Molecular Identification of Extrapulmonary Vaccine Adverse Events after BCG in Paraffin-Embedded Specimens.

According to the World Health Organization (WHO), around 1 million children worldwide are diagnosed with tuberculosis each year. The Bacillus Calmette-Guérin (BCG) vaccine has been used around the world for over 100 years. The complications of the BCG vaccination can occur in about 0,06% of children and include local or systemic adverse reactions. Due to the close analogy between the vaccine strain and other species of the Mycobacterium tuberculosis complex (MTBC), molecular methods are recommended for differential diagnosis of Vaccine adverse events (VAE) after BCG. The ability to quickly and specifically identify BCG is important in view of different treatment regimens. The aim of the study was to assess the usefulness of genetic testing for Mycobacterium bovis BCG in the paraffin-embedded specimens' methods. We describe two cases of VAE in immune-compromised children presenting with osteoarticular changes that had been clinically suspected of tuberculosis and led to molecular identification through GeneXpert, GenoType MTBC, and Spoligotyping. Results: Mycobacterium bovis BCG was detected in osteoarticular changes embedded in paraffin block of two patients. Conclusion: Genetic tests using paraffin-embedded materials allow for quick identification and differential diagnosis of patients with Tuberculosis and VAE after BCG. This is an important issue, especially in cases where the tissue has only been submitted for histopathological examination without microbiological diagnostics for tuberculosis.

Severe Respiratory Failure Due to Pulmonary BCGosis in a Patient Treated for Superficial Bladder Cancer.

Intra-vesical instillations with bacillus Calmette-Guerin (BCG) are the established adjuvant therapy for superficial bladder cancer. Although generally safe and well tolerated, they may cause a range of different, local, and systemic complications. We present a patient treated with BCG instillations for three years, who was admitted to our hospital due to fever, hemoptysis, pleuritic chest pain and progressive dyspnea. Chest computed tomography (CT) showed massive bilateral ground glass opacities, partly consolidated, localized in the middle and lower parts of the lungs, bronchial walls thickening, and bilateral hilar lymphadenopathy. PCR tests for SARS-CoV-2 as well as sputum, blood, and urine for general bacteriology-were negative. Initial empiric antibiotic therapy was ineffective and respiratory failure progressed. After a few weeks, a culture of M. tuberculosis complex was obtained from the patient's specimens; the cultured strain was identified as Mycobacterium bovis BCG. Anti-tuberculous treatment with rifampin (RMP), isoniazid (INH) and ethambutol (EMB) was implemented together with systemic corticosteroids, resulting in the quick improvement of the patient's clinical condition. Due to hepatotoxicity and finally reported resistance of the BCG strain to INH, levofloxacin was used instead of INH with good tolerance. Follow-up CT scans showed partial resolution of the pulmonary infiltrates. BCG infection in the lungs must be taken into consideration in every patient treated with intra-vesical BCG instillations and symptoms of protracted infection.

Tuberculous Pericarditis-Own Experiences and Recent Recommendations.

Tuberculous pericarditis (TBP) accounts for 1% of all forms of tuberculosis and for 1-2% of extrapulmonary tuberculosis. In endemic regions, TBP accounts for 50-90% of effusive pericarditis; in non-endemic, it only accounts for 4%. In the absence of prompt and effective treatment, TBP can lead to very serious sequelae, such as cardiac tamponade, constrictive pericarditis, and death. Early diagnosis of TBP is a cornerstone of effective treatment. The present article summarises the authors' own experiences and highlights the current status of knowledge concerning the diagnostic and therapeutic algorithm of TBP. Special attention is drawn to new, emerging molecular methods used for confirmation of M. tuberculosis infection as a cause of pericarditis.

From NTM (Nontuberculous mycobacterium) to Gordonia bronchialis-A Diagnostic Challenge in the COPD Patient.

