Varying efficacy of artesunate+amodiaquine and artesunate+sulphadoxine-pyrimethamine for the treatment of uncomplicated falciparum malaria in the Democratic Republic of Congo: a report of two in-vivo studies

BACKGROUND: Very few data on anti-malarial efficacy are available from the Democratic Republic of Congo (DRC). DRC changed its anti-malarial treatment policy to amodiaquine (AQ) and artesunate (AS) in 2005. METHODS: The results of two in vivo efficacy studies, which tested AQ and sulphadoxine-pyrimethamine (SP) monotherapies and AS+SP and AS+AQ combinations in Boende (Equatorial province), and AS+SP, AS+AQ and SP in Kabalo (Katanga province), between 2003 and 2004 are presented. The methodology followed the WHO 2003 protocol for assessing the efficacy of anti-malarials in areas of high transmission. RESULTS: Out of 394 included patients in Boende, the failure rates on day 28 after PCR-genotyping adjustment of AS+SP and AS+AQ were estimated as 24.6% [95% CI: 16.6-35.5] and 15.1% [95% CI: 8.6-25.7], respectively. For the monotherapies, failure rates were 35.9% [95% CI: 27.0-46.7] for SP and 18.3% [95% CI: 11.6-28.1] for AQ. Out of 207 patients enrolled in Kabalo, the failure rate on day 28 after PCR-genotyping adjustment was 0 [1-sided 95% CI: 5.8] for AS+SP and AS+AQ [1-sided 95% CI: 6.2]. It was 19.6% [95% CI: 11.4-32.7] for SP monotherapy. CONCLUSION: The finding of varying efficacy of the same combinations at two sites in one country highlights one difficulty of implementing a uniform national treatment policy in a large country. The poor efficacy of AS+AQ in Boende should alert the national programme to foci of resistance and emphasizes the need for systems for the prospective monitoring of treatment efficacy at sentinel sites in the country

Real-time PCR/MCA assay using fluorescence resonance energy transfer for the genotyping of resistance related DHPS-540 mutations in Plasmodium falciparum

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

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

Failure of artesunate-mefloquine combination therapy for uncomplicated Plasmodium falciparum malaria in southern Cambodia

<p>Abstract</p> <p>Background</p> <p>Resistance to anti-malarial drugs hampers control efforts and increases the risk of morbidity and mortality from malaria. The efficacy of standard therapies for uncomplicated <it>Plasmodium falciparum </it>and <it>Plasmodium vivax </it>malaria was assessed in Chumkiri, Kampot Province, Cambodia.</p> <p>Methods</p> <p>One hundred fifty-one subjects with uncomplicated falciparum malaria received directly observed therapy with 12 mg/kg artesunate (over three days) and 25 mg/kg mefloquine, up to a maximum dose of 600 mg artesunate/1,000 mg mefloquine. One hundred nine subjects with uncomplicated vivax malaria received a total of 25 mg/kg chloroquine, up to a maximum dose of 1,500 mg, over three days. Subjects were followed for 42 days or until recurrent parasitaemia was observed. For <it>P. falciparum </it>infected subjects, PCR genotyping of <it>msp1</it>, <it>msp2</it>, and <it>glurp </it>was used to distinguish treatment failures from new infections. Treatment failure rates at days 28 and 42 were analyzed using both per protocol and Kaplan-Meier survival analysis. Real Time PCR was used to measure the copy number of the <it>pfmdr1 </it>gene and standard 48-hour isotopic hypoxanthine incorporation assays were used to measure IC₅₀for anti-malarial drugs.</p> <p>Results</p> <p>Among <it>P. falciparum </it>infected subjects, 47.0% were still parasitemic on day 2 and 11.3% on day 3. The PCR corrected treatment failure rates determined by survival analysis at 28 and 42 days were 13.1% and 18.8%, respectively. Treatment failure was associated with increased <it>pfmdr1 </it>copy number, higher initial parasitaemia, higher mefloquine IC₅₀, and longer time to parasite clearance. One <it>P. falciparum </it>isolate, from a treatment failure, had markedly elevated IC₅₀for both mefloquine (130 nM) and artesunate (6.7 nM). Among <it>P. vivax </it>infected subjects, 42.1% suffered recurrent <it>P. vivax </it>parasitaemia. None acquired new <it>P. falciparum </it>infection.</p> <p>Conclusion</p> <p>The results suggest that artesunate-mefloquine combination therapy is beginning to fail in southern Cambodia and that resistance is not confined to the provinces at the Thai-Cambodian border. It is unclear whether the treatment failures are due solely to mefloquine resistance or to artesunate resistance as well. The findings of delayed clearance times and elevated artesunate IC₅₀suggest that artesunate resistance may be emerging on a background of mefloquine resistance.</p

