Mycobacterial Interspersed Repeat Unit-Variable Number Tandem Repeat Typing of Mycobacterium avium Strains Isolated from the Lymph Nodes of Free-Living Carnivorous Animals in Poland.

Non-tuberculous mycobacteria (NTM) are ubiquitous organisms, of which some, especially those of the Mycobacterium avium complex (MAC), may be opportunistic animal and human pathogens. Infection with NTM can interfere with tuberculosis (TB) diagnosis and induce zoonoses, especially in immunocompromised individuals. Diseases caused by NTM have become more readily recognized; however, they are likely still underestimated. In this study, we identified and genotyped Mycobacterium avium strains that were isolated during TB monitoring among free-living carnivorous animals from southeastern Poland. In 2011-2020, lymph node samples from 192 such animals were tested for mycobacteria. A total of 41 isolates of M. avium strains were detected with the use of IS901, IS900, IS1245, and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR) identification. Thirty-three were identified as M. avium subsp. avium. These strains were derived from 1 beech marten (Martes foina), 1 common buzzard (Buteo buteo), 2 European badgers (Meles meles), 3 wolves (Canis lupus), and 26 red foxes (Vulpes vulpes). One strain isolated from a wolf was identified as M. avium subsp. hominissuis. The results show the widespread occurrence of MAC bacilli in the studied environment and additionally comprise new data on the molecular characteristics of M. avium subspecies carried by free-living southeastern Polish carnivores.

Cutaneous tuberculosis-ambiguous transmission, bacterial diversity with biofilm formation in humoral abnormality: case report illustration.

Background: Cutaneous tuberculosis (CTB) and its paucibacillary forms are rare and difficult to diagnose, especially in immunocompromised patients with significant comorbidity. The aim of the study was to introduce the modern concept of the microbiome and diagnostic chain into clinical practice (patient-centered care) with the presentation of an atypical form of cutaneous tuberculosis with necrotizing non-healing ulcers leading to polymicrobial infection. Methods: The study material included samples from sputum, broncho-alveolar lavage and skin ulcer, taken from a patient developing cutaneous tuberculosis. The microbiological investigation was performed, and identification of the isolates was carried out using genotyping and the matrix-assisted laser desorption ionization-time of flight mass spectrometry. Results: The immunocompromised patient with humoral abnormality (plasma cell dyscrasia) and severe paraproteinemia developed multiorgan tuberculosis. Although cutaneous manifestation preceded systemic and pulmonary symptoms (approximately half a year), the mycobacterial genotyping confirmed the same MTB strain existence in skin ulcers and the respiratory system. Therefore, the infectious chain: transmission, the portal of entry, and bacterial spreading in vivo, were unclear. Microbial diversity found in wound microbiota (among others Gordonia bronchialis, Corynebacterium tuberculostearicum, Staphylococcus haemolyticus, and Pseudomonas oryzihabitans) was associated with the spread of a skin lesion. The in vitro biofilm-forming capacity of strains isolated from the wound may represent the potential virulence of these strains. Thus, the role of polymicrobial biofilm may be crucial in ulcer formation and CTB manifestation. Conclusions: Severe wound healing as a unique biofilm-forming niche should be tested for Mycobacterium (on species and strain levels) and coexisting microorganisms using a wide range of microbiological techniques. In immunodeficient patients with non-typical CTB presentation, the chain of transmission and MTB spread is still an open issue for further research.

An overview of tuberculosis outbreaks reported in the years 2011-2020.

