International Journal of Infectious Diseases 122 (2022) 820–828

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International Journal of Infectious Diseases

journal homepage: www.elsevier.com/locate/ijid

A new droplet digital PCR assay: improving detection of paucibacillary

smear-negative pulmonary tuberculosis
Zhenzhen Zhao 1,#, Tao Wu 2,#, Minjin Wang 1,#, Xiaojuan Chen 3, Tangyuheng Liu 1,
Yanjun Si 1, Yanhong Zhou 1, Binwu Ying 1,∗
1
Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, China
2
Department of Clinical Laboratory Medicine, People’s Hospital of Ningxia Hui Autonomous Region (First Affiliated Hospital of Northwest Minzu University),
Yinchuan, 750002, China
3
Department of Laboratory Medicine, The People’s Hospital of Leshan, Leshan, 614000, China

a r t i c l e i n f o a b s t r a c t

Article history: Objectives: Smear-negative pulmonary TB (PTB) is difficult to diagnose. Current diagnosis and treatment
Received 21 April 2022 monitoring methods have inherent limitations. Droplet digital PCR (ddPCR) is a new technique with high
Revised 30 June 2022
sensitivity. This study presents a novel ddPCR for rapid and sensitive identification of Mycobacterium tu-
Accepted 16 July 2022
berculosis (MTB).
Methods: MTB DNA was detected in respiratory specimens from suspected PTB cases using ddPCR assay,
Keywords: which was directed at two different locations within IS6110. We, for the first time, evaluated the clinical
Tuberculosis diagnostic ability of this ddPCR for paucibacillary smear-negative PTB.
Paucibacillary Results: A total of 605 PTB suspects were recruited, including 263 patients with confirmed PTB (84.03%
Smear-negative pulmonary tuberculosis
from smear-negative PTB) and 342 without PTB. The sensitivity and specificity of IS6110 ddPCR were
Digital droplet polymerase chain reaction
61.22% (95% confidence interval (CI) 55.00-67.10%) and 95.03% (95% CI 92.20-97.10%) for total PTB and
Xpert MTB/RIF
57.92% (95% CI 51.10-64.50%) and 94.57% (95% CI 91.20-96.90%) for smear-negative PTB. ddPCR assay out-
performed Xpert MTB/RIF (53.08% vs 28.46%, P = 0.020) in smear-negative PTB detection. Furthermore,
effective anti-TB treatment was linked to significantly lower IS6110 copies detected by ddPCR.
Conclusion: Herein, we developed and validated a highly sensitive and robust ddPCR assay for MTB quan-
tification in respiratory specimens, which improves diagnosis and therapeutic effect evaluation of smear-
negative PTB.
© 2022 Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.
This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)

Introduction nonspecific clinical symptoms. For example, mycobacterial cul-

TB caused by Mycobacterium tuberculosis (MTB) is the leading
cause of infectious agent-related deaths. The majority of TB infec-
tions are pulmonary TB (PTB), accounting for roughly 90% of all
active TB cases (Lawn and Zumla, 2011). The World Health Or-
ganization (WHO) reported an estimated 10.0 million new cases
of TB globally in 2019, with a significant proportion being smear-
negative PTB (World Health Organization, 2020); this proportion
can reach up to 85% in China.
Definite diagnosis of smear-negative PTB is currently difficult
because of the low efficacy of available diagnostic methods and
∗
Corresponding author: Binwu Ying, Department of Laboratory Medicine, West
China Hospital, Sichuan University, Chengdu, Sichuan, 610041, China
E-mail address: yingbinwu@scu.edu.cn (B. Ying).
#
These authors contributed equally to this work.
ture is time-consuming; therefore, it cannot guide early diag-
nosis and timely treatment (Heemskerk et al., 2018). Also, the
GeneXpert MTB/RIF (Xpert; Cepheid, Sunnyvale, CA, USA) has a
suboptimal sensitivity in patients with the paucibacillary smear-
negative PTB (Badal-Faesen et al., 2017; Horne et al., 2019). In
2017, WHO evaluated and recommended the Xpert MTB/RIF Ultra
(Xpert Ultra, Cepheid, Sunnyvale, CA, USA), a next-generation test
that would improve the clinical diagnosis of smear-negative PTB
(Wang et al., 2019). This method, however, comes at a high cost
and is not widely available in low- and middle-income settings,
including southwest China. According to the 2019 report of WHO,
the mortality rate for smear-negative PTB can be as high as 20%,
which could be attributed to misdiagnosis or delayed diagnosis
(World Health Organization, 2020). As a result, there is an urgent
need to develop a rapid and highly sensitive diagnostic method for
detecting smear-negative PTB.

