Introduction Of Artemisinin Combination Therapy Research Articles

Comparative efficacy of sulphadoxine-pyrimethamine and dihydroartemisinin-piperaquine against malaria infection during late-stage pregnancy in mice.

The introduction of artemisinin combination therapies (ACTs) against malaria infections opened up a window of possibilities to combat malaria in pregnancy. However, the usefulness of ACTs in all stages of pregnancy must be critically assessed. This study was designed to evaluate dihydroartemisinin-piperaquine (DHAP) as a suitable alternative to sulphadoxine-pyrimethamine (SP) in the treatment of malaria during third-trimester pregnancy in mice. Experimental animals were inoculated with a parasitic dose of 1x106Plasmodium berghei (ANKA strain) infected erythrocytes and randomly allocated into treatment groups. The animals received standard doses of chloroquine alone (CQ)[10 mg/kg], SP [25 mg/kg] and [1.25 mg/kg] and DHAP [4 mg/kg] and [18 mg/kg] combinations. Maternal and pupil survival, litter sizes, pup weight and still-births were recorded, while the effect of the drug combinations on parasite suppression, recrudescence and parasite clearance time were evaluated. The day 4 chemo-suppression of parasitemia by DHAP in infected animals was comparable to SP, and CQ treatment (P > 0.05). The mean recrudescence time was significantly delayed (p = 0.031) in the DHAP treatment group compared to the CQ treatment group, while, there was no recrudescence in animals treated with SP. The birth rate in the SP group was significantly higher than in the DHAP group (P < 0.05). There was 100% maternal and pup survival in both combination treatments comparable with the uninfected gravid controls. The overall parasitological activity of SP against Plasmodium berghei in late-stage pregnancy appeared better than DHAP. In addition, SP treatment resulted in better birth outcomes assessed compared to DHAP treatment.

Prevalence of arps10, fd, pfmdr-2, pfcrt and pfkelch13 gene mutations in Plasmodium falciparum parasite population in Uganda.

In Uganda, Artemether-Lumefantrine and Artesunate are recommended for uncomplicated and severe malaria respectively, but are currently threatened by parasite resistance. Genetic and epigenetic factors play a role in predisposing Plasmodium falciparum parasites to acquiring Pfkelch13 (K13) mutations associated with delayed artemisinin parasite clearance as reported in Southeast Asia. In this study, we report on the prevalence of mutations in the K13, pfmdr-2 (P. falciparum multidrug resistance protein 2), fd (ferredoxin), pfcrt (P. falciparum chloroquine resistance transporter), and arps10 (apicoplast ribosomal protein S10) genes in Plasmodium falciparum parasites prior to (2005) and after (2013) introduction of artemisinin combination therapies for malaria treatment in Uganda. A total of 200 P. falciparum parasite DNA samples were screened. Parasite DNA was extracted using QIAamp DNA mini kit (Qiagen, GmbH, Germany) procedure. The PCR products were sequenced using Sanger dideoxy sequencing method. Of the 200 P. falciparum DNA samples screened, sequencing for mutations in K13, pfmdr-2, fd, pfcrt, arps10 genes was successful in 142, 186, 141, 128 and 74 samples respectively. Overall, we detected six (4.2%, 6/142; 95%CI: 1.4-7.0) K13 single nucleotide polymorphisms (SNPs), of which 3.9% (2/51), 4.4% (4/91) occurred in 2005 and 2013 samples respectively. All four K13 SNPs in 2013 samples were non-synonymous (A578S, E596V, S600C and E643K) while of the two SNPs in 2005 samples, one (Y588N) is non-synonymous and the other (I587I) is synonymous. There was no statistically significant difference in the prevalence of K13 (p = 0.112) SNPs in the samples collected in 2005 and 2013. The overall prevalence of SNPs in pfmdr-2 gene was 39.8% (74/186, 95%CI: 25.1-50.4). Of this, 4.2% (4/95), 76.9% (70/91) occurred in 2005 and 2013 samples respectively. In 2005 samples only one SNP, Y423F (4.2%, 4/95) was found while in 2013, Y423F (38.5%, 35/91) and I492V (38.5%, 35/91) SNPs in the pfmdr-2 gene were found. There was a statistically significant difference in the prevalence of pfmdr-2 SNPs in the samples collected in 2005 and 2013 (p<0.001). The overall prevalence of arps10 mutations was 2.7% (2/72, 95%CI: 0.3-4.2). Two mutations, V127M (4.5%: 1/22) and D128H (4.5%: 1/22) in the arps10 gene were each found in P. falciparum parasite samples collected in 2013. There was no statistically significant difference in the prevalence of arps10 SNPs in the samples collected in 2005 and 2013 (p = 0.238). There were more pfmdr-2 SNPs in P. falciparum parasites collected after introduction of Artemisinin combination therapies in malaria treatment. This is an indicator of the need for continuous surveillance to monitor emergence of molecular markers of artemisinin resistance and its potential drivers in malaria affected regions globally.

