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Mycobacterial infections pose significant diagnostic challenges due to the genetic diversity of species, limitations of current detection methods, and the need for rapid and accurate identification tools. In this study, we developed and validated a novel molecular approach for the specific detection of the Mycobacterium genus and the Mycobacterium marinum species based on the identification of unique DNA sequences. Using comparative genomic alignments and in silico screening of curated genomic databases, we identified a 391 bp region of the mmpl gene specific to the Mycobacterium genus, and a 202 bp region of the espE_2 gene specific to M. marinum. Primers were designed for both targets and validated for specificity using in silico BLAST analysis and in vitro PCR and qPCR assays. Experimental validation involved DNA from 52 bacterial isolates, including 44 Mycobacterium species and 6 M. marinum strains. The mmpl target showed a sensitivity of 95% and specificity of 100% for Mycobacterium, while the espE_2 target achieved 100% sensitivity and specificity for M. marinum. We further demonstrated the applicability of our method using mock clinical samples spiked with bacteria and subjected to standard diagnostic workflows. Although qPCR sensitivity was reduced in complex matrices like sputum, likely due to DNA degradation and eukaryotic DNA interference, our method showed strong performance in buccal swabs and saliva. The assay offers a rapid, cost-effective, and adaptable alternative for the detection of mycobacteria, particularly in laboratories with limited resources. Future work will expand validation across a broader panel of strains and clinical specimens to enhance diagnostic confidence.

Multidrug-resistant tuberculosis (MDR-TB) poses a serious challenge to the global control of the disease. The purpose of this study was to characterize MDR-TB patients from Poland and to determine the extent of MDR-TB disease attributable to recent transmission. The study included all 46 patients diagnosed with MDR-TB in Poland in 2004 and followed up for 6 years (until 2011). For each patient, sociodemographic and clinical characteristics, treatment outcomes, and bacteriological data were collected by the review of medical and laboratory records. Mycobacterium tuberculosis isolates from all patients were characterized using spoligotyping, mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR) typing, IS 6110 restriction fragment length polymorphism (RFLP) analysis, and sequencing analysis of drug resistance-associated loci ( katG , mabA - inhA , rpoβ , rpsL , and embB ). The majority of patients were male (86.9 %), 40–64 years of age (60.8 %), with a history of TB treatment (84.8 %), and producing smear-positive sputa (86.9 %). Twenty-two (47.8 %) patients suffered from concomitant diseases and 28 (60.8 %) were alcohol abusers. Treatment outcome assessment revealed that 8 (17.4 %) patients were cured or completed therapy, while 15 (32.6 %) died of TB, 11 (23.9 %) defaulted, 8 (17.4 %) failed, and 1 (2.2 %) was transferred and lost to follow-up. Upon genotyping, 10 (21.7 %) isolates were allocated in four clusters. These were further subdivided by mutational profiling. Overall, in 6 (13 %) patients, MDR-TB was a result of recent transmission. For 4 (8.7 %) of these patients, a direct epidemiological link was established. The study shows that the transmission of MDR-TB occurs at a low rate in Poland. Of urgent need is the implementation of a policy of enforced treatment of MDR-TB patients in Poland.

Poland has been an officially bovine tuberculosis (bTB) free country for the last seven years. The problem currently observed is the increasing number of new cases of bTB in wild species, kept in a farmed herd and free-living herd: European bison (Bison bonasus), wild boar (Sus scrofa), wolves (Canis lupus) and red deer (Cervus elaphus). This article presents the case of Mycobacterium caprae transmission to an American bison (Bison bison) herd kept on a private farm in Eastern Poland.

Many studies on the molecular epidemiology of tuberculosis (TB) have shown that 10-20% of patients may be infected with more than one strain of (MTB), during a single episode of the disease. However, data on the frequency of this phenomenon are underestimated, and methodological difficulties mean that most cases are not detected. Below we present the first case of polyclonal/mixed infection documented in Poland. We described the case of a 33-year-old Ukrainian man, imprisoned in a Polish prison, who was diagnosed with pulmonary TB confirmed by culture. During the 6-month therapy, the patient was treated in three Polish hospitals, but no negative sputum test result was obtained. In the course of microbiological and molecular diagnostics, conducted from June 2021 to February 2022, divergent results were obtained from drug susceptibility testing and genotyping of MTB strains isolated from clinical materials from the patient. In the final stage of the tests, it was confirmed that the patient was infected with two strains of MTB - one of them was drug-sensitive and belonged to the T1 267 genotype, the other was pre-extensively drug resistant (pre-XDR) and belonged to the Beijing 265 genotype. The existence of clonally complex TB infections resulting in heteroresistance to basic antituberculosis drugs has important implications for patient care. Established molecular methods complemented by routine microbiological diagnostics allow rapid detection of these infections and appropriate adjustment of therapy.

