Establishing breakpoints for amoxicillin/clavulanate and ampicillin/sulbactam for rapid antimicrobial susceptibility testing directly from positive blood culture bottles.

Introduction. In 2018, EUCAST released guidelines on rapid antimicrobial susceptibility testing (RAST) directly from positive blood culture bottles for selected bacterial species and antimicrobial agents, but not for the commonly used agents amoxicillin/clavulanate (AMC) and ampicillin/sulbactam (SAM).Hypothesis/Gap statement. This work addresses the Enterobacterales RAST capability gap for betalactam/betalactamase inhibitor combinations.Aim. We aimed to determine RAST breakpoints for AMC and SAM for Escherichia coli and Klebsiella pneumoniae after 4 and 6 h of incubation directly from positive blood cultures.Methodology. Blood culture bottles were spiked with clinical isolates of E. coli (n=89) and K. pneumoniae (n=81). RAST was performed according to EUCAST guidelines and zones were read after 4 and 6 h. Breakpoints were defined to avoid very major errors.Results. The proportion of readable zone diameters after 4 h of incubation were 90.8 % in E. coli and 85.8 % in K. pneumoniae isolates. After 6 h of incubation all zone diameters could be read. The proposed breakpoints for E. coli after 6 h of incubation were ≥16 mm S (susceptible), 14-15 mm ATU (area of technical uncertainty) and <14 mm R (resistant) for AMC; ≥15 mm S, 12-14 mm ATU and <12 mm R for SAM; for K. pneumoniae these were ≥16 mm S, 14-15 mm ATU and <14 mm R for AMC; ≥13 mm S, 12 mm ATU, <12 mm R for SAM. Applying our newly set breakpoints, major errors were infrequent (2.6 %).Conclusion. We propose novel AMC and SAM breakpoints for RAST directly from positive blood cultures for reading after 4 and 6 h of incubation.

Musculoskeletal tuberculosis revisited: bone and joint tuberculosis in Austria.

BACKGROUND: To prevent further spread of the disease and secondary deformity, musculoskeletal tuberculosis (TB) remains a challenge in terms of early diagnosis and treatment. This study gives an overview on TB trends in Austria (pulmonary and extrapulmonary TB) (A) and analyses a retrospective series of musculoskeletal TB cases diagnosed and treated at an Austrian tertiary centre (B). METHODS: (A) We analysed data obtained from the Austrian national TB registry to provide information on TB patients´ demographics and manifestation sites between 1995 and 2019. (B) Furthermore, we performed an observational study of all patients with a confirmed diagnosis of musculoskeletal TB who were admitted to the Department of Orthopaedics and Trauma, Medical University of Graz (2005-2019). Demographic, diagnostic, clinical and follow-up data were retrieved from the medical records. RESULTS: (A) From 1995 to 2019, a significant linear reduction in overall Austrian tuberculosis incidence rates occurred (p < 0.001). In the period investigated, Austria recorded a total of 307 patients with musculoskeletal TB. (B) Our retrospective case-series included 17 individuals (9 males, 8 females; average follow-up 48.4 months; range 0-116). There was a biphasic age distribution with a peak in elderly native Austrians (median 69, range 63-92), and a second peak in younger patients with a migration background (median 29, range 18-39). Sites of manifestation were the spine (n = 10), peripheral joints (n = 5), and the soft tissues (n = 2). Diagnosis was based on histology (n = 13), PCR (n = 14), and culture (n = 12). Eleven patients underwent surgery (64.7%). Secondary deformities were frequent (n = 9), and more often observed in patients with spinal TB (n = 6). CONCLUSION: Musculoskeletal TB should be considered if untypical joint infections or nonspecific bone lesions occur in younger patients with a migration background or in patients with specific risk factors.

Pathogen Detection by Metagenomic Next-Generation Sequencing During Neutropenic Fever in Patients With Hematological Malignancies.

