The role of different components of the immune system against Plasmodium falciparum malaria: Possible contribution towards malaria vaccine development.

Plasmodium falciparum malaria still remains a major global public health challenge with over 220 million new cases and well over 400,000 deaths annually. Most of the deaths occur in sub-Saharan Africa which bears 90 % of the malaria cases. Such high P. falciparum malaria-related morbidity and mortality rates pose a huge burden on the health and economic wellbeing of the countries affected. Lately, substantial gains have been made in reducing malaria morbidity and mortality through intense malaria control initiatives such as use of effective antimalarials, intensive distribution and use of insecticide-treated nets (ITNs), and implementation of massive indoor residual spraying (IRS) campaigns. However, these gains are being threatened by widespread resistance of the parasite to antimalarials, and the vector to insecticides. Over the years the use of vaccines has proven to be the most reliable, cost-effective and efficient method for controlling the burden and spread of many infectious diseases, especially in resource poor settings with limited public health infrastructure. Nonetheless, this had not been the case with malaria until the most promising malaria vaccine candidate, RTS,S/AS01, was approved for pilot implementation programme in three African countries in 2015. This was regarded as the most important breakthrough in the fight against malaria. However, RTS,S/AS01 has been found to have some limitations, the main ones being low efficacy in certain age groups, poor immunogenicity and need for almost three boosters to attain a reasonable efficacy. Thus, the search for a more robust and effective malaria vaccine still continues and a better understanding of naturally acquired immune responses to the various stages, including the transmissible stages of the parasite, could be crucial in rational vaccine design. This review therefore compiles what is currently known about the basic biology of P. falciparum and the natural malaria immune response against malaria and progress made towards vaccine development.

A clinical trial of enteral Levetiracetam for acute seizures in pediatric cerebral malaria.

BACKGROUND: Acute seizures are common in pediatric cerebral malaria (CM), but usual care with phenobarbital risks respiratory suppression. We undertook studies of enteral levetiracetam (eLVT) to evaluate pharmacokinetics (PK), safety and efficacy including an open-label, randomized controlled trial (RCT) comparing eLVT to phenobarbital. METHODS: Children 24-83 months old with CM were enrolled in an eLVT dose-finding study starting with standard dose (40 mg/kg load, then 30 mg/kg Q12 hours) titrated upward until seizure freedom was attained in 75% of subjects. The RCT that followed randomized children to eLVT vs. phenobarbital for acute seizures and compared the groups on minutes with seizures based upon continuous electroencephalogram. Due to safety concerns, midway through the study children allocated to phenobarbital received the drug only if they continued to have seizures (either clinically or electrographically) after benzodiazepine treatment. Secondary outcomes were treatment failure requiring cross over, coma duration and neurologic sequelae at discharge. PK and safety assessments were also undertaken. RESULTS: Among 30 comatose CM children, eLVT was rapidly absorbed and well-tolerated. eLVT clearance was lower in patients with higher admission serum creatinine (SCr), but overall PK parameters were similar to prior pediatric PK studies. Within 4 h of the first dose, 90% reached therapeutic levels (> 20 µg/mL) and all were above 6 µg/mL. 7/7 children achieved seizure freedom on the initial eLVT dose. Comparing 23 eLVT to 21 phenobarbital patients among whom 15/21 received phenobarbital, no differences were seen for minutes with seizure, seizure freedom, coma duration, neurologic sequelae or death, but eLVT was safer (p = 0.019). Phenobarbital was discontinued in 3/15 due to respiratory side effects. CONCLUSION: Enteral LVT offers an affordable option for seizure control in pediatric CM and is safer than phenobarbital. TRIAL REGISTRATION: NCT01660672 . NCT01982812 .

Impact of Efavirenz-, Ritonavir-Boosted Lopinavir-, and Nevirapine-Based Antiretroviral Regimens on the Pharmacokinetics of Lumefantrine and Safety of Artemether-Lumefantrine in Plasmodium falciparum-Negative HIV-Infected Malawian Adults Stabilized on Antiretroviral Therapy.

