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Background: Chloroquine (CQ) resistance is conferred by mutations in the Plasmodium falciparum CQ resistance transporter (pfcrt). Following CQ withdrawal for anti-malarial treatment, studies across malaria-endemic countries have shown a range of responses. In some areas, CQ sensitive parasites re-emerge, and in others, mutant haplotypes persist. Active surveillance of resistance mutations in clinical parasites is essential to inform treatment regimens; this effort requires fast, reliable, and cost-effective methods that work on a variety of sample types with reagents accessible in malaria-endemic countries. Methods: Quantitative PCR followed by High-Resolution Melt (HRM) analysis was performed in a field setting to assess pfcrt mutations in two groups of clinical samples from Southwestern Uganda. Group 1 samples (119 in total) were collected in 2010 as predominantly Giemsa-stained slides; Group 2 samples (125 in total) were collected in 2015 as blood spots on filter paper. The Rotor-Gene Q instrument was utilized to assess the impact of different PCR-HRM reagent mixes and the detection of mixed haplotypes present in the clinical samples. Finally, the prevalence of the wild type (CVMNK) and resistant pfcrt haplotypes (CVIET and SVMNT) was evaluated in this understudied Southwestern region of Uganda. Results: The sample source (i.e. Giemsa-stained slides or blood spots) and type of LCGreen-based reagent mixes did not impact the success of PCR-HRM. The detection limit of 10− 5 ng and the ability to identify mixed haplotypes as low as 10 % was similar to other HRM platforms. The CVIET haplotype predominated in the clinical samples (66 %, 162/244); however, there was a large regional variation between the sample groups (94 % CVIET in Group 1 and 44 % CVIET in Group 2). Conclusions: The HRM-based method exhibits the flexibility required to conduct reliable assessment of resistance alleles from various sample types generated during the clinical management of malaria. Large regional variations in CQ resistance haplotypes across Southwestern Uganda emphasizes the need for continued local parasite genotype assessment to inform anti-malarial treatment policies. [ABSTRACT FROM AUTHOR]

Background: Artesunate plus sulfadoxine-pyrimethamine (ASP) is first-line treatment for uncomplicated Plasmodium falciparum malaria in most of India, except for six North-eastern provinces where treatment failure rates were high. In Ujjain, central India, the frequency of mutations associated with increased drug tolerance, but not overt resistance to sulfadoxine and pyrimethamine were 9% and > 80%, respectively, in 2009 and 2010, just prior to the introduction of ASP. The frequency of drug resistance associated mutations in Ujjain in 2015-2016 after 3-4 years of ASP use, are reported. Methods: Blood samples from patients with P. falciparum mono-infection verified by microscopy were collected on filter-paper at all nine major pathology laboratories in Ujjain city. Codons pfdhfr 16-185, pfdhps 436-632 and K13 407-689 were identified by sequencing. Pfcrt K76T and pfmdr1 N86Y were identified by restriction fragment length polymorphism. Results: Sulfadoxine-pyrimethamine resistance-associated pfdhfr 108 N and 59R alleles were found in 100/104 (96%) and 87/91 (96%) samples, respectively. Pfdhps 437G was found in 10/105 (10%) samples. Double mutant pfdhfr 59R + 108 N were found in 75/81 (93%) samples. Triple mutant pfdhfr 59R + 108 N and pfdhps 437G were found in 6/78 (8%) samples. Chloroquine-resistance-associated pfcrt 76T was found in 102/102 (100%). Pfmdr1 N86 and 86Y were identified in 83/115 (72%) and 32/115 (28%) samples, respectively. Conclusion: The frequency of P. falciparum with reduced susceptibility to sulfadoxine-pyrimethamine remained high, but did not appear to have increased significantly since the introduction of ASP. No polymorphisms in K13 associated with decreased artemisinin susceptibility were found. ASP probably remained effective, supporting continued ASP use. [ABSTRACT FROM AUTHOR]

