The International Journal of Mycobacteriology

: 2023  |  Volume : 12  |  Issue : 1  |  Page : 23--27

Identification of Mycobacterium tuberculosis in pulmonary and extrapulmonary specimens of Iranian hospitalized patients during 2017–2021

Mohammadreza Salehi1, Reza Shahsiah2, Kiarash Mohammad Khorasani1, Malihe Hasannezhad1, Arian Rahimi3, Bahare Yousefi4, Amir Aliramezani5,  
1 Department of Infectious Disease, School of Medicine, Imam Khomeini Hospital Complex, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Pathology, School of Medicine, Imam Khomeini Hospital Complex, Tehran University of Medical Sciences, Tehran, Iran
3 Department of Biochemistry and Molecular Biology, School of Life Sciences, USTC, Hefei, China
4 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
5 Department of Microbiology, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Kraków, Poland

Correspondence Address:
Amir Aliramezani
Department of Microbiology, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Krakow 30-387


Background: Tuberculosis (TB) is one of the most serious public health problems worldwide which is a chronic infectious disease and is still one of the major challenges for developing countries. This study was undertaken to identify Mycobacterium TB (MTB) in clinical specimens in hospitalized patients. Methods: The study was carried out on specimens from pulmonary and extrapulmonary suspected TB patients that were admitted to one of the largest tertiary hospitals located in Tehran, Iran from 2017 to 2021. The GeneXpert MTB/rifampin (RIF) method was applied to detect MTB and RIF resistance. Characteristics of demography, clinical features, and lifestyle were obtained from medical case records registered in the hospital. Results: Of 957 specimens, 92 (9.61%) were found positive for TB by GeneXpert assay. Of positive samples, 72 (78.26%) were considered pulmonary TB, and 20 (21.73%) of them are associated with extrapulmonary involvement. Four (4.3%) positive TB cases were categorized as rifampicin-resistant. Conclusion: This study showed a relatively high incidence rate of TB in distinct types of specimens in Iranian hospitalized patients but a low level of RIF resistance.

How to cite this article:
Salehi M, Shahsiah R, Khorasani KM, Hasannezhad M, Rahimi A, Yousefi B, Aliramezani A. Identification of Mycobacterium tuberculosis in pulmonary and extrapulmonary specimens of Iranian hospitalized patients during 2017–2021.Int J Mycobacteriol 2023;12:23-27

How to cite this URL:
Salehi M, Shahsiah R, Khorasani KM, Hasannezhad M, Rahimi A, Yousefi B, Aliramezani A. Identification of Mycobacterium tuberculosis in pulmonary and extrapulmonary specimens of Iranian hospitalized patients during 2017–2021. Int J Mycobacteriol [serial online] 2023 [cited 2023 Apr 1 ];12:23-27
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Tuberculosis (TB) is caused by one of the most important infectious agents, Mycobacterium TB (MTB), and it is among the top 10 leading causes of death globally.[1] The possible route of TB transmission from person to person is through the spreading of MTB into the air by an infected person through aerosol droplets.[2] TB mainly affects the pulmonary system (pulmonary TB [PTB]) and also organs of the body other than the lungs (Extra PTB [EPTB]), such as the brain, spine, skin, and lymph nodes. EPTB comprises about 15% to 20% of all incidences of TB in patients and it can be composed of up to 50% of TB cases in immunocompromised individuals.[3] This type of TB infection remains a diagnostic challenge not only due to the low number of bacteria but also due to invasive procedures often required for sample collection.[4] If there is any delay in the initiation of disease detection and treatment, the chance of transmissibility of MTB will be increased, dramatically.[5]

According to the World Health Organization (WHO) Global report, in 2018, an estimated 10 million (range: 9.0–11.1 million) new active TB disease cases were found and about 1.4 million deaths have occurred.[6] In addition, based on the WHO report in 2015, the incidence rate of TB was 16 cases per 100,000 population in Iran. This disease can affect about 16,000 Iranian people and killed about 2000 individuals each year.[7]

Rifampicin is considered the most effective first-line drug in the treatment of this bacterium.[8] Furthermore, it is believed that 484,000 (417,000–556,000) people developed TB that was resistant to rifampicin, and of these, nearly 78% (187,000) were multidrug-resistant (MDR)-TB cases (which is defined as infection with MTB strain resistant at least to rifampicin and isoniazid) that are much more difficult to treat in comparison with drug-susceptible TB.[9]

