Introduction Of Artemisinin-based Combination Therapy Research Articles

Comparative study of Plasmodium falciparum msp-1 and msp-2 Genetic Diversity in Isolates from Rural and Urban Areas in the South of Brazzaville, Republic of Congo.

Polymorphisms in the genes encoding the merozoite surface proteins msp-1 and msp-2 are widely used markers for characterizing the genetic diversity of Plasmodium falciparum. This study aimed to compare the genetic diversity of circulating parasite strains in rural and urban settings in the Republic of Congo after the introduction of artemisinin-based combination therapy (ACT) in 2006. A cross-sectional survey was conducted from March to September 2021 in rural and urban areas close to Brazzaville, during which Plasmodium infection was detected using microscopy (and nested-PCR for submicroscopic infection). The genes coding for merozoite proteins-1 and -2 were genotyped by allele-specific nested PCR. Totals of 397 (72.4%) and 151 (27.6%) P. falciparum isolates were collected in rural and urban areas, respectively. The K1/msp-1 and FC27/msp-2 allelic families were predominant both in rural (39% and 64%, respectively) and urban (45.4% and 54.5% respectively) areas. The multiplicity of infection (MOI) was higher (p = 0.0006) in rural areas (2.9) compared to urban settings (2.4). The rainy season and the positive microscopic infection were associated with an increase in MOI. These findings reveal a higher P. falciparum genetic diversity and MOI in the rural setting of the Republic of Congo, which is influenced by the season and the participant clinical status.

Polymorphism analysis of pfmdr1 gene in Plasmodium falciparum isolates 11\xa0years post-adoption of artemisinin-based combination therapy in Saudi Arabia

A total of 227 Plasmodium falciparum isolates from Jazan region, southwestern Saudi Arabia were amplified for the P. falciparum multi-drug resistance 1 (pfmdr1) gene to detect point mutations 11 years after the introduction of artemisinin-based combination therapy (ACT) in Saudi Arabia. The pfmdr1 86Y mutation was found in 11.5% (26/227) of the isolates while the N86 wild allele was detected in 88.5%. Moreover, 184F point mutations dominated (86.3%) the instances of pfmdr1 polymorphism while no mutation was observed at codons 1034, 1042 and 1246. Three pfmdr1 haplotypes were identified, NFSND (74.9%), NYSND (13.7%) and YFSND (11.4%). Associations of the prevalence of 86Y mutation and YFSND haplotype with participants’ nationality, residency and parasitaemia level were found to be significant (P < 0.05). The findings revealed significant decline in the prevalence of the pfmdr1 86Y mutation in P. falciparum isolates from Jazan region over a decade after the implementation of ACT treatment. Moreover, the high prevalence of the NFSND haplotype might be indicative of the potential emergence of CQ-sensitive but artemether-lumefantrine-resistant P. falciparum strains since the adoption of ACT. Therefore, continuous monitoring of the molecular markers of antimalarial drug resistance in Jazan region is highly recommended.

Trends and predictive factors for treatment failure following artemisinin-based combination therapy among children with uncomplicated malaria in Ghana: 2005\u20132018

BackgroundSince the introduction of artemisinin-based combination therapy (ACT) in Ghana in 2005 there has been a surveillance system by the National Malaria Control Programme (NMCP) and the University of Ghana Noguchi Memorial Institute for Medical Research (UG-NMIMR) to monitor the therapeutic efficacy of ACTs for the treatment of uncomplicated malaria in the country. We report trends and determinants of failure following treatment of Ghanaian children with artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) combinations.MethodsPer protocol analyses as well as cumulative incidence of day 28 treatment failure from Kaplan Meier survival analyses were used to describe trends of failure over the surveillance period of 2005–2018. Univariable and multivariable cox regression analyses were used to assess the determinants of treatment failure over the period.ResultsDay 28 PCR-corrected failure, following treatment with ASAQ, significantly increased from 0.0% in 2005 to 2.0% (95% CI: 1.1–3.6) in 2015 (p = 0.013) but significantly decreased to 0.4% (95% CI: 0.1–1.6) in 2018 (p = 0.039). Failure, following treatment with AL, decreased from 4.5% (95% CI: 2.0–9.4) in 2010 to 2.7% (95% CI: 1.4–5.1) in 2018, though not statistically significant (p = 0.426). Risk of treatment failure, from multivariable cox regression analyses, was significantly lower among children receiving ASAQ compared with those receiving AL (HR = 0.24; 95% CI: 0.11–0.53; p < 0.001); lower among children with no parasitaemia on day 3 compared with those with parasitaemia on day 3 (HR = 0.02; 95% CI: 0.01–0.13; p < 0.001); and higher among children who received ASAQ and had axillary temperature ≥ 37.5 °C on day 1 compared with those with axillary temperature < 37.5 °C (HR = 3.96; 95% CI: 1.61–9.75; p = 0.003).ConclusionsTreatment failures for both ASAQ and AL have remained less than 5% (below WHO’s threshold of 10%) in Ghana since 2005. Predictors of treatment failure that need to be considered in the management of uncomplicated malaria in the country should include type of ACT, day 3 parasitaemia, and day 1 axillary temperature of patients being treated.