BACKGROUND: In many countries tuberculosis (TB) remains a highly prevalent disease and a major contributor to infectious disease mortality. The fight against TB requires surveillance of the population of strains circulating worldwide and the analysis of the prevalence of certain strains in populations. Nowadays, whole genome sequencing (WGS) allows for accurate tracking of TB transmission. Currently, there is a lack of a comprehensive summary of the characteristics of TB outbreaks. METHODS: We systematically analyzed studies reporting TB outbreaks worldwide, monitored through WGS of Mycobacterium tuberculosis. We 1) mapped the reported outbreaks from 2011- 2020, 2) estimated the average size of the outbreaks, 3) indicated genetic lineages causing the outbreaks, and 4) determined drug-resistance patterns of M. tuberculosis strains involved in the outbreaks. RESULTS: Most data originated from Europe, Asia, and North America. We found that TB outbreaks were reported throughout the globe, on all continents, and in countries with both high and low incidences. The detected outbreaks contained a median of five M. tuberculosis isolates. Most strains causing the outbreaks belonged to lineage four, more rarely to lineage two. Reported outbreak isolates were often drug resistant. CONCLUSIONS: We conclude that more WGS surveillance of M. tuberculosis outbreaks is needed. Globally standardized procedures might improve the control of M. tuberculosis infections.

Cutaneous and Pulmonary Tuberculosis-Diagnostic and Therapeutic Difficulties in a Patient with Autoimmunity.

Cutaneous tuberculosis (CTB) is a very rare disease and accounts for only 1-2% of cases of extrapulmonary tuberculosis (EPTB). Due to the variety of its clinical manifestations, the uncharacteristic appearance of its lesions, resembling other dermatoses in the early stages, and the limited experience of clinicians due to the rarity of CTB, diagnosis is very difficult. Particularly noteworthy is the fact that most cases of EPTB, including skin tuberculosis (TB), can be a manifestation of systemic involvement. In this paper, we present a case of an immunocompromised patient who was diagnosed with CTB almost a year after the first dermatological lesions were located on the lower extremities. At the same time, due to respiratory symptoms, a diagnosis of pulmonary TB (PTB) was made, and radiological and microbiological confirmations were obtained.

Tuberculosis in Poland: Epidemiological and Molecular Analysis during the COVID-19 Pandemic.

The COVID-19 pandemic may have a negative impact on the proper implementation of TB control programmes and may increase TB incidence rates in the near future. The aim of this study was to perform an epidemiological and molecular analysis of Mycobacterium tuberculosis strains cultured from tuberculosis patients in Poland in 2020 and to compare the results of monitoring drug-resistant tuberculosis in Poland with previous studies in 2012 and 2016. The analysis was based on questionnaires and strains sent by regional laboratories during the 12 months of 2020. Molecular analysis was performed by spoligotyping 20% of the strains sensitive to the four primary antimycobacterial drugs and all of the drug-resistant strains. The number of strains sent for analysis dropped threefold, from 4136 in 2012 to 1383 in 2020. The incidence of tuberculosis among men was higher than among women. There was an increase in strains' resistance to antimycobacterial drugs in both newly diagnosed patients, from 4.4% in 2012 to 6.1% in 2020, and previously treated patients, from 11.7% to 12.3%. Four-year resistance increased to 1% and 2.1%, respectively. The spoligotype SIT1 was the most abundant among the resistant strains (17%), and SIT53 (13.9%) was the most common among susceptible strains.

Multidrug-Resistant Tuberculosis-Diagnostic Procedures and Treatment of Two Beijing-like TB Cases.

The Beijing/W genotype is one of the major molecular families of Mycobacterium tuberculosis complex (MTBC), responsible for approximately 50% of tuberculosis (TB) cases in Far East Asia and at least 25% of TB cases globally. Studies have revealed that the Beijing genotype family is associated with a more severe clinical course of TB, increased ability to spread compared to other genotypes, and an unpredictable response to treatment. Based on the profile of spacers 35-43 in the Direct Repeat (DR) locus of the MTBC genome determined by spoligotyping, classical (typical) and modern (Beijing-like) clones can be identified within the Beijing family. While the modern and ancient Beijing strains appear to be closely related at the genetic level, there are marked differences in their drug resistance, as well as their ability to spread and cause disease. This paper presents two cases of drug-resistant tuberculosis caused by rare mycobacteria from the Beijing family: the Beijing 265 and Beijing 541 subtypes. The genotypes of isolated strains were linked with the clinical course of TB, and an attempt was made to initially assess whether the Beijing subtype can determine treatment outcomes in patients.

XDR-TB Transmitted from Mother to 10-Month-Old Infant: Diagnostic and Therapeutic Problems.

