Deployment of ACT antimalarials for treatment of malaria: challenges and opportunities

Background

In late 2002, the health authorities of Mozambique implemented sulphadoxine-pyrimethamine (SP)/amodiaquine (AQ) as first-line treatment against uncomplicated falciparum malaria. In 2004, this has been altered to SP/artesunate in line with WHO recommendations of using Artemisinin Combination Therapies (ACTs), despite the fact that all the neighbouring countries have abandoned SP-drug combinations due to high levels of SP drug resistance. In the study area, one year prior to the change to SP/AQ, SP alone was used to treat uncomplicated malaria cases. The study described here investigated the immediate impact of the change to SP on the frequency of SP and CQ resistance-related haplotypes in the Plasmodium falciparum genes Pfdhfr, Pfdhps and Pfcrt before and a year after the introduction of SP.

Methods

Samples were collected during two cross sectional surveys in early 2002 and 2003 involving 796 and 692 children one year or older and adults randomly selected living in Maciana, an area located in Manhiça district, Southern Mozambique. Out of these, 171 and 173 P. falciparum positive samples were randomly selected to measure the frequency of resistance- related haplotypes in Pfdhfr, Pfdhps and Pfcrt based on results obtained by nested PCR followed by sequence-specific oligonucleotide probe (SSOP)-ELISA.

Results

The frequency of the SP-resistance associated Pfdhps double mutant (SGEAA) haplotype increased significantly from 14% to 35% (P < 0.001), while the triple mutant Pfdhfr haplotype (CIRNI) remained high and only changed marginally from 46% to 53% (P = 0.405) after one year with SP as first-line treatment in the study area. Conversely, the combined Pfdhfr/Pfdhps quintuple mutant haplotype increased from 8% to 26% (P = 0.005). The frequency of the chloroquine resistance associated Pfcrt -CVIET haplotype was above 90% in both years.

Conclusion

These retrospective findings add to the general concern on the lifespan of the combination of SP/artesunate in Mozambique. The high frequency of quintuple Pfdhfr/Pfdhps haplotypes found here as early as 2002 most likely cause ideal conditions for the development of artesunate tolerance in the P. falciparum populations and may even endanger the sensitivity to the second-line drug, Coartem^®.     

Declining trend of Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutant alleles after the withdrawal of Sulfadoxine-Pyrimethamine in North Western Ethiopia

Antimalarial drug resistance is one of the major challenges in global efforts of malaria control and elimination. In 1998, chloroquine was abandoned and replaced with sulfadoxine/pyrimethamine, which in turn was replaced with artemether/lumefantrine for the treatment of uncomplicated falciparum malaria in 2004. Sulfadoxine/pyrimethamine resistance is associated with mutations in dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes. The prevalence of mutation in Pfdhfr and Pfdhps genes were evaluated and compared for a total of 159 isolates collected in two different time points, 2005 and 2007/08, from Pawe hospital, in North Western Ethiopia. The frequency of triple Pfdhfr mutation decreased significantly from 50.8% (32/63) to 15.9% (10/63) (P<0.001), while Pfdhps double mutation remained high and changed only marginally from 69.2% (45/65) to 55.4% (40/65) (P = 0.08). The combined Pfdhfr/Pfdhps quintuple mutation, which is strongly associated with sulfadoxine/pyrimethamine resistance, was significantly decreased from 40.7% (24/59) to 13.6% (8/59) (P<0.0001). On the whole, significant decline in mutant alleles and re-emergence of wild type alleles were observed. The change in the frequency is explained by the reduction of residual drug-resistant parasites caused by the strong drug pressure imposed when sulfadoxine/pyrimethamine was the first-line drug, followed by lower fitness of these resistant parasites in the absence of drug pressure. Despite the decrease in the frequency of mutant alleles, higher percentages of mutation remain prevalent in the study area in 2007/08 in both Pfdhfr and Pfdhps genes. Therefore, further multi-centered studies in different parts of the country will be required to assess the re-emergence of sulfadoxine/pyrimethamine sensitive parasites and to monitor and prevent the establishment of multi drug resistant parasites in this region.     

