RESEARCH ARTICLE

Gene-Based Diagnosis of Tuberculosis from Oral Swabs with a

New Generation Pathogen Enrichment Technique
Young Ae Kang,a,b,c Bonhan Koo,d Ock-Hwa Kim,e,f Joung Ha Park,g Ho Cheol Kim,e Hyo Joo Lee,d Myoung Gyu Kim,d Youngwon Jang,e
Na Hyun Kim,e Yong Seo Koo,h Yong Shin,d Sei Won Lee,e Sung-Han Kimg

a Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, College of Medicine, Yonsei University, Seoul, Republic of Korea
b Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, Severance Hospital, Seoul, Republic of Korea
c Institute of Immunology and Immunological Disease, College of Medicine, Yonsei University, Seoul, Republic of Korea
d Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea
e Department of Pulmonary and Critical Care Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea
Division of Pulmonology and Critical Care Medicine, Department of Internal Medicine, Chungnam National University Sejong Hospital, Sejong, Republic of Korea
f

g Department of Infectious Diseases, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea
h Department of Neurology, Asan Medical Center, Seoul, Republic of Korea

Young Ae Kang and Bonhan Koo contributed equally to this article as first authors. Author order was determined based on their contribution.
Yong Shin and Sei Won Lee equally contributed to this study.

ABSTRACT A rapid and sensitive diagnosis is crucial for the management of tuberculo-
sis (TB). A simple and label-free approach via homobifunctional imidoesters with a micro-
fluidic platform (SLIM) assay showed a higher sensitivity than the Xpert MTB/RIF assay in
the diagnosis of pulmonary TB (PTB). Here, we evaluated the efficacy of the SLIM assay
for oral swab samples from cases of suspected PTB. Patients with clinically suspected PTB
were prospectively enrolled and oral swab samples were processed using the SLIM assay
and the attending physicians were blinded to the results of the SLIM assay. TB cases
were defined as those treated with anti-TB chemotherapy for at least 6 months at the dis-
cretion of the specialists based on their clinical features and conventional laboratory
results, including the Xpert assay. A total of 272 patients (with TB, n = 128 [47.1%]; with-
out TB, n = 144 [52.9%]; mean age, 59.8 years) were enrolled. Overall, the sensitivity of
the oral swab-based SLIM assay (65.6%) was higher than that of the sputum-based Xpert
assay (43.4%; P = 0.001). Specifically, the SLIM oral swab assay showed a notably higher
sensitivity in culture-negative TB cases compared with the Xpert assay (69.0% [95% CI:
49.2 to 84.7%] versus 7.4% [95% CI: 0.9 to 24.3%]; P = 0.001). The specificity of the SLIM
and the Xpert assays was 86.1% (95% CI: 79.3 to 91.3%) and 100% (95% CI: 97.2 to
100%), respectively. When only culture-confirmed cases were analyzed, the SLIM oral
swab was comparable to sputum Xpert in sensitivity (64.7% versus 54.3%, P = 0.26). The
oral swab-based SLIM assay showed a superior sensitivity for TB diagnosis over the spu-
tum-based Xpert assay, especially for culture-negative cases.
IMPORTANCE The development of a rapid, accessible, and highly sensitive diagnostic Editor Amit Singh, Indian Institute of Science
Bangalore
tool is a major challenge in the control and management of tuberculosis. Gene-based
Copyright © 2022 Kang et al. This is an open-
diagnostics is recommended for the rapid diagnosis of pulmonary tuberculosis (PTB), access article distributed under the terms of
but its sensitivity, such as Xpert MTB/RIF assay (Xpert), drops in cases with a low bac- the Creative Commons Attribution 4.0
International license.
terial load. It can only be applied to sputum samples, and it is quite difficult for some
Address correspondence to Yong Shin,
patients to produce an adequate amount of sputum. We evaluated the clinical validity [email protected], or Sei Won Lee,
of an oral swab-based microfluidic system, i.e., the SLIM assay. The SLIM assay showed [email protected].
a significantly higher sensitivity than the Xpert assay, especially in smear-negative TB The authors declare no conflict of interest.
cases. This non-sputum-based SLIM assay can be a useful diagnostic test by overcom- Received 22 January 2022
ing the limitations of conventional sputum-based tests in pulmonary TB. Accepted 22 April 2022
Published 19 May 2022
KEYWORDS tuberculosis, rapid diagnosis, oral swab, gene-based diagnosis

