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.])

Clinical factors associated with a Candida albicans Germ Tube Antibody positive test in Intensive Care Unit patients

Background: Poor outcomes of invasive candidiasis (IC) are associated with the difficulty in establishing the microbiological diagnosis at an early stage. New scores and laboratory tests have been developed in order to make an early therapeutic intervention in an attempt to reduce the high mortality associated with invasive fungal infections. Candida albicans IFA IgG has been recently commercialized for germ tube antibody detection (CAGTA). This test provides a rapid and simple diagnosis of IC (84.4% sensitivity and 94.7% specificity). The aim of this study is to identify the patients who could be benefited by the use of CAGTA test in critical care setting. Methods: A prospective, cohort, observational multicentre study was carried out in six medical/surgical Intensive care units (ICU) of tertiary-care Spanish hospitals. Candida albicans Germ Tube Antibody test was performed twice a week if predetermined risk factors were present, and serologically demonstrated candidiasis was considered if the testing serum dilution was >= 1: 160 in at least one sample and no other microbiological evidence of invasive candidiasis was found. Results: Fifty-three critically ill non-neutropenic patients (37.7% post surgery) were included. Twenty-two patients (41.5%) had CAGTA-positive results, none of them with positive blood culture for Candida. Neither corrected colonization index nor antifungal treatment had influence on CAGTA results. This finding could corroborate that the CAGTA may be an important biomarker to distinguish between colonization and infection in these patients. The presence of acute renal failure at the beginning of the study was more frequent in CAGTA-negative patients. Previous surgery was statistically more frequent in CAGTA-positive patients. Conclusions: This study identified previous surgery as the principal clinical factor associated with CAGTA-positive results and emphasises the utility of this promising technique, which was not influenced by high Candida colonization or antifungal treatment. Our results suggest that detection of CAGTA may be important for the diagnosis of invasive candidiasis in surgical patients admitted in ICU.This study has been supported by a Pfizer research gran