Four decades of epidemiological data reveal trajectories towards malaria elimination in Kheda district (Gujarat), western part of India

BackgroundMalaria is a main public health problem in India and was so particularly in the state of Gujarat in the western part of the country. This study assesses the effects...

Absence of Plasmodium falciparum artemisinin resistance gene mutations eleven years after the adoption of artemisinin-based combination therapy in Nigeria

BackgroundThe occurrence of artemisinin resistance (ART)-associated polymorphism of Plasmodium falciparum K13-propeller (pfk13) gene before and after the introduction of artemisinin-based combination therapy (ACT) in two regions of Nigeria was investigated in this study. Regular surveillance is necessary to make a definite conclusion on the emergence and pattern of possible resistance to ART.MethodsThis cross-sectional study was carried out in the Southwestern and Southeastern geopolitical zones of Nigeria. A total of 150, 217, and 475 participants were enrolled for the study in the Southwest (2004_Group A), Southwest (2015_Group B), and southeast (2015_Group C), respectively. Blood samples were collected from the study participants for DNA extraction and a nested PCR for P. falciparum identification. Samples that were positive for P. falciparum were genotyped for the pfk13 gene using the Sanger sequencing method. The single nucleotide polymorphisms were analysed using the Bioedit software.ResultsA total of 116, 125, and 83 samples were positive for P. falciparum, respectively for the samples collected from the Southwest (2004 and 2015) and southeast (2015). Parasite DNA samples collected from febrile children in 2004 (Group A; n = 71) and 2015 (Group B; n = 73) in Osogbo Western Nigeria and 2015_Group C (n = 36) in southeast Nigeria were sequenced successfully. This study did not observe mutations associated with the in vitro resistance in southeast Asia, such as Y493H, R539T, I543T, and C580Y. Two new polymorphisms V520A and V581I were observed in two samples collected in Osogbo, Southwest Nigeria. These two mutations occurred in the year 2004 (Group A) before the introduction of ACT. Six mutations were identified in 17% of the samples collected in southeast Nigeria. One of these mutations (D547G) was non-synonymous, while the remaining (V510V, R515R, Q613Q, E688E, and N458N) were synonymous. Also, one (2%) heterozygote allele was identified at codon 458 in the 2015 (Group C) samples.ConclusionsNone of the mutations observed in this study were previously validated to be associated with ART resistance. These results, therefore, suggest that artemisinin is likely to remain highly effective in treating malaria in the study areas that are malarious zone.

Longitudinal ex vivo and molecular trends of chloroquine and piperaquine activity against Plasmodium falciparum and P. vivax before and after introduction of artemisinin-based combination therapy in Papua, Indonesia

Drug resistant Plasmodium parasites are a major threat to malaria control and elimination. After reports of high levels of multidrug resistant P. falciparum and P. vivax in Indonesia, in 2005, the national first-line treatment policy for uncomplicated malaria was changed in March 2006, to dihydroartemisinin-piperaquine against all species.This study assessed the temporal trends in ex vivo drug susceptibility to chloroquine (CQ) and piperaquine (PIP) for both P. falciparum and P. vivax clinical isolates collected between 2004 and 2018, by using schizont maturation assays, and genotyped a subset of isolates for known and putative molecular markers of CQ and PIP resistance by using Sanger and next generation whole genome sequencing.The median CQ IC50 values varied significantly between years in both Plasmodium species, but there was no significant trend over time. In contrast, there was a significant trend for increasing PIP IC50s in both Plasmodium species from 2010 onwards. Whereas the South American CQ resistant 7G8 pfcrt SVMNT isoform has been fixed since 2005 in the study area, the pfmdr1 86Y allele frequencies decreased and became fixed at the wild-type allele in 2015. In P. vivax isolates, putative markers of CQ resistance (no pvcrt-o AAG (K10) insertion and pvmdr1 Y967F and F1076L) were fixed at the mutant alleles since 2005. None of the putative PIP resistance markers were detected in P. falciparum.The ex vivo drug susceptibility and molecular analysis of CQ and PIP efficacy for P. falciparum and P. vivax after 12 years of intense drug pressure with DHP suggests that whilst the degree of CQ resistance appears to have been sustained, there has been a slight decline in PIP susceptibility, although this does not appear to have reached clinically significant levels. The observed decreasing trend in ex vivo PIP susceptibility highlights the importance of ongoing surveillance.

