[Long-term care facilities during the COVID-19 pandemic : Considerations on the way back to normality]. / Langzeitpflegeeinrichtungen in der COVID-19-Pandemie : Überlegungen auf dem Weg zurück in die Normalität.

Long-term care facilities (LTCF) were and are particularly affected by the COVID-19 pandemic. The dimensions of the outbreaks and the high mortality among residents led to massive restrictions in LTCFs, especially in the area of social contacts and activities but also in areas of medical care. With the start of vaccinations and the improved testing options, the situation has now changed and existing restrictions must be evaluated to determine whether they are still appropriate. In an interprofessional and interdisciplinary group of experts, considerations have been formulated on how a way back to normality could look like in LTCFs.

Performance of the AsperGenius® PCR assay for detecting azole resistant Aspergillus fumigatus in BAL fluids from allogeneic HSCT recipients: A prospective cohort study from Essen, West Germany.

In this study a commercially available multiplex real-time PCR (AsperGenius®) was evaluated for its efficacy in detecting Aspergillus fumigatus and azole resistance markers in comparison with conventional culture methods and galactomannan (GM) testing from BAL fluids in allogeneic HSCT recipients. Between January 2015 and May 2017 100 allogeneic HSCT recipients with pulmonary infiltrates and suspicion of invasive fungal infection were recruited to the study from a tertiary care center in Germany. BAL fluid was routinely assessed using the following diagnostic tests: AsperGenius® PCR assay, GM testing (cut-off: 1.0) and conventional culture. Susceptibility testing of azoles was performed by using Etest and, in case presenting elevated MICs, PCR for mutations in the cyp51A gene was carried out. Criteria of EORTC/MSG were used to classify the patients for invasive fungal disease. According to the EORTC/MSG criteria 23 patients presented with probable invasive aspergillosis (IA). Aspergillus PCR showed a sensitivity of 65% for probable IA cases. A combination of PCR and GM results in BAL displayed a sensitivity of 96% (22/23) and 100% specificity. Mutations in the cyp51A gene were detected by PCR in three cases (3/23; 13%) which were also found resistant with the culture method. In one case a Y121F/T289A mutation and in two cases a L98H were found. The combination of a commercial Aspergillus PCR assay and GM testing from BAL demonstrated a high sensitivity and specificity for diagnosing IA in allogeneic HSCT recipients. The Aspergillus PCR assay was not superior in detecting azole resistant A. fumigatus compared to culture.

Prevalence and characterization of azole-resistant Aspergillus fumigatus in patients with cystic fibrosis: a prospective multicentre study in Germany.

Objectives: Aspergillus fumigatus is the most prevalent filamentous fungus in the respiratory tract of patients with cystic fibrosis (CF). The aim of this prospective multicentre study was to investigate the prevalence of azole-resistant A. fumigatus (ARAF) in respiratory secretions from CF patients across Germany and to characterize ARAF isolates by phenotypic and molecular methods. Methods: Twelve tertiary care centres from Germany participated in the study. In total, 2888 A. fumigatus isolates from 961 CF patients were screened for ARAF by using azole-containing agar plates. Antifungal susceptibility testing of isolates was performed by broth microdilution according to EUCAST guidelines. Analysis of mutations mediating resistance was performed using PCR and sequencing of the cyp51A gene. Furthermore, genotyping by microsatellite PCR was performed. Results: Of a total of 2888 A. fumigatus isolates, 101 isolates from 51 CF patients were found to be azole resistant (prevalence per patient 5.3%). The Essen centre had the highest prevalence (9.1%) followed by Munich (7.8%), Münster (6.0%) and Hannover (5.2%). Most ARAF isolates (n = 89) carried the TR34/L98H mutation followed by eight G54E/R, one TR46/Y121F/T289A and one F219S mutation. In two isolates no mutation was found. Genotyping results showed no major clustering. Forty-five percent of CF patients with ARAF had previously received azole therapy. Conclusions: This is the first multicentre study analysing the prevalence of ARAF isolates in German CF patients. Because of a resistance rate of up to 9%, susceptibility testing of A. fumigatus isolates from CF patients receiving antifungal treatment should be part of standard diagnostic work-up.