Drug-resistant TB (DR-TB) in children is a special epidemiological, clinical, and diagnostic problem, and its global incidence remains unknown. DR-TB in children is usually of a primary nature and is most often transmitted to the child from a household contact, so these cases reflect the prevalence of DR-TB in the population of adult patients. The risk of infection with Mycobacterium tuberculosis complex (MTBC) in children depends on age, duration of exposure, proximity of contact with the infected person, and the level of source virulence. Most cases of TB in children, especially in infants, are caused by household contacts, where the main sources of infection are parents, grandparents or older siblings. However, there are many documented cases of TB transmission outside the family. The most common source of infection is an adult who is profusely positive for mycobacteria, diagnosed too late, and inadequately treated. It has been estimated that a sputum-positive patient might infect 30-50% of their household members. For this reason, active epidemiological investigation and contact tracing in the environment of sputum-positive patients are the most appropriate methods of identifying infected family members. This paper presents a case report concerning the transmission of extensively drug-resistant TB, Beijing 265 genotype, from a mother to her 10-month-old daughter. It is the first case diagnosed in Poland, and one of very few described in the literature where treatment was effective in the mother and the infant recovered spontaneously.

COVID-19 in Patients with Active Tuberculosis.

Data on the coincidence of tuberculosis (TB) and COVID-19 are limited, and previous observations are based on the results of just a few studies, which has led to polarized views on the course of infection with SARS-CoV-2 in patients with active TB. We present the first two cases of TB and COVID-19 coinfection in the population of patients in Poland, diagnosed shortly after the outbreak of the global pandemic. In the first patient, TB was very advanced at the time of infection with SARS-CoV-2. From the third day of hospitalisation, respiratory failure was increasing, with no improvement after the use of high-flow oxygen therapy and mechanical ventilation. On the seventh day of hospitalization, the patient died. In the second presented case, therapeutic success was achieved despite the coincidence of COVID-19, infection with HIV, and extrapulmonary and pulmonary TB. The patient had symptoms of renal failure and the SARS-CoV-2 infection was mild and asymptomatic. Because both patients were in the care of a homeless shelter, a molecular epidemiological investigation was carried out. Different DNA profiles of Mycobacterium tuberculosis complex isolates detected in clinical materials from patients ruled out the transmission of tuberculosis. Based on our analysis, it is impossible to clearly define the influence of active TB on the course of SARS-CoV-2 infection. We can only suggest that coinfection is particularly dangerous for socially disadvantaged people, the elderly, and people with other comorbidities. In the coming years, a negative impact of the current pandemic on control programmes will be observed for many infectious diseases, including TB.

Epidemiological characterization of Mycobacterium caprae strains isolated from wildlife in the Bieszczady Mountains, on the border of Southeast Poland.

BACKGROUND: The majority of animal tuberculosis (TB) cases reported in wildlife in Poland over the past 20 years have concerned the European bison inhabiting the Bieszczady Mountains in Southeast Poland: an area running along the border of Southeast Poland. As no TB cases have been reported in domestic animals in this region since 2005, any occurrence of TB in the free-living animals inhabiting this area might pose a real threat to local livestock and result in the loss of disease-free status. The aim of the study was to describe the occurrence of tuberculosis in the wildlife of the Bieszczady Mountains and determine the microbiological and molecular characteristics of any cultured strains. Lymph node samples were collected for analysis from 274 free-living animals, including European bison, red foxes, badgers, red deer, wild boar and roe deer between 2011 and 2017. Löwenstein-Jensen and Stonebrink media were used for culture. Molecular identification of strains was performed based on hsp65 sequence analysis, the GenoType®MTBC (Hain Lifescience, Germany) test, spoligotyping and MIRU-VNTR analysis. RESULTS: Mycobacterium caprae was isolated from the lymph nodes of 21 out of 55 wild boar (38.2%; CI 95%: 26.5%, 51.4%) and one roe deer. Since 2014, no new TB cases have been reported in the Bieszczady European bison population. CONCLUSIONS: The identification of TB in wild boar in the Bieszczady is an alarming phenomenon, which requires further investigation. The Bieszczady mountains are a precious, unique area, home to many protected species. However, it is also the only area in Poland where TB cases have been reported in free-living animals. The occurrence of TB in wild boar inhabiting this area might pose a real threat to local livestock and many of the protected species (for example European bison that can share feeding places with wild boar). Given this situation, ongoing monitoring of the prevalence of TB should be conducted, and protective measures should be considered.

