Preanalytical, Analytical and Postanalytical Analyses on Corynebacterium spp. and Actinomycetaceae in Urine Samples of Patients with Suspected Urinary Tract Infection-A Hypothesis-Forming Observational Study.

A hypothesis-forming exploratory cross-sectional assessment was conducted to assess the occurrence and relevance of Gram-positive rod-shaped bacteria like Corynebacterium spp. and Actinomycetaceae in human urine samples. In total, 1170 urine samples from 1031 inpatients with suspected urinary tract infection were assessed for culture-based growth of Gram-positive rod-shaped bacteria applying API Coryne assays, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and in-house 16S rRNA gene sequencing. Overall, 502 different bacterial colonies from 346 urine samples taken from 324 inpatients were observed. The three quantitatively most abundant genera or genus clusters were Corynebacterium (254 isolates, 62%), Actinomyces/Winkia (79 isolates, 19%), and Actinotignum/Actinobaculum (29 isolates, 7%). Compared to sequencing, the diagnostic accuracy of all assessed competitor assays from the diagnostic routine was <80% for differentiation on the genus level and <30% for differentiation on the species level. Prolongated incubation for 4 days compared to 2 days resulted in additional detection of 15% of the totally recorded Gram-positive rod-shaped bacteria. An approximately 5-fold increased detection rate in mid-stream urine compared to urine acquired applying alternative sampling strategies was observed. In conclusion, in the rare event of the suspected clinical relevance of such findings, confirmatory testing with invasively sampled urine should be considered due to the high contamination rate observed in mid-stream urine. Confirmatory testing by DNA-sequencing methods should be considered if an exact identification of genus or species is regarded as relevant for the individual choice of the therapeutic strategy.

Epidemiology of vancomycin-resistant enterococci in the United Arab Emirates: a retrospective analysis of 12 years of national AMR surveillance data.

Introduction: Enterococci are usually low pathogenic, but can cause invasive disease under certain circumstances, including urinary tract infections, bacteremia, endocarditis, and meningitis, and are associated with peritonitis and intra-abdominal abscesses. Increasing resistance of enterococci to glycopeptides and fluoroquinolones, and high-level resistance to aminoglycosides is a concern. National antimicrobial resistance (AMR) surveillance data for enterococci from the Middle East and North Africa (MENA) and the Gulf region is scarce. Methods: A retrospective 12-year analysis of N = 37,909 non-duplicate diagnostic Enterococcus spp. isolates from the United Arab Emirates (UAE) was conducted. Data was generated by routine patient care during 2010-2021, collected by trained personnel and reported by participating surveillance sites to the UAE National AMR Surveillance program. Data analysis was conducted with WHONET. Results: Enterococcus faecalis was the most commonly reported species (81.5%), followed by Enterococcus faecium (8.5%), and other enterococci species (4.8%). Phenotypically vancomycin-resistant enterococci (VRE) were found in 1.8% of Enterococcus spp. isolates. Prevalence of VRE (%VRE) was highest for E. faecium (8.1%), followed by E. faecalis (0.9%). A significant level of resistance to glycopeptides (%VRE) for these two species has been observed in the majority of observed years [E. faecalis (0-2.2%), 2010: 0%, 2021: 0.6%] and E. faecium (0-14.2%, 2010: 0%, 2021: 5.8%). Resistance to fluoroquinolones was between 17 and 29% (E. faecalis) and was higher for E. faecium (between 42 and 83%). VRE were associated with higher patient mortality (RR: 2.97), admission to intensive care units (RR: 2.25), and increased length of stay (six excess inpatient days per VRE case), as compared to vancomycin-susceptible Enterococcus spp. Discussion: Published data on Enterococcus infections, in particular VRE-infections, in the UAE and MENA region is scarce. Our data demonstrates that VRE-enterococci are relatively rare in the UAE, however showing an increasing resistance trend for several clinically important antibiotic classes, causing a concern for the treatment of serious infections caused by enterococci. This study also demonstrates that VRE were associated with higher mortality, increased intensive care unit admission rates, and longer hospitalization, thus poorer clinical outcome and higher associated costs in the UAE. We recommend the expansion of current surveillance techniques (e.g., local VRE screening), stricter infection prevention and control strategies, and better stewardship interventions. Further studies on the molecular epidemiology of enterococci are needed.

