Challenges of Diphtheria Toxin Detection.

Diphtheria toxin (DT) is the main virulence factor of Corynebacterium diphtheriae, C. ulcerans and C. pseudotuberculosis. Moreover, new Corynebacterium species with the potential to produce diphtheria toxin have also been described. Therefore, the detection of the toxin is the most important test in the microbiological diagnosis of diphtheria and other corynebacteria infections. Since the first demonstration in 1888 that DT is a major virulence factor of C. diphtheriae, responsible for the systemic manifestation of the disease, various methods for DT detection have been developed, but the diagnostic usefulness of most of them has not been confirmed on a sufficiently large group of samples. Despite substantial progress in the science and diagnostics of infectious diseases, the Elek test is still the basic recommended diagnostic test for DT detection. The challenge here is the poor availability of an antitoxin and declining experience even in reference laboratories due to the low prevalence of diphtheria in developed countries. However, recent and very promising assays have been developed with the potential for use as rapid point-of-care testing (POCT), such as ICS and LFIA for toxin detection, LAMP for tox gene detection, and biosensors for both.

The whole genome sequence of Polish vaccine strain Mycobacterium bovis BCG Moreau.

Currently, tuberculosis immunoprophylaxis is based solely on Bacillus Calmette-Guérin (BCG) vaccination, and some of the new potential tuberculosis vaccines are based on the BCG genome. Therefore, it is reasonable to analyze the genomes of individual BCG substrains. The aim of this study was the genetic characterization of the BCG-Moreau Polish (PL) strain used for the production of the BCG vaccine in Poland since 1955. Sequencing of different BCG lots showed that the strain was stable over a period of 59 years. As a result of comparison, BCG-Moreau PL with BCG-Moreau Rio de Janeiro (RDJ) 143 single nucleotide polymorphisms (SNPs) and 32 insertion/deletion mutations (INDELs) were identified. However, the verification of these mutations showed that the most significant were accumulated in the BCG-Moreau RDJ genome. The mutations unique to the Polish strain genome are 1 SNP and 2 INDEL. The strategy of combining short-read sequencing with long-read sequencing is currently the most optimal approach for sequencing bacterial genomes. With this approach, the only available genomic sequence of BCG-Moreau PL was obtained. This sequence will primarily be a reference point in the genetic control of the stability of the vaccine strain in the future. The results enrich knowledge about the microevolution and attenuation of the BCG vaccine substrains. IMPORTANCE: The whole genome sequence obtained is the only genomic sequence of the strain that has been used for vaccine production in Poland since 1955. Sequencing of different BCG lots showed that the strain was stable over a period of 59 years. The comprehensive genomic analysis performed not only enriches knowledge about the microevolution and attenuation of the BCG vaccine substrains but also enables the utilization of identified markers as a reference point in the genetic control and identity tests of the stability of the vaccine strain in the future.

Effectiveness of experimental and commercial pertussis vaccines in the elimination of Bordetella pertussis isolates with different genetic profiles in murine model.

The aim of this study was to compare the elimination of Bordetella pertussis clinical isolates, representing different genotypes in relation to alleles encoding virulence factors (MLST-multi-locus antigen sequence typing), MLVA type (multi-locus variable-number tandem repeat analysis) and PFGE group (pulsed-field gel electrophoresis) from the lungs of naive mice or mice were immunised with the commercial whole-cell pertussis vaccine, the acellular pertussis vaccine and the experimental whole-cell pertussis vaccine. Molecular data indicate that the resurgence of pertussis in populations with high vaccine coverage is associated with genomic adaptation of B. pertussis, to vaccine selection pressure. Pertactin-negative B. pertussis isolates were suspected to contribute to the reduced vaccine effectiveness. It was shown that one of the isolates used is PRN deficient. The mice were intranasally challenged with bacterial suspension containing approximately 5 × 10 7 CFU/ml B. pertussis. The immunogenicity of the tested vaccines against PT (pertussis toxin), PRN (pertactin), FHA (filamentous haemagglutinin) and FIM (fimbriae types 2 and 3) was examined. The commercial whole-cell and acellular pertussis vaccines induced an immunity effective at eliminating the genetically different B. pertussis isolates from the lungs. However, the elimination of the PRN-deficient isolate from the lungs of mice vaccinated with commercial vaccines was delayed as compared to the PRN ( +) isolate, suggesting phenotypic differences with the circulating isolates and vaccine strains. The most effective vaccine was the experimental vaccine with the composition identical to that of the strains used for infection.

