Molecular markers of drug resistance and clinical outcome in falciparum malaria in Cambodia and the Democratic Republic of Congo

Background. Drug resistance is a major obstacle to the control of Plasmodium falciparum malaria. Monitoring the efficacy of antimalarials is a critical component to malaria control. One possible surveillance method is to use molecular markers. However, their relationship with clinical resistance needs to be established before they can be used. Methods. Clinical samples from an in vivo efficacy study of sulfadoxine-pyrimethamine (SP) based therapy in Rutshuru, Democratic Republic of Congo were used to estimate the effect of mutations in dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) on treatment failure. In addition, clinical samples from an in vivo efficacy study of mefloquineartesunate in Pailin, Cambodia were used to estimate the effect of changes in the P. falciparum multidrug resistance-1 (pfmdr1) gene on recrudescence. Lastly, a cross-sectional survey of Cambodia was conducted to determine the geographic distribution of the genetic changes in pfmdr1. Results. In Rutshuru, the effect of mutations at dhps-437 and dhps-540 on SP treatment failure differed by level of parasitemia: for children with low parasitemia, the presence of both mutations was associated with a 17% (95%CI: -3%, 36%) greater absolute risk of treatment failure compared to having neither mutation (baseline risk: 21%). For children with high parasitemia, the risk difference was 50% (95%CI: 35%, 64%; baseline risk: 8%). In Pailin, pfmdr1 copy number was strongly related to time to recrudescence after mefloquine-artesunate treatment (adjusted HR = 7.91, 95%CI: 2.38, 26.29). In the cross-sectional study of Cambodia, increased pfmdr1 copy number was found not only in Pailin, where mefloquine resistance has been well documented, but also in Chumkiri, an area not previously known for drug resistance. Conclusion. These studies demonstrated dhps mutations are associated with SP treatment failure in the DRC and pfmdr1 copy number is associated with recrudescence after mefloquine-artesunate treatment in Cambodia. The cross-sectional study demonstrates how the use of a molecular marker can be operationalized to detect resistant areas outside of current sentinel surveillance sites. Detecting dhps mutations in DRC and pfmdr1 copy number in Cambodia are potentially cost-effective methods to complement the current in vivo efficacy monitoring of drug resistance in these countries

Delayed Plasmodium falciparum clearance following artesunate-mefloquine combination therapy in Thailand, 1997-2007

Abstract Background There is concern that artesunate resistance is developing in Southeast Asia. The purpose of this study is to investigate the prevalence of parasitaemia in the few days following treatment with artesunate-mefloquine (AM), which is an indirect measure of decreased artesunate susceptibility. Methods This is a retrospective analysis of 31 therapeutic efficacy studies involving 1,327 patients treated with AM conducted by the Thai National Malaria Control Programme from 1997–2007. Results The prevalence of patients with parasitaemia on day 2 was higher in the east compared to the west (east: 20%, west: 9%, OR 2.47, 95% CI: 1.77, 3.45). In addition, the prevalence of day-2 parasitaemia increased over time (OR for each year = 1.10, 95% CI: 1.03, 1.19). After controlling for initial parasitaemia and age, year and region remained important determinants of day-2 parasitaemia (OR for region = 3.98, 95%CI 2.63, 6.00; OR for year = 1.28, 95%CI: 1.17, 1.39). The presence of parasitaemia on day 2 and day 3 were specific, but not sensitive predictors of treatment failure. Discussion Delayed resolution of parasitaemia after AM treatment increased in eastern Thailand between 1997 and 2007, which may be an early manifestation of decreased artesunate susceptibility. However, clinical and parasitological treatment failure after 28 days (which is related to both mefloquine and artesunate decreased susceptibility) is not changing over time. The presence of parasitaemia on day 2 is a poor indicator of AM 28-day treatment failure

Molecular Surveillance for Multidrug-Resistant Plasmodium falciparum, Cambodia

We conducted surveillance for multidrug-resistant Plasmodium falciparum in Cambodia during 2004–2006 by assessing molecular changes in pfmdr1. The high prevalence of isolates with multiple pfmdr1 copies found in western Cambodia near the Thai border, where artesunate–mefloquine therapy failures occur, contrasts with isolates from eastern Cambodia, where this combination therapy remains highly effective

