Adaptation and validation of a quantitative vanA/vanB DNA screening assay on a high-throughput PCR system.

Vancomycin resistant enterococci (VRE) are a leading cause of ICU-acquired bloodstream infections in Europe. The bacterial load in enteral colonization may be associated with a higher probability of transmission. Here, we aimed to establish a quantitative vanA/vanB DNA real-time PCR assay on a high-throughput system. Limits of detection (LOD), linear range and precision were determined using serial bacterial dilutions. LOD was 46.9 digital copies (dcp)/ml for vanA and 60.8 dcp/ml for vanB. The assay showed excellent linearity between 4.7 × 101 and 3.5 × 105 dcp/ml (vanA) and 6.7 × 102 and 6.7 × 105 dcp/ml (vanB). Sensitivity was 100% for vanA and vanB, with high positive predictive value (PPV) for vanA (100%), but lower PPV for vanB (34.6%) likely due to the presence of vanB DNA positive anerobic bacteria in rectal swabs. Using the assay on enriched VRE broth vanB PPV increased to 87.2%. Quantification revealed median 2.0 × 104 dcp/ml in PCR positive but VRE culture negative samples and median 9.1 × 104 dcp/ml in VRE culture positive patients (maximum: 107 dcp/ml). The automated vanA/B_UTC assay can be used for vanA/vanB detection and quantification in different diagnostic settings and may support future clinical studies assessing the impact of bacterial load on risk of infection and transmission.

Performance and Hypothetical Impact on Joint Infection Management of the BioFire Joint Infection Panel: a Retrospective Analysis.

Pathogen identification is key in septic arthritis. Culture-based techniques are challenging, especially when patients have been pretreated with antibiotics or when difficult-to-culture bacteria are encountered. The BioFire joint infection assay (BJA) is a multiplex PCR panel which detects 31 of the most prevalent bacterial and fungal pathogens causing septic arthritis. Here, 123 cryoconserved contemporary synovial fluid samples from 120 patients underwent BJA analysis. Results were compared to those of culture-based diagnostics (standard of care [SOC]). Clinical data were collected, and the possible impact of the molecular diagnostic application on patient management was evaluated. Fifteen of 123 synovial fluid cultures grew bacterial pathogens. All on-panel pathogens (9/15) were correctly identified by the BJA. The BJA identified four additional bacterial pathogens in four SOC-negative cases. BJA sensitivity and specificity were 100% (95% confidence interval [CI], 69.2% to 100%) and 100% (95% CI, 96.8% to 100%), respectively. Compared to the SOC, the BJA would have resulted in faster provision of species identification and molecular susceptibility data by 49 h and 99 h, respectively. Clinical data analysis indicates that in BJA-positive cases, faster species ID could have led to timelier optimization of antibiotic therapy. This retrospective study demonstrates high sensitivity and specificity of the BJA to detect on-panel organisms in bacterial arthritis. The usefulness of the BJA in prosthetic-joint infections is limited, as important pathogens (i.e., coagulase negative staphylococci and Cutibacterium acnes) are not covered. Evidence from patient data analysis suggests that the assay might prove valuable for optimizing patient management in acute arthritis related to fastidious organisms or for patients who received antibiotics prior to specimen collection.

High burden of RSV and influenza in patients presenting with suspected pneumonia in the emergency room of a German tertiary hospital in fall of 2022.

PURPOSE: Bacterial pneumonia, a major cause of respiratory tract infections (RTI), can be challenging to diagnose and to treat adequately, especially when seasonal viral pathogens co-circulate. The aim of this study was to give a real-world snapshot of the burden of respiratory disease and treatment choices in the emergency department (ED) of a tertiary care hospital in Germany in the fall of 2022. METHODS: Anonymized analysis of a quality control initiative that prospectively documented all patients presenting to our ED with symptoms suggestive of RTI from Nov 7th to Dec 18th, 2022. RESULTS: 243 patients were followed at the time of their ED attendance. Clinical, laboratory and radiographic examination was performed in 92% of patients (224/243). Microbiological work-up to identify causative pathogens including blood cultures, sputum or urine-antigen tests were performed in 55% of patients (n = 134). Detection of viral pathogens increased during the study period from 7 to 31 cases per week, while bacterial pneumonias, respiratory tract infections without detection of a viral pathogen and non-infectious etiologies remained stable. A high burden of bacterial and viral co-infections became apparent (16%, 38/243), and co-administration of antibiotic and antiviral treatments was observed (14%, n = 35/243). 17% of patients (41/243) received antibiotic coverage without a diagnosis of a bacterial etiology. CONCLUSION: During the fall of 2022, the burden of RTI caused by detectable viral pathogens increased unusually early. Rapid and unexpected changes in pathogen distribution highlight the need for targeted diagnostics to improve the quality of RTI management in the ED.