Correlation of Molecular Markers,Pfmdr1-N86Y and Pfcrt-K76T,with In Vitro Chloroquine Resistant Plasmodium falciparum,Isolated in the Malaria Endemic States of Assam and Arunachal Pradesh,Northeast India

The mechanism of chloroquine (CQ) resistance in Plasmodium falciparum is not clearly understood. However, CQ resistance has been shown to be associated with point mutations in Pfcrt and Pfmdr1. These genes encode for digestive vacuole transmembrane proteins Pfcrt and Pgh1, respectively. The present study was carried out to analyze the association of Pfcrt-K76T and Pfmdr1-N86Y mutations with CQ resistance in Northeast Indian P. falciparum isolates. 115 P. falciparum isolates were subjected to in vitro CQ sensitivity testing and PCR-RFLP analysis for the Pfmdr1-N86Y and Pfcrt-K76T mutations. 100 isolates of P. falciparum were found to be resistant to CQ by the in vitro susceptibility test (geometric mean EC₅₀ 2.21 µM/L blood) while 15 were found to be CQ sensitive (geometric mean EC₅₀ 0.32 µM/L blood). All the CQ resistant isolates showed the presence of Pfmdr1 and Pfcrt mutations. CQ sensitive isolates were negative for these mutations. Strong linkage disequilibrium was observed between the alleles at these two loci (Pfmdr1-N86Y and Pfcrt-K76T). The results indicate that Pfmdr1-N86Y and Pfcrt-K76T mutations can be used as molecular markers to identify CQ resistance in P. falciparum. The result necessitates the evaluation of CQ in vivo therapeutic efficacy in endemic areas for more effective malaria control strategies.     

Selection of antimalarial drug resistance after intermittent preventive treatment of infants and children (IPTi/c) in Senegal

Senegal has since 2003 used sulphadoxine-pyrimethamine (SP) for Intermittent Preventive Treatment (IPT) of malaria in risk groups. However, the large-scale IPT strategy may result in increasing drug resistance. Our study investigated the possible impact of SP-IPT given to infants and children on the prevalence of SP-resistant haplotypes in the Plasmodium falciparum genes Pfdhfr and Pfdhps, comparing sites with and without IPTi/c. P. falciparum positives samples (n = 352) were collected from children under 5 years of age during two cross-sectional surveys in 2010 and 2011 in three health districts (two on IPTi/c and one without IPTi/c intervention) located in the southern part of Senegal. The prevalence of SP-resistance-related haplotypes in Pfdhfr and Pfdhps was determined by nested PCR followed by sequence-specific oligonucleotide probe (SSOP)–ELISA. The prevalence of the Pfdhfr double mutant haplotypes (CNRN and CICN) was stable between years at < 10% in the control group (P = 0.69), while it rose significantly in the IPTi/c group from 2% in 2010 to 20% in 2011 (P = 0.008). The prevalence of the Pfdhfr triple mutant haplotype (CIRN) increased in both groups, but only significantly in the IPTi/c group from 41% to 65% in 2011 (P = 0.005). Conversely, the Pfdhps 437G mutation decreased in both groups from 44.6% to 28.6% (P = 0.07) and from 66.7% to 47.5% (P = 0.02) between 2010 and 2011 in the control and the IPTi/c groups, respectively. Combined with Pfdhfr, there was a weak trend for decreasing prevalence of quadruple mutants (triple Pfdhfr + Pfdhps 437G) in both groups (P = 0.15 and P = 0.34). During the two cross-sectional surveys, some significant changes were observed in the SP-resistance-related genes. However, since these changes were observed in the two groups, the IPTi/c strategy does only seem to have limited impact on resistance development and other factors as well. However, continuous monitoring will be needed, due to the up-scaling of the IPTi/c strategy in Senegal according to WHO recommendations.     

