Utilization of a Meningitis/Encephalitis PCR panel at the University Hospital Basel - a retrospective study to develop a diagnostic decision rule.

Background: The Biofire® FilmArray® Meningitis/Encephalitis (ME) PCR panel covers 14 viral, bacterial, and fungal pathogens and has been implemented in many institutions worldwide. Post-marketing studies indicate a reduced sensitivity and overutilization underscoring the need for a more targeted usage. The aim of our study is to describe the utilization of the ME panel and to develop a diagnostic-stewardship based decision rule. Materials: Adult patients, who underwent CSF analysis with the ME panel between August 2016 and June 2021 at the University Hospital Basel, were included. Demographic, clinical, microbiological, and laboratory data were extracted from the electronic health record. Factors associated with a positive ME panel result were identified, and a decision rule was developed to potentially optimize the diagnostic yield and reduce the number of unnecessary tests. Results: 1,236 adult patients received at least one panel in the observed period, of whom 106 panels tested positive (8.6%). The most frequently observed pathogens were Varicella Zoster Virus (VZV, 27%), Streptococcus pneumoniae (19%), Enterovirus (16%), Herpes simplex Virus 1/2 (16%), and Human Herpesvirus 6 (HHV-6, 13%). Fever, vomiting, headache, and photophobia were more frequently present in test positive patients as were significantly higher CSF leukocytes and protein concentrations. When simulating a decision rule based on CSF leukocytes and protein concentration, only 35% of all patients would have qualified for a ME panel tests, thereby increasing the positivity rate to 22.7%. 10 of 106 positive ME panels would have been missed, only involving HHV-6 and VZV (6 and 4 cases, respectively). As these subjects were either severely immunocompromised or had clinical features of shingles we propose extending the testing algorithm by including those criteria. Conclusion: The ME panel positivity rate at our institution was similar as previously published. Our results highlight the need for diagnostic-stewardship interventions when utilizing this assay by implementing a stepwise approach based on a limited number of clinical and laboratory features. This decision rule may improve the pretest probability of a positive test result, increase the quality of test utilization, and reduce costs.

Rapid detection of plasmid-mediated AmpC-producers by eazyplex® SuperBug AmpC assay compared to whole-genome sequencing.

Current methods for plasmid-mediated AmpC ß-lactamase (pAmpC) detection in routine microbiological laboratories are based on various phenotypic tests. Eazyplex®SuperBug AmpC assay is a molecular assay based on isothermal amplification for rapid detection of the most common pAmpC types from bacterial culture: CMY-2 group, DHA, ACC and MOX. Our aim was to evaluate the diagnostic performance of this assay. The assay was evaluated on 64 clinical isolates of Enterobacterales without chromosomal inducible AmpC, and with phenotypically confirmed AmpC production. The results were confirmed, and isolates further characterized by whole-genome sequencing (WGS). eazyplex®SuperBug AmpC assay correctly detected the two most common pAmpC types CMY-2 group (16/16) and DHA (19/19). Detection of ACC and MOX could not be evaluated on our set of isolates since there was only one isolate harbouring ACC and none with MOX. pAmpC encoding genes could be detected in only eight of 36 investigated Escherichia coli isolates. The remaining 28 E. coli isolates harboured previously described mutations in the blaEC promoter, leading to the overexpression of chromosomally encoded E. coli specific AmpC ß-lactamase. All results were 100% concordant with the results of WGS. eazyplex®SuperBug AmpC assay enabled rapid and reliable detection of pAmpC-encoding genes in Enterobacterales like Klebsiella spp. and Proteus spp. and the distinction between plasmid-mediated and chromosomally encoded AmpC in E. coli.

Tularemia on the rise in Switzerland? A one health approach is needed!