Azole Resistance in Aspergillus fumigatus in Patients with Cystic Fibrosis: A Matter of Concern?

Aspergillus fumigatus is the most frequent filamentous fungus isolated from respiratory specimens from patients with cystic fibrosis (CF). Triazoles are the most widely used antifungals in the treatment of allergic bronchopulmonary aspergillosis (ABPA) and invasive aspergillosis (IA) in CF patients. Treatment success could be severely compromised by the occurrence of azole-resistant A. fumigatus (ARAf), which is increasingly reported worldwide from both clinical samples and the environment. In previous studies, ARAf has been detected in up to 8% of CF patients. Isolates from CF patients requiring antifungal treatment should therefore be routinely subjected to antifungal susceptibility testing. The optimal treatment of ABPA or IA in CF patients with azole-resistant isolates has not been established; treatment options include liposomal amphotericin B i.v. and/or echinocandins i.v.

The lung microbiome in patients with pneumocystosis.

BACKROUND: Pneumocystis jirovecii pneumonia (PCP) is an opportunistic fungal infection that is associated with a high morbidity and mortality in immunocompromised individuals. In this study, we analysed the microbiome of the lower respiratory tract from critically ill intensive care unit patients with and without pneumocystosis. METHODS: Broncho-alveolar fluids from 65 intubated and mechanically ventilated intensive care unit patients (34 PCP+ and 31 PCP- patients) were collected. Sequence analysis of bacterial 16S rRNA gene V3/V4 regions was performed to study the composition of the respiratory microbiome using the Illumina MiSeq platform. RESULTS: Differences in the microbial composition detected between PCP+ and PCP- patients were not statistically significant on class, order, family and genus level. In addition, alpha and beta diversity metrics did not reveal significant differences between PCP+ and PCP- patients. The composition of the lung microbiota was highly variable between PCP+ patients and comparable in its variety with the microbiota composition of the heterogeneous collective of PCP- patients. CONCLUSIONS: The lower respiratory tract microbiome in patients with pneumocystosis does not appear to be determined by a specific microbial composition or to be dominated by a single bacterial species.

In Vitro Activity of Isavuconazole against Rasamsonia Species.

The in vitro susceptibilities to the novel triazole isavuconazole and six other antifungal agents of a large collection of Rasamsonia isolates (n = 47) belonging to seven species were determined. Isavuconazole and voriconazole had no in vitro activity (MIC, >32 mg/liter) against isolates of the Rasamsonia argillacea species complex. The echinocandins were the most potent antifungal drugs against all of the isolates tested (minimum effective concentration, ≤0.19 mg/liter).

Emergence of azole-resistant invasive aspergillosis in HSCT recipients in Germany.

OBJECTIVES: Aspergillus fumigatus is the most common agent of invasive aspergillosis (IA). In recent years, resistance to triazoles, the mainstay of IA therapy, has emerged in different countries worldwide. IA caused by azole-resistant A. fumigatus (ARAF) shows an exceedingly high mortality. In this study, IA due to ARAF isolates in HSCT recipients in Germany was investigated. METHODS: The epidemiology of azole resistance in IA was analysed in two German haematology departments. Between 2012 and 2013, 762 patients received HSCT in Essen (n = 388) and Cologne (n = 374). Susceptibility testing of A. fumigatus isolates was performed by Etest, followed by EUCAST broth microdilution testing if elevated MICs were recorded. In all ARAF isolates the cyp51A gene was sequenced and the genotype was determined by microsatellite typing using nine short tandem repeats. RESULTS: In total, A. fumigatus was recovered from 27 HSCT recipients. Eight patients had azole-resistant IA after HSCT, and seven of the cases were fatal (88%). All except one patient received antifungal prophylaxis (in five cases triazoles). TR34/L98H was the most common mutation (n = 5), followed by TR46/Y121F/T289A (n = 2). In one resistant isolate no cyp51A mutation was detected. Genotyping revealed genetic diversity within the German ARAF isolates and no clustering with resistant isolates from the Netherlands, India and France. CONCLUSIONS: This report highlights the emergence of azole-resistant IA with TR34/L98H and TR46/Y121F/T289A mutations in HSCT patients in Germany and underscores the need for systematic antifungal susceptibility testing of A. fumigatus.

