Resistance Rates of Mycobacterium tuberculosis Complex Strains: A Retrospective Study in Türkiye

TL;DR

This study analyzed drug resistance in tuberculosis strains in Türkiye from 1996 to 2023, finding that nearly 20% were resistant to at least one first-line drug.

Contribution

The study provides updated resistance rates of M. tuberculosis in western Türkiye over a 27-year period.

Findings

19.2% of M. tuberculosis isolates were resistant to at least one first-line drug.

2.9% of isolates were multidrug-resistant, showing resistance to two or more first-line drugs.

Abstract

Background and Objectives: Tuberculosis (TB) is one of the most common infectious diseases in developing countries. The resistance of the causative agent, Mycobacterium tuberculosis, to two or more first-line anti-TB drugs results in multidrug-resistant (MDR) TB, posing a serious challenge to the control of TB worldwide. This study was designed to determine the changes in drug resistance over time in TB strains isolated from patients in all departments of Uludağ University Hospital in western Türkiye. Materials and Methods: We retrospectively analyzed 104,598 clinical samples sent to our laboratory for the investigation of the presence of TB between 1996 and 2023. BACTEC 460 TB, BACTEC MGIT 960 culture systems and Löwenstein–Jensen medium were used for the culture of these samples. The susceptibility of M. tuberculosis complex strains grown in culture to isoniazid (INH) (0.1 μg/mL), rifampicin (RIF) (1.0 μg/mL), ethambutol (ETB) (5.0 μg/mL) and streptomycin (SM) (1.0 μg/mL) antibiotics was studied according to the manufacturer’s recommendation. Results: Out of 104,598 patient samples, 2752 (2.6%) were culture-positive, and the susceptibility test results of 1869 of these were analyzed. Of the isolates, 358 (19.2%) were found to be resistant to at least one first-line drug, i.e., INH, RIF, ETB, or SM. In addition, 2.9% were resistant to two or more first-line drugs. Conclusions: Drug susceptibility testing is essential to ensure the optimal treatment and control of drug-resistant TB strains. This study highlights the value of ongoing efforts to control tuberculosis drug resistance in the fight against this disease.

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1. Introduction

Tuberculosis (TB) is a chronic granulomatous infectious disease caused by Mycobacterium tuberculosis complex. Although the World Health Organization (WHO) has reported that the incidence of the disease has decreased worldwide in recent years, the goals of the WHO TB elimination program have not yet been achieved due to several factors, including the increase in human immunodeficiency virus (HIV) infections, increased migration, and deficiencies in public health system infrastructure [1]. Isoniazid (INH), rifampicin (RIF), ethambutol (ETM), and streptomycin (SM) are used as first-line TB drugs. Declared a threat to global health by the WHO in 1993, TB continues to be a major infectious disease worldwide; mortality rates due to increasing resistance to the antibiotics used in treatment are projected to equal cancer-related mortality rates by 2050 [1,2,3]. The resistance of M. tuberculosis to two or more anti-TB drugs, especially INH and RIF, is referred to as multidrug-resistant (MDR) TB, and these resistant strains pose a serious challenge to controlling TB worldwide [1,2,3,4,5,6,7,8,9,10,11]. Worldwide, MDR-TB and/or RIF-resistant TB were detected in an estimated 3.2% of new cases and 16.0% of previously treated cases in 2023 [3]. In this context, TB programs continue to plan to combat the emergence and consequences of drug resistance to anti-TB drugs. The most important obstacles to eliminating TB are delayed diagnosis and the spread of drug-resistant TB cases. Therefore, the early diagnosis of TB cases and testing drug susceptibility are important for the care of TB patients and the control of this disease [1]. It has been reported that there is a significant difference between the estimated number of MDR-positive TB cases and the number reported to WHO; it is reported that in 2023, an estimated 44% of cases were diagnosed and treated as MDR TB, while 66% of cases were undetected [2,3].

Since TB prevalence and resistant strain rates may vary between regions and over time, epidemiological information on regional anti-TB resistance rates is important in the global TB fight.