In patients with chronic obstructive pulmonary disease, respiratory infections are of various aetiology, predominantly viral and bacterial. However, due to structural and immunological changes within the respiratory system, such patients are also prone to mycobacterial and other relatively rare infections. We present the 70-year old male patient with chronic obstructive pulmonary disease (COPD) and coexisting bronchial asthma, diagnosed due to cough with purulent sputum expectoration lasting over three months. The first microbiological investigation of the sputum sample revealed the growth of mycobacteria. The identification test based on protein MPT64 production indicated an organism belonging to NTM (nontuberculous mycobacterium). However, further species identification by genetic testing verified the obtained culture as not belonging to the Mycobacterium genus. Based on observed morphology, the new characterisation identified an aerobic actinomycete, possibly a Nocardia spp. The isolated strain was recultured on standard microbiological media. The growth of colonies was observed on Columbia blood agar plates and solid Löewenstein-Jensen medium. The Gram and Zhiel-Nielsen stains revealed the presence of Gram-positive acid-fast bacilli. The extraction protocol and identification were performed in two repetitions; the result was G. bronchialis, with a confidence value of 99% and 95%, respectively. The gene sequencing method was applied to confirm the species affiliation of this isolate. The resulting sequence was checked against the 16S ribosomal RNA sequences database (Bacteria and Archaea). The ten best results indicated the genus Gordonia (99.04-100%) and 100% similarity of the 16S sequenced region was demonstrated for Gordonia bronchialis. The case described indicates that the correct interpretation of microbiological test results requires the use of advanced microbiology diagnosis techniques, including molecular identification of gene sequences. From a clinical point of view, Gordonia bronchialis infection or colonization may present a mild course, with no febrile episodes and no significant patient status deterioration and thus, it may remain undiagnosed more often than expected.

Mycobacterium chimaera as an Underestimated Cause of NTM Lung Diseases in Patients Hospitalized in Pulmonary Wards.

Mycobacterium chimaera is the newly described species belonging to Mycobacterium avium complex (MAC), with morphology and growth characteristics closely related to Mycobacterium intracellulare. The aim of this retrospective study was to analyze the frequency and clinical significance of M. chimaera identification in the population of patients with previous positive respiratory cultures for M. intracellulare or MAC. 200 strains of M. intracellulare or MAC, isolated from respiratory specimens of patients hospitalized in pulmonary wards, between 2011 and 2020, were retrospectively analyzed with GenoType NTM-DR test. 88 (44%) of strains were re-classified to M. chimaera species. Analysis of clinical data in 30 patients with positive M. chimaera isolates revealed that they were diagnosed with chronic obstructive pulmonary disease (COPD) - 27%, past tuberculosis - 20%, or interstitial lung diseases - 17%, respectively. Non-tuberculous mycobacterial lung disease (NTMLD) caused by M. chimaera has been recognized in 53% of patients, most often in those presenting with post-tuberculous lung lesions. M. chimaera was almost exclusively isolated from respiratory specimens of patients with underlying lung diseases, especially those with COPD and/or past tuberculosis. NTMLD due to M. chimaera was diagnosed predominantly in patients with past tuberculosis.

Tuberculosis diagnosed by thoracic surgeons.

INTRODUCTION: Tuberculosis is frequently omitted in the diagnostic workup and may be identified accidentally following thoracic surgeries, mostly those targeting lung cancer. OBJECTIVES: This study aimed to assess the clinical characteristics of patients who underwent thoracic surgery that resulted in the diagnosis of tuberculosis and to review lesions initially found on chest imaging in the context of the potential presence of tuberculosis. PATIENTS AND METHODS: We analyzed medical records of all patients hospitalized at the Department of Thoracic Surgery of the National Tuberculosis and Lung Diseases Research Institute, Warsaw, Poland, between the years 2014 and 2018 (n = 57) in whom tuberculosis was diagnosed. Two radiologists who knew the diagnosis retrospectively analyzed preprocedural chest computed tomography scans of the study patients. RESULTS: Tuberculosis was diagnosed by culture of specimens obtained during video­ assisted thoracoscopy (21 patients), thoracotomy (24 patients), mediastinoscopy (6 patients), transthoracic fine­ needle biopsy (3 patients), and transbronchial biopsy (1 patient). In the remaining 2 individuals, the diagnosis was established based on the microbiological examination of drained pleural fluid. In 42 patients (73.7%), the diagnosis of tuberculosis was unexpected to thoracic surgeons. Radiological findings suggestive of tuberculosis were present in 38 patients (66.7%). The radiologists who retrospectively analyzed the imaging records suggested tuberculosis in 31 persons (54.3%), whereas those who carried out the initial preprocedural evaluation, in 11 (19.3%). CONCLUSIONS: The majority of the study patients presented with radiological findings encountered in tuberculosis, which should have led to a less invasive diagnostic workup. This highlights the role of radiologists in the identification of the disease.

Factors predisposing to non-tuberculous mycobacterial lung disease in the patients with respiratory isolates of non-tuberculous mycobacteria.

