On predatory fungi feeding on free-living amoebae harbouring yeast-like endoparasites.

Amoebae of the genus Vannella isolated from an ornamental fish aquarium were found to be infected with fungi. Upon plate culture, amoeba-trapping hyphal filaments were developed, and the amoeba trophozoites were found to harbour yeast-like parasites in their cytoplasm. Transfection of hyphae to a laboratory strain of Vannella resulted in the formation of conidia indicating the possible presence of zygomycetes of the genus Acaulopage, while efforts to culture the endoparasite remained unsuccessful. Biomolecular analysis based on rDNA revealed the presence of two distinct types of fungi, confirming the filamentous form as Acaulopage sp. (Zoopagomycota, Zoopagales) and identifying the yeast-like endoparasite as Cladosporium sp. (Ascomycota, Cladosporiales). To our knowledge, this is the first report of amoebae infected with Cladosporium.

Influence of the Single Nucleotide Polymorphisms rs12252 and rs34481144 in IFITM3 on the Antibody Response after Vaccination against COVID-19.

The COVID-19 mRNA vaccine is the first mRNA vaccine approved for human administration by both the U.S. Food and Drug Administration and the European Medicines Agency. Studies have shown that the immune response and the decay of immunity after vaccination with the COVID-19 vaccines are variable within a population. Host genetic factors probably contribute to this variability. In this study, we investigated the effect of the single-nucleotide polymorphisms rs12252 and rs34481144 in the interferon-induced transmembrane protein (IFITM) 3 gene on the humoral immune response after vaccination against COVID-19 with mRNA vaccines. Blood samples were collected from 1893 healthcare workers and medical students at multiple time points post-vaccination and antibody titers against the SARS-CoV-2 S1 protein receptor binding domain were determined at all time points. All participants were genotyped for the rs34481144 and rs12252 polymorphisms in the IFITM3 gene. After the second and third vaccinations, antibody titer levels increased at one month and decreased at six months (p < 0.0001) and were higher after the booster vaccination than after the basic immunization (p < 0.0001). Participants vaccinated with mRNA-1273 had a higher humoral immune response than participants vaccinated with BNT162b2. rs12252 had no effect on the antibody response. In contrast, carriers of the GG genotype in rs34481144 vaccinated with BNT162b2 had a lower humoral immune response compared to A allele carriers, which reached statistical significance on the day of the second vaccination (p = 0.03) and one month after the second vaccination (p = 0.04). Further studies on the influence of rs12252 and rs34481144 on the humoral immune response after vaccination against COVID-19 are needed.

Monitoring of endoscope reprocessing with an adenosine triphosphate (ATP) bioluminescence method.

Background: The arising challenges over endoscope reprocessing quality proposes to look for possibilities to measure and control the process of endoscope reprocessing. Aim: The goal of this study was to evaluate the feasibility of monitoring endoscope reprocessing with an adenosine triphosphate (ATP) based bioluminescence system. Methods: 60 samples of eight gastroscopes have been assessed from routine clinical use in a major university hospital in Germany. Endoscopes have been assessed with an ATP system and microbial cultures at different timepoints during the reprocessing. Findings: After the bedside flush the mean ATP level in relative light units (RLU) was 19,437 RLU, after the manual cleaning 667 RLU and after the automated endoscope reprocessor (AER) 227 RLU. After the manual cleaning the mean total viable count (TVC) per endoscope was 15.3 CFU/10 ml, and after the AER 5.7 CFU/10 ml. Our results show that there are reprocessing cycles which are not able to clean a patient used endoscope. Conclusion: Our data suggest that monitoring of flexible endoscope with ATP can identify a number of different influence factors, like the endoscope condition and the endoscopic procedure, or especially the quality of the bedside flush and manual cleaning before the AER. More process control is one option to identify and improve influence factors to finally increase the overall reprocessing quality, best of all by different methods. ATP measurement seems to be a valid technique that allows an immediate repeat of the manual cleaning if the ATP results after manual cleaning exceed the established cutoff of 200 RLU.

Principal component analysis of MALDI TOF MS mass spectra separates M. abscessus (sensu stricto) from M. massiliense isolates.

