ORIGINAL ARTICLE

Rifampin resistance among individuals with extrapulmonary tuberculosis:

4 years of experience from a reference laboratory

S. Baghbanbashi1, S. Mohammad J. Mousavi1, H. Dabiri1, M. Hakemi-Vala1, H. Goudarzi1, G. Hamzehloo2, S. Amini2 and

M. J. Nasiri1
1) Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences and 2) Regional Tuberculosis Reference Laboratory, Tehran
University of Medical Sciences, Tehran, Iran

Abstract

Information is limited about the drug resistance patterns in extrapulmonary tuberculosis (EPTB) in Iran. This study aimed to determine the
prevalence of EPTB and to investigate the drug-resistance pattern in Mycobacterium tuberculosis strains collected from extrapulmonary samples
at the Tehran regional TB reference laboratory. Extrapulmonary specimens from individuals with suspected TB referred to the TB reference
laboratories in five cities of Iran were collected. Both standard conventional methods (culture and direct smear microscopy) and Xpert MTB/
RIF assay were used for the identification of mycobacteria. Drug susceptibility testing was done using Xpert MTB/RIF. The proportion method
on Lowenstein–Jensen medium was performed for confirmation. Between 2016 and 2020, a total of 12 050 clinical specimens from
individuals with suspected TB were collected, of which 10 380 (86%) were pulmonary specimens and 1670 (14%) were extrapulmonary.
Of the extrapulmonary specimens, 85 (5.0%) were positive for M. tuberculosis, and the remaining 1585 (95.0%) samples were negative by
standard methods. Of 85 M. tuberculosis isolates, drug susceptibility testing was performed for 32 isolates, of which 1 (3.1%, 95% CI
0.0%–9.4%) was rifampin resistant and 31 (96.9%, 95% CI 90.1%–100%) were pan-susceptible. The rifampin-resistant isolate was also
resistant to isoniazid, so was assigned as a multidrug-resistant TB. Our study indicated the frequency of drug-resistance among EPTB in
Iran. Establishing rapid diagnostic methods for detection of drug-resistance in EPTB, performing drug susceptibility testing for all EPTB
cases to provide effective treatment, and continuous monitoring of drug resistance, are suggested for prevention and control of drug
resistance in EPTB in Iran.
© 2021 The Author(s). Published by Elsevier Ltd.

Keywords: Drug resistance, extrapulmonary tuberculosis, Iran, Mycobacterium tuberculosis, rifampin

Original Submission: 27 October 2020; Revised Submission: 15 January 2021; Accepted: 15 January 2021
Article published online: 20 January 2021

[2,3]. In addition to pulmonary tuberculosis (PTB), this infec-

Corresponding author: M.J. Nasiri, Department of Microbiology,
tious agent can cause extrapulmonary tuberculosis (EPTB) in
School of Medicine, Shahid Beheshti University of Medical Sciences,
Tehran, Iran. other organs and tissues [4,5].
E-mail: [email protected] There are some obstacles in the diagnosis of EPTB that
cause difficulty. These include that a clinical sample requires
invasive procedures, it is hard to access infrequent bacterial
load, and signs and symptoms are non-specific [4,6,7]. For
the diagnosis of EPTB, several conventional and molecular
Introduction
methods, such as smear microscopy, culture identification
and Xpert MTB/RIF, have been widely used [5,8]. Similar to
Mycobacterium tuberculosis is an important public health prob- PTB, a major problem in EPTB is the increasing incidence of
lem because of the high mortality that it causes [1]. The main drug-resistant M. tuberculosis giving high mortality because
strategy to control this problem is the rapid diagnosis of EPTB is mostly found with a compromised immune system
M. tuberculosis infection and identification of high-risk cases [2,9].

New Microbe and New Infect 2021; 40: 100841

© 2021 The Author(s). Published by Elsevier Ltd
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
https://doi.org/10.1016/j.nmni.2021.100841
2 New Microbes and New Infections, Volume 40 Number C, --- 2021 NMNI

