The return of chloroquine-sensitive Plasmodium falciparum parasites in Jazan Region, Southwestern Saudi Arabia over a decade after the adoption of artemisinin-based combination therapy: analysis of genetic mutations in the pfcrt gene

This study investigated the polymorphism in the P. falciparum chloroquine resistance transporter (pfcrt) gene 11 years after chloroquine (CQ) cessation in Jazan region, southwestern Saudi Arabia. Two hundred and thirty-five P. falciparum isolates were amplified to detect mutations in the pfcrt gene. The pfcrt 76T molecular marker for CQ resistance was detected in 66.4% (156/235) of the isolates, while the K76 CQ-sensitive wild type was detected in 33.6%. The pfcrt 74I and pfcrt 75E point mutations were each found to be present in 56.2% of isolates, while only four isolates (1.7%) were found to carry the pfcrt 72S mutation. Moreover, four pfcrt haplotypes were identified: the CVIET triple-allele (56.2%), SVMET double-allele (1.7%), and CVMNT single-allele (8.5%) mutant haplotypes, and the CVMNK wild haplotype (33.6%). The analysis also revealed significant associations between the prevalence of mutant pfcrt alleles and haplotypes and the age group, governorate, and nationality of the patients as well as the parasitaemia level (P < 0.05). The findings provide evidence of the potential re-emergence of CQ-susceptible P. falciparum strains in Jazan region over a decade after CQ discontinuation, with about one third of the isolates analysed carrying the pfcrt K76 CQ-sensitive wild allele and the CVMNK ancestral wild haplotype. Although the reintroduction of CQ cannot be recommended at present in Saudi Arabia, these findings support the rationale for a potential future role for CQ in malaria treatment. Therefore, continuous molecular and in-vitro monitoring mutations of pfcrt polymorphism in Jazan region is highly recommended.

The return of chloroquine-sensitive Plasmodium falciparum parasites in Jazan Region, Southwestern Saudi Arabia over a decade after the adoption of artemisinin-based combination therapy: analysis of genetic mutations in the pfcrt gene

This study investigated the polymorphism in the P. falciparum chloroquine resistance transporter (pfcrt) gene 11 years after chloroquine (CQ) cessation in Jazan region, southwestern Saudi Arabia. Two hundred and thirty-five P. falciparum isolates were amplified to detect mutations in the pfcrt gene. The pfcrt 76T molecular marker for CQ resistance was detected in 66.4% (156/235) of the isolates, while the K76 CQ-sensitive wild type was detected in 33.6%. The pfcrt 74I and pfcrt 75E point mutations were each found to be present in 56.2% of isolates, while only four isolates (1.7%) were found to carry the pfcrt 72S mutation. Moreover, four pfcrt haplotypes were identified: the CVIET triple-allele (56.2%), SVMET double-allele (1.7%), and CVMNT single-allele (8.5%) mutant haplotypes, and the CVMNK wild haplotype (33.6%). The analysis also revealed significant associations between the prevalence of mutant pfcrt alleles and haplotypes and the age group, governorate, and nationality of the patients as well as the parasitaemia level (P < 0.05). The findings provide evidence of the potential re-emergence of CQ-susceptible P. falciparum strains in Jazan region over a decade after CQ discontinuation, with about one third of the isolates analysed carrying the pfcrt K76 CQ-sensitive wild allele and the CVMNK ancestral wild haplotype. Although the reintroduction of CQ cannot be recommended at present in Saudi Arabia, these findings support the rationale for a potential future role for CQ in malaria treatment. Therefore, continuous molecular and in-vitro monitoring mutations of pfcrt polymorphism in Jazan region is highly recommended.

Molecular surveillance of antimalarial drug resistance in the Democratic Republic of Congo: high variability of chloroquinoresistance and lack of amodiaquinoresistance

