Characterization of oral swab samples for diagnosis of pulmonary tuberculosis

Rachel C Wood; Alfred Andama; Gleda Hermansky; Stephen Burkot; Lucy Asege; Mukwatamundu Job; David Katumba; Martha Nakaye; Sandra Z Mwebe; Jerry Mulondo; Christine M Bachman; Kevin P Nichols; Anne-Laure M Le Ny; Corrie Ortega; Rita N Olson; Kris M Weigel; Alaina M Olson; Damian Madan; David Bell; Adithya Cattamanchi; William Worodria; Fred C Semitala; Akos Somoskovi; Gerard A Cangelosi; Kyle J Minch

doi:10.1371/journal.pone.0251422

. 2021 May 17;16(5):e0251422. doi: 10.1371/journal.pone.0251422

Characterization of oral swab samples for diagnosis of pulmonary tuberculosis

Rachel C Wood ^1,^#, Alfred Andama ^2,^3,^#, Gleda Hermansky ⁴, Stephen Burkot ^5,^¤a, Lucy Asege ², Mukwatamundu Job ², David Katumba ², Martha Nakaye ², Sandra Z Mwebe ², Jerry Mulondo ², Christine M Bachman ^5,^¤a, Kevin P Nichols ^4,^¤a, Anne-Laure M Le Ny ^4,^¤a, Corrie Ortega ^4,^¤a, Rita N Olson ¹, Kris M Weigel ¹, Alaina M Olson ¹, Damian Madan ^4,^¤a, David Bell ^5,^¤b, Adithya Cattamanchi ⁶, William Worodria ^2,³, Fred C Semitala ^2,³, Akos Somoskovi ^5,^¤c, Gerard A Cangelosi ^1,^*, Kyle J Minch ^4,^¤a

Editor: Selvakumar Subbian⁷

¹Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, Seattle, Washington, United States of America

²Infectious Diseases Research Collaboration, Kampala, Uganda

³Department of Medicine, Makerere University College of Health Sciences, Kampala, Uganda

⁴Intellectual Ventures Laboratory, Bellevue, Washington, United States of America

⁵Intellectual Ventures’ Global Good Fund, Bellevue, Washington, United States of America

⁶Division of Pulmonary and Critical Care Medicine and Center for Tuberculosis, Zuckerberg San Francisco General Hospital, University of California San Francisco, San Francisco, California, United States of America

⁷Rutgers Biomedical and Health Sciences, UNITED STATES

Competing Interests: At the time of study design and evaluation SB, CMB, DB, and AS received salary support from the Global Good Fund. At the time of study design and evaluation GH, KPN, AML, CO, DM, and KJM were employed by Intellectual Ventures Laboratory. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials. A number of the authors are currently affiliated with GH Labs and Roche Molecular Systems, but were not affiliated with them during the study, and these companies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

^¤a

Current address: GH Labs, Bellevue, Washington, United States of America.

^¤b

Current address: Independent Consultant, Issaquah, Washington, United States of America.

^¤c

Current address: Roche Molecular Systems, Inc., Pleasanton, California, United States of America.

^✉

* E-mail: gcang@uw.edu

Contributed equally.

Roles

Rachel C Wood: Investigation, Methodology, Validation, Writing – original draft, Writing – review & editing

Alfred Andama: Data curation, Formal analysis, Investigation, Supervision, Validation, Writing – original draft, Writing – review & editing

Gleda Hermansky: Investigation, Methodology, Validation, Writing – review & editing

Stephen Burkot: Data curation, Formal analysis, Visualization, Writing – review & editing

Lucy Asege: Investigation, Writing – review & editing

Mukwatamundu Job: Investigation, Writing – review & editing

David Katumba: Investigation, Writing – review & editing

Martha Nakaye: Investigation, Writing – review & editing

Sandra Z Mwebe: Investigation, Writing – review & editing

Jerry Mulondo: Investigation, Supervision, Writing – review & editing

Christine M Bachman: Methodology, Project administration, Writing – review & editing

Kevin P Nichols: Conceptualization, Supervision, Writing – review & editing

Anne-Laure M Le Ny: Supervision, Writing – review & editing

Corrie Ortega: Conceptualization, Investigation, Project administration, Writing – review & editing

Rita N Olson: Investigation, Methodology, Writing – review & editing

Kris M Weigel: Data curation, Investigation, Methodology, Writing – review & editing

Alaina M Olson: Investigation, Methodology, Writing – review & editing

Damian Madan: Conceptualization, Supervision, Writing – review & editing

David Bell: Conceptualization, Funding acquisition, Resources, Writing – review & editing

Adithya Cattamanchi: Conceptualization, Supervision, Writing – original draft, Writing – review & editing

William Worodria: Investigation, Writing – review & editing

Fred C Semitala: Supervision, Writing – review & editing

Akos Somoskovi: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing

Gerard A Cangelosi: Conceptualization, Project administration, Supervision, Writing – original draft, Writing – review & editing

Kyle J Minch: Conceptualization, Formal analysis, Investigation, Methodology, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

Selvakumar Subbian: Editor

PMCID: PMC8128230 PMID: 33999938

Abstract

Oral swab analysis (OSA) has been shown to detect Mycobacterium tuberculosis (MTB) DNA in patients with pulmonary tuberculosis (TB). In previous analyses, qPCR testing of swab samples collected from tongue dorsa was up to 93% sensitive relative to sputum GeneXpert, when 2 swabs per patient were tested. The present study modified sample collection methods to increase sample biomass and characterized the viability of bacilli present in tongue swabs. A qPCR targeting conserved bacterial ribosomal rRNA gene (rDNA) sequences was used to quantify bacterial biomass in samples. There was no detectable reduction in total bacterial rDNA signal over the course of 10 rapidly repeated tongue samplings, indicating that swabs collect only a small portion of the biomass available for testing. Copan FLOQSwabs collected ~2-fold more biomass than Puritan PurFlock swabs, the best brand used previously (p = 0.006). FLOQSwabs were therefore evaluated in patients with possible TB in Uganda. A FLOQSwab was collected from each patient upon enrollment (Day 1) and, in a subset of sputum GeneXpert Ultra-positive patients, a second swab was collected on the following day (Day 2). Swabs were tested for MTB DNA by manual IS6110-targeted qPCR. Relative to sputum GeneXpert Ultra, single-swab sensitivity was 88% (44/50) on Day 1 and 94.4% (17/18) on Day 2. Specificity was 79.2% (42/53). Among an expanded sample of Ugandan patients, 62% (87/141) had colony-forming bacilli in their tongue dorsum swab samples. These findings will help guide further development of this promising TB screening method.

Introduction

Tuberculosis disease (TB), caused by Mycobacterium tuberculosis (MTB), remains a major global cause of morbidity and mortality [1]. The standard sample for TB diagnosis is sputum, a viscous material derived from patient airways. Sputum collection presents safety risks to health personnel, and the material is notably difficult to standardize and process for detection of MTB DNA. Sputum can be difficult for some patients to produce, especially children and those who are HIV-infected. The availability of alternative, noninvasive samples, which can easily be collected outside of the clinic, would increase the efficiency of testing and reduce the exposure risk to health care professionals [2, 3].

We and others have shown that MTB DNA is deposited on the oral epithelium during active TB disease and can be detected by oral swab analysis (OSA) [4–9]. In OSA, the dorsum of the tongue is gently brushed with a sterile disposable swab. The swab head with collected material, consisting of bacterial biofilm and host cells, is deposited into a sample buffer and eluted as a non-viscous suspension suitable for nucleic acid amplification testing (NAAT) targeting MTB DNA. Tongue swabbing is fast, painless, and does not require accommodations for privacy or aerosol control. Sputum-scarce patients such as children and HIV-positive adults are easily swabbed in any setting, and self-sampling is straightforward [10]. Therefore, OSA may be especially useful for TB case finding in non-clinical and community settings.