Adaptation of Stenotrophomonas maltophilia in cystic fibrosis: molecular diversity, mutation frequency and antibiotic resistance.

Due to the continuous exposure to a challenging environment and repeated antibiotic treatment courses, bacterial populations in cystic fibrosis (CF) patients experience selective pressure causing the emergence of mutator phenotypes. In this study we investigated the genotypic diversity, mutation frequency and antibiotic resistance of S. maltophilia isolates chronically colonizing CF patients. S. maltophilia was isolated from a total of 90 sputum samples, collected sequentially from 19 CF patients admitted between January 2008 and March 2012 at the University Hospital Essen, Germany. DNA fingerprinting by repetitive-sequence-based PCR revealed that 68.4% (n=13) of CF patients harbored different S. maltophilia genotypes during the 4-year study course. Out of 90 S. maltophilia isolates obtained from chronically colonized CF patients, 17.8% (n=16) were hypomutators, 27.7% (n=25), normomutators, 23.3% (n=21), weak hypermutators and 31.2% (n=28) strong hypermutators. We also found that mutation rates of the most clonally related genotypes varied over time with the tendency to become less mutable. Mutator isolates were found to have no significant increase in resistance against eight different antibiotics versus nonmutators. Sequencing of the mismatch repair genes mutL, mutS and uvrD revealed alterations that resulted in amino acid changes in their corresponding proteins. Here, we could demonstrate that several different S. maltophilia genotypes are present in CF patients and as a sign of adaption their mutation status switches over time to a less mutator phenotype without increasing resistance. These results suggest that S. maltophilia attempts to sustain its biological fitness as mechanism for long-term persistence in the CF lung.

Value of multiplex PCR using cerebrospinal fluid for the diagnosis of ventriculostomy-related meningitis in neurosurgery patients.

PURPOSE: This prospective observational cohort study assessed the use of a multiplex real-time polymerase chain reaction (PCR) assay alone and in conjunction with biomarkers for the diagnosis of ventriculostomy-related meningitis in neurosurgery intensive care unit (ICU) patients with external ventricular drainage (EVD). METHODS: Concentrations of intrathecal biomarkers, including lactate and interleukin 6 (IL-6), were measured, and cerebrospinal fluid (CSF) was examined microbiologically by blood culture BACTEC bottles in 62 CSF samples from 41 patients with EVD. A portion of each sample was also tested with a commercially available PCR assay that simultaneously detects 25 species of bacteria and fungi [SeptiFast (SF)]. Receiver operating characteristic curve analysis was used to compare biomarker concentrations with SF and culture results. RESULTS: Seventeen (27 %) samples tested positive and 40 (65 %) tested negative for pathogens by both culture and SF. One pathogen was detected only by SF. Four samples tested positive by culture but negative by SF; in 3 of these, the isolates were considered to be contaminants. In comparison to CSF culture SF showed a larger area under the curve for IL-6 (0.90; 95 % CI 0.83-0.98) versus 0.70 (95 % CI 0.46-0.80) and for lactate (0.77; 95 % CI 0.63-0.93) versus 0.65 (95 % CI 0.50-0.80). In 94 % (17/18) of positive SF samples the results were obtained on the same day whereas the overall mean of the time-to-positivity of BACTEC bottles was 21.6 h. CONCLUSIONS: The diagnosis of EVD-related ventriculo-meningitis in neurosurgical ICU patients can be established in a rapid manner using a multiplex PCR assay on CSF samples in combination with intrathecal biomarkers.

[Panton-Valentine leukocidin in patients with chronic wounds. Results from a clinical investigation in 100 patients]. / Panton-Valentine Leukozidin in chronischen Wunden. Ergebnisse einer klinischen Untersuchung bei 100 Patienten.

