Factors associated with non-carbapenemase mediated carbapenem resistance of Gram-negative bacteria: a retrospective case-control study

Infections with carbapenemase-producing Gram-negative bacteria are related to increased morbidity and mortality, yet little is known regarding infections caused by non-beta-lactamase mediated carbapenem-resistant bacteria. Our objective was to identify risk factors for, and the clinical impact of infections caused by carbapenem-resistant carbapenemase-negative Enterobacterales and Pseudomonas aeruginosa. This retrospective matched case-control study was performed at the University Hospital of Basel, Switzerland, in 2016. We focused on other resistance mechanisms by excluding laboratory-confirmed carbapenemase-positive cases. Carbapenem resistance was set as the primary endpoint, and important risk factors were investigated by conditional logistic regression. The clinical impact of carbapenem resistance was estimated using regression models containing the resistance indicator as explanatory factor and adjusting for potential confounders. Seventy-five cases of infections with carbapenem-resistant, carbapenemase-negative bacteria were identified and matched with 75 controls with carbapenem-susceptible infections. The matched data set was well-balanced regarding age, gender, and comorbidity. Duration of prior carbapenem treatment (OR 1.15, [1.01, 1.31]) correlated with resistance to carbapenems. Our study showed that patients with carbapenem-resistant bacteria stayed 1.59 times (CI [0.81, 3.14]) longer in an ICU. The analyzed dataset did not provide evidence for strong clinical implications of resistance to carbapenems or increased mortality. The duration of prior carbapenem treatment seems to be a strong risk factor for the development of carbapenem resistance. The higher risk for a longer ICU stay could be a consequence of a carbapenem resistance. In contrast to carbapenemase-producers, the clinical impact of carbapenamase-negative, carbapenem-resistant strains may be limited. Trial registration: The study design was prospectively approved by the local Ethics Commission on 10.08.2017 (EKNZ BASEC 2017-00222)

Novel Organism Verification and Analysis (NOVA) study: identification of 35 clinical isolates representing potentially novel bacterial taxa using a pipeline based on whole genome sequencing

BACKGROUND Reliable species identification of cultured isolates is essential in clinical bacteriology. We established a new study algorithm named NOVA - Novel Organism Verification and Analysis to systematically analyze bacterial isolates that cannot be characterized by conventional identification procedures MALDI-TOF MS and partial 16 S rRNA gene sequencing using Whole Genome Sequencing (WGS). RESULTS We identified a total of 35 bacterial strains that represent potentially novel species. Corynebacterium sp. (n = 6) and Schaalia sp. (n = 5) were the predominant genera. Two strains each were identified within the genera Anaerococcus, Clostridium, Desulfovibrio, and Peptoniphilus, and one new species was detected within Citrobacter, Dermabacter, Helcococcus, Lancefieldella, Neisseria, Ochrobactrum (Brucella), Paenibacillus, Pantoea, Porphyromonas, Pseudoclavibacter, Pseudomonas, Psychrobacter, Pusillimonas, Rothia, Sneathia, and Tessaracoccus. Twenty-seven of 35 strains were isolated from deep tissue specimens or blood cultures. Seven out of 35 isolated strains identified were clinically relevant. In addition, 26 bacterial strains that could only be identified at the species level using WGS analysis, were mainly organisms that have been identified/classified very recently. CONCLUSION Our new algorithm proved to be a powerful tool for detection and identification of novel bacterial organisms. Publicly available clinical and genomic data may help to better understand their clinical and ecological role. Our identification of 35 novel strains, 7 of which appear to be clinically relevant, shows the wide range of undescribed pathogens yet to define

Diagnostic challenges within the Bacillus cereus-group: finding the beast without teeth

The Bacillus cereus-group (B. cereus sensu lato) includes common, usually avirulent species, often considered contaminants of patient samples in routine microbiological diagnostics, as well as the highly virulent B. anthracis. Here we describe 16 isolates from 15 patients, identified as B. cereus-group using a MALDI-TOF MS standard database. Whole genome sequencing (WGS) analysis identified five of the isolates as B. anthracis species not carrying the typical virulence plasmids pXO1 and pXO2, four isolates as B. paranthracis, three as B. cereus sensu stricto, two as B. thuringiensis, one as B. mobilis, and one isolate represents a previously undefined species of Bacillus (B. basilensis sp. nov.). More detailed analysis using alternative MALDI-TOF MS databases, biochemical phenotyping, and diagnostic PCRs, gave further conflicting species results. These cases highlight the difficulties in identifying avirulent B. anthracis within the B. cereus-group using standard methods. WGS and alternative MALDI-TOF MS databases offer more accurate species identification, but so far are not routinely applied. We discuss the diagnostic resolution and discrepancies of various identification methods

Probability of pharmacological target attainment with flucloxacillin in Staphylococcus aureus bloodstream infection: a prospective cohort study of unbound plasma and individual MICs.