High multiple mutations of Plasmodium falciparum-resistant genotypes to sulphadoxine-pyrimethamine in Lagos, Nigeria

BackgroundPlasmodium falciparum-resistance to sulphadoxine-pyrimethamine (SP) has been largely reported among pregnant women. However, the profile of resistance markers to SP dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) in the general population are varied and not frequently monitored. Currently, SP is used as partner drug for artemisinin combination therapy (SP-artesunate) in some sub-Saharan African countries or as a prophylactic drug in intermittent preventive treatment of malaria during pregnancy and infants and in seasonal malaria chemoprevention (SMC). Profiling of P. falciparum-resistant genotypes to SP is dynamic and critical in providing data that would be useful for malaria control programmes. This study assessed the profile of dhfr and dhps genes genotypes among individuals with malaria in Lagos, Nigeria.MethodsMolecular markers of SP resistance were identified by nested PCR and sequenced among malaria positive dried blood spots (DBS) that were collected from individuals attending health facilities from January 2013 to February 2014 and during community surveys from October 2010 to September 2011 across different Local Government Areas of Lagos State, Nigeria.ResultsA total of 242 and 167 samples were sequenced for dhfr and dhps, respectively. Sequence analysis of dhfr showed that 95.5% (231/242), 96.3% (233/242) and 96.7% (234/242) of the samples had N51I, C59R and S108N mutant alleles, respectively. The prevalence of dhps mutation at codons A437G, A613S, S436A, A581G, I431V and K540E were 95.8% (160/167), 41.9% (70/167), 41.3% (69/167), 31.1% (52/167), 25.1% (42/167), and 1.2% (2/167) respectively. The prevalence of triple mutations (CIRNI) in dhfr was 93.8% and 44.3% for the single dhps haplotype mutation (SGKAA). Partial SP-resistance due to quadruple dhfr-dhps haplotype mutations (CIRNI-SGKAA) and octuple haplotype mutations (CIRNI-VAGKGS) with rate of 42.6% and 22.0%, respectively has been reported.ConclusionsThere was increased prevalence in dhfr triple haplotype mutations when compared with previous reports in the same environment but aligned with high prevalence in other locations in Nigeria and other countries in Africa. Also, high prevalence of dhfr and dhps mutant alleles occurred in the study areas in Lagos, Nigeria five to eight years after the introduction of artemisinin combination therapy underscores the need for continuous monitoring.

Impact evaluation of malaria control interventions on morbidity and all-cause child mortality in Mali, 2000\u20132012

BackgroundMajor investments have been made since 2001, with intensification of malaria control interventions after 2006. Interventions included free distribution of insecticide-treated nets (ITN) to pregnant women and children under 5 years old, the introduction of artemisinin combination therapy (ACT) for malaria treatment, and indoor residual spraying of insecticides. Funders include the Government of Mali, the Global Fund to Fight AIDS, Tuberculosis and Malaria, and the US President’s Malaria Initiative.MethodsData from nationally representative household surveys conducted from 2000 to 2015 was used to performed the trend analysis for malaria intervention coverage, prevalence of morbidities among children under 5 years old [parasitemia and severe anaemia (< 8 g/dl)], and all-cause mortality of children under 5 (ACCM). Prevalence of contextual factors likely to contribute to ACCM were also assessed. The impact of these interventions was assessed on malaria morbidity and mortality using a plausibility argument. With the assumption that malaria contributes significantly to under-five mortality in settings with high malaria transmission, associations between malaria control interventions and all-cause under-five mortality (ACCM) were assessed taking into account other contextual factors related to child survival.ResultsIntervention coverage improved significantly from 2006 to 2012. Household ownership of ITN increased from 49% in 2006 to 84% in 2012. ITN use also increased over the same period, from 26% in 2006 to 69% in 2012 among children under 5 and from 28% in 2006 to 73% in 2012 among pregnant women. The coverage of intermittent preventive treatment in pregnancy (IPTp) using two or more doses of SP increased from 10% in 2006 to 29% in 2012. In 2010, 23% of febrile children under 5 received ACT, as opposed to 19% in 2012. The prevalence of Plasmodium falciparum infection increased from 2010 (38.6%) to 2012 (51.6%), followed by a decrease in 2015 (35.8%). The prevalence of severe anaemia decreased from 2010 (26.3%) to 2012 (20.6%) and continued to decline in 2015 (19.9%). An impressive decline in ACCM was observed, from 225 in 1997–2001 to 192 in 2002–2006 and 95 in 2008–2012. Changes in contextual factors such as climate, socio-economic, nutrition, and coverage of maternal and child health interventions over the evaluation period did not favour reductions in ACCM, and are therefore unlikely to explain the observed results.ConclusionsTaken as a whole, the evidence supports the conclusion that malaria control interventions substantially contributed to the observed decline in ACCM in Mali from 2000 to 2012, even in the context of continued high prevalence of parasitaemia explained by contextual factors such as climate change and political instability.