There is conflicting evidence of the impact of commonly used antiretroviral therapies (ARTs) on the pharmacokinetics of lumefantrine and the safety profile of artemether-lumefantrine. We compared the area under the concentration-time curve from 0 h to 14 days (AUC0-14 days) of lumefantrine and the safety profile of artemether-lumefantrine in malaria-negative human immunodeficiency virus (HIV)-infected adults in two steps. In step 1, a half-dose adult course of artemether-lumefantrine was administered as a safety check in four groups (n = 6/group): (i) antiretroviral naive, (ii) nevirapine-based ART, (iii) efavirenz-based ART, and (iv) ritonavir-boosted lopinavir-based ART. In step 2, a standard-dose adult course of artemether-lumefantrine was administered to a different cohort in three groups (n = 10 to 15/group): (i) antiretroviral naive, (ii) efavirenz-based ART, and (iii) ritonavir-boosted lopinavir-based ART. In step 1, lumefantrine's AUC0-14 days was 53% (95% confidence interval [CI], 0.27 to 0.82) lower in the efavirenz-based ART group than in the ART-naive group and was 2.4 (95% CI, 1.58 to 3.62) and 2.9(95% CI, 1.75 to 4.72) times higher in the nevirapine- and ritonavir-boosted lopinavir groups, respectively. In step 2, lumefantrine's AUC0-14 days was 1.9 (95% CI, 1.26 to 3.00) times higher in the ritonavir-boosted lopinavir group and not significantly different between the efavirenz- and ART-naive groups (0.99 [95% CI, 0.63 to 1.57]). Frequent cases of hematological abnormalities (thrombocytopenia and neutropenia) were observed in the nevirapine group in step 1, leading to a recommendation from the data and safety monitoring board not to include a nevirapine group in step 2. Artemether-lumefantrine was well tolerated in the other groups. The therapeutic implications of these findings need to be evaluated among HIV-malaria-coinfected adults. (This study has been registered at the Pan African Clinical Trials Registry under numbers PACTR2010030001871293 and PACTR2010030001971409.).

Pharmacokinetics and Safety Profile of Artesunate-Amodiaquine Coadministered with Antiretroviral Therapy in Malaria-Uninfected HIV-Positive Malawian Adults.

There are limited data on the pharmacokinetic and safety profiles of artesunate-amodiaquine in human immnunodeficiency virus-infected (HIV+) individuals receiving antiretroviral therapy. In a two-step intensive sampling pharmacokinetic trial, we compared the area under the concentration-time curve from 0 to 28 days (AUC0-28) of an active metabolite of amodiaquine, desethylamodiaquine, and treatment-emergent adverse events between antiretroviral therapy-naive HIV+ adults and those taking nevirapine and ritonavir-boosted lopinavir-based antiretroviral therapy. In step 1, malaria-uninfected adults (n = 6/arm) received half the standard adult treatment regimen of artesunate-amodiaquine. In step 2, another cohort (n = 25/arm) received the full regimen. In step 1, there were no safety signals or significant differences in desethylamodiaquine AUC0-28 among participants in the ritonavir-boosted lopinavir, nevirapine, and antiretroviral therapy-naive arms. In step 2, compared with those in the antiretroviral therapy-naive arm, participants in the ritonavir-boosted lopinavir arm had 51% lower desethylamodiaquine AUC0-28, with the following geometric means (95% confidence intervals [CIs]): 23,822 (17,458 to 32,506) versus 48,617 (40,787 to 57,950) ng · h/ml (P < 0.001). No significant differences in AUC0-28 were observed between nevirapine and antiretroviral therapy-naive arms. Treatment-emergent transaminitis was higher in the nevirapine (20% [5/25]) than the antiretroviral therapy-naive (0.0% [0/25]) arm (risk difference, 20% [95% CI, 4.3 to 35.7]; P = 0.018). The ritonavir-boosted lopinavir antiretroviral regimen was associated with reduced desethylamodiaquine exposure, which may compromise artesunate-amodiaquine's efficacy. Coadministration of nevirapine and artesunate-amodiaquine may be associated with hepatoxicity.

Pharmacokinetics of Piperaquine and Safety Profile of Dihydroartemisinin-Piperaquine Coadministered with Antiretroviral Therapy in Malaria-Uninfected HIV-Positive Malawian Adults.