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uberculosis (TB) caused by infection with Mycobacterium tuberculosis (MTB) still

T ranks as a leading cause of death worldwide (1). Rapid and accurate diagnosis of
pulmonary tuberculosis (PTB) in its early stage is vital for the successful control of the
transmission of TB and for improving the treatment outcomes. In 2011, the World
Health Organization (WHO) endorsed the use of the Xpert MTB/RIF assay (Xpert;
Cepheid, Sunnyvale, CA, USA), a novel, rapid, automated, cartridge-based real-time
PCR (PCR) method (2), for initial diagnosis of patients suspected of active PTB (3). In the
landmark study, the Xpert assay showed a high sensitivity of 98.2% in acid-fast bacilli
(AFB) smear-positive TB cases. However, the sensitivity was as low as 72.5% in smear-
negative cases (4), and data from real-world settings reported a sensitivity of only
around 60% to 74% (5, 6). Indeed, the sensitivity of Xpert for TB detection is inad-
equate when only a few bacilli are present in a clinical specimen.
The more recently developed Xpert-MTB/RIF Ultra assay showed a superior sensitiv-
ity to the Xpert assay (63% versus 46%) for diagnosing smear-negative PTB (7), but it
was still not high enough. In addition, the Xpert assays can only be applied to sputum
samples, which are occasionally hard to acquire from young children and asymptom-
atic patients with paucibacillary diseases. Furthermore, sputum collection is prone to
producing potentially infectious aerosols that present a hazard for health care workers
and fellow patients (8).
To overcome these limitations of the current TB diagnostics, we have developed a new
assay for diagnosing PTB that involves simple and rapid pathogen enrichment by homobi-
functional imidoesters (HIs) using a microfluidic system followed by conventional MTB PCR,
i.e., the SLIM assay (9). The SLIM assay showed significantly better performance over the
Xpert assay in terms of sensitivity (60%; 95% confidence interval [CI]: [47% to 72%] versus
37% [95% CI: 25% to 50%], P = 0.001) in the diagnosis of pulmonary TB (PTB) using sputum
samples without a significant decrease in specificity (10).
To expand the clinical applicability of the SLIM assay, we investigated its perform-
ance for the diagnosis of PTB from oral swab samples. Specifically, this real-world, prac-
tice-based study was performed in patients with clinically suspected PTB in a country
with an intermediate burden of TB and a low burden of the human immunodeficiency
virus (HIV).

RESULTS
Participants. A total of 272 patients suspected of PTB were enrolled in this study.
The patients’ mean age was 58.8 6 15.2 years and 174 (64.0%) were male. Malignant
diseases (34.6%) and diabetes mellitus (19.9%) were the most common underlying dis-
eases, followed by transplant status (4.8%) and liver cirrhosis (4.4%). Only one (0.4%)
patient had an HIV infection (Table 1). A total of 52 patients (19.9%) had a history of
previous pulmonary TB. Cough was the most common symptom (25%) Almost half of
the participants (49.3%) had no specific respiratory symptoms and only had radio-
graphic abnormalities.
A total of 128 patients were finally treated as TB cases and planned to take full-
course chemotherapy. They were categorized into smear-positive confirmed TB
(n = 35), smear-negative confirmed TB (n = 64), and possible TB (n = 29). Among them,
one participant (male, 57 years old) died of an underlying disease (neuroendocrine tu-
mor with liver metastasis) after 3 months of TB treatment and could not complete the
treatment. However, he was grouped as smear-negative culture-negative clinical TB
because he had a good and persistent response to the TB treatment. The remaining
144 patients did not meet the criteria of TB diagnosis according to the study definition
(Fig. 1).
Clinical validity of the assays. The results of the SLIM oral swab assay and the
Xpert assay according to the clinical diagnosis stratified by the AFB smear and MTB cul-
ture results are in Table 2, and their diagnostic performances compared with the other
tests are in Fig. 2 and Table 3. For confirmed TB, the sensitivity of the SLIM oral swab,
Xpert, AFB smear and culture for TB were 64.7% (64/99; 95% CI: 54.4 to 73.8%), 53.7%
(51/95; 95% CI: 43.2 to 64.0%), 35.4% (35/99; 95% CI: 26.0 to 46.6%) and 93.9% (93/99;