BACKGROUND: The toxin Panton-Valentine leukocidin (PVL) produced by S. aureus is known as a virulence factor that leads to severe infections of skin and soft tissue. However the effect of PVL on wound healing is not known yet. Therefore we examined the detection rate of PVL in patients with chronic wounds. PATIENTS AND METHODS: The study included 100 patients with chronic wounds of the lower limb. We determined in all S. aureus isolates the presence of the PVL gene using a PCR technique. RESULTS: Altogether 94% of the patients had a leg ulcer, while 6% had a foot ulcer; 65% were women. PVL was found in two patients. One of the strains was methicillin-resistant (MRSA) and the other was methicillin-sensitive (MSSA). CONCLUSION: In our investigation there was detection rate for PVL of 2% of all S. aureus isolates in patients with chronic wounds of the lower extremities. Although the role of PVL as a virulence factor of S. aureus in wound healing remains unclear, the detection of PVL should be taken as a cause for a consequent topical antimicrobial wound therapy because of the increased risk of serious infections.

Thermostability of seven hepatitis C virus genotypes in vitro and in vivo.

Hepatitis C virus (HCV) is transmitted primarily through percutaneous exposure to contaminated blood especially in healthcare settings and among people who inject drugs. The environmental stability of HCV has been extrapolated from studies with the bovine viral diarrhoea virus or was so far only addressed with HCV genotype 2a viruses. The aim of this study was to compare the environmental and thermostability of all so far known seven HCV genotypes in vitro and in vivo. Incubation experiments at room temperature revealed that all HCV genotypes showed similar environmental stabilities in suspension with viral infectivity detectable for up to 28 days. The risk of HCV infection may not accurately be reflected by determination of HCV RNA levels. However, viral stability and transmission risks assessed from in vitro experiments correlated with viral infectivity in transgenic mice containing human liver xenografts. A reduced viral stability for up to 2 days was observed at 37 °C with comparable decays for all HCV genotypes confirmed by thermodynamic analysis. These results demonstrate that different HCV genotypes possess comparable stability in the environment and that noninfectious particles after incubation in vitro do not cause infection in an HCV in vivo model. These findings are important for estimation of HCV cross-transmission in the environment and indicate that different HCV genotypes do not display an altered stability or resistance at certain temperatures.

In vitro activity of colistin as single agent and in combination with antifungals against filamentous fungi occurring in patients with cystic fibrosis.

Because published reports indicate that the antibiotic colistin (COL) has antifungal properties, this study investigated the antifungal in vitro activity of COL as single agent and in combination with the antifungal compounds voriconazole (VRC), caspofungin (CAS) and amphotericin B (AMB) against Scedosporium/Pseudallescheria spp., Exophiala dermatitidis and Geosmithia argillacea. In total, susceptibility was determined for 77 Scedosporium/Pseudallescheria spp., 82 E. dermatitidis and 17 G. argillacea isolates. The minimal inhibitory concentrations (MICs) of COL and the antifungals as single compound and in combination were determined with MIC test strips. Drug interactions were detected by crossing the MIC test strips at a 90º angle. The fractional inhibitory concentration index was used to categorise the drugs' interaction. The MIC50 value of COL was 12 µg ml(-1) for S. prolificans, 16 µg ml(-1) for P. apiosperma, 16 µg ml(-1) for P. boydii, 12 µg ml(-1) for E. dermatiditis and 6 µg ml(-1) for G. argillacea. VRC was the most active drug in combination without any antagonism with the exception of few P. boydii isolates. COL as single agent and in most combinations with antifungals exhibits in vitro antifungal activity against filamentous ascomycetes occurring in cystic fibrosis patients and may offer a novel therapeutic option, especially for multidrug-resistant S. prolificans.

Survival and inactivation of hepatitis E virus on inanimate surfaces.