BACKGROUND: The discrimination of the members of the Mycobacterium abscessus complex is of clinical interest because one of the subspecies, M. massiliense, exhibits higher rates of response to antibiotic treatment for lung infection than do the other members of that complex. M. abscessus complex contains three subspecies that are laborious to identify; therefore, a routine diagnostic tool would be worthwhile. RESULTS: We used principal component analysis, hierarchical cluster analysis, and single-peak analysis to examine peak lists derived from matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) mass spectra of 50 clinical M. abscessus complex isolates, including 28 M. abscessus (sensu stricto), 19 M. massiliense, and 3 M. bolletii isolates grown in mycobacterium growth indicator tube liquid medium and prepared with a bead-based protocol. Principal component analysis but not hierarchical cluster analysis separated M. abscessus (sensu stricto) isolates and M. massiliense isolates into two clusters. Furthermore, single-peak analysis displayed 4 discriminating peaks that separated M. abscessus (sensu stricto) from M. massiliense isolates. M. bolletii isolates did not exhibit specific peaks but resembled the M. abscessus (sensu stricto) peak profile and also grouped within this principal component analysis cluster. Principal component analysis of all peak lists with the exclusion of the four discriminating peaks again separated M. abscessus (sensu stricto) from M. massiliense isolates, thus relativizing the importance of these peaks for subspecies identification. CONCLUSIONS: Principal component analysis of peak lists derived from MALDI TOF mass spectra is a robust and convenient method of discriminating M. massiliense isolates from the other members of the M. abscessus complex.

Performance of Vitek MS in identifying nontuberculous mycobacteria from MGIT liquid medium and Lowenstein-Jensen solid medium.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is a fast and inexpensive method for bacterial identification. The aim of this study was to analyze the performance of Vitek MS in identifying 160 nontuberculous mycobacterial isolates of 24 species from Lowenstein-Jensen solid medium and BACTEC MGIT 960 liquid medium using a bead-based method. The system correctly identified 76.9% of the isolates (123 of 160) cultivated on solid medium and 76.9% (123 of 160) of positive liquid cultures. None of the isolates included in the study was misidentified. Although the overall performance of Vitek MS with the SARAMIS 4.12 database was comparable in identifying mycobacterial species grown on solid medium and in liquid medium, the identification rate varied notably between the various species analyzed, which currently limits the utility for identification in routine diagnostics for some species.

Delineation of Stenotrophomonas maltophilia isolates from cystic fibrosis patients by fatty acid methyl ester profiles and matrix-assisted laser desorption/ionization time-of-flight mass spectra using hierarchical cluster analysis and principal component analysis.

Stenotrophomonas maltophilia is an opportunist multidrug-resistant pathogen that causes a wide range of nosocomial infections. Various cystic fibrosis (CF) centres have reported an increasing prevalence of S. maltophilia colonization/infection among patients with this disease. The purpose of this study was to assess specific fingerprints of S. maltophilia isolates from CF patients (n = 71) by investigating fatty acid methyl esters (FAMEs) through gas chromatography (GC) and highly abundant proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and to compare them with isolates obtained from intensive care unit (ICU) patients (n = 20) and the environment (n = 11). Principal component analysis (PCA) of GC-FAME patterns did not reveal a clustering corresponding to distinct CF, ICU or environmental types. Based on the peak area index, it was observed that S. maltophilia isolates from CF patients produced significantly higher amounts of fatty acids in comparison with ICU patients and the environmental isolates. Hierarchical cluster analysis (HCA) based on the MALDI-TOF MS peak profiles of S. maltophilia revealed the presence of five large clusters, suggesting a high phenotypic diversity. Although HCA of MALDI-TOF mass spectra did not result in distinct clusters predominantly composed of CF isolates, PCA revealed the presence of a distinct cluster composed of S. maltophilia isolates from CF patients. Our data suggest that S. maltophilia colonizing CF patients tend to modify not only their fatty acid patterns but also their protein patterns as a response to adaptation in the unfavourable environment of the CF lung.

Circulating U2 small nuclear RNA fragments as a novel diagnostic tool for patients with epithelial ovarian cancer.

