embCAB sequence variation among ethambutol-resistant Mycobacterium tuberculosis isolates without embB306 mutation.

OBJECTIVES: Mechanisms of resistance to ethambutol in Mycobacterium tuberculosis remain inadequately described. Although there is mounting evidence that mutations of codon 306 in embB play a key role, a significant number of phenotypically ethambutol-resistant strains do not carry mutations in this codon. Here, other mutations in the embCAB operon are suggested to be involved in resistance development. METHODS: The entire embCAB operon ( approximately 10 kb) was analysed in 34 phenotypically ethambutol-resistant M. tuberculosis strains without mutations in embB306 and in 12 ethambutol-susceptible strains. Furthermore, 106 control strains were investigated for the presence of particular mutations only. RESULTS: Overall, 18 non-synonymous mutations in 15 distinct codons of the embCAB operon were identified in ethambutol-resistant strains but not in ethambutol-susceptible isolates. The majority occurred in the embB gene (10 distinct codons), in a 570 bp region also encompassing embB306. Mutations in embC and embA were found rarely and in most cases in combination with polymorphisms in embB. One synonymous mutation (embA 228 bp) and two non-synonymous mutations (embCVal981Leu and embCArg738Gln) were found in ethambutol-susceptible strains as well as resistant strains and were confirmed to represent phylogenetic markers for strains of the Beijing, Haarlem and Delhi/CAS genotypes, respectively. CONCLUSIONS: Besides mutations in embB306, mutations in embB406 and embB497 were confirmed as hot spots for genomic variation in ethambutol-resistant clinical isolates. Of all resistant strains 70.6% carry a mutation in a relatively short region in embB, which therefore represents a promising target for inclusion in molecular assays for rapid detection of ethambutol resistance.

Sequence analyses of just four genes to detect extensively drug-resistant Mycobacterium tuberculosis strains in multidrug-resistant tuberculosis patients undergoing treatment.

The rapid detection of Mycobacterium tuberculosis isolates resistant to second-line drugs is crucial for the institution of appropriate treatment regimens as early as possible. Although molecular methods have successfully been used for the rapid detection of resistance to first-line drugs, there are limited data on mutations that confer resistance to second-line drugs. To address this question, we analyzed Mycobacterium tuberculosis strains resistant to ofloxacin (n = 26) and to capreomycin and/or amikacin (n = 48) from Uzbekistan for variations in target genes (gyrA, gyrB, rrs, and tlyA). Strains susceptible to ofloxacin (n = 49) and capreomycin and/or amikacin (n = 39) were included as controls. Mutations in gyrA or gyrB were found in 96% (25/26 strains) of the ofloxacin-resistant strains, while none of the susceptible strains displayed mutations in those two genes. The most common mutation occurred in gyrA at codon 94 (17/26 strains [65.4%]), followed by mutations at codons 90 and 91. Two strains showed a mutation in gyrB, at codons 485 and 543, respectively; both mutations have not been reported previously. The most frequent mutation in strains resistant to both amikacin and capreomycin was A1401G in rrs (34/40 strains [85.0%]). Three strains had mutations in tlyA, of which two (at codons 18 and 118) were associated with resistance to capreomycin alone. Overall, none of the 10 resistant strains (5 amikacin-resistant and capreomycin-susceptible strains) and none of the 39 susceptible control strains had mutations in the genes investigated. Our results clearly demonstrate the potential of sequence analyses of short regions of relatively few target genes for the rapid detection of resistance to second-line drugs among strains isolated from patients undergoing treatment for multidrug-resistant tuberculosis. The mechanisms that confer amikacin resistance in this setting remain unclear.

Multidrug-resistant tuberculosis treatment outcomes in Karakalpakstan, Uzbekistan: treatment complexity and XDR-TB among treatment failures.

BACKGROUND: A pilot programme to treat multidrug-resistant TB (MDR-TB) was implemented in Karakalpakstan, Uzbekistan in 2003. This region has particularly high levels of MDR-TB, with 13% and 40% among new and previously treated cases, respectively. METHODOLOGY: This study describes the treatment process and outcomes for the first cohort of patients enrolled in the programme, between October 2003 and January 2005. Confirmed MDR-TB cases were treated with an individualised, second-line drug regimen based on drug susceptibility test results, while suspected MDR-TB cases were treated with a standardised regimen pending susceptibility results. PRINCIPAL FINDINGS: Of 108 MDR-TB patients, 87 were started on treatment during the study period. Of these, 33 (38%) were infected with strains resistant to at least one second-line drug at baseline, but none had initial ofloxacin resistance. Treatment was successful for 54 (62%) patients, with 13 (15%) dying during treatment, 12 (14%) defaulting and 8 (8%) failing treatment. Poor clinical condition and baseline second-line resistance contributed to treatment failure or death. Treatment regimens were changed in 71 (82%) patients due to severe adverse events or drug resistance. Adverse events were most commonly attributed to cycloserine, ethionamide and p-aminosalicylic acid. Extensively drug resistant TB (XDR-TB) was found among 4 of the 6 patients who failed treatment and were still alive in November 2006. CONCLUSIONS: While acceptable treatment success was achieved, the complexity of treatment and the development of XDR-TB among treatment failures are important issues to be addressed when considering scaling up MDR-TB treatment.