Anti-folate quintuple mutations in Plasmodium falciparum asymptomatic infections in Yaoundé, Cameroon

A major challenge in the fight to effectively control malaria is the emergence of resistant parasite to drugs used in therapy as well as for chemoprevention. In this study, single nucleotide polymorphisms (SNPs) associated with Plasmodium falciparum resistance to sulfadoxine-pyrimethamine (SP), one of the partner drugs in artemisinin-based therapies (ACTs) were studied in asymptomatic P. falciparum isolates from Cameroon. Dried Blood spots were collected from children with asymptomatic malaria enrolled during a household survey. The P. falciparum dihydrofolate reductase (Pfdhfr), dihydropteroate synthase (Pfdhps) and Kelch 13 genes were amplified and point mutations in these gene sequences were analyzed by sequencing. Among a total of 234 samples collected, 51 showed parasitaemia after microscopic examination of which 47 were P. falciparum mono-infections. Molecular analysis revealed 97.3% of mutant alleles at codons 51I, 59R and 108 N in Pfdhfr gene. In Pfdhps gene the most common mutation was 437G (83.3%); followed by 436A (47.6%) and 436F (28.6%). The association of mutations in the two genes (dhfr + dhps) showed 11 different haplotypes including three sextuple mutants (IRNI + AGKGA, IRNI + AAKGS, IRNI + AGKAS) and one septuple mutant (IRNI + AGKGS). For K13 gene no SNPs were seen in the studied asymptomatic malaria samples. The findings revealed presence of SP-resistant alleles in asymptomatic infected individuals with presence of sextuples and septuple SNPs. This emphasizes that regular profiling of antimalarial drugs resistance markers in such population is essential for malaria control and elimination programmes.     

Chloroquine clinical failures in P. falciparum malaria are associated with mutant Pfmdr-1, not Pfcrt in Madagascar

Molecular studies have demonstrated that mutations in the Plasmodium falciparum chloroquine resistance transporter gene (Pfcrt) play a major role in chloroquine resistance, while mutations in P. falciparum multidrug resistance gene (Pfmdr-1) act as modulator. In Madagascar, the high rate of chloroquine treatment failure (44%) appears disconnected from the overall level of in vitro CQ susceptibility (prevalence of CQ-resistant parasites <5%) or Pfcrt mutant isolates (<1%), strongly contrasting with sub-Saharan African countries. Previous studies showed a high frequency of Pfmdr-1 mutant parasites (>60% of isolates), but did not explore their association with P. falciparum chloroquine resistance. To document the association of Pfmdr-1 alleles with chloroquine resistance in Madagascar, 249 P. falciparum samples collected from patients enrolled in a chloroquine in vivo efficacy study were genotyped in Pfcrt/Pfmdr-1 genes as well as the estimation of the Pfmdr-1 copy number. Except 2 isolates, all samples displayed a wild-type Pfcrt allele without Pfmdr-1 amplification. Chloroquine treatment failures were significantly associated with Pfmdr-1 86Y mutant codon (OR = 4.6). The cumulative incidence of recurrence of patients carrying the Pfmdr-1 86Y mutation at day 0 (21 days) was shorter than patients carrying Pfmdr-1 86N wild type codon (28 days). In an independent set of 90 selected isolates, in vitro susceptibility to chloroquine was not associated with Pfmdr-1 polymorphisms. Analysis of two microsatellites flanking Pfmdr-1 allele showed that mutations occurred on multiple genetic backgrounds. In Madagascar, Pfmdr-1 polymorphism is associated with late chloroquine clinical failures and unrelated with in vitro susceptibility or Pfcrt genotype. These results highlight the limits of the current in vitro tests routinely used to monitor CQ drug resistance in this unique context. Gaining insight about the mechanisms that regulate polymorphism in Pfmdr1 remains important, particularly regarding the evolution and spread of Pfmdr-1 alleles in P. falciparum populations under changing drug pressure which may have important consequences in terms of antimalarial use management.     