In the last 10 years, an increase in tularemia cases has been observed in both humans and animals in Switzerland. In these, infection with Francisella tularensis, the causative agent of the zoonotic disease tularemia, can occur through arthropod vectors or contact to infected animals or exposure to contaminated environmental sources. Currently, we are only able to postulate potential aetiologies: (i) behavioral changes of humans with more exposure to endemic habitats of infected arthropod vectors; (ii) an increased rate of tularemia infected ticks; (iii) increasing number and geographical regions of tick biotopes; (iv) increasing and/or more diverse reservoir populations; (v) increasing presence of bacteria in the environment; (vi) raised awareness and increased testing among physicians; (vii) improved laboratory techniques including molecular testing. To approach these questions, a one-health strategy is necessary. A functioning collaboration between public health, human medicine, and diagnostic and veterinary units for the control of tularemia must be established. Furthermore, the public should be included within citizen-supported-science-projects.

How much should we sequence? An analysis of the Swiss SARS-CoV-2 surveillance effort.

During the SARS-CoV-2 pandemic, many countries directed substantial resources toward genomic surveillance to detect and track viral variants. There is a debate over how much sequencing effort is necessary in national surveillance programs for SARS-CoV-2 and future pandemic threats. We aimed to investigate the effect of reduced sequencing on surveillance outcomes in a large genomic data set from Switzerland, comprising more than 143k sequences. We employed a uniform downsampling strategy using 100 iterations each to investigate the effects of fewer available sequences on the surveillance outcomes: (i) first detection of variants of concern (VOCs), (ii) speed of introduction of VOCs, (iii) diversity of lineages, (iv) first cluster detection of VOCs, (v) density of active clusters, and (vi) geographic spread of clusters. The impact of downsampling on VOC detection is disparate for the three VOC lineages, but many outcomes including introduction and cluster detection could be recapitulated even with only 35% of the original sequencing effort. The effect on the observed speed of introduction and first detection of clusters was more sensitive to reduced sequencing effort for some VOCs, in particular Omicron and Delta, respectively. A genomic surveillance program needs a balance between societal benefits and costs. While the overall national dynamics of the pandemic could be recapitulated by a reduced sequencing effort, the effect is strongly lineage-dependent-something that is unknown at the time of sequencing-and comes at the cost of accuracy, in particular for tracking the emergence of potential VOCs.IMPORTANCESwitzerland had one of the most comprehensive genomic surveillance systems during the COVID-19 pandemic. Such programs need to strike a balance between societal benefits and program costs. Our study aims to answer the question: How would surveillance outcomes have changed had we sequenced less? We find that some outcomes but also certain viral lineages are more affected than others by sequencing less. However, sequencing to around a third of the original effort still captured many important outcomes for the variants of concern such as their first detection but affected more strongly other measures like the detection of first transmission clusters for some lineages. Our work highlights the importance of setting predefined targets for a national genomic surveillance program based on which sequencing effort should be determined. Additionally, the use of a centralized surveillance platform facilitates aggregating data on a national level for rapid public health responses as well as post-analyses.

S-Gene Target Failure as an Effective Tool for Tracking the Emergence of Dominant SARS-CoV-2 Variants in Switzerland and Liechtenstein, Including Alpha, Delta, and Omicron BA.1, BA.2, and BA.4/BA.5.

During the SARS-CoV-2 pandemic, the Dr. Risch medical group employed the multiplex TaqPathTM COVID-19 CE-IVD RT-PCR Kit for large-scale routine diagnostic testing in Switzerland and the principality of Liechtenstein. The TaqPath Kit is a widely used multiplex assay targeting three genes (i.e., ORF1AB, N, S). With emergence of the B.1.1.7 (Alpha) variant, a diagnostic flaw became apparent as the amplification of the S-gene target was absent in these samples due to a deletion (ΔH69/V70) in the Alpha variant genome. This S-gene target failure (SGTF) was the earliest indication of a new variant emerging and was also observed in subsequent variants such as Omicron BA.1 and BA4/BA.5. The Delta variant and Omicron BA.2 did not present with SGTF. From September 2020 to November 2022, we investigated the applicability of the SGTF as a surrogate marker for emerging variants such as B.1.1.7, B.1.617.2 (Delta), and Omicron BA.1, BA.2, and BA.4/BA.5 in samples with cycle threshold (Ct) values < 30. Next to true SGTF-positive and SGTF-negative samples, there were also samples presenting with delayed-type S-gene amplification (higher Ct value for S-gene than ORF1ab gene). Among these, a difference of 3.8 Ct values between the S- and ORF1ab genes was found to best distinguish between "true" SGTF and the cycle threshold variability of the assay. Samples above the cutoff were subsequently termed partial SGTF (pSGTF). Variant confirmation was performed by whole-genome sequencing (Oxford Nanopore Technology, Oxford, UK) or mutation-specific PCR (TIB MOLBIOL). In total, 17,724 (7.4%) samples among 240,896 positives were variant-confirmed, resulting in an overall sensitivity and specificity of 93.2% [92.7%, 93.7%] and 99.3% [99.2%, 99.5%], respectively. Sensitivity was increased to 98.2% [97.9% to 98.4%] and specificity lowered to 98.9% [98.6% to 99.1%] when samples with pSGTF were included. Furthermore, weekly logistic growth rates (α) and sigmoid's midpoint (t0) were calculated based on SGTF data and did not significantly differ from calculations based on comprehensive data from GISAID. The SGTF therefore allowed for a valid real-time estimate for the introduction of all dominant variants in Switzerland and Liechtenstein.

