Use of SEM/EDX methods for the analysis of ambient particulate matter adhering to the skin surface

Background The skin is exposed to numerous particulate and gaseous air pollutants. The ones that need particular attention are the particles that adhere to the skin surface, which can later cause direct skin damage. This study aimed to characterize air pollution (AP) particles adhered to the human skin by using scanning electron microscopy (SEM) combined with X-ray dispersive energy spectrometry (EDX). Methods Tape stripping was performed from six healthy volunteers exposed to urban AP to collect stratum corneum samples from the cheeks and forehead. The samples were analysed using SEM equipped with EDX system with a silicon drift detector at an accelerating voltage of 20 keV. After the preliminary examination, the particles were located and counted using 1000× magnification. Each particle was analysed, increasing magnification up to 5000× for precise dimension measurement and elemental composition analysis. At least 100 fields or a surface of approximately 1 mm2 were examined. Results Particles adhered to the skin were identified in all samples, with a particle load ranging from 729 to 4525. The average area and perimeter of all particles identified were 302 ± 260 μm2 and 51 ± 23 μm subsequently, while the equivalent circular diameter was, on average, 14 ± 6 μm. The particles were classified into ten groups based on morphology and elemental composition. Chlorides were the most numerous particle group (21.9%), followed by carbonaceous organic particles (20.3%), silicates (18%), carbonates (16.4%), metal-rich particles (14%), and a minor number of bioaerosols, quartz-like, and fly ash particles. Conclusion The SEM–EDX analysis provides evidence of the contamination of exposed skin to various airborne PM of natural or anthropogenic origin. This method may provide new insights into the link between exposure to AP and AP-induced skin damage

High rate of fluoroquinolone resistant Neisseria gonorrhoeae detected by molecular surveillance of antimicrobial resistance determinants in Bulgaria

AbstractAntimicrobial-resistant Neisseria gonorrhoeae is a major public health concern. The surveillance of antimicrobial resistance benefits from rapid and accurate molecular techniques in molecular diagnostics to facilitate individualised medicine and antimicrobial stewardship. To support the recommendations for empirical treatment of gonococcal infections in Bulgaria, we investigated N. gonorrhoeae-positive clinical specimens from 2018 to 2021 for the presence of genetic determinants associated with antimicrobial resistance. N. gonorrhoeae-positive samples stored at the National Center of Infectious and Parasitic Diseases during the four-year study period were retrospectively analysed by polymerase chain reaction and DNA sequencing assays for resistance determinants to fluoroquinolones, third-generation cephalosporins and macrolides. The detected determinants indicated a high rate of fluoroquinolone resistance (59%), very low level of decreased susceptibility to third-generation cephalosporins (3%) but no macrolide resistance (0%). These findings validate the utilisation of the international guidelines’ recommendations for empirical dual therapy with ceftriaxone/cefixime and azithromycin in Bulgaria. Because of the high fluoroquinolone resistance rate, ciprofloxacin should only be considered as treatment if phenotypic or molecular antimicrobial susceptibility data indicate susceptibility to ciprofloxacin. For the purposes of surveillance and individualised medicine, molecular assays for resistance determinants could complement culture-based phenotypic gonococcal antimicrobial resistance testing

Azithromycin treatment failure and macrolide resistance in Mycoplasma genitalium infections in Sofia, Bulgaria

Introduction: Mycoplasma genitalium is an established cause of sexually transmitted infections in men and women. Current guidelines recommend azithromycin and moxifloxacin as first- and second-line treatment, respectively. However, azithromycin treatment failure has been increasingly reported. The aim of this study was to determine the efficacy of azithromycin and alternative antibiotic regimens in a prospective cohort of M. genitalium-positive patients, and macrolide resistance mutations associated with azithromycin failure. Materials and methods: Consecutive eligible M. genitalium-positive patients attending the National Center of Infectious and Parasitic Diseases in Sofia, Bulgaria between 1 January 2018 and 31 December 2020 were treated with azithromycin and retested by polymerase chain reaction 21-28 days after completion of the treatment. Cure was defined as M. genitalium-negative result on the test of cure. Cases failing azithromycin were treated with moxifloxacin and retested another 21-28 days after treatment. Pre- and post-treatment samples were assessed for macrolide resistance mutations by conventional DNA sequencing. Results: Of 21 patients treated with azithromycin, 11 (52.4%) were cured. Pre- and post-treatment macrolide resistance mutations were detected in 10 (47.6%) patients, and all of them failed azithromycin. Moxifloxacin was effective in all cases failing azithromycin; and all were M. genitalium-negative at the test of cure after moxifloxacin treatment. Conclusions: In this study a high azithromycin failure rate (47.6%) in an M. genitalium-positive cohort in association with high levels of pretreatment macrolide resistance was reported. Moxifloxacin was highly effective in treating macrolide-resistant infections. These findings necessitate implementation of new diagnostic and therapeutic strategies such as sequential antimicrobial therapy for M. genitalium guided by a macrolide-resistance assay

Two-Year Monitoring of Water Samples from Dam of Iskar and the Black Sea, Bulgaria, by Molecular Analysis: Focus on <i>Mycobacterium</i> spp

The coast of the Bulgarian Black Sea is a popular summer holiday destination. The Dam of Iskar is the largest artificial dam in Bulgaria, with a capacity of 675 million m3. It is the main source of tap water for the capital Sofia and for irrigating the surrounding valley. There is a close relationship between the quality of aquatic ecosystems and human health as many infections are waterborne. Rapid molecular methods for the analysis of highly pathogenic bacteria have been developed for monitoring quality. Mycobacterial species can be isolated from waste, surface, recreational, ground and tap waters and human pathogenicity of nontuberculose mycobacteria (NTM) is well recognized. The objective of our study was to perform molecular analysis for key-pathogens, with a focus on mycobacteria, in water samples collected from the Black Sea and the Dam of Iskar. In a two year period, 38 water samples were collected—24 from the Dam of Iskar and 14 from the Black Sea coastal zone. Fifty liter water samples were concentrated by ultrafiltration. Molecular analysis for 15 pathogens, including all species of genus Mycobacterium was performed. Our results showed presence of Vibrio spp. in the Black Sea. Rotavirus A was also identified in four samples from the Dam of Iskar. Toxigenic Escherichia coli was present in both locations, based on markers for stx1 and stx2 genes. No detectable amounts of Cryptosporidium were detected in either location using immunomagnetic separation and fluorescence microscopy. Furthermore, mass spectrometry analyses did not detect key cyanobacterial toxins. On the basis of the results obtained we can conclude that for the period 2012–2014 no Mycobacterium species were present in the water samples. During the study period no cases of waterborne infections were reported