Identification of pyrimethamine- and chloroquine-resistant Plasmodium falciparum in Africa between 1984 and 1998: genotyping of archive blood samples

<p>Abstract</p> <p>Background</p> <p>Understanding the geographical distribution of drug resistance of <it>Plasmodium falciparum </it>is important for the effective treatment of malaria. Drug resistance has previously been inferred mainly from records of clinical resistance. However, clinical resistance is not always consistent with the parasite's genetic resistance. Thus, molecular identification of the parasite's drug resistance is required. In Africa, clinical resistance to pyrimethamine (Pyr) and chloroquine (CQ) was evident before 1980 but few studies investigating the genetic resistance to these drugs were conducted before the late 1990s. In this study, genotyping of genes involved in resistance to Pyr and CQ was performed using archive blood samples from Africa between 1984 and 1998.</p> <p>Methods</p> <p>Parasite DNA was extracted from <it>P. falciparum</it>-infected blood smears collected from travellers returning to Japan from Africa between 1984 and 1998. Genotypes of the dihydrofolate reductase gene (<it>dhfr</it>) and CQ-resistance transporter gene (<it>pfcrt) </it>were determined by polymerase chain reaction amplification and sequencing.</p> <p>Results</p> <p>Genotyping of <it>dhfr </it>and <it>pfcrt </it>was successful in 59 and 80 samples, respectively. One wild-type and seven mutant <it>dhfr </it>genotypes were identified. Three <it>dhfr </it>genotypes lacking the S108N mutation (NRSI, ICSI, IRSI; amino acids at positions 51, 59, 108, and 164 with mutations underlined) were highly prevalent before 1994 but reduced after 1995, accompanied by an increase in genotypes with the S108N mutation. The <it>dhfr </it>IRNI genotype was first identified in Nigeria in 1991 in the present samples, and its frequency gradually increased. However, two double mutants (ICNI and NRNI), the latter of which was exclusively found in West Africa, were more frequent than the IRNI genotype. Only two <it>pfcrt </it>genotypes were found, the wild-type and a Southeast Asian type (CVIET; amino acids at positions 72-76 with mutations underlined). The CVIET genotype was already present as early as 1984 in Tanzania and Nigeria, and appeared throughout Africa between 1984 and 1998.</p> <p>Conclusions</p> <p>This study is the first to report the molecular identification of Pyr- and CQ-resistant genotypes of <it>P. falciparum </it>in Africa before 1990. Genotyping of <it>dhfr </it>and <it>pfcrt </it>using archive samples has revealed new aspects of the evolutionary history of Pyr- and CQ-resistant parasites in Africa.</p

Marine Actinomycetes: A New Source of Compounds against the Human Malaria Parasite

Background Malaria continues to be a devastating parasitic disease that causes the death of 2 million individuals annually. The increase in multi-drug resistance together with the absence of an efficient vaccine hastens the need for speedy and comprehensive antimalarial drug discovery and development. Throughout history, traditional herbal remedies or natural products have been a reliable source of antimalarial agents, e.g. quinine and artemisinin. Today, one emerging source of small molecule drug leads is the world's oceans. Included among the source of marine natural products are marine microorganisms such as the recently described actinomycete. Members of the genus Salinispora have yielded a wealth of new secondary metabolites including salinosporamide A, a molecule currently advancing through clinical trials as an anticancer agent. Because of the biological activity of metabolites being isolated from marine microorganisms, our group became interested in exploring the potential efficacy of these compounds against the malaria parasite.[br/] Methods We screened 80 bacterial crude extracts for their activity against malaria growth. We established that the pure compound, salinosporamide A, produced by the marine actinomycete, Salinispora tropica, shows strong inhibitory activity against the erythrocytic stages of the parasite cycle. Biochemical experiments support the likely inhibition of the parasite 20S proteasome. Crystal structure modeling of salinosporamide A and the parasite catalytic 20S subunit further confirm this hypothesis. Ultimately we showed that salinosporamide A protected mice against deadly malaria infection when administered at an extremely low dosage.[br/] Conclusion These findings underline the potential of secondary metabolites, derived from marine microorganisms, to inhibit Plasmodium growth. More specifically, we highlight the effect of proteasome inhibitors such as salinosporamide A on in vitro and in vivo parasite development. Salinosporamide A (NPI-0052) now being advanced to phase I trials for the treatment of refractory multiple myeloma will need to be further explored to evaluate the safety profile for its use against malaria