Background: Due to the increasing resistance of Plasmodium falciparum to chloroquine (CQ) in Sudan, a shift from CQ to artesunate combined with sulfadoxine/pyrimethamine as a first-line treatment for uncomplicated falciparum malaria was adopted in 2004. This study aimed to determine the frequency distribution of K76T and N86Y mutations in P. falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes as key markers of resistance to CQ among P. falciparum isolates from patients in Nyala district of South Darfur state, west of Sudan.Methods: A descriptive, cross-sectional study was conducted among 75 P. falciparum isolates from Sudanese patients diagnosed with falciparum malaria mono-infection. Parasite DNA was extracted from dried blood spots and amplified using a nested polymerase chain reaction (PCR). Then, restriction fragment length polymorphism (RFLP) was used to detect the genetic polymorphisms in codons 76 of pfcrt and 86 of pfmdr1. PCR-RFLP products were analyzed using 1.5% gel electrophoresis to identify the genetic polymorphisms in the studied codons. The wild-type (pfcrt K76 and pfmdr1 N86), mutant (pfcrt 76T and pfmdr1 86Y) and mixed-type (pfcrt K76T and pfmdr1 N86Y) alleles were expressed as frequencies and proportions.Results: The wild-type pfcrt K76 allele was observed among 34.7% of isolates and the mutant 76T allele among 20% of isolates, while the mixed-type K76T allele was observed among 45.3% of isolates. On the other hand, 54.7% of isolates harbored the wild-type pfmdr1 N86 allele and 5.3% of isolates had the mutant 86Y allele, while the mixed-type N86Y allele was observed among 40% of isolates.Conclusion: The key molecular markers associated with CQ resistance (pfcrt 76T and pfmdr1 86Y) are still circulating in high frequency among P. falciparum isolates in South Darfur state, about twelve years after the official withdrawal of the drug as a treatment for uncomplicated falciparum malaria. [ABSTRACT FROM AUTHOR]

Background After years of disuse of chloroquine (CQ) as first-line anti-malarial drug in Ghana, reports from molecular studies conducted in parts of the country indicate varying prevalence of T76 mutation in the pfcrt gene. This situation has several health implications, one being that mutations that confer resistance to CQ have been reported to show substantial cross-resistance to other anti-malarial drugs. It is important to identify some of the factors contributing to the continuous presence of CQ resistance markers in the country. This study determined the prevalence of T76 mutation in pfcrt gene of Plasmodium falciparum isolates collected from selected areas of the Central region of Ghana and correlated with the level of CQ use in these areas. Methods Plasmodium falciparum DNA was extracted from collected blood-blot filter paper samples in the study sites. The prevalence of T76 point mutation in pfcrt gene was assessed using nested PCR followed by RFLP. CQ from pharmacy and chemical shops was obtained using mystery buying method. The extent of CQ use by the participants was determined by measuring the level of the drug in their urine samples using the Saker-Solomon method. Results Of the 214 P. falciparum isolates analysed, 71.9% were found to have T76 mutation of pfcrt gene. The study revealed that 14.49% of community pharmacies and chemical shops had stocks of CQ for sale while 16.9% of the participants had CQ in their urine samples. There is five times more risks of becoming infected with CQ resistant strain for staying in an area where CQ is stocked for sale [RR = 0.20, p < 0.0001] and thirteen times more risks of having CQ-resistant mutant from those who still use CQ than non-users [OR = 0.08, p < 0.0001]. Conclusion This study has shown that high variation in the prevalence of T76 mutations of P. falciparum is linked with the level of CQ stocking and usage within study area. [ABSTRACT FROM AUTHOR]