Epidemiology of Plasmids in Escherichia coli and Klebsiella pneumoniae with Acquired Extended Spectrum Beta-Lactamase Genes Isolated from Chronic Wounds in Ghana.

Little information is available on the local epidemiology of mobile genetic elements such as plasmids harboring acquired beta-lactamase genes in Western African Ghana. In the present study, we screened for plasmids in three Escherichia coli and four Klebsiella pneumoniae isolates expressing extended spectrum beta-lactamases (ESBL) mediated by the blaCTX-M-15 gene from chronically infected wounds of Ghanaian patients. Bacterial isolates were subjected to combined short-read and long-read sequencing to obtain the sequences of their respective plasmids. In the blaCTX-M-15-gene-carrying plasmids of the four ESBL-positive K. pneumoniae isolates, IncFIB/IncFII (n = 3) and FIA (n = 1) sequences were detected, while in the blaCTX-M-15-gene-carrying plasmids of the three ESBL-positive E. coli isolates, IncFIA/IncFIB (n = 2) and IncFIB (n = 1) sequences were found. The three IncFIB/IncFII sequence-containing plasmids were almost identical to a K. pneumoniae plasmid reported from France. They belonged to the clonal lineages ST17, ST36 and ST39 of K. pneumoniae, suggesting transversal spread of this obviously evolutionary successful plasmid in Ghana. Other resistance gene-encoding plasmids observed in the assessed Enterobacterales harbored IncFIA/IncR and IncFII sequences. International spread was confirmed by the high genetic similarity to resistance-mediating plasmids published from Asia, Australia, Europe and Northern America, including a blaCTX-M-15-gene-carrying plasmid isolated from a wild bird in Germany. In conclusion, the study contributed to the scarcely available information on the epidemiology of third-generation cephalosporine resistance-mediating plasmids in Ghana. Furthermore, the global spread of resistance-mediating plasmids provided hints on the evolutionary success of individual resistance-harboring plasmids by transversal spread among K. pneumoniae lineages in Ghana.

Correction: Tanida et al. Comparative Assessment of In-House Real-Time PCRs Targeting Enteric Disease-Associated Microsporidia in Human Stool Samples. Pathogens 2021, 10, 656.

In the original publication [...].

Comparison of Three In-House Real PCR Assays Targeting Kinetoplast DNA, the Small Subunit Ribosomal RNA Gene and the Glucose-6-Phosphate Isomerase Gene for the Detection of Leishmania spp. in Human Serum.

To perform PCR from serum for the diagnosis of visceral leishmaniasis is convenient and much less invasive than the examination of deeper compartments such as bone marrow. We compared three Leishmania-specific real-time PCRs with three different molecular targets (kinetoplast DNA, the small subunit-ribosomal RNA-(ssrRNA-)gene, the glucose-6-phosphate isomerase-(gpi-)gene) regarding their sensitivity and specificity in human serum. Residual sera from previous diagnostic assessments at the German National Reference Center for Tropical Pathogens Bernhard Nocht Institute for Tropical Medicine Hamburg and the Swiss Tropical and Public Health Institute were used. The sensitivities of kinetoplast DNA-PCR, ssrRNA-gene PCR, and gpi-PCR were 93.3%, 73.3%, and 33.3%, respectively, with 15 initial serum samples from visceral leishmaniasis patients, as well as 9.1%, 9.1%, and 0.0%, respectively, with 11 follow-up serum samples taken at various time points following anti-leishmanial therapy. Specificity was 100.0% in all assays as recorded with 1.137 serum samples from deployed soldiers and migrants without clinical suspicion of visceral leishmaniasis. Kinetoplast-DNA PCR from serum was confirmed as a sensitive and specific approach for the diagnosis of visceral leishmaniasis. The results also indicate the suitability of serum PCR for diagnostic follow-up after therapy, in particular regarding therapeutic failure in case of persisting positive PCR results.

Comparative Assessment of In-House Real-Time PCRs Targeting Enteric Disease-Associated Microsporidia in Human Stool Samples.

Microsporidiosis is an infection predominantly occurring in immunosuppressed patients and infrequently also in travelers. This study was performed to comparatively evaluate the diagnostic accuracy of real-time PCR assays targeting microsporidia with etiological relevance in the stool of human patients in a latent class analysis-based test comparison without a reference standard with perfect accuracy. Thereby, two one-tube real-time PCR assays and two two-tube real-time PCR assays targeting Enterocytozoon bieneusi and Encephalocytozoon spp. were included in the assessment with reference stool material (20), stool samples from Ghanaian HIV-positive patients (903), and from travelers, migrants and Colombian indigenous people (416). Sensitivity of the assays ranged from 60.4% to 97.4% and specificity from 99.1% to 100% with substantial agreement according to Cohen's kappa of 79.6%. Microsporidia DNA was detected in the reference material and the stool of the HIV patients but not in the stool of the travelers, migrants, and the Colombian indigenous people. Accuracy-adjusted prevalence was 5.8% (n = 78) for the study population as a whole. In conclusion, reliable detection of enteric disease-associated microsporidia in stool samples by real-time PCR could be demonstrated, but sensitivity between the compared microsporidia-specific real-time PCR assays varied.