To date, the rates of multidrug-resistant TB in pulmonary Drug susceptibility testing

specimens have been reported in several studies in Iran. Drug susceptibility testing of isolates to rifampicin was deter-
However, there is limited analysis of drug-resistance in EPTB. mined using the Xpert MTB/RIF assay [13]. Briefly, Xpert
This study aimed to determine the prevalence of EPTB and to sample reagent was added to 1 mL of specimens in the ratio 1:2,
investigate the drug-resistance pattern in M. tuberculosis strains and then the mixture was transferred to the Xpert test car-
collected from extrapulmonary samples at the Tehran regional tridge. Cartridges were inserted into the Xpert machine, and
TB reference laboratory. the automatically generated results were read after 90 minutes.
The proportion method on the Lowenstein–Jensen medium
was performed for confirmation [14]. Resistance was
Materials and methods
expressed as the percentage of colonies that grew on critical
concentrations of the drug (40 μg/mL for rifampicin, 0.2 μg/mL
Setting, study population and samples for isoniazid). The interpretation was made according to the
This retrospective study was conducted over 4 years (from usual criteria for resistance, i.e. 1% for all drugs. Mycobacterium
April 2016 to October 2020) in the regional reference labo- tuberculosis H37Rv strain (ATCC 27294) was used for quality
ratory of TB in Tehran, Iran. The laboratory quality was su- control testing in drug susceptibility testing.
pervised by the Swedish Institute for Infectious Disease
Control. Clinical specimens from individuals with suspected TB Statistical analysis
from five cities—Tehran, Mashhad, Ahvaz, Shiraz and Isfa- Statistical analysis was carried out using SPSS version 22 (SPSS Inc.,
han—were included in this study. Specimens were either from Armonk, NY, USA). The frequency was reported with 95% CI.
new adult cases or from patients with treatment failure or
relapse. Results
Clinical samples were collected in sterile containers from
each patient for microscopy and culture tests. All specimens
were held at 4°C until processed by standard laboratory pro- Microbiological findings
cedures. The majority of specimens were processed within 24 A total of 12 050 clinical specimens from individuals with sus-
hours at the reference laboratory. One specimen was collected pected TB were collected, of which 10 380 (86%) were pulmo-
from each patient. nary specimens and 1670 (14%) were extrapulmonary—pleura
l fluid 375 (22.4%), biopsy 349 (20.9%), gastric lavage 248 (14.8%),
Identification of mycobacteria osteoarticular 187 (11.2%), abscess 123 (7.4%), urine 115 (6.9%),
Both standard conventional methods (culture and direct smear cerebrospinal fluid 114 (6.8%), ascites 102 (6.1%) and blood 57
microscopy) and Xpert MTB/RIF assay (Cepheid, Sunnyvale, (3.4%). Specimens were either from new cases or from patients
CA, USA) were used for the identification of mycobacteria. with treatment failure or relapse.
For microscopy examination, a smear from samples was Of the extrapulmonary specimens, 85 (5.0%) were positive
stained by the Ziehl–Neelsen method. Known negative and for M. tuberculosis and the remaining 1585 (95.0%) samples
positive slides were prepared with every batch of the were negative by standard methods (Table 1).
specimens. Biopsy was the most common specimen among confirmed
A subsample from each patient was decontaminated by ETPB cases (36.4%) followed by abscess (24.7%), gastric lavage
Petroff’s method and inoculated into two tubes of (10.5%), pleural fluid (9.4%), ascites (7.4%), osteoarticular
Lowenstein–Jensen medium (Merck, Kenilworth, NJ, USA) (4.7%), urine (3.5%), blood (2.3%) and cerebrospinal fluid
[10]. The slope cultures were incubated at 37°C and examined (1.1%), respectively.
for growth once weekly up to 8 weeks. Each isolate was
examined regarding morphology, pigmentation and date of Drug susceptibility testing
growth. Bacterial isolates were identified as M. tuberculosis Of 85 M. tuberculosis isolates, drug susceptibility testing was
complex using standard biochemical tests, including the pro- performed for 32 isolates, of which one (3.1%, 95% CI
duction of niacin, nitrate reduction and catalase [11]. 0.0%–9.4%) was rifampin resistant and 31 (96.9%, 95% CI
A 1-mL unconcentrated specimen was used (without 90.1%–100%) were pan-susceptible. Resistant and eight sus-
centrifuge) for Xpert MTB/RIF assay [12]. ceptible isolates (randomly selected) were confirmed by the
A specimen was considered positive for M. tuberculosis when proportion method. As shown in Table 2, the rifampin-resistant
culture, and/or Ziehl staining, and/or Xpert MTB/RIF assay was isolate was also resistant to isoniazid, and so was assigned as a
positive. multi-drug resistant TB.
© 2021 The Author(s). Published by Elsevier Ltd, NMNI, 40, 100841
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NMNI Baghbanbashi et al. Rifampin resistance in extrapulmonary TB 3

TABLE 1. Mycobacterial identification from extrapulmonary specimens

Total number, No. of smear No. of culture No. of GeneXpert Total MTB
Specimens n (%) positives positives positives positiven (%)

Urine 115 (6.9) 3 2 2 3 (3.5)

Abscess 123 (7.4) 13 9 9 21 (24.7)
Osteoarticular 187 (11.2) 3 1 2 4 (4.7)
Biopsy 349 (20.9) 21 14 12 31 (36.4)
Cerebrospinal fluid 114 (6.8) 0 1 1 1 (1.1)
Gastric lavage 248 (14.8) 7 2 2 9 (10.5)
Blood 57 (3.4) 2 1 0 2 (2.3)
Pleural fluid 375 (22.4) 5 3 5 8 (9.4)
Ascites 102 (6.1) 1 6 3 6 (7.4)
Total 1670 (100) 55 38 32 85 (100)

Abbreviation: MTB, Mycobacterium tuberculosis.