Historic methicillin-resistant Staphylococcus aureus: expanding current knowledge using molecular epidemiological characterization of a Swiss legacy collection.

BACKGROUND: Few methicillin-resistant Staphylococcus aureus (MRSA) from the early years of its global emergence have been sequenced. Knowledge about evolutionary factors promoting the success of specific MRSA multi-locus sequence types (MLSTs) remains scarce. We aimed to characterize a legacy MRSA collection isolated from 1965 to 1987 and compare it against publicly available international and local genomes. METHODS: We accessed 451 historic (1965-1987) MRSA isolates stored in the Culture Collection of Switzerland, mostly collected from the Zurich region. We determined phenotypic antimicrobial resistance (AMR) and performed whole genome sequencing (WGS) using Illumina short-read sequencing on all isolates and long-read sequencing on a selection with Oxford Nanopore Technology. For context, we included 103 publicly available international assemblies from 1960 to 1992 and sequenced 1207 modern Swiss MRSA isolates from 2007 to 2022. We analyzed the core genome (cg)MLST and predicted SCCmec cassette types, AMR, and virulence genes. RESULTS: Among the 451 historic Swiss MRSA isolates, we found 17 sequence types (STs) of which 11 have been previously described. Two STs were novel combinations of known loci and six isolates carried previously unsubmitted MLST alleles, representing five new STs (ST7843, ST7844, ST7837, ST7839, and ST7842). Most isolates (83% 376/451) represented ST247-MRSA-I isolated in the 1960s, followed by ST7844 (6% 25/451), a novel single locus variant (SLV) of ST239. Analysis by cgMLST indicated that isolates belonging to ST7844-MRSA-III cluster within the diversity of ST239-MRSA-III. Early MRSA were predominantly from clonal complex (CC)8. From 1980 to the end of the twentieth century, we observed that CC22 and CC5 as well as CC8 were present, both locally and internationally. CONCLUSIONS: The combined analysis of 1761 historic and contemporary MRSA isolates across more than 50 years uncovered novel STs and allowed us a glimpse into the lineage flux between Swiss-German and international MRSA across time.

Phage Paride can kill dormant, antibiotic-tolerant cells of Pseudomonas aeruginosa by direct lytic replication.

Bacteriophages are ubiquitous viral predators that have primarily been studied using fast-growing laboratory cultures of their bacterial hosts. However, microbial life in nature is mostly in a slow- or non-growing, dormant state. Here, we show that diverse phages can infect deep-dormant bacteria and suspend their replication until the host resuscitates ("hibernation"). However, a newly isolated Pseudomonas aeruginosa phage, named Paride, can directly replicate and induce the lysis of deep-dormant hosts. While non-growing bacteria are notoriously tolerant to antibiotic drugs, the combination with Paride enables the carbapenem meropenem to eradicate deep-dormant cultures in vitro and to reduce a resilient bacterial infection of a tissue cage implant in mice. Our work might inspire new treatments for persistent bacterial infections and, more broadly, highlights two viral strategies to infect dormant bacteria (hibernation and direct replication) that will guide future studies on phage-host interactions.