There are limited data on the pharmacokinetic and safety profiles of dihydroartemisinin-piperaquine (DHA-PQ) among human immunodeficiency virus-infected (HIV-positive [HIV+]) individuals taking antiretroviral therapy (ART). In a two-step (parallel-group) pharmacokinetic trial with intensive blood sampling, we compared the area under the concentration-time curve from days 0 to 28 (AUC0-28 days) and the safety outcomes of piperaquine among malaria-uninfected HIV+ adults. In step 1, half the adult dose of DHA-PQ was administered for 3 days as an initial safety check to four groups (n = 6/group) of HIV+ adults (age ≥18 years): (i) antiretroviral-naive individuals, (ii) individuals on nevirapine-based ART, (iii) individuals on efavirenz-based ART, and (iv) individuals on ritonavir-boosted lopinavir-based ART. In step 2, a full adult treatment course of DHA-PQ was administered to a different cohort of participants in three groups: (i) antiretroviral-naive individuals, (ii) individuals on efavirenz-based ART, and (iii) individuals on nevirapine-based ART (n = 10 to 15/group). The ritonavir-boosted lopinavir-based ART group was dropped in step 2 due to the limited number of participants who were on this second-line ART and were eligible for recruitment. Piperaquine's AUC0-28 days in both steps was 43% lower among participants on efavirenz-based ART than among ART-naive participants. There were no significant differences in AUC0-28 days between the other ART groups and the ART-naive group in each of the two steps. Furthermore, no differences in treatment-emergent clinical and laboratory adverse events were observed across the groups in steps 1 and 2. Although it was well tolerated at the half and full standard adult treatment courses, the efavirenz-based antiretroviral regimen was associated with reduced piperaquine exposure, which may compromise dihydroartemisinin-piperaquine's effectiveness in programmatic settings. (The clinical trials presented in this study have been registered at the WHO's International Clinical Trials Registry Platform under ID numbers PACTR2010030001871293 and PACTR2010030001971409.).

A simultaneous population pharmacokinetic analysis of rifampicin in Malawian adults and children.

AIMS: Low rifampicin plasma concentrations can lead to treatment failure and increased risk of developing drug resistant tuberculosis. The objectives of this study were to characterize the population pharmacokinetics (popPK) of rifampicin in Malawian children and adults with tuberculosis, simulate exposures under revised WHO dosing guidelines that aim to reduce the risk of low exposures of rifampicin and examine predicted exposures using weight- and age-based dosing bands under new dosing recommendations. METHODS: Patients were recruited at least two weeks after initiation of the intensive phase of treatment and received RIF in FDC of anti-TB drugs. A total of 5-6 rich and 1-2 sparse samples were collected. nonmem (v7.2) was used to build a population-PK model. RESULTS: A 165 TB patients, 115 adults and 50 children, aged 7 months to 65 years and weighing 4.8 to 87 kg, were included in the one compartment model with first order absorption best described the data. The mean population estimate for CL/F was 23.9 (l h(-1)  70 kg(-1) ) with inter-individual variability of 46.6%. Exposure was unaffected by HIV status. Relative bioavailability in children was estimated at 49% lower compared to adults (100% relative bioavailability). Simulations showed significantly lower rifampicin exposure in children vs. adults. In children average AUC was 13.5 mg l(-1) h, which was nearly half that was observed in adults (26.3 mg l(-1)  h). Using age as a surrogate for weight in dosing bands gave similar results compared with the weight bands. Increasing dose to approximately 15 mg kg(-1) , increased AUC in children to an average of 22 mgl(-1)  h. bringing expected exposures in children closer to those predicted for adults. CONCLUSION: The popPK model developed can be used to optimize rifampicin exposures through dosing simulations. WHO dosing recommendations may not be achieved using currently licensed fixed dose combination formulations of TB therapy.

Persistence of Sulfadoxine-Pyrimethamine Resistance Despite Reduction of Drug Pressure in Malawi.