Studies on OSA for TB diagnosis have shown mixed results. In a blinded study conducted on 219 adult TB patients in South Africa, OSA exhibited 93% sensitivity and 92% specificity relative to sputum GeneXpert testing [4]. In a blinded study conducted on 201 children in South Africa, OSA matched or exceeded the diagnostic yield of induced sputum testing [6]. However, studies that used different methods from ours yielded more modest sensitivity values for OSA both in adults and children [7–9]. Moreover, in our South African studies, we collected two to three samples from each patient on separate days. Sensitivity and specificity were scored by calling a patient positive if either swab was positive [4–6]. A requirement for collecting and testing of multiple swabs per patient over multiple days would greatly reduce the utility of this approach.

In order to improve OSA-based TB testing, there is a need to optimize swab collection methods and to more fully understand the nature of the DNA biomarker at this anatomical site (for example, whether it is associated with viable MTB cells, with nonviable cells, or in cell-free form). Therefore, we conducted studies to: 1) compare the amounts of oral bacterial biomass collected by commercially-available swab products using a bacterial biomass proxy (qPCR measurement of conserved bacterial rDNA); 2) evaluate the accuracy of OSA-based TB PCR testing using the best oral swab product; and 3) assess whether viable MTB can be isolated and cultured from oral swab samples.

Methods

Study sites, populations, and sampling workflows

Healthy control participants in Seattle, King County, WA provided swabs for a rapid repeat swabbing analysis and swab comparison. For the rapid repeat analysis, 4 volunteers provided 10 swabs each with a maximum of 15 seconds between each swab collection. For the swab comparison, 3 volunteers each provided 5 swabs of each type.

For evaluations of sensitivity and specificity of OSA relative to sputum testing and of MTB viability in oral swab samples, we used a nested strategy as shown in Fig 1. Between October 2018 and April 2019 (6 months), patients with presumed pulmonary TB at Kiruddu Referral Hospital, Mulago National Referral Hospital TB ward, and Kisenyi Health Center IV out-patient clinic in Kampala, Uganda, were enrolled as previously described [11, 12]. We included adults (>18 years) who presented with respiratory symptoms and excluded participants who received anti-TB treatment or antibiotics with anti-TB activity such as fluoroquinolones in the prior 12 months or who refused or were unable to provide informed consent. After consent, all eligible participants completed a survey on demographics and medical history. Each patient provided at least 1 swab sample on Day 1 (n = 194 swabs from 144 patients, combining the sensitivity, specificity, and viability studies as shown in Fig 1). A subset of patients who were sputum GeneXpert positive were asked to return to provide a second sample on Day 2 (n = 41 swabs combining the sensitivity and viability studies). We matched enrollment of the first 50 GeneXpert positive individuals with 53 GeneXpert negative individuals. To assess viability of MTB cells in oral swab samples, an additional 91 GeneXpert positive individuals were enrolled (Fig 1).

Fig 1 — Patients were enrolled in a nested strategy for MTB detection by qPCR (50 Xpert positive individuals and 53 Xpert negative individuals) or mycobacterial culture (141 Xpert positive individuals). All Xpert positive individuals from the qPCR arm were included in the culture arm.

The study workflow is outlined in S1 Fig. To accommodate assessment of sensitivity, specificity, and viability, the sampling protocol was as follows: a sputum sample was collected and split into two portions: the first portion was processed for Xpert (Xpert MTB/RIF Ultra assay, Cepheid, Sunnyvale, CA, USA), and the second portion was processed for sputum culture only. Immediately after collecting sputum for Xpert testing, an oral swab (for culture) was collected and processed for culture. After waiting for ≥ 1 hour, an oral swab (for PCR) was collected. A second sputum sample was collected and processed for culture, and 2 additional oral swabs were collected. Two swabs were collected from patients who returned on day 2 (n = 41)–the first swab was processed for culture, and the second swab was processed for PCR. These swabs were collected in the absence of preliminary prompted sputum production.

The study and full protocol were reviewed and approved by the Makerere University School of Medicine Research and Ethics Committee, the Uganda National Council for Science and Technology, the University of California San Francisco Committee on Human Research, and the Human Subjects Division of the University of Washington (STUDY00001840). The study was performed according to the Standards for Reporting of Diagnostic Accuracy Studies (STARD) guidelines [13].

Swab collection

Participants were asked to refrain from eating, drinking, brushing teeth, and using mouthwash for at least 30 minutes prior to swab collection. For the rapid repeat swabbing analysis, the Puritan PurFlock Ultra® (25-3606-U) was used. This swab type was evaluated in a previous study [4]. For the swab comparison, two swab products were evaluated: Puritan PurFlock Ultra swabs and Copan FLOQSwabs® (520CS01). Study staff asked subjects to stick out their tongue and using a sterile, individually wrapped swab, study staff ran the swab over the length and breadth of the front 2/3 of the subject’s tongue. Pressure was sufficient to slightly bend the swab shafts. Samples were collected for 15–20 seconds while rotating the swab throughout.

Sputum sample processing and analysis

Trained study technologists at Mulago National Referral Hospital Tuberculosis Laboratory and the Makerere University Mycobacteriology Laboratory performed all TB testing using standard protocols [14]. For Xpert, Cepheid sample reagent was added to the sputum sample at a 2:1 ratio. The mixture was vortexed for 10–15 seconds and incubated for 15 minutes at room temperature. Two milliliters (2 mL) of the liquefied sample were then transferred to the Xpert cartridge for testing in a four-module GeneXpert instrument using manufacturer standard settings.

Mycobacterial culture of sputum was performed by trained staff blinded to Xpert results. Sputum specimens were digested and decontaminated using standard methods [14]. Briefly, samples were treated with an equal volume of sodium hydroxide (Griffchem, cat #: 1310-73-2) and N-acetyl-Cysteine (SIGMA-Aldrich, cat #: 616-91-1) mixture to a final concentration of 1.5% for 15 minutes, neutralized with twice the volume of sterile phosphate-buffered solution pH 6.8, and centrifuged for 20 minutes at 3000 x g. The resulting sediment was resuspended in 2 mL of sterile phosphate-buffered solution at pH 6.8. Lowenstein-Jensen (BD, cat #: 220909) slant and Mycobacterial Growth Indicator Tube (MGIT) media were inoculated with 0.5 mL suspension, and Ziehl-Neelson (ZN) staining for acid-fast bacillus (AFB) smear microscopy was performed following standard protocols [14]. Prior to inoculation, a standard cocktail of antibiotics containing PANTA (SIGMA Aldrich: polymyxin B, CAS #: 1405-20-5; amphotericin B, CAS #: 1397-89-3; nalidixic acid solubilized with trimethoprim, CAS #: 23256-42-0; and azlocillin, CAS # 37091-65-9) mixed with 10% OADC (oleic acid, bovine serum albumin, dextrose, and catalase: BD, cat #: 212240) was added to MGIT tubes to suppress bacterial contamination and provide an optimum growth medium for mycobacteria. MGIT tubes were incubated in a BACTEC MGIT 960 instrument (BD; Franklin Lakes, NJ, USA) for up to 42 days and LJ slants were incubated at 37°C for up to 56 days.

