An alternative method for SARS-CoV-2 detection with use modified fluorescent in situ hybridization.

The real-time reverse-transcriptase polymerase-chain-reaction (rRT-PCR) tests are the gold standard in detecting SARS-CoV-2 virus infection. However, despite high sensitivity and specificity, they have limitations that in some cases may result in false negative results. Therefore, it is reasonable to search for additional tools that could support microbiological diagnosis of SARS-CoV-2. The aim of the study was to develop a highly specific molecular test capable of detecting and visualizing SARS-CoV-2 infection. A universal probe and a set of 18 specific oligonucleotides with a FLAP sequence attached to them on both sides were designed to visualize SARS-CoV-2 virus infection based on the fluorescence in situ hybridization method (FISH). FISH conditions using the developed kit were standardized on the Vero CCL-81 cell line infected by SARS-CoV-2 virus. The method was tested on 290 nasopharyngeal swabs (collected in a doublet) from patients with clinical symptoms of SARS-CoV-2. Each one swab from the doublet was subjected to RNA isolation and amplification by rRT-PCR. From the second swab, a microscopic preparation was performed for FISH. The use of the rRT-PCR allowed obtaining 200 positive and 90 negative results, while our FISH method allowed for 220 positive results and 70 negative results. The differences obtained using both methods were statistically significant (p = 0.008). The obtained results support the use of FISH as an additional method in microbiological diagnostics of SARS-CoV-2.

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.

Oral microbiota study of the patients after hospitalisation for COVID-19, considering selected dental indices and antibiotic therapy using the next generation sequencing method (NGS).

Background: Poor oral hygiene and the increased incidence and severity of periodontitis may exacerbate SARS-CoV-2 infection. The aim was to evaluate the oral microbiota of 60 participants divided into groups: COVID-19 convalescents who received antibiotics during hospitalization (I), COVID-19 convalescents without antibiotic therapy (II) and healthy individuals (III). Materials and Methods: Dental examination was conducted, and oral health status was evaluated using selected dental indexes. Clinical samples (saliva, dorsal swabs, supragingival and subgingival plaque) were collected and used for metagenomic library to the next-generation sequencing (NGS) preparation. Results: Each of the clinical materials in particular groups of patients showed a statistically significant and quantitatively different bacterial composition. Patients from group I showed significantly worse oral health, reflected by higher average values of dental indexes and also a higher percentage of Veillonella, Tannerella, Capnocytophaga and Selenomonas genera in comparison to other groups. Additionally, a statistically significant decrease in the amount of Akkermansia type in both groups with COVID-19 was observed for all materials. Conclusions: The primary factor affecting the composition of oral microbiota was not the SARS-CoV-2 infection itself, but the use of antibiotic therapy. The increased percentage of pro-inflammatory pathogens observed in COVID-19 patients underscores the importance of preventing periodontal disease and improving oral hygiene in the future.

Stethoscopes or Maybe "Bacterioscopes" - Is hand Hygiene Solely Capable of Preventing Hospital-Associated Infections?

The stethoscope remains an indispensable diagnostic tool for medical students. Improper stethoscope hygiene may cause bacterial infections, including hospital-associated infections (HAIs), which challenge the Polish medical system. The study's main objective was to evaluate the hygiene habits declared by medical students. Moreover, microbiological control with the characteristics of potentially pathogenic microorganisms was performed. The study included 66 medical students from the Faculty of Medicine at the Jagiellonian University Medical College in Cracow, Poland. The participants filled in an anonymous questionnaire. Stethoscope contamination was assessed through isolation, identification, testing of antibiotic resistance, and clonality of the isolates bacterial pathogens. The survey showed that only 30.3% of students cleaned their stethoscopes after each patient, and 1.5% never did this. Of the 66 stethoscopes tested, 100% were positive for bacterial growth. Staphylococcus spp. was the most frequently isolated contaminant (50.5%). The questionnaire results demonstrated the necessity of the validated procedures for cleaning the stethoscopes. Stethoscopes used by medical students are contaminated with numerous bacterial species, including multidrug-resistant organisms. The clonal structure of the MRSA and MRSE populations acquired from stethoscopes has been demonstrated. Our results confirm the possibility that these medical devices mediate the spread of hazardous pathogens in the hospital environment. Practical exercises are essential to forming the correct hygiene habits involving stethoscopes, which enable practicing and checking the correctness of the established skills.