Background: Febrile neutropenia (FN) after chemotherapy is a major cause of morbidity during cancer treatment. The performance of metagenomic next-generation sequencing (mNGS) of circulating cell-free deoxyribonucleic acid from plasma may be superior to blood culture (BC) diagnostics for identification of causative pathogens. The aim of this study was to validate mNGS (DISQVER test) for the detection of pathogens in hematologic patients with FN. Methods: We collected paired whole blood specimens from central venous catheter and peripheral vein during FN for BC and mNGS testing. We repeated paired sampling at the earliest after 3 days of fever, which was defined as 1 FN episode. All clinical data were retrospectively reviewed by an infectious disease expert panel. We calculated percent positive agreement (PPA), percent negative agreement (PNA), percent overall agreement (POA), and sensitivity and specificity. Results: We analyzed a total of 98 unselected FN episodes in 61 patients who developed predominantly FN after conditioning therapy for allogeneic (n = 22) or autologous (n = 21) hematopoietic stem cell transplantation. Success rate of mNGS was 99% (97 of 98). Positivity rate of mNGS was 43% (42 of 97) overall and 32% (31 of 97) excluding viruses compared to 14% (14 of 98) in BC. The PPA, PNA, and POA between mNGS and BC were 84.6% (95% confidence interval [CI], 54.6% to 98.1%), 63.1% (95% CI, 51.9% to 73.4%), and 66% (95% CI, 55.7% to 75.3%), respectively. Sensitivity for bacteria or fungi was 40% (95% CI, 28.0% to 52.9%) and 18.5% (95% CI, 9.9% to 30.0%), respectively. Conclusions: Pathogen detection by mNGS (DISQVER) during unselected FN episodes shows 2-fold higher sensitivity and a broader pathogen spectrum than BC.

Acute suppurative thyroiditis due to Streptococcus anginosus leading to sepsis and acute respiratory distress syndrome: a case report

SUMMARY Acute suppurative thyroiditis (AST) is a rare but potentially life-threatening thyroid disease with a high mortality if left untreated. Thus, differentiation from other thyroid disorders is highly important in clinical practice. A 22-year-old male patient was admitted to a tertiary care hospital with cervical pain, palpitations, thyrotoxicosis, and an inhomogeneously enlarged right thyroid lobe. In view of the clinical findings, subacute thyroiditis (SAT) was suspected and treatment with glucocorticoids was started. After initial amelioration, the patient developed cervical erythema, fever, and recurrent pain. A CT scan showed extensive phlegmonous inflammation and abscess formation, suggestive of AST. We started immediate empiric antibiotic therapy and performed surgical drainage of the abscess formations. Subsequently, the patient developed hypoxic respiratory failure, leading to ICU admission and intermittent need for non-invasive ventilation. Blood and abscess cultures were positive for Streptococcus anginosus. If left untreated, AST represents a potentially life-threatening disease. Thus, in clinically doubtful cases, liberal further assessment by means of cervical CT scans or fine needle aspiration biopsy are strongly advised.

Acute suppurative thyroiditis due to Streptococcus anginosus leading to sepsis and acute respiratory distress syndrome: a case report.

Acute suppurative thyroiditis (AST) is a rare but potentially life-threatening thyroid disease with a high mortality if left untreated. Thus, differentiation from other thyroid disorders is highly important in clinical practice. A 22-year-old male patient was admitted to a tertiary care hospital with cervical pain, palpitations, thyrotoxicosis, and an inhomogeneously enlarged right thyroid lobe. In view of the clinical findings, subacute thyroiditis (SAT) was suspected and treatment with glucocorticoids was started. After initial amelioration, the patient developed cervical erythema, fever, and recurrent pain. A CT scan showed extensive phlegmonous inflammation and abscess formation, suggestive of AST. We started immediate empiric antibiotic therapy and performed surgical drainage of the abscess formations. Subsequently, the patient developed hypoxic respiratory failure, leading to ICU admission and intermittent need for non-invasive ventilation. Blood and abscess cultures were positive for Streptococcus anginosus. If left untreated, AST represents a potentially life-threatening disease. Thus, in clinically doubtful cases, liberal further assessment by means of cervical CT scans or fine needle aspiration biopsy are strongly advised.

Antifungal prophylaxis for prevention of COVID-19-associated pulmonary aspergillosis in critically ill patients: an observational study.

