Development of a rapid diagnostic test based on loop-mediated isothermal amplification to identify the most frequent non-typhoidal Salmonella serovars from culture.

Identification of Salmonella serovars is performed by conventional seroagglutination or sequencing. These methods are labor-intensive and require technical experience. An easy-to-perform assay allowing the timely identification of the most common non-typhoidal serovars (NTS) is needed. In this study, a molecular assay based on loop-mediated isothermal amplification (LAMP) targeting specific gene sequences of Salmonella Enteritidis, S. Typhimurium, S. Infantis, S. Derby, and S. Choleraesuis has been developed for rapid serovar identification from cultured colonies. A total of 318 Salmonella strains and 25 isolates of other Enterobacterales species that served as negative controls were analyzed. All S. Enteritidis (n = 40), S. Infantis (n = 27), and S. Choleraesuis (n = 11) strains were correctly identified. Seven out of 104 S. Typhimurium and 10 out of 38 S. Derby strains missed a positive signal. Cross-reactions of the gene targets were only rarely observed and restricted to the S. Typhimurium primer set (5 false-positives). Sensitivity and specificity of the assay compared to seroagglutination were as follows: 100% and 100% for S. Enteritidis, 93.3% and 97.7% for S. Typhimurium, 100% and 100% for S. Infantis, 73.7% and 100% for S. Derby, and 100% and 100% for S. Choleraesuis, respectively. With results available in just a few minutes of hands-on time and a test run time of 20 min, the LAMP assay developed here may be a useful tool for the rapid identification of common Salmonella NTS in daily routine diagnostics.

Performance of the eazyplex® BloodScreen GN as a simple and rapid molecular test for identification of Gram-negative bacteria from positive blood cultures.

The LAMP-based eazyplex® BloodScreen GN was evaluated for the detection of frequent Gram-negatives directly from positive blood culture (BC) bottles. A total of 449 BCs were analyzed. Sensitivities and specificities were 100% and 100% for Escherichia coli, 95.7% and 100% for Klebsiella pneumoniae, 100% and 100% for blaCTX-M, 100% and 100% for Klebsiella oxytoca, 100% and 99% for Proteus mirabilis, and 100% and 99.8% for Pseudomonas aeruginosa, respectively. The time to result ranged from 8 to 16 min, plus about 6 min for sample preparation. The eazyplex® BloodScreen GN is a reliable molecular assay for rapid BC testing.

Heater-Cooler Devices and Risk of Contamination during Cardiac Surgery.

BACKGROUND: Heater-cooler devices (HCD) have been implicated in a cardiosurgical contamination scenario causing prosthetic valve endocarditis. AIM: We characterized contamination of new HCDs and assessed the risk of intraoperative microorganism transmission from the HCD to the operating field. METHODS: We initially acquired four new FlexTherm and then four new Maquet HCU40 HCDs and assessed occurrence and speed of microbial contamination (including mycobacteria) assessing swab and water samples from the device. In parallel, we collected repeated samples from different sites in the operating room either by swab sticks or by exposing different sample plates to room air. We also reviewed microbiological results from the hospital and compared them to cardiosurgical wound infections and endocarditis cases. Finally, we simulated cardiosurgical conditions and assessed the devices' ability to expel air to the operative field. RESULTS: All new HCDs were clean before first use. Despite authority-mandated decontamination procedures, microbial growth (Fusarium solani, Sphingomonas paucimobilis, Pseudomonas aeruginosa, Mycobacterium chelonae, and gordonae) was identified in all HCDs over time and could not be permanently eliminated. Four of these mircoorganisms were also found in tap water. However, none of the HCD-organisms were found inside the laminar airflow operating area. Importantly, except for P. aeruginosa, none of the HCD organisms were found in patients with surgical wound infections or endocarditis. HCD-expelled air did not rise more than 40 cm above ground. CONCLUSION: HCDs cannot be expected to remain sterile despite extensive decontamination procedures. However, airborne transmission of microorganisms directly from the HCD to the operating field appears unlikely.

Use of MALDI-TOF mass spectrometry to detect nosocomial outbreaks of Serratia marcescens and Citrobacter freundii.