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TABLE 1 Baseline characteristics of the participants

Total Treated as TB Not TB
Characteristic (n = 272) (n = 128) (n = 144) P value
Age, yrs 6 SDa 58.8 6 15.2 56.4 6 16.0 60.9 6 14.1 0.014
Male sex, n (%) 174 (64.0) 85 (66.4) 89 (61.8) 0.43

Symptoms, n (%)
Cough 68 (25.0) 29 (22.7) 39 (27.1) 0.4
Sputum 47 (17.3) 17 (13.3) 30 (20.8) 0.1
Hemoptysis 12 (4.4) 4 (3.1) 8 (5.6) 0.33
Fever 28 (10.3) 12 (9.4) 16 (11.1) 0.64
Night sweat 6 (2.2) 2 (1.6) 4 (2.8) 0.69
Dyspnea 15 (5.5) 7 (5.5) 8 (5.6) 0.98
Chest pain 8 (2.9) 7 (5.5) 9 (6.3) 0.78
General weakness 16 (5.9) 2 (1.6) 6 (4.2) 0.29

Chest radiograph abnormality, only 134 (49.3) 56 (43.8) 78 (54.2) 0.09

Previous TB history, n (%) 52 (19.9) 21 (16.4) 31 (21.5) 0.28
AFB smear positive, n (%) 46 (16.9) 35 (27.3) 11 (7.6) ,0.0001
Mycobacterial culture, TB isolated, n (%) 93 (34.2) 93 (72.7) 0 (0) ,0.0001
IGRA, n (%) 89 (49.2) 45 (68.2) 44 (38.3) 0.0001

Underlying disease, n (%)

Malignant disease 94 (34.6) 31 (24.2) 63 (43.8) 0.001
Diabetes mellitus 54 (19.9) 24 (18.8) 30 (20.8) 0.67
Transplant recipient 13 (4.8) 8 (6.3) 5 (3.5) 0.28
Liver cirrhosis 12 (4.4) 5 (3.9) 7 (4.9) 0.7
Gastrectomy 7 (2.6) 1 (0.8) 6 (4.2) 0.13
Rheumatoid disease 3 (1.1) 0 (0.0) 3 (2.1) 0.25
HIV infection 1 (0.4) 1 (0.8) 0 (0.0) 0.47
aSD,standard deviation; HIV, human immunodeficiency virus; TB, tuberculosis; IGRA, interferon-gamma release
assay.