Effect of transmission reduction by insecticide-treated bednets (ITNs) on antimalarial drug resistance in western Kenya

Despite the clear public health benefit of insecticide-treated bednets (ITNs), the impact of malaria transmission-reduction by vector control on the spread of drug resistance is not well understood. In the present study, the effect of sustained transmission reduction by ITNs on the prevalence of Plasmodium falciparum gene mutations associated with resistance to the antimalarial drugs sulfadoxine-pyrimethamine (SP) and chloroquine (CQ) in children under the age of five years was investigated during an ITN trial in Asembo area, western Kenya. During the ITN trial, the national first line antimalarial treatment changed from CQ to SP. Smear-positive samples collected from cross sectional surveys prior to ITN introduction (baseline, n = 250) and five years post-ITN intervention (year 5 survey, n = 242) were genotyped for single nucleotide polymorphisms (SNPs) at dhfr-51, 59, 108, 164 and dhps-437, 540 (SP resistance), and pfcrt-76 and pfmdr1-86 (CQ resistance). The association between the drug resistance mutations and epidemiological variables was evaluated. There were significant increases in the prevalence of SP dhps mutations and the dhfr/dhps quintuple mutant, and a significant reduction in the proportion of mixed infections detected at dhfr-51, 59 and dhps-437, 540 SNPs from baseline to the year 5 survey. There was no change in the high prevalence of pfcrt-76 and pfmdr1-86 mutations. Multivariable regression analysis further showed that current antifolate use and year of survey were significantly associated with more SP drug resistance mutations. These results suggest that increased antifolate drug use due to drug policy change likely led to the high prevalence of SP mutations 5 years post-ITN intervention and reduced transmission had no apparent effect on the existing high prevalence of CQ mutations. There is no evidence from the current study that sustained transmission reduction by ITNs reduces the prevalence of genes associated with malaria drug resistance.     

Assessment of Markers of Antimalarial Drug Resistance in Plasmodium falciparum Isolates from Pregnant Women in Lagos,Nigeria

Background

The use of antimalarial drugs for prevention and treatment is a major strategy in the prevention of malaria in pregnancy. Although sulphadoxine-pyrimethamine (SP) is currently recommended for intermittent preventive treatment of malaria during pregnancy in Nigeria, previously used drugs for prophylaxis such as chloroquine (CQ) and pyrimethamine are accessible as they are purchased over the counter. This study describes the markers of absence or presence of resistance to quinoline (Pfcrt and Pfmdr 1) and type 1 antifolate antimalarial medicines (Pfdhfr).

Methods

Plasmodium falciparum-positive dried blood spots from pregnant women attending antenatal clinics for the first time during current pregnancy were investigated for the presence of mutations at codons 72–76 of Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene by real time polymerase chain reaction (PCR) using haplotype-specific probes. PCR followed by sequence analysis was used to identify mutations at codons 86, 184, 1034, 1042 and 1246 of P. falciparum multi-drug resistance-1 (Pfmdr1) gene; and codons 16, 50, 51, 59, 108, 140 and 164 of Pfdhfr gene.

Results

Two haplotypes of Pfcrt (n = 54) were observed: CVMNK 13(24.2%) and CVIET 41 (75.9%) of the samples. The SVMNT haplotype was absent in this population. The Pfmdr1 (n = 28) haplotypes were NYSND 15(53.6%), YYSND 5(17.9%), NFSND 6(21.4%) and YFSND 2(7.1%). The Pfdhfr (n = 15) were ACNCSVI 4(26.7%), and ACICNSVI 1(6.7%) and ACIRNVI 10 (66.7%). The rate of occurrence of Pfcrt 76T, Pfdhfr108N, Pfmdr186Yand184F were 75.9%, 73.3%, 25% and 28.1% respectively. The Pfmdr1 86Y was associated with low parasitaemia (median = 71 parasites/μl, P = 0.024) while Pfcrt 76T was associated with young maternal age (mean 24.1 ± 4.5 years; P = 0.006). The median parasitaemia were similar (P>0.05) in wild and mutant strains of Pfcrt 76, Pfmdr1 184 and Pfdhfr 108. There was no association between gravidity or gestational age of the women and presence of mutations in the Pfcrt, Pfmdr1 or Pfdhfr genes (P>0.05).

Conclusion

Markers of resistance to chloroquine and pyrimethamine were high, whereas cycloguanil-resistance marker was not present in the studied population. The low level of mutations in the Pfmdr1gene indicates likely efficacy of amodiaquine against malaria in pregnancy.     

