Counter-Selection of Antimalarial Resistance Polymorphisms by Intermittent Preventive Treatment in Pregnancy

Background: Innovative approaches are needed to limit antimalarial resistance evolution. Understanding the role of intermittent preventive treatment in pregnancy (IPTp) on the selection for resistance and the impact such selection has on pregnancy outcomes can guide future interventions. Methods: Plasmodium falciparum isolates (n = 914) from 2 randomized clinical trials were screened for pfmdr1 copy number variation and pfcrt, pfmdr1, pfdhfr, and pfdhps resistance markers. The trials were conducted between 2010 and 2013 in Benin, Gabon, Kenya, and Mozambique to establish the efficacy of IPTp-mefloquine (MQ) compared with IPTp-sulphadoxine-pyrimethamine (SP) in human immunodeficiency virus (HIV)-uninfected and to IPTp-placebo in HIV-infected women. Results: In HIV-uninfected women, the prevalence of pfcrt mutants, pfdhfr/pfdhps quintuple mutants, and pfmdr1 copy number was similar between women receiving IPT-SP and IPTp-MQ. However, prevalence of pfmdr1 polymorphism 86Y was lower in the IPTp-MQ group than in the IPTp-SP group, and within the IPTp-MQ group it was lower at delivery compared with recruitment. No effect of IPTp-MQ on resistance markers was observed among HIV-infected women. The carriage of resistance markers was not associated with pregnancy outcomes.

In vitro growth of Plasmodium falciparum in neonatal blood.

BACKGROUND: Children below the age of six months suffer less often from malaria than older children in sub-Saharan Africa. This observation is commonly attributed to the persistence of foetal haemoglobin (HbF), which is considered not to permit growth of Plasmodium falciparum and therefore providing protection against malaria. Since this concept has recently been challenged, this study evaluated the effect of HbF erythrocytes and maternal plasma on in vitro parasite growth of P. falciparum in Central African Gabon. METHODS: Umbilical cord blood and peripheral maternal blood were collected at delivery at the Albert Schweitzer Hospital in Gabon. Respective erythrocyte suspension and plasma were used in parallel for in vitro culture. In vitro growth rates were compared between cultures supplemented with either maternal or cord erythrocytes. Plasma of maternal blood and cord blood was evaluated. Parasite growth rates were assessed by the standard HRP2-assay evaluating the increase of HRP2 concentration in Plasmodium culture. RESULTS: Culture of P. falciparum using foetal erythrocytes led to comparable growth rates (mean growth rate = 4.2, 95% CI: 3.5 - 5.0) as cultures with maternal red blood cells (mean growth rate =4.2, 95% CI: 3.4 - 5.0) and those from non-malaria exposed individuals (mean growth rate = 4.6, 95% CI: 3.8 - 5.5). Standard in vitro culture of P. falciparum supplemented with either maternal or foetal plasma showed both significantly lower growth rates than a positive control using non-malaria exposed donor plasma. CONCLUSIONS: These data challenge the concept of HbF serving as intrinsic inhibitor of P. falciparum growth in the first months of life. Erythrocytes containing HbF are equally permissive to P. falciparum growth in vitro. However, addition of maternal and cord plasma led to reduced in vitro growth which may translate to protection against clinical disease or show synergistic effects with HbF in vivo. Further studies are needed to elucidate the pathophysiology of innate and acquired protection against neonatal malaria.

Intermittent preventive treatment of malaria in pregnancy with mefloquine in HIV-negative women: a multicentre randomized controlled trial

Background: Intermittent preventive treatment in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) is recommended by WHO to prevent malaria in African pregnant women. The spread of SP parasite resistance has raised concerns regarding long-term use for IPT. Mefloquine (MQ) is the most promising of available alternatives to SP based on safety profile, long half-life, and high efficacy in Africa. We evaluated the safety and efficacy of MQ for IPTp compared to those of SP in HIV-negative women. Methods and findings: A total of 4,749 pregnant women were enrolled in an open-label randomized clinical trial conducted in Benin, Gabon, Mozambique, and Tanzania comparing two-dose MQ or SP for IPTp and MQ tolerability of two different regimens. The study arms were: (1) SP, (2) single dose MQ (15 mg/kg), and (3) split-dose MQ in the context of long lasting insecticide treated nets. There was no difference on low birth weight prevalence (primary study outcome) between groups (360/2,778 [13.0%]) for MQ group and 177/1,398 (12.7%) for SP group; risk ratio [RR], 1.02 (95% CI 0.86-1.22; p=0.80 in the ITT analysis). Women receiving MQ had reduced risks of parasitemia (63/1,372 [4.6%] in the SP group and 88/2,737 [3.2%] in the MQ group; RR, 0.70 [95% CI 0.51-0.96]; p=0.03) and anemia at delivery (609/1,380 [44.1%] in the SP group and 1,110/2743 [40.5%] in the MQ group; RR, 0.92 [95% CI 0.85-0.99]; p=0.03), and reduced incidence of clinical malaria (96/551.8 malaria episodes person/year [PYAR] in the SP group and 130/1,103.2 episodes PYAR in the MQ group; RR, 0.67 [95% CI 0.52-0.88]; p=0.004) and all-cause outpatient attendances during pregnancy (850/557.8 outpatients visits PYAR in the SP group and 1,480/1,110.1 visits PYAR in the MQ group; RR, 0.86 [0.78-0.95]; p=0.003). There were no differences in the prevalence of placental infection and adverse pregnancy outcomes between groups. Tolerability was poorer in the two MQ groups compared to SP. The most frequently reported related adverse events were dizziness (ranging from 33.9% to 35.5% after dose 1; and 16.0% to 20.8% after dose 2) and vomiting (30.2% to 31.7%, after dose 1 and 15.3% to 17.4% after dose 2) with similar proportions in the full and split MQ arms. The open-label design is a limitation of the study that affects mainly the safety assessment. Conclusions: Women taking MQ IPTp (15 mg/kg) in the context of long lasting insecticide treated nets had similar prevalence rates of low birth weight as those taking SP IPTp. MQ recipients had less clinical malaria than SP recipients, and the pregnancy outcomes and safety profile were similar. MQ had poorer tolerability even when splitting the dose over two days. These results do not support a change in the current IPTp policy.