95% CI: 82.3 to 97.7%), respectively. The sensitivity of the SLIM oral swab assay was
higher than that of the Xpert assay, but it did not reach statistical significance (P =
0.12). The specificity of the SLIM oral swab, Xpert, AFB smear, and MTB culture were
86.1% (124/144; 95% CI: 86.1 to 91.3%), 100% (130/130; 95% CI: 97.2 to 100.0%), 92.0%
(127/138; 95% CI: 86.2 to 96.0%), and 100% (138/138; 95% CI: 97.4 to 100.0%),
respectively.
The sensitivity of the SLIM oral swab assay and the Xpert assay were further ana-
lyzed according to the four categories of TB: smear-positive confirmed, smear-negative
confirmed, possible, and treated as TB (Fig. 2B). The sensitivity of the SLIM oral swab
assay was not significantly different according to the TB categories and ranged from
62.5% to 69.0% (P = 0.55). In contrast, the sensitivity of the Xpert assay was the highest
in smear-positive confirmed TB (79.4%) and was significantly lower in smear-negative
confirmed TB (37.3%, P = 0.0002) and possible TB (7.4%, P , 0.0001). As such, whereas
the two assays did not show significant differences in sensitivity for smear-positive PTB
(P = 0.31), the SLIM oral swab assay had significantly higher sensitivity than the Xpert
assay in both smear-negative confirmed TB (P = 0.01) and possible TB (P , 0.0001). The
sensitivity of the SLIM oral swab relative to Xpert was significantly superior to that of
Xpert relative to the SLIM oral swab (P = 0.039, Table S1).
A combination of the SLIM oral swab assay and Xpert assay was evaluated for its
clinical usefulness. The SLIM oral swab found 31 additional patients with confirmed TB
and 18 with possible TB. Accordingly, this combination showed a sensitivity of 86.3%
(95% CI: 77.7% to 92.5%) and specificity of 85.4% (95% CI: 78.1% to 91.0%; Table S2).
Clinical characteristics of the patients with false-positive results on SLIM oral
swab. A total of 20 patients with positive SLIM oral swab results and the presence of
MTB DNA confirmed by Sanger sequencing were not finally diagnosed with PTB
according to the study definition. Among them, 10 (50.0%) patients had an inflamma-
tory scar on the chest CT suspected of old inactive TB, and six of them had a history of
TB treatment. Another five (25.0%) patients had lesions suspected of active PTB.

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FIG 1 Diagnostic flow of the study patients. Among 272 patients with clinically suspected TB, 128 were
finally treated for TB by respiratory or infection specialists who were blinded to the results of the SLIM
oral swab assay. Confirmed TB was defined as culture-positive TB patients with at least one positive
culture result for MTB from their sputum. Possible TB was defined as culture-negative TB patients with a
high clinical likelihood of active TB and a negative mycobacterial culture finding in three or more sputum
examinations, but with good clinical and radiographic responses to anti-TB treatment during follow-up
without any evidence of an alternative diagnosis. *, six participants with TB not isolated in sputum but
isolated from bronchial washing fluid were included. †, one participant who could not produce sputum
was included. NTM, nontuberculous Mycobacterium.

However, they were regularly followed up without treatment because the physician
considered that the clinical evidence for treatment was not sufficient. Three (15.0%)
patients had pneumonic infiltration and were treated with broad-spectrum antibiotics,
and the other two (10.0%) were diagnosed with NTM pulmonary disease. The detailed
characteristics and representative chest images of these patients are provided in the
online supplement (Table S3 and Fig. S2).

DISCUSSION
In this real-world practice setting study, we showed that the SLIM oral swab assay, a
non-sputum-based diagnostic test, can detect PTB with high sensitivity, comparable to
conventional sputum-based tests, such as mycobacterial culture and Xpert. The superi-
ority of the SLIM assay was particularly pronounced for smear-negative PTB, especially
culture-negative clinical PTB, which are cases with a low bacterial load.

TABLE 2 Comparison of sputum exam results according to the categories of TB

SLIM oral swab Xpert MTB/RIF
(n = 272) (n = 252a)

Case definition Positive Negative Total Positive Negative Total

Confirmed TB
Smear positive 24 11 35 27 7 34
Smear negative 40 24 64 24 37 61

Possible TB
Culture negative 20b 9 29 2 25 27
c
Not TB 20 124 144 0 130 130
aThe results of Xpert MTB/RIF were not available for 20 patients.
bOne patient without a sputum exam was included. See Fig. 2 for more details.

cSee Table S1 for more details. Xpert MTB/RIF was not available for one participant.

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FIG 2 Clinical validity of the SLIM oral swab assay and the Xpert MTB/RIF assay for the diagnosis of
TB. (A) Sensitivity and specificity of the five different types of assays for the diagnosis of confirmed
TB. (B) Sensitivity of the SLIM oral swab assay and Xpert MTB/RIF according to the categories of TB.