Regional spread of an atypical ESBL-producing Escherichia coli ST131H89 clone among different human and environmental reservoirs in Western Switzerland.

We describe the inter-regional spread of a novel ESBL-producing Escherichia coli subclone (ST131H89) in long-term care facility residents, general population, and environmental water sources in Western Switzerland between 2017 and 2020. The study highlights the importance of molecular surveillance for tracking emerging antibiotic-resistant pathogens in healthcare and community settings.

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).

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.

Immunogenicity of High-Dose Versus MF59-Adjuvanted Versus Standard Influenza Vaccine in Solid Organ Transplant Recipients: The Swiss/Spanish Trial in Solid Organ Transplantation on Prevention of Influenza (STOP-FLU Trial).

BACKGROUND: The immunogenicity of the standard influenza vaccine is reduced in solid-organ transplant (SOT) recipients, so new vaccination strategies are needed in this population. METHODS: Adult SOT recipients from 9 transplant clinics in Switzerland and Spain were enrolled if they were >3 months after transplantation. Patients were randomized (1:1:1) to a MF59-adjuvanted or a high-dose vaccine (intervention), or a standard vaccine (control), with stratification by organ and time from transplant. The primary outcome was vaccine response rate, defined as a ≥4-fold increase of hemagglutination-inhibition titers to at least 1 vaccine strain at 28 days postvaccination. Secondary outcomes included polymerase chain reaction-confirmed influenza and vaccine reactogenicity. RESULTS: A total of 619 patients were randomized, 616 received the assigned vaccines, and 598 had serum available for analysis of the primary endpoint (standard, n = 198; MF59-adjuvanted, n = 205; high-dose, n = 195 patients). Vaccine response rates were 42% (84/198) in the standard vaccine group, 60% (122/205) in the MF59-adjuvanted vaccine group, and 66% (129/195) in the high-dose vaccine group (difference in intervention vaccines vs standard vaccine, 0.20; 97.5% confidence interval [CI], .12-1); P < .001; difference in high-dose vs standard vaccine, 0.24 [95% CI, .16-1]; P < .001; difference in MF59-adjuvanted vs standard vaccine, 0.17 [97.5% CI, .08-1]; P < .001). Influenza occurred in 6% of the standard, 5% in the MF59-adjuvanted, and 7% in the high-dose vaccine groups. Vaccine-related adverse events occurred more frequently in the intervention vaccine groups, but most of the events were mild. CONCLUSIONS: In SOT recipients, use of an MF59-adjuvanted or a high-dose influenza vaccine was safe and resulted in a higher vaccine response rate. CLINICAL TRIALS REGISTRATION: Clinicaltrials.gov NCT03699839.

SARS-CoV-2 in an immunocompromised host: convalescent plasma therapy and viral evolution elucidated by whole genome sequencing.

The evolution of SARS-CoV-2 within immunocompromised hosts who fail to clear the virus over many months has been proposed as a route to the development of Variants of Concern (VoCs). We present a case of an immunocompromised male patient with a prolonged SARS-CoV-2 infection. During hospitalisation, 7 weeks after first diagnosis, his condition worsened to require continuous ventilation support. Resolution of symptoms was observed after convalescent plasma therapy. Whole genome sequencing of the virus showed Pango lineage B.1.221. Between the first sample and the second from bronchoalveolar lavage fluid 7 weeks later, we identified eight mutations, including minor variants, which could be used to estimate the chronology of mutations. This suggests an elevated mutation rate, in-host accumulation of mutations and further evidence for sources of VoCs. Prolonged SARS-CoV-2 infections in immunocompromised hosts increase the likelihood of hospital stays and morbidity, and also pose an increased risk to global public health.

Surgical site infections after kidney transplantation are independently associated with graft loss.