Antifolate drug resistance: Novel mutations and haplotype distribution in <Emphasis Type="Italic">dhps</Emphasis> and <Emphasis Type="Italic">dhfr</Emphasis> from Northeast India

Malaria is a major public health concern in Northeast India with a preponderance of drug-resistant strains. Until recently the partner drug for artemisinin combination therapy (ACT) was sulphadoxine pyrimethamine (SP). Antifolate drug resistance has been associated with the mutations at dihydropteroate synthase (dhps) and dihydrofolatereductase (dhfr) genes. This study investigated antifolate drug resistance at the molecular level. A total of 249 fever cases from Arunachal Pradesh, NE India, were screened for malaria, and of these, 75 were found to be positive for Plasmodium falciparum. Samples were sequenced and analysed with the help of BioEdit and ClustalW. Three novel point mutations were found in the dhps gene with 10 haplotypes along with the already reported mutations. A single haplotype having quadruple mutation was found in the dhfr gene. The study reports higher degree of antifolate drug resistance as evidenced by the presence of multiple point mutations in dhps and dhfr genes. The findings of this study strongly discourage the use SP as a partner drug in ACT.     

Genetics of chloroquine-resistant malaria: a haplotypic view

The development and rapid spread of chloroquine resistance (CQR) in Plasmodium falciparum have triggered the identification of several genetic target(s) in the P. falciparum genome. In particular, mutations in the Pfcrt gene, specifically, K76T and mutations in three other amino acids in the region adjoining K76 (residues 72, 74, 75 and 76), are considered to be highly related to CQR. These various mutations form several different haplotypes and Pfcrt gene polymorphisms and the global distribution of the different CQR- Pfcrt haplotypes in endemic and non-endemic regions of P. falciparum malaria have been the subject of extensive study. Despite the fact that the Pfcrt gene is considered to be the primary CQR gene in P. falciparum , several studies have suggested that this may not be the case. Furthermore, there is a poor correlation between the evolutionary implications of the Pfcrt haplotypes and the inferred migration of CQR P. falciparum based on CQR epidemiological surveillance data. The present paper aims to clarify the existing knowledge on the genetic basis of the different CQR- Pfcrt haplotypes that are prevalent in worldwide populations based on the published literature and to analyse the data to generate hypotheses on the genetics and evolution of CQR malaria.     

Geographical spread and structural basis of sulfadoxine-pyrimethamine drug-resistant malaria parasites

The global spread of sulfadoxine (Sdx, S) and pyrimethamine (Pyr, P) resistance is attributed to increasing number of mutations in DHPS and DHFR enzymes encoded by malaria parasites. The association between drug resistance mutations and SP efficacy is complex. Here we provide an overview of the geographical spread of SP resistance mutations in Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) encoded dhps and dhfr genes. In addition, we have collated the mutation data and mapped it on to the three-dimensional structures of DHPS and DHFR which have become available. Data from genomic databases and 286 studies were collated to provide a comprehensive landscape of mutational data from 2005 to 2019. Our analyses show that the Pyr-resistant double mutations are widespread in Pf/PvDHFR (P. falciparum ～61% in Asia and the Middle East, and in the Indian sub-continent; in P. vivax ～33% globally) with triple mutations prevailing in Africa (～66%) and South America (～33%). For PfDHPS, triple mutations dominate South America (～44%), Asia and the Middle East (～34%) and the Indian sub-continent (～27%), while single mutations are widespread in Africa (～45%). Contrary to the status for P. falciparum, Sdx-resistant single point mutations in PvDHPS dominate globally. Alarmingly, highly resistant quintuple and sextuple mutations are rising in Africa (PfDHFR-DHPS) and Asia (Pf/PvDHFR-DHPS). Structural analyses of DHFR and DHPS proteins in complexes with substrates/drugs have revealed that resistance mutations map proximal to Sdx and Pyr binding sites. Thus new studies can focus on discovery of novel inhibitors that target the non-substrate binding grooves in these two validated malaria parasite drug targets.     