At present, one of the major problems in managing TB is the lack of an accurate and rapid diagnostic test for MTB.[10] In Iran, TB diagnosis usually relies on acid-fast bacilli (AFB) staining and the conventional Lowenstein–Jensen (LJ) culture method in conjunction with an assessment of clinical symptoms and radiographic evidence to demonstrate TB.[11] Although AFB staining is a rapid and inexpensive method, it suffered from poor sensitivity. In addition, LJ culture is the gold standard technique for the detection of TB, but it is slow and may take at least 8 weeks.[12] Therefore, some of the cutting-edge technologies such as nucleic acid-based instruments are used for the detection of TB with very high sensitivity and specificity, and they can overcome the problems associated with the classical standard laboratory methods.[13]

Therefore, the present study was designed with the aim to detect the frequency of TB in clinical specimens of patients admitted to the hospital and suspected of having PTB and EPTB.



This cross-sectional study was conducted from August 2017 to July 2021 at Imam Khomeini Hospital Complex (IKHC), Tehran, Iran. This hospital is the largest hospital in the Middle Eastern region, and it is a major tertiary referral hospital, especially in infectious diseases prevention, control, and treatment with more than 1000 active beds that admit patients from other hospitals throughout Iran and also provide primary care for local patients. We went to the electronic hospital registry system and recorded all the patients for whom samples were sent for MTB polymerase chain reaction (PCR).

Medical information of the patients with positive MTB PCR, such as age, sex, occupation, address, and clinical signs and symptoms, were collected from the data recorded in the medical forms of IKHC patients' records archive. No ethical permission for TB data was required for this study.

The following treatment outcome (6–9 months for treatment) definitions are usually used in diagnosed TB patients; Treated: a patient whose sputum smear or culture was negative in the last month of treatment. Recurrence: a patient whose sputum smear or culture is positive after treatment. Expired: A patient who died during treatment.

Classification of pulmonary and extrapulmonary samples

It is well documented that direct extension of the disease from lung tissue leads to other organs' involvement in TB. Consequently, in this study, different samples such as sputum, bronchoalveolar lavage (BAL), pleural fluid, cerebrospinal fluid (CSF), plasma, ascites, abscess, secretions, skin, etc., were collected from suspected cases of TB infection based on clinical criteria (cough of 2 weeks and fever, weight loss of >3 kg or dyspnea, and having radiographic imaging features of TB).

Patients with intrathoracic involvement related to the lung parenchyma and pleural and intrathoracic lymph nodes were considered as PTB. On the other hand, patients with extension of TB to organs or tissues outside the thorax, including those patients who had pulmonary involvement, and also tuberculous intrathoracic lymphadenitis or tuberculous pleural effusion, without radiographic abnormalities in the lungs, constitutes a case of EPTB.

GeneXpert Mycobacterium tuberculosis/rifampin assay

The GeneXpert assay was performed on all specimens and the obtained results were interpreted according to the manufacturer's instructions. Briefly, 2 mL of GeneXpert MTB/rifampin (RIF) sample reagent was added to 1 mL of the specimen using a sterile pipette and the specimen was allowed to liquefy for 15 min and then 2 mL of the mixture was transferred into the GeneXpert cartridge. This cartridge contained the required reagents for nucleic acid amplification and RIF drug resistance detection. Results were displayed by the GeneXpert system automatically within about 2 h.

Statistical analysis

All results were statistically analyzed by SPSS software (SPSS Inc., Chicago, IL, USA) version 26, using t-test analysis. Statistical significance was P < 0.05 level for all analyses. Demographic characteristics, lifestyle factors, and clinical variables were compared between PTB and EPTB groups.


Of the 957 cases of suspected TB, 92 (9.61%) tested positive (males: 62 [67.3%] and females: 30 [32.7%]) for TB by GeneXpert. The overall male-to-female ratio of TB-positive patients was 2 (62/30). For pulmonary cases, the male-to-female ratio was 2.2 (50/22), but for extrapulmonary patients was 1.5 (12/8) which was statistically significant (P < 0.05). The overall mean age of the patients was 46.89 ± 19.49 years and the average hospital stay among our patients was 15.36 ± 13.16 days. The number of samples collected from patients and TB-positive cases was summarized in [Figure 1]. Among TB-positive patients, the yield of isolation of TB was: 6 (6.5%) trace, 20 (21.7%) very low, 24 (26%) low, 23 (25%) medium, and 27 (29.3%) high, respectively. Of those, only 4 (4.3%) were categorized as rifampicin resistant and 88 (95.7%) as sensitive to rifampicin.{Figure 1}