New insights into diversity of the upper respiratory tract microbiota and its relationship with SARS-CoV-2 viral load in the nasopharyngeal epithelial cells in patients with COVID-19.

INTRODUCTION: The effects of SARS­CoV­2 infection on the composition of the upper respiratory tract (URT) microbiota are yet to be established, and more attention to this topic is needed. OBJECTIVES: The study aimed to assess the bacterial profile and the possible association between the URT microbiota composition and the SARS­CoV­2 viral load. PATIENTS AND METHODS: Nasopharyngeal swabs were taken from 60 adult patients with SARS­CoV­2 infection who were divided into 3 groups based on the quantification cycle (Cq) value in the quantitative polymerase chain reaction test: group I (n = 20), Cq lower than or equal to 31 (high replication rate); group II (n = 20), Cq greater than 31 and lower than 38 (low replication rate), and group III (n = 20), Cq higher than or equal to 38 (virus eliminated from the nasopharyngeal epithelial cells). The obtained genetic libraries of 16S rRNA were sequenced and taxonomic diversity profiling was performed to determine the α- and ß­biodiversity in each group. RESULTS: A significantly lower abundance of Prevotella species was noted in group I, as compared with groups II and III. Akkermansia muciniphila, Faecalibacterium prausnitzii, Fusicatenibacterium saccharivorans, and Bacteroides dorei abundance was characteristic of and significantly greater in group I than in groups II and III. Overall, the microbiota composition was the most diverse in group I, whereas groups II and III were more homogenous in terms of taxonomic diversity. CONCLUSIONS: The arbitrary division of patients according to the SARS­CoV­2 viral load was reflected in diverse composition of their bacterial microbiota, which implies an association between these 2 factors. The patients with a low viral replication rate and those who eliminated the virus from the epithelial cells belonged to a group with a less diverse microbiota community than the patients with a high viral replication rate.

Repetitive transcranial direct current stimulation modulates the brain-gut-microbiome axis in obese rodents.

BACKGROUND: Complex interactions between the brain, gut and adipose tissue allow to recognize obesity as a neurometabolic disorder. The recent data have shown that gut microbiota can play a potential role in obesity development. Transcranial direct current stimulation (tDCS) is a safe and non-invasive technique to modulate the activity of cerebral cortex and other connected brain areas also in context of appetite control. The objective of this study was to evaluate the effects of repetitive anodal tDCS (AtDCS) of prefrontal cortex on feeding behavior, metabolic status and selected phyla of gut microbiota in rats with obesity induced by high-calorie diet (HCD). METHODS: 32 female Wistar rats were equally divided into 4 subgroups depending on diet effect (lean versus obese) and type of stimulation (active versus sham tDCS versus no stimulation). Feed intake, body weight, blood lipoproteins and leptin levels as well as Firmicutes and Bacteroidetes in intestines and stool were examined. RESULTS: HCD changed feeding behavior and metabolic parameters typically for obesity-related ranges and resulted in an abundance of Firmicutes at the expanse of Bacteroidetes in the large intestine and stool. AtDCS decreased appetite, body weight, and cholesterol levels. In addition, AtDCS reduced ratio of the average number of Firmicutes to average number of Bacteroidetes in all examined tissues. CONCLUSIONS: Repetitive AtDCS is not only effective for appetite restriction but can also modulate gut microbiome composition which demonstrates the existence of the brain-gut-microbiome axis and points at this technique as a promising complementary treatment for obesity. However, the effects should be further replicated in human studies.

Vaginal Secretion Epithelium Count as a Prognostic Indicator of High Abundance of Ureaplasmas in Women with a Normal Nugent Score.