https://doi.org/10.1016/j.ijid.2022.07.041
1201-9712/© 2022 Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
Z. Zhao, T. Wu, M. Wang et al.
Multiple droplet digital PCR (ddPCR) assays have now been de-
scribed in several medical fields, including prenatal diagnosis, in-
fectious pathogen detection, DNA methylation determination, gene
expression, and gene mutation analysis (Kelley et al., 2013; Silveira
et al., 2021; Tian et al., 2016; Vidal-Folch et al., 2018; Van Wesen-
beeck et al., 2018; Yu et al., 2020). Recently, some research data
on the applications of ddPCR technology to detect MTB pathogens
have been published (Nyaruaba et al., 2019). ddPCR has a high po-
tential for measuring MTB DNA in plasma of patients with PTB as
well as in cerebrospinal fluid from patients with TB meningitis (Li
et al., 2020; Ushio et al., 2016). As a new diagnostic tool, ddPCR
holds enormous promise for diagnosing TB; however, performance
data remain limited. Furthermore, the ability of ddPCR to detect
smear-negative paucibacillary PTB is yet to be investigated.
In this research, we developed a novel ddPCR assay to identify
MTB DNA rapidly and sensitively in clinics, using respiratory sam-
ples from suspected patients with PTB. Two primer pairs were de-
signed to detect the conserved regions of the insert sequence 6110
(IS6110) of MTB (Cho et al., 2020; Ustinova et al., 2019). Our re-
search, for the first time, evaluated the clinical diagnostic ability of
this novel ddPCR test of respiratory specimens for smear-negative
PTB.
Materials and methods
Study subjects
The ethical committee of West China Hospital, Sichuan Univer-
sity approved the study protocol (reference no. 829, 2019). Patients
suspected of having PTB who agreed to participate in this study
were recruited consecutively and prospectively at West China Hos-
pital, Sichuan University, between August 2020 and December
2020.
Clinical diagnosis of pulmonary TB
The following tests were performed to confirm the final clin-
ical diagnosis: chest computed tomography, laboratory examina-
tions including acid-fast bacilli smear microscopy, mycobacterial
culture, GeneXpert MTB/RIF assay, real-time quantitative PCR de-
tecting IS6110 (IS6110-qPCR), and blood interferon-γ release assay
(TB-IGRA). Respiratory physicians define PTB diagnosis by integrat-
ing the patient’s medical record, radiologic and laboratory findings,
and response to anti-TB chemotherapy (Eurosurveillance editorial
team, 2013). Patients’ clinical information, including age, gender,
clinical diagnosis, and examination results, were then collected ret-
rospectively.
Clinical sample collection and DNA extraction
Respiratory samples, sputum, and bronchoalveolar lavage fluid
(BALF) were consecutively collected from suspected PTB cases.
BALF sediments were collected by centrifuging at 30 0 0 rpm for 15
minutes at room temperature. Sputum and BALF sediments were
treated with 20-50 μl of proteinase K (10 mg/ml). For total DNA
analysis, 1 ml of treated specimens was mixed with 2 ml of lysis
buffer (BioMerieux, France) and allowed to sit at room tempera-
ture for 10 minutes. Total DNA was extracted using the automated
NucliSens EasyMAG nucleic acid extraction platform (BioMerieux,
France), following the manufacturer’s instructions (Loens et al.,
2007). A final elution volume of 35 μl was used to increase MTB-
derived DNA concentration. The DNA samples were analyzed for
IS6110-qPCR (QIAGEN, Hilden, Germany), and the rest were stored
at -80 °C in batches for ddPCR assay.
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International Journal of Infectious Diseases 122 (2022) 820–828
Laboratory study
The respiratory samples from study patients were sent for
smear microscopy, mycobacterial culture, and the Xpert assay, as
per the manufacturer’s instructions. For Xpert assay (Cepheid, Sun-
nyvale, CA, USA), samples were mixed with the sample reagent
of the Xpert kit for 15 minutes at room temperature and then
transferred into the cartridge. Specimens were inoculated in MGIT
tubes for 6 weeks for mycobacterial culture (BACTEC MGIT 960
system; BD, Sparks, MD, USA). Positive cultures were subjected to
Ziehl-Neelsen staining, identification of the MTB complex-specific
protein MPT64, and para-nitrobenzoic acid/thiophene-2-carboxylic
acid hydrazide test, which confirmed the presence of the MTB
pathogen. In addition, peripheral blood samples were taken from
the participants. The examination was performed according to the
manufacturer’s instructions for the TB-IGRA assay (Wantai Biologi-
cal Pharmacy Enterprise, Beijing, China). Regrettably, the Xpert Ul-
tra test was not available at the central laboratory at the West
China Hospital. Therefore, the assay performances of the ddPCR
and Xpert Ultra methods were unable to be compared in the cur-
rent study.
ddPCR assay
Specific primers and probes were designed for MTB ddPCR as-
say, each targeting two different locations in the IS6110 sequence
(designated as MTB-1 and MTB-2 [Table S1]). A synthetic DNA frag-
ment served as an internal control to monitor the ddPCR process
(Table S1). The ddPCR test was performed using the Droplet Digi-
tal PCR System (Pilot Gene Technologies, Hangzhou, China), accord-
ing to the manufacturer’s instructions. A 15 μl reaction mixture
was prepared as follows: 3 μl of 5 × ddPCR Supermix for probes
(Pilot Gene Technologies, Hangzhou, China), 1.0 mM primers, 250
nM probes, 0.5 μl of internal control DNA, and 3.75 μl of the
extracted DNA sample. The 15 μl mixture was placed in a mi-
crofluidic chip and loaded into a DG32 droplet generator (Pilot
Gene Technologies) for droplet generation. The PCR cycling proto-
col was 98°C for 5 minutes, then 40 cycles of 98°C for 15 seconds
and 60°C for 30 seconds. Next, the cycled chip was loaded onto
an iScanner 5 Droplet Reader (Pilot Gene Technologies) to auto-
matically measure each droplet’s fluorescence amplitude. The con-
centrations of target IS6110 were calculated using GenePMS Ver-
sion 1.0.1 (Pilot Gene Technologies) and expressed as copy numbers
per 15 μl of the reaction mixture. Supplementary Figure 1 further
explains the workflow for detecting MTB IS6110 using the devel-
oped ddPCR assay. In each batch of ddPCR experiments, no tem-
plate negative control and MTB DNA positive control were used.
Furthermore, 10% of all samples were randomly chosen and tested
twice.
Limit of blank and limit of detection of ddPCR assay
Three bacterial strains quantitative genomic DNA samples, in-
cluding MTB.H37Rv, MTB.H37Ra, and Mycobacterium bovis bacillus
Calmette-Guérin, were used to evaluate the analytical performance
of the ddPCR assay before analyzing patient samples. The limit of
blank (LOB) was defined as the highest number of IS6110 copies
found in 16 blank samples (Armbruster and Pry, 2008). The limit
of detection (LOD) was determined to be the lowest concentration
of IS6110 copies at which detection is possible (Armbruster and
Pry, 2008). LOD for this ddPCR assay was determined by measuring
four concentrations around LOD. Ten replicates were run for each
concentration, and LOD was defined as the lowest concentration
with a 100% positive result.
Z. Zhao, T. Wu, M. Wang et al. International Journal of Infectious Diseases 122 (2022) 820–828