This study aimed to retrospectively evaluate the susceptibility of TB strains to first-line anti-TB drugs (INH, RIF, ETM and SM) among TB strains isolated from Uludağ University Hospital in Bursa province, located in western Türkiye, between 1996 and 2023. Although there are many studies on MDR TB strains, our study is very important in terms of examining the changes in resistance over a very long period of 28 years, comparing these data with our country’s data and showing the impact of the implementation of WHO recommendations in the fight against TB.

2. Materials and Methods

2.1. Samples

In this study, the results of 104,598 samples sent to our laboratory with a preliminary diagnosis of TB between 1 January 1996 and 31 December 2023 were evaluated retrospectively. During this 28-year period, patients who applied to various clinics and polyclinics of our hospital, which serves as a tertiary health institution in Bursa and accepts patients from surrounding provinces, and who were suspected of pulmonary or extrapulmonary TB as a result of clinical evaluation and examinations were included in the study. Mycobacteriology laboratory reports from this period were examined retrospectively. Respiratory system samples (sputum, bronchial lavage and bronchoalveolar lavage) and non-respiratory system samples (paracentesis fluid, urine, cerebrospinal fluid, wound discharge, joint fluid and abscess) sent to our mycobacteriology laboratory for the investigation of the presence of TB were evaluated. Direct microscopic examination (smear) and culture tests were applied to all samples; the susceptibilities of M. tuberculosis complex strains grown in culture to first-line TB drugs (INH, RIF, ETM, SM) were examined. Since the susceptibility results of repeated samples from the same patient could affect the resistance rates and the analysis of this subject, they were excluded from the evaluation and 1878 susceptibility results were analyzed. The mycobacteriology laboratory where the study was conducted is included in the External Quality Assessment Program of the Turkish Public Health Agency.

2.2. Culture, Identification and Anti-TB Susceptibility Testing of Mycobacteria

A total of 104,598 clinical samples taken from patients with a preliminary diagnosis of TB were cultured according to Löwenstein–Jensen medium procedures; the BACTEC 460 TB (Becton Dickinson Diagnostic, Franklin Lakes, NJ, USA) culture system was used for samples between 1996 and 2003, and the BACTEC 960 MGIT (Becton Dickinson Diagnostic, USA) culture system was used for samples between 2004 and 2023. For mycobacteria typing, p-Nitro-α-acetylamino-β-hydroxypropiophenone (NAP) was used in the BACTEC 460 TB system, and p-nitrobenzoic acid (pNBA) was used in the BACTEC 960 MGIT system and the BD MGITM TBc Identification Test (Becton Dickinson Diagnostic, USA) kit. This study did not aim to compare the differences in positivity between the LJ and MGIT methods. Positivity in either method was considered sufficient for anti-TB tests and the results were analyzed accordingly. In the tests of susceptibility to major anti-TB drugs, INH, RIF, ETB, SM were applied at concentrations of 1.0, 0.1, 5.0 and 1.0 µg/mL, respectively, in accordance with the operating procedures of the systems.

2.3. Statistical Analysis

In the statistical analysis, four-year time periods were used instead of annual intervals to prevent short-term fluctuations on an annual basis from overshadowing general trends and to ensure that changes in the dataset over time could be analyzed correctly.

Since our aim was to determine a general increase or decrease trend over time, only one trend was focused on, separate comparisons were not made between matched groups, and multiple test correlations were not required.

Descriptive statistics are presented as frequency and percentage. The Chi-square trend test was used to evaluate changes in the culture positivity and MDR rates over time. This test is a suitable method for analyzing possible linear trends in rates. In our study, the statistical significance of trends over time was evaluated with this test, and the odds ratio (OR) values were given. Statistically, the significance level was accepted as α = 0.05. Statistical analyses were performed with IBM SPSS 29.0.2.0 (IBM Corp. Released 2023. IBM SPSS Statistics for Windows, Version 29.0.2.0 Armonk, NY, USA: IBM Corp.).