A Process Similar to Autophagy Is Associated with Cytocidal Chloroquine Resistance in Plasmodium falciparum

Resistance to the cytostatic activity of the antimalarial drug chloroquine (CQ) is becoming well understood, however, resistance to cytocidal effects of CQ is largely unexplored. We find that PfCRT mutations that almost fully recapitulate P. falciparum cytostatic CQ resistance (CQR^CS) as quantified by CQ IC₅₀ shift, account for only 10–20% of cytocidal CQR (CQR^CC) as quantified by CQ LD₅₀ shift. Quantitative trait loci (QTL) analysis of the progeny of a chloroquine sensitive (CQS; strain HB3)×chloroquine resistant (CQR; strain Dd2) genetic cross identifies distinct genetic architectures for CQR^CS vs CQR^CC phenotypes, including identification of novel interacting chromosomal loci that influence CQ LD₅₀. Candidate genes in these loci are consistent with a role for autophagy in CQR^CC, leading us to directly examine the autophagy pathway in intraerythrocytic CQR parasites. Indirect immunofluorescence of RBC infected with synchronized CQS vs CQR trophozoite stage parasites reveals differences in the distribution of the autophagy marker protein PfATG8 coinciding with CQR^CC. Taken together, the data show that an unusual autophagy – like process is either activated or inhibited for intraerythrocytic trophozoite parasites at LD₅₀ doses (but not IC₅₀ doses) of CQ, that the pathway is altered in CQR P. falciparum, and that it may contribute along with mutations in PfCRT to confer the CQR^CC phenotype.     

Association of molecular markers in Plasmodium falciparum crt and mdr1 with in vitro chloroquine resistance: A Philippine study

Specific mutations in the pfcrt and pfmdr1 genes have been reported to be associated with chloroquine-resistant falciparum malaria parasites worldwide. These genetic markers are considered to be useful tools for the elucidation of several aspects of the epidemiology of drug resistant malaria. In this study, Plasmodium falciparum isolates from three distinct areas of the Philippines were analyzed for drug-resistance-associated genetic mutations, and their association with the in vitro chloroquine (CQ) response. Two novel pfcrt 72–76 allelic types, CVMDT and SVMDT, were detected. The frequency of the pfcrt K76T mutation in the isolates that were successfully tested for in vitro CQ susceptibility was found to be 100% in Kalinga, 80% in Palawan, and 87% in Mindanao. The frequency of the pfmdr1 N86Y mutation was 39% in Kalinga, 35% in Palawan, and 93% in Mindanao isolates. No mutations were found at positions 1042 and 1246 of pfmdr1. However, there were no significant associations found between polymorphisms in these genes and in vitro CQ susceptibility. The results of this study indicate that mutations in pfcrt and pfmdr1 are not predictive of in vitro CQ resistance in Philippine isolates and may therefore not be suitable as molecular markers for surveillance.     

Sulfadoxine-pyrimethamine-based combinations for malaria: a randomised blinded trial to compare efficacy, safety and selection of resistance in Malawi

Background

In Malawi, there has been a return of Plasmodium falciparum sensitivity to chloroquine (CQ) since sulfadoxine-pyrimethamine (SP) replaced CQ as first line treatment for uncomplicated malaria. When used for prophylaxis, Amodiaquine (AQ) was associated with agranulocytosis but is considered safe for treatment and is increasingly being used in Africa. Here we compare the efficacy, safety and selection of resistance using SP or CQ+SP or artesunate (ART)+SP or AQ+SP for the treatment of uncomplicated falciparum malaria.

Methodology and Findings

455 children aged 1–5 years were recruited into a double-blinded randomised trial comparing SP to the three combination therapies. Using intention to treat analysis with missing outcomes treated as successes, and without adjustment to distinguish recrudescence from new infections, the day 28 adequate clinical and parasitological response (ACPR) rate for SP was 25%, inferior to each of the three combination therapies (p<0.001). AQ+SP had an ACPR rate of 97%, higher than CQ+SP (81%) and ART+SP (70%), p<0.001. Nineteen children developed a neutropenia of ≤0.5×10³ cells/µl by day 14, more commonly after AQ+SP (p = 0.03). The mutation pfcrt 76T, associated with CQ resistance, was detected in none of the pre-treatment or post-treatment parasites. The prevalence of the pfmdr1 86Y mutation was higher after treatment with AQ+SP than after SP, p = 0.002.