Table 1
The demographic and clinical information in enrolled participants.

Features PTB Non-PTB Total participants

Patients number 263 342 605

Gender(%)
Male 165(62.74) 192(56.14) 357(59.01)
Female 98(37.26) 150(43.86) 248(40.99)
Age, mean±SD (years) 50.13±18.16 56.00±15.75 53.44±17.07
Referred respiratory samples(%)
Sputum 137(52.09) 185(54.09) 322(53.23)
BALF 126(47.91) 157(45.91) 283(46.77)
IS6110-qPCR(%)
Positive 72(27.38) 2(0.58) 74(12.23)
Negative 191(72.62) 340(99.42) 531(87.77)
Xpert MTB/RIF(%)
Positive 59(38.06) - 59(26.34)
Negative 96(61.94) 69(100) 165(73.66)
Smear microscopy(%)
Positive 23(9.43) - 23(4.42)
Negative 221(90.57) 276(100) 497(95.58)
Mycobacterial culture(%)
Positive 45(33.83) - 45(25.86)
Negative 88(66.17) 41(100) 129(74.14)
Blood TB-IGRA(%)
Positive 127(68.65) 62(26.50) 189(45.11)
Negative 58(31.35) 172(73.50) 230(54.89)

BALF, bronchoalveolar lavage fluid; IGRA, blood interferon-gamma release assay; PTB, pul-
monary TB.
PTB was diagnosed based on the composite reference standard; SD, standard deviation.