Swab processing for mycobacteriological culture

Tongue swab samples, which are rich in fast-growing oral microorganisms, were decontaminated and cultured as follows. Working stocks of OMNIgene-SPUTUM (DNA Genotek, cat #: OM-SPD-250) were prepared every 30 days during the study. Stock solution (supplied at 2.0x stock concentration) was diluted 1:20 or 1:8 in sterile deionized water to create 0.1x or 0.25x working solutions, which were distributed in 1.0 mL aliquots into 2.0 mL gasketed screwcap tubes and stored protected from light at room temperature for 30 days. After 30 days, any unused aliquots were discarded, and fresh working solutions were prepared.

After tongue swab sample collection, swab heads were inserted into a screwcap tube containing OMNIgene-SPUTUM and rotated against sidewalls of tube for ~15 seconds. Swab heads were secured in capped tube and inverted 10 times to cover all surfaces with buffer. Swab samples were incubated at 25°C overnight (≥ 18 hours, depending on time of collection) in OMNIgene-SPUTUM. Samples were pulse spun 2–3 seconds in a single-speed tabletop microfuge (Qor Labs 10,000 rpm Mini Centrifuge). For mycobacteriological culture, samples were taken from the swab/OMNIgene-SPUTUM tube (1.0 mL total volume) and inoculated directly to MGIT+PANTA (750 μL inoculum), LJ (100 μL inoculum), or 7H10+PACT (SIGMA Aldrich, cat #: 262710; polymyxin B, amphotericin B, carbenicillin, and trimethoprim; 100 μL inoculum). All cultures were incubated at 37°C and monitored for growth for up to 56 days. The identity of growth-positive cultures was confirmed acid-fast by ZN staining and to the MTB complex level by SD Bioline immunoassay to detect MTB antigen MPT64 following manufacturer-supplied methods (SD MPT64TB Ag kit, South Korea). Samples that remained negative after 56 days were discarded.

Swab processing for qPCR

After collection, the head of the swab was inserted into a 2 mL tube containing 500 μL of a sterile lysis buffer (65 mM Tris pH 8.0, 50 mM EDTA, 50 mM sucrose, 100 mM NaCl, and 0.3% SDS) and snapped off. All swabs were stored at -80°C within 8 hours of collection.

The samples collected in Seattle and used for the rapid repeat analysis and the swab comparison were stored at -80°C and extracted using the QIAGEN QIAamp DNA mini kit. The samples were eluted into 300 μL (2 x 150 μL) of Buffer AE and stored at -20°C. To prepare for qPCR analysis targeting conserved bacterial rDNA, the samples were diluted 1:100.

The samples collected in Uganda were transported on dry ice to the Cangelosi lab in Seattle, WA. The laboratory team was blinded to the TB status of the Day 1 samples, though not the Day 2 samples (which were known to be from Xpert-positive participants). Before starting the extraction, each sample was split in half, and one half was stored at -80°C. The reserved half of the sample was kept as a precaution in case complications arose during the subsequent extraction and analysis. DNA was extracted using the QIAGEN QIAamp DNA mini kit, as described previously [4]. After elution into 300 μL of QIAGEN Buffer AE, 5 μL was used for qPCR analysis. For any sample that tested negative, 150 μL of the sample elution was ethanol precipitated [4, 5], resuspended in 15 μL of 3:1 molecular-grade H₂O and Buffer AE, and retested. The ethanol precipitation served to concentrate the sample allowing for qPCR analysis of a greater proportion of the sample.

Swab sample analysis by qPCR

A qPCR assay targeting a conserved bacterial 16S rDNA sequence was used to compare the swab types based on the relative quantity of bacterial biomass that they gathered. The primer pair Com1/769R was used to amplify a 270 bp amplicon [15]. Each 20 μL reaction consisted of 1 μL of each 20 μM primer, 10 μL of iTaq Universal SYBR Green Supermix from BioRad, 6 μL molecular-grade H₂O, and 2 μL 1:100 diluted template. The cycling conditions were as described [15].

The qPCR analysis used for the detection of MTB in the samples collected in Kampala was described previously, for both the unconcentrated and concentrated samples [4, 5]. The reaction targets IS6110, a multicopy insertion element unique to the M. tuberculosis complex. Each qPCR run included a positive control containing a known amount of cultured MTB strain H37Ra extracted alongside the samples, a negative control consisting of sterile buffer extracted with the samples, negative template PCR controls, and a standard curve made with purified H37Ra DNA.

Results

Characterization of biomass collection

Previously, we compared two swab brands for their abilities to detect MTB DNA on buccal (not tongue) surfaces in the mouths of adult TB patients. Based on Cq values from qPCR analysis, Puritan PurFlock Ultra swabs were found to collect about twice as much MTB DNA as Whatman OmniSwabs^® (p = 0.015) [4]. This observation raised the possibility that signal strength (and therefore sensitivity) of OSA might be limited by the amount of biomass collected by some swab products, such that alternative products could enable greater sensitivity.

In order to test this possibility in the context of tongue swabbing without having to enroll new TB patients, we used normal oral flora as a measure of bacteria collected from the tongue dorsum surface. Biomass of collected bacteria was estimated by using a pan-bacterial domain qPCR that detects conserved portions of bacterial small subunit rDNA. While no single primer set is truly universal within the domain Bacteria, the broad-spectrum primer set Com1/769R has sufficient breadth to compare non-specific bacterial loads in paired analyses [12].

The first question we asked was whether bacterial biomass in samples is depleted over the course of repeated samplings. Four healthy US participants were repeatedly sampled with Puritan PurFlock Ultra swabs 10 times in rapid succession (approximately 10 seconds between each sampling). If tongue surface biomass was depleted, then we expected to see diminishing signals (rising Cq values) over the course of repeated sampling and testing by conserved bacterial rDNA qPCR; however, no such depletion was observed, and these data suggest that these swabs collect only a fraction of biomass that is available for sampling at this site (Fig 2, p = 0.099, one-way repeated measures ANOVA).

Fig 2 — Ten samples were collected in rapid succession from the tongue dorsa of four healthy volunteers, and tested by conserved bacterial rDNA qPCR. Each individual is represented by a distinct type of symbol.

An alternative swab brand collects more bacterial biomass

Next, we asked whether an alternative swab brand collects more bacterial biomass from the tongue dorsum than the best product identified previously, Puritan PurFlock Ultra [4]. Three healthy US volunteers each provided 5 samples using Puritan PurFlock Ultra or Copan FLOQSwabs. Collected bacterial biomass was quantified again by conserved bacterial rDNA qPCR as in Fig 2. Copan FLOQSwabs collected 2-fold more bacterial biomass than PurFlock Ultra (Fig 3). A paired t-test showed that this difference in Cq value was significant (p = 0.0064).

Fig 3 — Five samples were collected from 3 healthy volunteers using Puritan Purflock swabs or Copan FLOQSwabs. Bacterial biomass was quantified by qPCR using primers against a conserved bacterial rDNA locus. Bars represent mean Cq +/- standard deviation. Significance calculated using paired t-test (p = 0.0064).

Clinical evaluation of alternative swabs

Based on their biomass capacity, Copan FLOQSwabs were selected for evaluation in a clinical study in Kampala, Uganda. Adult patients with suspected active pulmonary TB (N = 191) were identified and enrolled by the study staff, with samples collected as described in Methods and S1 Fig. Socio-demographic and clinical characteristics of this cohort are summarized in Tables 1 and 2.

Table 1. Socio-demographic characteristics and other patient information.