Proof-of-Concept Standardized Approach Using a Single-Disk Method Analogous to Antibiotic Disk Diffusion Assays for Routine Phage Susceptibility Testing in Diagnostic Laboratories.

Application of bacteriophages is increasingly being implemented in clinical therapies. Prior susceptibility testing should be regarded as mandatory, but standards are lacking. The objective of this research was to develop a highly standardized methodology to facilitate phage susceptibility testing (PST) in clinical microbiology routine laboratories. Therefore, EUCAST methods established for single disk-based antibiotic susceptibility testing (AST) were adapted. In a first step, basic parameters were evaluated using well-studied Escherichia phage T4-Escherichia coli combinations. In addition, test results were compared to those from conventional spot test and efficiency of plating (EOP) approaches. In a second step, the applicability of the methodology and the most promising test parameters were demonstrated for five other frequently isolated clinical bacterial species and their corresponding phages. At present, the method predominantly leads to qualitative rather than quantitative results. This disk-based approach provides a standardized, easy-to-handle, reproducible and reliable PST protocol by relying on well-established routine procedures in diagnostic laboratories. IMPORTANCE Application of bacteriophages in clinical therapies is attractive due to increasing rates of isolation of multidrug-resistant bacteria worldwide. As the phage effect is highly specific, prior susceptibility testing of target bacteria is mandatory. Of note, established standards are lacking. In this research, we adapted the single-disk method for antibiotic susceptibility testing to phage susceptibility testing (PST) in order to provide a standardized, easy-to-handle, reproducible, and reliable PST protocol for application in diagnostic routine laboratories.

Imaging and Clinical Parameters for Distinction between Infected and Non-Infected Fluid Collections in CT: Prospective Study Using Extended Microbiological Approach.

The aim of this investigation was to evaluate predictive CT imaging features and clinical parameters to distinguish infected from sterile fluid collections. Detection of infectious agents by advanced microbiological analysis was used as the reference standard. From April 2018 to October 2019, all patients undergoing CT-guided drainages were prospectively enrolled, if drainage material volume was at least 5 mL. Univariate analysis revealed attenuation (p = 0.001), entrapped gas (p < 0.001), fat stranding (p < 0.001), wall thickness (p < 0.001) and enhancement (p < 0.001) as imaging biomarkers and procalcitonin (p = 0.003) as clinical predictive parameters for infected fluid collections. On multivariate analysis, attenuation > 10 HU (p = 0.038), presence of entrapped gas (p = 0.027) and wall enhancement (p = 0.028) were independent parameters for distinguishing between infected and non-infected fluids. Gas entrapment had high specificity (93%) but low sensitivity (48%), while wall enhancement had high sensitivity (91%) but low specificity (50%). CT attenuation > 10 HU showed intermediate sensitivity (74%) and specificity (70%). Evaluation of the published proposed scoring systems did not improve diagnostic accuracy over independent predictors in our study. In conclusion, this prospective study confirmed that CT attenuation > 10 HU, entrapped gas and wall enhancement are the key imaging features to distinguish infected from sterile fluid collections on CT.

Risk Assessment for Molds in the Vicinity of a Child Requiring Peritoneal Dialysis Living in a Rural Northern German Area.

As well as severe immunosuppression, other predisposing factors may facilitate invasive mycosis caused by molds. Chronic kidney disease and the resulting peritoneal dialysis have been reported as factors putting patients at risk of fungal infections from environmental sources. We describe an environmental investigation undertaken to guide exposure prevention for a peritoneal dialysis patient with transient colonization of her nostrils by Lichtheimia corymbifera in a rural area of northern Germany. Systematic screening for airborne and surface-deposited molds enabled targeted recommendations to be made, although Lichtheimia corymbifera itself was not grown from the collected environmental samples. This communication is intended to illustrate how such an investigation can be performed on the basis of the environmental distribution of the molds and how preventive recommendations can be derived from the results.

