title: Multicenter evaluation of mycobacteria identification by PCR restriction enzyme analysis in laboratories from Latin America and the Caribbean.
abstract: The identification of mycobacterial species in clinical isolates is essential for making patient care decisions. Polymerase chain reaction (PCR) restriction enzyme analysis (PRA) is a simple and rapid identification method, based on amplification of 441 bp of the hsp65 gene and restriction with BstEII and HaeIII. As a contribution to the validation of PRA, a multicenter study was performed in eight laboratories located in Argentina, Brazil, Colombia, Chile, and Guadeloupe. Each laboratory received 18 coded isolates from the collection of the Institute of Tropical Medicine (Antwerp, Belgium), representing duplicates of nine laboratory strains: Mycobacterium terrae CIPT 140320001, Mycobacterium scrofulaceum CIPT 140220031, Mycobacterium flavescens ATCC 14474, Mycobacterium triviale ATCC 23292, Mycobacterium nonchromogenicum ATCC 19530, Mycobacterium chitae ATCC 19627, Mycobacterium abscessus ATCC 19977, Mycobacterium kansasii ATCC 12478, and Mycobacterium peregrinum ATCC 14467. A detailed protocol including amplification, enzymatic digestion, and gel preparation was provided to each laboratory. Two laboratories identified correctly all 18 (100%) isolates, one identified correctly 17 (94.5%), two identified 14 (77.7%), one identified 11 (61%), and two identified 8 (44.4%) isolates. Errors detected in laboratories with more than 77% accuracy were associated with electrophoresis running conditions and an unspecific amplicon produced by a single strain. Lower accuracy was mainly related to inappropriate use of DNA markers and insufficient training in interpretation of patterns. In conclusion, the PRA method was readily implemented in some Latin American and Caribbean laboratories of mycobacteria, but improvements in critical points, as gel running conditions and training in interpretiation of patterns, are needed in order to improve accuracy. In others, improvement in critical points is still necessary.
authors.affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo-Escola Paulista de Medicina, Rua Botucatu, 862 3 degrees andar, São Paulo 04023-062, Brazil. sylvia@ecb.epm.br
authors.forename: Sylvia Cardoso
authors.initials: SC
authors.lastname: Leão
authors.forename: Amelia
authors.initials: A
authors.lastname: Bernardelli
authors.forename: Angel
authors.initials: A
authors.lastname: Cataldi
authors.forename: Martin
authors.initials: M
authors.lastname: Zumarraga
authors.forename: Jaime
authors.initials: J
authors.lastname: Robledo
authors.forename: Teresa
authors.initials: T
authors.lastname: Realpe
authors.forename: Gloria Isabel
authors.initials: GI
authors.lastname: Mejía
authors.forename: Maria Alice
authors.initials: MA
authors.lastname: da Silva Telles
authors.forename: Erica
authors.initials: E
authors.lastname: Chimara
authors.forename: Maritza
authors.initials: M
authors.lastname: Velazco
authors.forename: Jorge
authors.initials: J
authors.lastname: Fernandez
authors.forename: Pamela Araya
authors.initials: PA
authors.lastname: Rodrigues
authors.forename: Martha Inirida
authors.initials: MI
authors.lastname: Guerrero
authors.forename: Clara Ines
authors.initials: CI
authors.lastname: León
authors.forename: Tania Bibiana
authors.initials: TB
authors.lastname: Porras
authors.forename: Nalin
authors.initials: N
authors.lastname: Rastogi
authors.forename: Khye Seng
authors.initials: KS
authors.lastname: Goh
authors.forename: Philip
authors.initials: P
authors.lastname: Suffys
authors.forename: Adalgisa
authors.initials: A
authors.lastname: da Silva Rocha
authors.forename: Diogo
authors.initials: D
authors.lastname: dos Santos Netto
authors.forename: Viviana
authors.initials: V
authors.lastname: Ritacco
authors.forename: Beatriz
authors.initials: B
authors.lastname: López
authors.forename: Lucia
authors.initials: L
authors.lastname: Barrera
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Palomino
authors.forename: Anandi
authors.initials: A
authors.lastname: Martin
authors.forename: Françoise
authors.initials: F
authors.lastname: Portaels
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D018834:Chaperonin 60; D018833:Chaperonins / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D019430:Guadeloupe; D006801:Humans; D009161:Mycobacterium / Q000235:genetics / Q000302:isolation & purification*; D009164:Mycobacterium Infections / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015183:Restriction Mapping; D013020:South America
chemical_list: D001426:Bacterial Proteins; D018834:Chaperonin 60; D004269:DNA, Bacterial; C074119:heat-shock protein 65, Mycobacterium; D018833:Chaperonins