• Artesunate + amodiaquine and artesunate + sulphadoxine-pyrimethamine for treatment of uncomplicated malaria in Democratic Republic of Congo: a clinical trial with determination of sulphadoxine and pyrimethamine-resistant haplotypes.

      Swarthout, T D; van den Broek, I; Kayembe, G; Montgomery, J; Pota, H; Roper, C; Médecins Sans Frontières, London, UK. (Wiley-Blackwell, 2006-10)
      We undertook a trial of artesunate + amodiaquine (AS + AQ) and artesunate + sulphadoxine-pyrimethamine (AS + SP) in 180 children of age 6-59 months with uncomplicated malaria in Democratic Republic of Congo. Children were randomly allocated to receive 3 days observed treatment of AS + AQ (n = 90) or 3 days of AS + SP (n = 90). Primary efficacy outcomes were 28-day parasite recurrence rates, and recrudescence rates were adjusted by genotyping to distinguish new infection and recrudescence. In addition, we determined the prevalence of molecular markers of resistance to sulphadoxine and pyrimethamine. Day 28 parasite recurrence rates were 16.9% (14/83; 95% CI: 9.5-26.7) in the AS + AQ group and 34.6% (28/81; 95% CI: 24.3-46.0) in the AS + SP group (P = 0.009). After PCR correction, recrudescence rates were 6.7% (5/74; 95% CI: 2.2-15.1) for AS + AQ and 19.7% (13/66; 95% CI: 10.9-31.3) for AS + SP (P = 0.02). There was no significant difference between the two arms in time to parasite clearance, fever clearance and gametocyte clearance. Parasite genotyping showed high frequencies of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) molecular SP-resistance markers, with 57% of the samples showing more than three mutations linked to SP resistance, and 27% with triple-dhfr/double-dhps haplotype, confirming that SP treatment failure rates are likely to be high. AS + AQ had significantly higher efficacy than AS + SP. These results contributed to the subsequent change to AS + AQ as first-line regimen in the country. Efforts to properly implement the new protocol and maintain adherence at acceptable levels should include health staff and patient sensitization. The 6.8% recrudescence rate indicates that AS + AQ should be monitored closely until a more effective artemisinin combination therapy regimen is needed and can be introduced.
    • Dihydroartemisinin-piperaquine and artemether-lumefantrine for treating uncomplicated malaria in African children: a randomised, non-inferiority trial

      Bassat, Quique; Mulenga, Modest; Tinto, Halidou; Piola, Patrice; Borrmann, Steffen; Menéndez, Clara; Nambozi, Michael; Valéa, Innocent; Nabasumba, Carolyn; Sasi, Philip; Bacchieri, Antonella; Corsi, Marco; Ubben, David; Talisuna, Ambrose; D'Alessandro, Umberto; Barcelona Centre for International Health Research (CRESIB), Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Universitat de Barcelona, Barcelona, Spain; Manhiça Health Research Centre (CISM), Manhiça, Mozambique; Tropical Disease Research Centre, Ndola, Zambia; Centre Muraz, Bobo-Dioulasso, Burkina Faso, IRSS/DRO, Bobo-Dioulasso, Burkina Faso; Epicentre/MSF, Mbarara, Uganda; Kenya Medical Research Institute, Kilifi, Kenya; University of Heidelberg, Heidelberg, Germany; Department of Clinical Pharmacology, Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania; Sigma Tau Industrie Farmaceutiche Riunite, Pomezia, Rome, Italy; Medicines for Malaria Venture, Geneva, Switzerland; Prince Leopold Institute of Tropical Medicine, Antwerp, Belgium (2009-11-17)
      BACKGROUND: Artemisinin combination therapies (ACTs) are currently the preferred option for treating uncomplicated malaria. Dihydroartemisinin-piperaquine (DHA-PQP) is a promising fixed-dose ACT with limited information on its safety and efficacy in African children. METHODOLOGY/PRINCIPAL FINDINGS: The non-inferiority of DHA-PQP versus artemether-lumefantrine (AL) in children 6-59 months old with uncomplicated P. falciparum malaria was tested in five African countries (Burkina Faso, Kenya, Mozambique, Uganda and Zambia). Patients were randomised (2:1) to receive either DHA-PQP or AL. Non-inferiority was assessed using a margin of -5% for the lower limit of the one-sided 97.5% confidence interval on the treatment difference (DHA-PQP vs. AL) of the day 28 polymerase chain reaction (PCR) corrected cure rate. Efficacy analysis was performed in several populations, and two of them are presented here: intention-to-treat (ITT) and enlarged per-protocol (ePP). 1553 children were randomised, 1039 receiving DHA-PQP and 514 AL. The PCR-corrected day 28 cure rate was 90.4% (ITT) and 94.7% (ePP) in the DHA-PQP group, and 90.0% (ITT) and 95.3% (ePP) in the AL group. The lower limits of the one-sided 97.5% CI of the difference between the two treatments were -2.80% and -2.96%, in the ITT and ePP populations, respectively. In the ITT population, the Kaplan-Meier estimate of the proportion of new infections up to Day 42 was 13.55% (95% CI: 11.35%-15.76%) for DHA-PQP vs 24.00% (95% CI: 20.11%-27.88%) for AL (p<0.0001). CONCLUSIONS/SIGNIFICANCE: DHA-PQP is as efficacious as AL in treating uncomplicated malaria in African children from different endemicity settings, and shows a comparable safety profile. The occurrence of new infections within the 42-day follow up was significantly lower in the DHA-PQP group, indicating a longer post-treatment prophylactic effect. TRIAL REGISTRATION: Controlled-trials.com ISRCTN16263443.
    • Drug resistance in Plasmodium falciparum from the Chittagong Hill Tracts, Bangladesh.

