Performance of a Commercial Multiplex Allele-Specific Polymerase Chain Reaction Kit to Genotype African-Type Glucose-6-Phosphate Dehydrogenase Deficiency

Abstract

8-Aminoquinoline antimalarial drugs (primaquine, tafenoquine) are required for complete cure of Plasmodium vivax malaria, but they are contraindicated in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. In the absence of spectrophotometry, which is a gold standard for measuring G6PD activity, G6PD genotyping is one of the alternatives to establish a database and distribution map of G6PD enzyme deficiency in Mauritania, which has become a new epicenter of P. vivax malaria in West Africa. The aim of our study was to assess the performance of multiplex allele-specific polymerase chain reaction (PCR) (African-type Diaplex C™ G6PD kit) against PCR–restriction fragment length polymorphism and sequencing. Of 146 mutations associated with G6PD A− genotypes in 177 blood samples from Mauritanian patients, all but two samples were identified correctly using multiplex allele-specific PCR (100% sensitivity and 99% specificity; “almost perfect agreement” between allele-specific PCR and PCR-restriction fragment length polymorphism/sequencing, with a kappa coefficient of 0.977). Despite a suboptimal PCR protocol for dried blood spots and the inability of the commercial assay to predict unequivocally the G6PD enzyme level in heterozygous females, the African-type Diaplex C™ G6PD genotyping kit seemed to be a valuable screening tool for male subjects and for research purposes in resource-limited countries where spectrophotometer and DNA sequencing are not available.

Single-Drop Blood Detection of Common G6PD Mutations in Thailand Based on Allele-Specific Recombinase Polymerase Amplification with CRISPR-Cas12a

“…G6PD genotyping can be achieved in several ways, including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), ,, Taq Man SNP assay, AS-PCR, reverse dot blot hybridization (RDB), amplification refractory mutation system, , gold nanoparticle-based test, high-resolution melting curve analysis (HRM), − and DNA sequencing. , In addition, a high-throughput multiplex AS-PCR-based assay is available for the diagnosis of G6PD mutations. DiaPlexC G6PD genotyping kits based on a single PCR step with gel electrophoresis are capable of identifying the G6PD mutations, which are specific to Asians , and Africans. , Recently, isothermal amplification methods using locked nucleic acids (LNAs) and the Yaku-Bonczyk principle primers for identifying substitution mutations for G6PD mutations at the point of care have been explored, as well. A LNA is a nucleic acid analogue that improves mismatch discrimination over DNA-only primers by locking the ribose moiety into a C3′-endo conformation using a 2′-O, 4′-C methylene bridge. , In accordance with the Yaku-Bonczyk principle, the AS primer design method was applied to increase the performance of primers that distinguish mutant from wild type DNA.…”

“…DiaPlexC G6PD genotyping kits based on a single PCR step with gel electrophoresis are capable of identifying the G6PD mutations, which are specific to Asians 30 , 31 and Africans. 32 , 33 Recently, isothermal amplification methods using locked nucleic acids (LNAs) and the Yaku-Bonczyk principle primers for identifying substitution mutations for G6PD mutations at the point of care have been explored, as well. A LNA is a nucleic acid analogue that improves mismatch discrimination over DNA-only primers by locking the ribose moiety into a C3′-endo conformation using a 2′-O, 4′-C methylene bridge.…”