Polymorphisms in Plasmodium falciparum dihydropteroate synthetase and dihydrofolate reductase genes in Nigerian children with uncomplicated malaria using high-resolution melting technique

In 2005, the Nigerian Federal Ministry of Health revised the treatment policy for uncomplicated malaria with the introduction of artemisinin-based combination therapies (ACTs). This policy change discouraged the use of Sulphadoxine-pyrimethamine (SP) as the second-line treatment of uncomplicated falciparum malaria. However, SP is used as an intermittent preventive treatment of malaria in pregnancy (IPTp) and seasonal malaria chemoprevention (SMC) in children aged 3–59 months. There have been increasing reports of SP resistance especially in the non-pregnant population in Nigeria, thus, the need to continually monitor the efficacy of SP as IPTp and SMC by estimating polymorphisms in dihydropteroate synthetase (dhps) and dihydrofolate reductase (dhfr) genes associated with SP resistance. The high resolution-melting (HRM) assay was used to investigate polymorphisms in codons 51, 59, 108 and 164 of the dhfr gene and codons 437, 540, 581 and 613 of the dhps gene. DNA was extracted from 271 dried bloodspot filter paper samples obtained from children (< 5 years old) with uncomplicated malaria. The dhfr triple mutant I51R59N108, dhps double mutant G437G581 and quadruple dhfr I51R59N108 + dhps G437 mutant haplotypes were observed in 80.8%, 13.7% and 52.8% parasites, respectively. Although the quintuple dhfr I51R59N108 + dhps G437E540 and sextuple dhfr I51R59N108 + dhps G437E540G581 mutant haplotypes linked with in-vivo and in-vitro SP resistance were not detected, constant surveillance of these haplotypes should be done in the country to detect any change in prevalence.

Efficacy of artemisinin-lumefantrine for treatment of uncomplicated malaria after more than a decade of its use in Kenya.

The resistance of Plasmodium falciparum to antimalarial drugs remains a major impairment in the treatment and eradication of malaria globally. Following the introduction of artemisinin-based combination therapy (ACT), there have been reports of delayed parasite clearance. In Kenya, artemether-lumefantrine (AL) is the recommended first-line treatment of uncomplicated malaria. This study sought to assess the efficacy of AL after a decade of use as the preferred method of managing malarial infections in Kenya. We assessed clinical and parasitological responses of children under 5 years between May and November 2015 in Chulaimbo sub-County, Kisumu, Kenya. Patients aged between 6 and 60 months with uncomplicated P. falciparum mono-infection, confirmed through microscopy, were enrolled in the study. The patients were admitted at the facility for 3 days, treated with a standard dose of AL, and then put under observation for the next 28 days for the assessment of clinical and parasitological responses. Of the 90 patients enrolled, 14 were lost to follow-up while 76 were followed through to the end of the study period. Seventy-five patients (98.7%) cleared the parasitaemia within a period of 48 h while one patient (1.3%) cleared on day 3. There was 100% adequate clinical and parasitological response. All the patients cleared the parasites on day 3 and there were no re-infections observed during the stated follow-up period. This study, therefore, concludes that AL is highly efficacious in clearing P. falciparum parasites in children aged ≥6 and ≤60 months. The study, however, underscores the need for continued monitoring of the drug to forestall both gradual ineffectiveness and possible resistance to the drug in all target users.

Ex-vivo Sensitivity of Plasmodium falciparum to Common Anti-malarial Drugs: The Case of Kéniéroba, a Malaria Endemic Village in Mali.