The malaria testing and treatment landscape in mainland Tanzania, 2016

BackgroundUnderstanding the key characteristics of malaria testing and treatment is essential to the control of a disease that continues to pose a major risk of morbidity and mortality in mainland Tanzania, with evidence of a resurgence of the disease in recent years. The introduction of artemisinin combination therapy (ACT) as the first-line treatment for malaria, alongside policies to promote rational case management following testing, highlights the need for evidence of anti-malarial and testing markets in the country. The results of the most recent mainland Tanzania ACTwatch outlet survey are presented here, including data on the availability, market share and price of anti-malarials and malaria diagnosis in 2016.MethodsA nationally-representative malaria outlet survey was conducted between 18th May and 2nd July, 2016. A census of public and private outlets with potential to distribute malaria testing and/or treatment was conducted among a representative sample of administrative units. An audit was completed for all anti-malarials, malaria rapid (RDT) diagnostic tests and microscopy.ResultsA total of 5867 outlets were included in the nationally representative survey, across both public and private sectors. In the public sector, availability of malaria testing was 92.3% and quality-assured (QA) ACT was 89.1% among all screened outlets. Sulfadoxine–pyrimethamine (SP) was stocked by 51.8% of the public sector and injectable artesunate was found in 71.4% of all screened public health facilities. Among anti-malarial private-sector stockists, availability of testing was 15.7, and 65.1% had QA ACT available. The public sector accounted for 83.4% of the total market share for malaria diagnostics. The private sector accounted for 63.9% of the total anti-malarial market, and anti-malarials were most commonly distributed through accredited drug dispensing outlets (ADDOs) (39.0%), duka la dawa baridi (DLDBs) (13.3%) and pharmacies (6.7%). QA ACT comprised 33.1% of the national market share (12.2% public sector and 20.9% private sector). SP accounted for 53.3% of the total market for anti-malarials across both private and public sectors (31.3 and 22.0% of the total market, respectively). The median price per adult equivalent treatment dose (AETD) of QA ACT in the private sector was $1.40, almost 1.5 times more expensive than the median price per AETD of SP ($1.05). In the private sector, 79.3% of providers perceived ACT to be the most effective treatment for uncomplicated malaria for adults and 88.4% perceived this for children.ConclusionsWhile public sector preparedness for appropriate malaria testing and case management is showing encouraging signs, QA ACT availability and market share in the private sector continues to be sub-optimal for most outlet types. Furthermore, it is concerning that SP continues to predominate in the anti-malarial market. The reasons for this remain unclear, but are likely to be in part related to price, availability and provider knowledge or preferences. Continued efforts to implement government policy around malaria diagnosis and case management should be encouraged.

Genetically Determined Response to Artemisinin Treatment in Western Kenyan Plasmodium falciparum Parasites.

Genetically determined artemisinin resistance in Plasmodium falciparum has been described in Southeast Asia. The relevance of recently described Kelch 13-propeller mutations for artemisinin resistance in Sub-Saharan Africa parasites is still unknown. Southeast Asia parasites have low genetic diversity compared to Sub-Saharan Africa, where parasites are highly genetically diverse. This study attempted to elucidate whether genetics provides a basis for discovering molecular markers in response to artemisinin drug treatment in P. falciparum in Kenya. The genetic diversity of parasites collected pre- and post- introduction of artemisinin combination therapy (ACT) in western Kenya was determined. A panel of 12 microsatellites and 91 single nucleotide polymorphisms (SNPs) distributed across the P. falciparum genome were genotyped. Parasite clearance rates were obtained for the post-ACT parasites. The 12 microsatellites were highly polymorphic with post-ACT parasites being significantly more diverse compared to pre-ACT (p < 0.0001). The median clearance half-life was 2.55 hours for the post-ACT parasites. Based on SNP analysis, 15 of 90 post-ACT parasites were single-clone infections. Analysis revealed 3 SNPs that might have some causal association with parasite clearance rates. Further, genetic analysis using Bayesian tree revealed parasites with similar clearance phenotypes were more closely genetically related. With further studies, SNPs described here and genetically determined response to artemisinin treatment might be useful in tracking artemisinin resistance in Kenya.

Prevalence of Plasmodium falciparum anti-malarial resistance-associated polymorphisms in pfcrt, pfmdr1 and pfnhe1 in Muheza, Tanzania, prior to introduction of artemisinin combination therapy.

