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doi.org/10.36721/PJPS.2022.35.6.SP.1719-1724.1
Pak. J. Pharm. Sci., Vol.35, No.6(Special), November 2022, pp.1719-1724
1719
Pattern of rifampicin resistance and gene xpert based molecular typing
of tuberculosis patients in tertiary care hospitals
Sidra Aslam1, Sultan Ali1, Shafia Tehseen Gul2 and Muhammad Imran Arshad1*
1Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan
2Department of Pathology, University of Agriculture, Faisalabad, Pakistan
Abstract: Mycobacterium tuberculosis associated morbidity, mortality and drug resistance is a global health issue. The
Gene Xpert is used for early diagnosis of TB and simultaneous detection of Rifampicin (RIF) resistance. We aimed to
determine situation analysis of clinical TB in tertiary care hospitals of Faisalabad and to find out frequency of TB and
drug resistance pattern by Gene Xpert. A total of 220 samples from suspected patients of TB were included in this study
and 214 samples were detected as positive by Gene Xpert. Samples were classified on the basis of gender, age group
(<30, 30-50 and >50 years), type of sample (sputum and pleural) and number of M. tuberculosis by ct value (cycle
threshold). The results of present study showed high positive frequency of TB in male patients and in 30-50 years of age
groups by Gene Xpert. High number of M. tuberculosis was found in low and medium category in TB patients. Out of
214 positive TB patients, rifampicin resistance was detected in 16 patients. In conclusion, our study identified that Gene
Xpert is an effective approach for diagnosing TB by detection of M. tuberculosis and rifampicin resistance in <2 hours
for rapid diagnosis and management of TB.
Keywords: Pleural tuberculosis, Gene Xpert assay, rifampicin resistance.
INTRODUCTION
Tuberculosis (TB) is a serious public health problem
worldwide that is caused by M. tuberculosis. TB affects
most commonly the tissues of the lungs known as
pulmonary TB but it can also involve any other tissue of
the body which is known as extra-pulmonary tuberculosis
(EPTB) (Liang et al., 2019). The most common
presentation of EPTB is pleural TB. Tuberculous pleurisy
is common cause of pleural effusion in many countries
(Shaw et al., 2019). TB is global health issue particularly
in developing countries. According to WHO, Pakistan
reported 5.8% cases of TB and extra-pulmonary TB
(EPTB) contributes 20% of all TB cases. It ranks as the
tenth leading cause of death among infectious diseases
worldwide (Sinshaw et al., 2019, Ullah et al., 2021).
Pakistan currently ranks fifth among the countries having
highest burden of TB and ranks fourth among the list of
countries having highest burden of drug resistant (DR) TB
(Ullah et al., 2021). Pakistan is one of major contributor
to TB burden in all over the world and almost 61% cases
of TB were determined in the Eastern Mediterranean
WHO region is Pakistan (WHO, 2021). The highest
percentage of TB patients was determined in Baluchistan
(79.4%) followed by Khyber Pakhtunkhwa (68.7%) and
Punjab (42.8%) (Ullah et al., 2021). Almost 10.1 million
cases are reported each year and 1.6 million deaths are
occurred yearly in the whole world (Phillips, 2018).
The estimation of about 10 million people illness is due to
TB and around a total of 1.4 million people die from it
globally in 2019. About 208000 TB patients co-infected
with human immunodeficiency virus (HIV) died
worldwide in 2019 (MacLean et al., 2020;WHO, 2021).
EPTB accounts for about one-fifth of TB cases are
reported. Pleural tuberculosis arises in up to 30% of TB
patients, which is second most common site of extra-
pulmonary involvement. Pleural TB is the most common
source of a lymphocytic pleural effusion in HIV positive
people (Mustafa et al., 2020). Rifampicin (RIF’s)
resistance is used as an indicator for drug resistance. The
strains which are RIF resistant almost 90 % of them also
show resistance to isoniazid (INH) (Atashi et al., 2017).
Drug resistance development is a wearisome problem in
the course of anti-tuberculous therapy. Resistance is of
many types either of multi drug resistance (MDR TB) or
extensively drug resistance (XDR TB). MDR TB is a type
of TB in which resistance is found against two foremost
first line anti-tuberculosis drugs such as INH and RIF
(Masenga et al., 2017). XDR TB is the form of MDR-TB
in which resistance is present to more than two anti-
tuberculosis drugs (Matabane et al., 2015). Patients can
develop MDR-TB either due to exposure to the resistant
strain or selection of resistant strain by inappropriate
treatment (Mulu et al., 2017).