MALDI-TOF mass spectrometry (MS) may be used as a rapid typing method for nosocomial pathogens. Here, we evaluated MALDI-TOF MS for discrimination of hospital outbreak-related clusters of Serratia marcescens and carbapenemase-producing Citrobacter freundii. Thirty-three S. marcescens isolates collected from neonatal intensive care unit (NICU) patients, and 23 C. freundii isolates including VIM-positive isolates from a hospital colonization outbreak were measured by Vitek MS. Consensus spectra of each isolate were clustered using SARAMIS software. Genotyping was performed by whole-genome sequencing (WGS). First, a set of 21 S. marcescens isolates from 2014 with seven genotypes including three monoclonal clusters was used for the evaluation of MALDI-TOF typing. MS clustering was largely in agreement with genotyping results when the similarity cut-off for clonal identity was set on 90%. MALDI-TOF cluster analysis was then investigated for the surveillance of S. marcescens in the NICU in 2017 and demonstrated the introduction of new strains into the hospital and nosocomial transmissions. MS analysis of the C. freundii outbreak in 2016 revealed a monoclonal cluster of VIM-positive isolates and the separation of epidemiologically non-related VIM-positive and negative isolates. Two additional VIM-positive Citrobacter isolates from food samples were closely related to the large monoclonal cluster. WGS confirmed the MS results. MALDI-TOF MS may be used as a first-line typing tool for S. marcescens and C. freundii to detect transmission events in the hospital because isolates of an identical WGS type were grouped into the same MS cluster.

A Nosocomial Foodborne Outbreak of a VIM Carbapenemase-Expressing Citrobacter freundii.

Background: A foodborne outbreak of VIM carbapenemase-expressing Citrobacter freundii (CPC) occurred between February 2016 and June 2016 at a major university hospital in Germany. Methods: An explosive increase in CPC isolated from rectal swabs of patients during weekly routine screening led to the declaration of an outbreak. A hospital-wide prevalence screening was initiated as well as screening of all patients on admission and before transfer to another ward, canteen staff, patient rooms, medical and kitchen inventory, and food. Swabs were streaked out on selective plates. All CPC isolates were analyzed using mass spectrometry, and selected isolates were analyzed using whole-genome sequencing. Results: A total of 76 were identified; most were unrelated cases in different wards. The CPC was isolated from retained samples of prepared vegetable salads and puddings and from a mixing machine used to prepare these foods only after an overnight culture. The immediate ban on serving potential source food resulted in a sharp decline and finally disappearance of novel cases. Repeated testing of presliced vegetables showed a high degree of contamination with C. freundii without a carbapenemase, indicating a possible source. Conclusions: An explosive increase in carbapenemase-expressing Enterobacteriaceae contamination may have been caused by a foodborne source, and presliced vegetables should be taken into account as a putative pathogen repository. These findings underline the importance of appropriate cooling, transport, reheating, and distribution of meals and indicate that probing of nonorganic surfaces is limited by low sensitivity, which may be increased by additional overnight cultivation in appropriate media.

Experience with PCR Testing for Enteric Bacteria and Viruses of Emergency Department Patients with Acute Gastroenteritis: Are There Implications for the Early Treatment of Clostridioides difficile Infection?

Early identification of acute gastroenteritis (AGE) pathogens via PCR may improve the management of patients presenting to the emergency department (ED). In this study, we evaluated the implementation of a testing algorithm for ED patients with AGE using the BD MAX automated PCR system. Data from 133 patients were analyzed. A total of 56 patients (42%) tested positive via PCR for at least one bacterial or viral pathogen. The median time to report PCR results was 6.17 h compared to 57.28 h for culture results for bacterial pathogens. The most common pathogen was Clostridioides difficile (n = 20, 15%). In total, 14 of the 20 C. difficile-positive patients were aged >65 years and 17 of the 20 patients (85%) were diagnosed with a clinically relevant infection based on typical symptoms and laboratory values. They received antibiotics, mostly oral vancomycin, starting a median of 11.37 h after ED admission. The introduction of PCR for the diagnosis of AGE infection in patients presenting to the ED may have the greatest impact on the rapid identification of C. difficile and the timely administration of antibiotics if necessary.

Competitive inhibition and mutualistic growth in co-infections: deciphering Staphylococcus aureus-Acinetobacter baumannii interaction dynamics.

Staphylococcus aureus (Sa) and Acinetobacter baumannii (Ab) are frequently co-isolated from polymicrobial infections that are severe and refractory to therapy. Here, we apply a combination of wet-lab experiments and in silico modeling to unveil the intricate nature of the Ab/Sa interaction using both, representative laboratory strains and strains co-isolated from clinical samples. This comprehensive methodology allowed uncovering Sa's capability to exert a partial interference on Ab by the expression of phenol-soluble modulins. In addition, we observed a cross-feeding mechanism by which Sa supports the growth of Ab by providing acetoin as an alternative carbon source. This study is the first to dissect the Ab/Sa interaction dynamics wherein competitive and cooperative strategies can intertwine. Through our findings, we illuminate the ecological mechanisms supporting their coexistence in the context of polymicrobial infections. Our research not only enriches our understanding but also opens doors to potential therapeutic avenues in managing these challenging infections.