The onset and rapid spread of chloroquine resistance and the introduction of amodiaquine for the treatment of uncomplicated malaria in Cameroon have influenced the proportion of Plasmodium falciparum sensitive and resistant alleles related to 4-aminoquinoline drugs. This study was undertaken to determine the prevalence of resistance markers to antimalarial 4-aminoquinolines in Douala in the Littoral Region, and Buea in the South West Region in June 2020. Dry blood spots were prepared from malaria microscopy positive cases and used for parasite DNA extraction by chelex-100 method. Plasmodium species identification was carried out by PCR amplification/agarose gel electrophoresis of 18srRNA. The Pfcrt and Pfmdr1 genes were amplified by PCR followed by restriction digestion. The prevalence of single nucleotide polymorphisms (SNPs) was compared between study sites and with previous studies carried out between 2003–2005 and 2009–2011 using the Chi square test. The results showed that Plasmodium falciparum was the dominant species occurring as mono-infections (84.6%). The wild type K76 allele of the Pfcrt gene was found in 74.9% of isolates while the wild N86, Y184 and D1246 alleles of the Pfmdr1 gene were found respectively in 87.2%, 89.6% and 100% of field isolates. The results showed a significant reduction in the mutant alleles compared to results obtained in 2003–2005 and 2009–2013. The KNYD haplotype was observed to be the most prevalent. The results indicated that there is a gradual erosion of the mutant Pfcrt and Pfmdr1 genotype and a gradual return to the sensitive P. falciparum genotype in Cameroon. [ABSTRACT FROM AUTHOR]

Background: A reversal of chloroquine (CQ) resistance following a period of withdrawal has raised the possibility of its re-introduction. This study evaluated the current prevalence of Pfcrt and Pfmdr1 alleles in Plasmodium falciparum isolates, 11 years after CQ withdrawal in Southeast Nigeria. Methods: Filter-paper blood samples were collected from 725 non-febrile individuals, comprising 250 children (≤ 12 years), 250 pregnant women and 225 other adults, between October 2014 and February 2015 in Nnewi town, Southeast Nigeria. Nested PCR followed by direct sequencing was employed for the genotyping of Pfcrt and Pfmdr1 genes. Results: A total of 103 parasites-positive samples were recovered, comprising of 48 (19.20%) among children, 20 (20.00%) among pregnant women and 35 (15.50%) among other adults cohort. The frequency of the mutant genotype of Pfcrt 76T, 75E and 74I was 94.50% each. Parasite isolates from children had a frequency of 100% for mutant alleles in all Pfcrt codons while isolates from pregnant women and other adults had a frequency of 91% each in all codons. Haplotype distribution of pfcrt gene were 5.45, 0.00 and 76.37% for CVMNK, SVMNT and CVIET, respectively. For Pfmdr1 gene, the frequency of 86Y, 184F and 1246Y mutant alleles were 8.54, 29.27 and 3.66%, respectively. Amongst the Pfmdr1 haplotypes analysed, NFD had the highest frequency of 24.4%, followed by YFD at 6.10%. NYF and NYY occurred the least (1.20%). Conclusion: The high level of Pfcrt mutations is suggestive of a sustained CQ pressure on P. falciparum isolates in the study area, despite the change of first line treatment from CQ to artemisinin combination therapy for 11 years. A new strategy to ensure the complete withdrawal of CQ from the country is recommended. [ABSTRACT FROM AUTHOR]

Abstract Background Chloroquine (CQ) resistance is conferred by mutations in the Plasmodium falciparum CQ resistance transporter (pfcrt). Following CQ withdrawal for anti-malarial treatment, studies across malaria-endemic countries have shown a range of responses. In some areas, CQ sensitive parasites re-emerge, and in others, mutant haplotypes persist. Active surveillance of resistance mutations in clinical parasites is essential to inform treatment regimens; this effort requires fast, reliable, and cost-effective methods that work on a variety of sample types with reagents accessible in malaria-endemic countries. Methods Quantitative PCR followed by High-Resolution Melt (HRM) analysis was performed in a field setting to assess pfcrt mutations in two groups of clinical samples from Southwestern Uganda. Group 1 samples (119 in total) were collected in 2010 as predominantly Giemsa-stained slides; Group 2 samples (125 in total) were collected in 2015 as blood spots on filter paper. The Rotor-Gene Q instrument was utilized to assess the impact of different PCR-HRM reagent mixes and the detection of mixed haplotypes present in the clinical samples. Finally, the prevalence of the wild type (CVMNK) and resistant pfcrt haplotypes (CVIET and SVMNT) was evaluated in this understudied Southwestern region of Uganda. Results The sample source (i.e. Giemsa-stained slides or blood spots) and type of LCGreen-based reagent mixes did not impact the success of PCR-HRM. The detection limit of 10− 5 ng and the ability to identify mixed haplotypes as low as 10 % was similar to other HRM platforms. The CVIET haplotype predominated in the clinical samples (66 %, 162/244); however, there was a large regional variation between the sample groups (94 % CVIET in Group 1 and 44 % CVIET in Group 2). Conclusions The HRM-based method exhibits the flexibility required to conduct reliable assessment of resistance alleles from various sample types generated during the clinical management of malaria. Large regional variations in CQ resistance haplotypes across Southwestern Uganda emphasizes the need for continued local parasite genotype assessment to inform anti-malarial treatment policies.

