Keywords
Mycobacterium tuberculosis - multiplex PCR - molecular diagnosis of tuberculosis
Introduction
Tuberculosis (TB), caused by members of Mycobacterium tuberculosis complex, remains one of the most important causes of morbidity and mortality worldwide,
with approximately 10 million new cases and 1.4 million deaths every year.[1 ] India bears 26% of this burden, that is, approximately 2.69 million cases every
year.[2 ] The most common presentation is pulmonary TB, accounting for approximately 85% of
all tubercular infections.[1 ]
The effective control and eventual elimination of TB depends on rapid diagnosis and
treatment initiation. Rapid and accurate diagnosis is essential not only for better
individual patient outcome but also from a public health perspective. To facilitate
this, the World Health Organization (WHO) has recommended the use of molecular assays
as initial tests for suspected TB patients and recommended commercial assays for this
purpose.[3 ] These commercial assays require an initial setup of the specific analysis platform
and continuous supply of assay kits. In such conditions, sputum smear microscopy is
often used as the initial test. Although it is highly specific, it may be variably
sensitive (sensitivity ranging from 20–80%).[4 ] Therefore, smear-negative samples need to be examined with another method; otherwise
a substantial number of TB cases may remain undiagnosed and untreated. Culture isolation
of bacilli remains the gold standard for diagnosis of TB because of its highest sensitivity,
but its utility in clinical setting is limited by long turnaround time to positive
result.[4 ]
The modality evaluated in this study is a multiplex polymerase chain reaction (PCR)
assay developed and validated by K. Gopinath and S. Singh.[5 ] It uses specific gene targets to detect and differentiate M. tuberculosis complex and non-tuberculosis mycobacteria (NTM) directly from clinical samples. The
sensitivity of the multiplex PCR in mycobacteriologically confirmed human immunodeficiency
virus (HIV)-positive cases and HIV-negative cases was 97.1 and 97.05%, respectively,
while the specificity was found to be 94.87% in both.[5 ]
This test was chosen as it is capable of simultaneous detection and differentiation
of genus Mycobacteria , M. tuberculosis complex, and M. avium complex directly from a single clinical sample. Moreover, it does not require a specific
platform for the assay, rather it can be performed in any laboratory equipped for
molecular diagnostics. The turnaround time is significantly lower than solid or liquid
culture and excellent performance for the detection of M. tuberculosis complex has been reported by the original authors.[5 ] This may be of particular significance in immunocompromised patients and in patients
with specific risk factors, in whom nontubercular mycobacterial lung disease may be
mistaken for pulmonary TB due to their similar clinical presentation, or it may be
misdiagnosed (various commercial Nucleic Acid Amplification tests [NAAT]), or missed
altogether (GeneXpert MTB/RIF).
In this study, we have assessed the diagnostic performance of multiplex PCR for the
detection of M. tuberculosis complex in presumptive pulmonary TB patients, in the setting of a tertiary level
teaching hospital in central India, in comparison to liquid culture using BACTEC mycobacteria
growth indicator tubes (MGIT) 960 TB system as the gold standard. We have further
assessed the performance of multiplex PCR for Ziehl Neelsen smear-negative samples,
to ascertain the usefulness of this assay in smear-negative samples. Our secondary
objective was to explore the prevalence of non-TB mycobacteria in patients presenting
at this institute of central India.
Materials and Methods
A hospital-based cross sectional study was conducted in the Department of Microbiology,
Pulmonary Medicine and General Medicine at a tertiary care hospital in central India
from November 2019 to July 2021, after due ethical clearance was obtained from Institutional
Ethics Committee. Optimal sample size for the study was calculated to be approximately
100 participants, using OpenEpi software. The formula used was
Sample size n = [DEFF × Np(1 − p)]/ [(d2/Z21 − α/2 × (N − 1) + p × (1 − p)].
During the study period, on 2 days of each week, patients presenting to the outpatient
department (OPD) with presumptive pulmonary TB were screened according to the inclusion
and exclusion criteria by reviewing the OPD records. From among the patients who fulfilled
all these criteria, recruitment was done in consecutive manner prospectively.