3. Results

The anatomical distribution and positivity rates of a total of 104,598 samples analyzed for the presence of TB between 1996 and 2023, separated according to whether they were pulmonary (respiratory tract samples) (52.1%) or extrapulmonary (47.9%), are presented in Table 1. When the distribution of a total of 2752 samples in which M. tuberculosis positivity was detected during the study was examined, it was determined that 1858 (68.2%) samples were of pulmonary origin and 874 (31.8%) samples were of extrapulmonary origin.

In this study, it was found that the culture positivity rates, which were 2.6% in all samples, varied between 1.5% and 4.8% in the 1996–2023 period and showed a statistically significant decreasing trend over the years (p < 0.001). In our study, it can be seen that the culture positivity rate is low (2.6%) due to the evaluation of a large-scale sample sent with clinical suspicion instead of a general screening of the population.

When the data were grouped into 4-year periods, a statistically significant decreasing trend was observed in the positivity rates of both pulmonary and extrapulmonary samples during the study period (pulmonary: p < 0.001; extrapulmonary: p < 0.001). However, a significant increase in the positivity of extrapulmonary samples was observed during the 2004–2007 period (Table 2, Figure 1). The distribution of the anti-TB drug susceptibility of the 1869 M. tuberculosis strains included in the study by years is shown in Table 3. In general, 358 (19.2%) of the isolates were found to be resistant to at least one first-line drug, and 2.9% (54 strains) were found to be resistant to two or more first-line drugs.

When the proportion of strains resistant to at least one drug was examined by year, a decreasing trend was observed between 2000 and 2003 compared to the previous period (1996–2002), increasing in the period 2004–2007 and decreasing again in the period 2008–2011 (p = 0.008) (Table 4). When the data were grouped and examined in 4-year periods, a statistically significant decreasing trend was found in the proportion of MDR strains over time (Table 4, Figure 2).

When the resistance rates for INH, RIF, ETM and SM were calculated by taking into account the cases showing resistance to one or more drugs, the highest resistance rate was observed against INH, at 12.6%; as expected, INH resistance was found to be the highest. This was followed by SM with 9.0%, ETM with 3.9% and RIF with 3.3%. The MDR rate was determined to be 2.9% (54 strains) (Table 5). A total of 15 cases (0.8%) resistant to INH+RIF+ETM+SM drugs were detected. Although this situation does not technically fully meet the definition of XDR-TB, it is important in terms of showing that MDR-TB strains exhibit a broader resistance profile. The data of these cases are presented in detail in Table 5. Since susceptibility tests were not performed for fluoroquinolones and second-line drugs such as amikacin, capreomycin or kanamycin in our study, it could not be definitively determined whether these 15 cases could be classified as XDR-TB.

When the change in resistance to anti-TB drugs was examined over time, no significant trend was observed for RIF, ETM and SM, while a statistically significant decreasing trend was observed in the most common INH resistance (Table 6 and Table 7).

4. Discussion

With the rapid emergence of methods for diagnosing TB and their increasing availability worldwide, the rate of early diagnosis has increased. Successful diagnostic and therapeutic practices, such as the direct observation, follow-up, and treatment of identified patients and others in contact with them, have led to a decrease in the incidence of TB globally, but TB continues to be a major public health problem worldwide [12,13].

The increasing resistance to TB drugs and the development of MDR-TB are among the most important challenges in achieving global TB control. The treatment of patients with MDR-TB is difficult and requires the use of drugs that are less effective, more toxic, and more expensive than those used in first-line treatment.

Although many studies have been conducted on TB incidence and drug resistance in Türkiye, comprehensive studies are needed in terms of both the number of isolates and the duration of the study to obtain data from which reliable conclusions can be drawn about temporal trends. In this study, the susceptibility results of a total of 1878 strains were evaluated by removing repeated patient samples from 2752 M. tuberculosis complex strains that were found to be positive in a total of 104,598 samples analyzed in our university’s mycobacteriology laboratory over a period of 28 years and whose anti-TB susceptibilities were studied.