	TB patients (Xpert or culture-positive) (n = 142)	Non-TB patients (Xpert and culture-negative) (n = 52)	p-value
Age in years (%)
Mean (SD)	32.1 (9.6)	34.2 (11)
Median (IQR)	30 (13.8)	34 (14)
Gender (%)
Female	46 (32.4)	24 (46.1)	0.08
Male	96 (67.6)	28 (53.9)	0.08
Smoker (%)	21 (14.8)
Enrollment Site (%)	n = 141
Kiruddu	19 (13.5)	16 (30.8)	0.006^*
Kisenyi	95 (67.4)	36 (69.2)	0.8
Mulago	27 (19.1)	0 (0)
Patient type (%)
In-patient	8 (5.6)	3 (5.8)	1
Out-patient	134 (94.4)	49 (94.2)	0.8

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*Significant at p < 0.05, z-score for 2 population proportions.

Table 2. Clinical characteristic of patients.

	TB patients (Xpert or culture-positive) (n = 142)	Non-TB patients (Xpert and culture-negative) (n = 52)	p-value
Current cough (%)	142 (100)	52 (100)	< 0.0001^*
Duration of current cough (days)
Mean (SD)	70.9 (52.9)	43.8 (57)
Median (IQR)	30 (60)	25.5 (46)
Coughing up blood (%)	38 (26.8)	10 (19.2)	0.3
Fever (%)	137 (96.5)	47 (90.4)	0.09
Night sweats (%)	135 (95)	40 (76.9)	0.0002^*
Weight loss (%)	139 (98)	46 (88.5)	0.006^*
Weight loss > 5kg (%)	118 (85), (n = 139)	24 (52.2), (n = 46)	< 0.0001^*
Decreased appetite (%)	128 (91.4), (n = 140)	39 (75)	0.003^*
Swollen lymph nodes (%)	12 (8.5), (n = 141)	2 (3.9)	0.3
Stomach pain or swelling (%)	21 (15), (n = 140)	7 (13.5)	0.8
Oxygen saturation (%)
Mean (SD)	96.5 (2.8)	97.7 (2.7)
Median (IQR)	97 (2)	98 (1)
ECOG performance score (%)
0	9 (6.3)	8 (15.4)	0.05^*
1	63 (44.4)	30 (57.7)	0.1
2	65 (45.8)	13 (25)	0.009^*
3	5 (3.5)	1 (1.9)	0.6
HIV infection (%)	36 (25.4)	19 (36.5)	0.1
Household TB contact (%)	10 (7)	1 (1.9)	0.2
Previous TB disease (%)	10 (7)	4 (7.7)	0.9

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*Significant at p < 0.05, z-score for 2 population proportions.

Of the 191 participants, 103 were included in the qPCR analysis, as shown in Fig 1. Of these, 50 (48.5%) tested positive for TB by sputum GeneXpert MTB/RIF Ultra, and 47 (45.6%) tested positive for TB by sputum culture. Negative results were obtained for 53 (51.5%) and 56 (54.4%) of these participants by Xpert and sputum culture, respectively. As described in Methods, swabs were collected after sputum collection on Day 1. Of the 50 sputum Xpert-positive individuals enrolled on Day 1, 18 returned on Day 2 for oral swab sampling without prior prompted sputum production. Two of these subjects subsequently delivered negative results by sputum culture. In contrast to previous studies [4, 5], here we measured sensitivity of OSA based on single swab results, rather than multiple swabs. Day 1 swabs exhibited 88.0% (44/50) and 91.5% (43/47) sensitivity relative to sputum Xpert and culture results, respectively (Table 3). Specificity of Day 1 swabs was 79.2% (42/53) and 66.1% (37/56) relative to sputum Xpert and culture, respectively. Day 2 swabs exhibited 94.4% (17/18) and 93.3% (15/16) sensitivity relative to sputum Xpert and culture, respectively (Table 3). The two sputum culture-negative Day 2 subjects who were positive by sputum GeneXpert were also positive by OSA.

Table 3. Sensitivity and specificity of OSA relative to sputum GeneXpert Ultra and culture.

	Day 1 swabs		Day 2 swabs
	Sensitivity	Specificity	Sensitivity	Specificity
Relative to Xpert	44/50 (88%)	42/53 (79.2%)	17/18 (94.4%)	N/A¹
Relative to culture	43/47 (91.5%)	37/56 (66.1%)	15/16 (93.8%)	0/2 (0%)

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¹Not applicable. All Day 2 subjects were Xpert-positive.

Factors associated with OSA results

No significant associations were observed between OSA positivity and smoking, previous TB infection, gender, having a household contact with TB, clinical site (Kiruddu Hospital vs. Kisenyi Health Centre), or patient type (in-patient vs. out-patient). However, among patients with positive oral swabs, an association was observed between OSA signal strength and HIV co-infection. Swabs from patients co-infected with HIV had higher Cq values, which indicates a weaker signal (Table 4).

Table 4. Association between OSA signal strength and HIV co-infection among patients with positive tongue swabs.

	HIV co-infected	HIV non-infected	p-value (t-test)
	Cq ± SD (N)	Cq ± SD (N)	p-value (t-test)
Day 1 swabs	34.35 ± 4.1 (9)	31.88 ± 2.9 (30)	0.047
Day 2 swabs	35.77 ± 4.2 (7)	31.09 ± 1.8 (8)	0.012

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Viability of MTB cells in tongue swab samples

We hypothesized that some fraction of TB patients harbor viable MTB cells that can be isolated, and cultured, from the oral cavity. To test this hypothesis, we analyzed samples from all 141 participants with sputum Xpert-confirmed TB (Fig 1). We designed this study to assess sensitivity, rather than specificity, of the oral swab for culture approach, and, accordingly, we enrolled only patients with positive sputum Xpert results.

Swab samples were collected, processed, and cultured for viable MTB as described in Methods and S1 Fig. Colonies were identified to the species level. At least one Day 1, post-sputum oral swab culture was positive in 82 of 141 (58.2%, Table 5) GeneXpert-positive patients. Of the 41 patients who returned on Day 2 for an additional swab collection without prior prompted sputum production, oral swab cultures were positive in 18 (43.9%, Table 5). This proportion increased to 50.0% (18 of 36) when excluding patients in whom all oral swab cultures were contaminated.

Table 5. Tuberculosis culture positivity among tongue swab samples from Xpert positive individuals.

	MTB positive/total (%)
Sputum culture	132/141 (93.6)
First Day 1 swab	71/141 (50.4)
Second Day 1 swab	61/141 (43.3)
Day 1 combined (either or both positive)	82/141 (58.2)
Day 2 swab	18/41 (43.9)
Any swab positive on Day 1 or 2	87/141 (61.7)
HIV+ individuals (any swab positive)	17/35 (48.6)

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While the collection design for Days 1 and 2 differed with respect to preceding sputum production, 87 of 141 (61.7%) patients had MTB-positive tongue swab cultures when considering all samples together. Patients with higher sputum smear grades or higher semi-quantitative sputum Xpert results were more likely to have cultivatable MTB from tongue swabs (Table 6; Pearson correlation coefficient for smear grade = 0.875, and Xpert semi-quantitative result = 0.983). Tongue swab cultures were positive for MTB in 17 of 35 (48.6%) HIV co-infected individuals, and this was not significantly different compared to HIV negative individuals (p > 0.1, Fisher’s Exact Test).

Table 6. Correlation between swab culture and smear microscopy grade or GeneXpert semiquantitative result.