All patients provided written informed consent for participation and patients' confidentiality
was maintained throughout the study. Samples were collected from patients who were
adults aged 18 years or older presenting with presumptive pulmonary TB as per the
definition laid down in the National Tuberculosis Elimination Program, or other patients
aged 18 years or older presenting for screening of TB (e.g., contacts of microbiologically
confirmed TB cases, people with HIV, diabetics, malnourished, cancer patients, patients
on immunosuppressant or steroid therapy). Patients who declined to provide written
informed consent or were currently on anti-TB therapy (ATT) or having history of administration
of ATT within the past 6 months (from date of enrolment) were excluded from this study.
Sputum samples were collected in accordance with the procedure recommended by the
National Tuberculosis Elimination Program.[6 ] Bronchoalveolar lavage fluid samples were collected as per the procedure recommended
by the American Thoracic Society Bronchoalveolar Lavage Guidelines[7 ] from patients who were unable to provide expectorated or induced sputum samples.
The recruitment process and study procedures are summarized in [Fig. 1 ].
Fig. 1 Flowchart of patient recruitment and study procedures. ATT, antituberculosis therapy;
PCR, polymerase chain reaction.
Sputum samples were processed using a combination of N-acetyl-L-cysteine, a mucolytic
agent, and sodium hydroxide, a decontaminating agent, under proper biosafety precautions,
following the procedure reported previously.[8 ]
Smears were prepared from processed samples and stained using the Ziehl Neelsen staining
method using 1% filtered carbol fuchsin as primary stain, 25% sulfuric acid as decolorizer,
and 0.1% methylene blue as counterstain.[6 ] The prepared smears were then observed under 1000× magnification with oil immersion
lens.[9 ]
For mycobacterial culture, all samples were inoculated into MGIT for culture isolation
in the BD BACTEC MGIT 960 instrument (Becton Dickinson and Company, Sparks, Maryland,
United States), following the procedures laid out by the manufacturer.[10 ]
For multiplex PCR assay, first, mycobacterial DNA was isolated from processed samples
using the chloroform isoamyl alcohol method.[11 ]
The PCR master mix was prepared and PCR was performed as per previously published
protocols.[5 ] The reaction mixture (25.5 μL) consisted of distilled water (13.7 μL), buffer (2.5
μL), dNTPs (4 μL), hsp forward primer (0.5 μL), hsp reverse primer (0.5 μL), ESAT-6 forward primer (0.5 μL), ESAT-6 reverse primer (0.5
μL), ITS MAC forward primer (0.5 μL), ITS MAC reverse primer (0.5 μL), Taq.Pol (0.3
μL), and template DNA (5 μL). The primer sequences and product lengths are summarized
in [Table 1 ]. One negative template control and two positive controls (previously isolated DNA
of M. tuberculosis H37Rv and M. avium complex strain) were included in each run of PCR. DNA amplification was performed
for 30 cycles after a hot start for 10 minutes at 94°C. Each cycle consisted of denaturation
for 1 minute at 94°C, annealing for 1 minute at 60°C and extension for 1 minute at
72°C. At the end, 10 minutes were allowed for final extension at 72°C.
Table 1
Primers used for multiplex PCR
S. No.
Name
Sequence
Product size
1
ESAT-6 F
GCG GAT CCC ATG ACA GAG CAG CAG TGG A
320bp
2
ESAT-6 R
CCC AAG CTT CCT ATG CGA ACA TCC CAG TGA CG
320bp
3
ITS MAC F
CCC TGA GAC AAC ACT CGG TC
144bp
4
ITS MAC R
ATT ACA CT TTC GAT GAA CGC
144bp
5
hsp-65 F
ACC AAC GAT GGT GTG TCC AT
441bp
6
hsp-65 R
CTT GTC GAA CCG CT ACC CT
441bp
Abbreviation: PCR, polymerase chain reaction.
After completion of PCR cycle, the amplified products were analyzed by electrophoresis
in ethidium bromide (5 µg/mL)-stained 1.8% (w/v) agarose gels and photographed using
gel documentation system. [Fig. 2 ] shows the results of one batch of samples processed in the above manner.