Statistical analysis assay was set as three copies per 15 μl reaction (Table S4). In ad-
dition, the LOD of this ddPCR test was also set to three copies per
SPSS software package version 21.0 software (SPSS Inc., Chicago, 15 μl reaction, using the genomic DNA samples of MTB. H37Ra and
IL, USA) and MedCalc version 15.2 (MedCalc Software, Mariakerke, M. bovis bacillus Calmette-Guérin (shown in Table S4). High agree-
Belgium) were used for statistical analysis. Categoric variables were ment of the LOD data obtained with the three strains demonstrates
analyzed using the chi-square test. Continuous data were tested the reproducibility of the ddPCR approach.
using the independent-sample t-test. The Spearman rank correla-
tion test was used for the correlation study of repeated tests. The
Subject characteristics
McNemar paired test was used to determine the significance of the
ddPCR assay compared with other routine TB diagnostic tests. The
A total of 628 patients with suspected PTB were recruited
two-sided P-value of <0.05 denoted statistical significance.
prospectively for this study. A total of 23 patients were excluded
due to a lack of anti-TB treatment outcomes. Eventually, 605 sub-
Results
jects were analyzed. Table 1 shows the demographic and clini-
cal information for enrolled participants. According to a compos-
Analytical evaluation of the ddPCR assay for MTB IS6110
ite reference standard, there were 263 cases of PTB, with the re-
maining 342 cases being non-PTB (depicted in Figure 1). There
Analytical specificity
were 62 cases (62/263, 23.57%) of pulmonary combined with ex-
A silico test using the basic local alignment search tool re-
trapulmonary TB disease among the 263 patients with PTB. Pa-
vealed that all the primers used were extremely specific to dif-
tients with bacterial lung infections made up the majority of the
ferent strains of the MTB complex. We also tested eight different
non-PTB control group, accounting for 50.88% (174/342). Only 23
non-TB mycobacteria species, 16 common respiratory tract bacte-
(23/244, 9.43%) of all PTB cases were smear microscopy positive for
ria, four candida species, host genomic DNA, and plasmid contain-
acid-fast bacilli, 45 (45/133, 33.83%) were positive on mycobacte-
ing the IS1081 sequence. All these tests were negative by ddPCR,
rial culture, and 59 (59/155, 38.06%) were positive on Xpert assay.
demonstrating that the primers were highly specific to MTB IS6110
Furthermore, the PTB group contained 137 (52.09%) sputa and 126
(Table S2).
(47.91%) BALF samples, whereas the non-PTB group contained 185
(54.09%) sputa and 157 (45.91%) BALF samples. There was no nom-
Limit of blank and LOD
inally significant difference between the two groups for specimen
There were no positive droplets for the MTB IS6110 target in
types (P = 0.625). Overall, males made up 59.01% of the study co-
15/16 blank samples (ddH2 O), but one positive droplet per 15 μl
hort, and the average age of all participants was 53 years. Patients
reaction mix was detected in one blank sample (Table S3). In this
with PTB were younger than patients without PTB (P <0.001).
view, the limit of blank was set to 1.5 copies/15 μl reaction. Hence,
in our test, a ddPCR result with fewer than two positive droplets
was considered “negative”. A series of four samples were prepared ddPCR-based IS6110 quantitation in respiratory samples
by diluting the reference strain, MTB.H37Rv; and these four differ-
ent low concentration samples (six, three, two, and one copies per Poisson statistics were used to calculate the quantitation of
15 μl mixture) were used to estimate LOD. MTB IS6110 sequences MTB target sequence IS6110, and a portion of the original mea-
were detected with 100% positivity at the level of three copies per surement results are shown in Supplementary Figure 2. Two ddPCR
15 μl reaction. However, only 50% positivity was obtained at the replicate tests revealed a high correlation (Supplementary Figure 3,
level of two copies per 15 μl reaction. LOD of this novel ddPCR r = 0.777, P <0.001).

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Z. Zhao, T. Wu, M. Wang et al. International Journal of Infectious Diseases 122 (2022) 820–828

Figure 1. A flow diagram of study design and patient enrollment.

Figure 2. Analysis of ddPCR in detecting MTB IS6110 sequence in respiratory specimens. (A) IS6110 copy numbers detected in PTB group and non-PTB group, respectively.
(B) IS6110 copy numbers detected in smear-positive, smear-negative, and non-PTB patient groups, respectively.
ddPCR, digital droplet PCR; PTB, pulmonary TB; MTB, Mycobacterium tuberculosis