BackgroundA report of the chloroquine and amodiaquine resistance pfcrt-SVMNT haplotype in Tanzania raises concern about high-level resistance to the artesunate-amodiaquine combination treatment widely employed in Africa. Mutations in the pfmdr1 multi-drug resistance gene may also be associated with resistance, and a highly polymorphic microsatellite (ms-4760) of the pfnhe1 gene involved in quinine susceptibility has not been surveyed in Tanzania.MethodsA total of 234 samples collected between 2003 – 2006 from an observational birth cohort of young children in Muheza, Tanzania were analysed. In these children, 141 cases of P. falciparum infections were treated with AQ and 93 episodes were treated with QN. Haplotypes of pfcrt and pfmdr1 were determined by a Taqman assay, and ms-4760 repeats in pfnhe1 were assessed by nested PCR amplification and direct sequencing. Parasite population diversity was evaluated using microsatellite markers on five different chromosomes.ResultsThe pfcrt-CVIET haplotype was present alone in 93.6% (219/234) of the samples over the study period; the wild-type chloroquine- and amodiaquine-sensitive haplotype pfcrt-CVMNK was present in 4.3% (10/234) of the samples; and both haplotypes were present in 2.1% (5/234) of the samples. No significant change in wild-type pfcrt-CVMNK prevalence was evident over the 4-year period of the study. The pfcrt-SVMNT haplotype associated with high-level amodiaquine resistance was not detected in this study. The pfmdr1 locus was genotyped in 178 of these samples. The pfmdr1-YYNY haplotype predominated in 67.4% (120/178) of infections and was significantly associated with the pfcrt-CVIET haplotype. All samples carried the wild-type pfmdr1-N1042 codon. The ms-4760 repeat on pfnhe1 locus displayed 12 distinct haplotypes with ms-4760-1 predominating in the population. Analysis of these haplotypes showed no association of a particular haplotype with quinine treatment outcome.ConclusionThe pfcrt-CVIET chloroquine resistance haplotype dominated in the collection of P. falciparum samples from Muheza. The pfcrt-SVMNT haplotype, which threatens the efficacy of amodiaquine and was reported in the same time period from Korogwe, Tanzania, 40 Km from Muheza, was not detected. Relative low prevalence of pfcrt-SVMNT in Africa may result from genetic or other factors rendering P. falciparum less supportive of this haplotype than in South America or other regions.Trial registrationTrial Protocol Number: 08-I-N064.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0642-2) contains supplementary material, which is available to authorized users.

Analysis of major genome loci underlying artemisinin resistance and pfmdr1 copy number in pre- and post-ACTs in western Kenya.

Genetic analysis of molecular markers is critical in tracking the emergence and/or spread of artemisinin resistant parasites. Clinical isolates collected in western Kenya pre- and post- introduction of artemisinin combination therapies (ACTs) were genotyped at SNP positions in regions of strong selection signatures on chromosome 13 and 14, as described in Southeast Asia (SEA). Twenty five SNPs were genotyped using Sequenom MassArray and pfmdr1 gene copy number by real-time PCR. Parasite clearance half-life and in vitro drug sensitivity testing were performed using standard methods. One hundred twenty nine isolates were successfully analyzed. Fifteen SNPs were present in pre-ACTs isolates and six in post-ACTs. None of the SNPs showed association with parasite clearance half-life. Post-ACTs parasites had significantly higher pfmdr1 copy number compared to pre-ACTs. Seven of eight parasites with multiple pfmdr1 were post-ACTs. When in vitro IC50s were compared for parasites with single vs. multiple gene copies, only amodiaquine and piperaquine reached statistical significance. Data showed SNPs on chromosome 13 and 14 had different frequency and trend in western Kenya parasites compared SEA. Increase in pfmdr1 gene copy is consistent with recent studies in African parasites. Data suggests genetic signature of artemisinin resistance in Africa might be different from SEA.

Temporal changes in Plasmodium falciparum anti-malarial drug sensitivity in vitro and resistance-associated genetic mutations in isolates from Papua New Guinea

BackgroundIn northern Papua New Guinea (PNG), most Plasmodium falciparum isolates proved resistant to chloroquine (CQ) in vitro between 2005 and 2007, and there was near-fixation of pfcrt K76T, pfdhfr C59R/S108N and pfmdr1 N86Y. To determine whether the subsequent introduction of artemisinin combination therapy (ACT) and reduced CQ-sulphadoxine-pyrimethamine pressure had attenuated parasite drug susceptibility and resistance-associated mutations, these parameters were re-assessed between 2011 and 2013.MethodsA validated fluorescence-based assay was used to assess growth inhibition of 52 P. falciparum isolates from children in a clinical trial in Madang Province. Responses to CQ, lumefantrine, piperaquine, naphthoquine, pyronaridine, artesunate, dihydroartemisinin, artemether were assessed. Molecular resistance markers were detected using a multiplex PCR ligase detection reaction fluorescent microsphere assay.ResultsCQ resistance (in vitro concentration required for 50% parasite growth inhibition (IC50) >100 nM) was present in 19% of isolates. All piperaquine and naphthoquine IC50s were <100 nM and those for lumefantrine, pyronaridine and the artemisinin derivatives were in low nM ranges. Factor analysis of IC50s showed three groupings (lumefantrine; CQ, piperaquine, naphthoquine; pyronaridine, dihydroartemisinin, artemether, artesunate). Most isolates (96%) were monoclonal pfcrt K76T (SVMNT) mutants and most (86%) contained pfmdr1 N86Y (YYSND). No wild-type pfdhfr was found but most isolates contained wild-type (SAKAA) pfdhps. Compared with 2005–2007, the geometric mean (95% CI) CQ IC50 was lower (87 (71–107) vs 167 (141–197) nM) and there had been no change in the prevalence of pfcrt K76T or pfmdr1 mutations. There were fewer isolates of the pfdhps (SAKAA) wild-type (60 vs 100%) and pfdhfr mutations persisted.ConclusionsReflecting less drug pressure, in vitro CQ sensitivity appears to be improving in Madang Province despite continued near-fixation of pfcrt K76T and pfmdr1 mutations. Temporal changes in IC50s for other anti-malarial drugs were inconsistent but susceptibility was preserved. Retention or increases in pfdhfr and pfdhps mutations reflect continued use of sulphadoxine-pyrimethamine in the study area including through paediatric intermittent preventive treatment. The susceptibility of local isolates to lumefantrine may be unrelated to those of other ACT partner drugs.Trial registrationAustralian New Zealand Clinical Trials Registry ACTRN12610000913077.