Background The loss of chloroquine (CQ) effectiveness has led to its withdrawal from national policies as a first-line treatment for uncomplicated malaria in several endemic countries, such as the Democratic Republic of Congo (DRC). The K76T mutation on the pfcrt gene has been identified as a marker of CQ resistance and the SVMNT haplotype in codons 72–76 on the same gene has been associated with resistance to amodiaquine (AQ). In the DRC, the prevalence of K76T has decreased from 100% in 2000 to 63.9% in 2014. The purpose of this study was to determine the prevalence of K76T mutations in circulating strains of P. falciparum , sixteen years after CQ withdrawal in the DRC and to investigate the presence of the SVMNT haplotype. Methods In 2017, ten geographical sites across the DRC were selected. Dried blood samples were collected from patients attending health centres. Malaria was first detected by a rapid diagnostic test (RDT) available on site (SD Bioline Malaria Ag P.f or CareStart Malaria Pf ) or thick blood smear and then confirmed by a P. falciparum species-specific real-time PCR assay. A pfcrt gene segment containing a fragment that encodes amino acids at positions 72-76 was amplified by conventional PCR before sequencing. Results A total of 1070 patients were enrolled. Of the 806 PCR-confirmed P. falciparum positive samples, 764 were successfully sequenced. The K76T mutation was detected in 218 samples (28.5%; 95% CI: 25.4%–31.9%), mainly (96%) with the CVIET haplotype. Prevalence of CQ resistance marker was unequally distributed across the country, ranging from 1.5% in Fungurume to 89.5% in Katana. The SVMNT haplotype, related to AQ resistance, was not detected. Conclusion Overall, the frequency of the P. falciparum CQ resistance marker has decreased significantly and no resistance marker to AQ was detected in the DRC in 2017. However, the between regions variability of CQ resistance remains high in the country. Further studies are needed for continuous monitoring of the CQ resistance level for its prospective re-use in malaria management. The absence of the AQ resistance marker is in line with the use of this drug in the current DRC malaria treatment policy.

Molecular surveillance of antimalarial drug resistance in Democratic Republic of Congo: high variability of chloroquinoresistance and lack of amodiaquinoresistance

Background The loss of chloroquine (CQ) effectiveness has led to its withdrawal from national policies as first-line treatment for uncomplicated malaria in several endemic countries such as in the Democratic Republic of Congo (DRC). The K76T mutation on the pfcrt gene has been identified as a marker of CQ resistance and the SVMNT haplotype in codons 72–76 on the same gene has been associated with resistance to amodiaquine (AQ). In DRC, the prevalence of K76T has decreased from 100% in 2000 to 63.9% in 2014. The purpose of the study was to determine the prevalence of K76T mutations in P. falciparum circulating strains, sixteen years after CQ withdrawal in DRC and to investigate the presence of SVMNT haplotype. Methods In 2017, ten geographical sites across DRC were selected. Dried blood samples were collected from patients attending health centers. Malaria was first detected by rapid diagnostic test (RDT) available on site (SD Bioline malaria Ag Pf or CareStart Malaria Pf) or thick blood smear and then confirmed by a P. falciparum species-specific real-time PCR assay. A pfcrt gene segment containing a fragment that encodes amino acids at positions 72-76 was amplified by conventional PCR before sequencing. Results A total of 1070 patients were enrolled. Of the 806 PCR-confirmed P. falciparum positive samples, 764 were successfully sequenced. The K76T mutation was detected in 218 (28.5%; 95% CI: 25.4% – 31.9%) samples, mainly (96%) with the CVIET haplotype. The CQ resistance prevalence was unequally distributed across the country ranging from 1.5% in Fungurume to 89.5% in Katana. The SVMNT haplotype, related to AQ resistance, was not detected. Conclusion Overall, the frequency of P. falciparum CQ resistance marker has decreased significantly and no resistance marker to AQ was detected in DRC in 2017. However, the between regions variability of CQ resistance remains high in the country. Further studies are needed for a continuous monitoring of the CQ resistance level for a prospective re-use in malaria management. The absence of AQ resistance is in line with the use of this drug in the current DRC malaria treatment policy.

Molecular surveillance of antimalarial drug resistance in Democratic Republic of Congo: high variability of chloroquinoresistance and lack of amodiaquinoresistance

Background The loss of chloroquine (CQ) effectiveness has led to its withdrawal from national policies as first-line treatment for uncomplicated malaria in several endemic countries such as in the Democratic Republic of Congo (DRC). The K76T mutation on the pfcrt gene has been identified as a marker of CQ resistance and the SVMNT haplotype in codons 72–76 on the same gene has been associated with resistance to amodiaquine (AQ). In DRC, the prevalence of K76T has decreased from 100% in 2000 to 63.9% in 2014. The purpose of the study was to determine the prevalence of K76T mutations in P. falciparum circulating strains, sixteen years after CQ withdrawal in DRC and to investigate the presence of SVMNT haplotype. Methods In 2017, ten geographical sites across DRC were selected. Dried blood samples were collected from patients attending health centers. Malaria was first detected by rapid diagnostic test (RDT) available on site (SD Bioline malaria Ag Pf or CareStart Malaria Pf) or thick blood smear and then confirmed by a P. falciparum species-specific real-time PCR assay. A pfcrt gene segment containing a fragment that encodes amino acids at positions 72-76 was amplified by conventional PCR before sequencing. Results A total of 1070 patients were enrolled. Of the 806 PCR-confirmed P. falciparum positive samples, 764 were successfully sequenced. The K76T mutation was detected in 218 (28.5%; IC95%: 25.4% – 31.9%) samples, mainly (96%) with the CVIET haplotype. The CQ resistance prevalence was unequally distributed across the country ranging from 1.5% in Fungurume to 89.5% in Katana. The SVMNT haplotype, related to AQ resistance, was not detected. Conclusion Overall, the P. falciparum CQ resistance rate has decreased significantly and no resistance marker to AQ was detected in DRC in 2017. However, the between regions variability of CQ resistance remains high in the country. Further studies are needed for a continuous monitoring of the CQ resistance level for a prospective re-use in malaria management. The absence of AQ resistance is in line with the use of this drug in the current DRC malaria treatment policy.