IS6110 copy numbers were significantly higher in the PTB
group than in the non-PTB group: median (range), 10.95 (0.00,
168,339.15) vs 0.00 (0.00, 36.60) copies/15 μl of reaction mixture,
P <0.001 (Figure 2A). Moreover, samples from patients with smear-
positive PTB have higher IS6110 copy numbers (median, 33,474.30
copies/15 μl mixture; range, 1.50-151,164.75 copies/15 μl mixture)
than samples from subjects with smear-negative PTB (median, 9.00
copies/15 μl; range, 0-168,339.15 copies/15 μl mixture), P <0.001
(Figure 2B). Furthermore, the number of IS6110 copies was signifi-
cantly reduced in the ddPCR re-examination results of six patients
with PTB who received effective anti-TB treatment (Table 2).
Evaluating the efficiency of ddPCR assay for MTB detection
The receiver operating characteristic (ROC) analysis was used to
evaluate the efficiency of the ddPCR assay in detecting MTB in res-
piratory tract specimens. Table 3 shows the classification perfor-
mance of the IS6110 ddPCR assay for PTB. Compared with the final
clinical diagnosis, the area under the ROC curve with the IS6110-
targeted ddPCR assay was 0.85 (95% confidence interval [CI] 0.82-
0.88; P <0.001) (Figure 3A) among overall PTB cases. The cut-off
point of IS6110 of 6.45 copy numbers per 15 μl of the reaction
mixture was calculated, taking into account the best specificity and

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Figure 3. Diagnostic performance of ddPCR for PTB using respiratory samples. (A) ROC curve of IS6110 ddPCR for the diagnosis of total PTB. (B) ROC curve of IS6110 ddPCR
for the smear-negative PTB diagnosis.
CI, confidence interval; ddPCR, digital droplet PCR; PTB, pulmonary TB; ROC, receiver operating characteristic

Table 2 nosis is difficult. Following further investigation, it was discovered

Detection of IS6110 measured by ddPCR in respiratory samples in PTB cases.
that the performance of the IS6110-ddPCR assay with respiratory
Patient First ddPCR Second Reduction Sample Time samples in patients with smear-negative PTB was also excellent,
number test ddPCR test percentage type interval(days) with an area under the curve of 0.84 (95% CI 0.80-0.87) (Figure 3B).
#1 1962.9 17.25 99.12% Sputum 8 The sensitivity of 57.92% (95% CI 51.10-64.50), the specificity of
#2 17.7 3.3 81.36% BALF 11 94.57% (95% CI 91.20-96.90), and PPV of 89.50% (95% CI 83.30-
#3 13086.3 3526.2 73.05% Sputum 62 94.00) were obtained using the IS6110 value of 6.45 copies/15 μl
#4 1433.1 1124.55 21.53% Sputum 5
reaction mixture (Table 3). Furthermore, The F-score value of 0.72
#5 82.35 2.1 97.45% BALF 5
#6 7.05 4.8 31.91% Sputum 6 was achieved by the ddPCR test on paucibacillary smear-negative
PTB.
BALF, bronchoalveolar lavage fluid; ddPCR, digital droplet PCR.
IS6110 measurement results by ddPCR expressed as copy numbers per 15 μl of the
Comparing different testing methods relevant to TB
reaction mixture.
Six TB patients received treatment with rifampin, isoniazid, pyrazinamide, and
ethambutol (HRZE). This study compared the ddPCR test with other routine TB
detection methods in total PTB cases and the smear-negative
PTB subgroup. The detailed performances of culture, Xpert, IS6110
the highest Youden index (the sum of sensitivity and specificity). qPCR, IGRA as well as IS6110 ddPCR are summarized in Figure 4
The sensitivity, specificity, and positive predictive value (PPV) for and Table S5. The ddPCR versus Xpert in total patients with PTB:
the IS6110 ddPCR (cut-off threshold of 6.45 copies/15 μl reaction the sensitivity, specificity, PPV, negative predictive value (NPV), and
applied) were 61.22% (95% CI 55.00-67.10%), 95.03% (95% CI 92.20- diagnostic accuracy of ddPCR test were 61.60%, 94.74%, 90.00%,
97.10%), and 90.40% (95% CI 85.10-94.30%), respectively (Table 3). 76.24%, and 80.33%, separately; whereas the corresponding values
In addition, the ddPCR assay had an F-score of 0.75 when detect- for Xpert assay were 38.06%, 100%, 100%, 41.82%, and 57.14%, re-
ing PTB cases using the respiratory samples. spectively (Figure 4C and Table S5); the sensitivity of the ddPCR
We were particularly concerned about using the ddPCR assay to assay slightly outperformed the Xpert assay (61.60% vs 38.06%,
detect paucibacillary smear-negative PTB because its effective diag- P = 0.654). Furthermore, compared with the gold standard for

Table 3
Diagnosis performance of IS6110 ddPCR assay for PTB in respiratory samples.

