AFONSO RAFAEL DA SILVA JUNIOR

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P ICHC, Hospital das Clínicas, Faculdade de Medicina

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Agora exibindo 1 - 4 de 4
  • article 32 Citação(ões) na Scopus
    Rapid identification of moulds and arthroconidial yeasts from positive blood cultures by MALDI-TOF mass spectrometry
    (2016) ALMEIDA JR., Joao N. de; SZTAJNBOK, Jaques; SILVA JUNIOR, Afonso Rafael da; VIEIRA, Vinicius Adriano; GALASTRI, Anne Layze; BISSOLI, Leandro; LITVINOV, Nadia; NEGRO, Gilda Maria Barbaro Del; MOTTA, Adriana Lopes; ROSSI, Flavia; BENARD, Gil
    Moulds and arthroconidial yeasts are potential life-threatening agents of fungemia in immunocompromised patients. Fast and accurate identification (ID) of these pathogens hastens initiation of targeted antifungal therapy, thereby improving the patients' prognosis. We describe a new strategy that enabled the identification of moulds and arthroconidial yeasts directly from positive blood cultures by MALDI-TOFmass spectrometry (MS). Positive blood cultures (BCs) with Gram staining showing hyphae and/or arthroconidia were prospectively selected and submitted to an in-house protein extraction protocol. Mass spectra were obtained by Vitek MS (TM) system, and identifications were carried out with in the research use only (RUO) mode with an extended database (SARAMIS (TM) [v.4.12] plus in-house database). Fusarium solani, Fusarium verticillioides, Exophiala dermatitidis, Saprochaete clavata, and Trichosporon asahii had correct species ID by MALDI-TOF MS analysis of positive BCs. All cases were related to critically ill patients with high mortality fungemia and direct ID from positive BCs was helpful for rapid administration of targeted antifungal therapy.
  • article 1 Citação(ões) na Scopus
    Performance of a Real-Time PCR Assay for the Detection of Five Candida Species in Blood Samples from ICU Patients at Risk of Candidemia
    (2023) FELIX, Gabriel N.; FREITAS, Vera L. T. de; SILVA JUNIOR, Afonso R. da; MAGRI, Marcello M. C.; ROSSI, Flavia; SEJAS, Odeli N. E.; ABDALA, Edson; MALBOUISSON, Luiz M. S.; GUIMARAES, Thais; BENARD, Gil; NEGRO, Gilda M. B. Del
    The gold standard for diagnosing invasive candidiasis still relies on blood cultures, which are inefficient and time-consuming to analyze. We developed an in-house qPCR assay to identify the 5 major Candida species in 78 peripheral blood (PB) samples from ICU patients at risk of candidemia. Blood cultures and (1,3)-beta-D-glucan (BDG) testing were performed concurrently to evaluate the performance of the qPCR. The qPCR was positive for DNA samples from all 20 patients with proven candidemia (positive PB cultures), showing complete concordance with Candida species identification in blood cultures, except for detection of dual candidemia in 4 patients, which was missed by blood cultures. Additionally, the qPCR detected Candida species in six DNA samples from patients with positive central venous catheters blood (CB) but negative PB cultures. BDG values were similarly high in these six samples and the ones with proven candidemia, strongly suggesting the diagnosis of a true candidemia episode despite the negative PB cultures. Samples from patients neither infected nor colonized yielded negative results in both the qPCR and BDG testing. Our qPCR assay was at least as sensitive as blood cultures, but with a shorter turnaround time. Furthermore, negative results from the qPCR provided strong evidence for the absence of candidemia caused by the five major Candida species.
  • article 35 Citação(ões) na Scopus
    Identification of Candida haemulonii Complex Species: Use of ClinProTools (TM) to Overcome Limitations of the Bruker Biotyper (TM), VITEK MS (TM) IVD, and VITEK MS (TM) RUO Databases
    (2016) GRENFELL, Rafaella C.; SILVA JUNIOR, Afonso R. da; NEGRO, Gilda M. B. Del; MUNHOZ, Regina B.; GIMENES, Viviane M. F.; ASSIS, Diego M.; ROCKSTROH, Anna C.; MOTTA, Adriana L.; ROSSI, Flavia; JULIANO, Luiz; BENARD, Gil; ALMEIDA JUNIOR, Joao N. de
    Candida haemulonli is now considered a complex of two species and one variety: C. haemulonii sensu strict, Candida duobushaemulonii and the variety C. haemulonii var. vulnera. Identification (ID) of these species is relevant for epidemiological purposes and for therapeutic management, but the different phenotypic commercial systems are unable to provide correct species ID for these emergent pathogens. Hence, we evaluated the MALDI-TOF MS performance for the ID of C. haemulonli species, analyzing isolates/strains of C. haemulonli complex species, Candida pseudohaemulonii and Candida auris by two commercial platforms, their databases and softwares. To differentiate C. haemulonli sensu sctricto from the variety vulnera, we used the ClinProTools (TM) models and a single-peak analysis with the software FlexAnalysis (TM). The Biotyper (TM) database gave 100% correct species ID for C. haemulonii sensu strict, C. pseudohaemulonii and C. auris, with 69% of correct species ID for C. duobushaemulonii. Vitek MS (TM) IVD database gave 100% correct species ID for C. haemulonii sensu stricto, misidentifying all C. duobushaemulonii and C. pseudohaemulonii as C. haemulonii, being unable to identify C. auris. The Vitek MS (TM) RUO database needed to be upgraded with in-house SuperSpectra to discriminate C. haemulonii sensu stricto, C. duobushaemulonii, C. pseudohaemulonii, and C. auris strains/isolates. The generic algorithm model from ClinProTools (TM) software showed recognition capability of 100% and cross validation of 98.02% for the discrimination of C. haemulonli sensu stricto from the variety vulnera. Single-peak analysis showed that the peaks 5670, 6878, or 13750 m/z can distinguish C. haemulonli sensu stricto from the variety vulnera.
  • article 12 Citação(ões) na Scopus
    Evaluation of Vitek MS for Differentiation of Cryptococcus neoformans and Cryptococcus gattii Genotypes
    (2019) SIQUEIRA, Lumena P. Machado; GIMENES, Viviane M. Favero; FREITAS, Roseli Santos de; MELHEM, Marcia de Souza Carvalho; BONFIETTI, Lucas Xavier; JR, Afonso Rafael da Silva; SANTOS, Leticia B. Souza; MOTTA, Adriana L.; ROSSI, Flavia; BENARD, Gil; JR, Joao N. de Almeida
    Cryptococcus neoformans and Cryptococcus gattii are the main pathogenic species of invasive cryptococcosis among the Cryptococcus species. Taxonomic studies have shown that these two taxa have different genotypes or molecular types with biological and ecoepidemiological peculiarities. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been proposed as an alternative method for labor-intensive methods for C. neoformans and C. gattii genotype differentiation. However, Vitek MS, one of the commercial MALDI-TOF MS instruments, has not been yet been evaluated for this purpose. Thus, we constructed an in-house database with reference strains belonging to the different C. neoformans (VNI, VNII, VNIII, and VNIV) and C. gattii (VGI, VGII, VGIII, and VGIV) major molecular types by using the software Saramis Premium (bioMerieux, Marcy-l'Etoile, France). Then, this new database was evaluated for discrimination of the different genotypes. Our in-house database provided correct identification for all C. neoformans and C. gattii genotypes; however, due to the intergenotypic mass spectral similarities, a careful postanalytic evaluation is necessary to provide correct genotype identification.