Genital tract ureaplasma infections are associated with numerous complications, ranging from inflammation, through infertility, to problematic pregnancy. In the course of ureaplasma infection, the risk of human papillomavirus infection increases. Diagnostic tests for urea-plasma infections are not always carried out, especially in women with the normal Nugent test results. The study attempts to check whether it is possible to find a prognostic indicator that could suggest a high abundance of ureaplasmas (≥ 104 CFU/ml) at the stage of the initial examination of vaginal discharge. Such a prognostic factor could qualify women for further tests to detect infections with these atypical bacteria. Six hundred twenty-seven white women with a score of 0-3 on the Nugent scale were tested, including 322 patients with a high abundance of ureaplasmas (≥ 104 CFU/ml) and 305 who tested negative for these bacteria. Ureaplasma infections were detected statistically significant in women who had few or no epithelial cells in the genital swab specimens compared to the results obtained for women with numerous or very numerous epithelial cells (p < 0.001). The risk of the high density of ureaplasmas was 38.7% higher with fewer or no epithelial cells than with high numbers. In patients aged 18-40 years with few or no epithelial cells, a high density of ureaplasmas (≥ 104 CFU/ml) was observed significantly more frequently (p = 0.003). Determining the number of epithelial cells in Gram-stained slides may be the prognostic indicator of ureaplasma infection. Testing for genital ureaplasma infection should be considered, especially in women of childbearing age (18-40 years), even if the Nugent test value is normal and pH ≤ 4.6.

Sarcoptes Infestation. What Is Already Known, and What Is New about Scabies at the Beginning of the Third Decade of the 21st Century?

Currently, there are three known subtypes of scabies: ordinary, crusted, and bullous. The worldwide prevalence of scabies remains high in the 21st century. To decrease the social, economic, and psychological impact on the enormous population infected, a lot of important work has been completed over the last 20 years concerning the management of scabies. For example, a standardization of guidelines for the treatment of scabies has been completed and programs have been designed for the prevention and treatment in endemic populations, called mass drug administrations. Unfortunately, these only apply to the ordinary form of scabies. Moreover, resistance to the drugs currently used in treatment is growing, which imposes the need to search for new treatments. For this purpose, new acaricides are being developed to enhance the therapeutic options for the patients' benefit and effectively treat this disease. There is also the necessity for prevention before the development of scabies. An effective vaccine has the potential to protect people before this disease, especially in endemic areas. Unfortunately, there are no such vaccines against Sarcoptes yet.

Investigation of Acinetobacter baumannii Activity in Vascular Surgery Units through Epidemiological Management Based on the Analysis of Antimicrobial Resistance, Biofilm Formation and Genotyping.

BACKGROUND/OBJECTIVES: The genus Acinetobacter demonstrates resistance to antibiotics and has been shown to spread in the hospital environment causing epidemic outbreaks among hospitalized patients. The objectives of the present study was to investigate the antibiotic resistance, biofilm formation, and clonality among Acinetobacter baumannii strains. MATERIALS AND METHODS: The study involved 6 (I Outbreak) and 3 (II Outbreak) A. baumannii strains isolated from patients hospitalized in vascular surgery unit. RESULTS: All tested A. baumannii strains were extensively drug resistant (XDR) and all the isolates were carbapenem-resistant and among them, all carried the blaOXA-51 gene, the blaOXA-24 gene, as well as the blaOXA-23 gene. All of the investigated strains had the ability to form a biofilm, but all of them produced less biofilm than the reference strain. Multi-locus sequence typing (MLST) showed that all strains belonged to the ST2 clone. Pulsed-field gel electrophoresis (PFGE) divided the tested outbreak strains into two clones (A and B). CONCLUSION: This study shows a nosocomial spread of XDR A. baumannii ST2 having the blaOXA-51 gene, the blaOXA-24 gene, as well as the blaOXA-23 gene, low biofilm formers, that was prevalent in the vascular surgery unit. To identify the current situation of vascular surgery departments targeted epidemiological investigation was needed. Effective implementation of infection control prevented the spread of the epidemic outbreaks.

Unexpected subungual Sarcoptes infestation of toenail - a case report and literature review.

Scabies is the skin infestation caused by Sarcoptes scabiei var hominis. It is one of the commonest dermatological infection which can affect people around the world. However, nails are relatively rarely involved, and the fingernails are mostly infected. The report a case of a 77-year-old woman, long-term pensioner of a nursing home, who had isolated toe subungual Sarcoptes infestation. In addition, the results of systematic review of toenails scabies was presented. Analysis of 21 subungual Sarcoptes infestation cases, revealed that patients in any age (median age 45+/-31.7-year-old) and sex can be affected. Most of the patients had concomitant diseases. Seventy-five percent of cases of nail involvement were treated with combined or sequential therapy. The most used drugs were ivermectin (IVR) and permethrin (PER) (each used in 47.6% cases), following γ-BHC (38.1%) and crotamiton (CRO) (23.8%). It seems that the crucial for adequate diagnosis in scabies affected nails is a precise anamnesis, early and accurate diagnosis that consists of examining not only skin lesions, but including assessing toenails, and differentiation of Sarcoptes infestation from other nail diseases as onychomycosis or psoriasis. Important to achieving a cure is at least frequent nail trimming, softening the nail plate with urea or in the difficult cases the mechanical removal of subungual plaque with using of a scabicide in the location allowing to penatrate it under the nail plate.