Abstract Background Artesunate plus sulfadoxine–pyrimethamine (ASP) is first-line treatment for uncomplicated Plasmodium falciparum malaria in most of India, except for six North-eastern provinces where treatment failure rates were high. In Ujjain, central India, the frequency of mutations associated with increased drug tolerance, but not overt resistance to sulfadoxine and pyrimethamine were 9% and > 80%, respectively, in 2009 and 2010, just prior to the introduction of ASP. The frequency of drug resistance associated mutations in Ujjain in 2015–2016 after 3–4 years of ASP use, are reported. Methods Blood samples from patients with P. falciparum mono-infection verified by microscopy were collected on filter-paper at all nine major pathology laboratories in Ujjain city. Codons pfdhfr 16–185, pfdhps 436–632 and K13 407–689 were identified by sequencing. Pfcrt K76T and pfmdr1 N86Y were identified by restriction fragment length polymorphism. Results Sulfadoxine–pyrimethamine resistance-associated pfdhfr 108 N and 59R alleles were found in 100/104 (96%) and 87/91 (96%) samples, respectively. Pfdhps 437G was found in 10/105 (10%) samples. Double mutant pfdhfr 59R + 108 N were found in 75/81 (93%) samples. Triple mutant pfdhfr 59R + 108 N and pfdhps 437G were found in 6/78 (8%) samples. Chloroquine-resistance-associated pfcrt 76T was found in 102/102 (100%). Pfmdr1 N86 and 86Y were identified in 83/115 (72%) and 32/115 (28%) samples, respectively. Conclusion The frequency of P. falciparum with reduced susceptibility to sulfadoxine–pyrimethamine remained high, but did not appear to have increased significantly since the introduction of ASP. No polymorphisms in K13 associated with decreased artemisinin susceptibility were found. ASP probably remained effective, supporting continued ASP use.

Abstract Background A reversal of chloroquine (CQ) resistance following a period of withdrawal has raised the possibility of its re-introduction. This study evaluated the current prevalence of Pfcrt and Pfmdr1 alleles in Plasmodium falciparum isolates, 11 years after CQ withdrawal in Southeast Nigeria. Methods Filter-paper blood samples were collected from 725 non-febrile individuals, comprising 250 children (≤ 12 years), 250 pregnant women and 225 other adults, between October 2014 and February 2015 in Nnewi town, Southeast Nigeria. Nested PCR followed by direct sequencing was employed for the genotyping of Pfcrt and Pfmdr1 genes. Results A total of 103 parasites-positive samples were recovered, comprising of 48 (19.20%) among children, 20 (20.00%) among pregnant women and 35 (15.50%) among other adults cohort. The frequency of the mutant genotype of Pfcrt 76T, 75E and 74I was 94.50% each. Parasite isolates from children had a frequency of 100% for mutant alleles in all Pfcrt codons while isolates from pregnant women and other adults had a frequency of 91% each in all codons. Haplotype distribution of pfcrt gene were 5.45, 0.00 and 76.37% for CVMNK, SVMNT and CVIET, respectively. For Pfmdr1 gene, the frequency of 86Y, 184F and 1246Y mutant alleles were 8.54, 29.27 and 3.66%, respectively. Amongst the Pfmdr1 haplotypes analysed, NFD had the highest frequency of 24.4%, followed by YFD at 6.10%. NYF and NYY occurred the least (1.20%). Conclusion The high level of Pfcrt mutations is suggestive of a sustained CQ pressure on P. falciparum isolates in the study area, despite the change of first line treatment from CQ to artemisinin combination therapy for 11 years. A new strategy to ensure the complete withdrawal of CQ from the country is recommended.