Fig. 2 Visualization of results of multiplex polymerase chain reaction one batch of samples
by agarose gel electrophoresis. (L, Ladder; NTC, negative template control; PC1, positive
control with Mycobacterium tuberculosis H37Rv; PC2, positive control with Mycobacterium avium complex; S1 to S27—clinical specimens; Mycobacterium tuberculosis complex detected in: S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14,
S15, S16, S17, S18, S20, S22, S23, S24, S25, S26, S27; No mycobacteria detected in:
S19, S21).
For statistical analysis, 2 × 2 contingency tables were prepared and analyzed separately
for all samples and for smear-negative samples using GraphPad and MedCalc tools. Sensitivity,
specificity, positive predictive value, and negative predictive value of multiplex
PCR were calculated by taking MGIT culture as the reference standard, and p -value was calculated with Fisher's exact test. Quantification of agreement was done
with Kappa results. Diagnostic test characteristics were determined with 95% confidence
intervals (CIs).
Results
In this study, total 114 samples were collected over a period of 14 months, out of
which 102 (89.47%) were sputum samples and 12 (10.53%) were bronchoalveolar lavage
fluid samples. The demographic and baseline clinical characteristics of the patients
are summarized in [Table 2 ].
Table 2
The demographic and baseline clinical characteristics of the patients
Characteristic
Number of samples (%)
Sex
Female
32 (28.07%)
Male
82 (71.93%)
Age
18–45 years
59 (51.75%)
46–60 years
33 (28.95%)
60 years +
22 (19.30%)
State of residence
Bihar
1 (0.88%)
Madhya Pradesh
109 (95.61%)
Uttar Pradesh
4 (3.51%)
Presenting symptoms
Cough with expectoration
78 (68.42%)
Fever
46 (40.35%)
Loss of appetite
39 (34.21%)
Weight loss
33 (28.95%)
Dry cough
28 (24.56%)
Dyspnea
23 (20.18%)
Other symptoms
21 (18.42%)
Chest pain
14 (12.28%)
Hemoptysis
11 (9.65%)
Altered consciousness
2 (1.75%)
HIV status
Negative
61 (53.51%)
Positive
2 (1.75%)
Not tested
51 (44.74%)
Sample obtained
Bronchoalveolar lavage fluid
12 (10.53%)
Sputum
102 (89.47%)
Abbreviation: HIV, human immunodeficiency virus.
Taking all three modalities into account, M. tuberculosis complex was detected in 50 out of 114 specimens, giving a positivity rate of 43.86%
for the entire specimen pool of the study. To elaborate further, out of 12 bronchoalveolar
lavage (BAL) fluid samples, 3 were positive for M. tuberculosis complex and 9 were negative. However, out of 112 sputum samples, 47 were positive
and 55 were negative. The detailed description of results of detection of M. tuberculosis complex from the two types of samples is depicted in [Fig. 3 ]. Thus, the positivity rate for BAL fluid samples was 25%, while for sputum samples
the positivity rate was 46%.
Fig. 3 Number of samples (total, BAL fluid, and sputum) testing positive and negative for
Mycobacterium tuberculosis complex. BAL, bronchoalveolar lavage.
Positivity rate for the detection of M. tuberculosis complex was slightly higher in males (43.90%) as compared with females (43.75%).
Among the three age groups, the positivity rate for the detection of M. tuberculosis complex was highest in the 18 to 45 years age group (50.85%), followed by the 46
to 60 years age group (39.39%) and was lowest in the 60+ years age group (31.82%).
Among the 49 positive samples, the highest number belonged to male patients from the
18 to 45 years age group (19 samples or 38.78%), followed by females from the same
age group and males from the 45 to 60 years age group (10 samples or 20.41% each).
Results of Ziehl Neelsen Staining : Out of the total sputum specimens (n = 102), 32 were positive for acid fast bacilli and 70 were negative. Among the positive
specimens, 6 were graded 3 + , 15 were graded 2 + , 8 were graded 1 + , and 3 were
graded scanty. Out of the total BAL specimens (n = 12), 10 were positive for acid fast bacilli and 2 were negative.