Cut-off value
(copies/15 μl AUC Sensitivity% Specificity% NLR
Methods mixture) F-score (95%CI) (95%CI) (95%CI) PLR (95%CI) (95%CI) PPV% (95%CI) NPV% (95%CI)
a
Total PTB vs 6.45 0.75 0.85 61.22 95.03 12.32 0.41 90.40 76.10
non-PTB (0.82-0.88) (55.00-67.10) (92.20-97.10) (7.70-19.80) (0.30-0.50) (85.10-94.30) (71.80-80.10)
(263 vs 342)
Smear-negative 6.45 0.72 0.84 57.92a 94.57 10.66 0.44 89.50 73.70
PTB vs non-PTB (0.80-0.87) (51.10-64.50) (91.20-96.90) (6.40-17.70) (0.40-0.50) (83.30-94.00) (68.80-78.20)
(221 vs 342)

AUC, Area under curve; CI, confidence interval; ddPCR, digital droplet PCR; NLR, negative likelihood ratio; NPV, negative predictive value; PLR, positive likelihood ratio;
PPV, positive predictive value; PTB, pulmonary TB.
a
Sensitivity comparison of IS6110-ddPCR assay between total PTB and smear-negative PTB groups (P = 0.470).

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Z. Zhao, T. Wu, M. Wang et al. International Journal of Infectious Diseases 122 (2022) 820–828

Figure 4. Comparing the diagnostic performance between ddPCR and routine tests in detecting total PTB cases. (A) ddPCR vs mycobacterial culture: sensitivity analysis of
55.22% vs 33.58% (P = 0.172); (B) ddPCR vs qPCR: sensitivity analysis of 61.60% vs 27.38% (P = 0.033); (C) ddPCR vs Xpert: sensitivity comparison of 58.06% vs 38.06%
(P = 0.654); (D) ddPCR vs IGRA: sensitivity analysis of 59.46% vs 68.65% (P <0.001). The cut-off value of IS6110-ddPCR assay used in respiratory samples is set as 6.45
copies/15 μl reaction mixture. Sensitivity comparisons of IS6110-ddPCR assay with culture, IS6110-qPCR, Xpert and IGRA tests using the McNemar test. P <0.05, ∗ ; P <0.01,
∗∗
; P <0.001, ∗ ∗ ∗ .
ddPCR, digital droplet PCR; IGRA, blood interferon-gamma release assay; NPV, negative predictive value; PTB, pulmonary TB; PPV, positive predictive value; qPCR, IS6110-qPCR.

confirming MTB infection, mycobacterial culture, the sensitivity of
ddPCR was slightly higher (55.22% vs 33.58%, respectively), but the
difference was not significant (P = 0.172). As expected, the ddPCR
assay outperformed the qPCR in detecting MTB infection (sensitiv-
ity comparison: 61.60% vs 27.38%, P = 0.033). Culture and Xpert as-
says showed perfect specificity of 100%, whereas ddPCR and qPCR
also achieved a decent specificity of more than 94% (Figure 4 and
Table S5).
The comparisons of analytical indicators of culture, IS6110
qPCR, Xpert, IGRA with IS6110 ddPCR, in the smear-negative
PTB group are shown in Figure 5 and Table S6. ddPCR versus
Xpert for smear-negative PTB identification: the sensitivity, speci-
ficity, PPV, NPV, and diagnostic accuracy of ddPCR were 53.08%,
97.10%, 97.18%, 52.34%, and 68.34%, respectively; whereas the cor-
responding indicators for Xpert assay were 28.46%, 100%, 100%,
42.59%, and 53.27% (Figure 5C and Table S6); the sensitivity of
ddPCR assay was significantly higher than that of the Xpert as-
say (P = 0.020). Furthermore, the sensitivity of the ddPCR assay
was significantly higher than that of culture (49.09% vs 23.64%,
P = 0.001) and the qPCR test (58.37% vs 19.91%, P <0.001). Fur-
thermore, all four detection methods demonstrated high specificity
of more than 94% in detecting smear-negative PTB (Figure 5 and
Table S6).
Discussion
Accurate and rapid identification of PTB in suspected pa-
tients is critical for timely treatment initiation, improving out-
comes, and preventing PTB transmission and epidemics. Further-
more, smear-negative PTB accounts for at least half of all ac-
tive PTB cases, posing significant transmission risks and mortal-
ity. Physicians continue to struggle with early diagnosis of smear-
negative PTB because of the inherent limitations of currently avail-
able PTB diagnostic methods. The ddPCR approach has recently
found widespread applications in clinical microbiology because of
its high sensitivity and specificity. Researchers used ddPCR to iden-
tify TB, and it proved to be extremely useful in detecting MTB nu-

