**1. Introduction**

5

Tuberculosis, despite the considerable effort aimed at controlling its global spread, remains a public health challenge worldwide. One-third of the human population is currently estimated to harbor a *Mycobacterium tuberculosis* infection. According to the World Health Organization (WHO), there were approximately 10 million tuberculosis cases in 2019, 465,000 of which were cases of tuberculosis caused by strains resistant to at least two first-line anti-tuberculosis drugs: rifampicin and isoniazid (MDR-TB) [1]. The most important reasons for the increasingly deteriorating epidemiological situation of tuberculosis worldwide are: poor results of tuberculosis control programs and insufficient implementation thereof, lack of funds for treatment in developing countries, the spread of HIV infection, and drug resistance in *Mycobacterium tuberculosis*, which is considered by WHO experts to be a major driver of tuberculosis in the modern world.

The WHO definition of a tuberculosis case requires microbiological confirmation of the disease, which involves the isolation of the causative agent, namely bacteria belonging to the *Mycobacterium tuberculosis* complex (MTC), determination of the species, and drug susceptibility testing. The monitoring of TB programs is aimed at the rapid identification

**Citation:** Zabost, A.; Filipczak, D.; Kupis, W.; Szturmowicz, M.; Olendrzy ´nski, Ł.; Winiarska, A.; Jagodzi ´nski, J.; Augustynowicz-Kope´c, E. Use of a FluoroType® System for the Rapid Detection of Patients with Multidrug-Resistant Tuberculosis—State of the Art Case Presentations. *Diagnostics* **2022**, *12*, 711. https://doi.org/10.3390/ diagnostics12030711

 Academic Editor: Stefano Gasparini

Received: 2 February 2022 Accepted: 11 March 2022 Published: 15 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

of patients, prompt initiation of appropriate treatment, and monitoring of the treatment progress, thus preventing further transmission of *Mycobacterium tuberculosis*. According to statistical data, one patient with tuberculosis infects about 15 individuals annually.

The achievement of the gold standard in the diagnosis of tuberculosis, which consists of microbiological confirmation in a culture, is a difficult process due to the long generation time for the genus Mycobacterium of about 18 h. Due to the slow growth of mycobacteria on bacteriological culture media, *Mycobacterium tuberculosis* infection is diagnosed after several days in the case of liquid media (Bactec MGIT; Becton Dickinson) or several weeks in the case of solid media (Löwensteina-Jensena (LJ)) [2].

Progress in molecular biology and the development of nucleic acid amplification assays have paved the way for improvements to methods for the direct detection of *Mycobacterium tuberculosis* in specimens from patients. These techniques make it possible to detect and identify the *Mycobacterium tuberculosis* complex at a higher sensitivity and within a shorter period of time compared to conventional methods, which is particularly important in cases that are difficult to diagnose. This mainly applies to the paucibacillary extrapulmonary form of tuberculosis.

In light of the growing problem of drug resistance in *Mycobacterium tuberculosis* across laboratories worldwide, the rapid identification of drug-resistant strains of the *Mycobacterium tuberculosis* complex poses the greatest challenge. Early detection and diagnosis of drug resistance make it possible to initiate an appropriate treatment regimen. The introduction of genetic tests in routine diagnostic procedures enables the quick detection of resistance to rifampicin due to the identification of a mutation in the *rpoB* gene. The implementation of FluoroType® MTBDR VER. 2.0 (Bruker) additionally allows us to define mutations in the *katG* and *inhA* genes that determine resistance to isoniazid. According to WHO and ECDC (European Centre for Disease Prevention and Control) recommendations, the progressive increase in the prevalence of drug-resistant tuberculosis requires accurate and rapid diagnostic tools [2].

The presented case series illustrates the implementation of such tools in the recognition of drug-resistant tuberculosis. Case 1 concerns the prompt molecular diagnosis of *M. tuberculosis* strains monoresistant to isoniazid directly from surgical lung specimens. Case 2 illustrates the possibility of the quick molecular diagnosis of MDR-TB from sputum.

#### **2. Case Presentation**

#### *2.1. Case 1*

A 62-year-old male was admitted to the Department of Thoracic Surgery of National Tuberculosis and Lung Diseases Research Institute due to a focal lesion localized in the left lung. Irregular shape consolidation in the upper zone of the left lung was found in a chest X-ray (Figure 1a). Chest CT (chest computed tomography) revealed several nodules of various shapes and sizes, with small calcifications, localized in the apicoposterior segmen<sup>t</sup> of the left lung. The largest nodule, measuring 28 × 23 mm, had spiculated borders (Figure 1b–d). These findings were ambiguous, requiring differentiation between tuberculosis and neoplasm.