OBJECTIVES MSSA bloodstream infections (BSIs) are associated with considerable mortality. Data regarding therapeutic drug monitoring (TDM) and pharmacological target attainment of the β-lactam flucloxacillin are scarce. PATIENTS AND METHODS We determined the achievement of pharmacokinetic/pharmacodynamic targets and its association with clinical outcome and potential toxicity in a prospective cohort of 50 patients with MSSA-BSI. Strain-specific MICs and unbound plasma flucloxacillin concentrations (at five different timepoints) were determined by broth microdilution and HPLC-MS, respectively. RESULTS In our study population, 48% were critically ill and the 30 day mortality rate was 16%. The median flucloxacillin MIC was 0.125 mg/L. The median unbound trough concentration was 1.7 (IQR 0.4-9.3), 1.9 (IQR 0.4-6.2) and 1.0 (IQR 0.6-3.4) mg/L on study day 1, 3 and 7, respectively. Optimal (100% fT>MIC) and maximum (100% fT>4×MIC) target attainment was achieved in 45 (90%) and 34 (68%) patients, respectively, throughout the study period. Conversely, when using the EUCAST epidemiological cut-off value instead of strain-specific MICs, target attainment was achieved in only 13 (26%) patients. The mean unbound flucloxacillin trough concentration per patient was associated with neurotoxicity (OR 1.12 per 1 mg/L increase, P = 0.02) and significantly higher in deceased patients (median 14.8 versus 1.7 mg/L, P = 0.01). CONCLUSIONS Flucloxacillin pharmacological target attainment in MSSA-BSI patients is frequently achieved when unbound flucloxacillin concentrations and strain-specific MICs are considered. However, currently recommended dosing regimens may expose patients to excessive flucloxacillin concentrations, potentially resulting in drug-related organ damage

Unexpectedly High False-Positive Rates for Haemophilus influenzae Using a Meningoencephalitis Syndromic PCR Panel in Two Tertiary Centers

False-positive results in the diagnostic of meningitis and encephalitis pose important challenges. This study aimed to determine false-positive rates for Haemophilus influenzae in cerebrospinal fluids evaluated by the BioFire FilmArray® Meningitis/Encephalitis Panel. We conducted a retrospective study of all H. influenzae-positive FilmArray®. Meningitis/Encephalitis Panel results from June 2016 to October 2019 in two Swiss university hospitals. Cases were classified as true positive, likely true-positive, and likely false-positive results according to cerebrospinal fluid culture, H. influenzae-specific quantitative real-time PCR (qPCR), and Gram staining, as well as culture of other materials. We performed 3,082 panels corresponding to 2,895 patients: 0.6% of the samples (18/3,082) were positive for H. influenzae. Culture and H. influenzae-specific qPCR were performed on 17/18 (94.4%) and 3/18 (16.7%) cerebrospinal fluid samples, respectively; qPCR was negative in all cases. Among 17 samples sent for culture, 10 concerned patients were not treated with antibiotics prior to lumbar puncture. Only 1/17 revealed growth of H. influenzae and was classified as a true positive. We further classified 3/18 (16.7%) cases with the identification of Gram-negative rods in the cerebrospinal fluid or positive blood cultures for H. influenzae as likely true-positive and 14/18 (77.8%) cases as likely false-positive. Diagnostic results should always be interpreted together with the clinical presentation, cerebrospinal fluid analysis, and other available microbiological results. All H. influenzae-positive results should be viewed with special caution and a H. influenzae-specific qPCR should be systematically considered

Factors impacting the pre-analytical quality of blood cultures-Analysis at a tertiary medical center

