High-resolution melting assay for genotyping variants of the CYP2C19 enzyme and predicting voriconazole effectiveness

Voriconazole is a triazole antifungal agent recommended as primary treatment for invasive aspergillosis, as well as some other mold infections. However, it presents some pharmacokinetic singularities that lead to a great variability intra- and interindividually, nonlinear pharmacokinetics, and a narrow therapeutic range. Most experts have recommended tracing the levels of voriconazole in patients when receiving treatment. This azole is metabolized through the hepatic enzyme complex cytochrome P450 (CYPP450), with the isoenzyme CYP2C19 being principally involved. Allelic variations (polymorphisms) of the gene that encodes this enzyme are known to contribute to variability in voriconazole exposure. Three different allelic variants, CYP2C19*17, CYP2C19*2, and CYP2C19*3, could explain most of the phenotypes related to the voriconazole metabolism and some of its pharmacokinetic singularities. We designed a rapid molecular method based on high-resolution melting to characterize these polymorphisms in a total of 142 samples, avoiding sequencing. Three PCRs were designed with similar cycling conditions to run simultaneously. The results showed that our method represents a fast, accurate, and inexpensive means to study these variants related to voriconazole metabolism. In clinical practice, this could offer a useful tool to individually optimize therapy and reduce expenses in patients with fungal infections.National Institute of Health Carlos III (AES13PI13/01817Research Project MPY 1367/13). L.B.-M. has a contract supported by theMinisterio de Ciencia e Innovación, Instituto de Salud Carlos III, cofinanced by the EuropeanDevelopment Regional Fund (EDRF) “A Way to Achieve Europe” and the SpanishNetwork for the Research in Infectious Diseases (REIPI; RD12/0015/0015). B.M.-R. is astudent in the Master’s Program entitled “Microbiología Aplicada a la Salud Pública eInvestigación en Enfermedades Infecciosas,” Alcalá de Henares University, Madrid,Spain. A.C. and C.C. were supported by the Northern Portugal Regional OperationalProgram (NORTE 2020) under the Portugal 2020 Partnership Agreement through theEuropean Regional Development Fund (FEDER; NORTE-01-0145-FEDER-000013) and theFundação Para a Ciência e Tecnologia (FCT; IF/00735/2014 [A.C.] and SFRH/BPD/96176/2013 [C.C.])