Results of MGIT Liquid Culture : Out of the total sputum specimens (n = 102), 43 were culture positive for M. tuberculosis complex and 59 were negative. Out of the total BAL specimens (n = 12), 3 were culture positive for M. tuberculosis complex and 9 were negative. Overall, 46 specimens were culture positive for M. tuberculosis complex and 68 were negative.
Results of Multiplex PCR : Out of 102 sputum specimens, 43 were positive for M. tuberculosis complex with the multiplex PCR and 59 were negative, while out of 12 BAL specimens,
9 were positive for M. tuberculosis complex with the multiplex PCR and 3 were negative. Overall, 46 specimens were multiplex
PCR positive for M. tuberculosis complex and 68 were negative. Importantly, no non-TB mycobacterial species were detected
in any of the specimens with the multiplex PCR.
A comparative description of the results of all three tests for M. tuberculosis complex for all samples is shown in [Table 3 ]. Overall, there were 19 samples with discordant results among the three tests, while
there were 6 samples showing discordance between multiplex PCR results and MGIT liquid
culture results.
Table 3
Comparison of results of different microbiological tests for M. tuberculosis
ZN Smear Result
mPCR Result
MGIT liquid culture
No. of samples (%)
Neg
Neg
Neg
64 (56.14%)
Neg
Neg
Pos
3 (2.63%)
Neg
Pos (MTB)
Neg
1 (0.88%)
Neg
Pos (MTB)
Pos
12 (10.53%)
Pos
Neg
Neg
1 (0.88%)
Pos
Pos (MTB)
Neg
2 (1.75%)
Pos
Pos (MTB)
Pos
31 (27.19%)
Total
114 (100.00%)
Abbreviations: MGIT, mycobacteria growth indicator tube; mPCR, multiplex polymerase
chain reaction; MTB, Mycobacterium tuberculosis complex; Neg, negative; Pos, positive; ZN, Ziehl Neelsen.
To fulfil the primary objective, the results of multiplex PCR for the detection of
M. tuberculosis complex from all samples (n = 114) were compared with MGIT liquid culture as the reference standard using a 2 × 2
table. The sensitivity was found to be 93.48% (95% CI: 82.10–98.63%), the specificity
was 95.59% (95% CI: 87.64–99.08%), positive predictive value was 93.48% (95% CI: 82.54–97.75%),
and negative predictive value was 95.59% (95% CI: 87.87–98.48%). Accuracy was 94.74%
(95% CI: 88.90–98.04%). Kappa was calculated to be 0.891 (standard error 0.043; 95%
CI: 0.806–0.976), signifying almost perfect agreement. The two-tailed p -value calculated by Fisher's exact test was less than 0.0001; thus, these findings
are statistically significant.
To fulfil the secondary objective, the results of multiplex PCR for the detection
of M. tuberculosis complex from smear-negative samples (n = 80) were compared with MGIT liquid culture as the reference standards using a 2 × 2
table. The sensitivity was found to be 80.00% (95% CI: 51.91–95.67%), the specificity
was 98.46% (95% CI: 91.72–99.96%), positive predictive value was 92.31% (95% CI: 62.80–98.84%),
negative predictive value was 95.52% (95% CI: 88.57–98.33%). Accuracy was 95.00% (95%
CI: 87.69–98.62%). Kappa was calculated to be 0.827 (standard error 0.084; 95% CI:
0.663–0.991), signifying almost perfect agreement. The two-tailed p -value calculated by Fisher's exact test was less than 0.0001; thus, these findings
are statistically significant.
The diagnostic performance of multiplex PCR for all samples and for smear-negative
samples is summarized in [Table 4 ].
