Comment on: S-nitrosoglutathione (GSNO) is cytotoxic to intracellular amastigotes and promotes healing of topically treated Leishmania major or Leishmania braziliensis skin lesions.

We read with great interest the letter by Kö ser et al., 1 responding to our article on the characterization of the embB gene in the area of Barcelona. 2 The point that Kö ser et al. 1 raise is very interesting and deserves further comment. Codon 378 has indeed been described by several authors as a phylogenetic polymorphism not related to resistance. 3,4 However, in our opinion the role of this codon remains unclear, since some studies describe the existence of ethambutol-resistant isolates with a mutation only in this codon. 5,6 Even one work 7 cited by Kö ser et al. 1 includes two isolates with a single Glu378Ala substitution and a decreased susceptibility to ethambutol. The design of our microarray was based on the existing literature , taking into account all the possible embB codons that have been implicated in ethambutol resistance. We finally included the ones that were also found in our setting. The microarray system was not designed to determine the association of each mutation with the phenotypic resistance (allelic exchange experiments would be required for this purpose), but to reflect the variety of embB substitutions prevalent in our area. Moreover, the frequency of mutations in embB378 is low (,2% in our study), so we consider that the probability of misassigning a result of embB378 mutation to phenotypic resistance rather than to an epidemiological cause is negligible and does not compromise the effectiveness of the microarray. Regarding the isolates, we included a collection of Mycobacterium tuberculosis complex (MTBC) clinical isolates (not identified to the species level). Isolate 233R has now been analysed and identified as M. tuberculosis/Mycobacterium canettii. It contains the embB378Glu variant; therefore, a homoplastic event may be present (experimental error was ruled out). Likewise, the MIRU-VNTR genotyping was performed not for epidemiological purposes (i.e. for the identification of lineages), but to establish the real frequency of embB mutations among circulating MTBC isolates in our geographical area. Finally, we stress that the main objective of this study was to highlight the relevance of mutations in embB codons apart from embB306, focusing on codon 406, which represents 20% of the embB mutations in our area. Our results show that this target (embB406) should be included in any genotypic method for rapid ethambutol resistance detection. References 1 Kö ser CU, Bryant JM, Comas I et al. Comment on: Characterization of the embB gene in Mycobacterium tuberculosis isolates …