Within the scope of the “Direct Observation Treatment Strategy” implemented in the public health system in Türkiye since 2006, data on all TB patients nationwide are recorded and patients are followed up individually. In this way, the number and location of cases can be monitored, and the decreasing trend in TB and resistance rates can be followed [14,15,16]. In studies in Türkiye, the M. tuberculosis complex isolation rates vary between 4.9% and 13.8% [17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43]. In our study, the isolation rate in all samples was found to be lower, at 2.6% (1.5–4.8%) (Table 1 and Table 2). It is thought that the culture positivity rate is low due to the evaluation of a large sample sent with clinical suspicion in our study. Since we are a tertiary university hospital, samples of patients from our province and neighboring provinces whose diagnosis has not yet been confirmed but who have received a preliminary diagnosis of TB are studied in our center. This situation causes undiagnosed TB cases to be included in the population, which naturally leads to a lower culture positivity rate. In conclusion, the fact that our study covers a large and heterogeneous population may have resulted in the culture positivity rate differing from the general population average.

Considering the national data in Türkiye, approximately two-thirds of TB cases were pulmonary TB cases throughout all years [43]. This situation is consistent with the fact that 1878 (68.7%) of the 2732 samples in which culture positivity was detected in our study were of pulmonary origin (Table 3 and Table 4).

In our study, it was determined that the positivity rate in pulmonary samples decreased from 16.4% in the 1996–1999 period to 4.7% in the 2020–2023 period (Table 2). This remarkable decrease can be explained by the successful TB control strategies implemented throughout Türkiye and especially in our province, and the effectiveness of directly observed treatment (DGT) applications.

On the other hand, although it is seen that the positivity rates in extrapulmonary samples generally decreased over time, a statistically significant increase is striking in the 2004–2007 period (p < 0.001, Table 2, Figure 1). During this period, the extrapulmonary positivity rate increased to 6.1%. During this period, an increased awareness of extrapulmonary TB in immunocompromised individuals (e.g., HIV-positive patients, organ transplant recipients, long-term corticosteroid users) may have led to the more frequent investigation of these cases and thus a temporary increase in diagnosis rates. In general, these findings further demonstrate the importance of following and examining changes in TB epidemiology over time.

Of the 1869 M. tuberculosis strains whose susceptibility to anti-TB drugs was analyzed, 19.2% were found to be resistant to at least one drug. This rate fluctuated over time, while a significant decreasing trend was observed in MDR (Table 5, Table 6 and Table 7). When similar studies conducted in Türkiye were examined, it was observed that MDR-TB rates decreased over time, similar to our study [43].

The fluctuating trend between the periods in question may be due to changes in many factors over time, such as the rational use of antibiotics affecting the development of antimicrobial resistance and surveillance programs implemented to monitor resistant strains. In addition, the statistically significant decrease in the rate of MDR strains detected on a four-year basis in our study suggests that long-term intervention strategies may be effective in controlling resistance. These findings emphasize the need for sustainable, multifaceted and interdisciplinary approaches to combating antimicrobial resistance.

The rate of susceptibility to first-line drugs varies in different regions of the world, including Türkiye. Studies on these rates conducted in different regions of Türkiye are summarized in Table 8.

When Table 8 is examined, it can be seen that there are significant fluctuations in drug resistance rates in M. tuberculosis strains isolated from different regions of Türkiye according to years and provinces. These fluctuations may be due not only to epidemiological changes but also to different reasons such as diagnostic methods and reporting processes. For example, while INH resistance was 11.9% in İzmir in 1999, this rate decreased to 4.2% in the 2002–2003 period. This decrease may be related to changes in patient populations in the relevant center during that period. Similarly, while the INH resistance reported in Edirne was 9.0% in 2003, it increased to 27.1% in 2004. This increase may be related to the implementation of a more active surveillance program or changes in laboratory studies.

