Don P. Mathanga

Absence of Putative Artemisinin Resistance Mutations Among Plasmodium falciparum in Sub-Saharan Africa: A Molecular Epidemiologic Study

Plasmodium falciparum parasites that are resistant to artemisinins have been detected in Southeast Asia. Resistance is associated with several polymorphisms in the parasites K13-propeller gene. The molecular epidemiology of these artemisinin resistance genotypes in African parasite populations is unknown. We developed an assay to quantify rare polymorphisms in parasite populations that uses a pooled deep-sequencing approach to score allele frequencies, validated it by evaluating mixtures of laboratory parasite strains, and then used it to screen P. falciparum parasites from >1100 African infections collected since 2002 from 14 sites across sub-Saharan Africa. We found no mutations in African parasite populations that are associated with artemisinin resistance in Southeast Asian parasites. However, we observed 15 coding mutations, including 12 novel mutations, and limited allele sharing between parasite populations, consistent with a large reservoir of naturally occurring K13-propeller variation. Although polymorphisms associated with artemisinin resistance in P. falciparum in Southeast Asia are not prevalent in sub-Saharan Africa, numerous K13-propeller coding polymorphisms circulate in Africa. Although their distributions do not support a widespread selective sweep for an artemisinin-resistant phenotype, the impact of these mutations on artemisinin susceptibility is unknown and will require further characterization. Rapid, scalable molecular surveillance offers a useful adjunct in tracking and containing artemisinin resistance.

Absence of putative Plasmodium falciparum artemisinin resistance mutations in sub-Saharan Africa: A molecular epidemiologic study

Plasmodium falciparum parasites that are resistant to artemisinins have been detected in Southeast Asia. Resistance is associated with several polymorphisms in the parasites K13-propeller gene. The molecular epidemiology of these artemisinin resistance genotypes in African parasite populations is unknown. We developed an assay to quantify rare polymorphisms in parasite populations that uses a pooled deep-sequencing approach to score allele frequencies, validated it by evaluating mixtures of laboratory parasite strains, and then used it to screen P. falciparum parasites from >1100 African infections collected since 2002 from 14 sites across sub-Saharan Africa. We found no mutations in African parasite populations that are associated with artemisinin resistance in Southeast Asian parasites. However, we observed 15 coding mutations, including 12 novel mutations, and limited allele sharing between parasite populations, consistent with a large reservoir of naturally occurring K13-propeller variation. Although polymorphisms associated with artemisinin resistance in P. falciparum in Southeast Asia are not prevalent in sub-Saharan Africa, numerous K13-propeller coding polymorphisms circulate in Africa. Although their distributions do not support a widespread selective sweep for an artemisinin-resistant phenotype, the impact of these mutations on artemisinin susceptibility is unknown and will require further characterization. Rapid, scalable molecular surveillance offers a useful adjunct in tracking and containing artemisinin resistance.

Comparative field performance and adherence to test results of four malaria rapid diagnostic tests among febrile patients more than five years of age in Blantyre, Malawi

BackgroundMalaria rapid diagnostics tests (RDTs) can increase availability of laboratory-based diagnosis and improve the overall management of febrile patients in malaria endemic areas. In preparation to scale-up RDTs in health facilities in Malawi, an evaluation of four RDTs to help guide national-level decision-making was conducted.MethodsA cross sectional study of four histidine rich-protein-type-2- (HRP2) based RDTs at four health centres in Blantyre, Malawi, was undertaken to evaluate the sensitivity and specificity of RDTs, assess prescriber adherence to RDT test results and explore operational issues regarding RDT implementation. Three RDTs were evaluated in only one health centre each and one RDT was evaluated in two health centres. Light microscopy in a reference laboratory was used as the gold standard.ResultsA total of 2,576 patients were included in the analysis. All of the RDTs tested had relatively high sensitivity for detecting any parasitaemia [Bioline SD (97%), First response malaria (92%), Paracheck (91%), ICT diagnostics (90%)], but low specificity [Bioline SD (39%), First response malaria (42%), Paracheck (68%), ICT diagnostics (54%)]. Specificity was significantly lower in patients who self-treated with an anti-malarial in the previous two weeks (odds ratio (OR) 0.5; p-value < 0.001), patients 5-15 years old versus patients > 15 years old (OR 0.4, p-value < 0.001) and when the RDT was performed by a community health worker versus a laboratory technician (OR 0.4; p-value < 0.001). Health workers correctly prescribed anti-malarials for patients with positive RDT results, but ignored negative RDT results with 58% of patients with a negative RDT result treated with an anti-malarial.ConclusionsThe results of this evaluation, combined with other published data and global recommendations, have been used to select RDTs for national scale-up. In addition, the study identified some key issues that need to be further delineated: the low field specificity of RDTs, variable RDT performance by different cadres of health workers and the need for a robust quality assurance system. Close monitoring of RDT scale-up will be needed to ensure that RDTs truly improve malaria case management.