BACKGROUND: Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis, and mainly transmitted via faecal-oral contamination or consumption of contaminated food products. However, limited data on the surface stability and HEV sensitivity to chemical disinfectants are available. AIM: To establish an HEV-based carrier assay to evaluate its surface stability and the virucidal activity of nine surface disinfectants. METHODS: A recently developed robust HEV-3 cell culture system for an HEV-based carrier assay. FINDINGS: Alcohol-based disinfectants were insufficient to eliminate HEV infectivity, whereas disinfectants based on aldehyde, peracetic acid, oxygen, and/or quaternary ammonium inactivated HEV. CONCLUSION: These findings have strong implications for the recommendation of evidence-based hygiene guidelines to reduce HEV transmission.

Evaluation of temperature, drying time and other determinants for the recovery of Gram-negative bacterial pathogens in disinfectant efficacy testing.

BACKGROUND: In the clinical setting, surface disinfection is an important measure to reduce the risk of cross transmission of micro-organisms and the risk of nosocomial infections. Standardized methods can be used to evaluate disinfection procedures, as well as the effectiveness of the active ingredients used for disinfection. However, despite standardization, the results of such methodologies are still determined by several factors, and incorrect results may lead to invalid assumptions about the effectiveness of a disinfectant, posing significant health risks for patients and health personnel. AIM: The objective of this study was to evaluate several determinants for the recovery of Pseudomonas aeruginosa and other test organisms to establish their influence on the results of standardized disinfection methodologies, and to find Gram-negative strains that can be used as suitable replacements for P. aeruginosa. METHODS: The effects of inoculum application method, drying time, temperature and carrier material on the survival and recovery of the test organisms were evaluated using Student's t-test, one-way analysis of variance and Tukey's multiple comparison test. FINDINGS AND CONCLUSIONS: Temperature, drying time, application method and carrier material were found to affect the recovery of P. aeruginosa cells significantly, and therefore influence the outcome of the methodologies used. This study also showed thatP. aeruginosa could be replaced with the Gram-negative species Acinetobacter baumannii, a test organism used in many standardized methodologies, which responds better under the same circumstances and has a behaviour similar to that of P. aeruginosa in disinfectant efficacy tests.

Virucidal activity of oral, hand, and surface disinfectants against respiratory syncytial virus.

BACKGROUND: Respiratory syncytial virus (RSV) is known as a major cause of respiratory tract infection in adults and children. Human-to-human transmission occurs via droplets as well as direct and indirect contact (e.g. contaminated surfaces or hands of medical staff). Therefore, applicable hygiene measures and knowledge about viral inactivation are of utmost importance. AIM: To elucidate the disinfection profile of RSV. METHODS: The study evaluated the virucidal efficacy of oral rinses specifically designed for children, World Health Organization (WHO)-recommended hand-rub formulations, and ethanol, as well as 2-propanol against RSV in a quantitative suspension test (EN14476). The stability of RSV on stainless steel discs was assessed and its inactivation by different surface disinfectants (EN16777) investigated. FINDINGS: All tested oral rinses except one reduced infectious viral titres to the lower limit of quantification. The two WHO-recommended hand-rub formulations as well as 30% ethanol and 2-propanol completely abolished the detection of infectious virus. Infectious RSV was recovered after several days on stainless steel discs. However, RSV was efficiently inactivated by all tested surface disinfectants based on alcohol, aldehyde, or hydrogen peroxide. CONCLUSION: Oral rinses, all tested hand-rub formulations as well as surface inactivation reagents were sufficient for RSV inactivation in vitro.

Results and relevance of molecular detection of pathogens by SeptiFast--a retrospective analysis in 75 critically ill children.