Application of loop-mediated isothermal amplification combined with colorimetric and lateral flow dipstick visualization as the potential point-of-care testing for Corynebacterium diphtheriae.

BACKGROUND: Diphtheria outbreaks occurred in endemic areas and imported and indigenous cases are reported in UE/EEA. Because of the high infectiveness and severity of the disease, early and accurate diagnosis of each suspected case is essential for the treatment and management of the case and close contacts. The aim of the study was to establish simple and rapid testing methods based on Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Corynebacterium diphtheriae and differentiation between toxigenic and non-toxigenic strains. METHODS: Corynebacterium diphtheriae and Corynebacterium ulcerans isolates from the National Institute of Public Health-National Institute of Hygiene collection were used for the development of LAMP assay for the diagnosis of diphtheria and nontoxigenic C. diphtheriae infections. Various colorimetric methods for visualization of results were investigated. Sensitivity and specificity of the assay were examined using a collection of DNA samples from various gram-positive and gram-negative bacteria. RESULTS: The LAMP assay for tox and dtxR genes was developed. The sensitivity and specificity of the assay were calculated as 100%. The detection limit was estimated as 1.42 pg/µl concentration of DNA template when the reaction was conducted for 60 min. However, the detection limit was lowered 10 times for every 10 min of reduction in the time of incubation during the reaction. Positive results were successfully detected colorimetrically using hydroxynaphthol blue, calcein, QuantiFluor, and lateral flow Milenia HybriDetect dipsticks. CONCLUSION: The assay developed in the study might be applied for point-of-care testing of diphtheria and other C. diphtheriae infections as well as for other infections caused by diphtheria-toxin producing Corynebacterium species. It is highly sensitive, specific, inexpensive, easy to use, and suitable for low-resource settings.

Analysis of the Amino Acid Sequence Variation of the 67-72p Protein and the Structural Pili Proteins of Corynebacterium diphtheriae for their Suitability as Potential Vaccine Antigens.

The aim of this study was to identify the potential vaccine antigens in Corynebacterium diphtheriae strains by in silico analysis of the amino acid variation in the 67-72p surface protein that is involved in the colonization and induction of epithelial cell apoptosis in the early stages of infection. The analysis of pili structural proteins involved in bacterial adherence to host cells and related to various types of infections was also performed. A polymerase chain reaction (PCR) was carried out to amplify the genes encoding the 67-72p protein and three pili structural proteins (SpaC, SpaI, SapD) and the products obtained were sequenced. The nucleotide sequences of the particular genes were translated into amino acid sequences, which were then matched among all the tested strains using bioinformatics tools. In the last step, the affinity of the tested proteins to major histocompatibility complex (MHC) classes I and II, and linear B-cell epitopes was analyzed. The variations in the nucleotide sequence of the 67-72p protein and pili structural proteins among C. diphtheriae strains isolated from various infections were noted. A transposition of the insertion sequence within the gene encoding the SpaC pili structural proteins was also detected. In addition, the bioinformatics analyses enabled the identification of epitopes for B-cells and T-cells in the conserved regions of the proteins, thus, demonstrating that these proteins could be used as antigens in the potential vaccine development. The results identified the most conserved regions in all tested proteins that are exposed on the surface of C. diphtheriae cells.The aim of this study was to identify the potential vaccine antigens in Corynebacterium diphtheriae strains by in silico analysis of the amino acid variation in the 67­72p surface protein that is involved in the colonization and induction of epithelial cell apoptosis in the early stages of infection. The analysis of pili structural proteins involved in bacterial adherence to host cells and related to various types of infections was also performed. A polymerase chain reaction (PCR) was carried out to amplify the genes encoding the 67­72p protein and three pili structural proteins (SpaC, SpaI, SapD) and the products obtained were sequenced. The nucleotide sequences of the particular genes were translated into amino acid sequences, which were then matched among all the tested strains using bioinformatics tools. In the last step, the affinity of the tested proteins to major histocompatibility complex (MHC) classes I and II, and linear B-cell epitopes was analyzed. The variations in the nucleotide sequence of the 67­72p protein and pili structural proteins among C. diphtheriae strains isolated from various infections were noted. A transposition of the insertion sequence within the gene encoding the SpaC pili structural proteins was also detected. In addition, the bioinformatics analyses enabled the identification of epitopes for B-cells and T-cells in the conserved regions of the proteins, thus, demonstrating that these proteins could be used as antigens in the potential vaccine development. The results identified the most conserved regions in all tested proteins that are exposed on the surface of C. diphtheriae cells.