		MTB culture positive/total for category, n = 87, (%)
GeneXpert Semiquantitative Result	Trace	1/4 (25.0)
	Very Low	5/13 (38.5)
	Low	16/30 (53.3)
	Medium	28/40 (70.0)
	High	37/47 (78.7)
Smear Microscopy Grade	Negative	14/33 (42.4)
	Scanty	6/14 (42.9)
	1+	24/31 (77.4)
	2+	32/42 (76.2)
	3+	11/14 (78.6)

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Discussion

Oral swabs have many potential advantages for TB screening and triage, especially in community settings where the collection of sputum or urine isn’t practical. However, evaluations of the method have yielded mixed indications of sensitivity [4–8]. In some studies, multiple swabs had to be tested to yield sensitivity values above 90% relative to sputum GeneXpert or culture [4, 5]. Oral swab samples are small in volume and unlikely to contain large numbers of bacilli in all cases. Therefore, we evaluated alternative sample collection methods designed to increase sample biomass.

Our laboratory analysis using conserved bacterial rDNA indicated that a previously-used tongue swabbing method collected only a small fraction of the bacterial biomass that exists on this surface. Assuming that MTB cells at this site are entrained in the tongue dorsum biofilm or the underlying epithelium, it seems feasible that sensitivity can be improved by increasing the amount of collected material. Through use of the bacterial biomass proxy, we identified the Copan FLOQSwab as a product that collects more biomass than the products used previously.

The clinical evaluation in Kampala yielded promising results with this product, albeit within a small sample set. Where previously it had taken 2 swabs per patient to achieve up to 93% sensitivity in adults, the current study exhibited up to 94% sensitivity relative to sputum culture using just one swab per patient. This was a different population from previous studies, and we did not do side-by-side clinical comparisons between the two swab brands. However, Copan FLOQSwabs performed well in this analysis and, we now use it exclusively for OSA.

Several limitations should be noted. First, even with increased biomass collection, sensitivity was less than 100% relative to both GeneXpert and culture. False-negative swabs were tested quantitatively for human DNA by using a sample adequacy control described recently [16]. They appeared to have been properly collected. Therefore, some patients who are sputum-positive can be missed by OSA. Second, specificity at 79% and 66% relative to sputum Xpert and culture, respectively, was markedly lower than the 92% observed previously [4]. Negative controls did not indicate laboratory contamination, so higher levels of false positivity in the present study may have had other causes. One possibility is that high-capacity FLOQSwabs are better able to collect MTB DNA that may be present in the oral cavities of people without TB disease in high-prevalence environments. Third, samples were tested by using manual qPCR. Automated methodologies are needed that exploit the specific advantages of swab samples relative to sputum. Fourth, this study was embedded within a larger study whose properties required the collection of Day 1 swabs after on-site collection of sputum. Production of sputum prior to swab sampling could have affected swab results by depositing fresh sputum onto the tongue. However, Day 2 swabs were collected without prior sputum production, and there was no evidence for reduced sensitivity under these conditions. Fifth, although Day 1 swab analyses were blinded with regard to TB status, Day 2 analyses were not blinded due to the study’s design. Finally, the sample size was small.

Despite these limitations, the results show promise in OSA as an easy-to-collect, noninvasive sample for TB screening and diagnosis. If larger studies continue to exhibit sensitivity in excess of 90% using a single swab, the method has the potential to meet at least some of the criteria needed for community-based triage testing [17].

Swabs from TB patients co-infected with HIV yielded higher Cq values (weaker signals) on average than those who were not co-infected with HIV. This confirms and extends an observation reported previously in a South African population [4].

The results from swab culture experiments supported the hypothesis that viable MTB can be cultured from a swab of the tongue dorsum, with sensitivity relative to sputum culture of 58% and 50% for swabs collected following sputum or in the absence of prompted sputum production, respectively. As with the molecular analysis, a limiting factor was that patients provided sputum before Day 1 swab sampling, which poses the risk of artificially loading the tongue dorsum with MTB. However, some patients returned and provided Day 2 swabs without prior sputum collection, and there was no significant difference in results between Day 1 and Day 2 swabs (P > 0.1, Fisher’s exact test). This analysis suggests that the detection of viable cells was not an artifact of study design. The results inform our understanding of OSA by confirming that at least part of the signal is associated with whole, viable MTB cells. This has implications for occupational safety of the method in addition to the further development of methods for sample handling and processing. These findings may also affect our understanding of the dynamics of TB transmission.

In summary, we used swab culture to improve our understanding of the physiology of MTB present on the tongue dorsum and a bacterial biomass proxy to identify a product with increased capacity to collect dorsum biofilm for MTB testing. The product performed well in a clinical assessment, exhibiting single-swab sensitivity in a range that approaches the needs of triage testing. Furthermore, the results in a Ugandan population confirmed and extended previous findings from the study in South Africa [4, 5]. With continued improvement, OSA could become an effective noninvasive, non-sputum sampling method for TB diagnosis and screening.

Supporting information

S1 File. Source table containing data informing all figures, tables, and analyses described in study.

(CSV)

Click here for additional data file.^{(46.4KB, csv)}

S1 Fig. Study design for sample collection and processing.

(TIF)

Click here for additional data file.^{(1.1MB, tif)}

Acknowledgments

We wish to thank Santina Castriciano of Copan Italia for providing swabs and technical guidance.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

Study funding provided by The Global Good Fund I, LLC (www.globalgood.com), and by grants from the Bill & Melinda Gates Foundation (INV-004527) and the National Institute of Allergy and Infectious Diseases (R01AI139254). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. At the time of study design and evaluation SB, CMB, DB, and AS received salary support from the Global Good Fund. At the time of study design and evaluation GH, KPN, AML, CO, DM, and KJM were employed by Intellectual Ventures Laboratory. The specific roles of these authors are articulated in the ‘author contributions’ section.

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PLoS One. doi: 10.1371/journal.pone.0251422.r001

Decision Letter 0

Selvakumar Subbian

9 Oct 2020

PONE-D-20-28429

Viable Mycobacterium tuberculosis from swabs of the tongue dorsum of pulmonary tuberculosis patients

PLOS ONE

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==============================

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5. Review Comments to the Author

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Reviewer #1: Abstract: “Sensitivities of the collection methods are not sufficient for use in routine culture based diagnostic tesating” is one of the main findings, but the abstract gives a different view, and conveys the message that this collection procedure will be an alternative to the rest stating that it might facilitate culture based diagnostic testing for TB. This needs to be rephrased in a manner so that the reader might get a true picture.

The positive findings of the study related to the comparison of the results with molecular based techniques are all reported previously and the abstract reiterates the same. The only positive finding is that viable bacilli is present in tongue, but this is expected and the authors have tried to merely substantiate this. But this message is not seen in the abstract.

Materials and methods: (Line 110 ) It is mentioned that all the cultures were incubated for 56 days, But is it the same for MGIT systems also?

Results and Discussion: (Line 133) It is not clear if the figure 61.4% includes single oral swab culture results or both, do specify this.

Table 3 legend shows swab culture, but is not reflected in the table title

(Line 163) This statement is contraindicative, says it is not statistically significant in the second half, but the sentence begins that the assessment is most discriminatory and how do you justify that the second day specimen is able to do so and the reason behind this.

Line 169 is confusing and explain what does oral sampling rate stands for.

Reviewer #2: This is an interesting but a simplistic and short study. In this reviewer's opinion it does not reach the level of a research article - a short communication would be more appropriate. The following are to be noted:

1. There is no clear description of the clinical application of the oral swab method.

2. No clear description/implications or insights for the understanding of TB pathogenesis presented/discussed.

3. The authors are respectfully advised to substantially expand the scope of the paper for it to fly as a full fledged research article.

4. On p3, lines 51-54 the tongue swab procedure is described stating three swabs were taken. The first and the third swabs are mentioned clearly but when was the 2nd one taken? It is not clear.