It can be observed that the INH, RIF and MDR rates have changed over time in three studies conducted in different years in İzmir province. In 1999, the MDR rate was 6.6%, decreased to 5.8% in 2001, but increased to 8.2% in the 2002–2003 period. In a study conducted by Karabay et al. [26], in Edirne in 2004, the MDR rate was found to be quite high at 11.6%. Similarly, high MDR rates are also noted in the provinces of Trabzon (14.7%) and Mersin (10.7%). The high rate of drug resistance in these regions may be due to problems in diagnosis and treatment processes or inappropriate treatment regimens. It is also striking that SM resistance is particularly high in some regions (e.g., Edirne 2004: 29.0%) [26].

On the other hand, MDR rates are observed to be below 2% in some provinces such as Balıkesir, Erzurum, Adana and Samsun. This situation may indicate differences in patient populations as well as effective surveillance systems and successful treatment programs. In the case of Balıkesir, INH resistance is 7.9% and RIF resistance is only 1.0%; these rates are below the national average.

In addition, the data from this study conducted in our country show that there has been a general decreasing trend in drug resistance in recent years. For example, INH and RIF resistance are lower in most studies conducted after 2010. This suggests that control programs such as the Directly Observed Treatment Strategy (DOTS) implemented in Türkiye are effective.

However, some limitations should be taken into consideration when evaluating these data. The fact that the data were collected in different years and centers may create differences in standard methods. In addition, the sample size is low in some provinces (e.g., Düzce n = 62, Mardin n = 81), which reduces generalizability.

As a result, TB drug resistance in Türkiye shows a heterogeneous distribution regionally and over time; monitoring these rates with continuous surveillance systems at the national level constitutes an important goal in planning the measures to be taken. When the results of our study and these studies are evaluated together, it can be said that although there are regional differences in the rates of resistance to anti-TB drugs, INH resistance rates are the highest in our country (Table 8) [17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43].

We believe that the results obtained in our study may reflect the TB drug resistance patterns in the Southern Marmara region of Türkiye and may be a valuable guide for epidemiologists nationwide. The large number of samples included in this study (104,598 samples) and the long period (28 years) increase the reliability and generalizability of the findings. In addition, the fact that the study was conducted in a tertiary health center that not only serves the city of Bursa but also accepts patients from surrounding provinces strengthens the representativeness of the data beyond the local level, potentially at regional and national scales.

Defining drug resistance patterns and continuously monitoring drug-resistant TB are critical steps in reducing resistance rates. Therefore, long-term local surveillance studies such as this are extremely important. Data obtained from such studies may contribute not only to regional health policy planning but also to national and even international TB epidemiology [9,43]. In this context, we believe that the findings of our study may provide valuable insights for TB drug resistance surveillance in Türkiye and help shape future control strategies.

There are some limitations of this study that should be noted here. The retrospective nature of this study is a limitation as it prevents the classification of resistant cases as previously treated and relapsed. Conducting multicenter studies by adding information about the clinical status and treatment of patients in similar studies will make significant contributions to the fight against TB.

5. Conclusions

The resistance rates of M. tuberculosis complex strains to primary anti-TB drugs are consistent with the average resistance rates reported by the Tuberculosis Control Department of the Turkish Ministry of Health. This study is important in terms of reflecting the M. tuberculosis complex drug resistance patterns in our region and also showing that successful diagnostic and therapeutic practices such as the direct observation, follow-up and treatment of detected patients and other people who come into contact with them lead to a decrease in TB cases and resistance rates in our country and worldwide. Although our study was conducted as a single-center study, our center is a regional hospital that accepts patients not only from the province where it is located but also from many neighboring provinces. Therefore, the samples evaluated in this study have the potential to reflect not only local but also regional resistance patterns. However, we believe that the findings should be supported by multicenter studies in order to be generalizable at a broader level.

In addition, it should be noted that changes in diagnostic methods may affect the observed resistance and positivity rates. In our study, the results of molecular diagnostic and resistance analysis methods, which have been used in our institution in recent years, were not evaluated. When compared with culture, it is possible that these methods may change the number of cases and therefore affect the positivity and resistance rates. Therefore, we believe that these new methods should be included in future studies and that the effects of possible changes in the diagnostic methods used should be taken into account.