How to Handle CT-Guided Abscess Drainages in Microbiological Analyses? Sterile Vials vs. Blood Culture Bottles for Transport and Processing.

The aim of this investigation was to compare microbiological analyses of 100 computed tomography-guided drainages from infectious foci (thoracic, abdominal, musculoskeletal), transported and analyzed by two widely established techniques, that are (i) sterile vials or (ii) inoculated blood culture bottles. The mean number of detected microorganisms from blood culture (aerobic/anaerobic) or conventional method (sterile vial, solid and broth media) per specimen were comparable with 1.29 and 1.41, respectively (p = 1.0). The conventional method showed a trend towards shorter time-to-result (median 28.62 h) in comparison to blood culture incubation (median 43.55 h) (p = 0.0722). Of note, detection of anaerobes (13% vs. 36%) and the number of detected microorganisms in polymicrobial infections (2.76 vs. 3.26) differed significantly with an advantage towards conventional techniques (p = 0.0015; p = 0.035), especially in abdominal aspirations. Despite substantially overlapping results from both techniques, the conventional approach includes some benefits which justify its role as standard approach.

Targeted Swabbing of Implant-Associated Biofilm Formation-A Staining-Guided Sampling Approach for Optimizing Routine Microbiological Diagnostics.

Background: Swabbing of implants removed from potentially infected sites represents a time saving and ubiquitously applicable alternative to sonication approaches. The latter bears an elevated risk of processing related contaminations due to the high number of handling steps. Since biofilms are usually invisible to the naked eye, adequate swabbing relies on the chance of hitting the colonized area on the implant. A targeted directed swabbing approach could overcome this detriment. Method: Three dyes were tested at different concentrations for their toxicity on biofilm-associated cells of S. epidermidis, the species most frequently identified as a causative agent of implant-associated infections. Results: Malachite green (0.2%) delivered the highest bacterial recovery rates combined with the best results in biofilm visualization. Its suitability for diagnostic approaches was demonstrated for smooth and rough implant surfaces. Biofilm-covered areas were successfully visualized. Conclusion: Subsequent targeted swab-sampling resulted in a significantly increased bacterial recovery rate compared to a dye-free "random swabbing" diagnostic approach.

Serology- and Blood-PCR-Based Screening for Schistosomiasis in Pregnant Women in Madagascar-A Cross-Sectional Study and Test Comparison Approach.

This work was conducted as a cross sectional study to define the disease burden of schistosomiasis in pregnant Madagascan women and to evaluate serological and molecular diagnostic assays. A total of 1154 residual EDTA blood samples from pregnant Madagascan women were assessed. The nucleic acid extractions were subjected to in-house real-time PCRs specifically targeting S. mansoni complex, S. haematobium complex, and African Schistosoma spp. on genus level, while the EDTA plasma samples were analyzed using Schistosoma-specific IgG and IgM commercial ELISA and immunofluorescence assays. The analyses indicated an overall prevalence of schistosomiasis in Madagascan pregnant women of 40.4%, with only minor regional differences and differences between serology- and blood PCR-based surveillance. The S. mansoni specific real-time PCR showed superior sensitivity of 74% (specificity 80%) compared with the genus-specific real-time PCR (sensitivity 13%, specificity 100%) in blood. The laborious immunofluorescence (sensitivity IgM 49%, IgG 87%, specificity IgM 85%, IgG 96%) scored only slightly better than the automatable ELISA (sensitivity IgM 38%, IgG 88%, specificity IgM 78%, IgG 91%). Infections with S. mansoni were detected only. The high prevalence of schistosomiasis recorded here among pregnant women in Madagascar calls for actions in order to reduce the disease burden.

Molecular Epidemiology of Carbapenem-Resistant Acinetobacter baumannii Isolates from Northern Africa and the Middle East.