Bronchoscopy was unremarkable. Open surgical biopsy revealed several pulmonary nodules, 10–12 mm in size, localized in segmen<sup>t</sup> two of the left lung. The intraoperative pathological examination documented the presence of an inflammatory lesion with signs of necrosis. No neoplastic cells were found. Subsequently, left segmentectomy1+2+3 was performed, with lymphadenectomy in groups 5, 7, and 11.

A lung fragment was collected to test for tuberculosis and non-tuberculous mycobacterial infections. Smear microscopy revealed acid-fast bacilli. The patient was in contact with a person suffering from tuberculosis. In order to determine the mycobacterial species, genetic testing with the GeneXpert MTB/RIF (Cepheid) system was carried out, which confirmed the presence of a *Mycobacterium tuberculosis* complex susceptible to rifampicin in the specimen tested. The same clinical specimen was used for molecular testing with the FluoroType® system. The test confirmed the presence of the genetic material of *Mycobac-* *terium tuberculosis* and identified, at the same time, the presence of the S315T1 mutation in the *katG* gene, which confers the resistance to isoniazid (INH). Neither the FluoroType® system nor the GeneXpert® system identified any mutations responsible for resistance to rifampicin.

**Figure 1.** Chest X-ray and CT (**a**). Chest X-ray, posteroanterior projection. Irregular shape consolidation in the upper zone of the left lung. (**b**). Chest CT, lung window, coronal image. Various shape and size lung nodules in the left upper lobe. (**c**) Chest CT, lung window, axial image. The largest lung nodule with spiculated borders is in the left upper lobe. (**d**) Chest CT, mediastinal window, axial image. Calcifications in lung nodules.

On histopathology, multiple necrotizing granulomas were found. Ziehl-Neelsen's staining for mycobacteria was positive. Resected lymph nodes showed signs of pneumoconiosis and a few non-necrotizing granulomas.

After 17 days, the liquid culture became positive for *Mycobacterium tuberculosis*, and the result was confirmed by a test that detects the production of the MPT64 protein. The strain grown in the culture was subjected to a drug susceptibility test using the Bactec MGIT system and molecular identification of drug resistance using the GenoType MTBDRplus assay (Bruker). Genetic testing confirmed the S315T1 mutation in the *katG* gene, previously detected using the FluoroType® system. A phenotypic assay for drug resistance confirmed that the strain was resistant to isoniazid only.

The patient was transferred to a tuberculosis inpatient department for tuberculosis treatment.

#### *2.2. Case 2*

A 41-year-old female, emaciated, with alcohol dependence syndrome, treated for sensitive tuberculosis in 2016, was admitted to hospital due to productive cough, progressive general weakness, shortness of breath, and difficulty in swallowing foods and liquids.

The chest radiograph showed extensive, parenchymal consolidations in both lungs, with low-attenuation areas in upper zones suggesting cavitations (Figure 2).

**Figure 2.** Chest X-ray anteroposterior in a horizontal position. Extensive, parenchymal consolidations in both lungs with low-attenuation areas in upper zones sugges<sup>t</sup> cavitations.

Laboratory tests revealed increased levels of CRP (C reactive protein), INR (international normalized ratio) and aminotransferases, and decreased levels of total protein, albumin, iron, folic acid, and calcium. As part of the workup, sputum was collected to test for tuberculosis and non-tuberculous mycobacterial infections. Smear microscopy revealed a very high count of acid-fast bacilli. Genetic testing with the GeneXpert MTB/RIF revealed the presence of a *Mycobacterium tuberculosis* complex and identified resistance to rifampicin (RMP). As the patient was suspected of having multidrug-resistant tuberculosis, molecular testing was also carried out with the FluoroType® system. The test confirmed the presence of the genetic material of *Mycobacterium tuberculosis*, the S531L mutation in the *rpoB* gene, and the S315T1 mutation in the *katG* gene, allowing us to identify multidrug resistance (MDR). After six days, a positive culture on the MGIT liquid medium was obtained, and a test that detects the production of MPT64 protein was carried out, which confirmed that the identified bacteria belonged to the *Mycobacterium tuberculosis* species. The GenoType molecular drug resistance assay confirmed the presence of the S531L mutation in the *rpoB* gene and the S315T1 mutation in the *katG* gene, allowing us to identify multidrug resistance. Further analysis of drug resistance in the grown strain, using the GenoType MTBDRsl assay, allowed us to classify the strain as an extensively drug-resistant (XDR) strain. A molecular analysis based on spoligotyping qualified the strain to the Beijing 1 molecular family. The patient was admitted to the tuberculosis inpatients department to start appropriate therapy; nevertheless, she died 10 days later.
