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Evaluation of sample pre-treatment protocols for detection of mycobacterial DNA directly in sputum samples

M Jonsson Nordvall(1,2) M Bergman-Jungeström(2,3) L Serrander(1,2,4) J Paues(2,4) M Lerm(2) T Schön(1,2,5)

1:Department of Clinical Microbiology, University Hospital, Linköping, Sweden; 2:Department of Biomedical and Clinical Sciences, Division of Inflammation and Infection, Linköping University, Sweden.; 3:Precision medicine laboratory, University Hospital, Linköping, Sweden.; 4:Department of Infectious Diseases, University Hospital, Linköping, Sweden.; 5:Department of Infectious Diseases, Kalmar County Hospital, Kalmar, Sweden.

Strategies to increase sensitivity for rapid detection of mycobacterial DNA are needed. Current methods are commonly used on decontaminated samples and necessary for culture but may also reduce mycobacterial DNA in sputum samples. The aim of this study was to optimize pre-treatment for increased yield of mycobacterial DNA.

Pooled sputum samples were spiked with 500 and 10⁴ CFU/ml of M. tuberculosis H37Rv (Mtb). Each concentration with and without decontamination was pre-treated by either inactivation in 80°C for 20 minutes, a saponin-based protocol or with sample reagent (SR) solution (GeneXpert, Cepheid, USA). The DNA yield was measured by Ct-values (m2000; Abbott, USA).

In total, 180 samples were processed including 18 for each pre-treatment with or without decontamination. Ct- values were was significantly lower without decontamination (mean Ct 27,9 at 10⁴ CFU/ml and 32,7 at 500 CFU/ml) compared to decontaminated samples (mean Ct 31,9 and 35,6 respectively). Highest DNA yield was obtained from samples heated to 80°C for 20 minutes without decontamination (mean Ct 24,3 at 10⁴ CFU/ml and 29,4 at 500 CFU/ml) compared to decontaminated samples (mean Ct 31,0 and 35,4 respectively). Compared to heating without decontamination, SR solution had lower DNA yield (Ct 33,3 at 500 CFU/ml) but higher than the saponin-based protocol (35,5 at 500 CFU/ml).

Overall, decontamination decreased mycobacterial DNA in sputum samples. Samples without decontamination in combination with heating at 80°C gave the highest Mtb DNA yield. Further optimization of pre-treatment including DNA extraction, consideration of human DNA influence in combination with targeted sequencing methods is planned.

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