The development of a rapid, accessible, and highly sensitive diagnostic tool is a

major challenge in the control and management of TB. Among a total of 10 million
new TB cases worldwide in 2019, as many as 2.9 million cases were estimated to have
been not diagnosed or detected (1), which may be the main source of its transmission
and morbidity. As an effort to overcome this diagnostic gap, the WHO recommended
the use of the Xpert assay as the initial test for TB (11). However, the sensitivity of the
Xpert assay is not high enough for paucibacillary TB cases (4, 12), and is thus limited
for use in smear-negative TB cases that require more sensitive diagnostic methods. To
meet this unmet clinical need, we applied the SLIM assay, a new generation pathogen
enriching technique, and demonstrated its efficacy in PTB and other infectious diseases
(9, 10, 13).
In conventional assays for DNA extraction, only a small volume (between 100 m L
and 200 m L) of clinical samples is used for the detection of pathogens due to the
capacity of the assays; in contrast, the SLIM assay can use both small volumes
(between 100 m L and 1 mL) and large volume (more than 1 mL) samples by enabling
simultaneous concentration and extraction of the pathogens in a single system. Due to

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TABLE 3 Diagnostic performance of the TB assays according to the categories of TB

Positive Negative
Sensitivity % Specificity % PPVa % NPV % likelihood likelihood
Case definition (n/N, 95% CI) (n/N, 95% CI) (n/N, 95% CI) (n/N, 95% CI) Ratio (95% CI) Ratio (95% CI)
Confirmed TB (n = 99b)
versus not TB (n = 144)
SLIM oral swab 65 (64/99, 54–74) 86 (124/144, 79–91) 76 (64/84, 68–83) 78 (124/159, 73–82) 4.66 (3.02–7.17) 0.41 (0.31–0.54)
Xpert MTB/RIF 54 (51/95, 43–64) 100 (130/130, 97–100) 100 (51/51) 75 (130/174, 70–79) Not applicable 0.46 (0.37–0.58)
AFB smear 35 (35/99, 26–46) 92 (127/138, 86–96) 76 (35/46, 63–86) 66 (127/191, 63–70) 4.44 (2.37–8.30) 0.70 (0.60–0.82)
MTB culture 94 (93/99, 87–98) 100 (138/138, 97–100) 100 (93/93) 96 (138/144, 91–98) Not applicable 0.06 (0.03–0.13)

Possible TB (n = 29)
versus not TB (n = 144)
SLIM oral swab 69 (20/29, 49–85) 86 (124/144, 79–91) 50 (20/40, 38–62) 93 (124/133, 89–96) 4.978 (3.09–7.98) 0.36 (0.21–0.62)
Xpert MTB/RIF 7 (2/27, 1–24) 100 (130/130, 97–100) 100 (2/2) 84 (130/155, 82–85) Not applicable 0.93 (0.83–1.03)
AFB smear 0 (0/28c, 0–12) 92 (127/138, 86–96) 0 (0/11) 82 (127/155, 81–83) 0.00 1.09 (1.04–1.14)
MTB culture 0 (0/28c, 0–12) 100 (138/138, 97–100) Not applicable 83 (138/166, 83–83) Not applicable 1.00 (1.00–1.00)
aPPV,positive predictive value; NPV, negative predictive value; CI, confidence interval; AFB, acid-fast bacilli; MTB, Mycobacterium tuberculosis.
participants with TB not isolated in sputum but isolated from bronchial washing fluid were included.
bSix

cOne participant without a sputum exam was included. See Fig. 2 for more details.