At the Bundeswehr Hospitals of Hamburg and Westerstede, patients repatriated from subtropical war and crisis zones of Northern Africa and the Middle East were medically treated, including microbiological assessment. Within a six-year interval, 16 Acinetobacter spp. strains, including 14 Acinetobacter baumannii (Ab) isolates with resistance against carbapenems and origins in Afghanistan (n = 4), Iraq (n = 2), Libya (n = 2), and Syria (n = 8) were collected. While clonal relationships of Libyan and Syrian strains had been assessed by superficial next generation sequencing (NGS) and "DiversiLab" repetitive elements sequence-based (rep-)PCR so far, this study provides core genome-based sequence typing and thus more detailed epidemiological information. In detail, sequencing allowed a definitive species identification and comparison with international outbreak-associated Ab strains by core genome multi locus sequence typing (cgMLST) and the identification of MLST lineages, as well as the identification of known resistance genes. The sequence analysis allowed for the confirmation of outbreak-associated clonal clusters among the Syrian and Afghan Ab isolates, indicating likely transmission events. The identified acquired carbapenem resistance genes comprised blaOXA-23, blaOXA-58, blaNDM-1, and blaGES-11, next to other intrinsic and acquired, partly mobile resistance-associated genes. Eleven out of 14 Ab isolates clustered with the previously described international clonal lineages IC1 (4 Afghan strains), IC2 (6 Syrian strains), and IC7 (1 Syrian strain). Identified Pasteur sequence types of the 14 Ab strains comprised ST2 (Syrian), ST25 (Libyan), ST32 (Iraqi), ST81 (Afghan), ST85 (Libyan), and ST1112 (Syrian), respectively. In conclusion, the study revealed a broad spectrum of resistance genes in Ab isolated from war-injured patients from Northern Africa and the Middle East, thereby broadening the scarcely available data on locally abundant clonal lineages and resistance mechanisms.

Binary surrogate endpoints in clinical trials from the perspective of case definitions.

INTRODUCTION: Surrogate endpoints are widely used in clinical trials, especially in situations where the endpoint of interest is not directly observable or to avoid long trial periods. A typical example for this case is frequently found in clinical trials in oncology, where overall survival (OS) as endpoint of interest and progression free survival (PFS) as surrogate endpoint are discriminated. METHODS: Based on the perspective of case definitions on surrogate endpoints, we provide a formal definition of such endpoints followed by a description of the structure of surrogate endpoints. RESULTS: Surrogate endpoints can be considered as case definitions for the endpoint of interest. Therefore, the performance of surrogate endpoints can be described using the classical terminology of diagnostic tests including sensitivity and specificity. Since such endpoints always focus on sensitivity with necessarily reduced specificity, efficacy estimates based on such endpoints are in general biased. CONCLUSION: The abovementioned has to be taken into account while interpreting the results of clinical trials and should not be ignored while planning or conducting a study.

Molecular Epidemiology of Carbapenem-Resistant Acinetobacter baumannii Isolated from War-Injured Patients from the Eastern Ukraine.

Recently, a total of 32 carbapenem- and fluoroquinolone-resistant Acinetobacter baumannii (Ab) isolates was isolated from war-injured patients who were treated at German Bundeswehr Hospitals, and preliminarily typed by "DiversiLab" repetitive elements sequence-based (rep-) PCR. Core genome-based sequence typing was also used to provide more detailed epidemiological information. From the clusters observed by rep-PCR, selected Ab strains were subjected to Next Generation Sequencing (NGS) in order to compare them with international outbreak-associated Ab strains and to identify MLST (multi-locus sequence type) lineages, as well as to identify known resistance genes. Accordingly, NGS indicated higher diversity than rep-PCR, but also confirmed likely transmission events. The identified acquired carbapenem-resistant genes comprised blaOXA-23, blaOXA-72 and blaGES-12, as well as various other intrinsic and acquired resistance-associated genetic elements. All isolates clustered with the previously identified international clonal lineages IC1, IC2, IC6 and IC7, with corresponding Pasteur sequence types ST1, ST2, ST78 and ST25, respectively. In conclusion, the assessment confirmed a broad spectrum of resistance-associated genes in Ab isolated from war-injured patients from the Eastern Ukraine, and provided the first insights into locally abundant clonal lineages.

Comparison of commercial and in-house real-time PCR platforms for 15 parasites and microsporidia in human stool samples without a gold standard.