Candida prevalence and oral hygiene due to orthodontic therapy with conventional brackets.

BACKGROUND: Conventional brackets are often used during orthodontic therapy of patients with malocclusion. The complex construction of such brackets greatly inhibits oral hygiene, which predisposes to increased carriage of microbiota. Orthodontic brackets could act as reservoir of yeast and predispose to oral candidosis. The aim of this study was to assess Candida prevalence and the role of oral hygiene during fixed appliance therapy. A further aim was to characterize the isolated yeasts according to their ability to form biofilms. METHODS: Seventeen participants (average age 17 ± 7 years) were monitored by taking oral rinses and elastomeric ligature samples, and by evaluating the approximal plaque index (API) and gingival bleeding index (GBI) before and after placement of the orthodontic conventional brackets for 12 weeks. Isolated yeasts were counted and biofilm formation was evaluated. RESULTS: One hundred and sixteen samples (67 oral rinses and 49 orthodontic elastomers) were collected. Ten patients (58.8% subjects) were Candida-carriers (two were colonized after bracket placement) and C. albicans was the most common species. The average number of yeasts in the oral cavity showed some fluctuation during the study, but in general had an upward trend (adj. R2 = 0.7967, p = 0.07025). A correlation was found between median number of yeasts and the periodontal indices (API, GBI). The average API values decreased in the Candida-carriers (adj. R2 = 0.95; p = 0.01709), while average GBI values increased in the noncarriers (adj. R2 = 0.92; p = 0.0256). CONCLUSIONS: Treatment with orthodontic appliances promotes Candida yeast colonization, which is variable over time in terms of strain and species, with dominance of C. albicans, and without increased biofilm-forming activity. The API value decreases over time in carriers, and the GBI value increases in uncolonized patients, which may have predictive significance for the development of oral candidiasis during orthodontic treatment.

Assessment to access to social and medical help- for homeless people in Cracow in the context to health problems

INTRODUCTION: Although homelessness is a worldwide problem also present in Poland and the Malopolska region, the data about it are not sufficient. AIM OF THE STUDY: Assessment of the general situation in Cracow and help options for homeless people. MATERIALS AND METHODS: A survey study conducted in 11 places offering refuge to homeless people in Cracow. RESULTS: In Cracow, there were 691 places to sleep in 11 institutions. Nine out of 11 were open all year long and most of them were available only for men. Five institutions offered meals, all of them had bathrooms, 7 places provided medical help. DISCUSSION: The expected number of homeless people in Poland and in Cracow seems to be underestimated. Basic needs of homeless people remain unsatisfied which decreases the possibility of transitioning out of homelessness. CONCLUSIONS: Help available for homeless people in Cracow needs improvements in all of the areas examined.

Intelligent and active furcellaran-gelatin films containing green or pu-erh tea extracts: Characterization, antioxidant and antimicrobial potential.

Biodegradable films from furcellaran and gelatin (FUR/GEL) with pu-erh (RTE) and green tea (GTE) water extracts (TEs) were obtained by a casting method. The films were examined in terms of physical properties (thickness, density, water solubility, water content, degree of swelling, color), antioxidant properties (DPPH, ABTS, TPC), antimicrobial properties (Escherichia coli, Staphylococcus aureus, Candida albicans and Henseniaspora uvarum) and mechanical properties (tensile strength, elongation at break). The values of tensile strength (TS) increased from 9.62 to 24.14 or 13.59 MPa after GTE or RTE incorporation, respectively. Parameters of elongation at break were not changed. Higher color parameters: a*, b* and ∆E were observed in biocomposite films with RTE. The FUR/GEL films with GTE and RTE can be used for intelligent food packaging, due to their sensibility of pH changes. Possible interaction between the film matrix and TEs was confirmed using FTIR. The antioxidant properties were significantly enhanced with increasing GTE and RTE concentration reaching 43.92 and 48.94% of DPPH and 15.76 and 14.98% of ABTS radical inhibition, respectively. The microbiological analysis confirmed antimicrobial properties of FUR/GEL with GTE against E.coli and S. aureus (inhibition zones of 16 and 22.5 mm, respectively).