this advantage, the sensitivity of the SLIM system for pathogen diagnosis is signifi-
cantly higher than that of conventional assays (9, 10, 13).
In our previous study, SLIM assays with 2 mL sputum had a higher sensitivity than the
Xpert assay for the diagnosis of culture-positive pulmonary TB (57% [95% CI: 39% to 73%]
Xpert versus 91% [95% CI: 78% to 97%]) (SLIM 2 mL) (10). In addition, the SLIM system can
minimize the time (,50 min for pathogen enrichment and DNA extraction), cost ($5 to
$6), instrument requirements (centrifuges and vortexes), and additional reagents (antibod-
ies) for sample processing considering the material required (9, 10).
Easier, safer, and more effective sampling methods are essential in TB diagnosis (14).
Many patients struggle to produce an adequate amount of sputum for testing. Non-spu-
tum-based samples, such as saliva, urine, blood, and exhaled breath concentrate, have
been tested, but these samples are typically less useful than sputum (15–17). Recent
studies have suggested the use of oral swab samples, which can easily be obtained
through noninvasive, non-aerosol-producing methods. Previous studies have shown
that MTB DNA can be detected in oral swabs from human and nonhuman primates (18–
20). Wood et al. (21) reported 90% sensitivity of oral swab samples, although the number
of participants was small, and more than half of them (60%) were smear-positive.
Luabeya et al. (22) reported that oral swab samples had 92.8% sensitivity and 91.5%
specificity. These two studies showed promising results, but both used two swabs
instead of one and included participants with chronic respiratory symptoms, which
might increase the sensitivity of those who could be distinguished as TB.
In our study, the SLIM assay was applied to detect PTB using a single oral swab sam-
ple per patient suspected of TB. This new method overcame the main limitation of the
currently available diagnostics. It increased the sensitivity, which is relatively low in
Xpert in cases of smear-negative PTB, and it showed the potential usefulness of non-
sputum-based assays, whose efficacies are comparable or even superior to conven-
tional sputum-based techniques. Non-sputum-based assays will be especially helpful
in mass-screening large groups of people (e.g., school, prison, military base), in which
obtaining sputum may be difficult, such as from children or those without symptoms
(23, 24). It will also be useful in the setting of a TB outbreak investigation, in which
many asymptomatic active cases may be present (25). The non-sputum-based sensitive
examination will reduce the unnecessary spread of sputum production during mass
screening by preselecting those few individuals who need a sputum exam.
There is also an unmet clinical need for diagnosing extrapulmonary TB and PTB in
patients who cannot produce an adequate amount of sputum. The sensitivity of Xpert
in the diagnosis of PTB is very low when using samples other than sputa, such as
exhaled breath condensate (0%) and saliva (38.5%) (15). For other clinical samples,

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such as pleural fluid from TB pleurisy, the sensitivity was quite low at 30% (26). Other
techniques showed the possibility of the detection of MTB DNA from plasma (27).
Thus, further validation of the SLIM assay with various types of clinical samples and
additional development of the system with automation can widen its clinical utility.
We expect that our ongoing studies on the application of the SLIM assay to various
specimens, such as cerebrospinal fluid (CSF), blood, and oral swabs will be able to pro-
vide useful data on this issue. These strengths, its high sensitivity, and application to
various samples indicate the future direction and potential uses of the SLIM assay in TB
diagnosis. The SLIM assay can increase the sensitivity and have a validated specificity
by integrating with the Xpert platform. It can also be used as a rapid diagnostic for TB
suspected patients without sputum. A more simplified method with automation will
be a critical step to increase its utility independent of an extensive laboratory facility.
This study and the SLIM assay still have some limitations to overcome. First, the
false-positive results should be further controlled. The reasons for this were not deter-
mined in this study. It may be related to the high sensitivity of the new method, the
detection of remnant bacilli or DNA from previous infections, contamination of sam-
ples during collection or analysis, or combinations of these things. Further work is
needed to discern these possibilities. As Xpert is a closed cartridge diagnostic system
that uses automated PCR testing to detect TB with high specificity, the development of
a closed SLIM assay may be a key factor in increasing the specificity of the SLIM assay
for TB. The development of the automated SLIM system would reduce the problems
that result from uncontrollable contaminants by minimizing the external exposure of
the sample. Because the microfluidic chip of the SLIM assay is small and requires only
an input system that can inject samples, buffers, and air, it can be easily combined
with an automated detection system, such as Xpert, which does not have a pathogen
concentration system, and it would be able to compensate for the shortcomings of
low sensitivity.
Second, a comparison with the Xpert MTB/RIF Ultra assay, which was not available
during the initiation step of the study period, should be carried out. Third, the sam-
pling techniques for oral swabs were not compared because this study was targeted to
examine the clinical utility of using a single sample in a real practice setting. The swab-
bing sites, times, and collection kit can affect the results (28, 29), and further study can
elucidate if these factors affect the results, especially in this assay designed to increase
the sensitivity.
Fourth, the sensitivity of Xpert in this study was relatively low compared with other
studies (30, 31). The inclusion of a small number of smear-positive patients and many
asymptomatic patients (49.3%) might be the reason. This study was performed in a metro-
politan city where medical facilities are easy to access, and regular health checkup services
are quite active. Therefore, this study setting makes it difficult to extrapolate our findings
to countries with a high burden of TB, such as South Africa. The disease prevalence can
affect the positive or negative predictive value (32). The utility of diagnostics should be
considered in various clinical and geographical settings.
In conclusion, the sensitivity of the oral swab-based SLIM assay for the diagnosis of PTB
was comparable to that of conventional sputum-based methods. The superiority of the
SLIM assay in terms of sensitivity was more pronounced in cases of smear-negative PTB, for
which tests with a higher sensitivity are critically needed. Further studies on the application
of automation and the reduction of false-positive results will expand the role of the SLIM
assay in various clinical settings by using both sputum and non-sputum-based samples.