The study was carried out in Polish goat population to estimate the prevalence of the nasal cavity infection with various staphylococcal species including methicillin-resistant Staphylococcus aureus (MRSA), investigate the potential permissive role of small ruminant lentivirus (SrLV) infection and determine the level of clonality of S. aureus nasal isolates. Nasal swabs and blood samples were collected from 1300 clinically healthy adult goats from 21 Polish goat herds. Blood samples were serologically screened for SRLV. Staphylococci were isolated from nasal swabs and identified using classical microbiological methods, MALDI-TOF, multiplex-PCR, and their clonality was assessed using PFGE. Antimicrobial resistance was determined on the basis of minimum inhibitory concentration and by demonstration of the presence of the mecA gene encoding the multiplex-PCR PBP2a protein and of the five main types of staphylococcal cassette chromosome mec. The apparent prevalence of staphylococcal and S. aureus infection of the nasal cavity was 29.1% (CI 95%: 26.9%, 31.5%) and 7.3% (Ci 95%: 6.1%, 8.8%), respectively. No relationship was found between the SRLV-infection and the presence of any staphylococcal species including S. aureus (p=0.143). Only 9.8% of S. aureus isolates were resistant to amoxicillin/clavulanic acid and 5.9% to chloramphenicol and ciprofloxacin. All tested isolates proved to be phenotypically and genotypically sensitive to methicillin, which yielded the apparent prevalence of MRSA of 0% (Cl 95%: 0%, 7.0%). S. aureus isolates show high genetic similarity within goat herds, however vary considerably between herds. Goats do not appear to be an important source of S. aureus for humans in Poland.

Pyrazinamide (PZA) is a prodrug that is converted to pyrazinoic acid by the enzyme pyrazinamidase, encoded by the pncA gene in Mycobacterium tuberculosis . Molecular identification of mutations in pncA offers the potential for rapid detection of pyrazinamide resistance (PZA r ). However, the genetic variants are highly variable and scattered over the full length of pncA , complicating the development of a molecular test. We performed a large multicenter study assessing pncA sequence variations in 1,950 clinical isolates, including 1,142 multidrug-resistant (MDR) strains and 483 fully susceptible strains. The results of pncA sequencing were correlated with phenotype, enzymatic activity, and structural and phylogenetic data. We identified 280 genetic variants which were divided into four classes: (i) very high confidence resistance mutations that were found only in PZA r strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZA r strains, (iii) mutations with an unclear role found in less than 70% of PZA r strains, and (iv) mutations not associated with phenotypic resistance (10%). Any future molecular diagnostic assay should be able to target and identify at least the very high and high-confidence genetic variant markers of PZA r ; the diagnostic accuracy of such an assay would be in the range of 89.5 to 98.8%. IMPORTANCE Conventional phenotypic testing for pyrazinamide resistance in Mycobacterium tuberculosis is technically challenging and often unreliable. The development of a molecular assay for detecting pyrazinamide resistance would be a breakthrough, directly overcoming both the limitations of conventional testing and its related biosafety issues. Although the main mechanism of pyrazinamide resistance involves mutations inactivating the pncA enzyme, the highly diverse genetic variants scattered over the full length of the pncA gene and the lack of a reliable phenotypic gold standard hamper the development of molecular diagnostic assays. By analyzing a large number of strains collected worldwide, we have classified the different genetic variants based on their predictive value for resistance which should lead to more rapid diagnostic tests. This would assist clinicians in improving treatment regimens for patients. Conventional phenotypic testing for pyrazinamide resistance in Mycobacterium tuberculosis is technically challenging and often unreliable. The development of a molecular assay for detecting pyrazinamide resistance would be a breakthrough, directly overcoming both the limitations of conventional testing and its related biosafety issues. Although the main mechanism of pyrazinamide resistance involves mutations inactivating the pncA enzyme, the highly diverse genetic variants scattered over the full length of the pncA gene and the lack of a reliable phenotypic gold standard hamper the development of molecular diagnostic assays. By analyzing a large number of strains collected worldwide, we have classified the different genetic variants based on their predictive value for resistance which should lead to more rapid diagnostic tests. This would assist clinicians in improving treatment regimens for patients.

Monika Kozińska, Dorota Filipczak, Ewa Augustynowicz-Kope𐟞partment of Microbiology, National Tuberculosis and Lung Diseases Research Institute, Warsaw, PolandCorrespondence: Monika Kozińska, Department of Microbiology, National Tuberculosis and Lung Diseases Research Institute, Plocka 26, Warsaw, 01-138, Poland, Tel/Fax +48224312182, Email m.kozinska@igichp.edu.plAbstract: Many studies on the molecular epidemiology of tuberculosis (TB) have shown that 10– 20% of patients may be infected with more than one strain of Mycobacterium tuberculosis (MTB), during a single episode of the disease. However, data on the frequency of this phenomenon are underestimated, and methodological difficulties mean that most cases are not detected. Below we present the first case of polyclonal/mixed infection documented in Poland. We described the case of a 33-year-old Ukrainian man, imprisoned in a Polish prison, who was diagnosed with pulmonary TB confirmed by culture. During the 6-month therapy, the patient was treated in three Polish hospitals, but no negative sputum test result was obtained. In the course of microbiological and molecular diagnostics, conducted from June 2021 to February 2022, divergent results were obtained from drug susceptibility testing and genotyping of MTB strains isolated from clinical materials from the patient. In the final stage of the tests, it was confirmed that the patient was infected with two strains of MTB - one of them was drug-sensitive and belonged to the T1 267 genotype, the other was pre-extensively drug resistant (pre-XDR) and belonged to the Beijing 265 genotype. The existence of clonally complex TB infections resulting in heteroresistance to basic antituberculosis drugs has important implications for patient care. Established molecular methods complemented by routine microbiological diagnostics allow rapid detection of these infections and appropriate adjustment of therapy.Keywords: Mycobacterium tuberculosis mixed infection, polyclonal infection, heteroresistance, genotyping methods, next-generation sequencing