Pertactin-deficient Bordetella pertussis isolates in Poland-a country with whole-cell pertussis primary vaccination.

The introduction of pertussis vaccination in the 1950s resulted in a significant decrease in the incidence of disease. However, since the 1990s many highly vaccinated countries have observed the re-emergence of the disease. One of the causes of this phenomenon might be related to the adaptation of Bordetella pertussis to vaccination. The purpose of the presented study was an investigation of the emergence and spread of vaccine antigen-deficient B. pertussis isolates in Poland and genomic characterization of the currently circulating pathogen population using PFGE, MLVA and MAST. The results revealed that all tested isolates expressed Ptx, FHA and ACT antigens but 15.4% (4/26) of isolates from 2010 to 2016 were Prn-deficient. Moreover, one TcfA-deficient isolate was collected in 2015. The genotyping showed a genetic distinction between the isolates circulating in 2010-2016 and isolates from previous periods. The majority of currently circulating isolates belonged to PFGE group IV (96.2%), type MT27 (73.1%), and carried ptxA1-ptxC2-ptxP3-prn2-tcfA2-fim2-1-fim3-1 alleles (61.5%). The unique genetic structure of the B. pertussis population in Poland has changed since 2010 and became similar to that observed in countries with aP vaccination. This could be a result of increasing use of aP vaccines (60% of primary vaccination in 2013) over wP vaccines, which have been broadly used for primary vaccination in Poland for decades.

MLST and RAPD molecular analysis of Staphylococcus aureus subsp. anaerobius isolated from goats in Poland.

Staphylococcus aureus subsp. anaerobius is an etiological agent of Morel's disease in small ruminants. The infection results in superficial abscesses located near lymph nodes. In the study, molecular analysis based on multilocus sequence typing (MLST) of seven housekeeping genes (arcC, aroE, glp, gmk, pta, tpi, yqiL) and random amplified polymorphic DNA (RAPD) was carried out on 19 S. aureus subsp. anaerobius strains isolated from two different goat herds from Poland. All of the 19 S. aureus subsp. anaerobius strains were found to belong to single MLST and RAPD types which support the high clonality level of this agent. However, the results obtained show clearly that the S. aureus subsp. anaerobius clone found in goats in Poland is different from those previously described. However, it is identical to the ATCC 38844 strain isolated from sheep in Spain, which has not been so far genotyped using MLST.

Multi-locus variable-number tandem repeat analysis of Bordetella pertussis isolates circulating in Poland in the period 1959-2013.

PURPOSE: Despite the long history of pertussis vaccination and high vaccination coverage in Poland and many other developed countries, pertussis incidence rates have increased substantially, making whooping cough one of the most prevalent vaccine-preventable diseases. Among the factors potentially involved in pertussis resurgence, the adaptation of the Bordetella pertussis population to country-specific vaccine-induced immunity through selection of non-vaccine-type strains still needs detailed studies. METHODOLOGY: Multi-locus variable-number tandem repeat analysis (MLVA), also linked to MLST and PFGE profiling, was applied to trace the genetic changes in the B. pertussis population circulating in Poland in the period 1959-2013 versus country-specific vaccine strains. RESULTS: Generally, among 174 B. pertussis isolates, 31 MLVA types were detected, of which 11 were not described previously. The predominant MLVA types of recent isolates in Poland were different from those of the typical isolates circulating in other European countries. The MT27 type, currently predominant in Europe, was rarely seen and detected in only five isolates among all studied. The features of the vaccine strains used for production of the pertussis component of a national whole-cell diphtheria-tetanus-pertussis (DTP) vaccine, as studied by MLVA and MLST tools, were found to not match those observed in the currently circulating B. pertussis isolates in Poland. CONCLUSIONS: Differences traced by MLVA in relation to the MLST and PFGE profiling confirmed that the B. pertussis strain types currently observed elsewhere in Europe, even if appearing in Poland, were not able to successfully disseminate within a human population in Poland that has been vaccinated with a whole-cell pertussis vaccine not used in other countries.

Microevolution of BCG substrains.