INTRODUCTION: An increasing incidence rate of respiratory isolates of non-tuberculous mycobacteria (NTM) has been noted recently in most European countries as well as in the US. Despite many publications, there is no consensus concerning the importance of different factors in promoting NTM lung disease (NTMLD). The aim of the present retrospective study was to analyse patients with positive NTM respiratory isolates in search of factors predisposing to NTMLD. MATERIAL AND METHODS: 73 patients, 23 males, 50 females, median age 62.2 years, in whom NTM have been cultured from respiratory specimen (sputum and/or bronchial washings), in the period 2010-2015, entered the study. RESULTS: NTMLD (according to ATS/IDSA) has been recognised in 36 patients, airways colonisation by NTM - in 37 patients. NTMLD was diagnosed more often in the patients infected with M. kansasii, M. abscessus and M. avium/M. intracellulare comparing to those infected with M.xenopi, M. gordonae and M. fortuitum (p < 0.0001). The proportion of females to males was significantly higher in the NTMLD group comparing to the colonisation group (p < 0.007). Previous tuberculosis or mycobacteriosis were noted significantly more frequently in the group of patients with NTMLD comparing to the colonisation group (28% vs 8%, p = 0.038). Univariate regression analysis revealed M. kansasii, female gender, and previous tuberculosis or mycobacteriosis as significant predictors of NTMLD. CONCLUSIONS: The risk factors of NTMLD recognition in the presented group of patients were the following: female gender, M. kansasii isolation, as well as past tuberculosis or mycobacteriosis.

Nontuberculous mycobacteria strains isolated from patients between 2013 and 2017 in Poland. Our data with respect to the global trends.

INTRODUCTION: During the last decades the prevalence of NTM infections has increased, especially in developed countries. The aim of the study was to provide an overview on all NTM isolated from clinical samples in Poland between 2013 and 2017. MATERIAL AND METHODS: The study comprised 2799 clinical specimens, mostly respiratory accessed in the reference laboratory of National Tuberculosis and Lung Diseases Research Institute in Warsaw and in the Wielkopolska Center of Pulmonology and Thoracic Surgery, Poland, 2013-2017. RESULTS: During the study period 35 species of NTM were isolated . The number of isolates increased almost 1.6-fold: from 420 in 2013 to 674 in 2017. M. kansasii, M. avium, M. xenopi, M. gordonae and M. intracellulare were the most common species. This NTM pattern was rather stable over the time. If the aggregated amount of all MAC species was taken into account they dominated over M. kansasii from 2015. M. avium and M. intracellulare were more often isolated from women, while M. kansasii, M. gordonae and M. xenopi predominated in men. Men and women were infected almost with the same frequency. In older patients 65+ women were in majority, quite opposite to those aged 25 to 64 years. CONCLUSION: In Poland, like in other countries increased the frequency of isolated NTM. M. kansasii and M. avium were the most frequently identified species from clinical samples. Men and women were infected with NTM with the same frequency.

[Interferon-Gamma Release Assays in the diagnosis of latent tuberculosis infection in clinical situations]. / Testy IGRA w diagnostyce zakazenia pratkami gruzlicy w wybranych sytuacjach klinicznych.

Until recently, the basic test to identify latent tuberculosis infection (LTBI) was the tuberculin skin test, despite its limitations in the form of low sensitivity and specificity. Currently, Interferon Gamma Release Assays from peripheral blood are used for a rapid diagnosis of LTBI and measurement of the interferon gamma (IFN-g) levels secreted by specific T cells stimulated with Mycobacterium tuberculosis antigens. Detection of LTBI is important in the control of people potentially at risk of TB disease, such as people remaining in close contact with BK (+) tb patient and for patients evaluated for biological treatment. The paper presents the value of IGRA in three selected clinical situations: in two cases of latent tuberculosis infection and in one case of active tuberculosis.

[Transmission of Mycobacterium tuberculosis among household contacts of patients with tuberculosis]. / Badanie transmisji Mycobacterium tuberculosis wsród osób blisko spokrewnionych chorych na gruzlice.