Surgical site infections (SSIs) are common health care-associated infections. SSIs after kidney transplantation (K-Tx) can endanger patient and allograft survival. Multicenter studies on this early posttransplant complication are scarce. We analyzed consecutive adult K-Tx recipients enrolled in the Swiss Transplant Cohort Study who received a K-Tx between May 2008 and September 2020. All data were prospectively collected with the exception of the categorization of SSI which was performed retrospectively according to the Centers for Disease Control and Prevention criteria. A total of 58 out of 3059 (1.9%) K-Tx recipients were affected by SSIs. Deep incisional (15, 25.9%) and organ/space infections (34, 58.6%) predominated. In the majority of SSIs (52, 89.6%), bacteria were detected, most frequently Escherichia coli (15, 28.9%), Enterococcus spp. (14, 26.9%), and coagulase-negative staphylococci (13, 25.0%). A BMI ≥25 kg/m2 (multivariable OR 2.16, 95% CI 1.07-4.34, P = .023) and delayed graft function (multivariable OR 2.88, 95% CI 1.56-5.34, P = .001) were independent risk factors for SSI. In Cox proportional hazard models, SSI was independently associated with graft loss (multivariable HR 3.75, 95% CI 1.35-10.38, P = .011). In conclusion, SSI was a rare complication after K-Tx. BMI ≥25 kg/m2 and delayed graft function were independent risk factors. SSIs were independently associated with graft loss.

SARS-CoV-2 Nucleocapsid Protein Mutations Found in Switzerland Disrupt N-Gene Amplification in Commonly Used Multiplex RT-PCR Assay.

At the end of 2021, we observed an increase in N-gene target failures (NGTF) with the TaqPathTM COVID-19 CE-IVD RT-PCR Kit from Thermo Fisher Scientific (TaqPath). We subsequently used whole-genome sequencing (Oxford Nanopore Technology) to identify potential issues with N-gene PCR efficacy. Among 168,101 positive samples with a cycle threshold (CT) value <30 from August 2021 to May 2022, 194 specimens without N-gene amplification by PCR were identified (0.12%). Most NGTF samples originated from a wave of infection attributable to the Delta variant (B.1.617.2) and its sublineages. Sequencing revealed the nucleotide substitution G28922T (A217S) in 151 samples (88.8%). The substitution G215C, a hallmark mutation for Delta lineages, was concurrently present in all of these samples. Ten samples (5.9%) carried the deletion 28,913-28,918 (del214/215), eight samples (4.7%) the deletion 28,913-28,915 (del214) and one sample (0.6%) the deletion 28,892-28,930 (del207-219). Samples showing intact N-gene amplification by PCR lacked these specific mutations, but delayed-type amplification (i.e., partial or pNGTF) was attributable to the exclusive presence of A217S. As the N gene is a common target in many RT-PCR methods for SARS-CoV-2, an in-depth analysis of single-target failures using a combination with viral whole genome sequencing may allow for the identification of diagnostic flaws and eventual new variants.

Investigation of an international water polo tournament in Czechia as a potential source for early introduction of the SARS-CoV-2 Omicron variant into Belgium, Switzerland and Germany, November 2021.

BackgroundThe earliest recognised infections by the SARS-CoV-2 Omicron variant (Pango lineage B.1.1.529) in Belgium and Switzerland suggested a connection to an international water polo tournament, held 12-14 November 2021 in Brno, Czechia.AimTo study the arrival and subsequent spread of the Omicron variant in Belgium and Switzerland, and understand the overall importance of this international sporting event on the number of infections in the two countries.MethodsWe performed intensive forward and backward contact tracing in both countries, supplemented by phylogenetic investigations using virus sequences of the suspected infection chain archived in public databases.ResultsThrough contact tracing, we identified two and one infected athletes of the Belgian and Swiss water polo teams, respectively, and subsequently also three athletes from Germany. In Belgium and Switzerland, four and three secondary infections, and three and one confirmed tertiary infections were identified. Phylogenetic investigation demonstrated that this sporting event played a role as the source of infection, but without a direct link with infections from South Africa and not as a superspreading event; the virus was found to already be circulating at that time in the countries involved.ConclusionThe SARS-CoV-2 Omicron variant started to circulate in Europe several weeks before its identification in South Africa on 24 November 2021. Accordingly, it can be assumed that travel restrictions are usually implemented too late to prevent the spread of newly detected SARS-CoV-2 variants to other regions. Phylogenetic analysis may modify the perception of an apparently clear result of intensive contact tracing.