Tuberculosis was, and still is, one of the main causes of morbidity and mortality in the world. Thus it still remains a public health priority. Nonetheless, without a newly developed vaccine, it is rather unlikely to be easily resolved. The only available vaccine against tuberculosis (BCG) has been used for nearly 100 years. Currently a variety of BCG substrains are used by many manufacturers in the world. All these substrains were obtained from a single parental strain of Mycobacterium bovis. Attempts to explain the complete mechanisms of attenuation, as well as tracing the microevolution resulting from the different distribution time and conditions of production of BCG vaccines in the different parts of the world, might explain the differences in the observed efficacy of vaccines produced with different substrains. The most important marker associated with attenuation of virulent M. bovis is the loss of the RD1 region observed in all BCG substrains. Among other attenuation markers, still not completely identified, accumulation of SNP mutations seems to be an important one. The different number of passages and culture conditions of the parental vaccine strain have led to there being about 50 different sister vaccine BCG substrains throughout the world. Among them, there are "early strains", distributed until 1927, and "later strains" with the RD2 deletion obtained during 1927­1961. It has also been found that 22 regions containing 52 genes were lost during the distribution of sister substrains during the period 1924­1966. Genetic differences due to selection pressure, revealing specific microevolutionary traits, may explain the variability in immunogenicity and residual virulence of each vaccine BCG substrain.

[Susceptibility of Lactobacillus rhamnosus strains isolated from probiotic products to antimicrobial agents]. / Lekowrazliwosc szczepów Lactobacillus rhamnosus wyizolowanych z produktów probiotycznych.

INTRODUCTION: Probiotics Generally Recognized as Safe (GRAS), when given in relevant dose, are able to induce strain-specific beneficial effects for health of humans or animals. METHODS: L. rhamnosus strains originating from four medicinal products, 2 dietary foods for special medical purposes and dietary supplement, were tested for susceptibility to antibiotics and chemotherapeutics following L. rhamnosus and L.rhamnosus GG strain identity confirmation with use of PCR, rep-PCR and AFLP methods. RESULTS AND CONCLUSIONS: L. rhamnosus working seeds of medicinal products and isolates originating from dietary foods for special medical purposes or dietary supplement were found correctly classified on the levels of species or L. rhamnosus GG strain identities. Antibiotics and chemotherapeutics susceptibility profiles of L. rhamnosus strains allowed for choice of treatment options in six out of seven products under study.

PCR-based genomic deletion analysis of RD-regions in the identification of mycobacteria isolated from adverse events following BCG vaccination or TB suspected cases.

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.

[Advances in the development of new vaccines against tuberculosis. 100 years after the introduction of BCG]. / Postepy w dziedzinie opracowania nowych szczepionek przeciw gruzlicy. 100 lat po wprowadzeniu BCG.

The BCG vaccine used in the world for nearly 100 years protects children against the most severe forms of tuberculosis, but its effectiveness in preventing the most commonly occurring tuberculosis and the one burdened with the highest risk of transmission in adults is very diverse. Contraindications for BCG vaccination include HIV infection and other conditions of immunosuppression. Tuberculosis is a global problem difficult to control because of three main reasons: poor diagnostics in developing countries, long-term therapy or discontinuation of treatment resulting in the emergence of drug-resistant mycobacteria, and the availability of a TB vaccine which only protects children from the most severe forms of tuberculosis. BCG has little to no efficacy in preventing the most common adult pulmonary TB. The development of a more effective vaccine against tuberculosis is undoubtedly still a public health priority in order to improve control of the disease throughout the world. Elimination of TB as a global public health goal by 2050 is particularly ambitious and its achievement depends on the development and application of new intervention measures and newly designed vaccines. Currently, 14 newly developed products are undergoing clinical trials. These include a prophylactic vaccine capable of replacing the current BCG, booster vaccines to increase the effects of BCG, and therapeutic vaccines. The aim of the study is to present the current state of knowledge on cutting-edge research into new vaccines against tuberculosis, their efficacy, immunogenicity and potential use in the future.

Consistency of Bordetella pertussis vaccine seed strains and potency of whole-cell pertussis vaccine still in use in Poland.