BACKGROUND: In 2007, Malawi replaced sulfadoxine-pyrimethamine (SP) with an artemisinin-based combination therapy as the first-line treatment for uncomplicated Plasmodium falciparum malaria in response to failing SP efficacy. Here we estimate the effect of reduced SP pressure on the prevalence of SP-resistant parasites and the characteristics of the associated selective sweeps flanking the resistance loci. METHODS: Samples obtained from individuals with clinical malaria during a period of high SP use (1999-2001), a transitional period (2007-2008), and a period of low SP use (2012) were genotyped for resistance markers at pfdhfr-ts codons 51, 59, and 108 and pfdhps codons 437, 540, and 581. Expected heterozygosity was estimated to evaluate the genetic diversity flanking pfdhfr-ts and pfdhps. RESULTS: An increase in the prevalence of the resistance haplotypes DHFR 51I/59R/108N and DHPS 437G/540E occurred under sustained drug pressure, with no change in haplotype prevalence 5 years after reduction in SP pressure. The DHPS 437G/540E/581G haplotype was observed in 2007 and increased in prevalence during a period of reduced SP pressure. Changes to the sweep characteristics flanking pfdhfr-ts and pfdhps were minimal. CONCLUSIONS: In contrast to the rapid and complete return of chloroquine-susceptible falciparum malaria after chloroquine was withdrawn from Malawi, a reemergence of SP efficacy is unlikely in the near future.

The A581G Mutation in the Gene Encoding Plasmodium falciparum Dihydropteroate Synthetase Reduces the Effectiveness of Sulfadoxine-Pyrimethamine Preventive Therapy in Malawian Pregnant Women.

BACKGROUND: The A581 G: mutation in the gene encoding Plasmodium falciparum dihydropteroate synthase (dhps), in combination with the quintuple mutant involving mutations in both dhps and the gene encoding dihydrofolate reductase (dhfr), the so-called sextuple mutant, has been associated with increased placental inflammation and decreased infant birth weight among women receiving intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP) during pregnancy. METHODS: Between 2009 and 2011, delivering women without human immunodeficiency virus infection were enrolled in an observational study of IPTp-SP effectiveness in Malawi. Parasites were detected by polymerase chain reaction (PCR); positive samples were sequenced to genotype the dhfr and dhps loci. The presence of K540 E: in dhps was used as a marker for the quintuple mutant. RESULTS: Samples from 1809 women were analyzed by PCR; 220 (12%) were positive for P. falciparum. A total of 202 specimens were genotyped at codon 581 of dhps; 17 (8.4%) harbored the sextuple mutant. The sextuple mutant was associated with higher risks of patent infection in peripheral blood (adjusted prevalence ratio [aPR], 2.76; 95% confidence interval [CI], 1.82-4.18) and placental blood (aPR 3.28; 95% CI, 1.88-5.78) and higher parasite densities. Recent SP use was not associated with increased parasite densities or placental pathology overall and among women with parasites carrying dhps A581 G: . CONCLUSIONS: IPTp-SP failed to inhibit parasite growth but did not exacerbate pathology among women infected with sextuple-mutant parasites. New interventions to prevent malaria during pregnancy are needed urgently.

Return of widespread chloroquine-sensitive Plasmodium falciparum to Malawi.

BACKGROUND: The return of chloroquine-sensitive Plasmodium falciparum to the limited area of Blantyre, Malawi, has been well demonstrated in several studies. METHODS: To characterize chloroquine susceptibility over a wide geographic area, infants and children aged 6-59 months were selected using 2-stage cluster sampling in 8 Malawian districts. Pyrosequencing of the pfcrt gene codon 76 region was performed for children with asexual parasitemia. RESULTS: Of 7145 children, 1150 had microscopic asexual parasitemia, and sequencing was performed in 685, of whom 1 had a chloroquine-resistant genotype. CONCLUSIONS: Systematic countrywide sampling demonstrates that the chloroquine pfcrt genotype has reached near-fixation, raising the possibility of reintroducing chloroquine for malaria prevention and treatment.

Chloroquine-azithromycin combination antimalarial treatment decreases risk of respiratory- and gastrointestinal-tract infections in Malawian children.

