Barth, Afonso Luis

Carneiro, Maiara dos Santos

Volpato, Fabiana Caroline Zempulski

Wink, Priscila Lamb

Drug Analytical Research

English

Lume 5.8

DRUG ANALYTICAL RESEARCH - ISSN: 2527-2616 XIII MEETING OF Pharmaceutical Sciences Graduate Program of UFRGS, 2021 – Brazil   www.seer.ufrgs.br/dar Drug Anal. Res., Porto Alegre, v. 5, n. 2, July/Dec. 202162Evaluation of filter paper to transport inactivated  Nontuberculous Mycobacteria for identification using the MALDI-TOF MS system Maiara dos Santos Carneiro *a,b; Fabiana Caroline Zempulki Volpato a; Priscila Lamb Wink a,c; Afonso Luis Barth a,b,c a Laboratório de Pesquisa em Resistência Bacteriana, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil; b PPGCF - Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil; c PPGCM – Programa de Pós-Graduação em Ciências Médicas, Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil *Doctoral student since 2017Research area: Investigation of Therapeutic Targets and Biomarkers (A3) Subject area: Therapeutic Targets and Biomarkers Keywords: Nontuberculous Mycobacteria;  Filter paper; MALDI-TOF MS; Means of transportation Introduction  The identification of Nontuberculous Mycobacteria (NTM) is troublesome due to the complexity involved in the sample preparation process and to the time needed to obtain satisfactory results. Usually, the differentiation of species belonging to the M. tuberculosis complex from Non-Tuberculous Mycobacteria (NTM) is carried out at the State Center for Health Surveillance in Rio Grande do Sul state. However, the  identification of NTM to species-level is only performed in the institution Professor Hélio Fraga in Rio de Janeiro state. Therefore, alternatives are needed to improve secure identification of the NTM regionally. The use of sterile filter paper previously impregnated with an inactivated bacterial biomass can be an option to transport  microorganisms between laboratories (1). In addition, identification by MALDI-TOF MS would significantly reduce the time to obtain result (2). This study aimed to evaluate the use of filter paper to transport NTM for identification using MALDI-TOF MS. Experimental section A total of 60 isolates of NTM (41 M. abscessus complex, 11 M. fortuitum complex, 3 M. avium complex, 2 M. smegmatis, 2 M. immunogenum, 1 M. intracellulare/M. chimaera) identified by a reference method (PRAhsp65) were evaluated in this study. MALDI-TOF MS identification of each isolate was performed as follows: a) directly from original bacterial colony in solid media; b) after the extraction procedure recommended by Bruker®; and c) in filter paper after procedures of inactivation, impregnation and extraction (the paper filter was kept for 2 days in room temperature to simulate transport time). The  score values were interpreted as follow: 2.00-3.00 - high-confidence identification (+++); 1.70-1.99 - low-confidence identification (+); 0.00-1.69 - no organism identification possible (-). Results and Discussion A total of 24 isolates were identified as (+++), 33 isolates as (+) and 3 isolates were not identified (-) directly from the original bacterial colony. Thirty-eight isolates were identified as (+++), 13 isolates as (+) and 9 isolates were not identified (-) after the extraction procedure. After filter paper disk transportation, a total of 49 isolates were identified as (+++), 10 isolates as (+) and 1 isolate were not identified (-). Considering the reliability of direct identification and after the extraction process, we obtained more high-confidence (+++) isolates after the extraction process, although 9 isolates were not identified after the extraction process, while only 3 isolates were not directly identified from original bacterial colony. Conclusions The mycobacterial cell wall, rich in mycolic acids, can make it difficult to directly identify these microorganisms by MALDI-TOF, so the manufacturer recommends the use of an extraction process to improve the sensitivity of the test. MNT present biohazard associated to their transport, as it has the ability to form biofilms and a safe method for identification is essential. Therefore, the inactivation of these microorganisms followed by impregnation in filter paper would make it possible for a large portion of MNT to be identified locally through reference laboratories that provide identification service using the MALDI-TOF MS. Moreover, inactivated MNT in filter paper does not present biohazard for transportation. Therefore, filter paper as a means to transport and storage of inactivated MNT can be considered a potential tool for faster, more accurate, biosafe and less expensive identification. Acknowledgments 63The authors would like to thank laboratory of microbiology of Hospital de Clínicas de Porto Alegre, Instituto Nacional de Pesquisa em Resistência Antimicrobiana (INPRA) and “Fundo de Incentivo a Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre (FIPE/HCPA). References 1. M Carneiro et al. Evaluation of filter paper as a means to transport inactivated bacteria for identification usingthe MALDI-TOF MS system. J Microbiol Methods. 2020 Apr;171:105863. doi: 10.1016/j.mimet.2020.105863.Epub 2020 Feb 5. PMID: 32035115.2. F Alcaide et al. How to: identify non-tuberculous Mycobacterium species using MALDI-TOF massspectrometry. Clin Microbiol Infect. 2018 Jun;24(6):599-603. doi: 10.1016/j.cmi.2017.11.012. Epub 2017 Nov22. PMID: 29174730.

Evaluation of filter paper to transport inactivated Nontuberculous Mycobacteria for identification using the MALDI-TOF MS system

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Evaluation of filter paper to transport inactivated Nontuberculous Mycobacteria for identification using the MALDI-TOF MS system

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Lume 5.8