It is very difficult to treat MDR-TB in low income
countries. Treatment modalities of TB are costly and
limited. Almost 3.3% of new TB patients and about 20 %
of earlier treated patients can develop MDR-TB which is
responsible to cause high death rates (Matabane et al.,
2015).
The resistance mechanisms in M. tuberculosis are due to
efflux pump activation or procurement of mutation. The
*Corresponding author: e-mail: drimranarshad@yahoo.com
Pattern of rifampicin resistance and gene xpert based molecular typing of tuberculosis patients in tertiary care hospitals
Pak. J. Pharm. Sci., Vol.35, No.6(Special), November 2022, pp.1719-1724
1720
INH resistance usually rises by mutation in Kat G which
is activator of INH or target of INH (inhA). The chief
resistance mechanism to INH can be either due to
mutation in the activator of drug kat G that leads to
inhibition of activation of INH or if there is a mutation in
the inhA or its promotor region can lead to inhibition of
INH (Vilchèze and Jacobs, 2015). The RIF’s resistance
happens by mutation in 81-bp RIF’s resistance
determining region of rpoB gene (RRDR) which encodes
beta subunit of RNA polymerase of bacteria (Pienaar et
al., 2018).
There is lack of rapid stoppage in the control of TB. There
are many risk factors like age, chronic diseases and
immunosuppressive conditions such as diabetes,
overcrowding, alcohol, drugs and tobacco smoke, which
have played an important role in the increased frequency
of pulmonary and pleural TB (Macías et al., 2019).
The early, quick, and perfect diagnosis and treatment is
necessary for the elimination of TB. In low income
countries, a standard technique like Ziehl-Neelsen (ZN)
staining is inexpensive and prompt for the identification
of acid-fast bacilli. Though, it has poor positive
predicative value (PPV) and variable sensitivity. The gold
standard method to diagnose TB is culturing but it takes
6-8 weeks, which give rise to prominently delay in
diagnosis. Moreover, there is shortage of infrastructure,
expert and experienced staff along with specialized lab
that is required for patient care and outcomes. These
factors can lead to aggravate the problem of EPTB
diagnosis (M. Hefzy et al., 2021).
There are several molecular methods used for the
detection of M. tuberculosis which are recommended by
WHO such as Line probe assays (LPA), Loop-mediated
isothermal amplification (LAMP), Next-generation Xpert
testing, PCR-based test, Truenat MTB, Truenat MTB
Plus, and Truenat MTB-Rif assay or Gene Xpert and
whole genome sequencing (MacLean et al., 2020). We
want to screen our population by GeneXpert. The use of
Gene Xpert has been increased from last several years. It
is an automatic, cartridge based nucleic acid amplification
test for the detection of M. tuberculosis. This test can
detect nucleic acid of M. tuberculosis and RIF’s
resistance in less than 2 hours. The WHO recommended
this assay to diagnose TB and EPTB along with RIF
resistance. It is also used as initial screening test for the
diagnosis of MDR-TB (Theron et al., 2014).
MATERIALS AND METHODS
A cross-sectional study with appropriate sampling of
pleural TB patients was conducted. A total of 220 samples
(n) of TB patients were collected from tertiary care
hospitals, Faisalabad. Among 220 samples, there were
138 samples of male and 82 samples of female. The
samples were divided into three age groups according to
their ages i.e.<30 year, 30-50 years and >50 years. The
Gene Xpert was performed on these samples in DHQ
hospital for the analysis of prevalence of TB in different
age groups. In the inclusion criteria: The age of patients
of both genders were between 15-80 years and also
patients had pleural TB and in exclusion criteria, the
patients of age <15 years and >80 years, those who were
not willing to participate in the study and those who had
already taken anti-tuberculosis therapy were excluded.
Sample collection
Samples of pleural fluid and sputum were collected from
patients presented in tertiary care hospitals, Faisalabad
with clinical symptoms of TB for confirmation of M.
tuberculosis by Gene Xpert technique.
Classification of patients for gene xpert
Classification on basis of age groups
Patients were classified into three groups on the basis of
gender regarding their age groups
Group 1: Included 24 cases (6 males and 18 females) of
TB patients with age <30 years.