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Z. Zhao, T. Wu, M. Wang et al. International Journal of Infectious Diseases 122 (2022) 820–828

Figure 5. Performance comparison between ddPCR assay and other tests for smear-negative PTB diagnosis. (A) ddPCR vs mycobacterial culture: sensitivity analysis of 49.09%
vs 23.64% (P = 0.001); (B) ddPCR vs qPCR: sensitivity analysis of 58.37% vs 19.91% (P <0.001); (C) ddPCR vs Xpert: sensitivity comparison of 53.08% vs 28.46% (P = 0.020);
(D) ddPCR vs IGRA: sensitivity comparison of 55.63% vs 68.75% (P = 0.005). The cut-off value of IS6110-ddPCR assay used in respiratory samples is set as 6.45 copies/15 μl
reaction mixture. Sensitivity comparisons of IS6110-ddPCR assay with culture, IS6110-qPCR, Xpert and IGRA tests using the McNemar test. P <0.05, ∗ ; P <0.01, ∗ ∗ ; P <0.001,
∗∗∗
.
ddPCR, digital droplet PCR; IGRA, blood interferon-gamma release assay; NPV, negative predictive value; PTB, pulmonary TB; PPV, positive predictive value; qPCR, IS6110-qPCR.

cleic acid targets with low abundance (Cho et al., 2020; Li et al.,
2020; Ushio et al., 2016). A few studies with limited clinical sam-
ples have focused on the quantification of MTB-derived DNA in
plasma (Ushio et al., 2016), whole blood (Yang et al., 2017), CSF (Li
et al., 2020), and formalin-fixed, paraffin-embedded samples (Cao
et al., 2020). However, no study involving a large number of clin-
ical samples has been reported to confirm the diagnostic value of
ddPCR for PTB, particularly for paucibacillary smear-negative PTB.
The development and analytical evaluation of a novel ddPCR as-
say for MTB target IS6110 in respiratory samples is presented here.
We designed a set of two novel primers for identifying the MTB-
specific sequence IS6110, which has multiple copies in the genomes
of most MTB strains. Similarly, Gaur et al., (2020) reported a stool
IS6110 PCR testing, in which the IS6110 element was amplified with
two independent sets of primers to separately amplify fragments
of 123bp and 343bp, to detect MTB DNA in fecal specimens from
subjects with smear-negative PTB (Gaur et al., 2020). Nevertheless,
the amplification of these two primer pairs underwent comple-
tion under two independent thermal cycling procedures. The steps
toward developing and evaluating a single-dye duplex ddPCR as-
say for detecting two targets (IS6110 and IS1081) are clearly de-
scribed by Nyaruaba et al (2020). Duplexing with the two targets
contributes to eliminating the false-negative results. Their research
provides significant thoughts to further refine the ddPCR assay de-
veloped in our investigation. Further, our findings show that the
IS6110 ddPCR test has a high sensitivity for both PTB and smear-
negative PTB (61.22% and 57.92%, respectively). In the respiratory
sample, our IS6110 ddPCR assay has comparable diagnostic sensi-
tivity to Xpert, the important molecular test for PTB early diagno-
sis (78% for clinically diagnosed PTB [Kawkitinarong et al., 2017]