Genetic mutations in Plasmodium falciparum and Plasmodium vivax dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) in Vanuatu and Solomon Islands prior to the introduction of artemisinin combination therapy

BackgroundPlasmodium falciparum and Plasmodium vivax are endemic in Vanuatu and the Solomon Islands. While both countries have introduced artemether-lumefantrine (AL) as first-line therapy for both P. falciparum and P. vivax since 2008, chloroquine and sulphadoxine-pyrimethamine (SP) were used as first-line therapy for many years prior to the introduction of AL. Limited data are available on the extent of SP resistance at the time of policy change.MethodsBlood spots were obtained from epidemiological surveys conducted on Tanna Island, Tafea Province, Vanuatu and Temotu Province, Solomon Islands in 2008. Additional samples from Malaita Province, Solomon Islands were collected as part of an AL therapeutic efficacy study conducted in 2008. Plasmodium vivax and P. falciparum dhfr and dhps genes were sequenced to detect nucleotide polymorphisms.ResultsAll P. falciparum samples analysed (n =114) possessed a double mutant pfdhfr allele (C59R/S108N). Additionally, mutation A437G in pfhdps was detected in a small number of samples 2/13, 1/17 and 3/26 from Tanna Island, Vanuatu and Temotu and Malaita Provinces Solomon Islands respectively. Mutations were also common in pvdhfr from Tanna Island, Vanuatu, where 33/51 parasites carried the double amino acid substitution S58R/S117N, while in Temotu and Malaita Provinces, Solomon Islands 32/40 and 39/46 isolates carried the quadruple amino acid substitution F57L/S58R/T61M/S117T in DHFR respectively. No mutations in pvdhps (n =108) were detected in these three island groups.ConclusionPrior to the introduction of AL, there was a moderate level of SP resistance in the P. falciparum population that may cause SP treatment failure in young children. Of the P. vivax isolates, a majority of Solomon Islands isolates carried quadruple mutant pvdhfr alleles while a majority of Vanuatu isolates carried double mutant pvdhfr alleles. This suggests a higher level of SP resistance in the P. vivax population in Solomon Islands compared to the sympatric P. falciparum population and there is a higher level of SP resistance in P. vivax parasites from Solomon Islands than Vanuatu. This study demonstrates that the change of treatment policy in these countries from SP to ACT was timely. The information also provides a baseline for future monitoring.

Double mutation in the pfmdr1 gene is associated with emergence of chloroquine-resistant Plasmodium falciparum malaria in Eastern India.

Malaria is a major public health problem in tropical and subtropical countries, including India. This study elucidates the cause of chloroquine treatment failure (for Plasmodium falciparum infection) before the introduction of artemisinin combination therapy. One hundred twenty-six patients were randomized to chloroquine treatment, and the therapeutic efficacy was monitored from days 1 to 28. An in vitro susceptibility test was performed with all isolates. Parasitic DNA was isolated, followed by PCR and restriction digestion of different codons of the pfcrt gene (codons 72 to 76) and the pfmdr1 gene (N86Y, Y184F, S1034C, N1042D, and D1246Y). Finally, sequencing was done to confirm the mutations. Forty-three (34.13%) early treatment failure cases and 16 (12.69%) late treatment failure cases were observed after chloroquine treatment. In vitro chloroquine resistance was found in 103 isolates (81.75%). Twenty-six (60.47%) early treatment failure cases and 6 (37.5%) late treatment failure cases were associated with the CVMNK-YYSNY allele (the underlined amino acids are those that were mutated). Moreover, the CVIEK-YYSNY allele was found in 8 early treatment failure (18.60%) and 2 late treatment failure (12.5%) cases. The presence of the wild-type pfcrt (CVMNK) and pfmdr1 (YYSNY) double mutant allele in chloroquine-nonresponsive cases was quite uncommon. In vivo chloroquine treatment failure and in vitro chloroquine resistance were strongly correlated with the CVMNK-YYSNY and CVIEK-YYSNY haplotypes (P < 0.01).

Battling Malaria in Rural Zambia with Modern Technology: A Qualitative Study on the Value of Cell Phones, Geographical Information Systems, Asymptomatic Carriers and Rapid Diagnostic Tests to Identify, Treat and Control Malaria.