OBJECTIVE: The aim of this study was estimation of usefulness of two molecular methods for Mycobacterium tuberculosis typing in the epidemiological research of tuberculosis (TB). We determined molecular patterns of M. tuberculosis strains isolated from 66 patients, members of 29 families living in 9 voivodeships of Poland. We also analysed drug susceptibility of the strains as well as some demographic characteristics of the patients. METHODS: The genotype analysis of the 66 clinical isolates was performed by using spoligotyping and IS6110-Mtb1/Mtb2 PCR. MAIN OBSERVATIONS: Of the 29 families examined in this study, in 23 each family member was infected with the same M. tuberculosis strain. Three drug-resistant strains and two members of the Beijing family were identified. RESULTS: We found that strains within each of the 23 families had the same genetic patterns, whereas those of the strains identified in the rest 6 families were different. Among those 6 families, in 3 differentiation of the strains was obtained with both spoligotyping and IS6110-Mtb1/Mtb2 PCR analysis, while in another 3 only with spoligotyping method. CONCLUSIONS: Based on the results from this study, the two genotyping methods used were demonstrated as an efficient approach for investigating the epidemiological relatedness of TB cases.

[Frequency of drug resistant tuberculosis in Poland in 2000 as compared to 1997]. / Czestosc wystepowania gruzlicy lekoopornej w Polsce w 2000 R. w porównaniu z 1997 r.

Prevalence of drug resistance to one drug and multidrug resistance--MDR in different categories of tuberculosis patients is an important information about the susceptibility pattern of Mycobacterium tuberculosis isolates against antimycobacterial drugs. Poland joined WHO/IUATLD global project on TB drug resistance surveillance, and carried out in 1996/1997 the first prospective survey, simultaneously on primary and acquired drug resistance. This study is repeated in 2000 according to the WHO/IUATLD protocol. The programme covered the whole country. A total of 16 regional centers participated in the co-operative study. 3705 questionnaires and cultures were obtained from patients who excreted TB bacilli during the 12-months from 1 st. January to 31st December 2000. Drug resistance tests to INH, RMP, SM, EMB were performed on Lowenstein-Jensen medium according to the proportion method or/and Bactec 460 TB system. 3705 TB patients (3037 new and 668 treated cases) bacteriologically confirmed by culture were included in one-year study. Primary resistance to any drug was found in 6.12% (CI 5.27-6.56) of new cases. 35 patients (1.15%, CI 0.77-1.35) were infected with MDR strains. Acquired resistance to any drug was found in 16.6% (CI 5.27-6.56), 8.53% (CL 6.41-9.6) of the patients who excreted MDR strains. We have found increased resistance from 3.6% in 1997 to 6.12% (p < 0.001) in 2000 and MDR from 0.6% in 1997 to 1.15% (p < 0.001) in 2000 in untreated tuberculosis patients in Poland. The rate of resistance in the group of treated TB patients was very similar in 1997 (17.0%) and in 2000 (16.6%); except 20% increase of MDR cases--(7.0% in 1997, and 8.53% in 2000). We observed an increase in drug resistant tuberculosis first time during 40 years long period of its monitoring. Regular monitoring of drug resistance in TB patients in Poland is recommended.

[Sensitivity of microscopy for detection of mycobacterium tuberculosis and MOTT (mycobacteria other than tuberculosis) on the basis of analysis 22.218 clinical materials submitted in 1998-2001 to the Department of Microbiology in National Tuberculosis and Lung Diseases Research Institute in Warsaw, Poland]. / Czulosc metody bakterioskopowej w wykrywaniu pratków gruzlicy i MOTT na podstawie analizy 22.218 badan diagnostycznych przeprowadzonych w Zakladzie Mikrobiologii IGiCHP w okresie 1998-2001.

The results of 22.218 respiratory specimens sent to our laboratory were studied to determine the sensitivity of the Ziehl-Neelsen (Z-N) stain and microscopy-fluorescence method for detection of M.tbc and MOTT. There were no AIDS patients among analyzed cases. Smears were positive for acid fast bacilli (AFB) 60.0% (480 of 800) of specimens growing M. tuberculosis and 25.1% (219 of 872) of specimens growing the six common species of MOTT. Smear positivity by species was 28.1% (141 of 502) for M. kansasii, 29.4% (43 of 146) for MAIC, 28.7% (35 of 122) for M. xenopi. No smear was positive for M. gordone (43 cultures), M. fortuitum (33 cultures), M. scrofulaceum (26 cultures). The rate of nonculturable mycobacteria on L-J medium was 0.2%; mean rate of contamination was 4.6%. We also analyzed the relation between the number of AFB seen on the smear and time of the growth of M. tuberculosis and relation between abundance of the culture growth and AFB seen on smears. These study suggest that the sensitivity of microscopy for Mycobacterium tuberculosis is comparable with the data of others authors. Sensitivity of microscopy is lower in MOTT detection than for M.tbc (p < 0.001). Time of growth and abundance of the M. tuberculosis cultures were adequate to AFB seen in microscopy.