5. Table 1 shows Day2 culture positivity for the one swab sample taken is 43.9%. The authors have not discussed why they think it is so much lower than the first swab on Day 1 (50.4).

Reviewer #3: The manuscript “Viable Mycobacterium tuberculosis from swabs of the tongue dorsum of

pulmonary tuberculosis patients” is quite interesting and informative.

The paper is simple and concise but few more additions seems imperative before it can be considered for publication. The study emphasizes on the use of non-sputum method in the context of use in paucibacillary situation as an alternate to sputum. There are few clarifications needed.

1. Apart from culturing of MTB, was there any molecular diagnostic test performed on these tongue swabs? The gene Xpert results used for selection criteria is performed on the sputum samples and not the tongue swabs. If this method is going to be suggested as an alternate to sputum collection a parallel rapid diagnostic test to first identify positive and then its co-relation to their growth as culture will be more valid.

2. The bacilli were cultured in MGIT, LJ and 7H10. It would be very useful to give a comprehensive table in place of table 2 given currently to understand the revival and contamination rate in different MTB media assessed here.

3. It would be interesting to add the drug resistance data on the isolates and any indications to cultivability.

Reviewer #4: The authors in this manuscript describe an approach for isolation of viable Mycobacterium tuberculosis from swabs of the tongue dorsum of pulmonary tuberculosis patients. The study looks interesting, but the authors should address the following concerns.

• The results indicate that viable mtb bacilli do exist in the tongue dorsum, but the culture positivity is quite low. How will it improve the current TB diagnosis portfolio? Where do the authors want to position this test? Molecular tests are gaining advantage and use of DNA isolated from these samples as an input in endorsed molecular tests will provide a platform for positioning this technique.

• It will be useful to see the contamination rates of OminiGene vs. the NALC-NaOH method for the sputum versus swabs, as the former is reported to provide lower contamination rates, but here the rates were quite high (130/477 were contaminated). It will be useful to standardize the decontamination procedure for tongue swabs.

• Was the sample stored after reconstitution in OMNIgene tube? Did that make a difference in culture positivity? What advantage does the author foresee for this method? It will be interesting to see the viability assessment of bacilli stored in the OMNIgene tube over a period of time.

• Line 192. What bio-safety measures do the authors suggest, in view of their results. Also, a follow-up experiment might be useful to see how does the mtb load decrease in the dorsum during the course of therapy.

• What was interesting was the number of samples positive by swab culture (42.4%) in smear-ve category; what category of Xpert result did these samples lie in? A head to head comparison of Xpert versus Smear grade in all samples might be useful.

**********

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Reviewer #1: Yes: Azger Dusthackeer

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Reviewer #3: No

Reviewer #4: No

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PLoS One. 2021 May 17;16(5):e0251422. doi: 10.1371/journal.pone.0251422.r002

Author response to Decision Letter 0

18 Feb 2021

PONE-D-20-28429 Manuscript Re-submission

Text included here is also contained within the associated "Response to Reviewers" document. We appreciate the reviewers’ thorough read-through and comments on our manuscript, and in the rewrite/resubmission process we paid particular attention to the consistent critiques that the study was overly would benefit from additional data and a revised scope. We have substantially updated the manuscript, and responded to individual reviewer comments, below.

Point-by-point response to reviewer comments:

Reviewer #1:

1. Abstract: “Sensitivities of the collection methods are not sufficient for use in routine culture based diagnostic testing” is one of the main findings, but the abstract gives a different view, and conveys the message that this collection procedure will be an alternative to the rest stating that it might facilitate culture based diagnostic testing for TB. This needs to be rephrased in a manner so that the reader might get a true picture.

Response: Please review revised abstract in which we have focused more on the qPCR/molecular results that are now central to this manuscript. The information regarding culture positivity is included as an arm of the study, but without reference to using oral swab cultures for diagnosis.

2. Materials and methods: (Line 110 ) It is mentioned that all the cultures were incubated for 56 days, But is it the same for MGIT systems also?

Response: MGIT cultures were incubated for 42 days. We have revised the text to make this clear (Line 171).

3. Results and Discussion: (Line 133) It is not clear if the figure 61.4% includes single oral swab culture results or both, do specify this.

Response: The original text as written indicated “While the collection design for Days 1 and 2 differed with respect to preceding sputum production, 87 of 141 (61.7 %) patients had MTB-positive tongue swab cultures when considering all samples together.” We have revised this language to clarify: “While the collection design for Days 1 and 2 differed with respect to preceding sputum production, 87 of 141 (61.7 %) patients had MTB-positive tongue swab cultures when considering all samples together.” (Lines 335 – 337).

4. Table 3 legend shows swab culture, but is not reflected in the table title

Response: In the revised manuscript these data are included as Table 6, and we have revised the table title to clarify based on this point: “Table 6. Correlation between swab culture and smear microscopy grade or GeneXpert semiquantitative result.”

5. (Line 163) This statement is contraindicative, says it is not statistically significant in the second half, but the sentence begins that the assessment is most discriminatory and how do you justify that the second day specimen is able to do so and the reason behind this.

Response: The first half of the sentence was intended a comment that the Day 2 samples (collected without prior prompted sputum production) are more likely to be reflective of a patient’s natural disease state, while the observation that there was not a significant difference between Day 2 (no prior prompted sputum) and Day 1 samples (prior prompted sputum) is a comment on study design. We have modified the language in the revised draft, which can be found on lines 396-400.

6. Line 169 is confusing and explain what does oral sampling rate stands for.

Response: Thank you for raising this. This was a word omission on our part, and should have read “…oral sampling culture contamination rate…”. With the focus on the qPCR/molecular results in the revised manuscript we have removed this text.

1. There is no clear description of the clinical application of the oral swab method.

Response: We have addressed this concern in the revised manuscript with a greater focus on the diagnostic potential of qPCR from oral swab sampling. The integration of this information can be found across the manuscript; however, we specifically point out Lines 63 – 81 in the Introduction, the sociodemographic and clinical characteristics of the patients included in the study (Tables 1 & 2), comparison to GeneXpert of swab-based molecular MTB diagnosis (Tables 3 & 4), the text throughout the Results section associated with these tables, and the expanded text in the Discussion (for example, Lines 362 – 367 and Lines 406 – 413).

2. No clear description/implications or insights for the understanding of TB pathogenesis presented/discussed.

Response: In the revised manuscript we have shifted the emphasis from insights on TB pathogenesis attendant with the phenomenon of culturable MTB on the tongue dorsum, to a molecular diagnostic focus. We believe the culture/viability data still support the observation that knowledge of viable MTB on the tongue contributes to our knowledge base of the pathogen, but as the reviewer points out in the absence of additional data we limit further speculation on TB pathogenesis.

3. The authors are respectfully advised to substantially expand the scope of the paper for it to fly as a full fledged research article.

Response: Thank you for this comment and suggestion. In line with other reviewers and editor suggestion, we hope that our revised manuscript and expanded scope satisfy this request.

4. On p3, lines 51-54 the tongue swab procedure is described stating three swabs were taken. The first and the third swabs are mentioned clearly but when was the 2nd one taken? It is not clear.

Response: We have added a supplemental figure, “Fig S1” with a complete workflow of sample collection.

5. Table 1 shows Day2 culture positivity for the one swab sample taken is 43.9%. The authors have not discussed why they think it is so much lower than the first swab on Day 1 (50.4).

Response: The information raised in this comment can now be found in Table 5 and is described in the text on Lines 335 – 336 and Lines 393 – 401, in which we suggest that while it there is not a statistically significant difference in culture tongue swab culture positivity with or without preceding sputum production, it is possible that this procedural detail in the sample collection workflow (see Figure S1) explains the discrepancy.