BACKGROUND: Coronavirus disease 19 (COVID-19)-associated pulmonary aspergillosis (CAPA) emerged as important fungal complications in patients with COVID-19-associated severe acute respiratory failure (ARF). Whether mould active antifungal prophylaxis (MAFP) can prevent CAPA remains elusive so far. METHODS: In this observational study, we included all consecutive patients admitted to intensive care units with COVID-19-associated ARF between September 1, 2020, and May 1, 2021. We compared patients with versus without antifungal prophylaxis with respect to CAPA incidence (primary outcome) and mortality (secondary outcome). Propensity score adjustment was performed to account for any imbalances in baseline characteristics. CAPA cases were classified according to European Confederation of Medical Mycology (ECMM)/International Society of Human and Animal Mycoses (ISHAM) consensus criteria. RESULTS: We included 132 patients, of whom 75 (57%) received antifungal prophylaxis (98% posaconazole). Ten CAPA cases were diagnosed, after a median of 6 days following ICU admission. Of those, 9 CAPA cases were recorded in the non-prophylaxis group and one in the prophylaxis group, respectively. However, no difference in 30-day ICU mortality could be observed. Thirty-day CAPA incidence estimates were 1.4% (95% CI 0.2-9.7) in the MAFP group and 17.5% (95% CI 9.6-31.4) in the group without MAFP (p = 0.002). The respective subdistributional hazard ratio (sHR) for CAPA incidence comparing the MAFP versus no MAFP group was of 0.08 (95% CI 0.01-0.63; p = 0.017). CONCLUSION: In ICU patients with COVID-19 ARF, antifungal prophylaxis was associated with significantly reduced CAPA incidence, but this did not translate into improved survival. Randomized controlled trials are warranted to evaluate the efficacy and safety of MAFP with respect to CAPA incidence and clinical outcomes.

3D magnetically controlled spatiotemporal probing and actuation of collagen networks from a single cell perspective.

Cells continuously sense and react to mechanical cues from their surrounding matrix, which consists of a fibrous network of biopolymers that influences their fate and behavior. Several powerful methods employing magnetic control have been developed to assess the micromechanical properties within extracellular matrix (ECM) models hosting cells. However, many of these are limited to in-plane sensing and actuation, which does not allow the matrix to be probed within its full 3D context. Moreover, little attention has been given to factors specific to the model ECM systems that can profoundly influence the cells contained there. Here we present methods to spatiotemporally probe and manipulate extracellular matrix networks at the scale relevant to cells using magnetic microprobes (µRods). Our techniques leverage 3D magnetic field generation, physical modeling, and image analysis to examine and apply mechanical stimuli to fibrous collagen matrices. We determined shear moduli ranging between hundreds of Pa to tens of kPa and modeled the effects of proximity to rigid surfaces and local fiber densification. We analyzed the spatial extent and dynamics of matrix deformation produced in response to magnetic torques on the order of 10 pNm, deflecting fibers over an area spanning tens of micrometers. Finally, we demonstrate 3D actuation and pose extraction of fluorescently labelled µRods.

Cat at home? Cat scratch disease with atypical presentations and aggressive radiological findings mimicking sarcoma, a potential diagnostic pitfall.

Background and purpose - Cat scratch disease (CSD) is a self-limiting disease caused by Bartonella (B.) henselae. It is characterized by granulomatous infection, most frequently involving lymph nodes. However, it can present with atypical symptoms including musculoskeletal manifestations, posing a diagnostic challenge. We describe the prevalence and demographics of CSD cases referred to a sarcoma center, and describe the radiological, histological, and molecular findings.Patients and methods - Our cohort comprised 10 patients, median age 27 years (12-74) with clinical and radiological findings suspicious of sarcoma.Results - 7 cases involved the upper extremities, and 1 case each involved the axilla, groin, and knee. B. henselae was found in 6 cases tested using polymerase chain reaction and serology in 5 cases. 9 cases were soft tissue lesions and 1 lesion involved the bone. 1 patient had concomitant CSD with melanoma metastasis in enlarged axillary lymph nodes. On MRI, 5 soft tissue lesions were categorized as probably inflammatory. In 3 cases, with still detectable lymph node structure and absent or initial liquefaction, the differential diagnosis included lymph node metastasis. A sarcoma diagnosis was suggested in 4 cases. The MRI imaging features of the bone lesion were suspicious of a bone tumor or osteomyelitis.Interpretation - Atypical imaging findings cause a diagnostic challenge and the differential diagnosis includes malignant neoplasms (such as sarcoma or carcinoma metastasis) and other infections. The distinction between these possibilities is crucial for treatment and prognosis.