Discussion by Maurya et al, multidrug-resistant TB was observed in 13.4%

of EPTB cases in India [25]. Another study from Ethiopia indi-
cated that of 151 M. tuberculosis isolates from EPTB, 9% of
According to the current data, the prevalence of EPTB among
isolates were multidrug-resistant TB [26].
clinical samples collected at the Tehran regional TB reference
As mentioned before, a major problem in the management
laboratory was 85/1670 (5%). The proportion of EPTB among
of EPTB is the increasing rate of drug-resistant M. tuberculosis,
EPTB clinical samples in other countries varies, being 9.9% in
because most patients are in an immunocompromised condi-
Ethiopia, 19% in Pakistan, 36.7% in Netherlands, 53% in England
tion [2,9]. As a result, using rapid first-line diagnostic methods
and Wales, and 59% in Germany [15–19]. The discrepancy
for drug resistance, such as Xpert MTB/RIF, is important for
between the current study and previous reports might be the
starting sufficient treatment and to reduce the death rate.
result of methodological differences, such as the difference in
However, according to the previous studies, an acceptable ac-
the inclusion criteria.
curacy for EPTB has not been established. Previous in-
In the current study population, biopsy and abscess speci-
vestigators reported that the accuracy of Xpert MTB/RIF in
mens were commonly seen EPTB types, comprising 36.4% and
non-respiratory specimens for the diagnosis of various forms
24.7% of the EPTB samples, respectively. A similar observation
of EPTB varies considerably with specimen type and bacillary
was reported by an earlier study in Iran, in which biopsy
load [27]. For example, Xpert MTB/RIF is highly sensitive in
(26.5%), abscess (20.4%) and pleural fluid (14.2%) were the
lymph node samples, moderately sensitive in meningitis, and
most commonly involved samples for EPTB [12]. In a previous
shows low sensitivity for testing pleural fluid [27]. Therefore,
study conducted in Turkey, lymph nodes (39.4%), and pleura
the sensitivity and specificity of Xpert MTB/RIF for EPTB
(23.6 %) were the most common sites of EPTB involvement
specimens is variable, and Xpert MTB/RIF cannot be recom-
[20]. Another study conducted on 363 culture-proven EPTB
mended to replace standard conventional tests for diagnosis of
cases in the USA showed that lymphatic TB was the most
EPTB [12]. Reflecting the needs of healthcare providers, there
frequent form (45.1%) followed by bone and joint TB (15.6%)
is a requirement for future research.
and pleural TB (14.3%) [21].
There were some limitations to this study. First, the po-
Our study demonstrated that the rate of drug resistance
tential influence of age, sex, previous treatment and human
among EPTB was relatively low. Out of 85 M. tuberculosis iso-
immunodeficiency virus on drug resistance could not be ana-
lates included in this study, one (3.1%) was multidrug resistant.
lysed because of the limited information obtained from the
This proportion is lower than the earlier results reported from
clinical records of the patients. Second, although, clinical sam-
Thailand, India and South Korea [22–24]. In a study conducted
ples were collected from five cities of Iran, it cannot fully
represent the prevalence of drug resistance in EPTB, because
TABLE 2. The drug-resistance pattern of the extrapulmonary the magnitude of drug resistance is not yet reported in several
tuberculosis isolates regions of the country. Finally, the sample size for positive
M. tuberculosis isolates was low and further studies with larger
Type of resistance No. of resistant isolates
sample sizes from more cities are recommended.
Pan-susceptible 31 In conclusion, our study indicated the frequency of drug-
Mono-resistance 0
Multidrug resistance 1 resistance among EPTB in Iran. Our results suggest that
establishing rapid diagnostic methods for detection of drug-

© 2021 The Author(s). Published by Elsevier Ltd, NMNI, 40, 100841

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
4 New Microbes and New Infections, Volume 40 Number C, --- 2021 NMNI

resistance in EPTB, performing DST for all EPTB cases to isolates from patients of five provinces of Iran. Asian Pac J Trop Med
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