Molecular Characterisation of the Mycobacterium Bovis Causing Bovine Tuberculosis Outbreaks in Poland.

INTRODUCTION: Since 2009, Poland has been recognised as a country officially free of bovine tuberculosis (bTB), although in each year of the last five there were from 8 to 18 outbreaks of the disease. In 2008-2016, the largest number of cattle infected with bovine mycobacteria were eliminated in the Masovian Province (the central region of Poland) and the largest number of outbreaks of this zoonosis were recorded in this area. The close proximity of farms where bTB was found led to the suspicion that tuberculosis could have been transmitted between the affected herds. The aim of the study was the molecular characterisation of the pertinent M. bovis/caprae strains and determination of the epidemiological relationship of various bTB outbreaks. MATERIAL AND METHODS: The material for microbiological tests came from 119 cattle (Bos taurus) from nine herds located in five provinces, neighbouring the Masovian Province. RESULTS: Laboratory tests of tissue material gave results confirming tuberculosis in 54 (45%) animals. All strains belonged to the Mycobacterium bovis species. A two-step analysis of genetic affinity allowed 50 strains to be identified as phylogenetically closely related and separated between three genetic clusters consisting of 2 to 27 strains. CONCLUSION: Based on the results of genotyping, bTB outbreaks were found in three herds, and three transmission chains were identified among these herds.

Mycobacterium caprae - the first case of the human infection in Poland.

The strain of tuberculous mycobacteria called Mycobacterium caprae infects many wild and domestic animals; however, because of its zoonotic potential and possibility of transmission between animals and humans, it poses a serious threat to public health. Due to diagnostic limitations regarding identification of MTB strains available data regarding the incidence of M. caprae, human infection does not reflect the actual size of the problem. Despite the fact that the possible routes of tuberculosis transmission are known, the epidemiological map of this zoonosis remains underestimated. The progress in diagnostic techniques, application of advanced methods of mycobacterium genome differentiation and cooperation between scientists in the field of veterinary medicine and microbiology, have a profound meaning for understanding the phenomenon of bovine tuberculosis and its supervise its incidence. This is the first bacteriologically confirmed case of human infection of M. caprae in Poland.

Selected factors of the innate immunity in Polish patients suffering from pulmonary tuberculosis.

We conducted a prospective study of 453 Polish patients suffering from pulmonary tuberculosis and 267 healthy controls. Selected polymorphisms of the genes encoding for collectins, ficolins and MBL-associated serine protease 2 were investigated as were serum concentrations of mannose-binding lectin, surfactant protein D, ficolin-1 and ficolin-3. The number of MBL2 gene exon 1 variant allele carriers was significantly higher in patients, compared with controls. The homozygosity for SFTPA2 +26 C > A SNP variant allele occurred less commonly within TB, while homozygosity for the FCN1 -542 G > A major allele was less frequent within the control group. Two patients were found MASP-2-deficient. Serum concentrations of MBL, SP-D and ficolin-1 were higher amongst patients while the converse was found for ficolin-3. Ficolin-1 had high specificity to differentiate between individuals with tuberculosis and healthy persons and therefore may be considered potential disease marker.

Molecular methods in diagnostics of post-BCG vaccine adverse events.

