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.

Evaluation of precipitation time of the aluminum salts adsorbed potentially frozen vaccines used in the Polish National Immunization Schedule for their pre-qualification before the administration.

Purpose: Vaccines adsorbed on aluminum adjuvants irreversibly lose potency after freezing and their safety is affected. To prevent the administration of such vaccines, the World Health Organization developed the Shake Test designed to determine whether adsorbed vaccines have been frozen or not. However, the Shake Test is difficult and time-consuming when routinely conducted at the place of vaccination. In this study, a modified shake test for prequalification of potentially frozen vaccines was elaborated. Materials and Methods: Vaccines used in the Polish Immunization Schedule were investigated and the analysis includes an assessment of precipitation time and the influence of the container type, amount and type of aluminum compound, and a volume of vaccine dose on the precipitation time. Results: Significant differences between the precipitation time of frozen and non-frozen vaccines routinely used in the Polish Immunization Schedule were observed. The precipitation time of all non-frozen vaccines was above 30 minutes. The longest precipitation time of frozen vaccines was 10 minutes. Conclusion: The finding of the study can be used in practice by the personnel administering vaccines to patients. Step-by-step recommendations for the preparation of the test have been proposed in the article.

Comparison of anti-SARS-CoV-2 IgG and IgA antibody responses post complete vaccination, 7 months later and after 3rd dose of the BNT162b2 vaccine in healthy adults.

BACKGROUND: The mRNA Covid-19 vaccine (BNT162b2) is administered in two doses with 21 days interval. On 4th October 2021 European Medicines Agency approved administration of a booster dose in at least 6 months after the second dose for people aged 18 years and older. OBJECTIVES: In the present study we compare the anti-SARS-COV-2 IgG and IgA antibody responses post complete vaccination, 7 months later and after the 3rd (booster) dose of the BNT162B2 vaccine in healthy adults. STUDY DESIGN: The levels of vaccine IgG and IgA antibodies to SARS-CoV-2 were assessed in serum samples obtained from individuals vaccinated with two doses and a booster of BNT162b2 vaccine. Samples were tested using the SARS-CoV-2 receptor-binding domain (RCB) IgG and IgA semi-quantitative commercial ELISA assay. RESULTS: The geometric mean of the anti-SARS-COV-2 IgG and IgA antibody level 7 months after vaccination of 90 healthy adults with BNT162B2 vaccine decreased significantly from 12.0 to 5.4 and 5.6 to 2.3, respectively. After the third dose of the same vaccine, the antibody level increased again, to values higher than at the beginning after the second dose. CONCLUSIONS: Significant decrease of antibody levels within a few months after full vaccination could result in the higher risk of SARS-CoV-2 infection, especially when new variants of the virus emerge. The booster could be crucial for protection against new SARS-CoV-2 variants. The antibody level seems to decrease slower in vaccinated individuals with history of COVID-19 and in younger individuals.

Diphtheria-tetanus-pertussis vaccine: past, current & future.

The diphtheria-tetanus-pertussis (DTP) vaccine can prevent diphtheria, tetanus and pertussis. The component antigens of the DTP vaccine had long been monovalent vaccines. The pertussis vaccine was licensed in 1914. The same year, the mixtures of diphtheria toxin and antitoxin were put into use. In 1926, alum-precipitated diphtheria toxoid was registered, and in 1937 adsorbed tetanus toxoid was put on the market. The development of numerous effective DTP vaccines quickly stimulated efforts to combine DTP with other routine vaccines for infants. This overview covers the most important information regarding the invention of DTP vaccines, their modifications and the needs that should be focused on in the future.

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.

Detection and Identification of Bacillus anthracis: From Conventional to Molecular Microbiology Methods.

Rapid and reliable identification of Bacillus anthracis is of great importance, especially in the event of suspected deliberate release of anthrax spores. However, the identification of B. anthracis is challenging due to its high similarity to closely related species. Since Amerithrax in 2001, a lot of effort has been made to develop rapid methods for detection and identification of this microorganism with special focus on easy-to-perform rapid tests for first-line responders. This article presents an overview of the evolution of B. anthracis identification methods from the time of the first description of the microorganism until the present day.

Molecular diagnosis of COVID-19 ­ present experiences / Molekularna diagnostyka COVID-19 ­ dotychczasowe doswiadczenia.

Severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2), a new highly emerging and pathogenic for human RNA virus, is responsible for the present COVID-19 pandemic. Molecular diagnostic methods, including real-time reverse transcription-PCR (RT-PCR) assay are the recommended methods for the identification and laboratory confirmation of COVID-19 cases. RT-PCR allows for detection the RNA of the virus in clinical specimens from patients suspected of COVID-19 with high specificity and sensitivity. Testing is still crucial for rapid detection of infected persons, implementation of appropriate measures to suppress further virus transmission and mitigate its impact. In response to demand of a molecular diagnostic test for SARS-CoV-2, within a first few months ongoing pandemic many commercial kits has become available on the market. However, these tests have varied in number and type of molecular targets, time of reaction as well as quality. In this study we compared different commercial tests for the detection of SARS-CoV-2 in clinical samples sending to Laboratory of Department of Virology, NIPH-NIH.