Investigating the Presence of Plasmodium falciparum Chloroquine Resistant Transporter (Pfcrt) Drug-resistance Alleles in Some Northern Nigerian States

This study was designed to investigate the presence of Pfcrt drug- resistance alleles (CQ resistant biomarker) and attempted to analyse the outcome in some states of northern Nigeria. A total of four hundred and thirteen (413) Plasmodium falciparum positive blood samples were collected from Kaduna, Jigawa, Katsina and Kebbi states during the period of April-August 2013. The samples were genotyped at codon 76 using specific primers for Pfcrt gene. The data was analysed using Chi-square to determine significance association. Four hundred and thirteen (413) P. falciparum positive samples were genotyped at codon 76 of pfcrt gene. Sixty eight 68(16.5%) samples contained single K76 (Chloroquine sensitive) alleles, 49(11.9%) contained 76T, while 16(3.9%) contained mixed K76T alleles. K76 alleles were highest in Kaduna state 17(32.1%) and lowest in Kebbi state 10(7.4%), 76T was highest in Jigawa state 11 (25.6%) and lowest in Kebbi state 7(5.2%) while K76T was highest in Jigawa state 5(11.5%) and lowest in Kebbi state 2(1.5%) with significant difference between the states P<0.05. K76 was higher among females 43(17.6%), 76T was also higher females 30(12.2%) while K76T was higher among males 7 (4.2%). K76 was higher among age group of 16-25 years 17(22.4%) and least among 26-40years age group 13 (13.5%). 76T was also higher among 26-40 years age group 17(17.7%) and least among age group >40 years 1(2.0%) and K76T was higher among age group 16-25 years 6(7.9%) and least in >40 years of age 1(2.0%) with high significant difference P<0.05 between the age groups. The results of this study genetically confirms the use of CQ for malaria treatment in the area and attributed the varied distribution across the states, to high irrigation activities, self medication leading to dosage non compliance and improper diagnosis due to use of low sensitive RDT in most government hospitals. The need for enlightenment of the populace cannot over emphasize.

Pfcrt mutant haplotypes may not correspond with chloroquine resistance

Introduction: Chloroquine resistance in Plasmodium falciparum is associated with mutations in pfcrt and pfmdr1 genes. The frequency distribution of pfcrt K76T and pfmdr1 N86Y mutations and their association with chloroquine susceptibility was studied in an endemic area along the Indo-Bangladesh border. Methodology: A single-arm prospective study of clinical and parasitological responses in P. falciparum malaria patients to chloroquine was conducted in vivo. PCR-RFLP assay was used to detect pfcrt K76T and pfmdr1 N86Y mutations in P. falciparum. The PCR products of pfcrt gene were sequenced, translated and aligned for haplotyping. Results: Out of 63 cases, 44 (69.8%) responded adequately to chloroquine treatment. Pfcrt K76T mutation was recorded in 100% of the treatment failure cases, whereas pfmdr1 N86Y mutation was found in 52.6% of the cases only. Early treatment failure (84.2%) occurred more frequently than late treatment failure (15.8%). Kaplan–Meier survival analysis showed that the probability estimate for treatment success after 7 and 15 days was 0.84 (95% CI = 0.72-0.92) and 0.70 (95% CI = 0.57-0.80), respectively. Sequence analysis of 72 to 76 pfcrt gene codons revealed the presence of two mutant (CVMNT, CVIET) and two wild (CVMNK, CVIEK) haplotypes. The mutant CVIET haplotype was predominantly distributed (42.1%). Conclusions: The presence of mutations in pfcrt K76T and pfmdr1 N86Y genes is not sufficient to explain the therapeutic efficacy of chloroquine to P. falciparum. Study suggests that pfcrt K76T mutant haplotypes are widely distributed and are spreading diligently, which needs to be taken into account in devising an antimalarial policy.