Screening of Klebsiella pneumoniae Isolates for Carbapenemase and Hypervirulence-Associated Genes by Combining the Eazyplex® Superbug CRE and hvKp Assays.

The acquisition of hypervirulence-associated genes by carbapenemase-producing Klebsiella pneumoniae is being increasingly observed, and easy-to-use diagnostic tests are needed for the surveillance of the hypervirulent K. pneumoniae (hvKp). In this pilot study, 87 K. pneumoniae isolates from invasive infections collected in 2022 and 2023 were analysed using the LAMP-based eazyplex® Superbug CRE and hvKp assays for the simultaneous identification of carbapenemases and virulence genes (rmpA/A2, iuC, iroC, ybt, clb). Nine isolates showed a Kleborate virulence score of 4 or 5 (10.3%). The time for the results of the eazyplex® assays ranged from 6.5 to 13 min, and the total turnaround time, including sample preparation, was less than 30 min. Five isolates, three of which produced New Delhi metallo-beta lactamase (NDM), were subjected to whole-genome sequencing (WGS) analysis for further characterisation. The eazyplex® test results for beta-lactamase and virulence genes were confirmed. The eazyplex® hvKp, currently only available as a Research Use Only assay, may be a useful tool for the rapid identification of hvKp without significant additional workload when combined with the eazyplex® Superbug CRE assay for the detection of carbapenemases.

SARS-CoV-2 Testing of Emergency Department Patients Using cobas® Liat® and eazyplex® Rapid Molecular Assays.

Rapid testing for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) of patients presenting to emergency departments (EDs) facilitates the decision for isolation on admission to hospital wards. Differences in the sensitivity of molecular assays have implications for diagnostic workflows. This study evaluated the performance of the cobas® Liat® RT-PCR, which is routinely used as the initial test for ED patients in our hospitals, compared with the eazyplex® RT-LAMP. A total of 378 oropharyngeal and nasal swabs with positive Liat® results were analysed. Residual sample aliquots were tested using NeuMoDx™, cobas® RT-PCR, and the eazyplex® assay. Patients were divided into asymptomatic (n = 157) and symptomatic (n = 221) groups according to the WHO case definition. Overall, 14% of positive Liat® results were not confirmed by RT-PCR. These samples were mainly attributed to 26.8% of asymptomatic patients, compared to 3.8% of the symptomatic group. Therefore, positive Liat® results were used to provisionally isolate patients in the ED until RT-PCR results were available. The eazyplex® assay identified 62% and 90.6% of RT-PCR-confirmed cases in asymptomatic and symptomatic patients, respectively. False-negative eazyplex® results were associated with RT-PCR Ct values > 30, and were more frequent in the asymptomatic group than in the symptomatic group (38.1% vs. 5.1%, respectively). Both the Liat® and eazyplex® assays are suitable for testing symptomatic patients. Their use in screening asymptomatic patients depends on the need to exclude any infection or identify those at high risk of transmission.

Performance of the RT-LAMP-based eazyplex® SARS-CoV-2 as a novel rapid diagnostic test.

BACKGROUND: Diagnostic assays for severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) that are easy to perform and produce fast results are essential for timely decision making regarding the isolation of contagious individuals. OBJECTIVE: We evaluated the CE-approved eazyplex® SARS-CoV-2, a ready-to-use real time RT-LAMP assay for identification of the SARS-CoV-2 N and ORF8 genes from swabs in less than 30 min without RNA extraction. STUDY DESIGN: Oropharyngeal and nasal swabs from 100 positive and 50 negative patients were inoculated into 0.9 % saline and tested by NeuMoDx™ RT-PCR. An aliquot was diluted fivefold in Copan sputum liquefying (SL) solution and directly analyzed by eazyplex® SARS-CoV-2. In addition, 130 patient swabs were prospectively tested with both methods in parallel. Analytical sensitivity of the assay was determined using virus stock dilutions. RESULTS: Positive percent agreement (PPA) between the eazyplex® SARS-CoV-2 and RT-PCR was 74 % for samples with Ct values < 35. When using a Ct cut-off ≤ 28 the PPA increased to 97.4 %. In the prospective part of the study overall PPA of the eazyplex® kit was 66.7 % but increased to 100 % when only Ct values ≤ 28 were considered. There were no false positive results. The median time to positivity was 12.5 min for the N gene and 16.75 min for ORF8. Analytical sensitivity was 3.75 TCID50/mL. 105 virus copies/mL were reproducibly detected. CONCLUSION: The eazyplex® SARS-CoV-2 is a rapid assay that accurately identifies samples with high viral loads. It may be useful for near-patient testing outside of a molecular diagnostic laboratory.

