DNA was extracted from cultures using Instigate Matrix (Bio-Rad, USA) and sent to the Swiss Tropical and Public Health Institute for molecular analyses. Strain genotyping Spoligotyping and 24 locus MIRU-VNTR were used to define strain clusters as previously described [28, 29]. The online MIRU-VNTRplus platform was used for cluster identification ( http://www.miru-vntrplus.org). Clusters were defined for strains sharing identical spoligotype and 24 locus MIRU-VNTR patterns. Strains were assigned to one of the six previously described
lineages by real-time PCR identification of specific single nucleotide polymorphisms (SNPs) find more [5, 31–33]. Drug resistance mutations The following genes (or gene regions) were sequenced to capture drug resistance conferring SNPs: rpoB katG inhA promoter, ahpC promoter, embB pncA rpsL rrs gidB, and gyrA (see Additional file 1: Table S1 for primers and PCR conditions). Sequencing was performed by Macrogen (The Netherlands). Observed mutations were compared to the online
Tuberculosis Drug Resistance Mutation Database (TBDream, http://www.tbdreamdb.com). Ethical approval The PNG Institute for Medical Research Review Board, and the PNG National Medical Research Advisory Council’s Ethics Committee approved the study protocol. The Ethikkommission beider Basel in Switzerland Selleckchem AL3818 was informed about the study. Written informed consent was Selleck Temozolomide obtained from all patients enrolled in the study. Authors’ information Co-senior author: Sebastien Gagneux and Hans-Peter Beck. Acknowledgments We thank all the study participants whose samples were used for analyses. We are indebted to the TB laboratory 6-phosphogluconolactonase team in Madang. This work was supported by the Swiss National Science Foundation (North–South Program, grant number IZ70Z0_123988) and partially subsidized by
a grant from the Stanley-Thomas Johnson Foundation and the Medicor Foundation, Lichtenstein. Electronic supplementary material Additional file 1: Table 1.Primers and PCR conditions. (DOC 58 KB) References 1. World Health Organization: Tuberculosis country profile. Guinea: Papua New Guinea; 2011. 2. Hillemann D, Rüsch-Gerdes S, Richter E: Evaluation of the Genotype MTBDRplus assay for rifampin and isoniazid susceptibility testing of Mycobacterium tuberculosis strains and clinical specimens. J Clin Microbiol 2007, 45:2635–2640.PubMedCrossRef 3. Boehme CC, Nicol MP, Nabeta P, Michael JS, Gotuzzo E, Tahirli R, Gler MT, Blakemore R, Worodria W, Gray C, Huang L, Caceres T, Mehdiyev R, Raymond L, Whitelaw A, Sagadevan K, Alexander H, Albert H, Cobelens F, Cox H, Alland D, Perkins MD: Feasibility, diagnostic accuracy, and effectiveness of decentralised use of the Xpert MTB/RIF test for diagnosis of tuberculosis and multidrug resistance: a multicentre implementation study. Lancet 2011, 377:1495–1505.