INTRODUCTION: Bacillus Calmette-Guérin (BCG) is the only tuberculosis vaccine available and although it has been routinely used for more than 80 years, its protective effect varies depending on the age and the form of tuberculosis. Due to the close analogy between the vaccine strain and other species of the Mycobacterium tuberculosis complex, molecular methods are recommended for differential diagnosis of post-BCG complications. The aim of the study was to assess usefulness of molecular methods in diagnosis of post-BCG vaccine adverse events (VAEs). MATERIAL AND METHODS: M. tuberculosis complex strains obtained in 2011-2017 from 68 ill children were subjected to molecular analysis. RESULTS: Molecular analysis of 68 strains showed 100% agreement between the results in the GenoType MTBC method and the multiplex PCR method. For the strains isolated from 45 patients with suspected VAE, M. bovis BCG was obtained, whereas the strains isolated from the remaining 23 children were identified as M. tuberculosis. The analysis confirmed the close relationship between the result of identification and the type of material as well as the patient's age. CONCLUSIONS: The use of genetic methods enables quick and detailed diagnostics of infections caused by M. bovis BCG, which allows for the confirmation or exclusion of VAE.

Tuberculosis transmission in the population of patients from the Krakow Region (Poland) based on the epidemiological and molecular methods.

BACKGROUND: The transmission of tuberculosis may affect the incidence rate of the disease in Poland. Genetic methods are of assistance in tracing the infection transmission, identifying its sources, determining the risk groups, and focusing on the preventive actions. OBJECTIVES: The objectives of this study lie in an assessment of tuberculosis transmission by genetic methods with the assistance of the standard epidemiologic interview. METHODS: The genome DNA of 275 Mycobacterium tuberculosis (Mtb) strains from tuberculosis patients, inhabitants of the city of Krakow, was subjected to a genetic analysis via the spoligotyping method and the IS6110-Mtb1-Mtb2 polymerase chain reaction (PCR) method. If the DNA profiles were identical in both of the PCRs, they were considered identical and classified within one molecular family. RESULTS: Among 275 strains, 104 genetic patterns (spoligotypes) were identified. Two hundred and three strains were divided into 66 molecular families (clusters) and analyzed with the IS6110- Mtb1-Mtb2 PCR method. Eighteen clusters were separated. In the Mtb1-Mtb2 clusters, 21 patients were in the risk groups (the homeless, prisoners, and nursing home residents). We did not confirm any direct or temporary contacts between the patients constituting the Mtb1-Mtb2 clusters (apart from the risk groups). However, the patients in these clusters often lived in the same parts of Krakow. CONCLUSIONS: The standard epidemiologic interview in tuberculosis patients should be combined with genetic methods. Active transmission of tuberculosis occurs largely among the individuals maintaining probably periodic contacts. The patients who are in the risk groups may play an important role in the transmission of tuberculosis.

Molecular characterisation of Mycobacterium caprae strains isolated in Poland.

Bovine tuberculosis (bovine TB, bTB) is caused by bovine bacilli: Mycobacterium bovis and M caprae The studies conducted in Poland, in the National Bovine Tuberculosis Reference Laboratory in the Department of Microbiology of the National Veterinary Research Institute in Pulawy, show that animal tuberculosis in Poland is also caused by M caprae We here describe the identification and genotypic assessment of 52 isolates of M caprae obtained from Polish cattle and wild animals over the last five years. We show that strains isolated from bison have significant genotypic diversity and are distinct compared with the genotypes of strains isolated from cattle. Similarly, isolates from cattle herds can be highly genotypically variable. Formal designation of the members of the Mycobacterium tuberculosis complex is controversial in Poland; there is a gap in veterinary legislation with regard to bTB and no explicit mention of M caprae causing tuberculosis in animal.

From Latent Tuberculosis Infection to Tuberculosis. News in Diagnostics (QuantiFERON-Plus).

It is estimated that one third of the world's population have latent tuberculosis infection and that this is a significant reservoir for future tuberculosis cases. Most cases occur within two years following initial infection. The identification of individuals with latent tuberculosis infection is difficult due to the lack of an ideal diagnostic assay and incomplete understanding of latent infection. Currently, there are three tests: the oldest tuberculin skin test, T-SPOT.TB and the latest QuantiFERON-Plus for the detection of Mycobacterium tuberculosis infection. The interpretation of the test results must be used in the conjunction with a patient's epidemiological history, risk assessment, current clinical status, radiography and microbiological methods to ensure accurate diagnosis.

The incidence of tuberculosis transmission among family members and outside households.

