scholarly journals 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

2021 ◽  
Author(s):  
Aymen M. Madkhali ◽  
Ahmed A. Abdulhaq ◽  
Wahib M. Atroosh ◽  
Ahmad Hassn Ghzwani ◽  
Khalid Ammash Zain ◽  
...  
Keyword(s):  
Saudi Arabia ◽  
Point Mutations ◽  
Chloroquine Resistance ◽  
Wild Type ◽  
Chloroquine Resistance Transporter ◽  
Pfcrt Gene ◽  
Wild Allele ◽  
Parasitaemia Level ◽  
Single Allele

Abstract 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.

scholarly journals 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

2021 ◽  
Author(s):  
Aymen M. Madkhali ◽  
Ahmed A. Abdulhaq ◽  
Wahib M. Atroosh ◽  
Ahmad Hassn Ghzwani ◽  
Khalid Ammash Zain ◽  
...  
Keyword(s):  
Saudi Arabia ◽  
Point Mutations ◽  
Chloroquine Resistance ◽  
Wild Type ◽  
Chloroquine Resistance Transporter ◽  
Pfcrt Gene ◽  
Wild Allele ◽  
Parasitaemia Level ◽  
Single Allele

Abstract 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.

scholarly journals Polymorphism Analysis of pfmdr1 Gene in Plasmodium falciparum Isolates 11 Years Post-adoption of Artemisinin-based Combination Therapy in Saudi Arabia

2021 ◽  
Author(s):  
Hesham M. Al-Mekhlafi ◽  
Aymen M. Madkhali ◽  
Ahmed A. Abdulhaq ◽  
Wahib M. Atroosh ◽  
Ahmad Hassn Ghzwani ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Saudi Arabia ◽  
Combination Therapy ◽  
Point Mutations ◽  
Multi Drug Resistance ◽  
Polymorphism Analysis ◽  
Wild Allele ◽  
High Prevalence ◽  
Parasitaemia Level

Abstract 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.

scholarly journals Polymorphism analysis of pfmdr1 gene in Plasmodium falciparum isolates 11 years post-adoption of artemisinin-based combination therapy in Saudi Arabia

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Hesham M. Al-Mekhlafi ◽  
Aymen M. Madkhali ◽  
Ahmed A. Abdulhaq ◽  
Wahib M. Atroosh ◽  
Ahmad Hassn Ghzwani ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Saudi Arabia ◽  
Combination Therapy ◽  
Point Mutations ◽  
Multi Drug Resistance ◽  
Polymorphism Analysis ◽  
Wild Allele ◽  
High Prevalence ◽  
Parasitaemia Level

AbstractA 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.

scholarly journals Pfmdr1 Alleles and Response to Ultralow-Dose Mefloquine Treatment in Gabonese Patients

2002 ◽  
Vol 46 (1) ◽  
pp. 166-170 ◽  
Author(s):  
Denise P. Mawili-Mboumba ◽  
Jürgen F. J. Kun ◽  
Bertrand Lell ◽  
Peter G. Kremsner ◽  
Francine Ntoumi
Keyword(s):  
Point Mutations ◽  
Chloroquine Resistance ◽  
Low Grade ◽  
Wild Type ◽  
Multidrug Resistance Gene ◽  
Ultralow Dose ◽  
Group A ◽  
The Relationship

ABSTRACT The identification of parasite molecular markers involved in resistance to antimalarial compounds is of great interest for monitoring the development and spread of resistance in the field. Polymorphisms in Plasmodium falciparum multidrug resistance gene 1 (pfmdr1) have been associated with chloroquine resistance and mefloquine susceptibility. In the present study, carried out in Lambaréné, Gabon, we investigated the relationship between the presence of mutations at codons 86, 184, 1034, 1042, and 1246 in the pfmdr1 gene and the success of ultralow-dose mefloquine treatment (1.1 mg/kg of body weight). Sixty-nine patients were included in the study, and depending on the level of in vivo resistance to mefloquine, they were classified as sensitive responders (S), patients with low-grade resistance (RI), and nonresponders (NR). We found that the prevalences of the Tyr-86 mutation among isolates from patients in groups S, RI, and NR were 100, 96, and 90%, respectively, and that the prevalence of the Phe-184 mutation among the isolates was 80% in each group. A prevalence of about 10% point mutations at codons 1042 and 1246 was detected only in isolates from patients in groups RI and NR. There was no statistically significant association between the presence of the Tyr-86 mutation and the in vivo response (P = 0.79). Among the parasite isolates from patients with drug-resistant infections, 83% had the wild-type pfmdr1 genotype (S1034-N1042-D1246). No link between the presence of this genotype and parasite resistance was detected (P = 0.42). Among the isolates analyzed, 85 had double mutations (Y86-F184 or Y86-Y1246) and 11 had triple mutations (Y86-D1042-Y1246, Y86-F184-Y1246, or Y86-F184-D1042). These findings are not consistent with those of previous in vitro studies and suggest that further evaluation of pfmdr1 gene polymorphism and in vivo mefloquine sensitivity are needed.