Socio-cultural predictors of health-seeking behaviour for febrile under-five children in Mwanza-Neno district, Malawi

BackgroundPrompt access to effective treatment for malaria is unacceptably low in Malawi. Less than 20% of children under the age of five with fever receive appropriate anti-malarial treatment within 24 hours of fever onset. This study assessed socio-cultural factors associated with delayed treatment of children with fever in Mwanza district, Malawi.MethodologyIt was a qualitative study using focus group discussions and key informant interviews.ResultsA total of 151 caregivers and 46 health workers participated in the focus group discussions. The majority of caregivers were able to recognize fever and link it to malaria. Despite high knowledge of malaria, prompt treatment and health-seeking behaviour were poor, with the majority of children first being managed at home with treatment regimens other than effective anti-malarials. Traditional beliefs about causes of fever, unavailability of anti-malarial drugs within the community, barriers to accessing the formal health care system, and trust in traditional medicine were all associated with delays in seeking appropriate treatment for fever.ConclusionThe study has demonstrated important social cultural factors that negatively influence for caregivers of children under five. To facilitate prompt and appropriate health-seeking behaviour, behavioral change messages must address the prevailing local beliefs about causes of fever and the socio-economic barriers to accessing health care.

Lack of decline in childhood malaria, Malawi, 2001-2010.

Despite increased control activities, malaria did not substantially decline.

Adherence to Treatment With Artemether-Lumefantrine for Uncomplicated Malaria in Rural Malawi

BACKGROUND In 2007, Malawi replaced the first-line medication for uncomplicated malaria, sulfadoxine-pyrimethamine-a single-dose regimen-with artemether-lumefantrine (AL)-a 6-dose, 3-day regimen. Because of concerns about the complex dosing schedule, we assessed patient adherence to AL 2 years after routine implementation. METHODS Adults and children with uncomplicated malaria were recruited at 3 health centers. We conducted both pill counts and in-home interviews on medication consumption 72 hours after patients received AL. Complete adherence was defined as correctly taking all 6 AL doses, as assessed by pill count and dose recall. We used logistic regression to identify factors associated with complete adherence. RESULTS Of 386 patients, 65% were completely adherent. Patients were significantly more likely to be completely adherent if they received their first dose of AL as directly observed therapy at the health center (odds ratio [OR], 2.4; P < .01), received instructions using the medication package as a visual aid (OR, 2.5; P = .02), and preferred AL over other antimalarials (OR, 2.7; P < .001). In contrast, children <5 years of age were significantly less likely to be adherent (OR, 0.5; P = .05). CONCLUSIONS Adherence to AL treatment for uncomplicated malaria was moderate, and children, who are the most likely to die of malaria, were less adherent than adults. Efforts to improve adherence should be focused on this vulnerable group. Interventions including the introduction of child-friendly antimalarial formulations, direct observation of the first dose, use of the AL package as a visual aid for instructions, and enhancing patient preference for AL could potentially increase AL adherence and overall effectiveness.

School-Age Children Are a Reservoir of Malaria Infection in Malawi

Malaria surveillance and interventions in endemic countries often target young children at highest risk of malaria morbidity and mortality. We aimed to determine whether school-age children and adults not captured in surveillance serve as a reservoir for malaria infection and may contribute to malaria transmission. Cross-sectional surveys were conducted in one rainy and one dry season in southern Malawi. Demographic and health information was collected for all household members. Blood samples were obtained for microscopic and PCR identification of Plasmodium falciparum. Among 5796 individuals aged greater than six months, PCR prevalence of malaria infection was 5%, 10%, and 20% in dry, and 9%, 15%, and 32% in rainy seasons in Blantyre, Thyolo, and Chikhwawa, respectively. Over 88% of those infected were asymptomatic. Participants aged 6–15 years were at higher risk of infection (OR=4.8; 95%CI, 4.0–5.8) and asymptomatic infection (OR=4.2; 95%CI, 2.7–6.6) than younger children in all settings. School-age children used bednets less frequently than other age groups. Compared to young children, school-age children were brought less often for treatment and more often to unreliable treatment sources. Conclusion: School-age children represent an underappreciated reservoir of malaria infection and have less exposure to antimalarial interventions. Malaria control and elimination strategies may need to expand to include this age group.

