Voluntary counselling and testing points (VCTs) in Poland as a complementary and accessible alternative to public healthcare facilities: A comparative analysis of epidemiological data from the VCTs and the National Institute of Public Health NIH-National Research Institute (2015-2022).

OBJECTIVE: Voluntary counselling and testing points (VCTs) offer anonymous and free HIV tests in Poland. They also play an essential role in educational initiatives focused on the prevention and diagnosis of HIV and other sexually transmitted infections. However, no comprehensive data is available that summarizes the results of the work carried out by these VCTs. Therefore, our aim was to conduct a comparative analysis of epidemiological data obtained from VCTs and data reported by the epidemiological surveillance undertaken by the National Institute of Public Health NIH-National Research Institute (NIPH NIH-NRI) covering the period from 2015 to 2022. METHODS: This retrospective analysis was conducted on data from 258 071 people attending VCTs in Poland in 2015-2022. RESULTS: On average, 32 259 individuals underwent testing each year, with a notable increase in the number of people being tested in November. The average positivity rate was 1.39% (3576/258 071). The Masovian voivodeship conducted the most tests and had the highest number of positive results. The comparative analysis of the frequency of detecting positive results in VCTs and those reported in NIPH NIH-NRI data revealed that, on average, 31.49% (3576/11 356) of positive results in Poland between 2015 and 2022 were identified through tests conducted at VCTs. CONCLUSION: The positive results identified in VCTs constituted approximately one-third of all results reported by the National Institute of Public Health NIH-National Research Institute, highlighting the importance of VCTs. Moreover, the high availability of testing in the Masovian voivodeship resulted in better detection of HIV. The educational actions performed during European Testing Week increased the number of tests performed in November.

Development of a prototypic, field-usable diagnostic tool for the detection of gram-positive cocci-induced mastitis in cattle.

BACKGROUND: Bovine mastitis is one of the most widespread diseases affecting cattle, leading to significant losses for the dairy industry. Currently, the so-called gold standard in mastitis diagnosis involves determining the somatic cell count (SCC). Apart from a number of advantages, this method has one serious flaw: It does not identify the etiological factor causing a particular infection, making it impossible to introduce targeted antimicrobial therapy. This can contribute to multidrug-resistance in bacterial species. The diagnostic market lacks a test that has the advantages of SCC and also recognizes the species of pathogen causing the inflammation. Therefore, the aim of our study was to develop a lateral flow immunoassay (LFIA) based on elongation factor Tu for identifying most prevalent Gram-positive cocci responsible for causing mastitis including Streptococcus uberis, Streptococcus agalactiae and Staphylococcus aureus. RESULTS: As a result, we showed that the assay for S. uberis detection demonstrated a specificity of 89.02%, a sensitivity of 43.59%, and an accuracy of 80.3%. In turn, the second variant - assay for Gram-positive cocci reached a specificity of 95.59%, a sensitivity of 43.28%, and an accuracy of 78.33%. CONCLUSIONS: Our study shows that EF-Tu is a promising target for LFIA and we have delivered evidence that further evaluation could improve test parameters and fill the gap in the mastitis diagnostics market.

Molecular and phenotypic identification of bacterial species isolated from cows with mastitis from three regions of Poland.