Background: Plasmodium falciparum malaria is a public health problem worldwide. Malaria treatment policy has faced periodic changes due to emergence of drug resistant parasites. In Sudan chloroquine has been replaced by artesunate and sulfadoxine/pyrimethamine (AS/SP) in 2005 and to artemether–lumefantrine (AL) in 2017, due to the development of drug resistance. Different molecular markers have been used to monitor the status of drug resistant P. falciparum. This study aimed to determine the frequency of malaria drug resistance molecular markers in Southeast Sudan. Methods: The samples of this study were day zero dried blood spot samples collected from efficacy studies in the Blue Nile State from November 2015 to January 2016. A total of 130 samples were amplified and sequenced using illumina Miseq platform. The molecular markers included were Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, Pfk13, exonuclease and artemisinin resistant (ART‐R) genetic background (Pfmdr2, ferroredoxine, Pfcrt and Pfarps10). Results: Resistance markers for chloroquine were detected in 25.8% of the samples as mutant haplotype Pfcrt 72-76 CVIET and 21.7% Pfmdr1 86Y. Pfdhfr mutations were detected in codons 51, 59 and 108. The ICNI double-mutant haplotype was the most prevalent (69%). Pfdhps mutations were detected in codons 436, 437, 540, 581 and 613. The SGEGA triple-mutant haplotype was the most prevalent (43%). In Pfdhfr/Pfdhps combined mutation, quintuple mutation ICNI/SGEGA is the most frequent one (29%). Six of the seven treatment failure samples had quintuple mutation and the seventh was quadruple. This was significantly higher from the adequately responsive group (P < 0.01). Pfk13 novel mutations were found in 7 (8.8%) samples, which were not linked to artemisinin resistance. Mutations in ART‐R genetic background genes ranged from zero to 7%. Exonuclease mutation was not detected. Conclusion: In this study, moderate resistance to chloroquine and high resistance to SP was observed. Novel mutations of Pfk13 gene not linked to treatment failure were described. There was no resistance to piperaquine the partner drug of dihydroartemisinin/piperaquine (DHA-PPQ). [ABSTRACT FROM AUTHOR]

Background: Chloroquine (CQ) was the cornerstone of anti-malarial treatment in Africa for almost 50 years, but has been widely withdrawn due to the emergence and spread of resistance. Recent reports have suggested that CQsusceptibility may return following the cessation of CQ usage. Here, we monitor CQ sensitivity and determine the prevalence of genetic polymorphisms in the CQ resistance transporter gene (pfcrt) of Plasmodium falciparum isolates recently imported from Africa to China. Methods: Blood samples were collected from falciparum malaria patients returning to China from various countries in Africa. Isolates were tested for their sensitivity to CQ using the SYBR Green I test ex vivo, and for a subset of samples, in vitro following culture adaptation. Mutations at positions 72-76 and codon 220 of the pfcrt gene were analyzed by sequencing and confirmed by PCR-RFLP. Correlations between drug sensitivity and pfcrt polymorphisms were investigated. Results: Of 32 culture adapted isolates assayed, 17 (53.1%), 6 (18.8%) and 9 (28.1%) were classified as sensitive, moderately resistant, and highly resistant, respectively. In vitro CQ susceptibility was related to point mutations in the pfcrt gene, the results indicating a strong association between pfcrt genotype and drug sensitivity. A total of 292 isolates were typed at the pfcrt locus, and the prevalence of the wild type (CQ sensitive) haplotype CVMNK in isolates from East, South, North, West and Central Africa were 91.4%, 80.0%, 73.3%, 53.3% and 51.7%, respectively. The only mutant haplotype observed was CVIET, and this was almost always linked to an additional mutation at A220S. Conclusions: Our results suggest that a reduction in drug pressure following withdrawal of CQ as a first-line drug may lead to a resurgence in CQ sensitive parasites. The prevalence of wild-type pfcrt CQ sensitive parasites from East, South and North Africa was higher than from the West and Central areas, but this varied greatly between countries. Further surveillance is required to assess whether the prevalence of CQ resistant parasites will continue to decrease in the absence of widespread CQ usage. [ABSTRACT FROM AUTHOR]