Longitudinal Evaluation of Plasma Cytokine Levels in Patients with Invasive Candidiasis.

Interleukin (IL) 17A plays a decisive role in anti-Candida host defense. Previous data demonstrated significantly increased IL-17A values in candidemic patients. We evaluated levels and time courses of IL-17A, and other cytokines suggested to be involved in Candida-specific immunity (IL-6, IL-8, IL-10, IL-17F, IL-22, IL-23, interferon-γ, tumor necrosis factor-α, Pentraxin-related protein 3, transforming growth factor-ß) in patients with invasive candidiasis (IC) compared to bacteremic patients (Staphylococcus aureus, Escherichia coli) and healthy controls (from previous 4 days up to day 14 relative to the index culture (-4; 14)). IL-17A levels were significantly elevated in all groups compared to healthy controls. In IC, the highest IL-17A values were measured around the date of index sampling (-1; 2), compared to significantly lower levels prior and after sampling the index culture. Candidemic patients showed significantly higher IL-17A values compared to IC other than candidemia at time interval (-1; 2) and (3; 7). No significant differences in IL-17A levels could be observed for IC compared to bacteremic patients. Candidemic patients had higher IL-8, IL-10, IL-22, IFN-γ, PTX3 and TNF-α values compared to non-candidemic. Based on the limited discriminating competence between candidemia and bacteremia, IL-17A has to be considered a biomarker for blood stream infection rather than invasive Candida infection.

Risk of reinfection after two- or multiple-stage knee revision surgery using superficial vancomycin coating and conventional spacers.

This study investigates the effect of superficial vancomycin coating (SVC) in two- or more-stage exchange procedures of prosthetic knee joint infections. We hypothesized that spacer treatment with SVC result in lower reinfection rates than conventional spacers after prosthetic reimplantation. Our secondary aim was to determine the demographic and treatment factors associated with reinfection rates. This retrospective cohort study compromised 96 cases with prosthetic knee infections. Twenty-four cases were treated with a temporary SVC spacer and 72 cases with conventional spacers. Prosthetic reinfection occurred after a median observation period of 1.7 ± 4.0 years in 24 cases (25%). The prevalence of having a reinfection was not significantly different between the two treatment groups (13% [3 cases] in the SVC group vs. 29% [21 cases] in the conventional spacer group [p = .104]). In seven cases (7.3%), two in the SVC group (8.3%) and five (6.9%) in the conventional spacer group (p ≥ .999), histological, respectively microbiological evaluations from the intraoperative specimens revealed persistent infection at the second stage. Nevertheless, in all seven cases no significant higher risk of periprosthetic reinfection was observed during follow-up (p = .750). Our secondary investigation of cofactors revealed that spacers additionally stabilized by nails were significantly associated with a 3.9-fold higher hazard ratio of sustaining a reinfection of revision prosthesis (p = .005).

Invasive pulmonary aspergillosis complicating COVID-19 in the ICU - A case report.

It is not yet known, if critically ill COVID-19 patients are prone to fungal infections. We report a 69-year-old patient without typical risk factors for invasive pulmonary aspergillosis (IPA), who developed IPA two weeks after onset of symptoms. Our report shows that IPA may occur in critically ill COVID-19 patients.

Implementation of rapid antimicrobial susceptibility testing combined with routine infectious disease bedside consultation in clinical practice (RAST-ID): a prospective single-centre study.

OBJECTIVES: Recently, EUCAST released guidelines for rapid antimicrobial susceptibility testing (RAST) directly from positive blood culture bottles. The aim of our prospective single-centre clinical study was to assess the proportion of readable results and errors compared with routine antimicrobial susceptibility testing and the clinical consequences drawn by infectious disease (ID) physicians from RAST results during same-day bedside consultation. METHODS: All positive blood cultures suitable for RAST from January to December 2019 were included and RAST results at 4 and 6 h compared with standard disc diffusion. The real-life impact of RAST on clinical decisions was assessed during same-day ID bedside consultation. RESULTS: The proportion of readable RAST results was significantly higher after 6 h of incubation compared with after 4 h (881/930 versus 642/847; P < 0.0001). Major and very major errors were rare (17/642 after 4 h and 12/881 after 6 h; P = 0.087). ID consultation was performed in 134 patients after the RAST result. Antimicrobial treatment was changed in 73 patients and 84 additional measures (i.e. imaging studies, surgery, additional resistance testing) were ordered in 62 patients. CONCLUSIONS: RAST according to EUCAST methods was easy to implement with a low number of major and very major errors after 6 h of incubation. ID physicians performing bedside consultations frequently used this information to change antimicrobial treatment and recommended additional measures.