BackgroundIn 2006, the National Malaria Control Program in Mali recommended artemisinin-based combination therapy as the first-line treatment for uncomplicated malaria. Since the introduction of artemisinin-based combination therapy, few reports are available on the level of resistance of Plasmodium falciparum to the most common anti-malarial drugs in Mali.MethodsFrom 2016 to 2017, we assessed the ex-vivo drug sensitivity of P. falciparum isolates in Kéniéroba, a village located in a rural area of southern Mali. We collected P. falciparum isolates from malaria-infected children living in Kéniéroba. The isolates were tested for ex-vivo sensitivity to commonly used anti-malarial drugs, namely chloroquine, quinine, amodiaquine, mefloquine, lumefantrine, dihydroartermisinin, and piperaquine. We used the 50% inhibitory concentration determination method, which is based on the incorporation of SYBR® Green into the parasite’s genetic material.ResultsPlasmodium falciparum isolates were found to have a reduced ex-vivo sensitivity to quinine (25.7%), chloroquine (12.2%), amodiaquine (2.7%), and mefloquine (1.3%). In contrast, the isolates were 100% sensitive to lumefantrine, dihydroartermisinin, and piperaquine. A statistically significant correlation was found between 50% inhibitory concentration values of quinine and amodiaquine (r = 0.80; p < 0.0001).ConclusionsPlasmodium falciparum isolates were highly sensitive to dihydroartermisinin, lumefantrine, and piperaquine and less sensitive to amodiaquine (n = 2), mefloquine (n = 1), and quinine (n = 19). Therefore, our data support the previously reported increasing trend in chloroquine sensitivity in Mali.

Prevalence of Plasmodium falciparum Chloroquine Resistance Transporter (Pfcrt76T) Mutation Associated with Antimalarial Drug Resistance in Two Different Epidemiological Setting (Banfora and Saponé) in Burkina Faso Few Years after the Implementation of Artemisnine Based Combination Therapy (ACTs)

Introduction: In spite of considerable progress, malaria remains a public health problem in many areas, particularly in sub-Saharan Africa. One major complexity of malaria disease is caused by the development and the spread of vector and parasite resistance to insecticides and antimalarial drugs respectively. The Pfcrt76T gene mutation has been validated as a marker conferring resistance to chloroquine and other antimalarial drugs. The extension of Plasmodium falciparum resistance to commonly used antimalarial drugs (chloroquine, sulfadoxine-pyrimethamine) led to the adoption and the use of artemisinin-based combinations in Burkina Faso since 2005.&#x0D; Aims: The present study was initiated to assess the prevalence of the Pfcrt76T mutation in two different malaria epidemiological setting after a decade of introduction of artemisinin-based combination therapies (ACTs) in Burkina Faso. &#x0D; Methodology: The study population consisted of 181 uncomplicated malaria patients recruited in Banfora and Saponé health districts in 2012 and 2013. Blood samples were collected from finger prick on filter paper, dried and sent to the Molecular Biology Laboratory at Centre National de Recherche et de Formation sur le Paludisme (CNRFP) for molecular analyzes. DNA of Plasmodium falciparum was extracted with DNA extraction kit (Qiagen®) and the Pfcrt76T mutation was determined based on Polymerase Chain Reaction / Restriction Fragment Length Polymorphism technique (RFLP).&#x0D; Results: The results of this study showed that the frequency of the pfcrt76T mutant allele (33.7%) was statistically lower than the Pfcrt76K wild-type allele (57.4%) in the study area. Moreover, the prevalence of Pfcrt76T mutation was neither associated with the patient age nor with the parasite density while a significant difference was observed between the two epidemiological setting, Banfora and Saponé.&#x0D; Conclusion: The findings of this study has shown a drop in the prevalence of mutant parasites Pfcrt76T in both the study area eight years after the introduction of ACTs compared to previous studies.

