Recent uptake of intermittent preventive treatment during pregnancy with sulfadoxine-pyrimethamine is associated with increased prevalence of Pfdhfr mutations in Bobo-Dioulasso, Burkina Faso.

BACKGROUND: The impact of sulfadoxine-pyrimethamine (SP) used as intermittent preventive treatment during pregnancy (IPTp-SP) on mutant parasite selection has been poorly documented in Burkina Faso. This study sought first to explore the relationship between IPTp-SP and the presence of mutant parasites. Second, to assess the relationship between the mutant parasites and adverse pregnancy outcomes. METHODS: From September to December 2010, dried blood spots (DBS) were collected during antenatal care visits and at delivery from 109 pregnant women with microscopically confirmed falciparum malaria infection. DBS were analysed by PCR-restriction fragment length polymorphism (PCR-RFLP) for the polymorphisms at codons 51, 59, 108, and 164 of the Pfdhfr gene and codons 437 and 540 in the Pfdhps gene. RESULTS: Both the Pfdhfr and Pfdhps genes were successfully genotyped in 92.7% (101/109) of the samples. The prevalence of Pfdhfr mutations N51I, C59R and S108N was 71.3, 42.6 and 64.4%, respectively. Overall, 80.2% (81/101) of samples carried the Pfdhps A437G mutation. None of the samples had the Pfdhfr I164L and the Pfdhps K540E mutations. The prevalence of the triple mutation N51I + C59R + S108N was 25.7% (26/101). The use of IPTp-SP was associated with a threefold increased odds of Pfdhfr C59R mutation [crude OR 3.29; 95% CI (1.44-7.50)]. Pregnant women with recent uptake of IPTp-SP were at higher odds of both the Pfdhfr C59R mutation [adjusted OR 4.26; 95% CI (1.64-11.07)] and the Pfdhfr intermediate-to-high resistance, i.e., ≥ 2 Pfdhfr mutations [adjusted OR 3.45; 95% CI (1.18-10.07)]. There was no statistically significant association between the presence of the Pfdhfr intermediate-to-high resistance and parasite densities or both maternal haemoglobin level and anaemia. CONCLUSION: The data indicate that despite the possibility that IPTp-SP contributes to the selection of resistant parasites, it did not potentiate pregnancy-associated malaria morbidity, suggesting the continuation of SP use as IPTp in Burkina Faso.

Non-falciparum malaria infections in pregnant women in West Africa.

BACKGROUND: Non-Plasmodium falciparum malaria infections are found in many parts of sub-Saharan Africa but little is known about their importance in pregnancy. METHODS: Blood samples were collected at first antenatal clinic attendance from 2526 women enrolled in a trial of intermittent screening and treatment of malaria in pregnancy (ISTp) versus intermittent preventive treatment (IPTp) conducted in Burkina Faso, The Gambia, Ghana and Mali. DNA was extracted from blood spots and tested for P. falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale using a nested PCR test. Risk factors for a non-falciparum malaria infection were investigated and the influence of these infections on the outcome of pregnancy was determined. RESULTS: P. falciparum infection was detected frequently (overall prevalence by PCR: 38.8 %, [95 % CI 37.0, 40.8]), with a prevalence ranging from 10.8 % in The Gambia to 56.1 % in Ghana. Non-falciparum malaria infections were found only rarely (overall prevalence 1.39 % [95 % CI 1.00, 1.92]), ranging from 0.17 % in the Gambia to 3.81 % in Mali. Ten non-falciparum mono-infections and 25 mixed falciparum and non-falciparum infections were found. P. malariae was the most frequent non-falciparum infection identified; P. vivax was detected only in Mali. Only four of the non-falciparum mono-infections were detected by microscopy or rapid diagnostic test. Recruitment during the late rainy season and low socio-economic status were associated with an increased risk of non-falciparum malaria as well as falciparum malaria. The outcome of pregnancy did not differ between women with a non-falciparum malaria infection and those who were not infected with malaria at first ANC attendance. CONCLUSIONS: Non-falciparum infections were infrequent in the populations studied, rarely detected when present as a mono-infection and unlikely to have had an important impact on the outcome of pregnancy in the communities studied due to the small number of women infected with non-falciparum parasites.

Parasite clearance following treatment with sulphadoxine-pyrimethamine for intermittent preventive treatment in Burkina-Faso and Mali: 42-day in vivo follow-up study.