BACKGROUND: Ovarian cancer is the leading cause of death among malignancies in women. Despite advances in treatment, >50% of patients relapse. For disease monitoring, the identification of a blood-based biomarker would be of prime interest. In this regard, noncoding RNAs, such as microRNA (miRNA) or small nuclear RNA (snRNA), have been suggested as biomarkers for noninvasive cancer diagnosis. In the present study, we sought to identify differentially expressed miRNA/snRNA in sera of ovarian cancer patients and investigate their potential to aid in therapy monitoring. METHODS: miRNA/snRNA abundance was investigated in serum (n = 10) by microarray analysis and validated in an extended serum set (n = 119) by reverse-transcription quantitative PCR. RESULTS: Abundance of U2-1 snRNA fragment (RNU2-1f) was significantly increased in sera of ovarian cancer patients (P < 0.0001) and paralleled International Federation of Gynecology and Obstetrics stage as well as residual tumor burden after surgery (P < 0.0001 and P = 0.011, respectively). Moreover, for patients with suboptimal debulking, preoperative RNU2-1f concentration was associated with radiographic response after chemotherapy and with platinum resistance (P = 0.0088 and P = 0.0015, respectively). Interestingly, according to the RNU2-1f abundance dynamics, persistent RNU2-1f positivity before surgery and after chemotherapy identified a subgroup of patients with high risk of recurrence and poor prognosis. CONCLUSIONS: This is the first report to suggest that a circulating snRNA can serve as an auxiliary diagnostic tool for monitoring tumor dynamics in ovarian cancer. Our results provide a rationale to further investigate whether this high-risk patient group may benefit from additional therapies that are directly applied after chemotherapy.

Direct identification of bacteria in urine samples by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and relevance of defensins as interfering factors.

Standard methods for the identification of uropathogens that are based on the determination of metabolic activity require cultivation on agar plates, which often takes more than 1 day. If microbial growth on agar plates is slow, or if metabolic activity is impaired by adverse interactions resulting from the patient's condition or from medical treatment, the application of standard methods may lead to delayed or erroneous identification of bacteria. In recent studies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be able to rapidly identify bacteria obtained from cultures. We tested the applicability of this analytical technique for the rapid identification of bacteria collected directly from urine samples and compared the results with those of conventional identification methods, such as the Vitek system, the MicroScan WalkAway system and the API system, and in some cases with the gas chromatographic determination of the bacterial long-chain fatty acid pattern. We analysed a total of 107 urine samples with bacterial counts ranging from 10(2) to ≥10(5) c.f.u. ml(-1). Mass spectrometric identification of bacteria was accomplished for 62 of these samples. In the mass spectra obtained from 40 of the 45 urine samples for which no identification result was achieved, a triplet of very intense peaks corresponding to the human α-defensins 1, 2 and 3 occurred at m/z values of around 3440 Da. This signal suppressed the intensity of the bacterial protein peaks and thus impaired database matching. Our results show that MALDI-TOF MS allows the reliable direct identification of bacteria in urine samples at concentrations as low as 10(3) c.f.u. ml(-1). In a subset of samples, human defensins may occur and impair the mass spectrometric identification of bacteria.

Determination of trimethylbismuth in the human body after ingestion of colloidal bismuth subcitrate.

Biological methylation and hydride formation of metals and metalloids are ubiquitous environmental processes that can lead to the formation of chemical species with significantly increased mobility and toxicity. Whereas much is known about the interaction of metal(loid)s with microorganisms in environmental settings, little information has been gathered on respective processes inside the human body as yet. Here, we studied the biotransformation and excretion of bismuth after ingestion of colloidal bismuth subcitrate (215 mg of bismuth) to 20 male human volunteers. Bismuth absorption in the stomach and upper intestine was very low, as evidenced by the small quantity of bismuth eliminated via the renal route. Total bismuth concentrations in blood increased rapidly in the first hour after ingestion. Most of the ingested bismuth was excreted via feces during the study period. Trace levels of the metabolite trimethylbismuth [(CH(3))(3)Bi] were detected via low temperaturegas chromatography/inductively coupled plasma-mass spectrometry in blood samples and in exhaled air samples. Concentrations were in the range of up to 2.50 pg/ml (blood) and 0.8 to 458 ng/m(3) (exhaled air), with high interindividual variation being observed. Elimination routes of bismuth were exhaled air (up to 0.03 per thousand), urine (0.03-1.2%), and feces. The site of (CH(3))(3)Bi production could not be identified in the present study, but the intestinal microflora seems to be involved in this biotransformation if accompanying ex vivo studies are taken into consideration.

Role of intestinal microbiota in transformation of bismuth and other metals and metalloids into volatile methyl and hydride derivatives in humans and mice.