INTRODUCTION: A test comparison of in-house and commercial real-time PCR (qPCR) kits for the detection of human parasites and microsporidia in stool samples was conducted without a gold standard. Three different commercial kits were included in the comparison, with a range of 3-15 different PCR targets, while 14 targets were covered by in-house testing, so not all 16 target pathogens were covered by all assays. METHODS: Residual materials from nucleic acid extractions of stool samples with very high likelihood of being colonized or infected by at least one enteric parasite species or microsporidia were tested. In all, 500 DNA samples were analyzed, but due to limited sample volume, only 250 of the 500 samples were tested per assay. Each sample was assessed with the qPCR platforms being compared and cycle threshold (Ct) values were included in a descriptive comparison. RESULTS: Depending on the assay applied, qPCR detected per 250 tested samples Giardia duodenalis (184-205), Blastocystis spp. (174-183), Trichuris trichiura (118-120), Ascaris lumbricoides (79-96), Necator americanus (78-106), Hymenolepis nana (40-42), Cryptosporidium spp. (27-36), Dientamoeba fragilis (26-28), Schistosoma spp. (13-23), Enterobius vermicularis (8-14), Entamoeba histolytica (7-16), Strongyloides stercoralis (6-38), Cyclospora spp. (6-13), Taenia spp. (1-4), microsporidia (1-5), and Ancylostoma spp. (1-2). Inter-assay agreement kappa was almost perfect (0.81-1) for Dientamoeba fragilis, Hymenolepis nana, Cryptosporidium spp., and Ascaris lumbricoides, substantial (0.61-0.8) for Necator americanus, Blastocystis spp., Ancylostoma spp., Giardia duodenalis, Schistosoma spp., Trichuris trichiura, and Enterobius vermicularis, moderate (0.41-0.6) for Entamoeba histolytica, fair (0.21-0.4) for microsporidia, slight (0-0.2) for Cyclospora spp. and Strongyloides stercoralis, and poor (<0) for Taenia spp. CONCLUSIONS: Varying inter-assay agreement makes interpretation of microsporidia and parasite PCR in stool samples challenging. Intra-assay agreement had been controlled during the developing of the assays. Future studies, e.g., with optimized nucleic acid procedures and including microscopically characterized samples, are advisable.

On detection thresholds-a review on diagnostic approaches in the infectious disease laboratory and the interpretation of their results.

Diagnostic testing in the infectious disease laboratory facilitates decision-making by physicians at the bedside as well as epidemiological assessments and surveillance at study level. Problems may arise if test results are uncritically considered as being the same as the unknown true value. To allow a better understanding, the influence of external factors on the interpretation of test results is introduced with the example of prevalence, followed by the presentation of strengths and weaknesses of important techniques in the infectious disease laboratory like microscopy, cultural diagnostics, serology, mass spectrometry, nucleic acid amplification and hypothesis-free metagenomic sequencing with focus on basic, high-technology and potential future approaches. Special problems like multiplex testing as well as uncertainty of test evaluations, if no gold standard is available, are also stressed with a final glimpse on emerging future technologies for the infectious disease laboratory. In the conclusions, suitability for point-of-care-testing and field laboratory applications is summarized. The aim is to illustrate the limitations of diagnostic accuracy to both clinicians and study planners and to stress the importance of close cooperation with experts in laboratory disciplines so as to avoid potentially critical misunderstandings due to inappropriate interpretation of diagnostic test results.

Next-generation sequencing for hypothesis-free genomic detection of invasive tropical infections in poly-microbially contaminated, formalin-fixed, paraffin-embedded tissue samples - a proof-of-principle assessment.

BACKGROUND: The potential of next-generation sequencing (NGS) for hypothesis-free pathogen diagnosis from (poly-)microbially contaminated, formalin-fixed, paraffin embedded tissue samples from patients with invasive fungal infections and amebiasis was investigated. Samples from patients with chromoblastomycosis (n = 3), coccidioidomycosis (n = 2), histoplasmosis (n = 4), histoplasmosis or cryptococcosis with poor histological discriminability (n = 1), mucormycosis (n = 2), mycetoma (n = 3), rhinosporidiosis (n = 2), and invasive Entamoeba histolytica infections (n = 6) were analyzed by NGS (each one Illumina v3 run per sample). To discriminate contamination from putative infections in NGS analysis, mean and standard deviation of the number of specific sequence fragments (paired reads) were determined and compared in all samples examined for the pathogens in question. RESULTS: For matches between NGS results and histological diagnoses, a percentage of species-specific reads greater than the 4th standard deviation above the mean value of all 23 assessed sample materials was required. Potentially etiologically relevant pathogens could be identified by NGS in 5 out of 17 samples of patients with invasive mycoses and in 1 out of 6 samples of patients with amebiasis. CONCLUSIONS: The use of NGS for hypothesis-free pathogen diagnosis from contamination-prone formalin-fixed, paraffin-embedded tissue requires further standardization.