BACKGROUND: Bovine mastitis is a widespread disease affecting dairy cattle worldwide and it generates substantial losses for dairy farmers. Mastitis may be caused by bacteria, fungi or algae. The most common species isolated from infected milk are, among others, Streptococcus spp., Escherichia coli, Staphylococcus aureus and non-aureus staphylococci and mammaliicocci. The aim of this paper is to determine the frequency of occurrence of bacterial species in milk samples from cows with mastitis from three regions of Poland: the north-east, the south-west and the south. To this end 203 milk samples taken from cows with a clinical form (CM) of mastitis (n = 100) and healthy animals (n = 103) were examined, which included culture on an appropriate medium followed by molecular detection of E. coli, S. aureus, Streptococcus agalactiae and Streptococcus uberis, as one of the most common species isolated from mastitis milk. RESULTS: The results obtained indicated that S. uberis was the most commonly cultivated CM species (38%, n = 38), followed by S. aureus (22%, n = 22), E. coli (21%, n = 21) and S. agalactiae (18%, n = 18). Similar frequencies in molecular methods were obtained for S. uberis (35.1%) and S. aureus (28.0%). The variation of sensitivity of both methods may be responsible for the differences in the E. coli (41.0%, p = 0.002) and S. agalactiae (5.0%, p = 0.004) detection rates. Significant differences in composition of species between three regions of Poland were noted for E. coli incidence (p < 0.001), in both the culture and molecular methods, but data obtained by the PCR method indicated that this species was the least common in north-eastern Poland, while the culture method showed that in north-eastern Poland E. coli was the most common species. Significant differences for the molecular method were also observed for S. uberis (p < 0.001) and S. aureus (p < 0.001). Both species were most common in southern and south-western Poland. CONCLUSIONS: The results obtained confirm the need to introduce rapid molecular tests for veterinary diagnostics, as well as providing important epidemiological data, to the best of our knowledge data on Polish cows in selected areas of Poland is lacking.

Antimicrobial Activity of N,N-Diethyldithiocarbamate against Ureaplasma parvum and Ureaplasma urealyticum.

Ureaplasma species (Ureaplasma spp.) are commonly found as commensals in the human urogenital tracts, although their overgrowth can lead to infection in the urogenital tract and at distal sites. Furthermore, ureaplasmas lack a cell wall and do not synthesize folic acid, which causes all ß-lactam and glycopeptide antibiotics, and sulfonamides and diaminopyrimidines, to be of no value. The antibiotics used in therapy belong to the fluoroquinolone, tetracycline, chloramphenicol and macrolide classes. However, the growing incidence of antibiotic-resistant Ureaplasma spp. in the population becomes a problem. Thus, there is a need to search for new drugs effective against these bacteria. Since 1951, the FDA-approved, well-tolerated, inexpensive, orally administered drug disulfiram (DSF) has been used in the treatment of chronic alcoholism, but recently, its antimicrobial effects have been demonstrated. The main biological metabolite of DSF, i.e., N,N-diethyldithiocarbamate (DDC), is generally believed to be responsible for most of the observed pharmacological effects of DSF. In the presented studies, the effect of DDC at concentrations of 2 µg/mL, 20 µg/mL and 200 µg/mL on the growth and survival of Ureaplasma urealyticum and Ureaplasma parvum was tested for the first time. The results indicated that all the used DDC concentrations showed both bacteriostatic and bactericidal activity against both tested strains.

The Two-Track Investigation of Fibronectin Binding Protein A of Staphylococcus aureus from Bovine Mastitis as a Potential Candidate for Immunodiagnosis: A Pilot Study.

Bovine mastitis is the most common disease affecting dairy cattle worldwide and it generates substantial losses for cattle breeders. One of the most common pathogens identified in infected milk samples is Staphylococcus aureus. Currently, there is no fast test for recognizing bacteria species on the market. The aim of this study was to bioinformatically and laboratory detect and characterize the fibronectin binding protein A (FnBPA) of S. aureus (SA) in milk samples obtained from cows diagnosed with mastitis. More than 90,000,000 amino acid sequences were subjected to bioinformatic detection in the search for a potential biomarker for bovine SA. The analysis of FnBPA included the detection of signal peptides and nonclassical proteins, antigenicity, and the prediction of epitopes. To confirm the presence of the fnbA gene in four SA isolates, amplification with specific primers was performed. FnBPA was detected by immunoblotting. The immunoreactivity and selectivity were performed with monoclonal anti-FnBPA antibodies and SA-negative serum. The bioinformatic analysis showed that FnBPA is a surface, conservative, immunoreactive, and species-specific protein with antigenic potential. Its presence was confirmed in all of the SA isolates we studied. Immunoblotting proved its immunoreactivity and specificity. Thus, it can be considered a potential biomarker in mastitis immunodiagnostics.