According to different body sites for TB-positive patients, 70 (76%) samples were taken from sputum, 7 (7.6%) from BAL, 4 (4.3%) from pleural fluid, 3 (3.2%) from CSF, 3 (3.2%) from plasma, 2 (2.1%) from the ascites, 1 (1%) from knee secretions, 1 (1%) from lymph node secretions, and 1 (1%) from the skin. Therefore, based on the pulmonary and extrapulmonary criteria mentioned before, 72 (78.26%) samples were considered pulmonary and 20 (21.73%) of them were categorized as an EPTB. Of the 20 cases of EPTB, 8 (40%) had concurrent PTB. The clinical and radiographical features and lifestyle factors associated with the patients who had PTB and EPTB in this study were shown in [Table 1]. The outcome of treatment showed that 82.2% of the patients were completely treated, 9.59% of the patients were confronted with recurrence of TB and 8.22% of them expired.{Table 1}


TB is a serious disease which accounts for a major global public health problem worldwide. MTB has infected nearly one-third of the world's population, and TB remains one of the leading causes of death globally.[14] Although the lung remains the common site of TB infection, there are increasing reports of EPTB, showing the potential spread capacity of MTB to other organs in the body.[15] EPTB occurs in 10% to 42% of adult TB patients depending on race or ethnic background, age, immune status, presence or absence of underlying disease, and genotype of the MTB strain. Sometimes it is accompanied by pulmonary disease and it can affect any organ in the body and has numerous clinical manifestations and thus requires a high index of clinical suspicion.[3]

According to the WHO report, among the Middle Eastern countries, Iran has a high prevalence of MDR-TB with 1.3% in 2015.[16] MDR-TB is defined as TB caused by strains of MTB that are resistant to at least RIF and isoniazid. Mono-resistance to RIF is rare; however, over 90% of RIF-resistant isolates also exhibit resistance to isoniazid. Therefore, the detection of RIF resistance may serve as an alternate indicator for MDR-TB.[17] Although in some studies conducted in Iran resistance to RIF was not reported,[18] we found 4 (4.3%) RIF-resistant TB isolates among the patients. They were isolated from four immunocompromised male patients who were admitted to different wards (intensive care unit [ICU], respiratory, infectious disease, and medical) in our hospital from sputum, BAL, and CSF samples. These results show the increasing frequency of MDR-TB in developing countries such as Iran, and it could a major threat to the life of human beings and requires the use of highly sensitive and specific techniques for early detection of TB. Of the estimated 74,000 cases of MDR-TB in 2015 in Iran, only 60% were diagnosed (due to limited access to rapid and quality diagnosis) and treated.[19] Therefore, rapid diagnosis of TB prevents the development of drug resistance and improves the outcome of illness, whereas delayed diagnosis increases illness and death, and incorrect diagnosis causes unnecessary treatment.

In our study, the CSF was the most common site of EPTB. All CSF-positive samples were collected from patients admitted to ICUs. Two of these patients were discharged after receiving appropriate therapy but one of them passed away due to TB consequences. It is widely believed that central nervous system infection accounts for about 5% of all cases of TB, although the exact incidence and prevalence of TB meningitis in most parts of the world are not known, precisely. This type of infection is the most severe form of EPTB and remains one of the major global health threats with a high mortality rate.[20]

In general, conventional techniques for detecting TB have low sensitivity in distinct clinical specimens. Modern molecular approaches have changed the field of diagnosis of TB and it contains high sensitivity and specificity results.[20] GeneXpert MTB/RIF uses qualitative nested real-time PCR to detect the specific sequence for MTB as well as that for rifampicin-resistance mutation in the rpoB gene and can supplement standard diagnostic tools. This assay is highly rapid, sensitive, and specific in the diagnosis of both PTB and EPTB, and the sensitivity of this test is 95% in smear positive and also 50% to 80% in smear-negative clinical samples, respectively.[21] Although culture is considered the gold standard for the definitive diagnosis of TB, it has low sensitivity for specimens from extrapulmonary sites.[16] This assay was as sensitive for the diagnosis of MDR-TB and simultaneously detected TB and RIF resistance in a short turnaround time of about 2 h. This extremely helpful diagnostic tool should be implemented for screening and management of MDR-TB in TB-endemic countries and can provide accurate results and can allow rapid initiation of MDR-TB treatment.[22]