Conclusions

The combination AQ+SP was highly efficacious, despite the low efficacy of SP alone; however, we found evidence that AQ may exert selective pressure for resistance associated mutations many weeks after treatment. This study confirms the return of CQ sensitivity in Malawi and importantly, shows no evidence of the re-emergence of pfcrt 76T after treatment with CQ or AQ. Given the safety record of AQ when used as a prophylaxis, our observations of marked falls in neutrophil counts in the AQ+SP group requires further scrutiny.

Trial Registration

Controlled-Trials.com ISRCTN22075368     

Evolution of Genetic Polymorphisms of Plasmodium falciparum Merozoite Surface Protein (PfMSP) in Thailand

Plasmodium falciparum malaria is a major public health problem in Thailand due to the emergence of multidrug resistance. The understanding of genetic diversity of malaria parasites is essential for developing effective drugs and vaccines. The genetic diversity of the merozoite surface protein-1 (PfMSP-1) and merozoite surface protein-2 (PfMSP-2) genes was investigated in a total of 145 P. falciparum isolates collected from Mae Sot District, Tak Province, Thailand during 3 different periods (1997-1999, 2005-2007, and 2009-2010). Analysis of genetic polymorphisms was performed to track the evolution of genetic change of P. falciparum using PCR. Both individual genes and their combination patterns showed marked genetic diversity during the 3 study periods. The results strongly support that P. falciparum isolates in Thailand are markedly diverse and patterns changed with time. These 2 polymorphic genes could be used as molecular markers to detect multiple clone infections and differentiate recrudescence from reinfection in P. falciparum isolates in Thailand.     

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.     

Sequence signatures involved in targeting the male-specific lethal complex to X-chromosomal genes in Drosophila melanogaster

Background

Drug resistance in the malaria parasite Plasmodium falciparum severely compromises the treatment and control of malaria. A knowledge of the critical mutations conferring resistance to particular drugs is important in understanding modes of drug action and mechanisms of resistances. They are required to design better therapies and limit drug resistance. A mutation in the gene (pfcrt) encoding a membrane transporter has been identified as a principal determinant of chloroquine resistance in P. falciparum, but we lack a full account of higher level chloroquine resistance. Furthermore, the determinants of resistance in the other major human malaria parasite, P. vivax, are not known. To address these questions, we investigated the genetic basis of chloroquine resistance in an isogenic lineage of rodent malaria parasite P. chabaudi in which high level resistance to chloroquine has been progressively selected under laboratory conditions.

Results

Loci containing the critical genes were mapped by Linkage Group Selection, using a genetic cross between the high-level chloroquine-resistant mutant and a genetically distinct sensitive strain. A novel high-resolution quantitative whole-genome re-sequencing approach was used to reveal three regions of selection on chr11, chr03 and chr02 that appear progressively at increasing drug doses on three chromosomes. Whole-genome sequencing of the chloroquine-resistant parent identified just four point mutations in different genes on these chromosomes. Three mutations are located at the foci of the selection valleys and are therefore predicted to confer different levels of chloroquine resistance. The critical mutation conferring the first level of chloroquine resistance is found in aat1, a putative aminoacid transporter.

Conclusions

Quantitative trait loci conferring selectable phenotypes, such as drug resistance, can be mapped directly using progressive genome-wide linkage group selection. Quantitative genome-wide short-read genome resequencing can be used to reveal these signatures of drug selection at high resolution. The identities of three genes (and mutations within them) conferring different levels of chloroquine resistance generate insights regarding the genetic architecture and mechanisms of resistance to chloroquine and other drugs. Importantly, their orthologues may now be evaluated for critical or accessory roles in chloroquine resistance in human malarias P. vivax and P. falciparum.     