BACKGROUND: Chloroquine-azithromycin is being evaluated as combination therapy for malaria. It may provide added benefit in treating or preventing bacterial infections that occur in children with malaria. OBJECTIVE: We aim to evaluate the effect of treating clinical malaria with chloroquine-azithromycin on the incidence of respiratory-tract and gastrointestinal-tract infections compared to treatment with chloroquine monotherapy. METHODS: We compared the incidence density and time to first events of respiratory-tract and gastrointestinal-tract infections among children assigned to receive chloroquine-azithromycin or chloroquine for all symptomatic malaria episodes over the course of 1 year in a randomized longitudinal trial in Blantyre, Malawi. RESULTS: The incidence density ratios of total respiratory-tract infections and gastrointestinal-tract infections comparing chloroquine-azithromycin to chloroquine monotherapy were 0.67 (95% confidence interval [CI], .48, .94) and 0.74 (95% CI, .55, .99), respectively. The time to first lower-respiratory-tract and gastrointestinal-tract infections were significantly longer in the chloroquine-azithromycin arm compared to the chloroquine arm (P = .04 and P = .02, respectively). CONCLUSIONS: Children treated routinely with chloroquine-azithromycin had fewer respiratory and gastrointestinal-tract infections than those treated with chloroquine alone. This antimalarial combination has the potential to reduce the burden of bacterial infections among children in malaria-endemic countries.

Enabling factors facilitating the use of neem-based remedies for the management of malaria in the Lower Shire District of Chikwawa, Malawi.

Background: Malaria remains a major public health threat in Malawi, affecting mostly children under five and pregnant women. Despite the availability of chemotherapy and chemoprophylaxis, resistance to sulfadoxine pyrimethamine and the high cost and complicated regimen of artemether-lumefantrine have accelerated the use of home-based remedies for the management of malaria in Chikwawa district, Malawi. This study aimed to determine factors that facilitate the use of herbal remedies within communities in the management of malaria in the presence of free health care services, with the intention of assessing the feasibility of developing improved herbal products as anti-malarial prophylaxis. Materials and Methods: Data on factors driving the use of neem-based preparations commonly used in the management of malaria were collected through qualitative interviews and focus group discussions. Qualitative data were analysed drawing on the Framework Analysis approach. Results: Neem and moringa were identified as the principal plants used for the management of malaria, with neem being the most frequently used. Factors favouring the communal use of neem-based remedies included the habit of resorting to herbal remedies as first aid treatment, lack of drugs and proper medical care in modern health facilities, and the need for preventive anti-malarial remedies during the high-transmission season. The perceived effectiveness of neem-based herbal remedies was based on their fast action against the symptoms of malaria, thereby providing immediate relief to the patient, which might explain their wide-scale use for malaria treatment. Conclusions: Local communities prefer to use neem and/or moringa remedies for their primary healthcare needs in the management of malaria because of their ease of access, preparation and administration without frequent adverse events, as opposed to ACTs. These remedies are already being used as prophylaxis in unimproved/non-standardised formulation. This suggests that standardised herbal preparations would be culturally acceptable at community level. Evidence-based research is required to validate parasitological and clinical efficacy and determine safety of these anti-malarial herbs.

A longitudinal trial comparing chloroquine as monotherapy or in combination with artesunate, azithromycin or atovaquone-proguanil to treat malaria.

BACKGROUND: The predominance of chloroquine-susceptible falciparum malaria in Malawi more than a decade after chloroquine's withdrawal permits contemplation of re-introducing chloroquine for targeted uses. We aimed to compare the ability of different partner drugs to preserve chloroquine efficacy and prevent the re-emergence of resistance. METHODOLOGY/PRINCIPAL FINDINGS: Children with uncomplicated malaria were enrolled at a government health center in Blantyre, Malawi. Participants were randomized to receive chloroquine alone or combined with artesunate, azithromycin or atovaquone-proguanil for all episodes of uncomplicated malaria for one year. The primary outcome was incidence of clinical malaria. Secondary endpoints included treatment efficacy, and incidence of the chloroquine resistance marker pfcrt T76 and of anemia. Of the 640 children enrolled, 628 were included in the intention-to-treat analysis. Malaria incidence (95% confidence interval) was 0.59 (.46-.74), .61 (.49-.76), .63 (.50-.79) and .68 (.54-.86) episodes/person-year for group randomized to receive chloroquine alone or in combination with artesunate, azithromycin or atovaquone-proguanil respectively and the differences were not statistically significant. Treatment efficacy for first episodes was 100% for chloroquine monotherapy and 97.9% for subsequent episodes of malaria. Similar results were seen in each of the chloroquine combination groups. The incidence of pfcrt T76 in pure form was 0%; mixed infections with both K76 and T76 were found in two out of 911 infections. Young children treated with chloroquine-azithromycin had higher hemoglobin concentrations at the study's end than did those in the chloroquine monotherapy group. CONCLUSION/SIGNIFICANCE: Sustained chloroquine efficacy with repeated treatment supports the eventual re-introduction of chloroquine combinations for targeted uses such as intermittent preventive treatment. TRIAL REGISTRATION: ClinicalTrials.gov NCT00379821.