Identification of microorganisms by a rapid PCR panel from positive blood cultures leads to faster optimal antimicrobial therapy - a before-after study.

BACKGROUND: The BioFire® FilmArray® Blood Culture Identification Panel 1 (BF-FA-BCIP) detects microorganisms with high accuracy in positive blood cultures (BC) - a key step in the management of patients with suspected bacteraemia. We aimed to compare the time to optimal antimicrobial therapy (OAT) for the BF-FA-BCIP vs. standard culture-based identification. METHODS: In this retrospective single-centre study with a before-after design, 386 positive BC cases with identification by BF-FA-BCIP were compared to 414 controls with culture-based identification. The primary endpoint was the time from BC sampling to OAT. Secondary endpoints were time to effective therapy, length of stay, (re-)admission to ICU, in-hospital and 30-day mortality. Outcomes were assessed using Cox proportional hazard models and logistic regressions. RESULTS: Baseline characteristics of included adult inpatients were comparable. Main sources of bacteraemia were urinary tract and intra-abdominal infection (19.2% vs. 22.0% and 16.8% vs. 15.7%, for cases and controls, respectively). Median (95%CI) time to OAT was 25.5 (21.0-31.2) hours with BF-FA-BCIP compared to 45.7 (37.7-51.4) hours with culture-based identification. We observed no significant difference for secondary outcomes. CONCLUSIONS: Rapid microorganism identification by BF-FA-BCIP was associated with a median 20-h earlier initiation of OAT in patients with positive BC. No impact on length of stay and mortality was noted. TRIAL REGISTRATION: Clinicaltrials.gov, NCT04156633, registered on November 5, 2019.

Bacterial genome-wide association study substantiates papGII of Escherichia coli as a major risk factor for urosepsis.

BACKGROUND: Urinary tract infections (UTIs) are among the most common bacterial infections worldwide, often caused by uropathogenic Escherichia coli. Multiple bacterial virulence factors or patient characteristics have been linked separately to progressive, more invasive infections. In this study, we aim to identify pathogen- and patient-specific factors that drive the progression to urosepsis by jointly analysing bacterial and host characteristics. METHODS: We analysed 1076 E. coli strains isolated from 825 clinical cases with UTI and/or bacteraemia by whole-genome sequencing (Illumina). Sequence types (STs) were determined via srst2 and capsule loci via fastKaptive. We compared the isolates from urine and blood to confirm clonality. Furthermore, we performed a bacterial genome-wide association study (bGWAS) (pyseer) using bacteraemia as the primary clinical outcome. Clinical data were collected by an electronic patient chart review. We concurrently analysed the association of the most significant bGWAS hit and important patient characteristics with the clinical endpoint bacteraemia using a generalised linear model (GLM). Finally, we designed qPCR primers and probes to detect papGII-positive E. coli strains and prospectively screened E. coli from urine samples (n = 1657) at two healthcare centres. RESULTS: Our patient cohort had a median age of 75.3 years (range: 18.00-103.1) and was predominantly female (574/825, 69.6%). The bacterial phylogroups B2 (60.6%; 500/825) and D (16.6%; 137/825), which are associated with extraintestinal infections, represent the majority of the strains in our collection, many of which encode a polysaccharide capsule (63.4%; 525/825). The most frequently observed STs were ST131 (12.7%; 105/825), ST69 (11.0%; 91/825), and ST73 (10.2%; 84/825). Of interest, in 12.3% (13/106) of cases, the E. coli pairs in urine and blood were only distantly related. In line with previous bGWAS studies, we identified the gene papGII (p-value < 0.001), which encodes the adhesin subunit of the E. coli P-pilus, to be associated with 'bacteraemia' in our bGWAS. In our GLM, correcting for patient characteristics, papGII remained highly significant (odds ratio = 5.27, 95% confidence interval = [3.48, 7.97], p-value < 0.001). An independent cohort of cases which we screened for papGII-carrying E. coli at two healthcare centres further confirmed the increased relative frequency of papGII-positive strains causing invasive infection, compared to papGII-negative strains (p-value = 0.033, chi-squared test). CONCLUSIONS: This study builds on previous work linking papGII with invasive infection by showing that it is a major risk factor for progression from UTI to bacteraemia that has diagnostic potential.