scholarly journals Kenyan Plasmodium falciparum Field Isolates: Correlation between Pyrimethamine and Chlorcycloguanil Activity In Vitro and Point Mutations in the Dihydrofolate Reductase Domain

1998 ◽  
Vol 42 (1) ◽  
pp. 164-169 ◽  
Author(s):  
A. Nzila-Mounda ◽  
E. K. Mberu ◽  
C. H. Sibley ◽  
C. V. Plowe ◽  
P. A. Winstanley ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Dihydrofolate Reductase ◽  
Drug Response ◽  
Point Mutations ◽  
Distinct Group ◽  
Wild Type ◽  
Field Isolates ◽  
Vitro Data ◽  
In Vitro Data

ABSTRACT Sixty-nine Kenyan Plasmodium falciparum field isolates were tested in vitro against pyrimethamine (PM), chlorcycloguanil (CCG), sulfadoxine (SD), and dapsone (DDS), and their dihydrofolate reductase (DHFR) genotypes were determined. The in vitro data show that CCG is more potent than PM and that DDS is more potent than SD. DHFR genotype is correlated with PM and CCG drug response. Isolates can be classified into three distinct groups based on their 50% inhibitory concentrations (IC50s) for PM and CCG (P< 0.01) and their DHFR genotypes. The first group consists of wild-type isolates with mean PM and CCG IC50s of 3.71 ± 6.94 and 0.24 ± 0.21 nM, respectively. The second group includes parasites which all have mutations at codon 108 alone or also at codons 51 or 59 and represents one homogeneous group for which 25- and 6-fold increases in PM and CCG IC50s, respectively, are observed. Parasites with mutations at codons 108, 51, and 59 (triple mutants) form a third distinct group for which nine- and eightfold increases in IC50s, respectively, of PM and CCG compared to the second group are observed. Surprisingly, there is a significant decrease (P < 0.01) of SD and DDS susceptibility in these triple mutants. Our data show that more than 92% of Kenyan field isolates have undergone at least one point mutation associated with a decrease in PM activity. These findings are of great concern because they may indicate imminent PM-SD failure, and there is no affordable antimalarial drug to replace PM-SD (Fansidar).

scholarly journals Molecular Basis of In Vivo Resistance to Sulfadoxine-Pyrimethamine in African Adult Patients Infected withPlasmodium falciparum Malaria Parasites

1998 ◽  
Vol 42 (7) ◽  
pp. 1811-1814 ◽  
Author(s):  
Leonardo K. Basco ◽  
Rachida Tahar ◽  
Pascal Ringwald
Keyword(s):  
Dihydrofolate Reductase ◽  
Point Mutations ◽  
Gene Mutations ◽  
Adult Patients ◽  
Wild Type ◽  
Dihydropteroate Synthase ◽  
Reductase Gene ◽  
Parasite Populations

ABSTRACT In vitro sulfadoxine and pyrimethamine resistance has been associated with point mutations in the dihydropteroate synthase and dihydrofolate reductase domains, respectively, but the in vivo relevance of these point mutations has not been well established. To analyze the correlation between genotype and phenotype, 10 Cameroonian adult patients were treated with sulfadoxine-pyrimethamine and followed up for 28 days. After losses to follow-up (n = 1) or elimination of DNA samples due to mixed parasite populations with pyrimethamine-sensitive and pyrimethamine-resistant profiles (n = 3), parasite genomic DNA from day 0 blood samples of six patients were analyzed by DNA sequencing. Three patients who were cured had isolates characterized by a wild-type or mutant dihydrofolate reductase gene (with one or two mutations) and a wild-type dihydropteroate synthase gene. Three other patients who failed to respond to sulfadoxine-pyrimethamine treatment carried isolates with triple dihydrofolate reductase gene mutations and either a wild-type or a mutant dihydropteroate synthase gene. Three dihydrofolate reductase gene codons (51, 59, and 108) may be reliable genetic markers that can accurately predict the clinical outcome of sulfadoxine-pyrimethamine treatment in Africa.

Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system

1989 ◽  
Vol 9 (10) ◽  
pp. 4467-4472
Author(s):  
M Altmann ◽  
N Sonenberg ◽  
H Trachsel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Point Mutations ◽  
Translation Initiation Factor ◽  
Apparent Temperature ◽  
Initiation Factor ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Free System ◽  
Gal1 Promoter

The gene encoding translation initiation factor 4E (eIF-4E) from Saccharomyces cerevisiae was randomly mutagenized in vitro. The mutagenized gene was reintroduced on a plasmid into S. cerevisiae cells having their only wild-type eIF-4E gene on a plasmid under the control of the regulatable GAL1 promoter. Transcription from the GAL1 promoter (and consequently the production of wild-type eIF-4E) was then shut off by plating these cells on glucose-containing medium. Under these conditions, the phenotype conferred upon the cells by the mutated eIF-4E gene became apparent. Temperature-sensitive S. cerevisiae strains were identified by replica plating. The properties of one strain, 4-2, were further analyzed. Strain 4-2 has two point mutations in the eIF-4E gene. Upon incubation at 37 degrees C, incorporation of [35S]methionine was reduced to 15% of the wild-type level. Cell-free translation systems derived from strain 4-2 were dependent on exogenous eIF-4E for efficient translation of certain mRNAs, and this dependence was enhanced by preincubation of the extract at 37 degrees C. Not all mRNAs tested required exogenous eIF-4E for translation.