Clinical manifestations of new versus recrudescent malaria infections following anti-malarial drug treatment.

BACKGROUND: Distinguishing new from recrudescent infections in post-treatment episodes of malaria is standard in anti-malarial drug efficacy trials. New infections are not considered malaria treatment failures and as a result, the prevention of subsequent episodes of malaria infection is not reported as a study outcome. However, in moderate and high transmission settings, new infections are common and the ability of a short-acting medication to cure an initial infection may be outweighed by its inability to prevent the next imminent infection. The clinical benefit of preventing new infections has never been compared to that of curing the initial infection. METHODS: Children enrolled in a sulphadoxine-pyrimethamine efficacy study in Blantyre, Malawi from 1998-2004 were prospectively evaluated. Six neutral microsatellites were used to classify new and recrudescent infections in children aged less than 10 years with recurrent malaria infections. Children from the study who did not experience recurrent parasitaemia comprised the baseline group. The odds of fever and anaemia, the rate of haemoglobin recovery and time to recurrence were compared among the groups. RESULTS: Fever and anemia were more common among children with parasitaemia compared to those who remained infection-free throughout the study period. When comparing recrudescent vs. new infections, the incidence of fever was not statistically different. However, children with recrudescent infections had a less robust haematological recovery and also experienced recurrence sooner than those whose infection was classified as new. CONCLUSIONS: The results of this study confirm the paramount importance of providing curative treatment for all malaria infections. Although new and recrudescent infections caused febrile illnesses at a similar rate, recurrence due to recrudescent infection did have a worsened haemological outcome than recurrence due to new infections. Local decision-makers should take into account the results of genotyping to distinguish new from recrudescent infections when determining treatment policy on a population level. It is appropriate to weigh recrudescent malaria more heavily than new infection in assessing treatment efficacy.

Return of chloroquine-susceptible falciparum malaria in Malawi was a reexpansion of diverse susceptible parasites.

The spread of drug-resistant Plasmodium falciparum malaria has been a major impediment to malaria control and threatens prospects for elimination. We recently demonstrated the return of chloroquine-susceptible malaria in Malawi after chloroquine use was abandoned. In this study, we trace the origins of chloroquine-resistant and chloroquine-susceptible parasites in Malawi by sequencing the P. falciparum chloroquine resistance transporter gene (pfcrt) and by genotyping microsatellites flanking this gene in isolates from infections that occurred in Malawi from 1992 through 2005. Malaria parasites from 2005 harbored the expected wild-type pfcrt haplotype associated with chloroquine susceptibility and have maintained high levels of diversity without linkage disequilibrium, which suggests that the return of chloroquine susceptibility is not the result of a back mutation in a formerly resistant parasite or a new selective sweep. Chloroquine-susceptible parasites that predominate in Malawi likely represent a reexpansion of the susceptible parasites that survived in the population despite widespread drug pressure in the region.

Malaria treatment efficacy among people living with HIV: the role of host and parasite factors.