BACKGROUND: Intermittent Preventive Treatment in pregnancy (IPTp) with sulphadoxine-pyrimethamine (SP) is widely used for the control of malaria in pregnancy in Africa. The emergence of resistance to SP is a concern requiring monitoring the effectiveness of SP for IPTp. METHODS: This was an in-vivo efficacy study to determine the parasitological treatment response and the duration of post-treatment prophylaxis among asymptomatic pregnant women receiving SP as part of IPTp in Mali and Burkina-Faso. The primary outcome was the PCR-unadjusted % of patients with parasites recurrence by day 42 defined as a positive diagnostic test by malaria smear at any visit between days 4 and 42. Treatment failure was based on the standard World Health Organization criteria. The therapeutic response was estimated using the Kaplan-Meier curve. RESULTS: A total of 580 women were enrolled in Mali (N=268) and Burkina-Faso (N=312) and followed weekly for 42 days. Among these, 94.3% completed the follow-up. The PCR-unadjusted cumulative risk of recurrence by day 42 was 4.9% overall, and 3.2% and 6.5% in Mali and Burkina Faso respectively (Hazard Ratio [HR] =2.14, 95%, CI [0.93-4.90]; P=0.070), and higher among the primi- and secundigravida (6.4%) than multigravida (2.2%, HR=3.01 [1.04-8.69]; P=0.042). The PCR-adjusted failure risk was 1.1% overall (Mali 0.8%, Burkina-Faso 1.4%). The frequencies (95% CI) of the dhfr double and triple mutant and dhps 437 and 540 alleles mutant genotype at enrolment were 24.2% (23.7-25.0), 4.7% (4.4-5.0), and 21.4% (20.8-22.0) and 0.37% (0.29-0.44) in Mali, and 7.1% (6.5-7.7), 44.9% (43.8-46.0) and 75.3% (74.5-76.2) and 0% in Burkina-Faso, respectively. There were no dhfr 164L or dhps 581G mutations. CONCLUSION: SP remains effective at clearing existing infections when provided as IPTp to asymptomatic pregnant women in Mali and Burkina. Continued monitoring of IPTp-SP effectiveness, including of the impact on birth parameters in this region is essential.

Comparative Susceptibility of Plasmodium ovale and Plasmodium falciparum Field Isolates to Reference and Lead Candidate Antimalarial Drugs in Ghana.

Malaria treatments resulted in the decline of the deadliest Plasmodium falciparum globally while species, such as P. ovale, infections have been increasingly detected across sub-Saharan Africa. Currently, no experimental drug sensitivity data are available to guide effective treatment and management of P. ovale infections, which is necessary for effective malaria elimination. We conducted a prospective study to evaluate P. ovale epidemiology over 1 year and determined ex vivo susceptibility of the field isolates to existing and lead advanced discovery antimalarial drugs. We report that while P. falciparum dominated both symptomatic and asymptomatic malaria cases, P. ovale in mono or co-infections caused 7.16% of symptomatic malaria. Frontline antimalarials artesunate and lumefantrine inhibited P. ovale as potently as P. falciparum. Chloroquine, which has been withdrawn in Ghana, was also highly inhibitory against both P. ovale and P. falciparum. In addition, P. ovale and P. falciparum displayed high susceptibility to quinine, comparable to levels observed with chloroquine. Pyrimethamine, which is a major drug for disease massive prevention, also showed great inhibition of P. ovale, comparable to effects on P. falciparum. Furthermore, we identified strong inhibition of P. ovale using GNF179, a close analogue of KAF156 imidazolopiperazines, which is a novel class of antimalarial drugs currently in clinical phase II testing. We further demonstrated that the Plasmodium phosphatidylinositol-4-OH kinase (PI4K)-specific inhibitor, KDU691, is highly inhibitory against P. ovale and P. falciparum field isolates. Our data indicated that existing and lead advanced discovery antimalarial drugs are suitable for the treatment of P. ovale infections in Ghana. IMPORTANCE Current malaria control and elimination tools such as drug treatments are not specifically targeting P.ovale. P. ovale can form hypnozoite and cause relapsing malaria. P. ovale is the third most dominant species in Africa and requires radical cure treatment given that it can form liver dormant forms called hypnozoites that escape all safe treatments. The inappropriate treatment of P. ovale would sustain its transmission in Africa where the medical need is the greatest. This is a hurdle for successful malaria control and elimination. Here, we provided experiment data that were lacking to guide P. ovale treatment and disease control policy makers using reference antimalarial drugs. We also provided key experimental data for 2 clinical candidate drugs that can be used for prioritization selection of lead candidate's identification for clinical development.