In regions with high malaria endemicity, the withdrawal of chloroquine (CQ) as first-line treatment of Plasmodium falciparum infections has typically led to the restoration of CQ susceptibility through the reexpansion of the wild-type (WT) allele K76 of the chloroquine resistance transporter gene (pfcrt) at the expense of less fit mutant alleles carrying the CQ resistance (CQR) marker K76T. In low-transmission settings, such as South America, drug resistance mutations can attain 100% prevalence, thereby precluding the return of WT parasites after the complete removal of drug pressure. In French Guiana, despite the fixation of the K76T allele, the prevalence of CQR isolates progressively dropped from >90% to <30% during 17 y after CQ withdrawal in 1995. Using a genome-wide association study with CQ-sensitive (CQS) and CQR isolates, we have identified a single mutation in pfcrt encoding a C350R substitution that is associated with the restoration of CQ susceptibility. Genome editing of the CQR reference strain 7G8 to incorporate PfCRT C350R caused a complete loss of CQR. A retrospective molecular survey on 580 isolates collected from 1997 to 2012 identified all C350R mutant parasites as being CQS. This mutation emerged in 2002 and rapidly spread throughout the P. falciparum population. The C350R allele is also associated with a significant decrease in piperaquine susceptibility in vitro, suggesting that piperaquine pressure in addition to potential fitness costs associated with the 7G8-type CQR pfcrt allele may have selected for this mutation. These findings have important implications for understanding the evolutionary dynamics of antimalarial drug resistance. [ABSTRACT FROM AUTHOR]

Background: In Nigeria, despite the change in National malaria drug policy to artemisinin combination therapy (ACT) in 2005 due to widespread chloroquine resistance, chloroquine (CQ) is still widely used in the treatment of malaria because it is cheap, affordable and accessible. The use of ACT for the management of uncomplicated malaria is currently being promoted. The employment of genetic markers to track circulating chloroquine-resistant parasites are useful in elucidating likely poor efficacy of chloroquine, especially in settings where it is not recommended for the treatment of uncomplicated falciparum malaria. This study determined the prevalence of pfcrt haplotypes and point mutations in pfmdr1 genes four years after the change in antimalarial treatment policy from CQ to the ACTs in Lagos, a commercial city in South-West, Nigeria. Methods: This was a cross sectional study on uncomplicated malaria in children less than 12 years that presented with fever and other symptoms suggestive of malaria. Parasite DNA was extracted from 119 patients out of 251 children who were positive for Plasmodium falciparum by microscopy and amplified. The occurrence of haplotypes was investigated in pfcrt gene using probe-based qPCR and single nucleotide polymorphisms in pfmdr1 gene using nested PCR. Results: One hundred and nine (109) of the 119 children with P falciparum infection (91.6%) harbourd parasites with the mutant pfcrt haplotype (CVIET). Out of this, 4.2% comprised a mixture of genotypes encoding CVMNK and CVIET, while 4.2% had the wild type (CVMNK). Furthermore, the frequency of point mutations in pfmdr1 was 62.2% and 69.0% for codons Y86 and F184 respectively. There were no mutations at codons 1034, 1042 and 1246 of the Pfmdr1 genes. Conclusion: The high frequency of the CQ-resistant haplotypes (CVIET) and mutations in Pfmdr1 associated with CQ resistance in P. falciparum among these children suggest that CQ-resistant parasites are still in circulation. Continuous use of chloroquine may continue to increase the level of mutations in pfcrt and pfmdr1genes. There is need to strengthen current case management efforts at promoting ACT use as well as urgently restricting access to chloroquine by the National drug regulatory agency, National Agency for Food Drug Administration and Control (NAFDAC). [ABSTRACT FROM AUTHOR]

