Adjuvant Effect of Whole-Cell Pertussis Component on Tetanus Toxoid Potency in Murine Model.

There is currently an increasing interest in the development of new-generation purified antigen-based vaccines with a higher safety profile compared to conventional inactivated vaccines. The main problem of subunit vaccines is their lower immunogenicity compared to whole-cell vaccines and inducing weaker and shorter-lasting immune responses. In this paper, the results of the assay of the potency of the tetanus component combined with the diphtheria component and whole-cell pertussis vaccine (DTwP), diphtheria and tetanus vaccine (DT), and in monovalent tetanus vaccine (T) are presented. In the mice model, an adjuvant impact of the whole-cell pertussis component on the immune response against tetanus was observed. It was noticed that the potency of tetanus component in the DTwP vaccine was significantly higher than tetanus potency in DT and T vaccines, despite the same bounding ability unit of the tetanus toxoid in the vaccine formulations. The levels of induction of tetanus antibodies by the tested vaccines were also examined. There were no differences in the induction of humoral responses against tetanus by tested vaccines. This publication discusses the possible mechanisms of impact of the whole-cell pertussis component on the other vaccine antigens and the positive and negative aspects of using the whole-cell pertussis component as an adjuvant.

New Corynebacterium Species with the Potential to Produce Diphtheria Toxin.

Only three Corynebacterium species are known to produce a lethal exotoxin called diphtheria toxin. These are C. diphtheriae, C. ulcerans and C. pseudotuberculosis. The diphtheria toxin gene (tox) is carried in a family of closely related corynebacteriophages and therefore the toxin can be produced only through lysogenisation, in which the corynephage encoding tox is stably inserted into the chromosome. However, 'nontoxigenic tox gene-bearing' (NTTB) strains, which are genotypically tox-positive but do not express the protein, have been described. The emergence of NTTB strains was first observed during the 1990s diphtheria epidemic in Eastern Europe and nowadays such isolates have been detected in many countries in the world. Recently, novel species of Corynebacterium genus have been described which might have the potential of producing the diphtheria toxin due to the possession of the diphtheria toxin gene but it has not produced toxin in laboratory tests. The circulation of NTTB strains could be related to the increased risk for diphtheria disease arising from the risk of re-emerging toxin expression. The article presents the mechanism of diphtheria toxin expression and action, recently described novel species of NTTB corynebacteria as well as the taxonomic changes within the C. diphtheriae group.

Detection of SARS-CoV-2 Using Reverse Transcription Helicase Dependent Amplification and Reverse Transcription Loop-Mediated Amplification Combined with Lateral Flow Assay.

Rapid and accurate detection and identification of pathogens in clinical samples is essential for all infection diseases. However, in the case of epidemics, it plays a key role not only in the implementation of effective therapy but also in limiting the spread of the epidemic. In this study, we present the application of two nucleic acid isothermal amplification methods-reverse transcription helicase dependent amplification (RT-HDA) and reverse transcription loop-mediated amplification (RT-LAMP)-combined with lateral flow assay as the tools for the rapid detection of SARS-CoV-2, the etiological agent of COVID-19, which caused the ongoing global pandemic. In order to optimize the RT-had, the LOD was 3 genome copies per reaction for amplification conducted for 10-20 min, whereas for RT-LAMP, the LOD was 30-300 genome copies per reaction for a reaction conducted for 40 min. No false-positive results were detected for RT-HDA conducted for 10 to 90 min, but false-positive results occurred when RT-LAMP was conducted for longer than 40 min. We concluded that RT-HDA combined with LFA is more sensitive than RT-LAMP, and it is a good alternative for the development of point-of-care tests for SARS-CoV-2 detection as this method is simple, inexpensive, practical, and does not require qualified personnel to perform the test and interpret its results.

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.

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.