      van den Broek, I; van der Wardt, S; Talukder, L; Chakma, S; Brockman, A; Nair, S; Anderson, T C; Médecins sans Frontières-Holland, Gulshan, Dhaka, Bangladesh. ingrid.van.den.braek@london.msf.org (Wiley-Blackwell, 2004-06)
      OBJECTIVE: To assess the efficacy of antimalarial treatment and molecular markers of Plasmodium falciparum resistance in the Chittagong Hill Tracts of Bangladesh. METHODS: A total of 203 patients infected with P. falciparum were treated with quinine 3 days plus sulphadoxine/pyrimethamine (SP) combination therapy, and followed up during a 4-week period. Blood samples collected before treatment were genotyped for parasite mutations related to chloroquine (pfcrt and pfmdr1 genes) or SP resistance (dhfr and dhps). RESULTS: Of 186 patients who completed follow-up, 32 patients (17.2%) failed to clear parasitaemia or became positive again within 28 days after treatment. Recurring parasitaemia was related to age (chi(2) = 4.8, P < 0.05) and parasite rates on admission (t = 3.1, P < 0.01). PCR analysis showed that some of these cases were novel infections. The adjusted recrudescence rate was 12.9% (95% CI 8.1-17.7) overall, and 16.6% (95% CI 3.5-29.7), 15.5% (95% CI 8.3-22.7) and 6.9% (95% CI 0.4-13.4) in three age groups (<5 years, 5-14, > or =15). The majority of infections carried mutations associated with chloroquine resistance: 94% at pfcrt and 70% at pfmdr. Sp-resistant genotypes were also frequent: 99% and 73% of parasites carried two or more mutations at dhfr and dhps, respectively. The frequency of alleles at dhfr, dhps and pfmdr was similar in cases that were successfully treated and those that recrudesced. CONCLUSIONS: The clinical trial showed that quinine 3-days combined to SP is still relatively effective in the Chittagong Hill Tracts. However, if this regimen is continued to be widely used, further development of SP resistance and reduced quinine sensitivity are to be expected. The genotyping results suggest that neither chloroquine nor SP can be considered a reliable treatment for P. falciparum malaria any longer in this area of Bangladesh.
    • Evaluation of three parasite lactate dehydrogenase-based rapid diagnostic tests for the diagnosis of falciparum and vivax malaria

