Multicenter evaluation of mycobacteria identification by PCR restriction enzyme analysis in laboratories from Latin America and the Caribbean

Sylvia Cardoso Leão, Amelia Bernardelli, Angel Cataldi, Martin Zumarraga, Jaime Robledo, Teresa Realpe, Gloria Isabel Mejía, Maria Alice Da Silva Telles, Erica Chimara, Maritza Velazco, Jorge Fernandez, Pamela Araya Rodrigues, Martha Inirida Guerrero, Clara Ines León, Tania Bibiana Porras, Nalin Rastogi, Khye Seng Goh, Philip Suffys, Adalgisa Da Silva Rocha, Diogo Dos Santos NettoViviana Ritacco, Beatriz López, Lucia Barrera, Juan Carlos Palomino, Anandi Martin, Françoise Portaels

Research output: Contribution to journalArticlepeer-review

35 Scopus citations

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.

Original languageEnglish
Pages (from-to)193-199
Number of pages7
JournalJournal of Microbiological Methods
Volume61
Issue number2
DOIs
StatePublished - May 2005
Externally publishedYes

Bibliographical note

Funding Information:
This works was supported by INCO-CA project no. ICA4-CT-2001-10087 from the European Commission. V.R. is a member of the research career, CONICET, Argentina. E.C. is the recipient of a fellowship from CAPES, Brazil.

Keywords

  • Mycobacterium
  • PCR
  • Species identification

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