Effectiveness of Intermittent Preventive Treatment With Sulfadoxine-Pyrimethamine During Pregnancy on Maternal and Birth Outcomes in Machinga District, Malawi

BACKGROUND Malaria during pregnancy is associated with low birth weight and increased perinatal mortality, especially among primigravidae. Despite increasing prevalence of malarial parasite resistance to sulfadoxine-pyrimethamine (SP), SP continues to be recommended for intermittent preventive treatment in pregnancy (IPTp). METHODS Women without human immunodeficiency virus infection were enrolled upon delivery. Data on the number of SP doses received during pregnancy were recorded. The primary outcome was placental infection demonstrated by histologic analysis. Secondary outcomes included malaria parasitemia (in peripheral, placental, cord blood specimens) at delivery and composite birth outcome (small for gestational age, preterm delivery, or low birth weight). RESULTS.: Of 703 women enrolled, 22% received <2 SP doses. Receipt of ≥ 2 SP doses had no impact on histologically confirmed placental infection. IPTp-SP was associated with a dose-dependent protective effect on composite birth outcome in primigravidae, with an adjusted prevalence ratio of 0.50 (95% confidence interval [CI], .30-.82), 0.30 (95% CI, .19-.48), and 0.18 (95% CI, .05-.61) for 1, 2, and ≥ 3 doses, respectively, compared with 0 doses. CONCLUSIONS IPTp-SP did not reduce the frequency of placental infection but was associated with improved birth outcomes. Few women received no SP, so the true effect of IPTp-SP may be underestimated. Malawian pregnant women should continue to receive IPTp-SP, but alternative strategies and antimalarials for preventing malaria during pregnancy should be investigated.

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.

Impact of Sulfadoxine-Pyrimethamine Resistance on Effectiveness of Intermittent Preventive Therapy for Malaria in Pregnancy at Clearing Infections and Preventing Low Birth Weight

BACKGROUND Owing to increasing sulfadoxine-pyrimethamine (SP) resistance in sub-Saharan Africa, monitoring the effectiveness of intermittent preventive therapy in pregnancy (IPTp) with SP is crucial. METHODS Between 2009 and 2013, both the efficacy of IPTp-SP at clearing existing peripheral malaria infections and the effectiveness of IPTp-SP at reducing low birth weight (LBW) were assessed among human immunodeficiency virus-uninfected participants in 8 sites in 6 countries. Sites were classified as high, medium, or low resistance after measuring parasite mutations conferring SP resistance. An individual-level prospective pooled analysis was conducted. RESULTS Among 1222 parasitemic pregnant women, overall polymerase chain reaction-uncorrected and -corrected failure rates by day 42 were 21.3% and 10.0%, respectively (39.7% and 21.1% in high-resistance areas; 4.9% and 1.1% in low-resistance areas). Median time to recurrence decreased with increasing prevalence of Pfdhps-K540E. Among 6099 women at delivery, IPTp-SP was associated with a 22% reduction in the risk of LBW (prevalence ratio [PR], 0.78; 95% confidence interval [CI], .69-.88; P < .001). This association was not modified by insecticide-treated net use or gravidity, and remained significant in areas with high SP resistance (PR, 0.81; 95% CI, .67-.97; P = .02). CONCLUSIONS The efficacy of SP to clear peripheral parasites and prevent new infections during pregnancy is compromised in areas with >90% prevalence of Pfdhps-K540E. Nevertheless, in these high-resistance areas, IPTp-SP use remains associated with increases in birth weight and maternal hemoglobin. The effectiveness of IPTp in eastern and southern Africa is threatened by further increases in SP resistance and reinforces the need to evaluate alternative drugs and strategies for the control of malaria in pregnancy.