During the last decade much progress has been made in reducing malaria transmission in Macha, Southern Province, Zambia. Introduction of artemisinin combination therapies as well as mass screenings of asymptomatic carriers is believed to have contributed the most. When an endemic malaria situation is moving towards a non-endemic situation the resident population loses acquired immunity and therefore active case detection and efficient surveillance is crucial to prevent epidemic outbreaks. Our purpose was to evaluate the impact of cell phone surveillance and geographical information systems on malaria control in Macha. Furthermore, it evaluates what screening and treatment of asymptomatic carriers and implementation of rapid diagnostic tests in rural health care has led to. Ten in-depth semi-structured interviews, field observations and data collection were performed at the Macha Research Trust and at surrounding rural health centers. This qualitative method was inspired by rapid assessment procedure. The cell phone surveillance has been easily integrated in health care, and its integration with Geographical Information Systems has provided the ability to follow malaria transmission on a weekly basis. In addition, active case detection of asymptomatic carriers has been fruitful, which is reflected in it soon being applied nationwide. Furthermore, rapid diagnostic tests have provided rural health centers with reliable malaria diagnostics, thereby decreasing excessive malaria treatments and selection for drug resistance. This report reflects the importance of asymptomatic carriers in targeting malaria elimination, as well as development of effective surveillance systems when transmission decreases. Such an approach would be cost-efficient in the long run through positive effects in reduced child mortality and relief in health care.

Changes in Malaria Parasite Drug Resistance in an Endemic Population Over a 25-Year Period With Resulting Genomic Evidence of Selection

Background. Analysis of genome-wide polymorphism in many organisms has potential to identify genes under recent selection. However, data on historical allele frequency changes are rarely available for direct confirmation.Methods. We genotyped single nucleotide polymorphisms (SNPs) in 4 Plasmodium falciparum drug resistance genes in 668 archived parasite-positive blood samples of a Gambian population between 1984 and 2008. This covered a period before antimalarial resistance was detected locally, through subsequent failure of multiple drugs until introduction of artemisinin combination therapy. We separately performed genome-wide sequence analysis of 52 clinical isolates from 2008 to prospect for loci under recent directional selection.Results. Resistance alleles increased from very low frequencies, peaking in 2000 for chloroquine resistance-associated crt and mdr1 genes and at the end of the survey period for dhfr and dhps genes respectively associated with pyrimethamine and sulfadoxine resistance. Temporal changes fit a model incorporating likely selection coefficients over the period. Three of the drug resistance loci were in the top 4 regions under strong selection implicated by the genome-wide analysis.Conclusions. Genome-wide polymorphism analysis of an endemic population sample robustly identifies loci with detailed documentation of recent selection, demonstrating power to prospectively detect emerging drug resistance genes.

Reduction of multiplicity of infections but no change in msp2 genetic diversity in Plasmodium falciparum isolates from Congolese children after introduction of artemisinin-combination therapy

BackgroundIn this first study conducted after the introduction of artemisinin-combination therapy (ACT), the major objective was to evaluate Plasmodium falciparum genetic diversity and multiplicity of infection in isolates from Congolese children between one and nine years of age enrolled and followed up for one year. The secondary objective was to characterize the msp2 profiles of P. falciparum isolates collected from successive malaria episodes in ten children who had four or more clinical episodes during the follow up.MethodsThree-hundred and thirteen children residing in southern part of Brazzaville participated in this study. Blood samples were obtained from all children at enrollment and checked for P. falciparum infection. Based on the one year follow-up data, two clinical groups were considered according to the number of malaria episodes presented over the follow up period: “protected”(children who did not experience any episode) and “unprotected” (those who experienced more that two episodes). Therefore, the msp2 genetic diversity of P. falciparum isolates collected at enrollment in the two groups was characterized by allele-specific nested PCR and compared. The msp2 profiles of P. falciparum isolates collected from successive malaria episodes was also characterized by allele-specific nested PCR.ResultsForty-three percent of FC27 and fifty-seven percent of 3D7 in protected vs fifty-six percent of FC27 and forty-four percent of 3D7 in isolates from unprotected children were detected. Seven and two alleles belonging to the FC27, and six and three alleles belonging to 3D7 families were distinguished in isolates from protected and unprotected children respectively. The mean multiplicity of infection (MOI) values at inclusion for the msp2 locus was 1.29 and 1.43 for protected and unprotected children respectively. 43 isolates were obtained from the ten children who had four or more clinical episodes during the follow up. A total of 63 alleles or fragments corresponding to 57% (36/63) FC27 and 43% (27/63) 3D7 were detected. The variant 400bp of FC27 was the most prevalent. 46% (20/43), 42% (18/43), 2% (1/43) and 2% (1/43) of isolates were found to have 1, 2, 3 and 4 parasite genotypes respectively and the mean MOI was 1.78.ConclusionThis study shows that the introduction of ACT in the Republic of Congo has reduced the MOI but not the genetic diversity of P. falciparum isolates from children living in Southern districts of Brazzaville.

Molecular markers in plasmodium falciparum linked to resistance to anti-malarial drugs in samples imported from Africa over an eight-year period (2002-2010): impact of the introduction of artemisinin combination therapy.

