Culturing Mycobacterium tuberculosis remains the gold standard for the laboratory diagnosis of pulmonary tuberculosis, with 9 million new cases and 1.5 million deaths mainly in developing countries. Reviewing data reported over 20 years yields a state-of-the-art procedure for the routine culture of M. tuberculosis in both developed and developing countries. Useful specimens include sputum, induced sputum, and stools collected in quaternary ammonium preservative-containing sterile cans. The usefulness of other non-invasive specimens remains to be evaluated. Specimens can be collected in a diagnosis kit also containing sampling materials, instructions, laboratory requests, and informed consent. Automated direct LED fluorescence microscopy after auramine staining precedes inoculation of an egg-lecithin-containing culture solid medium under microaerophilic atmosphere, inverted microscope reading or scanning video-imaging detection of colonies and colonies identification by recent molecular methods. This procedure should result in a diagnosis of pulmonary tuberculosis as fast as 5 days. It may be implemented in both developed and developing countries with automated steps replaceable by manual steps depending on local resources.

BackgroundCulture of Mycobacterium tuberculosis is the gold standard method for the laboratory diagnosis of pulmonary tuberculosis, after effective decontamination.ResultsWe evaluated squalamine and chlorhexidine to decontaminate sputum specimens for the culture of mycobacteria. Eight sputum specimens were artificially infected with 105 colony-forming units (cfu)/mL Mycobacterium tuberculosis and Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans as contaminants. In the second step, we tested chlorhexidine-based decontamination on 191 clinical specimens, (Chlorhexidine, 0.1, 0.5 and 0.7 %). In a last step, growth of contaminants and mycobacteria was measured in 75 consecutive sputum specimens using the routine NALC-NaOH decontamination protocol or with 0.7 % chlorhexidine decontamination and an inoculation on Coletsos medium.In the artificially model, contaminants grew in 100 % of the artificially infected sputum specimens decontaminated using 100 mg/mL squalamine, in 62.5 % of specimens decontaminated using N-Acetyl-L-Cysteine-Sodium Hydroxide (NALC-NaOH), and in 0 % of specimens decontaminated using 0.1 %, 0.35 %, or 1 % chlorhexidine (P < 0.05). These specimens yielded <102 cfu M. tuberculosis using NALC-NaOH and > 1.4.102 cfu M. tuberculosis when any concentration of chlorhexidine was used (P < 0.05).In the second step we found that 0.7 %-chlorhexidine yielded 0 % contamination rate, 3.2 % for 0.5 %-chlorhexidine and 28.3 % for 0.1 %-chlorhexidine. As for the 75 specimens treated in parallel by both methods we found that when using the standard NALC-NaOH decontamination method, 8/75 (10.7 %) specimens yielded M. tuberculosis colonies with a time to detection of 17.5 ± 3 days and an 8 % contamination rate. Additionally, 14 specimens yielded mycobacteria colonies (12 M. tuberculosis, and 2 Mycobacterium bolletii) (18.7 %) (P = 0.25), which has yielded a 100 % sensitivity for the chlorhexidine protocol. Time to detection was of 15.86 ± 4.7 days (P = 0.39) and a 0 % contamination rate (P < 0.05) using the 0.7 %-chlorhexidine protocol.ConclusionIn our work we showed for the first time that chlorhexidine based decontamination is superior to the standard NALC-NaOH method in the isolation of M. tuberculosis from sputum specimens. We currently use 0.7 %-chlorhexidine for the routine decontamination of sputum specimens for the isolation of M. tuberculosis and non-tuberculosis mycobacteria on egg-lecithin containing media.