Artesunate-Amodiaquine and Artemether-Lumefantrine Therapies and Selection of Pfcrt and Pfmdr1 Alleles in Nanoro,Burkina Faso

The adoption of Artemisinin based combination therapies (ACT) constitutes a basic strategy for malaria control in sub-Saharan Africa. Moreover, since cases of ACT resistance have been reported in South-East Asia, the need to understand P. falciparum resistance mechanism to ACT has become a global research goal. The selective pressure of ACT and the possibility that some specific Pfcrt and Pfmdr1 alleles are associated with treatment failures was assessed in a clinical trial comparing ASAQ to AL in Nanoro. Dried blood spots collected on Day 0 and on the day of recurrent parasitaemia during the 28-day follow-up were analyzed using the restriction fragments length polymorphism (PCR-RFLP) method to detect single nucleotide polymorphisms (SNPs) in Pfcrt (codon76) and Pfmdr1 (codons 86, 184, 1034, 1042, and 1246) genes. Multivariate analysis of the relationship between the presence of Pfcrt and Pfmdr1 alleles and treatment outcome was performed. AL and ASAQ exerted opposite trends in selecting Pfcrt K76T and Pfmdr1-N86Y alleles, raising the potential beneficial effect of using diverse ACT at the same time as first line treatments to reduce the selective pressure by each treatment regimen. No clear association between the presence of Pfcrt and Pfmdr1 alleles carried at baseline and treatment failure was observed.     

A Genome Wide Association Study of Plasmodium falciparum Susceptibility to 22 Antimalarial Drugs in Kenya

Background

Drug resistance remains a chief concern for malaria control. In order to determine the genetic markers of drug resistant parasites, we tested the genome-wide associations (GWA) of sequence-based genotypes from 35 Kenyan P. falciparum parasites with the activities of 22 antimalarial drugs.

Methods and Principal Findings

Parasites isolated from children with acute febrile malaria were adapted to culture, and sensitivity was determined by in vitro growth in the presence of anti-malarial drugs. Parasites were genotyped using whole genome sequencing techniques. Associations between 6250 single nucleotide polymorphisms (SNPs) and resistance to individual anti-malarial agents were determined, with false discovery rate adjustment for multiple hypothesis testing. We identified expected associations in the pfcrt region with chloroquine (CQ) activity, and other novel loci associated with amodiaquine, quinazoline, and quinine activities. Signals for CQ and primaquine (PQ) overlap in and around pfcrt, and interestingly the phenotypes are inversely related for these two drugs. We catalog the variation in dhfr, dhps, mdr1, nhe, and crt, including novel SNPs, and confirm the presence of a dhfr-164L quadruple mutant in coastal Kenya. Mutations implicated in sulfadoxine-pyrimethamine resistance are at or near fixation in this sample set.

Conclusions/Significance

Sequence-based GWA studies are powerful tools for phenotypic association tests. Using this approach on falciparum parasites from coastal Kenya we identified known and previously unreported genes associated with phenotypic resistance to anti-malarial drugs, and observe in high-resolution haplotype visualizations a possible signature of an inverse selective relationship between CQ and PQ.     

Evaluation of the acquired immune responses to Plasmodium vivax VIR variant antigens in individuals living in malaria-endemic areas of Brazil

Background

Chloroquine (CQ) or sulfadoxine-pyrimethamine (SP) monotherapy for Plasmodium falciparum often leads to therapeutic failure in Indonesia. Combining CQ with other drugs, like SP, may provide an affordable, available and effective option where artemisinin-combined therapies (ACT) are not licensed or are unavailable.

Methods

This study compared CQ (n = 29 subjects) versus CQ + SP (with or without primaquine; n = 88) for clinical and parasitological cure of uncomplicated falciparum malaria in the Menoreh Hills region of southern Central Java, Indonesia. Gametocyte clearance rates were measured with (n = 56 subjects) and without (n = 61) a single 45 mg dose of primaquine (PQ).

Results

After 28 days, 58% of subjects receiving CQ had cleared parasitaemia and remained aparasitaemic, compared to 94% receiving CQ combined with SP (p < 0.001). Msp-2 genotyping permitted reinfection-adjusted cure rates for CQ and CQ combined with SP, 70% and 99%, respectively (p = 0.0006).

Conclusion

Primaquine exerted no apparent affect on cure of asexual stage parasitaemia, but clearly accelerated clearance of gametocytes. CQ combined with SP was safe and well-tolerated with superior efficacy over CQ for P. falciparum parasitaemia in this study.