Do closed waste containers lead to less air contamination than opened? A clinical case study at Jena University Hospital, Germany.

Nosocomial infections are a growing challenge at hospitals. This clinical study aimed to investigate the influence of waste container construction ((open (O), closed (C), and hands-free opening (HF)) on microbial air contamination in a hospital setting. The results are intended to help develop guidelines for waste containers for the collection of non-infectious waste at hospitals and medical facilities. The clinical experiment was conducted at the University Hospital Jena, Germany. Air Impactor samples were performed and microbiologically evaluated for bacteria and fungi both quantitatively and qualitatively. The results were statistically determined using generalized estimating equations. Quantitatively, the lowest bacterial counts in ambient air were found around closed waste containers (114.74 CFU/m3) in comparison to HF (129.28 CFU/m3) and O (126.28 CFU/m3). For fungi, the surrounding air of C (2.08 CFU/m3) and HF (1.97 CFU/m3) waste containers showed a lower impact of fungal air contamination than for O (2.32 CFU/m3). Overall, it was shown that C are more preferable to HF and O waste containers from the point of view of microbial air contamination at hospitals.

Influence of the Incubator as Direct Patient Environment on Bacterial Colonization of Neonates.

BACKGROUND: Preventing healthcare-associated infections (HAI) in neonatal intensive care units is a challenge of highest priority. For further insight into the incubator as direct patient environment and potential source for contamination, we present data correlating microbiological samples of very low birthweight infants in the form of colonization results of surveillance screenings with samples of their associated incubator in this study. METHODS: Samples were taken via rectal and throat swabs of neonates as well as Polywipe® sponges for the incubator. If the same bacterial species was found in corresponding neonate and incubator samples, whole genome sequencing via Illumina technology was performed. RESULTS: 52 microbiological species matches were found, and 30 matches were sequenced where we found 26 clonal pairs (12 E. faecalis, 10 S. aureus, 2 E. coli, 1 E. cloacae, and 1 E. faecium). CONCLUSION: The combinations of measurements of weekly screenings swabs, probing of surfaces with Polywipes®, and whole genome sequencing showed transmissions of microorganism and risk for potential non-physiological colonization of neonatal infants.

Simple differentiation of Salmonella Typhi, Paratyphi and Choleraesuis from Salmonella species using the eazyplex TyphiTyper LAMP assay.

Introduction. Identification of typhoidal Salmonella (TS) serovars and their discrimination from non-typhoidal Salmonella (NTS) is conventionally performed by seroagglutination. This method is labour-intensive, requires technical experience and can be inconclusive in some cases. Molecular assays may be reliable alternative diagnostic tools.Aim. This study was designed to evaluate the eazyplex TyphiTyper based on loop-mediated isothermal amplification (LAMP) for fast identification of TS and S. Choleraesuis in culture.Methodology. A total of 121 Salmonella strains and 33 isolates of other Enterobacterales species were tested by the eazyplex TyphiTyper. Simulated and clinical blood cultures (BCs) were used to examine the performance of the assay for diagnosis of systemic infection. Sample preparation took about 5 min and the test running time was 20 min. Amplification was measured by real-time fluorescence detection.Results. All TS and S. Choleraesuis strains were correctly identified. The most common NTS S. Typhimurium (n=34) and S. Enteritidis (n=15) were detected as Salmonella species without any false positive result for TS targets. Cross-reactions of NTS with TS were only rarely observed. Direct testing of positive BCs gave correct results. Sensitivities and specificities of the assay were as follows: 100 and 99.3 % for S. Typhi, 100 and 98.7 % for S. Paratyphi A, 100 and 97.3 % for S. Paratyphi B, 100 and 100 % for S. Paratyphi C, 100 and 100 % for S. Choleraesuis, and 100 and 100 % for Salmonella species, respectively.Conclusion. The eazyplex TyphiTyper is very easy to perform and allows the rapid identification of TS and S. Choleraesuis isolates.

Use of the variplex™ SARS-CoV-2 RT-LAMP as a rapid molecular assay to complement RT-PCR for COVID-19 diagnosis.