Identification of an effect of HIV-associated immunosuppression on response to antimalarial therapy would help guide management of malaria infection in areas of high HIV prevalence. Therefore, we conducted an observational study of people living with HIV infection in Blantyre, Malawi. Participants who developed malaria were treated with sulfadoxine-pyrimethamine (SP) and followed for 28 days. Molecular markers for SP resistance were measured. One hundred seventy-eight episodes of malaria were assessed. The 28-day cumulative treatment failure rate was 29.1%. In univariate analysis, CD4 cell count was not associated with treatment failure (hazard ratio 0.6, 95% confidence interval 0.3-1.2). Among children, the risk of treatment failure increased with infection with SP-resistant parasites and anemia. Decreased CD4 cell count was not associated with impaired response to antimalarial therapy or diminished ability to clear SP-resistant parasites, suggesting that acquired immunity to malaria is retained in the face of HIV-associated immunosuppression.

Return of chloroquine antimalarial efficacy in Malawi.

BACKGROUND: In 1993, Malawi became the first country in Africa to replace chloroquine with the combination of sulfadoxine and pyrimethamine for the treatment of malaria. At that time, the clinical efficacy of chloroquine was less than 50%. The molecular marker of chloroquine-resistant falciparum malaria subsequently declined in prevalence and was undetectable by 2001, suggesting that chloroquine might once again be effective in Malawi. METHODS: We conducted a randomized clinical trial involving 210 children with uncomplicated Plasmodium falciparum malaria in Blantyre, Malawi. The children were treated with either chloroquine or sulfadoxine\#8211;pyrimethamine and followed for 28 days to assess the antimalarial efficacy of the drug. RESULTS: In analyses conducted according to the study protocol, treatment failure occurred in 1 of 80 participants assigned to chloroquine, as compared with 71 of 87 participants assigned to sulfadoxine\#8211;pyrimethamine. The cumulative efficacy of chloroquine was 99% (95% confidence interval [CI], 93 to 100), and the efficacy of sulfadoxine\#8211;pyrimethamine was 21% (95% CI, 13 to 30). Among children treated with chloroquine, the mean time to parasite clearance was 2.6 days (95% CI, 2.5 to 2.8) and the mean time to the resolution of fever was 10.3 hours (95% CI, 8.1 to 12.6). No unexpected adverse events related to the study drugs occurred. CONCLUSIONS: Chloroquine is again an efficacious treatment for malaria, 12 years after it was withdrawn from use in Malawi. (ClinicalTrials.gov number, NCT00125489 [ClinicalTrials.gov].).

Association between the pharmacokinetics and in vivo therapeutic efficacy of sulfadoxine-pyrimethamine in Malawian children.

Sulfadoxine-pyrimethamine (SP) has been widely used in recent years to treat acute uncomplicated Plasmodium falciparum malaria. Risk factors for SP therapeutic failure include young age, subtherapeutic SP concentrations, and resistance-conferring genetic mutations in parasite target enzymes. A substantial proportion of patients are able to clear genetically highly resistant P. falciparum genotypes. To determine whether blood SP concentrations independently affect the patient's ability to clear resistant genotypes, we compared SP pharmacokinetics of cases of adequate clinical and parasitological response (ACPR) with cases of treatment failure (TF). When patients with ACPR and TF were compared, mean values were similar for the day 3 blood pyrimethamine (205 ng/ml versus 172 ng/ml; P = 0.25) and estimated maximum sulfadoxine (79 +/- 6.52 versus 69 +/- 6.27 mug/ml; P = 0.60) concentrations, for sulfadoxine terminal-phase elimination half-lives (7.15 versus 6.41 days; P = 0.42), and for the extents of sulfadoxine absorption (areas under the concentration-time curve of 932 +/- 100 versus 888 +/- 78.9 mug day ml(-1); P = 0.72). Among patients infected with the quintuple resistant parasites, day 3 blood pyrimethamine concentrations were higher in those who cleared the infection than in those who did not (305 +/- 35.4 versus 228 +/- 21.7 ng/ml; P = 0.037). Within this subgroup, this finding remained significant after adjusting for endogenous folate levels, age, site, and resistance-conferring mutations (odds ratio: 1.011 [1.003 to 1.024]; P = 0.018). However, as a subgroup analysis, our biologically plausible observation that higher blood pyrimethamine concentrations enhance the ability of patients to clear resistant P. falciparum should be interpreted with caution and needs further validation.