Reviewer #3: The manuscript “Viable Mycobacterium tuberculosis from swabs of the tongue dorsum of

pulmonary tuberculosis patients” is quite interesting and informative.

Response: Consistent with comments from other reviewers, we have substantially revised the manuscript to include molecular/qPCR results to address this concern.

Response: With the revised focus on molecular detection of MTB from tongue swab, we felt that the content of the former table 2 distracted from the new focus. We have removed this table from the manuscript.

3. It would be interesting to add the drug resistance data on the isolates and any indications to cultivability.

Response: We agree that these are interesting questions; however, in our study setting, focused drug resistance (as determined by Xpert Rif analysis) is quite low (2 of 141 individuals, 1.4 %), and with the sample size it is difficult to draw significant conclusions on this question. This may be an excellent subject for follow-up studies, perhaps with a larger sample size and/or in settings with a greater burden of drug resistant MTB.

1. The results indicate that viable mtb bacilli do exist in the tongue dorsum, but the culture positivity is quite low. How will it improve the current TB diagnosis portfolio? Where do the authors want to position this test? Molecular tests are gaining advantage and use of DNA isolated from these samples as an input in endorsed molecular tests will provide a platform for positioning this technique.

Response: We appreciate this comment and agree with the reviewer’s assessment that molecular tests are gaining advantage in MTB diagnosis. Our revised manuscript is updated accordingly. We have retained data regarding viability of MTB collected with tongue swabs (Tables 5 & 6, with associated text Lines 319 – 346), however our focus is on the operational rather than diagnostic implications of these data (see, for example, Lines 401 – 405).

2. It will be useful to see the contamination rates of OminiGene vs. the NALC-NaOH method for the sputum versus swabs, as the former is reported to provide lower contamination rates, but here the rates were quite high (130/477 were contaminated). It will be useful to standardize the decontamination procedure for tongue swabs.

Response: While we retain our data on the viability of MTB from tongue swabs, our revised manuscript focuses on the ongoing efforts to develop (and ultimately standardize) oral swab analysis for molecular detection of MTB. We agree that if any tongue swab method were to be adopted for routine use, standardization of methodsis a critical component.

3. Was the sample stored after reconstitution in OMNIgene tube? Did that make a difference in culture positivity? What advantage does the author foresee for this method? It will be interesting to see the viability assessment of bacilli stored in the OMNIgene tube over a period of time.

Response: We have clarified the description of the methods to address this point (see Materials & Methods, Lines 183 - 196). The swab samples were stored in OMNIgene-SPUTUM for 18-24 hours at 25 °C prior to sample processing and culture inoculation.

4. Line 192. What bio-safety measures do the authors suggest, in view of their results. Also, a follow-up experiment might be useful to see how does the mtb load decrease in the dorsum during the course of therapy.

Response: While a data-driven answer to the question of MTB load over the course of therapy was outside the design and scope of this analysis, we agree that this is an interesting question for follow up work, as it may offer insights into the efficacy of a given therapy particularly in cases of drug resistance. While we were unable to conduct those experiments with our patient cohort we hypothesize that oral swab culture positivity rates over the course of treatment would track with sputum culture positivity rates.

5. What was interesting was the number of samples positive by swab culture (42.4%) in smear-ve category; what category of Xpert result did these samples lie in? A head to head comparison of Xpert versus Smear grade in all samples might be useful.

Response: We agree that these data reinforce the observation that MTB culture is more sensitive than smear microscopy, and can now point to tongue swab as an additional sampling matrix that follows this pattern; however, we feel that a head-to-head comparison of sputum Xpert results versus sputum smear microscopy falls outside of the scope of the current manuscript. We note, here, that for the majority of individuals (8 of 14) from this smear negative group had “very low” or “low” Xpert results, and the remainder (6 of 14) were split evenly between medium and high Xpert results.

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PLoS One. doi: 10.1371/journal.pone.0251422.r003

Decision Letter 1

Selvakumar Subbian

11 Apr 2021

PONE-D-20-28429R1

Characterization of oral swab samples for diagnosis of pulmonary tuberculosis

PLOS ONE

Dear Dr. Minch,

==============================

ACADEMIC EDITOR: Although two of the reviewers are contended with the revised manuscript, one of the reviewers has raised some minor issues that needs to be addressed by a revision. I encourage the authors to take this opportunity to check for any other issues, such as typos and grammatical errors.

==============================

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

Reviewer #4: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #3: Partly

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #3: No

Reviewer #4: Yes

**********

6. Review Comments to the Author

Reviewer #1: The manuscript could be accepted in the present form. Authors have addressed the queries raised and the response is acceptable and the edits have been made as per their replies.

Reviewer #3: The paper titled “Characterization of oral swab samples for diagnosis of pulmonary tuberculosis” is a neat study. In the present form it lacks being presented as a full-length article only based on methodology and its results. However, with the variety of papers that characterise oral swabs for diagnosis published in last 5 years, the authors need to justify how this study is different and how this study is a value addition to the previous ones. The scope of the paper in comparison to previously published ones is still lacking. The results need to be discussed in detail in context of previous published studies and not just the opinion of the authors about the same in the present context. There is no insight on the recommendations for culture of mycobacteria and preferred method for maximum recovery of viable bacteria among the three.

Line 49 Make it uniform % (87/114) to make it easy to understand.

Line 36 refers to geneXpert while line 45, 47 and 288 refers to GeneXpert Ultra, please give correct detail. Is it MTB/RIF or Ultra? Throughout the manuscript it reads MTB/Rif – eg Line 120 as the first portion was processed for Xpert (Xpert MTB/RIF assay, Cepheid, Sunnyvale, CA, USA).The authors got to be specific.

Line 51 – Referring to oral swabs as a screening in asymptomatic patients? While line 63 says “oral epithelium during 64 active TB disease” Line 71 says “useful for diagnosis and screening in non-clinical and community settings” Please clarify

Line 101 adults (>18 years) – please provide interquartile range for this.

Line 190-192 Clarify - the incubation periods for LJ and MGIT or 7H10 are not the same.

Line 238-240 - Based on Cq values from qPCR 239 analysis, Puritan PurFlock Ultra swabs were found to collect about twice as much MTB DNA as Whatman OmniSwabs® 240 (p = 0.015). Kindly discuss this in comparison to previous reported studies.

Line 365 This was a different population from previous studies, 366 and we did not do side-by-side clinical comparisons between the two swab brands. Please add further discussion to the swab type variation in comparison to previous studies.

Fig S1 refers to MGIT or LJ or both. Please confirm reference to 56 days wait for confirming culture negative.

Reviewer #4: The manuscript has improved in clarity from the previous version. The authors have addressed all the comments in the revised manuscript.

**********

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Reviewer #1: No

Reviewer #3: No

Reviewer #4: No

PLoS One. 2021 May 17;16(5):e0251422. doi: 10.1371/journal.pone.0251422.r004

Author response to Decision Letter 1

23 Apr 2021

Point-by-point response to reviewer comments

NOTE: Line numbers in our responses refer to the mark-up version. Major changes are also highlighted in the mark-up version.