From high to low malaria transmission in Zanzibar\u2014challenges and opportunities to achieve elimination

BackgroundSubstantial global progress in the control of malaria in recent years has led to increased commitment to its potential elimination. Whether this is possible in high transmission areas of sub-Saharan Africa remains unclear. Zanzibar represents a unique case study of such attempt, where modern tools and strategies for malaria treatment and vector control have been deployed since 2003.MethodsWe have studied temporal trends of comprehensive malariometric indices in two districts with over 100,000 inhabitants each. The analyses included triangulation of data from annual community-based cross-sectional surveys, health management information systems, vital registry and entomological sentinel surveys.ResultsThe interventions, with sustained high-community uptake, were temporally associated with a major malaria decline, most pronounced between 2004 and 2007 and followed by a sustained state of low transmission. In 2015, the Plasmodium falciparum community prevalence of 0.43% (95% CI 0.23–0.73) by microscopy or rapid diagnostic test represented 96% reduction compared with that in 2003. The P. falciparum and P. malariae prevalence by PCR was 1.8% (95% CI 1.3–2.3), and the annual P. falciparum incidence was estimated to 8 infections including 2.8 clinical episodes per 1000 inhabitants. The total parasite load decreased over 1000-fold (99.9%) between 2003 and 2015. The incidence of symptomatic malaria at health facilities decreased by 94% with a trend towards relatively higher incidence in age groups > 5 years, a more pronounced seasonality and with reported travel history to/from Tanzania mainland as a higher risk factor. All-cause mortality among children < 5 years decreased by 72% between 2002 and 2007 mainly following the introduction of artemisinin-based combination therapies whereas the main reduction in malaria incidence followed upon the vector control interventions from 2006. Human biting rates decreased by 98% with a major shift towards outdoor biting by Anopheles arabiensis.ConclusionsZanzibar provides new evidence of the feasibility of reaching uniquely significant and sustainable malaria reduction (pre-elimination) in a previously high endemic region in sub-Saharan Africa. The data highlight constraints of optimistic prognostic modelling studies. New challenges, mainly with outdoor transmission, a large asymptomatic parasite reservoir and imported infections, require novel tools and reoriented strategies to prevent a rebound effect and achieve elimination.

Absence of Plasmodium falciparum K13 Propeller Domain Polymorphisms among Field Isolates Collected from the Brazilian Amazon Basin between 1984 and 2011.

Artemisinin resistance, presently confined to Southeast Asia and associated with mutations in the Plasmodium falciparum K13 (PfK13) propeller domain, represents a serious threat to global malaria control. This study aimed to provide baseline information for future artemisinin resistance surveillance, by analyzing the PfK13 propeller domain in P. falciparum field isolates collected from the Brazilian Amazon Basin between 1984 and 2011. A total of 152 P. falciparum mono-infections were assessed, of which 118 (78%) were collected before and 34 (22%) after the introduction of artemisinin-based combination therapy (ACT) in 2006. An 849-base pair fragment encoding the PfK13 propeller was amplified by nested polymerase chain reaction and sequenced in both directions. The sequences were compared with the reference sequence of P. falciparum 3D7. All samples showed wild-type sequences, thus, no mutations were observed. The results are in agreement with other recent reports and do not provide evidence for presence of PfK13 propeller domain polymorphisms associated with artemisinin resistance among P. falciparum field isolates in the Brazilian Amazon Basin neither before nor after the implementation of ACT.

Prevalence of Plasmodium falciparum antimalarial drug resistance genes in Southeastern Gabon from 2011 to 2014.

PurposeThe introduction of artemisinin-based combination therapies (ACTs) in treating uncomplicated malaria and sulfadoxine–pyrimethamine (SP) as intermittent preventive treatment during pregnancy drastically decreased the burden of malarial disease around the world. However, ACTs are known to select for drug resistance markers. In Gabon, artemether–lumefantrine induced an increase in the prevalence of N86-Pfmdr1, which is associated with treatment failure. However, little data are available regarding resistance markers in Southeastern Gabon. This study aimed to evaluate the evolution of resistance haplotypes in the Pfcrt, Pfdhps, Pfdhfr, and PfK13 genes from 2011 to 2014 in Southeastern Gabon.MethodsA total of 233 Plasmodium falciparum DNA samples were collected from febrile pediatric patients in South Gabon: Franceville, an urban area; Koulamoutou, a semi-urban area; and Lastourville, a rural area. Pfcrt, Pfdhps, Pfdhfr, and the propeller domain of PfK13 were sequenced for all isolates.ResultsThe overall prevalence (3.7%–11.5%) of the wild-type haplotype Pfcrt 72-76 CVMNK was not significantly different between 2011 and 2014 in Southeast Gabon. For Pfdhfr (codons 51, 59, 108, 164), the IRNI triple-mutant haplotype was the most prevalent (>89.0%). The ICNI and NCNI mutant haplotypes and the NCSI wild-type haplotype showed a minor prevalence. There were no differences in the distributions of these haplotypes across the 4 years and the three study sites. For Pfdhps, the AAKAA and SGKAA mutant haplotypes and the SAKAA wild-type haplotype were similarly present in the three areas during the study period. The AGKAA double mutant was first observed in 2013 in Franceville and in 2014 in Koulamoutou and Lastourville. Interestingly, only the A578S mutation (0.4%) and two new A494V (0.4%) and V504A (0.9%) mutations were found in PfK13.ConclusionDespite the withdrawal of chloroquine, the frequency of the resistant allele 76T remained high in the south of Gabon. Moreover, a high level of resistant haplotypes against IPTp-SP was found.