Improving Biocompatibility of Polyurethanes Apply in Medicine Using Oxygen Plasma and Its Negative Effect on Increased Bacterial Adhesion.

Polyurethanes (PUs) are versatile polymers used in medical applications due to their high flexibility and fatigue resistance. PUs are widely used for synthetic blood vessels, wound dressings, cannulas, and urinary and cardiovascular catheters. Many scientific reports indicate that surface wettability is crucial for biocompatibility and bacterial adhesion. The use of oxygen plasma to modify PUs is advantageous because of its effectiveness in introducing oxygen-containing functional groups, thereby altering surface wettability. The purpose of this study was to investigate the effect of the modification of the oxygen plasma of polyurethane on its biocompatibility with lung tissue (A549 cell line) and the adhesion of Gram-positive bacteria (S. aureus and S. epidermidis). The results showed that the modification of polyurethane by oxygen plasma allowed the introduction of functional groups containing oxygen (-OH and -COOH), which significantly increased its hydrophilicity (change from 105° ± 2° to 9° ± 2°) of PUs. Surface analysis by atomic force microscopy (AFM) showed changes in PU topography (change in maximum height from ∼110.3 nm to ∼32.1 nm). Moreover, biocompatibility studies on A549 cells showed that on the PU-modified surface, the cells exhibited altered morphology (increases in cell surface area and length, and thus reduced circularity) without concomitant effects on cell viability. However, serial dilution and plate count and microscopic methods confirmed that plasma modification significantly increased the adhesion of S. aureus and S. epidermidis bacteria. This study indicate the important role of surface hydrophilicity in biocompatibility and bacterial adhesion, which is important in the design of new medical biomaterials.

Did safety-engineered device implementation contribute to reducing the risk of needlestick and sharps injuries? Retrospective investigation of 20 years of observation in a specialist tertiary referral hospital.

OBJECTIVES: In Poland, there are numerous cases of injuries caused by sharp instruments annually, still significantly more than in other European Union countries. The aim of this study was to analyze work-related injuries among healthcare workers in a selected hospital before and after the implementation of safety-engineered devices (SED). MATERIAL AND METHODS: Retrospective analysis of medical documentation regarding occupational needlestick and sharps injuries (NSSI) in a tertiary referral surgical hospital in 1998-2018. The study group consisted of nurses and doctors who had been injured and reported the incident. The frequency of injury reports, injury rate, and characterization of circumstances surrounding NSSI are presented. RESULTS: Over the period of 20 years, a total of 257 NSSI incidents were reported. The average injury rate was statistically significant for nurses (p = 0.004) and was higher before the introduction of SED. Moreover, the number of injuries among nurses showed a downward trend during the study period. However, for doctors, there was no statistically significant difference in the median puncture rate (p = 0.099), and the number of injuries showed an increasing trend. CONCLUSIONS: In this study, the authors' have demonstrated not only the occurrence of injuries and punctures in the daily work of medical personnel but also the potential for their reduction through the use of safety equipment at every workstation where healthcare services are provided using sharp medical instruments. Int J Occup Med Environ Health. 2024;37(2):234-43.

Standardization of the protocol for oral cavity examination and collecting of the biological samples for microbiome research using the next-generation sequencing (NGS): own experience with the COVID-19 patients.