BACKGROUND: Blood cultures (BC) are critical for the diagnosis of bloodstream infections, pathogen identification, and resistance testing. Guidelines recommend a blood volume of 8-10 mL per bottle as lower volumes result in decreased sensitivity. We aimed to evaluate factors for non-adherence to recommended volumes and assess the effects on diagnostic performance. METHODS: From February to April 2020, we measured collected blood volumes by weighing all BC containers from inpatient samples at the University Hospital Basel. Information on BC volumes was merged with clinical and microbiological data, as well as nursing staff schedules. We analyzed factors associated with (i) BC sampling volume, (ii) reaching recommended volumes (≥8 mL), (iii) BC positivity, and (iv) time to positivity using linear and generalized linear mixed effect models. RESULTS: We evaluated a total of 4'118 BC bottles collected from 686 patients. A total of 1'495 (36.3%) of all bottles contained the recommended filling volume of ≥8 mL. Using a central venous and arterial catheter for drawing blood resulted in an increase of filling volume by 0.26 mL (95% CI 0.10, 0.41) and 0.50 mL (95% CI 0.31, 0.69) compared to peripheral venipuncture, respectively. Each additional nursing staff working at the time of blood drawing was associated with 6% higher odds of achieving the recommended filling volume. We found no significant correlation between the filling volume and the positivity rate. CONCLUSION: Our results indicate critical pre-analytical quality markers linked to BC collection procedures to reach recommended collection volumes. No significant impact on the positivity rate was found

High prevalence of ESBL-Producing E. coli in private and shared latrines in an informal urban settlement in Dar es Salaam, Tanzania

Abstract Background Data about the burden of extended-spectrum beta-lactamase (ESBL)-producing microorganisms in Africa are limited. Our study aimed to estimate the prevalence of human faecal ESBL carriage in the community of an informal urban settlement in Dar es Salaam (Tanzania, East Africa) by using environmental contamination of household latrines with ESBL as a surrogate marker. Methods Within the context of a large survey in February 2014 assessing 636 randomly selected household latrines for faecal contamination by the detection of growth of E. coli and total faecal coliform bacteria, a randomly selected subset of the samples were screened for ESBL. Results Seventy latrines were screened for ESBL. An average of 11.4 persons (SD ±6.5) were sharing one latrine. Only three (4.3%) latrines had hand-washing facilities and 50 showed faeces on the floor. ESBL-producing Enterobacteriaceae were confirmed in 17 (24.3%) of the 70 latrine samples: 16 E. coli and 1 Klebsiella pneumoniae. Five ESBL E. coli strains were detected on door handles. The most prevalent ESBL type was CTX-M-1 group (76.5%). Pulsed-field gel electrophoresis typing of a subset of ESBL-producing E. coli isolates revealed both diverse singular types and a cluster of 3 identical isolates. There was no significant difference of the latrine and household characteristics between the group with ESBL (n = 17) and the group with non-ESBL E. coli (n = 53) (p > 0.05). Conclusions Almost a quarter of private and shared latrines in an informal urban settlement in Tanzania are contaminated with ESBL-producing microorganisms, suggesting a high prevalence of human ESBL faecal carriage in the community. Shared latrines may serve as a reservoir for transmission in urban community settings in Tanzania

Evaluation of the clinical relevance of the Biofire©^{©} FilmArray pneumonia panel among hospitalized patients

PURPOSE: Panel PCR tests provide rapid pathogen identification. However, their diagnostic performance is unclear. We assessed the performance of the Biofire$^{©}$ FilmArray pneumonia (PN)-panel against standard culture in broncho-alveolar lavage (BAL) samples. METHODS: Setting: University Hospital Basel (February 2019 to July 2020), including hospitalized patients with a BAL (± pneumonia). We determined sensitivity and specificity of the PN-panel against standard culture. Using univariate logistic regression, we calculated odds ratios (OR) for pneumonia according to PN-panel and culture status, stratifying by chronic pulmonary disease. We calculated ORs for pneumonia for different pathogens to estimate the clinical relevance. RESULTS: We included 840 adult patients, 60% were males, median age was 68 years, 35% had chronic pulmonary disease, 21% had pneumonia, and 36% had recent antibiotic use. In 1078 BAL samples, bacterial pathogens were detected in 36% and 16% with PN-panel and culture, respectively. The overall sensitivity and specificity of the PN-panel was high, whereas the positive predictive value was low. The OR of pneumonia was 1.1 (95% CI 0.7-1.6) for PN-panel-positive only; 2.6 (95% CI 1.3-5.3) for culture-positive only, and 1.6 (95% CI 1.0-2.4) for PN-panel and culture-positive. The detection rate of Haemophilus influenzae, Staphylococcus aureus, and Moraxella catarrhalis in the PN-panel was high but not associated with pneumonia. CONCLUSION: While sensitivity and specificity of PN-panel are high compared to culture, pathogen detection did not correlate well with a pneumonia diagnosis. Patients with culture-positive BAL had the highest OR for pneumonia-thus the impact of the PN-panel on clinical management needs further evaluation in randomized controlled trials