Table 4
Diagnostic performance of multiplex PCR for all samples and for smear negative samples
Statistic
Value (95% CI) for all samples
Value (95% CI) for smear negative samples
Sensitivity
93.48% (82.10–98.63%)
80.00% (51.91–95.67%)
Specificity
95.59% (87.64–99.08%)
98.46% (91.72–99.96%)
Positive predictive value
93.48% (82.54–97.75%)
92.31% (62.80–98.84%)
Negative predictive value
95.59% (87.87–98.48%)
95.52% (88.57–98.33%)
Accuracy
94.74% (88.90–98.04%)
95.00% (87.69–98.62%)
Kappa
0.891 (0.806–0.976)
0.827 (0.663–0.991)
p -Value
< 0.0001
< 0.0001
Abbreviations: CI, confidence interval; PCR, polymerase chain reaction
Discussion
In the current era, molecular methods of diagnosis have an undeniable and unequivocal
role in TB diagnosis, due to their rapidity and accuracy. Although the commercially
available platforms recommended by the WHO possess good performance characteristics,
their large-scale implementation has been restricted in low-resource settings.[12 ] In such settings, development of molecular assays provides a more economical and
practically feasible alternative since the assay can be customized as per the local
infrastructure and human resources.
In the current study, a total of 114 samples were collected out of which 102 were
sputum samples and 12 were BAL fluid samples. Low number of BAL fluid samples can
be attributed to the change in hospital policy for bronchoscopy due to the ongoing
COVID-19 pandemic. At the onset of the pandemic, bronchoscopy as a routine procedure
was suspended, and it was only performed for limited indications.
The positivity rates for the detection of M. tuberculosis complex in this study were largely in accordance with the nationally and internationally
reported data, in terms of age and sex distribution. Worldwide, adult men, women,
and children account for 56, 32, and 12% cases, respectively.[1 ] Similarly, in India, adult males, females, and children account for 61.7, 32.65,
and 5.65% cases of TB cases, respectively.[2 ] Majority (109 out of 114, or 95.61%) of the patients are residents of the central
Indian state of Madhya Pradesh. Remaining (< 5%) are from neighboring states. This
distribution indicates that the patients presenting to our hospital are from central
India, and the findings of this study would likely be a true representation of the
local population.
In this study, we have been able to demonstrate good sensitivity and specificity of
multiplex PCR for the detection of M. tuberculosis complex from pulmonary samples, regardless of their smear results. The sensitivity
(93.48% [95% CI: 82.10–98.63%]) and specificity (95.59% [95% CI: 87.64–99.08%]) of
multiplex PCR (with MGIT culture as the reference) from this study were fairly good,
considering the significant reduction in terms of time taken to positive result by
multiplex PCR as compared with MGIT culture. Even for smear-negative samples, we have
been able to demonstrate satisfactory diagnostic performance of multiplex PCR, with
sensitivity of 80.00% (95% CI: 51.91–95.67%), specificity of 98.46% (95% CI: 91.72–99.96%),
positive predictive value of 92.31% (95% CI: 62.80–98.84%), and negative predictive
value of 95.52% (95% CI: 88.57–98.33%). Thus, the assay is a potentially valuable
tool to diagnose smear-negative and paucibacillary samples rapidly and accurately.
These findings are supported by other recently published reports. For example, one
report from Meriki et al[13 ] found the sensitivity and specificity of duplex PCR assay for all samples to be
93.5 and 94%, respectively; and for smear-negative culture positive samples, the sensitivity
was 87.5%. Furthermore, a systematic review and meta-analysis to assess the overall
accuracy of RT-PCR assay for TB diagnosis in different samples for individuals with
active pulmonary and extrapulmonary infection[14 ] reported pooled sensitivity of 0.96 and pooled specificity of 0.92.
There were 19 samples which have discordant results. Most of them (n = 12) were samples that were smear-negative, but tubercle bacilli were detected by
both multiplex PCR and MGIT liquid culture. This is not surprising, given the variable
sensitivity of smear microscopy for the detection of acid-fast bacilli. Next, there
were three samples that were smear-negative, as well as multiplex PCR negative, liquid
culture grew M. tuberculosis complex. It is possible that the bacillary load was too low to be detected by multiplex
PCR, or there were some unrecognized PCR inhibitors in the samples.[13 ] These samples reiterate the role of culture as an important part of TB diagnosis.