In Poland, where the wP vaccine has been used since 1960, pertussis rates increased in the mid-1990s. In 2012, the rate of pertussis recognised by surveillance was unexpectedly found to be two-fold higher than in the previous decade. Quality measures on potency and vaccine working seeds were introduced, to confirm the possible impact of manufacturing inconsistency or potency lowering on the observed increase in pertussis. Shewhart charts on potency values for lots released between 2001 and 2013 did not reveal any significant fluctuations. Working seeds of three vaccine strains used within last decade for wP manufacturing belong to the PFGE group III and were highly related. According to PFGE and SDS-PAGE data, all vaccine strains were found consistent according profiling on the genomic and protein levels. According to the sequencing data, they harboured ptxA2, ptxC1, prn1, fim2-1, fim3-1, tcfA2, ptxP1 and were assigned as MLST-2 type. Other factors apart from vaccine manufacturing inconsistency might be responsible for the increase in pertussis noted in 2012 in Poland.

[Phenotypic and genotypic characterization of probiotic bacterial strains used in medicinal products]. / Fenotypowa i genotypowa charakterystyka probiotycznych szczepów bakteryjnych stosowanych w produktach leczniczych.

INTRODUCTION: The optimization of quality testing strategy of products containing probiotics might allow to general improvement of its safer use in humans. The goal of the study was the evaluation of quality expressed by identity, colony forming unit (CFU) and antibiotic sensitivity ofprobiotics used in medicinal products available in Poland using the appropriate and validated procedures. METHODS: The medicinal products containing L. rhamnosus, L. acidophilus, L. delbrueckii subsp. bulgaricus and B. animalis subsp. lactis, L. helveticus, and L. gasseri were tested for species identity performed with validated rep-PCR (BOXA 1R) method. The antimicrobial susceptibility of working seeds and strains isolated to 26 antibiotics were tested by disk diffusion and E-test methods using relevant references as recommended by EUCAST. The numbers of probiotic strains, expressed as cfu count per package, was done using plating plunge method. RESULTS: All strains tested, except B. lactis, were found to be resistant to trimethoprim-sulphamethoxazole, nalidixic acid, metronidazole, and colistin. B. lactis was resistant to aminoglycosides. L. rhamnosus strains were found to be resistant to vancomycin, (MIC > 256 microg/ml) similarly to ATCC strains (L. rhamnosus GG 53103 and 244). The sensitivity to other antibiotics was strain specific. The rep-PCR method was found species and strain specific. All products tested fulfilled declared countent as measured by cfu count/package. CONCLUSIONS: Quality of medicinal products containing probiotics was found undoubted and confirmed. The optimized strategy of quality monitoring of probiotics used in medicinal products can be used in dietary supplements and foodstuffs intended for particular nutritional uses.

Multiplex-PCR as an identity assay for Mycobacterium bovis BCG Moreau descendants.

In the study, we assessed the identity of locally produced BCG vaccine via screening for the presence of genetic markers specific for particular Mycobacterium bovis BCG substrains - RD8, RD2, senX3-regX3, RD14, RD16, ΔRD1, DU2, a second copy of IS6110, mutation D322G in phoR, and deletions in fadD26-ppsA and Rv3887c regions. In order to increase the specificity of the multiplex-PCR test for locally produced BCG vaccine, we have modified previously developed primer sets by the introduction of a primer pair specific for deletion in Rv3887c. The modified multiplex-PCR specifically and reproducibly distinguished both BCG Moreau sublineages, and allowed, with no decrease in power, differentiation of BCG substrains of different origin. The growing knowledge of genetic differences among BCG vaccine strains enables improvements in the specificity of identity tests that will be useful both for routine release of vaccines and potential applications in clinical practice. Modified multiplex-PCR accompanied by PFGE analysis can serve as specific tools to monitor consistency in BCG manufacture.

[Reproducibility of Fim2 and Fim3 antigens determination in Bordetella pertussis by serotyping method]. / Ocena porównywalnosci oznaczen obecnosci antygenów Fim2 i Fim3 Bordetella pertussis metoda serotypowania.