Reviewer #3:

1. The paper titled “Characterization of oral swab samples for diagnosis of pulmonary tuberculosis” is a neat study. In the present form it lacks being presented as a full-length article only based on methodology and its results. However, with the variety of papers that characterise oral swabs for diagnosis published in last 5 years, the authors need to justify how this study is different and how this study is a value addition to the previous ones. The scope of the paper in comparison to previously published ones is still lacking. The results need to be discussed in detail in context of previous published studies and not just the opinion of the authors about the same in the present context. There is no insight on the recommendations for culture of mycobacteria and preferred method for maximum recovery of viable bacteria among the three.

o We thank the reviewer for this summary of critiques, and point to several areas in the manuscript where their concerns are addressed. We note that key differences that distinguish this work from previous studies are articulated in the abstract (Lines 38-41, highlighted in the Track Changes version): “In previous analyses, qPCR testing of swab samples collected from tongue dorsa was up to 93 % sensitive relative to sputum GeneXpert, when 2 swabs per patient were tested. The present study modified sample collection methods to increase sample biomass and characterized the viability of bacilli present in tongue swabs.” We note further that we discuss this study/these results in comparison to previous work in several other locations in the manuscript (see, for example, lines 88-95 and in several highlighted passages in the Discussion).

o The reviewer also flags that there is no insight on the recommendation for culture of mycobacteria. As discussed in the cover letter associated with the manuscript resubmission, following the reviews of our original submission, we substantially revised the manuscript to focus on the molecular detection of MTB from oral swabs, rather than broad diagnostic application of culture-based methods. With that revised focus, in the present manuscript we do make note of considerations for occupational safety, and suggest that the demonstration that at least some DNA collected from the tongue is associated with whole, viable MTB cells has implications for sample handling and processing (Lines 407-411).

2. Line 49 Make it uniform % (87/114) to make it easy to understand.

o The text has been updated.

3. Line 36 refers to geneXpert while line 45, 47 and 288 refers to GeneXpert Ultra, please give correct detail. Is it MTB/RIF or Ultra? Throughout the manuscript it reads MTB/Rif – eg Line 120 as the first portion was processed for Xpert (Xpert MTB/RIF assay, Cepheid, Sunnyvale, CA, USA).The authors got to be specific.

o The comment on Line 36 refers to results published in a different study, and in that study the authors used sputum GeneXpert (as stated in the abstract). Within the current manuscript, we implemented the nomenclature suggested by the reviewer (GeneXpert MTB/Rif Ultra) throughout the manuscript. This information is also reflected in columns G and H in Table S1. For consistency, we have added the designator “Ultra” to line 126.

4. Line 51 – Referring to oral swabs as a screening in asymptomatic patients? While line 63 says “oral epithelium during 64 active TB disease” Line 71 says “useful for diagnosis and screening in non-clinical and community settings” Please clarify

o Line 63 is a description of previously published data. In this study our study enrollment criteria were to include patients “…who presented with respiratory symptoms…” (Line 107), and so we do not make comment on the screening of asymptomatic patients in the abstract. To minimize confusion we changed the wording that the reviewer flags to: “useful for TB case finding in non-clinical and community settings” (Line 76).

5. Line 101 adults (>18 years) – please provide interquartile range for this.

o The requested data are included in Table 1. The text on Lines 96-104 is a description of our prospective study design and inclusion criteria.

6. Line 190-192 Clarify - the incubation periods for LJ and MGIT or 7H10 are not the same.

o As described in Lines 198-199, and in Fig S1, in this study all cultures from tongue swab were incubated for up to 56 days. We acknowledge that guidance documents indicate different incubation periods for the various media, however, in this study investigating the tongue as a source matrix for viable MTB we adopted a conservative, long, incubation/observation period of 56 days uniform across all media types. As described in the section titled “Sputum sample processing and analysis,” for sputum culture, we incubated (sputum) MGIT cultures for up to 42 days and (sputum) LJ cultures for up to 56 days (lines 177-179).

7. Line 238-240 - Based on Cq values from qPCR 239 analysis, Puritan PurFlock Ultra swabs were found to collect about twice as much MTB DNA as Whatman OmniSwabs® 240 (p = 0.015). Kindly discuss this in comparison to previous reported studies.

o This sentence refers to results reported previously (reference 4, Luabeya et al 2019), as noted in the preceding sentence. The complete passage reads “Previously, we compared two swab brands for their abilities to detect MTB DNA on buccal (not tongue) surfaces in the mouths of adult TB patients. Based on Cq values from qPCR analysis, Puritan PurFlock Ultra swabs were found to collect about twice as much MTB DNA as Whatman OmniSwabs® (p = 0.015) [4].”

8. Line 365 This was a different population from previous studies, 366 and we did not do side-by-side clinical comparisons between the two swab brands. Please add further discussion to the swab type variation in comparison to previous studies.

o These comparisons and context are presented throughout the current manuscript. Please see, for example:

� The paragraph on Lines 77-88

� Lines 370-372 “…previously it had taken 2 swab per patient… the current study exhibited up to 94 % sensitivity relative to sputum culture using just one swab per patient.”

9. Fig S1 refers to MGIT or LJ or both. Please confirm reference to 56 days wait for confirming culture negative.

o This comment is addressed in response #6, above.

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PLoS One. doi: 10.1371/journal.pone.0251422.r005

Decision Letter 2

Selvakumar Subbian

27 Apr 2021

Characterization of oral swab samples for diagnosis of pulmonary tuberculosis

PONE-D-20-28429R2

Dear Dr. Cangelosi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Selvakumar Subbian, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0251422.r006

Acceptance letter

Selvakumar Subbian

7 May 2021

PONE-D-20-28429R2

Characterization of oral swab samples for diagnosis of pulmonary tuberculosis

Dear Dr. Cangelosi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Selvakumar Subbian

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 File. Source table containing data informing all figures, tables, and analyses described in study.

(CSV)

Click here for additional data file.^{(46.4KB, csv)}

S1 Fig. Study design for sample collection and processing.

(TIF)

Click here for additional data file.^{(1.1MB, tif)}

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Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

Characterization of oral swab samples for diagnosis of pulmonary tuberculosis

Rachel C Wood

Alfred Andama

Gleda Hermansky

Stephen Burkot

Lucy Asege

Mukwatamundu Job

David Katumba

Martha Nakaye

Sandra Z Mwebe

Jerry Mulondo

Christine M Bachman

Kevin P Nichols

Anne-Laure M Le Ny

Corrie Ortega

Rita N Olson

Kris M Weigel

Alaina M Olson

Damian Madan

David Bell

Adithya Cattamanchi

William Worodria

Fred C Semitala

Akos Somoskovi

Gerard A Cangelosi

Kyle J Minch

Roles

Abstract

Introduction

Methods

Study sites, populations, and sampling workflows

Fig 1. Patient numbers for oral swab study.

Swab collection

Sputum sample processing and analysis

Swab processing for mycobacteriological culture

Swab processing for qPCR

Swab sample analysis by qPCR

Results

Characterization of biomass collection

Fig 2. Rapid repeat sampling of individual participants.

An alternative swab brand collects more bacterial biomass

Fig 3. Comparison of total bacterial biomass collected from tongue dorsa by two swab products.

Clinical evaluation of alternative swabs

Table 1. Socio-demographic characteristics and other patient information.

Table 2. Clinical characteristic of patients.

Table 3. Sensitivity and specificity of OSA relative to sputum GeneXpert Ultra and culture.

Factors associated with OSA results

Table 4. Association between OSA signal strength and HIV co-infection among patients with positive tongue swabs.

Viability of MTB cells in tongue swab samples

Table 5. Tuberculosis culture positivity among tongue swab samples from Xpert positive individuals.

Table 6. Correlation between swab culture and smear microscopy grade or GeneXpert semiquantitative result.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Selvakumar Subbian

Roles

Author response to Decision Letter 0

Decision Letter 1

Selvakumar Subbian

Roles

Author response to Decision Letter 1

Decision Letter 2

Selvakumar Subbian

Roles

Acceptance letter

Selvakumar Subbian

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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