The aminoglycosides kanamycin and amikacin and the macrocyclic peptide capreomycin are key drugs for the treatment of multidrug-resistant tuberculosis (MDR-TB). The increasing rates of resistance to these drugs and the possible cross-resistance between them are concerns for MDR-TB therapy. Mutations in the 16S rRNA gene (rrs) have been associated with resistance to each of the drugs, and mutations of the tlyA gene, which encodes a putative rRNA methyltransferase, are thought to confer capreomycin resistance in Mycobacterium tuberculosis bacteria. Studies of possible cross-resistance have shown variable results. In this study, the MICs of these drugs for 145 clinical isolates from Georgia and the sequences of the rrs and tlyA genes of the isolates were determined. Of 78 kanamycin-resistant strains, 9 (11.5%) were susceptible to amikacin and 16 (20.5%) were susceptible to capreomycin. Four strains were resistant to capreomycin but were susceptible to the other drugs, whereas all amikacin-resistant isolates were resistant to kanamycin. Sequencing revealed six types of mutations in the rrs gene (A514C, C517T, A1401G, C1402T, C1443G, T1521C) but no mutations in the tlyA gene. The A514C, C517T, C1443G, and T1521C mutations showed no association with resistance to any of the drugs. The A1401G and C1402T mutations were observed in 65 kanamycin-resistant isolates and the 4 capreomycin-resistant isolates, respectively, whereas none of the susceptible isolates showed either of those mutations. The four mutants with the C1402T mutations showed high levels of resistance to capreomycin but no resistance to kanamycin and amikacin. Detection of the A1401G mutation appeared to be 100% specific for the detection of resistance to kanamycin and amikacin, while the sensitivities reached 85.9% and 94.2%, respectively.

BackgroundMultiplex ligation-dependent probe amplification (MLPA) is a powerful tool to identify genomic polymorphisms. We have previously developed a single nucleotide polymorphism (SNP) and large sequence polymorphisms (LSP)-based MLPA assay using a read out on a liquid bead array to screen for 47 genetic markers in the Mycobacterium tuberculosis genome. In our assay we obtain information regarding the Mycobacterium tuberculosis lineage and drug resistance simultaneously. Previously we called the presence or absence of a genotypic marker based on a threshold signal level. Here we present a more elaborate data analysis method to standardize and streamline the interpretation of data generated by MLPA. The new data analysis method also identifies intermediate signals in addition to classification of signals as positive and negative. Intermediate calls can be informative with respect to identifying the simultaneous presence of sensitive and resistant alleles or infection with multiple different Mycobacterium tuberculosis strains.ResultsTo validate our analysis method 100 DNA isolates of Mycobacterium tuberculosis extracted from cultured patient material collected at the National TB Reference Laboratory of the National Center for Tuberculosis and Lung Diseases in Tbilisi, Republic of Georgia were tested by MLPA. The data generated were interpreted blindly and then compared to results obtained by reference methods. MLPA profiles containing intermediate calls are flagged for expert review whereas the majority of profiles, not containing intermediate calls, were called automatically. No intermediate signals were identified in 74/100 isolates and in the remaining 26 isolates at least one genetic marker produced an intermediate signal.ConclusionBased on excellent agreement with the reference methods we conclude that the new data analysis method performed well. The streamlined data processing and standardized data interpretation allows the comparison of the Mycobacterium tuberculosis MLPA results between different experiments. All together this will facilitate the implementation of the MLPA assay in different settings.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-572) contains supplementary material, which is available to authorized users.

25 26 Mycobacterium tuberculosis (Mtb) lineage identification and typing of clinical isolates in 27 general is performed only retrospectively. The results are rarely linked to drug susceptibility 28 testing (DST) or patient data. Consequently, the association between Mtb lineage, (multi)drug 29 resistance and treatment history is not fully explored at the local level. Here we evaluated a 30 new SNP based typing assay. We furthermore assessed the added value of genotyping of Mtb 31 isolates for epidemiological purposes and guidance of tuberculosis (TB) control. Mtb lineage, 32 DST profile and treatment history were determined for 399 samples at the National TB 33 Reference Laboratory (NRL) in Tbilisi, Georgia by local staff. Data was shared electronically 34 and analysis was performed remotely. Out of 399 isolates, 74 (74/399, 18.5%) were at least 35 multidrug resistant (MDR)-TB, of which 63 (63/74, 85.1%) were members of three different 36 Mtb Beijing lineages. Previous treatment was reported in 38/74 (51.4%) MDR(+) patients. The 37 availability of this data allows associations with lineages. Notably, multidrug resistant TB was 38 more strongly associated with the Beijing lineage than treatment history. Of all MDR-TB 39Beijing strains 56.7% (42/74) were members of a genetic cluster. This is most easily explained 40 by (ongoing) MDR-TB transmission rather than drug resistance amplification. This knowledge 41 is useful when designing intervention strategies for MDR-TB. Our study provides an example 42 that on-site integrated Mtb genotyping is realistic and could support TB control activities. 43 44 48 TB control activities (1, 2). Justifying investment in effective TB control strategies in a 49 country can be achieved in part by defining and monitoring the (MDR) TB epidemic to 50 identify appropriate interventions. 51 52 Molecular tools can positively impact on earlier detection of Mtb and identification of drug 53 resistance (3, 4). Genotyping of Mtb isolates has revealed associations between drug 54resistance and Mtb lineage (5-8), identified routes of transmission (9, 10) and described the 55 dynamics of epidemic clones (3,(11)(12)(13)(14). Further developments in multiplex assays as well as 56 the expanded use of next generation sequencing assays will increasingly allow Mtb strains to 57 be simultaneously screened for resistance associated mutations and the bacterial lineage they 58 represent. 60A robust link has been found between previous treatment for TB and multidrug resistance 61 (15), and is identified as a risk factor for MDR-TB by the WHO (16) but other factors are 62 also important, for example the bacterial lineage. This is especially true when transmission of 63 resistant strains is more common than the acquisition of resistance during treatment. 64 Members of the East Asia lineage (Mtb lineage 2) (17, 18) have repeatedly been associated 65 with multidrug resistance in high burden MDR-TB countries (11, 19) but less so in low 66 burden (MDR)-TB countries (20-22). The relative importance and interdepen...