bed nets and additional treatment of malaria episodes could have contributed to the low overall proportion of placental infections, with a risk of bias toward the null hypothesis. However, only 9 women (3%), equally distributed in the 2 groups of the CM trial, received quinine or Coartem after enrollment. Based on a sensitivity analysis (see Table, SDC 3, http://links.lww.com/QAI/A485), we do not believe they significantly altered the final results.
Possible classification bias inherent to open-label design was minimized in our trial, thanks to its objective primary end point and the blind laboratory processing of samples. However, this design could have caused bias regarding assessment of side effects and other outcomes than microscopy that were not blinded for the investigator.
In summary, this trial demonstrated that CTX, used alone, provided sufficient protection against placental parasitemia in immunocompromised women eligible to CTX prophylaxis for their HIV. WHO recommendations for malaria prevention in HIV-infected pregnant women are stratified by CD4 count, as were PMTCT recommendations until recently. New PMTCT guidelines simplified the strategies and recommended immediate ART for all HIV-infected pregnant women, regardless of CD4 count.42 We suggest parallel simplification of malaria prevention guidelines: CTX prophylaxis should similarly be prescribed for all HIV-infected pregnant women, regardless of CD4 count.
However, in a context of rapid progression of parasite resistance to sulfa drugs, CTX efficacy is likely to be compromised in a short to medium term. We also demonstrated here that MQ-IPTp is an attractive alternative for HIV-infected women because of its very high antimalarial efficacy and the absence of serious adverse reactions. It remains for now the only drug available for second-generation IPTp regimens. We would, however, recommend a larger study in HIV-infected pregnant women to confirm the acceptability of MQ use considering its frequent but relatively minor side effects.
The authors thank the PAludisme-COtrimoxazole-MEfloquine (PACOME) study team: Natacha Aïhonnou, Jocelyn Akakpo, Jeanne Baï Amassé, Clarisse Briga, Claire Degnonvi, Florelle Kottin, Rodolphe Ladekpo, Charles Ahouansou, Azizath Moussilou, and Justine Ahlonsou for malaria PCR. The authors are grateful to the DSMB (Olivier Bouchot, Elisabeth Elefant, Olivier Lortholary, and Jean-Yves Mary) for their valuable advice. The authors kindly acknowledge all the study participants midwives and physicians and the Programme National de Lutte contre le Sida. The authors thank Patricia Kahn, Médecins Sans Frontières, New York, NY, for English editing of the article.
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