Physical and chemical changes in Alhydrogel™ damaged by freezing.

Accidental freezing of aluminum-based vaccines occurs during their storage and transportation, in both developed and developing countries. Freezing damages the freeze-sensitive aluminum adjuvanted vaccines, through separation of lattice between aluminum adjuvant and antigen, leading to formation of aluminum aggregates, and loss of potency. In this study, we examined Alhydrogel™ ([AlO(OH)]xnH2O, aluminum hydroxide, hydrated for adsorption) stored under recommended conditions, and exposed to freezing temperature until solid-frozen. The main purpose of our research was to determine the destruction areas of the solid-frozen Alhydrogel™ using selected methods of scanning electron microscopy, energy dispersive X-ray spectroscopy, Raman spectroscopy, Fourier-transform infrared spectroscopy and transmission electron microscopy working in diffraction mode. The Zeta potential evaluation, measurements of albumin adsorption power, thermogravimetric analysis and estimation of the mass loss after drying indicated significant structural (physical) and chemical differences between the freeze-damaged and non-frozen vaccine adjuvant. The presented results are important to better understand the type and nature of damages occurring in freeze-damaged aluminum-based vaccines. These results can be used in future studies to improve the temperature stability of aluminum adjuvanted vaccines.

Isothermal DNA amplification combined with lateral flow dipsticks for detection of biothreat agents.

The recently developed methods of nucleic acids isothermal amplification are promising tools for point-of-care diagnostics and in the field detection of pathogenic microorganisms. However, application of these methods outside a laboratory faces some challenges such as the rapid and sensitive detection of amplified products and the absence of cross-reactivity with genetically related microorganisms. In the presented study we compared three methods of isothermal DNA amplification loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA) and thermophilic helicase-dependent isothermal DNA amplification (tHDA), for detection of highly dangerous pathogens, such as Bacillus anthracis, Francisella tularensis and Yersinia pestis, and combined them with lateral flow dipsticks for the rapid visualization of amplified products. We observed low specificity of the three methods for B. antharcis, medium for Y. pestis and high for F. tularensis detection. Sensitivity and the detection limit were high and comparable for all the methods. We concluded that the lateral flow dipsticks have been a very useful tool for product detection of the isothermal amplification methods and enable reading the results without the use of any equipment. However, our results showed that the use of isothermal amplification methods is strongly related to the risk of false positive results.

Development and Evaluation of a Latex Agglutination Test for the Identification of Francisella tularensis Subspecies Pathogenic for Human.

Francisella tularensis are highly infectious bacteria causing a zoonotic disease called tularemia. Identification of this bacterium is based on antigen detection or PCR. The paper presents a latex agglutination test (LAT) for rapid identification of clinically relevant F. tularensis subspecies. The test can be performed within three minutes with live or inactivated bacteria. The possibility to test the inactivated samples reduces the risk of laboratory acquired infection and allows performing the test under BSL-2 conditions.

Injectional anthrax in human: A new face of the old disease.

Unusual human behavior leads to the emergence of new forms of infectious diseases and new routes of infection. In recent years, a new form of anthrax, called injectional anthrax, emerged and was related to 2 human anthrax outbreaks in Europe. The infection was caused by heroin contaminated with anthrax spores. The new form of anthrax differs from the earlier known "natural" forms of the disease in symptoms, length of the incubation period and recommended treatment. Despite medical treatment, the mortality rate in injectional anthrax is about 35%. This article presents an overview of the forms of anthrax infection in humans, with focus on injectional anthrax syndrome, as well as actual recommendations for treatment, including antibiotic therapy, surgery and possibilities of administering anthrax antitoxin. As a source of contamination of heroin have not been identified and new cases of injectional anthrax might occur again in any country in the future.

Changes in MLST profiles and biotypes of Corynebacterium diphtheriae isolates from the diphtheria outbreak period to the period of invasive infections caused by nontoxigenic strains in Poland (1950-2016).

BACKGROUND: Corynebacterium diphtheriae is a re-emerging pathogen in Europe causing invasive infections in vaccinated persons and classical diphtheria in unvaccinated persons. In the presented study we analysed genetic changes in C. diphtheriae isolates collected in Poland from the period before the introduction of the mass anti-diphtheria vaccination to the present time when over 98% of the population is vaccinated. METHODS: A total of 62 C. diphtheriae isolates collected in the 1950s-1960s, 1990s and 2000-2016 in Poland were investigated. Examined properties of the isolates included toxigenic status, presence of tox gene, biotype, MLST type (ST) and type of infection. RESULTS: A total of 12 sequence types (STs) were identified among the analysed C. diphtheriae isolates. The highest variability of STs was observed among isolates from diphtheria and asymptomatic carriers collected in the XX century. Over 95% of isolates collected from invasive and wound infections in 2004-2016 belonged to ST8. Isolates from the XX century represented all four biotypes: mitis, gravis, intermedius and belfanti, but the belfanti biotype appeared only after the epidemic in the 1990s. All except three isolates from the XXI century represented the biotype gravis. CONCLUSIONS: During a diphtheria epidemic period, non-epidemic clones of C. diphtheriae might also disseminate and persist in a particular area after the epidemic. An increase of the anti-diphtheria antibody level in the population causes not only the elimination of toxigenic strains from the population but may also influence the reduction of diversity of C. diphtheriae isolates. MLST types do not reflect the virulence of isolates. Each ST can be represented by various virulent variants representing various pathogenic capacities, for example toxigenic non-invasive, nontoxigenic invasive and nontoxigenic non-invasive.