ObjectiveAntineutrophil cytoplasmic antibodies (ANCAs) are considered a risk factor for granulomatosis with polyangiitis (GPA) exacerbation, especially when staphylococcal superantigens (SAgs) are present in nasal swabs. Their role in monitoring disease activity remains controversial. This study determined the relationship of ANCAs with disease activity and presence of SAgs in GPA patients.MethodsAmong a total of 115 GPA patients hospitalized in the period 2009–2016, we investigated the presence of SAgs and ANCA concentration. Blood samples and nasal swabs were taken at each visit (referred further to as episodes). Disease activity was assessed using the Birmingham Vasculitis Activity Score (BVAS).ResultsWe analyzed 362 episodes. ANCAs were detected in 215 (59.4%), while SAgs were detected in 126 (34.8%) episodes. We found a significant correlation between the presence of ANCAs and disease activity (p = 0.0032), as well as between their level and GPA severity (r = 0.25363, p = 0.000001). We also determined that an ANCA values ≥ 138 Ru/ml were an indicator of active disease with high specificity and low sensitivity (84.4% and 37.3%, respectively). The relationship between ANCA presence and the presence of SAgs was not confirmed; however, when SAgs were analyzed based on the different types, ANCA levels were found to be significantly higher in the group with SAg type B (p = 0.031).ConclusionsThere was no detectable evidence for the association between ANCA level and the presence of SAgs. Although monitoring ANCA levels as a marker of disease activity may be clinically relevant, GPA management cannot proceed on the basis of ANCA levels alone.Key Points• ANCA concentration usually correlates with GPA activity, although in half of patients, ANCAs persist despite effective treatment and clinical remission.• ANCA values of 138 Ru/ml seem to be an indicator of active disease with high specificity, but low sensitivity.• Although there is a relevance for ANCA monitoring as a marker of disease activity, GPA management cannot be based on ANCA levels alone.• The suspected clinical correlation between ANCA formation and SAg presence in nasal swabs is not obvious and requires further investigations.

Molecular typing and whole genome sequencing (WGS) information is used for (inter-) national outbreak investigations. To assist the implementation of these techniques for tuberculosis (TB) surveillance and outbreak investigations at European level there is a need for inter-country collaboration and standardization. This demands more information on molecular typing practices and capabilities of individual countries. We aimed to review the use of molecular/genomic typing for TB surveillance in European Union and European Economic Area countries in 2016; assess its public health value; and collect experiences on typing data use for cross-border cluster investigations. A web-based questionnaire was provided to all TB National Focal Points. The questionnaire consisted of three parts: i) Use and integration of molecular and genomic typing data into TB surveillance; ii) Cross-border cluster investigation and international collaboration, and iii) Perception and evaluation of public health benefits of molecular and genomic typing for TB surveillance. Of 26 responding countries, 20 used molecular typing for TB surveillance, including nine applying WGS. The level of integration into the national surveillance was heterogeneous. Among six countries not using typing for TB surveillance, more than half planned its implementation soon. Overall, most countries perceived an added public health value of molecular typing for TB control. Concerning international cluster investigations, countries had little experience and did not have standard protocols to exchange typing data. Our study shows a wide use of molecular and genomic typing data for TB surveillance in EU/EEA countries and reveals that transition to WGS-based typing is ongoing or is considered in most countries. However, our results also show a high heterogeneity in the use and integration of typing data for TB surveillance. Standardization of typing data use for TB surveillance is needed and formal procedures should be developed to facilitate international collaboration.

Early identification of mycobacterial species is crucial for early diagnosis. PCR-multiplex method performed on randomly chosen 54 mycobacteria isolates originating from clinical samples was found to be an inexpensive, quick and reliable alternative for commercially available diagnostics tests. Although the results of gene probes identification performed by NTLDR were generally consistent with multiplex PCR, two mixed Mycobacterium bovis BCG/Mycobacterium tuberculosis infections and a single misdiagnosis of M. tuberculosis with M. bovis were found. The routine application of multiplex-PCR has the potential to make diagnostics surveillance studies feasible.