INTRODUCTION: The risk of Mycobacterium tuberculosis complex (MTBC) infection is correlated with the concentration of infectious particles and exposure time. In closed populations, healthy people staying in very frequent, close and prolonged contact with a smear-positive person, become infected and represent another link in the chain of transmission of the disease. Therefore, in the fight against tuberculosis, an important element is quick identification of the patient and potentially infected people from his environment. In epidemiological investigation of tuberculosis (TB), family members are brought under special control as they are particularly exposed to transmission of infectious diseases. MATERIAL AND METHODS: The study included 150 patients with bacteriologically confirmed tuberculosis who were members of 59 families. In the years 2003-2013 this population represented all TB cases detected in Poland in a family environment.Three PCR-based genotyping methods: spoligotyping, IS6110-Mtb1-Mtb2 PCR and MIRU-VNTR typing were used. RESULTS: Of 150 patients, 138 could be assigned to intra-household transmission on the basis of identical DNA fingerprints upon a combined typing approach. For 12 patients in 6 households, the M. tuberculosis isolates were clearly distinct in individual analysis - IS6110-Mtb1-Mtb2 PCR, spoligotyping or MIRU-VNTR typing or in three genotyping methods, suggesting that these patients were infected by the sources in the community. CONCLUSIONS: The analysis confirmed the transmission of tuberculosis among members of 53 families. In the remaining 6 families the source of infection were people outside the households. In all families with young children, strains isolated from them have identical DNA patterns as strains obtained from their adult caregivers. To confirm the transmission of TB in the study population of patients, epidemiological analysis required the addition of a genotyping methods characterised by high discriminatory power.

Transmission of tuberculosis among people living in the border areas of Poland, the Czech Republic, and Slovakia.

INTRODUCTION: In 2007, Poland, the Czech Republic, and Slovakia joined the Schengen Agreement, abolishing restrictions on people crossing the borders. Currently, these areas are places of population movements for economic, family, and touristic reasons. This favors the transmission of infectious diseases, including tuberculosis, and requires enhanced control over the spread of the source of infection in the population of patients living in the border areas. OBJECTIVES: The aim of this study was to investigate the genetic relatedness among Mycobacterium tuberculosis complex strains isolated from patients living in 3 border areas: Poland, the Czech Republic, and Slovakia. PATIENTS AND METHODS The study group consisted of 209 patients with tuberculosis diagnosed and treated between 2007 and 2011 in health care facilities in the Silesia Province in Poland (121 patients [58%]), Zilina in Slovakia (57 [27%]), and the Moravian-Silesian Region in the Czech Republic (31 [15%]). Genotyping of strains was performed using spoligotyping and IS6110-Mtb1-Mtb2 polymerase chain reaction. RESULTS: Among 209 strains, 23 molecular families (clusters) were identified. Seventeen clusters were identified as national. Six international clusters consisted of 30 strains isolated from patients of various nationalities. CONCLUSIONS: We identified 6 potential outbreaks of tuberculosis transmission between patients of different nationalities. The circumstances favorable to potential contacts of patients included mainly travelling to the neighboring countries, hospital stays, and addictions. However, there was no evidence of an epidemiological link between these patients, so it may be assumed that if they had come in contact with one another, it was accidental. We observed that the greater incidence of tuberculosis on the Polish territory did not affect the incidence in the Czech Republic or Slovakia over the analysis period.

Drug Resistance and Population Structure of Mycobacterium tuberculosis Beijing Strains Isolated in Poland.

In total, 1095 Mycobacterium tuberculosis clinical isolates from 282 patients with drug-resistant and 813 with drug-sensitive tuberculosis (TB) in Poland during 2007-2011 were analysed. Seventy-one (6.5%) patients were found to have strains of Beijing genotype as defined by spoligotyping. The majority of patients were Polish-born; among foreign-born a large proportion came from Chechnya and Vietnam. Analysis showed strong associations between Beijing genotype infection and MDR, pre-XDR and XDR resistance, with a considerable relative risk among new patients, suggesting that this is due to increased spread of drug-resistant strains rather than acquisition of resistance during treatment.