The most significant clinical features of PTB and EPTB-positive patients in our study were chronic cough (64.1%), fever (56.5%), decreased weight (52.1%), decreased appetite (44.5%), fatigue (31.5%), and night sweats (28.2%) (P < 0.05). Furthermore, pulmonary nodules (35.8%) and cavitation (20.6%) (P < 0.05) are the major radiographical findings associated with TB-positive patients in this study. In addition, EPTB patients have experienced some clinical factors such as night sweats (40%), ascites (20%), enlarged lymph nodes (40%), urinary symptoms (20%), abdominal pain (30%), headache (25%), and neurological symptoms (15%). These findings are in accordance with the study carried out by Loddenkemper et al. revealed that the most frequent symptoms of active disease are fever, anorexia or reduced appetite, weight loss, night sweats, anemia, and persistent cough.[23]

In this study, we analyzed 957 clinical samples using the GeneXpert MTB/RIF test to evaluate different types of specimens of patients who were suspected of having PTB and/or EPTB. Among positive cases, 72 (78.26%) were from pulmonary and 20 (21.73%) were from extrapulmonary samples. The highest number of positive cases was related to sputum in pulmonary and CSF in extrapulmonary samples. Our findings indicate that the GeneXpert MTB/RIF test performs well in detecting EPTB from different samples. In this way, Habous et al. found that the GeneXpert MTB/RIF assay has a high sensitivity (82.6%) and specificity (100%) for the detection of PTB and EPTB in nonrespiratory samples.[24] In addition, a study conducted by Mechal et al. also showed that the GeneXpert MTB/RIF test has a high sensitivity (78.8%) and specificity (90.3%) for the diagnosis of EPTB samples.[25]


The findings of this study illustrated the incidence of PTB and EPTB among patients admitted to the largest hospital complex in Iran. Modern molecular diagnostic techniques such as GeneXpert MTB/RIF are crucial parts of the WHO new TB control strategy. This diagnostic method could improve the management of TB patients through the rapid introduction of anti-TB treatment in a different types of specimens and controlling the disease by early contact tracing.

Ethical clearance

As mentioned in the setting subheading, no ethical permission for TB data was required for this study. Furthermore, the study was approved by the Ethics Committee of Tehran University of Medical Sciences (IR.TUMS.IKHC.REC.1398.141).