MATERIALS AND METHODS

Participants. Adult patients (.18 years of age) who were clinically suspected of active PTB were
prospectively enrolled in two tertiary university-affiliated hospitals in Seoul, Republic of Korea (Asan
Medical Center and Severance Hospital) from May 2019 to October 2020. The suspicion of PTB was
based on the participants’ symptoms, history, and radiographic findings suggestive of TB (33, 34), and
the enrollment was decided by three respiratory and infection specialists (SHK, YAK, and SWL) who each
had experience in TB treatment for more than 15 years. Patients who could not understand the study
design or the instructions for the sputum exam were excluded.

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Study design and case definition. After receiving informed consent from each patient and before start-
ing the treatment, two trained researchers (YC and YAK) performed oral swabs using the OMNIgene.ORAL
OMR-110 kit (DNA Genotek, Ottawa, Canada) according to the manufacturer’s instructions. In brief, the swabs
were brushed in a back-and-forth motion along the participants’ palate, upper gum line, and tongue dorsum
for about 10 s (5 to 6 times for each site, a total of 20 times), taking care not to reach back into the mouth. The
swab was then inserted into a tube containing stabilizing liquid and the samples were immediately sent to the
laboratory and kept at 280°C until analysis. All other steps were performed according to the routine practice
of clinically suspected TB for these enrolled participants. Acid-fast bacilli (AFB) smears and mycobacterial cul-
tures (both liquid and solid culture) were examined at least two times and the Xpert assay was performed
according to the routine practice. AFB smear and cultures were examined by fluorochrome staining using au-
ramine-rhodamine and culturing in a 3% Ogawa medium and mycobacteria growth-indicator tube medium
(MGIT; Becton Dickson, NJ, USA) (34). If the patients could not produce a sufficient sputum sample, sputum
induction with 3% normal saline nebulization was performed. The sputum samples were collected after taking
the oral swab samples at the same visit from each patient.
TB cases were defined as those treated with anti-TB chemotherapy for at least 6 months according to the
American Thoracic Society (ATS), Infectious Disease Society of America (IDSA), and Korean guidelines at the
discretion of the respiratory and infectious specialists (SHK, YAK, and SWL) (34, 35) and the fulfillment of full-
term treatment. ‘Confirmed TB’ was defined as culture-positive TB patients with at least one positive culture
result for MTB from their sputum. Confirmed TB patients were considered smear-positive if they had at least
one positive smear result (inclusive of scanty positive smears). ‘Possible TB’ was defined as culture-negative
TB patients with a high clinical likelihood of active TB and good clinical and radiographic responses to anti-TB
treatment during follow-up without any evidence of an alternative diagnosis, but the mycobacterial culture
was not confirmed at the initial and follow-up sputum examinations. The specialists who decided on the TB
treatments were blinded to the results of the SLIM assay. We intended to enroll more than 270 participants
according to the estimation of the difference in sensitivity of 0.15 (36), a variance of 0.35, 95% CI, desired
power of 0.8, and exclusion of 10% in the final analysis. The institutional review boards of Asan Medical
Center (2018-0020) and Severance Hospital (4-2018-0029) approved this study, and the protocol of this study
was registered at clinicaltrials.gov (NCT03423550).
Oral sample analysis. Oral swabs were used for the SLIM assay (SLIM oral swab), and Fig. S1 depicts the
overall workflow of the SLIM assay. The principle and the detailed structure of the SLIM assay have been