To date, publications have shown that compositions of oral microbiota differ depending on their habitats (e.g. tongue, tonsils, pharynx). The absence of set standards for the choice of the areas and conditions of material collection makes the oral microbiome one of the most difficult environments for a comparative analysis with other researchers, which is a meaningful limitation during an assessment of the potential effects of microorganisms as biomarkers in the courses of various human diseases. Therefore, standardisation of basic conditions of a dental examination and collection of material for the next generation sequencing (NGS) is worth attempting. The standardisation of the dental exam and collection of the clinical materials: saliva, swab from the tongue ridge, hard palate, palatine tonsils and oropharynx, supragingival plaque and subgingival plaque. Protocol involved the patients (n = 60), assigned to 3 groups: I-COVID-19 convalescents who received antibiotics, n = 17, II-COVID-19 convalescents, n = 23 and III-healthy individuals, n = 20. The collected biological samples were used to conduct NGS (16S rRNA). The conditions of patient preparation for collecting biological materials as well as the schedule of dental examination, were proposed. Based on the research conducted, we have indicated the dental indicators that best differentiate the group of COVID-19 patients (groups I and II) from healthy people (group III). These include the DMFT, D and BOP indices. The use of alpha and beta diversity analysis provided an overall insight into the diversity of microbial communities between specific niches and patient groups. The most different diversity between the studied group of patients (group II) and healthy people (group III) was noted in relation to the supragingival plaque. The order of activities during the dental exam as well as while collecting and securing clinical materials is particularly important to avoid technical errors and material contamination which may result in erroneous conclusions from the analyses of the results of sensitive tests such as the NGS. It has been shown that the dental indices: DMFT, D number, PI and BOP are the best prognostic parameters to assess the oral health. Based on beta diversity the most sensitive niche and susceptible to changes in the composition of the microbiota is the supragingival plaque. The procedures developed by our team can be applied as ready-to-use forms in studies conducted by other researchers.

Revealing bactericidal events on graphene oxide nano films deposited on metal implant surfaces.

At the time when pathogens are developing robust resistance to antibiotics, the demand for implant surfaces with microbe-killing capabilities has significantly risen. To achieve this goal, profound understanding of the underlying mechanisms is crucial. Our study demonstrates that graphene oxide (GO) nano films deposited on stainless steel (SS316L) exhibit superior antibacterial features. The physicochemical properties of GO itself play a pivotal role in influencing biological events and their diversity may account for the contradictory results reported elsewhere. However, essential properties of GO coatings, such as oxygen content and the resulting electrical conductivity, have been overlooked so far. We hypothesize that the surface potential and electrical resistance of the oxygen content in the GO-nano films may induce bacteria-killing events on conductive metallic substrates. In our study, the GO applied contains 52 wt% of oxygen, and thus exhibits insulating properties. When deposited as a nano film on an electrically conducting steel substrate, GO flakes generate a Schottky barrier at the interface. This barrier, consequently, impedes the transfer of electrons to the underlying conductive substrate. As a result, this creates reactive oxygen species (ROS), leading to bacterial death. We confirmed the presence of GO coatings and their hydrolytic stability by using X-ray photoelectron spectroscopy (XPS), µRaman spectroscopy, scanning electron microscopy (SEM), and Kelvin probe force microscopy (KPFM) measurements. The biological evaluation was performed on the MG63 osteoblast-like cell line and two selected bacteria species: S. aureus and P. aeruginosa, demonstrating both the cytocompatibility and antibacterial behavior of GO-coated SS316L substrates. We propose a two-step bactericidal mechanism: electron transfer from the bacteria membrane to the substrate, followed by ROS generation. This mechanism finds support in changes observed in contact angle, surface potential, and work function, identified as decisive factors. By addressing overlooked factors and effectively bridging the gap between understanding and practicality, we present a transformative approach for implant surfaces, combating microbial resistance, and offering new application possibilitie.

Do NAAT-Based Methods Increase the Diagnostic Sensitivity of Streptococcus agalactiae Carriage Detection in Pregnant Women?