Group 2: Included 134 cases (87 males and 47 females) of
TB patients with age ranged from 30-50 years.
Group 3: Included 62 cases (45 males and 17 females) of
TB patients with age >50 years.
Classification on the basis of type of sample
Two types of samples were collected i.e. sputum and
pleural. The 174 sputum samples and 46 pleural samples
were collected.
Protocol of gene xpert for detection of M tuberculosis
The Gene Xpert test is an automatic assay for detection of
DNA of M. tuberculosis complex and resistance related
mutations. It assimilates and systemizes processing of
sample, amplification of nucleic acid and recognition of
target sequence in samples by means of real time PCR.
Sputum sample was added in the bottle containing reagent
of Gene Xpert and then mixing was done in vortex
mixture and incubated at room temperature for 15
minutes. Same procedure was done for pleural sample.
Cartridge of Gene Xpert was labelled and by using
pipette, sample from bottle was picked and poured in the
cartridge. Lid of cartridge was tightly closed. Then
cartridge was loaded in the Gene Xpert system. The result
of Gene Xpert was observed by comparing the detection
of M. tuberculosis with ct range. The result was displayed
in numbers of M. tuberculosis as High (ct: <16), Medium
(ct:16-22), Low (ct: 22-28) or Very Low (ct:
>28)(Elbrolosy et al., 2021).
Ethical approval
The study was approved by the Institutional Biosafety
Committee (IBC) of University of Agriculture, Faisalabad
(935/ ORIC, dated 19/2/2021) and Human research ethic
Sidra Aslam et al
Pak. J. Pharm. Sci., Vol.35, No.6(Special), November 2022, pp.1719-1724
1721
review committee of tertiary care hospitals, Faisalabad
(TUF/2020/161, dated 23/12/2020).
STATISTICAL ANALYSIS
The data of Gene Xpert (M. tuberculosis and RIF
resistance detection) was analyzed by calculating
percentage positivity or frequency of TB patients and
comparison of variables by using Graphpad Prism5
software.
RESULTS
In our study, a total of 220 samples were included, out of
220 samples, 214 samples were determined as positive for
TB by Gene Xpert. We determined the frequency of TB
according to their age, gender, sample type and number of
M. tuberculosis regarding the ct value. We also detected
RIF’s resistance in positive TB patients by Gene Xpert.
The positive results for M. tuberculosis by Gene Xpert are
displayed in table 1.
Gender wise occurrence of TB by gene xpert assay
The frequency of TB in male and female patients was
detected by Gene Xpert. The confirmed positive male
pleural TB patients by Gene Xpert were 133 and positive
female TB patients were 81. The higher frequency
(62.61%) in male TB patients was observed and lower
frequency (37.38%) in female TB patients was
determined by Gene Xpert as shown in the fig. 1.
Fig. 1: Frequency of M. tuberculosis with respect to
Gender by Gene Xpert
Fig. 2: Frequency of TB positive patients with respect to
age groups by Gene Xpert
Occurrence of TB with respect to different age groups
The frequency of TB in different age groups was
observed. The positive TB patients were divided into
three groups regarding their age. There was more number
of positive TB patients (132) in 30-50 years of age group
as compared to other two groups i.e. <30 years (22) and
>50 years (60). When other two groups were compared
with each other, more frequency was found in >50 years
of age group i.e. 60 as compared to <30 years of age, in
which 22 patients were detected as positive. So, the
higher positivity (62.14%) was observed in 30-50 years
followed by >50 years of age (28.03%) and lowest
positivity (9.81%) was detected in <30 years of age group
as presented in fig. 2.
Fig. 3: Detection of positive TB patients in
correspondence to ct value of Gene Xpert
Fig. 4: Frequency of positive TB patients in sputum and
pleural samples by RIF assay
Fig. 5: Detection of rifampicin resistance in positive TB
patients by Gene Xpert
Presence of M. tuberculosis in correspondence to the ct
value in gene xpert
Based upon the ct value of Gene Xpert, the result can be
categorized into very low, low, medium and high number
of M. tuberculosis and the ct value is inversely
proportional to presence of M. tuberculosis. In low
Pattern of rifampicin resistance and gene xpert based molecular typing of tuberculosis patients in tertiary care hospitals
Pak. J. Pharm. Sci., Vol.35, No.6(Special), November 2022, pp.1719-1724
1722
category, the positive patients were 70 and 66 positive
patients were found in medium category in
correspondence of ct value of Gene Xpert. Less number
of positive TB patients was detected in high category i.e.