A thirteen-year analysis of Plasmodium falciparum populations reveals high conservation of the mutant pfcrt haplotype despite the withdrawal of chloroquine from national treatment guidelines in Gabon.

BACKGROUND: Chloroquine resistance (CR) decreased after the removal of chloroquine from national treatment guidelines in Malawi, Kenia and Tanzania. In this investigation the prevalence of the chloroquine resistance (CQR) conferring mutant pfcrt allele and its associated chromosomal haplotype were determined before and after the change in Gabonese national treatment guidelines from chloroquine (CQ) to artesunate plus amodiaquine (AQ) in 2003. METHODS: The prevalence of the wild type pfcrt allele was assessed in 144 isolates from the years 2005 - 07 by PCR fragment restriction digest and direct sequencing. For haplotype analysis of the chromosomal regions flanking the pfcrt locus, microsatellite analysis was done on a total of 145 isolates obtained in 1995/96 (43 isolates), 2002 (47 isolates) and 2005 - 07 (55 isolates). RESULTS: The prevalence of the mutant pfcrt allele decreased from 100% in the years 1995/96 and 2002 to 97% in 2005 - 07. Haplotype analysis showed that in 1995/96 79% of the isolates carried the same microsatellite alleles in a chromosomal fragment spanning 39 kb surrounding the pfcrt locus. In 2002 and 2005 - 07 the prevalence of this haplotype was 62% and 58%, respectively. Pfcrt haplotype analysis showed that all wild type alleles were CVMNK. CONCLUSION: Four years after the withdrawal of CQ from national treatment guidelines the prevalence of the mutant pfcrt allele remains at 97%. The data suggest that the combination of artesunate plus AQ may result in continued selection for the mutant pfcrt haplotype even after discontinuance of CQ usage.

In vitro activity of antifungal drugs against Plasmodium falciparum field isolates.

The increasing resistance of the malaria parasite Plasmodium falciparum to currently available drugs necessitates a continuous effort to develop new antimalarial agents. We therefore aimed to assess the in vitro activity of the antifungal drugs clotrimazole, fluconazole, ketoconazole, itraconazole, voriconazole, flucytosine, amphotericin B, and caspofungin against field isolates of P. falciparum from Lambaréné, Gabon. Using the histidin-rich protein 2 (HRP-2) assay we determined the drug susceptibility (EC(50), EC(90)) of 16 field isolates obtained from outpatients attending the Albert Schweitzer Hospital in Lambaréné, Gabon. For fluconazole, itraconazole and caspofungin the in vitro growth inhibition of these drugs is reported for the first time. Our data indicate that clotrimazole, fluconazole, itraconazole and caspofungin show median EC(50) values of 3.1 µg/mL, 1.9 µg/mL, 1.1 µg/mL and 1.1 µg/mL respectively. Ketoconazole, voriconazole, flucytosine and amphotercin B showed no relevant growth inhibition within the range of drug concentrations used in this study.

High prevalence of asymptomatic Plasmodium falciparum infection in Gabonese adults.

Plasmodium falciparum, the most common malarial parasite in sub-Saharan Africa, accounts for a high number of deaths in children less than five years of age. In malaria-endemic countries with stable transmission, semi-immunity is usually acquired after childhood. For adults, severe malaria is rare. Infected adults have either uncomplicated malaria or asymptomatic parasitemia. During a period of one year, we screened 497 afebrile males to investigate the prevalence of asymptomatic P. falciparum parasitemia in villages near Lambaréné, Gabon by use of three different methods. A total of 52% of the individuals had parasites detected by a subtelomeric variable open reading frame polymerase chain reaction (stevor-PCR), 27% of the rapid diagnostic test results were positive, and 12% of the thick blood smears with low parasitemias had P. falciparum. Most positive cases were only detected by the stevor-PCR. Asymptomatic P. falciparum parasitemia in adults living in a malaria-endemic country is frequent.