Decreasing Burden of Malaria in Pregnancy in Malawian Women and Its Relationship to Use of Intermittent Preventive Therapy or Bed Nets

The World Health Organization recommends insecticidal bednets and intermittent preventive treatment to reduce malaria in pregnancy. Longitudinal data of malaria prevalence and pregnancy outcomes are valuable in gauging the impact of these antimalarial interventions.We recruited 8,131 women delivering in a single Malawian hospital over 9 years. We recorded demographic data, antenatal prescription of intermittent preventive therapy during pregnancy with sulfadoxine-pyrimethamine and bed net use, and examined finger-prick blood for malaria parasites and hemoglobin concentration. In 4,712 women, we examined placental blood for malaria parasites and recorded the infant's birth weight. Peripheral and placental parasitemia prevalence declined from 23.5% to 5.0% and from 25.2% to 6.8% respectively. Smaller declines in prevalence of low birth weight and anemia were observed. Coverage of intermittent preventive treatment and bednets increased. Number of sulfadoxine-pyrimethamine doses received correlated inversely with placental parasitemia (Odds Ratio (95% CI): 0.79 (0.68, 0.91)), maternal anemia (0.81, (0.73, 0.90)) and low birth weight from 1997-2001 (0.63 (0.53, 0.75)), but not from 2002-2006. Bednet use protected from peripheral parasitemia (0.47, (0.37, 0.60)) and placental parasitemia (0.41, (0.31, 0.54)) and low birth weight (0.75 (0.59, 0.95)) but not anemia throughout the study. Compared to women without nets who did not receive 2-dose sulfadoxine-pyrimethamine, women using nets and receiving 2-dose sulfadoxine-pyrimethamine were less likely to have parasitemia or low birth weight babies. Women receiving 2-dose sulfadoxine-pyrimethamine alone had little evidence of protection whereas bednets alone gave intermediate protection.Increased bednet coverage explains changes in parasitemia and birth weight among pregnant women better than sulfadoxine-pyrimethamine use. High bed net coverage, and sulfadoxine-pyrimethamine resistance, may be contributing to its apparent loss of effectiveness

Effect of trimethoprim-sulphamethoxazole on the risk of malaria in HIV-infected Ugandan children living in an area of widespread antifolate resistance

<p>Abstract</p> <p>Background</p> <p>Daily trimethoprim-sulfamethoxazole (TS) protects against malaria, but efficacy may be diminished as anti-folate resistance increases. This study assessed the incidence of falciparum malaria and the prevalence of resistance-conferring <it>Plasmodium falciparum </it>mutations in HIV-infected children receiving daily TS and HIV-uninfected children not taking TS.</p> <p>Materials and methods</p> <p>Subjects were 292 HIV-infected and 517 uninfected children from two cohort studies in Kampala, Uganda observed from August 2006 to December 2008. Daily TS was given to HIV-infected, but not HIV-uninfected children and all participants were provided an insecticide-treated bed net. Standardized protocols were used to measure the incidence of malaria and identify markers of antifolate resistance.</p> <p>Results</p> <p>Sixty-five episodes of falciparum malaria occurred in HIV-infected and 491 episodes in uninfected children during the observation period. TS was associated with a protective efficacy of 80% (0.10 vs. 0.45 episodes per person year, p < 0.001), and efficacy did not vary over three consecutive 9.5 month periods (81%, 74%, 80% respectively, p = 0.506). The prevalences of <it>dhfr </it>51I, 108N, and 59R and <it>dhps </it>437G and 540E mutations were each over 90% among parasites infecting both HIV-infected and uninfected children. Prevalence of the <it>dhfr </it>164L mutation, which is associated with high-level resistance, was significantly higher in parasites from HIV-infected compared to uninfected children (8% vs. 1%, p = 0.001). Sequencing of the <it>dhfr </it>and <it>dhps </it>genes identified only one additional polymorphism, <it>dhps </it>581G, in 2 of 30 samples from HIV-infected and 0 of 54 samples from uninfected children.</p> <p>Conclusion</p> <p>Despite high prevalence of known anti-folate resistance-mediating mutations, TS prophylaxis was highly effective against malaria, but was associated with presence of <it>dhfr </it>164L mutation.</p

The evolution of pyrimethamine resistant dhfr in Plasmodium falciparum of south-eastern Tanzania: comparing selection under SP alone vs SP+artesunate combination