Reduction of malaria prevalence after introduction of artemisinin-combination-therapy in Mbeya Region, Tanzania: results from a cohort study with 6773 participants

BackgroundA marked decline in malaria morbidity and mortality has been reported after the introduction of artemisinin-based combination therapy (ACT) in high malaria prevalence countries in Africa. Data on the impact of ACT and on the prevalence of malaria has so far been scarce for Southwest Tanzania.MethodsBetween 2005 and 2011, a large general population cohort in the Mbeya Region in the south-west of Tanzania has been surveyed within the EMINI-study (Evaluation and Monitoring of the Impact of New Interventions). Participants were examined once per year, including rapid diagnostic testing for malaria. ACT was introduced in the region according to national guidelines in the time period 2006/2007, replacing sulfadoxine/pyrimethamine as first-line therapy. In four study sites, 6773 individuals who participated in the first two of three consecutive survey visits in the period from 2006 to 2009 were included in this analysis. The prevalence of Plasmodium infection prior to and after the introduction of ACT was compared by logistic regression, with consideration of climatic variability, age, sex, socio-economic status and bed net use as potential confounders.ResultsA significant reduction over time in the prevalence of Plasmodium falciparum infection from 2.5 to 0.3% was shown across the four study sites. The decline was not explained by other factors included in the analysis, therefore, the decline over time most likely reflects the impact of introduction of ACT in the study area.ConclusionsThe longitudinal study showed a significant and relevant decline in the prevalence of P. falciparum infection after introduction of ACT, which could not be explained by potential confounders. The data suggests that artemisinin-based combinations are not only an effective instrument for reduction of immediate morbidity and mortality, but also for reduction of transmission rates.

Insecticide-treated nets and malaria prevalence, Papua New Guinea, 2008-2014.

ObjectiveTo investigate changes in malaria prevalence in Papua New Guinea after the distribution of long-lasting Insecticide-treated nets, starting in 2004, and the introduction of artemisinin-based combination therapy in 2011.MethodsTwo malaria surveys were conducted in 2010–2011 and 2013–2014. They included 77 and 92 randomly selected villages, respectively. In each village, all members of 30 randomly selected households gave blood samples and were assessed for malaria infection by light microscopy. In addition, data were obtained from a malaria survey performed in 2008–2009.ResultsThe prevalence of malaria below 1600 m in altitude decreased from 11.1% (95% confidence interval, CI: 8.5–14.3) in 2008–2009 to 5.1% (95% CI 3.6–7.4) in 2010–2011 and 0.9% (95% CI 0.6–1.5) in 2013–2014. Prevalence decreased with altitude. Plasmodium falciparum was more common than P. vivax overall, but not everywhere, and initially the prevalence of P. vivax infection decreased more slowly than P. falciparum infection. Malaria infections were clustered in households. In contrast to findings in 2008–2009, no significant association between net use and prevalence was found in the later two surveys. The prevalence of both fever and splenomegaly also decreased but their association with malaria infection became stronger.ConclusionLarge-scale insecticide-treated net distribution was associated with an unprecedented decline in malaria prevalence throughout Papua New Guinea, including epidemic-prone highland areas. The decline was accompanied by broader health benefits, such as decreased morbidity. Better clinical management of nonmalarial fever and research into residual malaria transmission are required.