described previously (9, 10, 13, 37). Briefly, the SLIM assay is based on a combination of a microfluidic plat-
form with low-cost thin film and homobifunctional imidoesters (HIs) reagents for MTB enrichment and DNA
extraction from the oral swab samples. HIs have two imido ester groups and act as cross-linkers. The imido
ester groups of HI form an amidine bond with an amine group on the surface of the thin film. The remaining
imido ester groups enable enrichment of MTB cells and extraction of MTB DNA based on electrostatic interac-
tions with negatively charged MTB cells and DNA due to the positive charge of HIs and covalent bonding
with the amine group of fragmented DNA due to the imido ester groups of HIs. The detailed processing tech-
niques were described in the online supplement and all experiments were performed by a researcher (BK)
blinded to the final diagnosis.
The extracted DNA was used to detect the IS6110 transposase and catalase-peroxidase (KatG)
gene. The amplified DNA product from conventional PCR was purified, and TB was confirmed using
Sanger sequencing. Diagnosis of TB was carried out according to the schematic flow. All conven-
tional PCR (endpoint PCR) reactions were performed using the extracted DNA as a template from
272 oral swab samples, and all results were reported as “positive” or “negative” to determine TB
(Fig. S3 and S4). Based on these results, the sensitivity and specificity were calculated as described in
the next section.
Statistical analysis. Baseline characteristics were compared by Student's t test for continuous varia-
bles or chi-square tests for categorical variables. To compare the clinical validity between tests,
McNemar’s chi-square test was used. All data are expressed as mean 6 standard deviation unless noted
otherwise. All statistical analyses were performed using IBM SPSS Statistics for Windows, version 21.0
(IBM Corp., Armonk, NY, USA).
Data availability. Individual participant data collected during the study, after deidentification, and
the study protocols and statistical analysis code are available beginning 3 months and ending 2 years
following article publication to researchers who provide a methodological sound proposal, with ap-
proval by an independent review committee. Data are available for analysis to achieve the aims stated
in an approved proposal. Proposals should be directed to [email protected]. To gain access, data
requestors will need to sign a data access or material transfer agreement approved by Asan Medical
Center and Severance Hospital.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
SUPPLEMENTAL FILE 1, PDF file, 0.6 MB.

ACKNOWLEDGMENTS
This work was supported by a grant from the Korea Health Technology R&D Project
through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry
of Health & Welfare, Republic of Korea (KMDF_PR_20200901_0049), grants from the Asan
Institute for Life Sciences (2019-7043) and the Ministry of Science, ICT, Future Planning

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Gene-Based Rapid TB Diagnosis Microbiology Spectrum

(MSIP) through the National Research Foundation of Korea (NRF) (2020R1A2C2007148)

and a grant of the Korea Health Promotion R&D Project, funded by the Ministry of Health
& Welfare, Republic of Korea (HS21C0096). The funding organizations played no role in
the design of the study, choice of enrolled patients, review, and interpretation of the
data, preparation of the manuscript, or final approval of the manuscript.
We thank the patients and their families who were willing to participate in this
study. We also appreciate the staff at Asan Medical Center and Severance Hospital who
were involved in this study.
We declare no conflict of interest.

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