Injuries as exposure events in providing medical services by nursing staff

INTRODUCTION: Among medical staff, nurses are particularly vulnerable to occupational exposure since they provide direct patient care and perform medical activities which often involve using sharps. AIM: The objective of the study was to examine the frequency of injuries and their causes in nursing. METHODS: A diagnostic survey was employed using an original questionnaire. The study was carried out from 3rd March to 2nd April 2017. The study group comprised part-time nursing students. 107 respondents participating in the study worked in out-patient (28%) and in-patient (72%) healthcare. Most of the respondents were aged 41­50 (34.6%). RESULTS: 61.7% of the respondents were injured at work. The injury reporting rate was: 19.7% always, 22.7% often, 30.3% rarely, and 27.3% never. The most commonly mentioned types of injuries included: prick (51.5%), cut (28.8%), scratch (10.6%), prick and cut (9.1%). The incidence of injuries varied. 48.5% of the studied people declared fewer than 5 incidents, 31.8% quoted 5­10 injuries, 6.1% recalled 11­20, 13.6% did not remember such a situation. For nurses with longer seniority, there is a significant increase in injuries (p=0.029). Sources of injuries were most often: injection needle (35.9%), ampoule with medicine (23.3%), pen (7.8%). CONCLUSIONS: Not all occupational exposure cases are reported by nurses. Seniority determines injury incidence among nurses. The longer the seniority, the more common the injuries. The needle causes injuries most frequently.

Analysis of the world epidemiological situation among vancomycin-resistant Enterococcus faecium infections and the current situation in Poland

Vancomycin-resistant Enterococcus faecium (VREfm) strains have become an important hospital pathogen due to their rapid spread, high mortality rate associated with infections and limited therapeutic options. Vancomycin resistance is predominantly mediated by VanA or VanB phenotypes, which differ as regards maintaining sensitivity to teicoplanin in the VanB phenotype. The majority of VREfm cases in the United States, Europe, Korea, South America and Africa are currently caused by the VanA phenotype. However, the epidemics in Australia and Singapore are chiefly brought about by the VanB phenotype. The rate of VREfm isolate spread varies greatly. The greatest percentage of VREfm is now recorded in the USA, Ireland and Australia. Supervision of VRE is implemented to varying degrees. Therefore, the epidemiological situation in some countries is difficult to assess due to limited data or lack thereof.

Do bacteria isolated from ICU patients 'ESKAPE' antibiotic treatment? In vitro susceptibility of the Enterobacteriaceae family to tigecycline.

BACKGROUND: Enterobacteriaceae are currently causing the majority of healthcare-associated infections (HAI) and simultaneously expressing increasing levels of antibiotic resistance. The purpose of this study is to assess the in vitro sensitivity of MDR strains from the family Enterobacteriaceae to tigecycline in relation to their origin from patients hospitalized in intensive care units (ICUs) and non-ICUs. METHODS: The study involved 156 clinically significant strains of the Enterobacteriaceae family isolated from patients with complicated intraabdominal infections (cIAIs) and/or complicated skin and skin structure infections (cSSSIs) hospitalized in ICUs and other surgical departments. Tigecycline MICs were determined by Etest. RESULTS: The highest percentage of tigecycline non-susceptible (intermediate + resistant strains) in vitro strains among the Enterobacteriaceae species were observed for Serratia spp. 77.3%, followed by Citrobacter spp. (76.9%) and Enterobacter spp. (70%); whereas K. pneumoniae and E. coli showed 73-73.8% tigecycline susceptibility rates. CONCLUSION: Tigecycline demonstrates a high level of antimicrobial in vitro activity when tested against E. coli and K. pneumoniae, even those with the ESBL-phenotype. Tigecycline retained activity against merely 22-30% of Enterobacter, Citrobacter and Serratia genera.