BACKGROUND: Sepsis is a common cause of death in children. Early detection of bloodstream pathogens is crucial for the appropriate antibio­tic treatment. Blood cultures (BC) are the gold standard test used for detection. Recently, additional molecular detection methods of microbial DNA by multiplex PCR (SeptiFast, SF) have become available. AIM: Our retrospective study was aimed to compare results of BC to those of SF regarding results and therapeutic relevance. METHOD: We identified a total of 110 SF samples in 75 patients with suspected systemic infection by retrospective chart review. Each patient underwent SF and BC testing simultaneously. RESULTS: The initial analysis displayed no statistical significant difference in positive SF results compared to BC (p=0.19): in 26 of 110 samples (24%) microbial DNA was found. 19 BC (17%) showed microbial growth. 14 samples were positive in SF but negative in BC (13%). In patients who were pretreated with antibiotics (n=97) pathogens were identified in 24 samples by SF (25%) but only in 11 samples by BC (11%). Based on the clinical presentation and the spectrum of bacterial isolates 3 BC were considered contaminated. Considering this, SF yielded pathogens significantly more often than BC in the overall study population (p=0.04). SF results were available at least 31 h before BC results. Based on SF result antibiotic therapy was adjusted in 14 patients (13%). CONCLUSION: Molecular detection of pathogens by SF was faster and more frequently positive than BC. We have therefore demonstrated that SF might be superior to BC in testing for bloodstream pathogens. Prospective multicentric studies are required to determine whether this hypothesis can be maintained.

[Microbiological diagnosis of implant-associated infections : Retrospective analysis of 133 patients in an arthroplasty center]. / Mikrobiologische Diagnostik bei implantatassoziierten Infektionen : Retrospektive Analyse von 133 Patienten in einem Endoprothetikzentrum.

BACKGROUND: Because standardized microbiological cultures of puncture fluids and tissue samples often do not provide pathogen detection in implant-associated infections, sonication and polymerase chain reaction (PCR) are used additionally today. OBJECTIVES: Pathogen spectra and previous microbiological standards are examined for agreement of results using the new methods sonication and PCR. MATERIALS AND METHODS: In this descriptive, retrospective observational study, we evaluated the data of 133 patients in whom a joint prosthesis, osteosynthesis material or a spacer was removed during revision surgery with suspected implant-associated infection and sent for sonication. RESULTS: Pathogen detection was achieved by culture of peri-implant material in 40.1% and by sonication in 42.5%. In each case, coagulase-negative staphylococci were detected most frequently. Overall, the results were consistent in 71.7% of cases. In the discrepant cases, more anaerobes could be detected by sonication, especially for osteosynthesis material and knee prostheses. PCR analyses in 21 cases showed pathogen detection in 14.3% and agreement with the results of peri-implant tissue culture and sonication in 57.1% and 66.7%, respectively. CONCLUSIONS: The present results indicate a gain in sensitivity of sonication, especially for anaerobes that are difficult to grow, and a gain in specificity through sonication. PCR analyses should be reserved for specific questions.

Virucidal activity of nasal sprays against severe acute respiratory syndrome coronavirus-2.

The highest viral loads of severe acute respiratory syndrome coronavirus-2 are detectable in the oral cavity, so a potential reduction of infectious virus by nasal and oral sprays could reduce transmission. Therefore, the inactivation capacity of nine nasal and oral sprays was evaluated according to EN 14476. One nasal spray based on sodium hypochlorite and one oral spray containing essential oils reduced viral titres by two to three orders of magnitude. Although clinical data are still sparse, nasal and oral sprays display a more convenient application for elderly people or those who are unable to rinse/gargle.

Evaluation of the substitution of poliomyelitis virus for testing virucidal activities of instrument and surface disinfection.

The Global Polio Eradication initiative has the goal to eradicate poliomyelitis worldwide. This means that poliomyelitisvirus type 1 strain LSc 2ab (PV-1) can no longer be used for the evaluation of virucidal activity of chemical disinfectants. This study evaluated murine parvovirus ATCC VR 1346 (minute virus of mice) as suitable surrogate for PV-1 when testing virucidal activity of biocides in instrument and surface disinfectants. Suspension testing in different laboratories with two commercially available active biocidal substances based on glutaraldehyde (0.01-0.25%) and peracetic acid (0.005-0.1%) with an exposure time of 30 min was performed. Both pathogens showed comparable susceptibility and dose-dependent reduction of virus titres following German and European Guidelines.