BACKGROUND: Molecular assays based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) may be useful for rapid diagnosis of the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) because of the easy performance and the option to bypass RNA extraction. OBJECTIVES: This study was designed to evaluate the clinical performance of the CE-labeled variplexTM real time SARS-CoV-2 RT-LAMP assay in comparison to commercial RT-PCRs. STUDY DESIGN: RNA extracted from pharyngeal swabs was tested by variplex™ RT-LAMP and Corman's LightMix™ E gene RT-PCR as reference. Samples of respiratory secretions from Coronavirus infection disease (COVID-19) and negative control patients were analyzed by variplex™ without RNA extraction and tested in parallel with the Allplex™ and VIASURE BD MAX RT-PCRs. RESULTS: Using isolated RNA variplex™ RT-LAMP showed a sensitivity of 75 % compared to LightMix E gene RT-PCR but contrary to the latter it produced no false-positive results. For the evaluation of samples from respiratory secretions concordance analysis showed only a moderate agreement between the variplex™ RT-LAMP conducted on unprocessed samples and Allplex™ and VIASURE RT-PCRs (Cohen's κ ranging from 0.52-0.56). Using the approach to define a sample as true-positive when at least two assays gave a positive result the clinical sensitivities were as follows: 76.3 % for variplex™, 84.2 % for Allplex™ and 68.4 % for VIASURE. However, when results of RT-PCR and RT-LAMP were combined diagnostic sensitivity was increased to 92-100 %. CONCLUSION: The variplex RT-LAMP may serve as a rapid test to be combined with a RT-PCR assay to increase the diagnostic accuracy in patients with suspected COVID-19 infection.

Acute diarrhoea due to a Shiga toxin 2e-producing Escherichia coli O8 : H19.

Introduction. Identification of non-O157 Shiga-toxin-producing Escherichia coli (STEC) infections may be underestimated in microbiological diagnosis. Case presentation. A 58-year-old woman developed diarrhoea with watery and subsequently mucous stool. Initial multiplex PCR testing revealed a positive result for stx2 . Culture isolation of a STEC was successful only after repeated inoculation of chromogenic E. coli media. Molecular characterization was performed and identified the isolate as stx2e-positive STEC of serotype O8 : H19. The strain harboured lpfA, but not eae. Conclusion. This case highlights the usefulness of initial multiplex PCR for diagnosis of non-O157 STEC infection.

Antibiotic treatment algorithm development based on a microarray nucleic acid assay for rapid bacterial identification and resistance determination from positive blood cultures.

Rapid diagnosis of bloodstream infections remains a challenge for the early targeting of an antibiotic therapy in sepsis patients. In recent studies, the reliability of the Nanosphere Verigene Gram-positive and Gram-negative blood culture (BC-GP and BC-GN) assays for the rapid identification of bacteria and resistance genes directly from positive BCs has been demonstrated. In this work, we have developed a model to define treatment recommendations by combining Verigene test results with knowledge on local antibiotic resistance patterns of bacterial pathogens. The data of 275 positive BCs were analyzed. Two hundred sixty-three isolates (95.6%) were included in the Verigene assay panels, and 257 isolates (93.5%) were correctly identified. The agreement of the detection of resistance genes with subsequent phenotypic susceptibility testing was 100%. The hospital antibiogram was used to develop a treatment algorithm on the basis of Verigene results that may contribute to a faster patient management.

Comparison of multilocus sequence typing, RAPD, and MALDI-TOF mass spectrometry for typing of ß-lactam-resistant Klebsiella pneumoniae strains.

Extended spectrum of ß-lactam (ESBL) resistance of Klebsiella pneumoniae has become an increasing problem in hospital infections. Typing of isolates is important to establish the intrahospital surveillance of resistant clones. In this study, the discriminatory potential of randomly amplified polymorphic DNA and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analyses were compared with multilocus sequence typing (MLST) by using 17 ß-lactam-resistant K. pneumoniae isolates of different genotypes. MLST alleles were distributed in 8 sequence types (STs). Among ESBL strains of the same ST, the presence of different ß-lactamase genes was common. RAPD band patterns also revealed 8 types that corresponded to MLST-defined genotypes in 15 out of 17 cases. MALDI-TOF analysis could differentiate 5 clusters of strains. The results of this work show that RAPD may be usable as a rapid screening method for the intrahospital surveillance of K. pneumoniae, allowing a discrimination of clonally related strains. MALDI-TOF-based typing was not strongly corresponding to genotyping and warrants further investigation.