Development of Cooperative Primer-Based Real-Time PCR Assays for the Detection of Plasmodium malariae and Plasmodium ovale.

Plasmodium malariae and Plasmodium ovale are increasingly gaining public health attention as the global transmission of falciparum malaria is decreasing. However, the absence of reliable Plasmodium species-specific detection tools has hampered accurate diagnosis of these minor Plasmodium species. In this study, SYBR Green-based real-time PCR assays were developed for the detection of P. malariae and P. ovale using cooperative primers that significantly limit the formation and propagation of primers-dimers. Both the P. malariae and P. ovale cooperative primer-based assays had at least 10-fold lower detection limit compared with the corresponding conventional primer-based assays. More important, the cooperative primer-based assays were evaluated in a cross-sectional study using 560 samples obtained from two health facilities in Ghana. The prevalence rates of P. malariae and P. ovale among the combined study population were 18.6% (104/560) and 5.5% (31/560), respectively. Among the Plasmodium-positive cases, P. malariae and P. ovale mono-infections were 3.6% (18/499) and 1.0% (5/499), respectively, with the remaining being co-infections with Plasmodium falciparum. The study demonstrates the public health importance of including detection tools with lower detection limits in routine diagnosis and surveillance of nonfalciparum species. This will be necessary for comprehensively assessing the effectiveness of malaria interventions and control measures aimed toward global malaria elimination.

A Non-Inferiority, Individually Randomized Trial of Intermittent Screening and Treatment versus Intermittent Preventive Treatment in the Control of Malaria in Pregnancy.

BACKGROUND: The efficacy of intermittent preventive treatment for malaria with sulfadoxine-pyrimethamine (IPTp-SP) in pregnancy is threatened in parts of Africa by the emergence and spread of resistance to SP. Intermittent screening with a rapid diagnostic test (RDT) and treatment of positive women (ISTp) is an alternative approach. METHODS AND FINDINGS: An open, individually randomized, non-inferiority trial of IPTp-SP versus ISTp was conducted in 5,354 primi- or secundigravidae in four West African countries with a low prevalence of resistance to SP (The Gambia, Mali, Burkina Faso and Ghana). Women in the IPTp-SP group received SP on two or three occasions whilst women in the ISTp group were screened two or three times with a RDT and treated if positive for malaria with artemether-lumefantrine (AL). ISTp-AL was non-inferior to IPTp-SP in preventing low birth weight (LBW), anemia and placental malaria, the primary trial endpoints. The prevalence of LBW was 15.1% and 15.6% in the IPTp-SP and ISTp-AL groups respectively (OR = 1.03 [95% CI: 0.88, 1.22]). The mean hemoglobin concentration at the last clinic attendance before delivery was 10.97g/dL and 10.94g/dL in the IPTp-SP and ISTp-AL groups respectively (mean difference: -0.03 g/dL [95% CI: -0.13, +0.06]). Active malaria infection of the placenta was found in 24.5% and in 24.2% of women in the IPTp-SP and ISTp-AL groups respectively (OR = 0.95 [95% CI 0.81, 1.12]). More women in the ISTp-AL than in the IPTp-SP group presented with malaria parasitemia between routine antenatal clinics (310 vs 182 episodes, rate difference: 49.4 per 1,000 pregnancies [95% CI 30.5, 68.3], but the number of hospital admissions for malaria was similar in the two groups. CONCLUSIONS: Despite low levels of resistance to SP in the study areas, ISTp-AL performed as well as IPTp-SP. In the absence of an effective alternative medication to SP for IPTp, ISTp-AL is a potential alternative to IPTp in areas where SP resistance is high. It may also have a role in areas where malaria transmission is low and for the prevention of malaria in HIV positive women receiving cotrimoxazole prophylaxis in whom SP is contraindicated. TRIAL REGISTRATION: ClinicalTrials.gov NCT01084213 Pan African Clinical trials Registry PACT201202000272122.