CARACTERIZAÇÃO MOLECULAR DE ISOLADOS DO VÍRUS VARICELA ZOSTER EM AMOSTRAS DE LÍQUIDO CEFALORRAQUIDIANO DE PACIENTES COM QUADRO DE MENINGITE, ENCEFALITE OU MENINGOENCEFALITE AGUDA

Introduction: Varicella Zoster Virus (VZV) is classified as human herpesvirus type (HHV-3).Its double stranded DNA genome comprises 125 bp.Currently, nine clades are described.It is the etiologic agent of varicella and, in case of reactivation, of herpes zoster.The most severe complications, in both clinical presentations, are the ones evolving central nervous system (CNS), among them, cases of acute meningitis, encephalitis and/or meningoencephalitis (MEM).Difficulty in accessing diagnostic tests with adequate sensitivity and specificity in the Brazilian public health system for VZV is a limiting factor for the diagnostic characterization and molecular epidemiology of VZV in different regions of our country.As a consequence, there are few studies in this regard in Brazil, despite its importance for the global and local public health scenario.Objectives: 1-To evaluate the performance of an in-house real-time PCR test and to validate it for its application in the detection of VZV DNA in CSF; 2-To characterize the identified VZV isolates to differentiate VZV vaccine strains from wildtype strains; 3-To characterize the identified VZV isolates to determine the phylogenetic classification of their clades.Material and Methods: The samples analyzed in this study are part of a larger study that included 600 cases of acute MEM from the city of São Paulo, Brazil and were collected between February 2018 and December 2019.For validation and evaluation of the analytical sensitivity and performance of the in-house test, a panel of VZV controls in the CSF, previously tested by reference diagnostic platforms and synthetic controls with known concentration, were used.For the differentiation of VZV vaccine strains from wild-type strains, the methodology used was based on the detection of SNPs in the 107252 region of the VZV genome, by real-time PCR.For the phylogenetic classification of the clades, amplification of the VZV DNA sequences of four ORFs of the viral genome, namely, 22, 38, 58 and 62, was used by Sanger method (SM) and, complementarily, by the massive parallel sequencing (MPS) method.Results: Among all analyzed samples, 30 cases of VZV MEM were identified, 5% (30/600) of included cases in the initial study.The performance of the in-house test for VZV was satisfactory and its analytical sensibility was < 5 copies per reaction.VZV vaccine strains were not identified.In 40% (12/30) of all analyzed samples, we were able to identify the evolved clades, as follows: clade 1 (5/12), clade 2 (3/12), clade 3 (1/12), clade 5 (2/12) andclade 6 (1/12), by SM.The adapted SMP method was responsible for identifying only a single VZV isolate among all (28) available samples.By using SMP we were able to identify samples suggestive of the presence of co-infection with other pathogens, among the analyzed samples.Conclusions: The in-house real-time PCR test, performed satisfactorily in terms of efficiency and analytical sensitivity, thus constituting an adequate diagnostic tool for the identification of VZV in CSF samples, in places with less access to commercial platforms already available for VZV detection.In the analyzed samples, we were not able to identify the presence of VZV vaccine strains and we were able to identify the presence of clade 2, for the first time in Latin America, and clade 6, for the first time in our country.The adapted MPS method did not meet the expectations of diagnostic complementation in relation to the SM in the analyzed samples.