BackgroundDrug resistance is a major problem to control Plasmodium falciparum infection in endemic countries. During last decade, African countries have changed first-line treatment to artemisinin-based combinations therapy (ACT); sulphadoxine-pyrimethamine (SP) is recommended for Intermittent Preventive Therapy (IPT). Molecular markers related to P falciparum resistance were analysed for the period of transition from SP to ACT, in isolates imported from Africa.MethodsA first group of samples was taken in the period between June 2002 and June 2006 (n = 113); a second group in the period between November 2008 and August 2010 (n = 46). Several alleles were analysed by nested PCR-RFLP: 51, 59, 108, 164, in the pfdhfr gene; 436, 437, 540, 581, in the pfdhps gene; 86, 1246, in the pfmdr1 gene and 76, in the pfcrt gene. The prevalence of alleles in the groups was compared with the chi-squared or Fisher's exact tests.ResultsThe pfdhfr N51I, C59R and S108N were over to 90% in the two groups; all samples had the I164. In the pfdhps, 437 G and 581 G, increased up to 80% and 10.9% (p = 0.024), respectively in the second group. The 540 G decreases (24% to 16.%) and the 436A disappears at the end of the follow-up (p = 0.004) in the second group. The 76I-pfcrt stayed over 95% in the two groups. Prevalence of 86Y-pfmdr1 decreased over eight years.ConclusionsPharmacological pressure affects the resistance strains prevalence. As for SP, the disappearance of 436A and the decrease in 540 G suggest that these mutations are not fixed. On the other hand, studies carried out after ACT introduction show there was a selection of strains carrying the SNPs N86Y, D1246Y in pfmdr1. In this work, the prevalence of pfmdr1- D1246Y is increasing, perhaps as a result of selective pressure by ACT. Continued surveillance is essential to monitor the effectiveness of treatments.

Dissolution Profiles of Twelve Brands of Sulphadoxine Pyrimethamine in the Nigerian Market

With the introduction of artemisinin-combination therapies (ACTs) as the standard recommended treatment for falciparum malaria by the World Health Organization (WHO) and its adoption by most malaria-endemic countries like Nigeria, the only available treatment option for pregnant women with malaria became sulphadoxine–pyrimethamine (SP). Several brands of SP have emerged in the local Nigerian and African market. An in vitro dissolution study was conducted on twelve brands of SP in three different dissolution media. The twelve brands of SP were obtained from both the public and private medicine outlets in Nigeria. They were subjected to dissolution in three dissolution media at pH values of 1.2, 4.5, and 6.8.The dissolution samples were assayed for the drug compounds using a double-beam ultraviolet spectrophotometer. The dissolution test results were subjected to analysis of variance (ANOVA) and analyses of difference (f1) and similarity factors (f2). Five of the selected twelve brands failed to meet the USP dissolution requirements for immediate-release tablets. Seven of twelve brands had dissimilar dissolution profiles.

Highly Effective Therapy for Maternal Malaria Associated With a Lower Risk of Vertical Transmission

Background. The epidemiology of congenital malaria was investigated in a hospital-based malaria surveillance study in Papua, Indonesia.Methods. From April 2005 to January 2010, 4878 delivering women and their newborns underwent prospective clinical review and malaria screening by peripheral blood microscopy.Findings. Congenital malaria occurred in 8 per 1000 (38/4884) live births, with Plasmodium falciparum accounting for 76.3% (29) and P. vivax for 15.8% (6) of infections. Maternal malaria at delivery (adjusted odds ratio [AOR], 9.5; 95% confidence interval [CI], 4.2–21.5; P < .001), age ≤ 16 years (AOR, 4; 95% CI, 1.4–12.1; P = .011), and prior malaria during pregnancy (AOR, 2.2; 95% CI, 1.1–4.4, P = .022) were independent risk factors for vertical transmission. Of 29 mothers and neonates with contemporaneous peripheral parasitemia, 17% (5) had discordant parasite species, suggesting possible antenatal malaria transmission. Newborns with malaria were at significantly greater risk of low birth weight (AOR, 2.8; 95% CI, 1.2–6.6; P = .002). Following introduction of dihydroartemisinin-piperaquine for uncomplicated malaria in the second and third trimesters of pregnancy, congenital malaria incidence fell from 3.2% to 0.2% (odds ratio, 0.07; 95% CI, .03–.15; P < .001).Conclusions. Congenital malaria is an important cause of neonatal morbidity in this region co-endemic for P. falciparum and P. vivax malaria. The introduction of artemisinin-combination therapy was associated with a significant risk reduction in the vertical transmission of malaria.