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


1Glaziou P, Floyd K, Raviglione MC. Global epidemiology of tuberculosis. Semin Respir Crit Care Med 2018;39:271-85.
2Gupta N, Bhat SN, Reddysetti S, Afees Ahamed MA, Jose D, Sarvepalli AS, et al. Clinical profile, diagnosis, treatment, and outcome of patients with tubercular versus nontubercular causes of spine involvement: A retrospective cohort study from India. Int J Mycobacteriol 2022;11:75-82.
3Ule Belotti NC, Madela NK, Tonelli Nardi SM, Mariano DC, de Souza NG, Oliveira RS, et al. Evaluation of xpert Mycobacterium tuberculosis rifampicin for tuberculosis diagnosis in a reference laboratory. Int J Mycobacteriol 2022;11:435-41.
4Tellier R, Li Y, Cowling BJ, Tang JW. Recognition of aerosol transmission of infectious agents: A commentary. BMC Infect Dis 2019;19:101.
5Peters JS, Andrews JR, Hatherill M, Hermans S, Martinez L, Schurr E, et al. Advances in the understanding of Mycobacterium tuberculosis transmission in HIV-endemic settings. Lancet Infect Dis 2019;19:e65-76.
6Dedefo MG, Sirata MT, Ejeta BM, Wakjira GB, Fekadu G, Labata BG. Treatment outcomes of tuberculosis retreatment case and its determinants in West Ethiopia. Open Respir Med J 2019;13:58-64.
7Tavakoli A. Incidence and prevalence of tuberculosis in Iran and neighboring countries. Zahedan J of Res in Med Sci 2017;19:e9238.
8Colangeli R, Jedrey H, Kim S, Connell R, Ma S, Chippada Venkata UD, et al. Bacterial factors that predict relapse after tuberculosis therapy. N Engl J Med 2018;379:823-33.
9Seung KJ, Keshavjee S, Rich ML. Multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis. Cold Spring Harb Perspect Med 2015;5:a017863.
10Habous M, Elimam M, AlDabal L, Chidambaran B, AlDeesi Z. Pattern of primary tuberculosis drug resistance and associated risk factors at Dubai health authority in Dubai. Int J Mycobacteriol 2020;9:391-6.
11Rodriguez-Takeuchi SY, Renjifo ME, Medina FJ. Extrapulmonary tuberculosis: Pathophysiology and imaging findings. Radiographics 2019;39:2023-37.
12Coronel JE, Del Carpio CC, Dianderas EJ, Florentini EA, Kemper GL, Sheen P, et al. Evaluation of microbiological variants of sputum processing and concentration of Mycobacteria to optimize the microscopic and imaging diagnosis of tuberculosis. Int J Mycobacteriol 2019;8:75-82.
13Chen Y, Qian C, Liu C, Shen H, Wang Z, Ping J, et al. Nucleic acid amplification free biosensors for pathogen detection. Biosens Bioelectron 2020;153:112049.
14Hoffner S. Multidrug-resistant tuberculosis: The problem and some priorities in controlling it. Int J Mycobacteriol 2016;5 Suppl 1:S59.
15Khosravi AD, Alami A, Meghdadi H, Hosseini AA. Identification of Mycobacterium tuberculosis in clinical specimens of patients suspected of having extrapulmonary tuberculosis by application of nested PCR on five different genes. Front Cell Infect Microbiol 2017;7:3.
16Behzadifar M, Martini M, Behzadifar M, Bragazzi NL. Tuberculosis in Iran: A historical overview from al-Tabari, Rhazes, Avicenna and Jorjani to Abolhassan Ziyā-Zarifi. Old and new pioneers in the fight against tuberculosis: challenges, pitfalls and hopes. J Prev Med Hyg 2020;61:E13-5.
17Takawira FT, Mandishora RS, Dhlamini Z, Munemo E, Stray-Pedersen B. Mutations in rpoB and katG genes of multidrug resistant Mycobacterium tuberculosis undetectable using genotyping diagnostic methods. Pan Afr Med J 2017;27:145.
18Bahraminia F, Azimi T, Zangiabadian M, Nasiri MJ, Goudarzi M, Dadashi M, et al. Rifampicin-resistant tuberculosis in Iran: A systematic review and meta-analysis. Iran J Basic Med Sci 2021;24:720-5.
19Tavanaee Sani A, Shakiba A, Salehi M, Bahrami Taghanaki HR, Ayati Fard SF, Ghazvini K. Epidemiological characterization of drug resistance among Mycobacterium tuberculosis isolated from patients in Northeast of Iran during 2012-2013. Biomed Res Int 2015;2015:747085.
20Eshraghi SS, Heidarzadeh S, Soodbakhsh A, Pourmand M, Ghasemi A, GramiShoar M, et al. Pulmonary nocardiosis associated with cerebral abscess successfully treated by co-trimoxazole: A case report. Folia Microbiol (Praha) 2014;59:277-81.
21Chakravorty S, Simmons AM, Rowneki M, Parmar H, Cao Y, Ryan J, et al. The New Xpert MTB/RIF ultra: Improving detection of Mycobacterium tuberculosis and resistance to rifampin in an assay suitable for point-of-care testing. mBio 2017;8:e00812-17.
22Lange C, Dheda K, Chesov D, Mandalakas AM, Udwadia Z, Horsburgh CR Jr. Management of drug-resistant tuberculosis. Lancet 2019;394:953-66.
23Loddenkemper R, Lipman M, Zumla A. Clinical aspects of adult tuberculosis. Cold Spring Harb Perspect Med 2015;6:a017848.
24Habous M, Elimam MA, Kumar R, Deesi ZA. Evaluation of GeneXpert Mycobacterium tuberculosis/rifampin for the detection of Mycobacterium tuberculosis complex and rifampicin resistance in nonrespiratory clinical specimens. Int J Mycobacteriol 2019;8:132-7.
25Mechal Y, Benaissa E, El Mrimar N, Benlahlou Y, Bssaibis F, Zegmout A, et al. Evaluation of GeneXpert MTB/RIF system performances in the diagnosis of extrapulmonary tuberculosis. BMC Infect Dis 2019;19:1069.