Isavuconazole Treatment in a Mixed Patient Cohort with Invasive Fungal Infections: Outcome, Tolerability and Clinical Implications of Isavuconazole Plasma Concentrations.

Isavuconazole (ISA) is a triazole antifungal agent recommended for treatment of invasive aspergillosis or mucormycosis. The objective of this study was to evaluate ISA levels in a real world setting in a mixed patient cohort including patients with non-malignant diseases and extracorporeal treatments, and to correlate findings with efficacy and safety outcomes. We investigated 33 ISA treatment courses in 32 adult patients with hematological and other underlying diseases and assessed the clinical response, side effects and ISA trough plasma concentrations. ISA treatment led to complete and partial response in 87% of patients and was well tolerated. The median ISA plasma concentration was 3.05 µg/mL (range 1.38-9.1, IQR 1.93-4.35) in patients without renal replacement therapy (RRT) or extracorporeal membrane oxygenation (ECMO) and significantly lower in patients with RRT including cases with additional ECMO or Cytosorb® adsorber therapy (0.88 µg/mL, range 0.57-2.44, IQR 0.71-1.21). After exclusion of values obtained from four patients with ECMO or Cytosorb® adsorber the median concentration was 0.91 µg/mL (range 0.75-2.44, IQR 0.90-1.36) in the RRT group. In addition to previous recommendations we propose to monitor ISA trough plasma concentrations in certain circumstances including RRT, other extracorporeal treatments and obesity.

Impact of ITS-Based Sequencing on Antifungal Treatment of Patients with Suspected Invasive Fungal Infections.

Molecular techniques including the sequencing of fungal-specific DNA targets are increasingly used in the diagnosis of suspected invasive fungal infections. In contrast to established biomarkers like galactomannan or 1-3-ß-d-glucan, the clinical impact of these methods remains unknown. We retrospectively investigated the impact of ITS1-sequencing on antifungal treatment strategies in 71 patients (81 samples) with suspected invasive fungal infections. ITS-sequencing either confirmed already ongoing antifungal therapy (19/71 patients, 27%), led to a change in antifungal therapy (11/71, 15%) or supported the decision to withhold antifungal treatment (34/71, 48%) (in seven of 71 patients, ITS-sequencing results were obtained postmortem). ITS-sequencing results led to a change in antifungal therapy in a relevant proportion of patients, while it confirmed therapeutic strategies in the majority. Therefore, ITS-sequencing was a useful adjunct to other fungal diagnostic measures in our cohort.

Mechanophenotyping of 3D multicellular clusters using displacement arrays of rendered tractions.

Epithelial tissues mechanically deform the surrounding extracellular matrix during embryonic development, wound repair, and tumor invasion. Ex vivo measurements of such multicellular tractions within three-dimensional (3D) biomaterials could elucidate collective dissemination during disease progression and enable preclinical testing of targeted antimigration therapies. However, past 3D traction measurements have been low throughput due to the challenges of imaging and analyzing information-rich 3D material deformations. Here, we demonstrate a method to profile multicellular clusters in a 96-well-plate format based on spatially heterogeneous contractile, protrusive, and circumferential tractions. As a case study, we profile multicellular clusters across varying states of the epithelial-mesenchymal transition, revealing a successive loss of protrusive and circumferential tractions, as well as the formation of localized contractile tractions with elongated cluster morphologies. These cluster phenotypes were biochemically perturbed by using drugs, biasing toward traction signatures of different epithelial or mesenchymal states. This higher-throughput analysis is promising to systematically interrogate and perturb aberrant mechanobiology, which could be utilized with human-patient samples to guide personalized therapies.

T2Candida magnetic resonance in patients with invasive candidiasis: Strengths and limitations.

