**3. Discussion**

The fundamental and best method of preventing tuberculosis is based on breaking the mycobacterial transmission chains in the population. Early detection of sputum-positive patients requires appropriate organization of medical services and the correct use of the available diagnostic methods. Patients who shed mycobacteria that are resistant to first-line drugs pose an obstacle to the rapid and effective eradication of tuberculosis. The risk of transmission increases with multidrug-resistant tuberculosis, as the period of patients' infectivity is prolonged in these cases. The rates of primary and acquired drug resistance and the rates of MDR strains are a measure of the quality of tuberculosis surveillance in a

given country. It is estimated that 3.3% of new patients and 18.0% of previously treated patients worldwide are individuals with MDR tuberculosis. The largest number of cases of MDR tuberculosis is recorded in Eastern Europe and Central Asia, where they account for over 20% of new patients and over 50% of previously treated patients [1]. In Poland, according to the Central Registry of Tuberculosis, 39 patients with MDR tuberculosis were registered in 2019, accounting for 1.0% of all bacteriologically confirmed tuberculosis cases [3].

Analysis of the prevalence of drug-resistant tuberculosis helps in the detection and monitoring of the spread of MDR and XDR strains and illustrates the effectiveness of tuberculosis surveillance in a given country. Multidrug-resistant tuberculosis is more difficult to cure than drug-susceptible tuberculosis; it requires longer, two-year treatment, and permanent conversion is achieved in fewer than 50% of new patients and in 30% of previously treated patients. In the course of a correct workup, rapid detection of *Mycobacterium tuberculosis* is important. This is currently possible through the use of genetic methods, which are able to identify patients with multidrug-resistant tuberculosis within a few hours of specimen collection. Early generations of the commercial tests used to detect *Mycobacterium tuberculosis* (ProbeTec ET DTB and COBAS TaqMan MTB) were characterized by relatively low sensitivities and directly detected the genetic material of *Mycobacterium tuberculosis* in patient specimens without providing information on drug resistance. The currently available kits, FluoroType® MTBDR VER. 2.0 and Xpert® MTB/RIF Ultra, are characterized by a much higher sensitivity and, at the same time, offer the possibility of rapidly identifying patients with drug-resistant tuberculosis (Table 1) [4,5].

**Table 1.** Comparison of genetic tests for the detection of *Mycobacterium tuberculosis* complex.


Xpert® MTB/RIF are closed, virtually fully automated semi-quantitative assays based on real-time PCR. They offer the possibility of the simultaneous detection of *Mycobacterium tuberculosis* DNA in specimens from patients with suspected tuberculosis, directly or after their processing, and the detection of mutations determining rifampicin resistance in the *rpoB* gene. This assay is able to provide a result for a single sample within just two hours [6]. Limitations of the GeneXpert® MTB/RIF Ultra assay include the lack of the option to identify mono-resistance to isoniazid and the fact that resistance to rifampicin is not always a surrogate for MDR. Augustynowicz-Kope´c et al. demonstrated that, in the Polish population, resistance to rifampicin in the group of newly diagnosed patients is a surrogate marker of MDR tuberculosis in a mere 30% of the cases [7]. The situation is different in the group of previously treated patients, where the predictive precision is approximately 80%, and resistance to rifampicin may be a surrogate marker of MDR [7,8]. A meta-analysis published in 2019 demonstrated that INH mono-resistant tuberculosis was more common in younger patients than drug-resistant tuberculosis (median age: 41 vs. 46 years) and affected more foreigners (37.0% vs. 28.6%) and more previously treated patients (13.6% vs. 9.4%). Cases of INH resistant tuberculosis were more commonly reported in countries with high incidence rates for tuberculosis than those with lower incidence rates (65.9% vs. 60.0%). The prevalence of isoniazid mono-resistance ranges from 1.1% in Slovakia to 66.7% in Iceland [9]. The same analysis revealed that one year after treatment initiation, cases of INH mono-resistant tuberculosis had lower treatment success rates compared to cases with drug-susceptible tuberculosis (67.7% vs. 75.8%), which allowed the authors to hypothesize that INH mono-resistance negatively impacted the treatment outcomes. Therefore, the results of this analysis emphasize the need to identify patients with INH mono-resistant

tuberculosis, especially in view of the fact that the rapid tests in recent years focused more on detecting resistance to rifampin as a surrogate marker of MDR tuberculosis [9,10].

A test that identifies patients with INH or RMP mono-resistance and patients with MDR-TB is FluoroType®MTBDR VER.2.0. The test enables rapid identification of *Mycobacterium tuberculosis* in clinical specimens and identification of the most common mutations associated with rifampicin and isoniazid resistance. The sensitivity and specificity of the FluoroType® system for the detection of rifampicin resistance in *Mycobacterium tuberculosis* strains are comparable to those obtained with the GenoType MTBDRplus and GeneXpert® systems [11,12]. The isoniazid resistance detection sensitivity with this system is 91.7% at a specificity of 100% [11]. In the first analysis of the usefulness of the FluoroType® system in detecting the genetic material of *Mycobacterium tuberculosis* in clinical specimens, the sensitivity and specificity of the FluoroType® system were shown to correlate with smear microscopy results, at 98% for AFB-positive specimens and 92% for AFB-negative specimens. At the same time, FluoroType®MTBDR VER.2.0 was shown to be characterized by high accuracy in detecting mutations in *rpoB*, *katG*, and *inhA*: 98%, 97%, and 97%, respectively. Such high accuracy in identifying mutations determining rifampicin and isoniazid resistance results in 100% sensitivity and specificity in detecting MDR-TB strains with the FluoroType® system. One advantage of the FluoroType® system is that 94 samples can be analyzed simultaneously within three hours and that the results are analyzed automatically, which removes the subjectivity of their interpretation [13].

In the cases presented here, the use of the FluoroType® system enabled the rapid and correct identification of drug-resistant tuberculosis. In the first case, a microbiological workup of the 62-year-old male identified a strain of *Mycobacterium tuberculosis* monoresistant to INH. In the second case of the 41-year-old female, the FluoroType® system enabled the identification of MDR-TB directly from the specimen. In both cases, using only the GeneXpert system in the microbiological diagnostic algorithm for tuberculosis would not have provided the opportunity to correctly identify drug-resistant tuberculosis.
