Detecting mutations in PfCRT and PfMDR1 genes among Plasmodium falciparum isolates from Saudi Arabia by pyrosequencing

• Published in: Parasitology research (1987) Vol. 109; no. 2; pp. 291 - 296
• Main Authors: Bin Dajem, Saad M., Al-Sheikh, Adel Ali H., Bohol, Marie Fe, Alhawi, Mohammad, Al-Ahdal, Mohammed N., Al-Qahtani, Ahmed
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.08.2011; Springer
• Subjects: Amino Acid Substitution - genetics; Analysis; Antimalarials - pharmacology; Biological and medical sciences; Biomedical and Life Sciences; Biomedicine; Chloroquine - pharmacology; Codon; DNA; DNA Primers - genetics; DNA, Protozoan - chemistry; DNA, Protozoan - genetics; Drug Resistance; Drug resistance in microorganisms; Fundamental and applied biological sciences. Psychology; Gene mutations; General aspects; General aspects and techniques. Study of several systematic groups. Models; Genes; Genetic aspects; Human protozoal diseases; Humans; Immunology; Infectious diseases; Invertebrates; Malaria; Malaria, Falciparum - parasitology; Medical Microbiology; Medical sciences; Membrane Transport Proteins - genetics; Microbiology; Multidrug Resistance-Associated Proteins - genetics; Mutation, Missense; Original Paper; Parasitic diseases; Parasitic Sensitivity Tests - methods; Parasitology - methods; Plasmodium falciparum; Plasmodium falciparum - genetics; Plasmodium falciparum - isolation & purification; Polymerase Chain Reaction; Protozoal diseases; Protozoan Proteins - genetics; Saudi Arabia; Sequence Analysis, DNA - methods; Saudi Arabia; Asia; PfMDR1 Gene; Lumefantrine; Malaria; Artemisinin; Chloroquine Resistance; Infection; Protozoa; Plasmodium falciparum; Apicomplexa; Protozoal disease; Gene; Parasite; Parasitosis; Mutation
• ISSN: 0932-0113; 1432-1955
• DOI: 10.1007/s00436-011-2251-5

The emergence of chloroquine resistance in Plasmodium falciparum is a significant public health problem where malaria is endemic. We aimed to evaluate the efficacy of pyrosequencing to assess chloroquine resistance among P. falciparum isolates from the southwestern region of Saudi Arabia by analyzing the K76T and N86Y mutations in the P. falciparum chloroquine resistance transporter (PfCRT) and P. falciparum multidrug resistance 1 (PfMDR1) genes, respectively. Blood samples ( n  = 121) from microscopically positive P. falciparum cases were collected. DNA was extracted, and fragments from each of the genes were amplified by PCR using new sets of primers. The amplicons were sequenced using a pyrosequencer. All of the 121 samples were amplified for assessment of the PfCRT K76T and PfMDR1 N86Y mutations. All of the samples amplified for the PfCRT 76T mutation harbored the ACA codon (121/121; 100%), indicating the presence of the 76T mutation. For the PfMDR1 N86Y mutation, 72/121 samples (59.5%) had the sequence AAT at that position, indicating the presence of the wild-type allele (86N). However, 49/121 samples (40.5%) had a TAT codon, indicating the mutant allele (Y) at position 86. This study shows that pyrosequencing could be useful as a high throughput, rapid, and sensitive assay for the detection of specific single nucleotide polymorphisms in drug-resistant P. falciparum strains. This will help health authorities in malaria-endemic regions to adopt new malaria control strategies that will be applicable for diagnostic and drug resistance assays for malaria and other life-threatening pathogens that are endemic in their respective countries.