PFMDR1 AND IN VIVO RESISTANCE TO ARTESUNATE-MEFLOQUINE IN FALCIPARUM MALARIA ON THE CAMBODIAN–THAI BORDER

Artemisinin combination therapies (ACTs) have recently been adopted as first-line therapy for Plasmodium falciparum infections in most malaria-endemic countries. In this study, we estimated the association between artesunate-mefloquine therapy failure and genetic changes in the putative transporter, pfmdr1. Blood samples were acquired from 80 patients enrolled in an 2004 in vivo efficacy study in Pailin, Cambodia, and genotyped for pfmdr1 copy number and haplotype. Having parasites with three or more copies of pfmdr1 before treatment was strongly associated with recrudescence (hazard ratio [HR] = 8.30; 95% CI: 2.60–26.43). This relationship was maintained when controlling for initial parasite density and hematocrit (HR = 7.91; 95% CI: 2.38–26.29). Artesunate-mefloquine treatment selected for increased pfmdr1 copy number, because isolates from recurrent episodes had higher copy numbers than the paired enrollment samples (Wilcoxon rank test, P = 0.040). pfmdr1 copy number should be evaluated further as a surveillance tool for artesunate-mefloquine resistance in Cambodia

Maternal–Fetal Microtransfusions and HIV-1 Mother-to-Child Transmission in Malawi

Background: Between 25% and 35% of infants born to HIV-infected mothers become HIV-1 infected. One potential route of mother-to-child transmission (MTCT) could be through a breakdown in the placental barrier (i.e., maternal–fetal microtransfusions). Methods and Findings: Placental alkaline phosphatase (PLAP) is a 130-kD maternal enzyme that cannot cross the intact placental barrier. We measured PLAP activity in umbilical vein serum as an indicator of maternal–fetal microtransfusion, and related this to the risk of HIV-1 MTCT. A case-cohort study was conducted of 149 women randomly selected from a cohort of HIV-1-infected pregnant Malawians; these women served as a reference group for 36 cases of in utero MTCT and 43 cases of intrapartum (IP) MTCT. Cord PLAP activity was measured with an immunocatalytic assay. Infant HIV status was determined by real-time PCR. The association between cord PLAP activity and HIV-1 MTCT was measured with logistic regression using generalized estimating equations. Among vaginal deliveries, PLAP was associated with IP MTCT (risk ratio, 2.25 per $log_{10}$ ng/ml PLAP; 95% confidence interval, 0.95–5.32) but not in utero MTCT. In a multivariable model adjusted for HIV-1 RNA load, chorioamnionitis, and self-reported fever, the risk of IP MTCT almost tripled for every $log_{10}$ increase in cord PLAP activity (risk ratio, 2.87; 95% confidence interval, 1.05–7.83). Conclusion: These results suggest that during vaginal deliveries, placental microtransfusions are a risk factor for IP HIV-1 MTCT. Future studies are needed to identify factors that increase the risk for microtransfusions in order to prevent IP HIV-1 MTCT

dhfr and dhps genotype and sulfadoxine-pyrimethamine treatment failure in children with falciparum malaria in the Democratic Republic of Congo

To determine the relationship between mutations in dhfr and dhps and SP treatment failure in Plasmodium falciparum malaria in the Democratic Republic of the Congo (DRC

A Randomized Controlled Pilot Trial of Azithromycin or Artesunate Added to Sulfadoxine-Pyrimethamine as Treatment for Malaria in Pregnant Women

New anti-malarial regimens are urgently needed in sub-Saharan Africa because of the increase in drug resistance. We investigated the safety and efficacy of azithromycin or artesunate combined with sulfadoxine-pyrimethamine used for treatment of malaria in pregnant women in Blantyre, Malawi

Effects of community-level bed net coverage on malaria morbidity in Lilongwe, Malawi

Background The protective effect of insecticide-treated bed nets against individual-level malaria transmission is well known, however community-level effects are less understood. Protective effects from community-level bed net use against malaria transmission have been observed in clinical trials, however, the relationship is less clear outside of a controlled research setting. The objective of this research was to investigate the effect of community-level bed net use against malaria transmission outside of a bed net clinical trial setting in Lilongwe, Malawi following national efforts to scale-up ownership of long-lasting, insecticide-treated bed nets. Methods An annual, cross-sectional, household-randomized, malaria transmission intensity survey was conducted in Lilongwe, Malawi (2011–2013). Health, demographic, and geographic-location data were collected. Participant blood samples were tested for Plasmodium falciparum presence. The percentage of people sleeping under a bed net within 400-m and 1-km radii of all participants was measured. Mixed effects logistic regression models were used to measure the relationship between malaria prevalence and surrounding bed net coverage. Each year, 800 people were enrolled (400 <5 years; 200 5–19 years; 200 ≥20 years; total n = 2400). Results From 2011 to 2013, malaria prevalence declined from 12.9 to 5.6%, while bed net use increased from 53.8 to 78.6%. For every 1% increase in community bed net coverage, malaria prevalence decreased among children under 5 years old [adjusted odds ratio: 0.98 (0.96, 1.00)]. Similar effects were observed in participants 5–19 years [unadjusted odds ratio: 0.98 (0.97, 1.00)]; the effect was attenuated after adjusting for individual-level bed net use. Community coverage was not associated with malaria prevalence among adults ≥20 years. Supplemental analyses identified more pronounced indirect protective effects from community-level bed net use against malaria transmission among children under 5 years who were sleeping under a bed net [adjusted odds ratio: 0.97 (0.94, 0.99)], compared to children who were not sleeping under a bed net [adjusted odds ratio: 0.99 (0.97, 1.01)]. Conclusions Malawi’s efforts to scale up ownership of long-lasting, insecticide-treated bed nets are effective in increasing reported use. Increased community-level bed net coverage appears to provide additional protection against malaria transmission beyond individual use in a real-world context