      Ashley, Elizabeth A; Touabi, Malek; Ahrer, Margareta; Hutagalung, Robert; Htun, Khayae; Luchavez, Jennifer; Dureza, Christine; Proux, Stephane; Leimanis, Mara; Lwin, Myo Min; Koscalova, Alena; Comte, Eric; Hamade, Prudence; Page, Anne-Laure; Nosten, François; Guerin, Philippe J; Epicentre, Paris, France; Department of Microbiology, Hammersmith Hospital, Imperial College NHS Trust, London, UK; Médecins sans Frontières, Switzerland; Médecins sans Frontières, Myanmar; Research Institute for Tropical Medicine, Alabang, Muntinlupa City, Philippines; Shoklo Malaria Research Unit, Mae Sot, Thailand; Mahidol-Oxford Tropical Medicine Research Unit (MORU), Mahidol University, Bangkok, Thailand; Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, UK; Médecins sans Frontières Malaria Working Group, UK; Malaria Consortium, London, UK (2009-10-27)
      BACKGROUND: In areas where non-falciparum malaria is common rapid diagnostic tests (RDTs) capable of distinguishing malaria species reliably are needed. Such tests are often based on the detection of parasite lactate dehydrogenase (pLDH). METHODS: In Dawei, southern Myanmar, three pLDH based RDTs (CareStart Malaria pLDH (Pan), CareStart Malaria pLDH (Pan, Pf) and OptiMAL-IT)were evaluated in patients presenting with clinically suspected malaria. Each RDT was read independently by two readers. A subset of patients with microscopically confirmed malaria had their RDTs repeated on days 2, 7 and then weekly until negative. At the end of the study, samples of study batches were sent for heat stability testing. RESULTS: Between August and November 2007, 1004 patients aged between 1 and 93 years were enrolled in the study. Slide microscopy (the reference standard) diagnosed 213 Plasmodium vivax (Pv) monoinfections, 98 Plasmodium falciparum (Pf) mono-infections and no malaria in 650 cases. The sensitivities (sens) and specificities (spec), of the RDTs for the detection of malaria were- CareStart Malaria pLDH (Pan) test: sens 89.1% [CI95 84.2-92.6], spec 97.6% [CI95 96.5-98.4]. OptiMal-IT: Pf+/- other species detection: sens 95.2% [CI95 87.5-98.2], spec 94.7% [CI95 93.3-95.8]; non-Pf detection alone: sens 89.6% [CI95 83.6-93.6], spec 96.5% [CI95 94.8-97.7]. CareStart Malaria pLDH (Pan, Pf): Pf+/- other species: sens 93.5% [CI95 85.4-97.3], spec 97.4% [95.9-98.3]; non-Pf: sens 78.5% [CI95 71.1-84.4], spec 97.8% [CI95 96.3-98.7]. Inter-observer agreement was excellent for all tests (kappa > 0.9). The median time for the RDTs to become negative was two days for the CareStart Malaria tests and seven days for OptiMAL-IT. Tests were heat stable up to 90 days except for OptiMAL-IT (Pf specific pLDH stable to day 20 at 35 degrees C). CONCLUSION: None of the pLDH-based RDTs evaluated was able to detect non-falciparum malaria with high sensitivity, particularly at low parasitaemias. OptiMAL-IT performed best overall and would perform best in an area of high malaria prevalence among screened fever cases. However, heat stability was unacceptable and the number of steps to perform this test is a significant drawback in the field. A reliable, heat-stable, highly sensitive RDT, capable of diagnosing all Plasmodium species has yet to be identified.
    • Low efficacy of the combination artesunate plus amodiaquine for uncomplicated falciparum malaria among children under 5 years in Kailahun, Sierra Leone.

      Grandesso, F; Hagerman, A; Kamara, S; Lam, E; Checchi, F; Balkan, S; Scollo, G; Durand, R; Guthmann, J P; Epicentre, Paris, France. (Wiley-Blackwell, 2006-07)
      OBJECTIVE: In 2004, Sierra Leone adopted artesunate plus amodiaquine as first-line antimalarial treatment. We evaluated the efficacy of this combination in Kailahun, where a previous study had shown 70.2% efficacy of amodiaquine in monotherapy. METHODS: Method and outcome classification of the study complied with WHO guidelines. Children 6-59 months with uncomplicated malaria were followed-up for 28 days. PCR genotyping was used to distinguish recrudescence from reinfection. Reinfections were reclassified as cured. RESULTS: Of 172 children who were referred to the study clinic, 126 satisfied inclusion criteria and were enrolled. No early treatment failures were reported. The day 14, efficacy was 98.2% (95% CI: 93.8-99.8). Of 65 recurrent parasitaemias analysed by PCR, 17 were recrudescences. The PCR-adjusted day 28 efficacy was 84.5% (95% CI: 76.4-90.7). All true failures occurred in the last 8 days of follow-up. Of 110 children who completed the 28-day follow-up, 54 (49.1%) experienced a novel infection. CONCLUSION: The efficacy of this combination was disappointing. The high reinfection rate suggested little prophylactic effect. In Kailahun a more efficacious combination might be necessary in the future. The efficacy of AS + AQ needs to be monitored in Kailahun and in the other regions of Sierra Leone.
    • Molecular genotyping in a malaria treatment trial in Uganda - unexpected high rate of new infections within 2 weeks after treatment.