Background Increased resistance by Plasmodium falciparum parasites led to the withdrawal of the antimalarial drugs chloroquine and sulphadoxine-pyrimethamine in Ethiopia. Since 2004 artemether-lumefantrine has served to treat uncomplicated P. falciparum malaria. However, increasing reports on delayed parasite clearance to artemisinin opens up a new challenge in anti-malarial therapy. With the complete withdrawal of CQ for the treatment of Plasmodium falciparum malaria, this study assessed the evolution of CQ resistance by investigating the prevalence of mutant alleles in the pfmdr1 and pfcrt genes in P. falciparum and pvmdr1 gene in Plasmodium vivax in Southern and Eastern Ethiopia. Methods Of the 1,416 febrile patients attending primary health facilities in Southern Ethiopia, 329 febrile patients positive for P. falciparum or P. vivax were recruited. Similarly of the 1,304 febrile patients from Eastern Ethiopia, 81 febrile patients positive for P. falciparum or P. vivax were included in the study. Of the 410 finger prick blood samples collected from malaria patients, we used direct sequencing to investigate the prevalence of mutations in pfcrt and pfmdr1. This included determining the gene copy number in pfmdr1 in 195 P. falciparum clinical isolates, and mutations in the pvmdr1 locus in 215 P. vivax clinical isolates. Results The pfcrt K76 CQ-sensitive allele was observed in 84% of the investigated P.falciparum clinical isolates. The pfcrt double mutations (K76T and C72S) were observed less than 3%. The pfcrt SVMNT haplotype was also found to be present in clinical isolates from Ethiopia. The pfcrt CVMNK-sensitive haplotypes were frequently observed (96%). The pfmdr1 mutation N86Y was observed only in 14.9% compared to 85.1% of the clinical isolates that carried sensitive alleles. Also, the sensitive pfmdr1 Y184 allele was more common, in 96% of clinical isolates. None of the investigated P. falciparum clinical isolates carried S1034C, N1042D and D1246Y pfmdr1 polymorphisms. All investigated P. falciparum clinical isolates from Southern and Eastern Ethiopia carried only a single copy of the mutant pfmdr1 gene. Conclusion The study reports for the first time the return of chloroquine sensitive P. falciparum in Ethiopia. These findings support the rationale for the use of CQ-based combination drugs as a possible future alternative. [ABSTRACT FROM AUTHOR]

Background: Resistance of Plasmodium falciparum to anti-malarial drugs has hampered efforts to eradicate malaria. Recent reports of a decline in the prevalence of chloroquine-resistant P. falciparum in several countries, including Malawi and Zambia, is raising the hope of reintroducing chloroquine in the near future, ideally in combination with another anti-malarial drug for the treatment of uncomplicated malaria. In Côte d'Ivoire, the decrease in the clinical efficacy of chloroquine, in addition to a high proportion of clinical isolates carrying the Thr-76 mutant allele of the pfcrt gene, had led to the discontinuation of the use of chloroquine in 2004. Previous studies have indicated the persistence of a high prevalence of the Thr-76 mutant allele despite the withdrawal of chloroquine as first-line anti-malarial drug. This present study is conducted to determine the prevalence of the Thr-76T mutant allele of the Pfcrt gene after a decade of the ban on the sale and use of chloroquine in Côte d'Ivoire. Results: Analysis of the 64 sequences from all three study sites indicated a prevalence of 15% (10/64) of the Thr-76 mutant allele against 62% (40/64) of the Lys-76 wild-type allele. No mutation of the allele Thr-76 was observed at Anonkoua Kouté while this mutant allele was in 31% (5/16) and 25% (5/20) of isolate sequences from Port-Bouët and Ayamé respectively. Conclusion: More than a decade after the discontinuation of the use of chloroquine in Côte d'Ivoire, the proportion of parasites sensitive to this anti-malarial seems to increase in Anonkoua-kouté, Port-bouët and Ayamé. [ABSTRACT FROM AUTHOR]