Plasmodium falciparum ATP6 Not under Selection during Introduction of Artemisinin Combination Therapy in Peru

Bacon et al. describe polymorphisms in a series of drug resistance genes or candidates before and after the introduction of artemisinin combination therapy (ACT) in Peru (1). Longitudinal studies of this type offer a dynamic view of molecular resistance determinants, but confounding factors that change over the course of the study must be removed during analysis. As noted in their paper, isolates collected in 1999 were obtained from two clinics. Sixty-four came from Padre Cocha (5 km from Iquitos) and 40 from Caballococha (311 km from Iquitos); data were pooled for statistical calculations. The 2006-2007 isolates were collected in Iquitos only. This change in composition of the study population between the two time points introduces a significant confounder that appears not to have been recognized in the analyses undertaken, undermining the value of these interesting data. The issue appears critical in the case of the Plasmodium falciparum ATP6 (PfATP6) sequence analysis. In Table 6 of the article, the 1999 data were expressed as a percentage with a total denominator of 104. Using the information provided in the tables in the supplemental material, we undertook a reanalysis of the PfATP6 sequence data, calculating percentages with the number of samples per region as the denominator, thereby removing this confounding factor. (Note that, given the data available, we expressed haplotypes 4 and 5 as a combined total; the single haplotype 5 sample in the study differs from haplotype 4 in the synonymous polymorphism at C1031.) Our reanalysis reveals that, contrary to the reported findings, the frequencies of both the G844 deletion allele and haplotype 6 (G844 deletion in isolation) did not change during the study period in the Iquitos samples (Table ​(Table1).1). The reported increase in haplotype 6 stemmed from the failure to take into account the confounding factor noted above. In Caballococha, the G844 deletion was completely associated with a second mutation, S466N (rendering it haplotype 4/5), unlike the majority of Iquitos area G844 deletion mutants that had the deletion in isolation (haplotype 6). Hence, the reason haplotype 6 became relatively more common is simply because the composition of the study population changed between the two time points; no Caballococha samples entered the 2006-2007 analysis. The result represents an artifact introduced by the analytical approach chosen. TABLE 1. Reanalysis of genotypic data presented by Bacon et al. (1) There is still no consensus as to how artemisinins exert their antimalarial action, a situation that has not been helped by the absence of a paper describing PfATP6 transfection data presented at a conference in early 2008 (2). The problems in data analysis in the paper by Bacon et al. are unfortunate since the authors' carefully collected longitudinal data are a precious resource that can inform this controversial area of research. Reanalysis of the data, taking into account the confounding factor of location, shows that allelic and haplotype frequencies were highly comparable before and after ACT introduction in this area and indicates that PfATP6 was not subjected to selection pressure over this time.

High prevalence of sulfadoxine/pyrimethamine resistance alleles in Plasmodium falciparum parasites from Bangladesh

In Bangladesh, despite the official introduction of artemisinin combination therapy in 2004, chloroquine+sulfadoxine/pyrimethamine has been used for the treatment of uncomplicated malaria. To assess the distribution of pfcrt, pfmdr1, dhfr, and dhps genotypes in Plasmodium falciparum, we conducted hospital- and community-based surveys in Bandarban, Bangladesh (near the border with Myanmar) in 2007 and 2008. Using nested PCR followed by digestion, 139 P. falciparum isolates were genotyped. We found fixation of a mutation at position 76 in pfcrt and low prevalence of a mutation at position 86 in pfmdr1. In dhfr, the highest pyrimethamine resistant genotype quadruple mutant was found in 19% of isolates, which is significantly higher prevalence than reported in a previous study in Khagrachari (1%) in 2002. Microsatellite haplotypes flanking dhfr of the quadruple mutants in Bangladesh were identical or very similar to those found in Thailand and Cambodia, indicating a common origin for the mutant in these countries. These observations suggest that the higher prevalence of the dhfr quadruple mutant in Bandarban is because of parasite migration from Myanmar. However, continuous use of sulfadoxine/pyrimethamine would have also played a role through selection for the dhfr quadruple mutant. These results indicate an urgent need to collect molecular epidemiological information regarding dhfr and dhps genes, and a review of current sulfadoxine/pyrimethamine usage with the aim of avoiding the widespread distribution of high levels of resistant parasites in Bangladesh.

Preliminary formulation of a fixed-dose paediatric combination of artesunate and amodiaquine hydrochloride.

ISince the introduction of artemisinin combination therapy (ACT), it has been recognised that challenges exist in presenting drugs as fixed dose combinations due to potential incompatibilities of the different chemical compounds. The aim of this study was to develop stable prototype formulations combining Artesunate (ART) and Amodiaquine hydrochloride (AMQ). Two fast-disintegrating granular formulations, containing ART and AMQ respectively were produced by wet granulation. Samples were stored as single component formulations or blends in glass vials for periods up to 13 weeks at refrigerated storage conditions (10°C), room temperature (24-26°C) and ambient humidity (in the dark and light conditions), 25°C/75% RH, and 50°C/75% RH. The active agent content of the two drugs was determined using HPLC-UV at 1, 4 and 13 weeks from the start of the study. Statistical analysis of data were undertaken to determine the factors influencing the stability of the formulations, both alone and in combination. The chemical stability of ART was markedly affected by relative humidity, with greatest levels of degradation occurring at 13 weeks after storage at 50°C/75% RH. No significant loss of active agent content was observed for AMQ at any storage conditions over the duration of the study. The results indicate that stable fixed dose granular formulations of ART and AMQ can be produced that are stable under accelerated conditions. These formulations should, however, be protected from extremes of relative humidity using suitable packaging materials to avoid degradation of ART.