High-sensitivity detection of Mycobacterium tuberculosis DNA in tongue swab samples

Tongue swab (TS) sampling combined with qPCR to detect Mycobacterium tuberculosis (MTB) DNA is a promising alternative to sputum testing for tuberculosis (TB) diagnosis. In prior studies, the sensitivity of tongue swabbing has usually been lower than sputum. In this study, we evaluated two strategies to improve sensitivity. In one, centrifugation was used to concentrate tongue dorsum bacteria from 2-mL suspensions eluted from high-capacity foam swab samples. The pellets were resuspended as 500-uL suspensions, and then mechanically lysed prior to dual-target qPCR to detect MTB insertion elements IS6110 and IS1081. Fractionation experiments demonstrated that most of the MTB DNA signal in clinical swab samples (99.22% +/- 1.46%) was present in the sedimentable fraction. When applied to archived foam swabs collected from 124 South Africans with presumptive TB, this strategy exhibited 83% sensitivity (71/86) and 100% specificity (38/38) relative to sputum MRS (microbiological reference standard; sputum culture and/or Xpert Ultra). The second strategy used sequence-specific magnetic capture (SSMaC) to concentrate DNA released from MTB cells. This protocol was evaluated on archived Copan FLOQSwabs flocked swab samples collected from 128 South African participants with presumptive TB. Material eluted into 500 uL buffer was mechanically lysed. The suspensions were digested by proteinase K, hybridized to biotinylated dual-target oligonucleotide probes, and then concentrated ~20-fold using magnetic separation. Upon dual-target qPCR testing of concentrates, this strategy exhibited 90% sensitivity (83/92) and 97% specificity (35/36) relative to sputum MRS. These results point the way toward automatable, high-sensitivity methods for detecting MTB DNA in TS.