Ex vivo susceptibility and genotyping of Plasmodium falciparum isolates from Pikine, Senegal

BackgroundThe monitoring of Plasmodium falciparum sensitivity to anti-malarial drugs is a necessity for effective case management of malaria. This species is characterized by a strong resistance to anti-malarial drugs. In Senegal, the first cases of chloroquine resistance were reported in the Dakar region in 1988 with nearly 7% population prevalence, reaching 47% by 1990. It is in this context that sulfadoxine–pyrimethamine temporarily replaced chloroquine as first line treatment in 2003, pending the introduction of artemisinin-based combination therapy in 2006. The purpose of this study is to assess the ex vivo sensitivity to different anti-malarial drugs of the P. falciparum population from Pikine.MethodsFifty-four samples were collected from patients with non-complicated malaria and aged between 2 and 20 years in the Deggo health centre in Pikine in 2014. An assay in which parasites are stained with 4′, 6-di-amidino-2-phenylindole (DAPI), was used to study the ex vivo sensitivity of isolates to chloroquine, amodiaquine, piperaquine, pyrimethamine, and dihydroartemisinin. High resolution melting was used for genotyping of pfdhps, pfdhfr, pfmdr1, and pfcrt genes.ResultsThe mean IC50s of chloroquine, amodiaquine, piperaquine, dihydroartemisinin, and pyrimethamine were, respectively, 39.44, 54.02, 15.28, 2.23, and 64.70 nM. Resistance mutations in pfdhfr gene, in codon 437 of pfdhps gene, and an absence of mutation at position 540 of pfdhps were observed. Mutations in codons K76T of pfcrt and N86Y of pfmdr1 were observed at 51 and 11% population prevalence, respectively. A relationship was found between the K76T and N86Y mutations and ex vivo resistance to chloroquine.ConclusionAn increase in sensitivity of isolates to chloroquine was observed. A high sensitivity to dihydroartemisinin was observed; whereas, a decrease in sensitivity to pyrimethamine was observed in the parasite population from Pikine.

Molecular surveillance of Plasmodium falciparum drug resistance in the Republic of Congo: four and nine years after the introduction of artemisinin-based combination therapy

BackgroundResistance to anti-malarial drugs hinders efforts on malaria elimination and eradication. Following the global spread of chloroquine-resistant parasites, the Republic of Congo adopted artemisinin-based combination therapy (ACT) in 2006 as a first-line treatment for uncomplicated malaria. To assess the impacts after implementation of ACT, a molecular surveillance for anti-malarial drug resistance was conducted in Congo 4 and 9 years after the introduction of ACT.MethodsBlood samples of 431 febrile children aged 1–10 years were utilized from two previous studies conducted in 2010 (N = 311) and 2015 (N = 120). All samples were screened for malaria parasites using nested PCR. Direct sequencing was used to determine the frequency distribution of genetic variants in the anti-malarial drug-resistant Plasmodium falciparum genes (Pfcrt, Pfmdr1, Pfatp6, Pfk13) in malaria-positive isolates.ResultsOne-hundred and nineteen (N = 70 from 2010 and N = 49 from 2015) samples were positive for P. falciparum. A relative decrease in the proportion of chloroquine-resistant haplotype (CVIET) from 100% in 2005, 1 year before the introduction and implementation of ACT in 2006, to 98% in 2010 to 71% in 2015 was observed. Regarding the multidrug transporter gene, a considerable reduction in the frequency of the mutations N86Y (from 73 to 27%) and D1246Y (from 22 to 0%) was observed. However, the prevalence of the Y184F mutation remained stable (49% in 2010 compared to 54% in 2015). Isolates carrying the Pfatp6 H243Y was 25% in 2010 and this frequency was reduced to null in 2015. None of the parasites harboured the Pfk13 mutations associated with prolonged artemisinin clearance in Southeast Asia. Nevertheless, 13 new Pfk13 variants are reported among the investigated isolates.ConclusionThe implementation of ACT has led to the decline in prevalence of chloroquine-resistant parasites in the Republic of Congo. However, the constant prevalence of the PfMDR1 Y184F mutation, associated with lumefantrine susceptibility, indicate a selective drug pressure still exists. Taken together, this study could serve as the basis for epidemiological studies monitoring the distribution of molecular markers of artemisinin resistance in the Republic of Congo.

Molecular Epidemiology of Malaria in Cameroon and Côte d'Ivoire. XXXI. Kelch 13 Propeller Sequences in Plasmodium falciparum Isolates before and after Implementation of Artemisinin-Based Combination Therapy.