T2Candida enables detection of five Candida species in whole blood within approximately 5 hours. Routinely drawn EDTA blood samples were prospectively stored and tested with T2Candida in patients with invasive candidiasis identified by routine index blood or sterile site cultures. T2Candida was compared to diagnostic blood and sterile site cultures and also performed with samples obtained prior and after collection of index cultures. T2Candida was evaluated with 133 samples of 32 patients with candidemia and 22 patients with deep-seated invasive candidiasis. In the candidemic group 28/32 (87.5%) patients had at least one positive T2Candida result at any time point. A total of 17/25 (68%) candidemic patients had a positive T2Candida sample that was drawn concurrently to the index blood culture. In the per patient analysis 17/18 (94.4%) candidemic patients with matched T2Candida samples and peripheral blood cultures at any timepoint had a positive T2Candida test. T2Candida revealed discordant Candida species identification in two candidemic patients. Six of 22 (27.3%) deep-seated IC patients had a positive T2Candida result. Despite advanced time-to-results the clinical value of T2Candida in diagnosing candidemia seems to be limited by missing blood culture positive cases. Positivity rates of T2Candida increased when serial T2Candida samples were tested. In patients with suspected deep-seated invasive candidiasis T2Candida might act as a blood based adjunct to sterile site cultures.

Breast Cancer Cells Transition from Mesenchymal to Amoeboid Migration in Tunable Three-Dimensional Silk-Collagen Hydrogels.

Invading cancer cells adapt their migration phenotype in response to mechanical and biochemical cues from the extracellular matrix. For instance, mesenchymal migration is associated with strong cell-matrix adhesions and an elongated morphology, while amoeboid migration is associated with minimal cell-matrix adhesions and a rounded morphology. However, it remains challenging to elucidate the role of matrix mechan-ics and biochemistry, since these are both dependent on ECM protein concentration. Here, we demonstrate a composite silk fibroin and collagen I hydrogel where stiffness and microstructure can be systematically tuned over a wide range. Using an overlay assay geometry, we show that the invasion of metastatic breast cancer cells exhibits a biphasic dependence on silk fibroin concentration at fixed collagen I concentration, first increasing as the hydrogel stiffness increases, then decreasing as the pore size of silk fibroin decreases. Indeed, mesenchymal morphology exhibits a similar biphasic depen-dence on silk fibroin concentration, while amoeboid morphologies were favored when cell-matrix adhesions were less effective. We used exogenous biochemical treatment to perturb cells towards increased contractility and a mesenchymal morphology, as well as to disrupt cytoskeletal function and promote an amoeboid morphology. Overall, we envision that this tunable biomaterial platform in a 96-well plate format will be widely applicable to screen cancer cell migration against combinations of designer biomaterials and targeted inhibitors.

Prospective evaluation of three rapid molecular tests for seasonal influenza in patients presenting at an emergency unit.

BACKGROUND: For infection control measures, rapid accurate diagnostics on admission of patients with suspected seasonal influenza is crucial. OBJECTIVE: Prospective comparison of three rapid molecular tests for detection of influenza A/B RNA. STUDY DESIGN: Outpatients presenting at the Medical emergency department of Graz University Hospital with influenza-like illness and a requirement for hospitalization (n = 312) were studied. Nasopharyngeal swabs were collected with the 3 mL-version of the UTM™ Viral Transport Medium (Copan). Specimens were tested for influenza A and B RNA using the Alere™ i Influenza A & B (Abbott), the cobas® Influenza A/B (Roche), and the Xpert® Xpress Flu/RSV (Cepheid) tests. Results were compared to those obtained from the same specimen by the Influenza A/B R-GENE® (bioMerieux) test based on real-time PCR as reference method. RESULTS: Overall sensitivities of the Abbott, Roche, and Cepheid tests were 90.5%, 96.0%, and 97.0%, overall specificities 99.4%, 97.6%, and 98.2% respectively. With the Abbott and the Cepheid tests, all specimens gave valid results, while the Roche test showed invalid results in 37 (12.1%) specimens. Total time to result for the Abbott, Roche, and Cepheid tests was 18 min, 22 min, and 32 min respectively. CONCLUSIONS: The Abbott test lacked sensitivity, the Roche test was impaired by a high number of invalid results. Overall, despite the longest total time to result, the Cepheid test showed the best performance to detect influenza virus RNA in symptomatic patients presenting at an emergency unit in this study.