Review 2: "High-Sensitivity Detection of Mycobacterium Tuberculosis DNA in Tongue Swab Samples"

Lucian (Luke) Davis

doi:doi:10.1162/2e3983f5.31414a09

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

by Alaina M. Olson, Rachel C. Wood, Kris M. Weigel, Alexander J. Yan, Katherine A. Lochner, Rane B. Dragovich, Angelique K. Luabeya, Paul Yager, Mark Hatherill, and Gerard A. Cangelosi

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Published on Jul 27, 2024
www.medrxiv.org

Description

Abstract 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-µL 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 µL 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.Importance Improved testing for tuberculosis (TB) is needed. Using a more accessible sample type than sputum may enable the detection of more cases, but it is critical that alternative samples be tested appropriately. Here, we describe two new, highly accurate methods for testing tongue swabs for TB DNA.

RR\ID Evidence Scale rating by reviewer:

Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review: Up to 1/3 of persons with TB are never diagnosed, and molecular testing of tongue swabs offers an exciting alternative to sputum for improving the feasibility and safety of TB diagnosis if a sufficiently sensitive, point-of-care(POC) test can be developed. Olson and colleagues demonstrate that improvements in swab design and swab processing techniques can likely increase the amount of biomass recovered for testing. Their study has multiple strengths including 1) the use of swab samples from persons undergoing clinical evaluation with TB in a high-burden setting to maximize applicability even at the design stage for these assays; 2) rigorous experimentation with swab collection type with attention to both yield and feasibility to reveal the potential for low-cost foam cell swabs as an alternative to nylon flocked swabs; and 3) innovative experimentation with sample processing techniques with an eye to enhancing DNA recovery to show that Mtb can be concentrated in sediment and captured using magnetic bead capture with target sequence hybridization. These findings complement recently published articles from Steadman et al Clin Infect Dis 2024 and Yan et al PLoS ONE 2024.

There are also a few limitations of the study including 1) a lack of description of the participants and how they were recruited, preventing a full assessment of the risk of bias and the generalizability of the sample; 2) a lack of statistical power to determine if these methods can enhance sensitivity on tongue swab testing, and 3) the fact that these innovations provide only a proof-of-concept rather than an implementable protocol, as several of the technologies that likely enabled the high sensitivity are not currently feasible in POC settings and also create infectious aerosols (centrifugation, bead-beating, and vortexing).

Review 2: "High-Sensitivity Detection of Mycobacterium Tuberculosis DNA in Tongue Swab Samples"

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