Clonal Clusters, Molecular Resistance Mechanisms and Virulence Factors of Gram-Negative Bacteria Isolated from Chronic Wounds in Ghana.

Wound infections are common medical problems in sub-Saharan Africa but data on the molecular epidemiology are rare. Within this study we assessed the clonal lineages, resistance genes and virulence factors of Gram-negative bacteria isolated from Ghanaian patients with chronic wounds. From a previous study, 49 Pseudomonas aeruginosa, 21 Klebsiellapneumoniae complex members and 12 Escherichia coli were subjected to whole genome sequencing. Sequence analysis indicated high clonal diversity with only nine P. aeruginosa clusters comprising two strains each and one E. coli cluster comprising three strains with high phylogenetic relationship suggesting nosocomial transmission. Acquired beta-lactamase genes were observed in some isolates next to a broad spectrum of additional genetic resistance determinants. Phenotypical expression of extended-spectrum beta-lactamase activity in the Enterobacterales was associated with blaCTX-M-15 genes, which are frequent in Ghana. Frequently recorded virulence genes comprised genes related to invasion and iron-uptake in E. coli, genes related to adherence, iron-uptake, secretion systems and antiphagocytosis in P. aeruginosa and genes related to adherence, biofilm formation, immune evasion, iron-uptake and secretion systems in K. pneumonia complex. In summary, the study provides a piece in the puzzle of the molecular epidemiology of Gram-negative bacteria in chronic wounds in rural Ghana.

Comparison of two commercial and one in-house real-time PCR assays for the diagnosis of bacterial gastroenteritis.

INTRODUCTION: The aim of the study was a comparative evaluation of in-house real-time PCR and commercial real-time PCR (Fast Track Diagnostics (FTD), ampliCube/Mikrogen) targeting enteropathogenic bacteria from stool in preparation of Regulation (EU) 2017/746 on in vitro diagnostic medical devices. METHODS: Both 241 stool samples from patients and 100 samples from German laboratory control schemes ("Ringversuche") were used to comparatively assess in-house real-time PCR, the FTD bacterial gastroenteritis kit, and the ampliCube gastrointestinal bacterial panels 1&2 either with the in-house PCRs as gold standard and as a test comparison without gold standard applying latent class analysis. Sensitivity, specificity, intra- and inter-assay variation and Cohen's kappa were assessed. RESULTS: In comparison with the gold standard, sensitivity was 75-100% for strongly positive samples, 20-100% for weakly positive samples, and specificity ranged from 96 to 100%. Latent class analysis suggested that sensitivity ranges from 81.2 to 100% and specificity from 58.5 to 100%. Cohen's kappa varied between moderate and nearly perfect agreement, intra- and inter-assay variation was 1-3 to 1-4 Ct values. CONCLUSION: Acceptable agreement and performance characteristics suggested replaceability of the in-house PCR assays by the commercial approaches.

Evaluation of the automated cartridge-based ARIES SARS-CoV-2 Assay (RUO) against automated Cepheid Xpert Xpress SARS-CoV-2 PCR as gold standard.

INTRODUCTION: To evaluate the automated cartridge-based PCR approach ARIES SARS-CoV-2 Assay targeting the ORF-sequence and the N-gene of SARS-CoV-2. METHODS: In line with the suggestions by Rabenau and colleagues, the automated ARIES SARS-CoV-2 Assay was challenged with strongly positive samples, weakly positive samples and negative samples. Further, intra-assay and inter-assay precision as well as the limit-of-detection (lod) were defined with quantified target RNA and DNA. The Cepheid Xpert Xpress SARS-Cov-2 Assay was used as gold standard. RESULTS: Concordance between the ARIES assay and the Cepheid assay was 100% for strongly positive samples and for negative samples, respectively. For weakly positive samples as confirmed applying the Cepheid assay, a relevant minority of 4 out of 15 samples (26.7%) went undetected by the ARIES assay. Intra- and inter-assay precision were satisfactory, while the lod was in the 103 DNA copies/reaction-range, in the 103 virus copies/reaction-range, or in the 103-104 free RNA copies/reaction-range in our hands. CONCLUSIONS: The automated ARIES assay shows comparable test characteristics as the Cepheid assay focusing on strongly positive and negative samples but a slightly reduced sensitivity with weakly positive samples. Decisions on diagnostic use should include considerations on the lod.