[Whole cell protein profiling characterization of Corynebacterium diphtheriae clinical isolates collected from various infections].

INTRODUCTION: Corynebacterium diphtheriae can cause various infections such as diphtheria, wound infections, septic arthritis, bacteraemia and endocarditis. Different virulence properties of the isolates might be related to different virulence factors expressed by the isolates. The objective of this study was to explore whether whole cell protein profiling might be useful in prediction of pathogenic properties of C. diphtheriae isolates. METHODS: C. disphtheriae isolates collected from diphtheria, invasive and local infections and from asymptomatic carriers in Poland, France, New Caledonia and Canada in 1950-2014 were investigated using whole cell protein profile analysis. RESULTS: All the examined isolates were divided into two clades: A and B with similarity about 47%, but clade B was represented by only one isolate. The clade A was divided in two subclades A.I NS .II with similarity 53,2% and then into four groups: A.Ia, A.Ib, A.Ic and A.Id. The comparative analysis did not distinguish clearly toxigenic and nontoxigenic isolates as well as invasive and noninvasive isolates. CONCLUSIONS: Whole cell protein profile analysis of C. diphtheria exhibits good concordance with other genotyping methods but this method is not able to distinguish clearly invasive from non-invasive isolates.

[Vaccination during pregnancy].

Despite the enormous development of vaccinology in recent decades, vaccinations of preg- nant women are still controversy. According to data from the literature, most of them are not only effective but also safe. The paper discusses the issues of vaccination among preg- nant women, with special accent on the recommendations of the most important Institu- tions of Public Health for this group of women.

[Selection of disposable personal protective equipment for work with samples containing highly pathogenic microorganisms]. / Dobór jednorazowych srodków ochrony osobistej do pracy z materialem zawierajacym wysoce niebezpieczne patogeny.

Emerging microbiological threats, such as SARS, Ebola, MERS-CoV, anthrax, cause necessity of considering how effectively protect laboratory workers against dangerous pathogens which might be present in clinical samples. The article presents requirements for personal protective equipment (PPE) in microbiological laboratories and examples of selection and application of disposable PPE.

Increasing role of arthropod bites in tularaemia transmission in Poland - case reports and diagnostic methods.

The study describes four cases of tularaemia - one developed after contact with rabbits and three developed after an arthropod bite. Due to non-specific clinical symptoms, accurate diagnosis of tularaemia may be difficult. The increasing contribution of the arthropod vectors in the transmission of the disease indicates that special effort should be made to apply sensitive and specific diagnostic methods for tularaemia, and to remind health-care workers about this route of Francisella tularensis infections. The advantages and disadvantages of various diagnostic methods - molecular, serological and microbiological culture - are discussed. The PCR as a rapid and proper diagnostic method for ulceroglandular tularaemia is presented.

Screening for anthrax occurrence in soil of flooded rural areas in Poland after rainfalls in spring 2010.

INTRODUCTION AND OBJECTIVE: Anthrax spores remain viable and infectious in soil for decades. Flood water can percolate towards the surface the spores buried in soil. Moreover, the flood water might transport spores to areas previously unaffected. After the water recedes the spores located on the surface of the ground can be consumed by grazing animals and cause outbreaks of anthrax. MATERIALS AND METHOD: Soil samples were collected in areas of Poland most affected by floods in 2010 (Lubelskie, Swietokrzyskie, Podkarpackie and Mazowieckie provinces). After heating with the aim to kill vegetative forms of bacteria, the samples were cultured on PLET agar and the resulted colonies were investigated in terms of motility and presence of anthrax specific chromosomal (SG-749, plcR) and plasmid markers (capB, pagA). RESULTS: In total, 424 spore-forming, aerobically growing isolates were collected from the tested soil samples. Eighty-nine of them were non-motile. All the isolates were negative in PCR for anthrax specific chromosomal and plasmid markers. CONCLUSIONS: Spores of B. anthracis that could be related to risk of anthrax outbreaks were not detected in soil samples tested in this study. The negative results presented may not be proof that Poland is country free of anthrax. The results, however, may suggest a relatively low risk of anthrax outbreaks being triggered in the sampled areas.

Nontoxigenic highly pathogenic clone of Corynebacterium diphtheriae, Poland, 2004-2012.

Twenty-five cases of nontoxigenic Corynebacterium diphtheriae infection were recorded in Poland during 2004-2012, of which 18 were invasive. Alcoholism, homelessness, hepatic cirrhosis, and dental caries were predisposing factors for infection. However, for 17% of cases, no concomitant diseases or predisposing factors were found.