INTRODUCTION: Serotyping is a commonly used method to characterize the presence of Fimbriae 2 and 3 in Bordetella pertussis strains for epidemiological purposes and optimal choice of strain composition of the pertussis whole-cell vaccine. Monoclonal antisera against Fim2 and Fim3 are recommended to be used for microplate serotyping instead ofpolyclonal. Reliable evaluation offimbriae expressed by B. pertussis strains influence interpretation of vaccine-driven strain evolution. METHODS: To evaluate the impact of tests conditions on the reproducibility of serotyping, results of serotyping based on a standardized protocol for microplate agglutination with monoclonal antisera performed in three different accredited laboratories were compared. For the study isolates of three vaccine strains of B. pertussis deposited within seed lot system originating from different liofilization lots were compared. RESULTS: Lack of the complete agreement on serotyping results among three labs might relates to the differences of media used, subjective reading, test conditions, and specificity of the reagents. CONCLUSIONS: Serotyping results should be interpreted with caution and the type of media and culture conditions used should be precisely recommended after validation studies. Inconsistent results should be confirmed using an alternative technique, eg. ELISA or by reference laboratory.

The rate of adverse events following BCG vaccination in Poland.

PURPOSE OF THE STUDY: The purpose of the study was to evaluate the capacity of the surveillance system to respond to the schedule changes in the view of TB vaccination uptake. Complications of Bacillus Calmette-Guerin (BCG) vaccination in Poland are as elsewhere uncommon. In Poland, BCG vaccination with a vaccine produced with Mycobacterium bovis BCG Moreau has been a part of the National Immunization Program since 1955. In the beginning the immunization schedule involved several BCG revaccinations in children and youths, with the first dose given to neonates up to 1 month old followed by revaccinations at 2, 4, 7, 12, 15, and 18 years of life. In 90s, the number of BCG doses was reduced and since 2006, according to recommendations made by the WHO, a single BCG dose is given to neonates only. METHODS: In the study we have analyzed data on adverse events following BCG vaccination registered within a period of 1994-2010, with attention to the periods before and after 2006, when different BCG vaccination schedules were used for immunization. RESULTS: The frequency of adverse events following BCG vaccination in Poland oscillated within 1994-2000 and 2001-2010 periods around 0.2 per thousand and 0.6 per thousand respectively, and in half consisted of local lesions at the injection sites and in half--appeared in the form of the regional lymphadenopathy. The analysis of surveillance data revealed similar rates of adverse events following BCG vaccination in the periods of different BCG vaccination schedules, eg. before and after 2006. CONCLUSIONS: Improvements in the data collecting manner from passive to active one and the introduction of the routine laboratory confirmation of the infection might evaluate the real prevalence of Mycobacterium bovis BCG infections and improve the treatment of adverse events following BCG vaccination cases.

Descendant of daughter Brazilian BCG Moreau substrain in Poland.

In this study we assessed the genomic stability of Mycobacterium bovis BCG Moreau seed lots used in Poland for BCG vaccine production since 1955 by pulsed field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) and random amplification of polymorphic DNA (RAPD). BCG vaccine lots were more closely related the original lot -M. bovis BCG Rio de Janeiro Moreau compared with seeds used before 1980, which is consistent with seed lot distribution recorded in the archives. We confirmed the presence of RD8, RD2, senX3-regX3, RD14, DU2-I, whiB3, trcR, the second copy of IS6110 inserted in the promoter region of phoP, mutation D322G in phoR, ΔRD1, and ΔfadD26-ppsA in M. bovis BCG Moreau used for BCG production in Poland. However, unlike the Rio de Janeiro parent BCG, the BCG Moreau substrain used in Poland does not harbour a deletion in Rv3887c, a region that is involved in the membrane transport protein that is part of the ESX-2 type VII secretion system. Differences in the distribution of BCG Moreau for its subsequent use for manufacturing influenced the microevolution of BCG Moreau used in Brazil and Poland.

The capacity of Mycobacterium tuberculosis complex species and M. bovis BCG substrains specific identification - implications for optimized PCR-based diagnostics in adverse events following vaccination suspected cases.

The capacities of differentiation of Mycobacterium bovis BCG from other members of M. tuberculosis complex species using PCR-RFLP, multiplex PCR, and PCR-based genomic deletion analysis approaches were compared. In the study, mycobacteria isolated from patients suspected of adverse events following vaccination with BCG, primarily classified according presence of RD1 marker as virulent and avirulent mycobacteria, were used. The PCR-based genomic deletion analysis was found the best option for mycobacteria diagnostics improvement, as it was capable precisely differentiate virulent and avirulent mycobacteria or virulent species of M. tuberculosis complex. The routine confirmation of mycobacteria species in the cases of adverse events following BCG vaccination is highly expected, especially in clinical practice of patients with primary immunodeficiency.