IVDR: Analysis of the Social, Economic, and Practical Consequences of the Application of an Ordinance of the In Vitro Diagnostic Ordinance in Switzerland.

IVDR regulation represents a major challenge for diagnostic microbiology laboratories. IVDR complicates a broad range of aspects and poses a risk given the high diversity of pathogens (including rare but highly virulent microbes) and the large variety of samples submitted for analysis. The regular emergence of new pathogens (including Echovirus E-11, Adenovirus 41, Monkeypox virus, Alongshan virus, and Enterovirus D68, as recent examples in Europe in the post SARS-CoV-2 era) is another factor that makes IVDR regulation risky, because its detrimental effect on production of in-house tests will negatively impact knowledge and expertise in the development of new diagnostic tests. Moreover, such regulations negatively impact the availability of diagnostic tests, especially for neglected pathogens, and has a detrimental effect on the overall costs of the tests. The increased regulatory burden of IVDR may thereby pose an important risk for public health. Taken together, it will have a negative impact on the financial balance of diagnostic microbiology laboratories (especially small ones). The already-high standards of quality management of all ISO-accredited and Swissmedic-authorized laboratories render IVDR law of little value, at least in Switzerland, while tremendously increasing the regulatory burden and associated costs. Eventually, patients will need to pay for diagnostic assays outside of the framework of their insurance in order to obtain a proper diagnostic assessment, which may result in social inequity. Thus, based on the risk assessment outlined above, the coordinated commission for clinical microbiology proposes adjusting the IvDO ordinance by (i) introducing an obligation to be ISO 15189 accredited and (ii) not implementing the IvDO 2028 milestone.

High-Fluence Accelerated PACK-CXL for Bacterial Keratitis Using Riboflavin/UV-A or Rose Bengal/Green in the Ex Vivo Porcine Cornea.

Purpose: To investigate and compare the efficacy of high-fluence accelerated photoactivated chromophore for keratitis-corneal cross-linking (PACK-CXL) using either riboflavin/ultraviolet (UV)-A light or rose bengal/green light to treat Staphylococcus aureus or Pseudomonas aeruginosa infections in an ex vivo porcine cornea model. Methods: One hundred and seventeen ex vivo porcine corneas were injected with clinical isolates of S. aureus or P. aeruginosa, divided into eight groups, and cultured for 24 hours. Then, either riboflavin with UV-A light irradiation (30 mW/cm2; 8 minutes, 20 seconds; 15 J/cm2) or rose bengal with green light irradiation (15 mW/cm2, 16 minutes, 40 seconds; 15 J/cm2) was applied; unirradiated infected groups served as controls. All corneas were incubated for another 24 hours. Next, corneal buttons were obtained and vortexed to release the bacterial cells. The irradiated and unirradiated solutions were then plated and incubated on agar plates. The amount of colony-forming units was quantified and the bacterial killing ratios (BKRs) resulting from different PACK-CXL protocols relative to non-treated controls were calculated. Results: Riboflavin/UV-A light PACK-CXL resulted in median BKRs of 52.8% and 45.8% in S. aureus and P. aeruginosa, respectively, whereas rose bengal/green light PACK-CXL resulted in significantly greater BKRs of 76.7% and 81.0%, respectively (both P < 0.01). Conclusions: Both accelerated PACK-CXL protocols significantly decreased S. aureus and P. aeruginosa bacterial loads. Comparing the riboflavin/UV-A light and rose bengal/green light PACK-CXL approaches in the same experimental setup may help develop strain-specific and depth-dependent PACK-CXL approaches that could be used alongside the current standard of care. Translational Relevance: Our study used an animal model to gain insight into the efficacy of high-fluence accelerated PACK-CXL using either riboflavin/UV-A light or rose bengal/green light to treat Staphylococcus aureus or Pseudomonas aeruginosa infections.