The present study shows that feces samples of 14 human volunteers and isolated gut segments of mice (small intestine, cecum, and large intestine) are able to transform metals and metalloids into volatile derivatives ex situ during anaerobic incubation at 37 degrees C and neutral pH. Human feces and the gut of mice exhibit highly productive mechanisms for the formation of the toxic volatile derivative trimethylbismuth [(CH(3))(3)Bi] at rather low concentrations of bismuth (0.2 to 1 mumol kg(-1) [dry weight]). An increase of bismuth up to 2 to 14 mmol kg(-1) (dry weight) upon a single (human volunteers) or continuous (mouse study) administration of colloidal bismuth subcitrate resulted in an average increase of the derivatization rate from approximately 4 pmol h(-1) kg(-1) (dry weight) to 2,100 pmol h(-1) kg(-1) (dry weight) in human feces samples and from approximately 5 pmol h(-1) kg(-1) (dry weight) to 120 pmol h(-1) kg(-1) (dry weight) in mouse gut samples, respectively. The upshift of the bismuth content also led to an increase of derivatives of other elements (such as arsenic, antimony, and lead in human feces or tellurium and lead in the murine large intestine). The assumption that the gut microbiota plays a dominant role for these transformation processes, as indicated by the production of volatile derivatives of various elements in feces samples, is supported by the observation that the gut segments of germfree mice are unable to transform administered bismuth to (CH(3))(3)Bi.

Laser Doppler imager (LDI) scanner and intradermal injection for in vivo pharmacology in human skin microcirculation: responses to acetylcholine, endothelin-1 and their repeatability.

AIMS: The purpose of this study was to evaluate the repeatability of forearm skin blood flow responses to intradermal injections of acetylcholine (ACh) and endothelin-1 (ET-1) using a double injection technique (DIT) and a laser Doppler imager (LDI) scanner in the human skin microcirculation. METHODS: We used a laser Doppler imager (Moor LDI V3.01) to continuously monitor the change in skin blood flow during intradermal administration of physiological saline (0.9% NaCl), acetylcholine (ACh 10(-7), 10(-8), 10(-9) M) and endothelin-1 (ET-1 10(-14), 10(-16), 10(-18) M) in 10 healthy male subjects. Subjects were examined on 3 different days for assessment of interday and interobserver repeatability. Injections of either drug were randomly placed on different sites of the forearm. Laser Doppler images were collected before and after injection at 2.5 min intervals for 30 min. Data were analysed after the completion of each experiment using Moor Software V.3.01. Results are expressed as changes from baseline in arbitrary perfusion units (PU). RESULTS: ACh caused a significant vasodilation (P < 0.0001 anova, mean +/- SE: 766 +/- 152 PU, ACh 10(-9) M; 1868 +/- 360 PU, ACh 10(-8) M; 4188 +/- 848 PU, ACh 10(-7) M; mean of days 1 and 2, n = 10), and ET-1 induced a significant vasoconstrictive response (P < 0.0001 anova, -421 +/- 83 PU, ET-1 10(-18) M; -553 +/- 66 PU, ET-1 10(-16) M; -936 +/- 90 PU, ET-1 10(-14) M; mean of days 1 and 2, n = 10). There was no difference on the response to either drug on repeated days. Bland-Altman analyses showed a close agreement of responses between days with repeatability coefficients of 1625.4 PU for ACh, and 386.0 PU for ET-1 (95% CI: ACh, -1438 to 1747 PU, ET-1, -399 to 358 PU) and between observers with repeatability coefficients of 1057.2 PU for ACh and 255.8 PU for ET-1 (95% CI: ACh, -1024 to 1048 PU, ET-1, -252 to 249 PU). The variability between these responses was independent of average flux values for both ACh and ET-1. There was a significant correlation between responses measured in the same site, in the same individual on two different days by the same observer (ACh, r = 0.94, P < 0.0001; ET-1, r = 0.90, P < 0.0006), and between responses measured by two different observers (ACh, r = 0.94, P < 0.0001; ET-1, r = 0.91, P < 0.0003). CONCLUSION: We have shown that interday and intraobserver responses to intradermal injections of ET-1 and ACh, assessed using the DIT in combination with an LDI scanner, exhibited good reproducibility and may be a useful tool for studying the skin microcirculation in vivo.