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Chlorhexidine decontamination of sputum for culturing Mycobacterium tuberculosis

Abstract : Background: Culture of Mycobacterium tuberculosis is the gold standard method for the laboratory diagnosis of pulmonary tuberculosis, after effective decontamination. Results: We 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 > 1.4.10(2) 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. Conclusion: In 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.
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Shady Asmar, Michel Drancourt. Chlorhexidine decontamination of sputum for culturing Mycobacterium tuberculosis. BMC Microbiology, BioMed Central, 2015, pp.1-6. ⟨10.1186/s12866-015-0479-4⟩. ⟨hal-01198958⟩

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