52 and 26 positive patients were identified in very low
category as shown as fig. 3.
Detection of high number of M. tuberculosis in different
samples by gene xpert
The presence of M. tuberculosis was detected in different
samples such as sputum samples and pleural fluid by
Gene Xpert. The high number of M. tuberculosis was
detected in sputum samples 171 in comparison of pleural
samples 43 as shown in fig. 4.
Detection of rifampicin (RIF) resistance in TB patients
by gene xpert
RIF resistance by Gene Xpert in positive pleural TB
patients was determined. Out of 220 samples, 214
samples were detected as positive for M. tuberculosis by
Gene Xpert. RIF resistance was detected in 16 patients
and 198 patients were negative for RIF resistance as
shown in fig. 5.
DISCUSSION
TB is a public health risk and mortality rate is increasing
day by day particularly in the low-income settings. To
decrease the death rate, it is important to detect M.
tuberculosis early and to start appropriate treatment and
timely diagnosis of TB is considered as a major pillar to
control the disease. The diagnosis of EPTB is a
significantly severe problem and accuracy of recent tests
is inadequate (Silva et al., 2021; WHO, 2017). The timely
diagnosis and proper treatment of TB can enhance cure
rate, decrease rate of transmission, illness and death. The
keystones for the diagnosis of TB are smear microscopy
by acid fast staining and the culturing. Culturing is
considered as gold standard technique but it is time
taking. There is need of suitable infrastructure and expert
staff for culturing (Dunn et al., 2016).
However acid-fast staining is quick and cheap but it has
variable sensitivity and limited specificity. It cannot
differentiate between non-tuberculous Mycobacteria and
M. tuberculosis. In contrast, WHO has recommended
Gene Xpert which is a fully automated and quick method
for the diagnosis of TB (WHO, 2013). The importance of
Gene Xpert in diagnosis of M. tuberculosis is recognized
due to its feasibility and suitability as a reliable, quick and
economic test (Metcalf et al., 2018).
Our data evidenced that in 30 to 50 years of age groups,
more number of positive TB patients was detected by
Gene Xpert than the other two groups. According to
Smiljić et al. (2019) and Zhang et al. (2011), all age
group can be effected with TB but more number of TB
patients were found in 25-44 years of age groups.
Tostmann et al. (2008) described in their study the
common age group of 15 to 34 years for TB and partially
within 25-44 years of age group. Based on Gene Xpert
detection of M. tuberculosis, more number of patients fall
in low and medium category in correspondence to ct
value.
The result of Gene Xpert revealed more frequency of
male TB patients than female patients. While study by
Kabir et al. (2021) also observed more ratio of male TB
patients as compared to female TB patients and these
results also resembles to the studies by Goroh et
al.(2020), Hernández-Garduño et al. (2004) and Linguissi
et al.(2015), in which males were more effected by M.
tuberculosis than females.
In sputum sample, more number of positive TB patients
was detected as compared to pleural sample by
performing Gene Xpert. While study by Mechal et
al.(2019) found that both pulmonary and extra-pulmonary
samples had almost the same sensitivity and specificity by
Gene Xpert.
The study found that there is more RIF resistance in
sputum sample in contrast to pleural samples. According
to Zong et al.(2019), detection of RIF by Gene Xpert
showed same sensitivity between low and high TB
prevalence countries. While study by Rahman et al.
(2017) who found the 5.2% RIF resistance by Gene Xpert
and between treated and untreated TB patients, prevalence
of RIF was high.
CONCLUSION
The Gene Xpert is an innovative and useful technique for
early detection of M. tuberculosis. Gene Xpert is highly
sensitive and specific test for diagnosis of M. tuberculosis
and its resistance.
Table 1: Classification of positive TB patients for Gene Xpert on the basis of gender regarding age groups.
Age/ sample
Positive male (n)
Positive female (n)
Total
<30 years (n=24)
5
17
22
30-50 years (n=134)
85
47
132
>50 years (n=62)
43
17
60
Total
133
81
214
Sidra Aslam et al
Pak. J. Pharm. Sci., Vol.35, No.6(Special), November 2022, pp.1719-1724
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