Detection of Tropheryma whipplei in stool samples by one commercial and two in-house real-time PCR assays.

OBJECTIVE: Tropheryma whipplei, the causative agent of Whipple's disease, can also be identified in stool samples of humans without systemic disease. It is much more frequently detected in human stool samples in tropical environments than in industrialized countries. PCR-screening has been applied for point prevalence studies and environmental assessments in tropical settings, but results depend on the applied assay. We compared one commercial qPCR kit with two well-described in-house assays for detection of T. whipplei from stool. METHODS: Residual materials from nucleic acid extractions of stool samples from two groups with presumably different prevalences and increased likelihood of being colonized or infected by T. whipplei were tested. One group comprised 300 samples from study participants from western Africa (group 1); the second group was of 300 returnees from tropical deployments (group 2). Each sample was assessed with all three qPCR assays. Cycle threshold (Ct ) values were descriptively compared. RESULTS: Based solely on mathematical modeling, the three PCR assays showed considerably different detection rates of T. whipplei DNA in stool samples (kappa 0.67 (95% confidence interval [0.60, 0.73])). Considering the calculated test characteristics, prevalence of 28.3% for group 1 and 5.0% for group 2 was estimated. Discordant test results were associated with later Ct values. The study did not validate the assays for the detection of T. whipplei in Whipple's disease and for diagnostic purposes since clinical specificity and sensitivity were not investigated. CONCLUSIONS: In spite of the observed diagnostic uncertainty, PCR-based screening approaches can be used for epidemiological purposes and environmental samples to define the source and reservoir in resource-limited tropical settings if prevalence is calculated using diagnostic accuracy-adjusted methods.

Antibiotics release from cement spacers used for two-stage treatment of implant-associated infections after total joint arthroplasty.

Two-stage revision arthroplasty is the treatment of choice for periprosthetic infection, a serious complication after knee or hip arthroplasty. Our prospective clinical trial aimed to investigate the concentrations of gentamicin and vancomycin in wound exudate and tissue in two-stage revision arthroplasty. Wound exudate and periprosthetic membrane samples were collected from 18 patients (10 hip and eight knee patients), who were due for two-stage treatment after a periprosthetic joint infection. Samples were taken during insertion of antibiotic-impregnated spacers and after their removal. The concentrations of gentamicin and vancomycin in wound exudates and adjacent tissue were analyzed using high-performance liquid chromatography mass spectrometry. Average time period of spacer implantation was 13.6 weeks (9.3-22.6 weeks). The concentration of vancomycin in wound exudate decreased from a median of 43.28 µg/mL (0.28-261.22) after implantation to 0.46 µg/mL (0.13-37.47) after the removal of the spacer. In the adjacent tissue, vancomycin concentration was mainly undetectable prior to spacer implantation (0.003 µg/g [0.003-0.261]) and increased to 0.318 µg/g [0.024-484.16] at the time of spacer removal. This was also observed for gentamicin in the tissue of patients who previously had cement-free implants (0.008 µg/g [0.008-0.087] vs. 0.164 µg/g [0.048-71.75]) while in the tissue of patients with previously cemented prosthesis, baseline concentration was already high (8.451 µg/g [0.152-42.926]). Despite the rapid decrease in antibiotics release from spacer cement observed in vitro, in vivo antibiotics are much longer detectable, especially in the adjacent soft tissue. © 2018 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials Published By Wiley Periodicals, Inc. J Biomed Mater Res B Part B, 2019. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1587-1597, 2019.