In two samples that were smear-positive, and M. tuberculosis complex was detected by multiplex PCR, but the MGIT liquid culture failed to show
any growth; and there was one sample that was smear-positive but M. tuberculosis complex was not detected by either multiplex PCR or liquid culture. The presence
of dead bacilli due to previously treated TB in these samples was ruled out by reviewing
clinical records of these patients. As per previous reports, this discordance may
be attributed to presence of nonculturable M. tuberculosis , which has low viability making them impossible to culture.[15 ]
[16 ]
Furthermore, there was one sample that was smear-negative, multiplex PCR positive,
and liquid culture negative. Again, the presence of dead bacilli due to past treatment
of TB was ruled out by review of clinical records. One of the possible reasons behind
negative culture result may be excessive harshness of the decontamination procedure
used for the concerned specimen. It has also been reported in existing literature
that culture recovery of mycobacteria from salivary sputum specimens reduces after
centrifugation due to the low buoyant density of bacilli, which might also be another
possible reason for negative culture.[17 ]
In this study, no other mycobacterial species apart from M. tuberculosis complex was detected by multiplex PCR or by liquid culture. This was a major limitation
as we were not able to assess the diagnostic performance of the multiplex PCR for
detection and differentiation of non-TB mycobacterial species, or its practical utility
in laboratory diagnosis of non-TB mycobacteria in suspected TB patients and subsequent
impact on treatment. Detection of NTM from a range of clinical samples using multiplex
PCR has been previously reported from several studies in North India. One study[18 ] from North India reported detection of NTM in 24 out of 572 clinical samples, while
another study[19 ] from the same region reported detection of NTM in 13 out of 436 clinical samples.
Other modalities have also been used for the detection of NTM, for example, one study[20 ] using The GenoType Mycobacterium common mycobacteria/additional species assay (Hain Lifescience, Nehren, Germany)
found that out of 1,080 clinical samples, NTM was detected in 60 samples.
One of the plausible reasons for lack of detection of NTM in the present study could
be the inadequate sample size due to low prevalence of NTM in this particular geographical
region. In previous reports, a wide range of NTM prevalence has been reported from
various parts of India,[21 ] but there has been only one study that reported NTM prevalence of 1.3% from sputum
specimens of suspected TB patients in Madhya Pradesh. Most common species identified
were M. abscessus and M. intracellulare .[22 ] Studies with larger and more diverse participant pool, along with wider geographical
coverage, are needed to fully illustrate the prevalence and species distribution of
NTM in central India. Inclusion of elderly females and extrapulmonary specimens may
prove to be especially valuable for this purpose.
Overall, multiplex PCR can be considered equivalent to MGIT culture in terms of clinical
decision making and yield of positivity with added advantage of rapidity. The most
important advantages offered by multiplex PCR over MGIT culture are that of reduced
turnaround time and simultaneous detection of other mycobacterial species. The maximum
benefit of implementation of molecular assays can be achieved in the setting of tertiary
level centers where the setup for molecular work already exists for other diagnostic
or research work, or where such a setup is under development. Such centers usually
cater to a large peripheral population, and there is availability of skilled human
resources. Assays provide a more sustainable and worthwhile alternative to commercial
assays because the reagents and equipment are not specific to one disease or organism,
rather there is unlimited potential for customization of the assays and their application
in diagnosis of other infectious diseases. Furthermore, dependency on commercial kits
and consumables can also be reduced, and the interruption of commercial supplies would
not affect patient care services.
Conclusion
In this study, we were able to demonstrate the good performance characteristics of
the multiplex PCR for the detection of M. tuberculosis complex from clinical samples of patients with presumptive pulmonary TB, with MGIT
liquid culture as the reference standard.
Multiplex PCR shows robust diagnostic performance with significant advantage over
MGIT liquid culture in terms of turnaround time; hence, it can be considered equivalent
to MGIT culture in terms of clinical decision making and yield of positivity, owing
to the good sensitivity and specificity for the detection of M. tuberculosis complex.