The aim of the study was to evaluate particular polymerase chain reaction primers targeting selected representative genes and the influence of a preincubation step in a selective broth on the sensitivity of group B Streptococcus (GBS) detection by nucleic acid amplification techniques (NAAT). Research samples were vaginal and rectal swabs collected in duplicate from 97 pregnant women. They were used for enrichment broth culture-based diagnostics, bacterial DNA isolation, and amplification, using primers based on species-specific 16S rRNA, atr and cfb genes. To assess the sensitivity of GBS detection, additional isolation of samples preincubated in Todd-Hewitt broth with colistin and nalidixic acid was performed and then subjected to amplification again. The introduction of the preincubation step increased the sensitivity of GBS detection by about 33-63%. Moreover, NAAT made it possible to identify GBS DNA in an additional six samples that were negative in culture. The highest number of true positive results compared to the culture was obtained with the atr gene primers, as compared to cfb and 16S rRNA primers. Isolation of bacterial DNA after preincubation in enrichment broth significantly increases the sensitivity of NAAT-based methods applied for the detection of GBS from vaginal and rectal swabs. In the case of the cfb gene, the use of an additional gene to ensure the appropriate results should be considered.

In Vitro and In Silico Studies of Functionalized Polyurethane Surfaces toward Understanding Biologically Relevant Interactions.

The solid-aqueous boundary formed upon biomaterial implantation provides a playground for most biochemical reactions and physiological processes involved in implant-host interactions. Therefore, for biomaterial development, optimization, and application, it is essential to understand the biomaterial-water interface in depth. In this study, oxygen plasma-functionalized polyurethane surfaces that can be successfully utilized in contact with the tissue of the respiratory system were prepared and investigated. Through experiments, the influence of plasma treatment on the physicochemical properties of polyurethane was investigated by atomic force microscopy, attenuated total reflection infrared spectroscopy, differential thermal analysis, X-ray photoelectron spectroscopy, secondary ion mass spectrometry, and contact angle measurements, supplemented with biological tests using the A549 cell line and two bacteria strains (Staphylococcus aureus and Pseudomonas aeruginosa). The molecular interpretation of the experimental findings was achieved by molecular dynamics simulations employing newly developed, fully atomistic models of unmodified and plasma-functionalized polyurethane materials to characterize the polyurethane-water interfaces at the nanoscale in detail. The experimentally obtained polar and dispersive surface free energies were consistent with the calculated free energies, verifying the adequacy of the developed models. A 20% substitution of the polymeric chain termini by their oxidized variants was observed in the experimentally obtained plasma-modified polyurethane surface, indicating the surface saturation with oxygen-containing functional groups.

The Frequency of Occurrence of Resistance and Genes Involved in the Process of Adhesion and Accumulation of Biofilm in Staphylococcus aureus Strains Isolated from Tracheostomy Tubes.

Background: Bacterial biofilm on the surface of tracheostomy tubes (TTs) is a potential reservoir of potentially pathogenic bacteria, including S. aureus. For this reason, our study aimed to investigate biofilm production in vitro and the presence of icaAD and MSCRAMM genes in clinical S. aureus strains derived from TTs, with respect to antibiotic resistance and genetic variability. Methods: The clonality of the S. aureus strains was analyzed by the PFGE method. The assessment of drug resistance was based on the EUCAST recommendations. The isolates were evaluated for biofilm production by the microtiter plate method and the slime-forming ability was tested on Congo red agar (CRA). The presence of icaAD genes was investigated by PCR and MSCRAMM genes were detected by multiplex PCR. Results: A total of 60 patients were enrolled in the study. One TT was obtained from each patient (n = 60). Twenty-one TTs (35%) were colonized with S. aureus. A total of 24 strains were isolated as 3 patients showed colonization with 2 SA clones (as confirmed by PFGE). PFGE showed twenty-two unique molecular profiles. Two isolates (8%) turned out to be MRSA, but 50% were resistant to chloramphenicol, 25% to erythromycin and 8% to clindamycin (two cMLSB and four iMLSB phenotypes were detected). The microtiter plate method with crystal violet confirmed that 96% of the strains were biofilm formers. Representative strains were visualized by SEM. All isolates had clfAB, fnbA, ebpS and icaAD. Different MSCRAMM gene combinations were observed. Conclusions: the present study showed that the S. aureus isolated from the TTs has a high diversity of genotypes, a high level of antibiotic resistance and ability to produce biofilm.