scholarly journals Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein by nsP2 protease

2001 ◽  
Vol 82 (4) ◽  
pp. 765-773 ◽  
Author(s):  
Andres Merits ◽  
Lidia Vasiljeva ◽  
Tero Ahola ◽  
Leevi Kääriäinen ◽  
Petri Auvinen
Keyword(s):  
Mammalian Cells ◽  
Active Sites ◽  
Insect Cells ◽  
Semliki Forest Virus ◽  
Point Mutations ◽  
Proteolytic Processing ◽  
Wild Type ◽  
In Trans ◽  
Polyprotein Precursor

The RNA replicase proteins of Semliki Forest virus (SFV) are translated as a P1234 polyprotein precursor that contains two putative autoproteases. Point mutations introduced into the predicted active sites of both proteases nsP2 (P2) and nsP4 (P4), separately or in combination, completely abolished virus replication in mammalian cells. The effects of these mutations on polyprotein processing were studied by in vitro translation and by expression of wild-type polyproteins P1234, P123, P23, P34 and their mutated counterparts in insect cells using recombinant baculoviruses. A mutation in the catalytic site of the P2 protease, C478A, (P2CA) completely abolished the processing of P12CA34, P12CA3 and P2CA3. Co-expression of P23 and P12CA34 in insect cells resulted in in trans cleavages at the P2/3 and P3/4 sites. Co-expression of P23 and P34 resulted in cleavage at the P3/4 site. In contrast, a construct with a mutation in the active site of the putative P4 protease, D6A, (P1234DA) was processed like the wild-type protein. P34 or its truncated forms were not processed when expressed alone. In insect cells, P4 was rapidly destroyed unless an inhibitor of proteosomal degradation was used. It is concluded that P2 is the only protease needed for the processing of SFV polyprotein P1234. Analysis of the cleavage products revealed that P23 or P2 could not cleave the P1/2 site in trans.

Induction of RNA editing at heterologous sites by sequences in apolipoprotein B mRNA

1993 ◽  
Vol 13 (12) ◽  
pp. 7288-7294
Author(s):  
D M Driscoll ◽  
S Lakhe-Reddy ◽  
L M Oleksa ◽  
D Martinez
Keyword(s):  
Rna Editing ◽  
Apolipoprotein B ◽  
Point Mutations ◽  
Upstream Region ◽  
Wild Type ◽  
Cross Link ◽  
Apo B ◽  
Optimal Distance ◽  
Luciferase Mrna

An RNA editing mechanism modifies apolipoprotein B (apo-B) mRNA in the intestine by converting cytosine at nucleotide (nt) 6666 to uracil. To define the sequence requirements for editing, mutant apo-B RNAs were analyzed for the ability to be edited in vitro by enterocyte extracts. Editing was detected by a sensitive and linear primer extension assay. An upstream region (nt 6648 to 6661) which affected the efficiency of editing was identified. RNAs with mutations in this efficiency sequence were edited at 22 to 160% of wild-type levels. Point mutations in a downstream 11-nt mooring sequence (nt 6671 to 6681) abolished editing, confirming previous studies (R. R. Shah, T. J. Knott, J. E. Legros, N. Navaratnam, J. C. Greeve, and J. Scott, J. Biol. Chem. 266:16301-16304, 1991). The optimal distance between the editing site and the mooring sequence is 5 nt, but a C positioned 8 nt upstream is edited even when nt 6666 contains U. The efficiency and mooring sequences were inserted individually and together adjacent to a heterologous C in apo-B mRNA. The mooring sequence alone induced editing of the C at nt 6597 both in vitro and in transfected rat hepatoma cells. Editing at nt 6597 was specific, was independent of editing at nt 6666, and was stimulated to wild-type levels when the efficiency sequence was also inserted. Introduction of the mooring sequence into a heterologous mRNA, luciferase mRNA, induced editing of an upstream cytidine. Although UV cross-linking studies have previously shown that proteins of 60 to 66 kDa cross-link to apo-B mRNA, these proteins did not cross-link to the luciferase translocation mutants.

Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae

1988 ◽  
Vol 8 (6) ◽  
pp. 2523-2535
Author(s):  
J H Hegemann ◽  
J H Shero ◽  
G Cottarel ◽  
P Philippsen ◽  
P Hieter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Chromosome ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Vector System ◽  
Chromosome Loss ◽  
Wild Type ◽  
Sequence Elements ◽  
Chromosome Fragments

Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.

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