Blood folate concentrations and in vivo sulfadoxine-pyrimethamine failure in Malawian children with uncomplicated Plasmodium falciparum malaria.

Folate antagonizes the antimalarial action of sulfadoxine-pyrimethamine (SP) in vitro, but its role in vivo is not well understood. We measured blood folate concentrations and SP therapeutic outcomes in Malawian children. Children with late treatment failure and those with adequate clinical and parasitologic responses had similar demographic characteristics, prevalence of parasite mutations conferring resistance to SP, and blood concentrations of anti-malarial drugs following treatment. However, a higher folate concentration was associated with late treatment failure. Patients from a low malaria transmission site had higher blood folate concentrations than those in a higher transmission site (mean +/- SEM = 39 +/- 9.3 ng/mL versus 29 +/- 10 ng/mL; P < 0.0001), and there was a higher rate of late treatment failure in the low transmission area (54.4% versus 40.2%; P = 0.010). This study also provides the first evidence of the independent role of physiologic folate concentrations in in vivo SP therapeutic efficacy, and the critical role of pyrimethamine concentrations in the therapeutic efficacy of SP when one controls physiologic folate levels and the frequency of critical dihydrofolate reductase/dihydropteroate synthase mutations.

Sustained clinical efficacy of sulfadoxine-pyrimethamine for uncomplicated falciparum malaria in Malawi after 10 years as first line treatment: five year prospective study.

OBJECTIVE: To measure the efficacy of sulfadoxine-pyrimethamine treatment of falciparum malaria in Malawi from 1998 to 2002, after a change from chloroquine to sulfadoxine-pyrimethamine as first line treatment in that country in 1993. DESIGN: Prospective open label drug efficacy study. SETTING: Health centre in large peri-urban township adjacent to Blantyre, Malawi. PARTICIPANTS: People presenting to a health centre with uncomplicated Plasmodium falciparum malaria. MAIN OUTCOME MEASURES: Therapeutic efficacy and parasitological resistance to standard sulfadoxine-pyrimethamine treatment at 14 days and 28 days of follow up. RESULTS: Therapeutic efficacy remained stable, with adequate clinical response rates of 80% or higher throughout the five years of the study. Analysis of follow up to 28 days showed modest but significant trends towards diminishing clinical and parasitological efficacy over time within the study period. CONCLUSION: Contrary to expectations, sulfadoxine-pyrimethamine has retained good efficacy after 10 years as the first line antimalarial drug in Malawi. African countries with very low chloroquine efficacy, high sulfadoxine-pyrimethamine efficacy, and no other immediately available alternatives may benefit from interim use of sulfadoxine-pyrimethamine while awaiting implementation of combination antimalarial treatments.

Plasmodium falciparum: PCR detection and genotyping of isolates from peripheral, placental, and cord blood of pregnant Malawian women and their infants.

Polymerase chain reaction (PCR) is both a sensitive means of detecting malaria parasitaemia, and a simple tool for identifying genetic differences in parasites infecting human subjects. We compared PCR to microscopy in detection of Plasmodium falciparum infection in peripheral, placental and cord blood samples collected from 131 pregnant Malawian women and their infants in 1997-99. Infections detected by species-specific PCR were genotyped at the merozoite surface protein 1 and 2 loci, and minimum numbers of infecting genotypes determined. PCR was of similar sensitivity to microscopy in detecting peripheral and placental infection, and placental blood PCR was 100% specific compared to placental histology. Cord blood parasitaemia was more frequently detected by PCR than microscopy, 20% versus 6%. Genotype numbers in peripheral blood (mean 2.36; range 1-5), placental blood (mean 2.41; range 1-6) and cord (mean 2.14; range 1-4) were similar. The frequency of detection of each allelic family did not differ between sites. Genotypes from different sites in each patient were compared. In 69% of women, genotypes were detected in peripheral blood and not placenta, or vice versa, suggesting possible differential sequestration of different parasite populations. Cord blood genotypes were usually a subset of those in peripheral and placental blood, but, in some cases, genotypes were found in cord blood that were absent from the mother. Transplacental infection before term, and clearance of maternal infection, is postulated.