      Mugittu, K; Priotto, G; Guthmann, J P; Kiguli, J; Adjuik, M; Snounou, G; Beck, H P; Mshinda, H; Olliaro, P; Taylor, W R J; Ifakara Health Research and Development Centre, Ifakara, Tanzania. (2007-02)
      Polymerase chain reaction (PCR) genotyping of malaria parasites in drug efficacy trials helps differentiate reinfections from recrudescences. A combination therapy trial of one (n = 115) or three (n = 117) days artesunate (1AS, 3AS 4 mg/kg/day) plus sulphadoxine-pyrimethamine (SP) vs. SP alone (n = 153) was conducted in Mbarara, a mesoendemic area of western Uganda. All paired recurrent Plasmodium falciparum parasitaemias on days 7, 14, 21 and 28 post-treatment were genotyped by PCR amplification and analysis of glutamate-rich protein (glurp) and merozoite surface proteins (msp) 1 and 2 genes to distinguish recrudescent from new infections. A total of 156 (1AS = 61, 3AS = 35, SP alone = 60) of 199 paired recurrent samples were successfully analysed and were resolved as 79 recrudescences (1AS = 32, 3AS = 8, SP = 39) and 77 as new infections (1AS = 29, 3AS = 27, SP = 21). The ratios of proportions of new to recrudescent infections were 0.2, 0.9, 1.4 and 1.9 on days 7, 14, 21 and 28, respectively (P < 0.001, chi(2) test for linear trend). Unexpected high new infection rates were observed early in follow-up on days 7 [5/26 (19.2%)] and 14 [24/51 (47.1%)]. These results impact significantly on resistance monitoring and point to the value of genotyping all recurrent infections in antimalarial trials.
    • Real-time PCR/MCA assay using fluorescence resonance energy transfer for the genotyping of resistance related DHPS-540 mutations in Plasmodium falciparum

      Mens, Petra F; van Overmeir, Chantal; Bonnet, Maryline; Dujardin, Jean-Claude; d'Alessandro, Umberto; Prince Leopold Institute of Tropical Medicine, Department of Parasitology, Antwerp, Belgium; Koninklijk Instituut voor de Tropen/Royal Tropical Institute, KIT Biomedical Research, Amsterdam, The Netherlands; Epicentre, Paris, France (2008-03-17)
      BACKGROUND: Sulphadoxine-pyrimethamine has been abandoned as first- or second-line treatment by most African malaria endemic countries in favour of artemisinin-based combination treatments, but the drug is still used as intermittent preventive treatment during pregnancy. However, resistance to sulphadoxine-pyrimethamine has been increasing in the past few years and, although the link between molecular markers and treatment failure has not been firmly established, at least for pregnant women, it is important to monitor such markers. METHODS: This paper reports a novel sensitive, semi-quantitative and specific real-time PCR and melting curve analysis (MCA) assay using fluorescence resonance energy transfer (FRET) for the detection of DHPS-540, an important predictor for SP resistance. FRET/MCA was evaluated using 78 clinical samples from malaria patients and compared to PCR-RFLP. RESULTS: Sixty-two samples were in perfect agreement between both assays. One sample showed a small wild type signal with FRET/MCA that indicates a polyclonal infection. Four samples were not able to generate enough material in both assays to distinguish mutant from wild-type infection, six samples gave no signal in PCR-RFLP and five samples gave no amplification in FRET/MCA. CONCLUSION: FRET/MCA is an effective tool for the identification of SNPs in drug studies and epidemiological surveys on resistance markers in general and DHPS-540 mutation in particular.
    • Short report: molecular markers associated with Plasmodium falciparum resistance to sulfadoxine-pyrimethamine in the Democratic Republic of Congo.

      Cohuet, S; Bonnet, M; Van Herp, M; Van Overmeir, C; D'Alessandro, U; Guthmann, J P; Epicentre, Paris, France; Médecins Sans Frontières, Brussels, Belgium; Prince Leopold Institute of Tropical Medicine, Antwerp, Belgium. (2006-07)
      Sulfadoxine-pyrimethamine (SP) is the first line antimalarial treatment in the Democratic Republic of Congo. Using polymerase chain reaction, we assessed the prevalence of mutations in the dihydrofolate reductase (dhfr) (codons 108, 51, 59) and dihydropteroate synthase (dhps) (codons 437, 540) genes of Plasmodium falciparum, which have been associated with resistance to pyrimethamine and sulfadoxine, respectively. Four hundred seventy-four patients were sampled in Kilwa (N = 138), Kisangani (N = 112), Boende (N = 106), and Basankusu (N = 118). The proportion of triple mutations dhfr varied between sites but was always > 50%. The proportion of dhps double mutations was < 20%, with some sites as low as 0.9%. A quintuple mutation was present in 12.8% (16/125) samples in Kilwa; 11.9% (13/109) in Kisangani, 2.9% (3/102) in Boende, and 0.9% (1/112) in Basankusu. These results suggest high resistance to pyrimethamine alone or combined with sulfadoxine. Adding artesunate to SP does not seem a valid alternative to the current monotherapy.