BACKGROUND\ud \ud Sulphadoxine-pyrimethamine (SP) resistance is now widespread throughout east and southern Africa and artemisinin compounds in combination with synthetic drugs (ACT) are recommended as replacement treatments by the World Health Organization (WHO). As well as high cure rates, ACT has been shown to slow the development of resistance to the partner drug in areas of low to moderate transmission. This study looked for evidence of protection of the partner drug in a high transmission African context. The evaluation was part of large combination therapy pilot implementation programme in Tanzania, the Interdisciplinary Monitoring Programme for Antimalarial Combination Therapy (IMPACT-TZ) METHODS: The growth of resistant dhfr in a parasite population where SP Monotherapy was the first-line treatment was measured for four years (2002-2006), and compared with the development of resistant dhfr in a neighbouring population where SP + artesunate (SP+AS) was used as the first-line treatment during the same interval. The effect of the differing treatment regimes on the emergence of resistance was addressed in three ways. First, by looking at the rate of increase in frequency of pre-existing mutant dhfr alleles under monotherapy and combination therapy. Second, by examining whether de-novo mutant alleles emerged under either treatment. Finally, by measuring diversity at three dhfr flanking microsatellite loci upstream of the dhfr gene.\ud \ud RESULTS\ud \ud The reduction in SP selection pressure resulting from the adoption of ACT slowed the rate of increase in the frequency of the triple mutant resistant dhfr allele. Comparing between the two populations, the higher levels of genetic diversity in sequence flanking the dhfr triple mutant allele in the population where the ACT regimen had been used indicates the reduction in SP selection pressure arising from combination therapy.\ud \ud CONCLUSION\ud \ud The study demonstrated that, alleles containing two mutations at the dhfr have arisen at least four times independently while those containing triple mutant dhfr arose only once, and were found carrying a single unique Asian-type flanking sequence, which apparently drives the spread of pyrimethamine resistance associated dhfr alleles in east Africa. SP+AS is not recommended for use in areas where SP cure rates are less than 80% but this study reports an observed principle of combination protection from an area where pyrimethamine resistance was already high

Molecular markers of anti-malarial drug resistance in Lahj Governorate, Yemen: baseline data and implications

<p>Abstract</p> <p>Background</p> <p>This is an investigation of anti-malarial molecular markers coupled with a therapeutic efficacy test of chloroquine (CQ) against falciparum malaria in an area of unstable malaria in Lahj Governorate, Yemen. The study was aimed at assessment of therapeutic response to CQ and elucidation of baseline information on molecular markers for <it>Plasmodium falciparum </it>resistance against CQ and sulphadoxine/pyrimethamine (SP).</p> <p>Methods</p> <p>Between 2002 and 2003 the field test was conducted according to the standard WHO protocol to evaluate the therapeutic efficacy of CQ in 124 patients with falciparum malaria in an endemic area in Lahj Governorate in Yemen. Blood samples collected during this study were analysed for <it>P. falciparum </it>chloroquine resistance transporter gene (<it>pfcrt</it>)-76 polymorphisms, mutation <it>pfcrt-</it>S163R and the antifolate resistance-associated mutations dihydrofolate reductase (<it>dhfr</it>)-C59R and dihydropteroate synthase (<it>dhps</it>)-K540E. Direct DNA sequencing of the <it>pfcrt </it>gene from three representative field samples was carried out after DNA amplification of the 13 exons of the <it>pfcrt </it>gene.</p> <p>Results</p> <p>Treatment failure was detected in 61% of the 122 cases that completed the 14-day follow-up. The prevalence of mutant <it>pfcrt </it>T76 was 98% in 112 amplified pre-treatment samples. The presence of <it>pfcrt </it>T76 was poorly predictive of <it>in vivo </it>CQ resistance (PPV = 61.8%, 95% CI = 52.7-70.9). The prevalence of <it>dhfr </it>Arg-59 mutation in 99 amplified samples was 5%, while the <it>dhps </it>Glu-540 was not detected in any of 119 amplified samples. Sequencing the <it>pfcrt </it>gene confirmed that Yemeni CQ resistant <it>P. falciparum </it>carry the old world (Asian and African) CQ resistant haplotype CVIETSESI at positions 72,73,74,75,76,220,271, 326 and 371.</p> <p>Conclusion</p> <p>This is the first study to report baseline information on the characteristics and implications of anti-malarial drug resistance markers in Yemen. It is also the first report of the haplotype associated with CQR <it>P. falciparum </it>parasites from Yemen. Mutant <it>pfcrt</it>T76 is highly prevalent but it is a poor predictor of treatment failure in the study population. The prevalence of mutation <it>dhfr</it>Arg59 is suggestive of emerging resistance to SP, which is currently a component of the recommended combination treatment of falciparum malaria in Yemen. More studies on these markers are recommended for surveillance of resistance in the study area.</p