Artemisinin-resistant malaria has not been reported from Africa, but resistance can possibly spread from Asia or arise independently in Africa. The emergence of artemisinin resistance in Africa can be monitored by molecular assay of Kelch 13 (K13) propeller sequences. A total of 251 archived DNA samples of Plasmodium falciparum isolates collected in 2002, 2003, and 2006 in Yaounde, Cameroon, and 47 samples collected in 2006 and 2013 in Abidjan, Côte d'Ivoire, were analyzed for K13-propeller sequence polymorphism. Only one isolate carried a mutant K13-propeller allele (E602D). None of the isolates carried the key mutant alleles (Y493H, R539T, I543T, and C580Y) associated with artemisinin resistance in Cambodia. The presence of the mutant allele was not correlated with in vitro response to dihydroartemisinin determined by the classical hypoxanthine incorporation assay. There was no evidence of K13 mutations associated with artemisinin resistance before and soon after the introduction of artemisinin-based combination therapies in Cameroon and Côte d'Ivoire.

Investigating antimalarial drug interactions of emetine dihydrochloride hydrate using CalcuSyn-based interactivity calculations.

The widespread introduction of artemisinin-based combination therapy has contributed to recent reductions in malaria mortality. Combination therapies have a range of advantages, including synergism, toxicity reduction, and delaying the onset of resistance acquisition. Unfortunately, antimalarial combination therapy is limited by the depleting repertoire of effective drugs with distinct target pathways. To fast-track antimalarial drug discovery, we have previously employed drug-repositioning to identify the anti-amoebic drug, emetine dihydrochloride hydrate, as a potential candidate for repositioned use against malaria. Despite its 1000-fold increase in in vitro antimalarial potency (ED50 47 nM) compared with its anti-amoebic potency (ED50 26–32 uM), practical use of the compound has been limited by dose-dependent toxicity (emesis and cardiotoxicity). Identification of a synergistic partner drug would present an opportunity for dose-reduction, thus increasing the therapeutic window. The lack of reliable and standardised methodology to enable the in vitro definition of synergistic potential for antimalarials is a major drawback. Here we use isobologram and combination-index data generated by CalcuSyn software analyses (Biosoft v2.1) to define drug interactivity in an objective, automated manner. The method, based on the median effect principle proposed by Chou and Talalay, was initially validated for antimalarial application using the known synergistic combination (atovaquone-proguanil). The combination was used to further understand the relationship between SYBR Green viability and cytocidal versus cytostatic effects of drugs at higher levels of inhibition. We report here the use of the optimised Chou Talalay method to define synergistic antimalarial drug interactivity between emetine dihydrochloride hydrate and atovaquone. The novel findings present a potential route to harness the nanomolar antimalarial efficacy of this affordable natural product.

High Frequency of Blackwater Fever Among Children Presenting to Hospital With Severe Febrile Illnesses in Eastern Uganda.

In the Fluid Expansion as a Supportive Treatment (FEAST) trial, an unexpectedly high proportion of participants from eastern Uganda presented with blackwater fever (BWF). We describe the prevalence and outcome of BWF among trial participants and compare the prevalence of 3 malaria-protective red blood cell polymorphisms in BWF cases vs both trial (non-BWF) and population controls. Of 3170 trial participants, 394 (12.4%) had BWF. The majority (318 [81.0%]) presented in eastern Uganda and were the subjects of further analysis. BWF cases typically presented with both clinical jaundice (254/318 [80%]) and severe anemia (hemoglobin level <5 g/dL) (238/310 [77%]). Plasmodium falciparum parasitemia was less frequent than in non-BWF controls, but a higher proportion were positive for P. falciparum histidine rich protein 2 (192/246 [78.0%]) vs 811/1154 [70.3%]; P = .014), suggesting recent antimalarial treatment. Overall, 282 of 318 (88.7%) received transfusions, with 94 of 282 (33.3%) and 9 of 282 (3.4%) receiving 2 or 3 transfusions, respectively. By day 28, 39 of 318 (12.3%) BWF cases and 154 of 1554 (9.9%) non-BWF controls had died (P = .21), and 7 of 255 (3.0%) vs 13/1212 (1%), respectively, had severe anemia (P = .036). We found no association with G6PD deficiency. The prevalence of both the sickle cell trait (10/218 [4.6%]) and homozygous α+thalassemia (8/216 [3.7%]) were significantly lower among cases than among population controls (334/2123 [15.7%] and 141/2114 [6.6%], respectively), providing further support for the role of malaria. We report the emergence of BWF in eastern Uganda, a condition that, according to local investigators, was rare until the last 7 years. We speculate that this might relate to the introduction of artemisinin-based combination therapies. Further studies investigating this possibility are urgently required.