826
Z. Zhao, T. Wu, M. Wang et al.
and 50% for smear-negative PTB [Badal-Faesen et al., 2017]). Fur-
thermore, our findings show a significant decrease in IS6110 copies
in re-examinations tested by ddPCR after anti-TB therapy. Follow-
ing in the footsteps of Devonshire et al. (2016), our findings indi-
cate that the ddPCR method has the potential to accurately quan-
tify MTB pathogen load and monitor treatment efficacy, poten-
tially leading to better PTB patient management. Our findings sig-
nify that the new ddPCR test potentially offers both diagnostic and
treatment response monitoring advantages in real time to inform
clinical decision-making.
In the current investigation, we compared the performance of
IS6110 ddPCR with routine TB diagnostic assays performed concur-
rently. In terms of sensitivity, ddPCR outperformed mycobacterial
culture (1.64-fold, P = 0.174), IS6110 qPCR (2.25-fold, P = 0.033),
and Xpert assay (1.53-fold, P = 0.654) in all patients with PTB.
IS6110 ddPCR had significantly higher diagnostic sensitivity in
smear-negative paucibacillary PTB cases than mycobacterial cul-
ture (2.07-fold, P = 0.001), IS6110 qPCR (2.93-fold, P <0.001), and
Xpert (1.87-fold, P = 0.020). This study supports the notion that
the newly developed IS6110 ddPCR assay is more sensitive than the
currently available bacteriologic and molecular tests for detecting
MTB in respiratory samples. IS6110 ddPCR had significantly lower
sensitivity than TB-IGRA, the most commonly used immunologic
test (whole PTB, 59.46% vs 68.65%, P <0.001; smear-negative PTB,
55.63% vs 68.75%, P = 0.005). Compared with TB-IGRA, which has
low specificity and frequently elevates in patients without TB, the
IS6110 ddPCR in the respiratory sample outperforms TB-IGRA in
PTB diagnosis.
Globally, TB continues to be a major public health issue, with
over 1.7 million individuals dying from the disease each year. Al-
most 40% of patients with active TB go undiagnosed, in part be-
cause most diagnostic algorithms in use today have poor diagnos-
tic utility (Green et al., 2009). The ddPCR method has been shown
to have several advantages, including high sensitivity, superior pre-
cision, absolute quantification without a calibration curve, and re-
sistance to PCR inhibitors (Salipante and Jerome, 2020). The advan-
tages of ddPCR are particularly evident when measuring low copies
(<200 copies) of nucleic acids (Sanders et al., 2011). The ddPCR
assay developed in this study is a sensitive, specific, and robust
molecular test that outperforms current etiologic tests. It can be
used in clinical practice to detect MTB-specific nucleic acids in res-
piratory samples, allowing the early and accurate detection of pa-
tients with paucibacillary smear-negative PTB. Considering the high
sensitivity of ddPCR, this assay may be capable of detecting pedi-
atric, extrapulmonary, or HIV-associated TB, all of which are now
major causes of morbidity and mortality. However, further testing
is required to confirm this.
There are some limitations to our study that could be addressed
in future research. First, all patients enrolled in this study were
from southwest China. Hence, the utility of using the ddPCR assay
in TB diagnosis in real-life clinical practice requires careful evalu-
ation through multicenter studies with larger sample size. Second,
due to the small number of samples studied, the diagnostic per-
formance comparison analysis between ddPCR and other methods
was not completely parallel. Third, although we have sufficiently
optimized the ddPCR settings and reaction conditions during the
development process, the specificity of this novel ddPCR method
remains nominally inferior to the current gold standard method for
detecting paucibacillary PTB (Table S5 and Table S6). The follow-
ing approaches could be considered to improve the specificity as-
pect: (i) some methodologic improvements could further increase
the analytical specificity of the ddPCR assay for the MTB deter-
mination, involving varying amplification cycle numbers, adjusting
primers annealing temperatures, optimizing final concentrations of
the primers and probes, and so on (Strati et al., 2021; Zaytseva
et al., 2022); (ii) adjusting the fluorescence threshold with software
827
International Journal of Infectious Diseases 122 (2022) 820–828
adjusting and bioinformatic pipelines can significantly reduce the
production of false-positive partitions in no template control sam-
ples, such a strategy deserves consideration (Kojabad et al., 2021);
Besides these, introducing other highly conserved MTB genome re-
gions as targets (e.g., IS1081, gyrB) to our ddPCR assay, where only
the MTB-specific IS6110 sequence was chosen as a target, whereas
positive MTB identification is judged when two or more targets are
detected.
In conclusion, the presently developed and validated ddPCR as-
say is highly sensitive and robust for quantifying MTB DNA in
respiratory specimens, offering improved performance in smear-
negative PTB diagnosis. This ddPCR assay may aid in the evaluation
of disease therapeutic effect. The clinical utility of this ddPCR as-
say for PTB diagnosis should be evaluated in a larger multicenter
patient cohort in the future.
Author contributions
All authors contributed to writing or revising the manuscript,
provided intellectual input, and gave final approval. Concept and
design: B.Y.; acquisition, analysis, or interpretation of data: Z.Z.,
T.W., M.W., X.C., T.L., Y.S., and Y.Z.; drafting of the manuscript: Z.Z.,
T.W., M.W., and B.Y.; critical revision of the manuscript for impor-
tant intellectual content: B.Y. and Y.Z.; statistical analysis: Z.Z., T.W.,
M.W., and B.Y.
Declaration of Competing Interest
The authors have no competing interests to declare.
Funding
This study was supported by grants of the Science and Tech-
nology Project of Tibet Autonomous Region (XZ201901-GB-08), Na-
tional Natural Science Foundation of China (82060389), “1·3·5”
project for disciplines of excellence- Clinical Research Incubation
Project, West China Hospital, Sichuan University (2020HXFH015),
National Natural Science Foundation of China (82102484), and Clin-
ical and Translational Medicine Research Foundation of Chinese
Academy of Medical Sciences (2020-I2M-C&T-B-097).
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ijid.2022.07.041.
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