A Randomized Controlled Pilot Trial of Azithromycin or Artesunate Added to Sulfadoxine-Pyrimethamine as Treatment for Malaria in Pregnant Women

New anti-malarial regimens are urgently needed in sub-Saharan Africa because of the increase in drug resistance. We investigated the safety and efficacy of azithromycin or artesunate combined with sulfadoxine-pyrimethamine used for treatment of malaria in pregnant women in Blantyre, Malawi.This was a randomized open-label clinical trial, conducted at two rural health centers in Blantyre district, Malawi. A total of 141 pregnant women with uncomplicated Plasmodium falciparum malaria were recruited and randomly allocated to 3 treatment groups: sulfadoxine-pyrimethamine (SP; 3 tablets, 500 mg sulfadoxine and 25 mg pyrimethamine per tablet); SP plus azithromycin (1 g/dayx2 days); or SP plus artesunate (200 mg/dayx3 days). Women received two doses administered at least 4 weeks apart. Heteroduplex tracking assays were performed to distinguish recrudescence from new infections. Main outcome measures were incidence of adverse outcomes, parasite and fever clearance times and recrudescence rates. All treatment regimens were well tolerated. Two women vomited soon after ingesting azithromycin. The parasite clearance time was significantly faster in the SP-artesunate group. Recrudescent episodes of malaria were less frequent with SP-azithromycin [Hazard Ratio 0.19 (95% confidence interval 0.06 to 0.63)] and SP-artesunate [Hazard Ratio 0.25 (95% confidence interval 0.10 to 0.65)] compared with SP monotherapy. With one exception (an abortion in the SP-azithromycin group), all adverse pregnancy outcomes could be attributed to known infectious or obstetrical causes. Because of the small sample size, the effect on birth outcomes, maternal malaria or maternal anemia could not be evaluated.Both SP-artesunate and SP-azithromycin appeared to be safe, well tolerated and efficacious for the treatment of malaria during pregnancy. A larger study is needed to determine their safety and efficacy in preventing poor birth outcomes.ClinialTrials.gov NCT00287300

Maternal–Fetal Microtransfusions and HIV-1 Mother-to-Child Transmission in Malawi

BACKGROUND: Between 25% and 35% of infants born to HIV-infected mothers become HIV-1 infected. One potential route of mother-to-child transmission (MTCT) could be through a breakdown in the placental barrier (i.e., maternal–fetal microtransfusions). METHODS AND FINDINGS: Placental alkaline phosphatase (PLAP) is a 130-kD maternal enzyme that cannot cross the intact placental barrier. We measured PLAP activity in umbilical vein serum as an indicator of maternal–fetal microtransfusion, and related this to the risk of HIV-1 MTCT. A case-cohort study was conducted of 149 women randomly selected from a cohort of HIV-1-infected pregnant Malawians; these women served as a reference group for 36 cases of in utero MTCT and 43 cases of intrapartum (IP) MTCT. Cord PLAP activity was measured with an immunocatalytic assay. Infant HIV status was determined by real-time PCR. The association between cord PLAP activity and HIV-1 MTCT was measured with logistic regression using generalized estimating equations. Among vaginal deliveries, PLAP was associated with IP MTCT (risk ratio, 2.25 per log(10) ng/ml PLAP; 95% confidence interval, 0.95–5.32) but not in utero MTCT. In a multivariable model adjusted for HIV-1 RNA load, chorioamnionitis, and self-reported fever, the risk of IP MTCT almost tripled for every log(10) increase in cord PLAP activity (risk ratio, 2.87; 95% confidence interval, 1.05–7.83). CONCLUSION: These results suggest that during vaginal deliveries, placental microtransfusions are a risk factor for IP HIV-1 MTCT. Future studies are needed to identify factors that increase the risk for microtransfusions in order to prevent IP HIV-1 MTCT