The impact of agar depth on antimicrobial susceptibility testing by disc diffusion.

Introduction. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) specifies the agar depth (4±0.5 mm) when performing antimicrobial susceptibility testing (AST). Since the infrastructure to produce standardized agar may be lacking in settings with limited resources, we wanted to examine to what extent variation in agar depth affects the inhibition zone diameters of quality control (QC) strains and AST of clinical isolates.Methods. The inhibition zone diameters on Mueller-Hinton II agar with different depths (2-6 mm) were tested for various QC strain-antimicrobial agent combinations using Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213. The relationship between zone diameters at different agar depths and MICs was investigated for 35 clinical isolates (E. coli, Klebsiella pneumoniae, S. aureus and P. aeruginosa) from Sierra Leone using MICs as the reference.Results. The inhibition zone diameters were within the acceptance ranges as defined by the EUCAST for the majority of QC strains and antimicrobials, independent of the agar depth. At extreme agar depths, inhibition zones were more frequently out of range. The accuracy of AST varied for clinical isolates at different agar depths for categorical agreement (85.8-94.6%), major error rate (0.4-2.1%) and very major error rate (VME: 3.3-12.5%).Conclusions. Even if the QC strains were in the acceptance range at different agar depths, this does not rule out unacceptably high VME rates (>3%) in clinical isolates.

Exploration of compounds to inhibit the Panton-Valentine leukocidin of Staphylococcus aureus.

The Panton-Valentine leukocidin (PVL) of Staphylococcus aureus is associated with necrotizing infections. After binding to complement 5a receptor (C5aR/CD88) and CD45 it causes cytolysis in polymorphonuclear neutrophils (PMNs) as well as inflammasome activation in monocytes. The objective of this study was to test if (ant)agonists of C5aR and CD45 can attenuate the effect of PVL on PMNs and monocytes. We tested the effect of various concentrations of six C5aR (ant)agonists (avacopan, BM213, DF2593A, JPE-1375, PMX205 and W-54011) and one CD45 antagonist (NQ301) to attenuate the cytotoxic effect of PVL on human PMNs and monocytes in vitro. Shifts in the half-maximal effective concentration (EC50) of PVL to achieve a cytotoxic effect on PMNs and modulation of inflammatory cytokine response from monocytes were determined by flow cytometry and IL-1ß detection. Pre-treatment of PMNs with avacopan, PMX205 and W-54,011 resulted in 3.6- to 4.3-fold shifts in the EC50 for PVL and were able to suppress IL-1ß secretion by human monocytes in the presence of PVL. BM213, DF2593A and NQ301 were unable to change the susceptibility of PMNs towards PVL or reduce inflammasome activation in monocytes. Avacopan, PMX205 and W-54,011 showed protection against PVL-induced cytotoxicity and suppressed IL-1ß secretion by monocytes. Clinical studies are needed to prove whether these substances can be used therapeutically as repurposed drugs.

Can pre-analytical procedures improve microbiological culture yield in patients with periprosthetic infections?

BACKGROUND: The detection of causative pathogens plays a crucial role in the diagnosis and targeted treatment of periprosthetic joint infections (PJI). While there have been improvements in analytic methods in the past, pre-analytical procedures have not yet been sufficiently investigated. The objective of this study was to compare the culture yield of four different pre-analytical procedures. METHODS: Patients with perioperative diagnosis of PJI were included in a single center cross-sectional study (2021-2022). Tissue samples (n = 20) of each patient were randomly and equally distributed to each of the four study arms. Tissue samples were either send to the laboratory without culture medium (group A) or were transported in thioglycolate medium immediately after sampling at three different temperatures (room temperature, 4 °C, 37° for 24 h; group B-D). Culture media were investigated for growth on days 1, 3, 7, 12, 14. All organisms, the number of positive samples and the time to positivity were recorded and compared between the study arms. Single positive cultures were considered as contamination. RESULTS: In total, 71 patients were included. The proportions of culture negative samples (10-15%) and polymicrobial infections (51-54%) were comparable between the four arms. Seven patients (10%) were culture-negative in group A, but showed growth in thioglycolate media (group B-D). Furthermore, 13% of patients showed growth in all groups, but additional organisms were cultured in thioglycolate. There was growth beyond day 7 of culturing only in thioglycolate, but not in group A. A storage temperature of 4 °C showed a longer time to positivity compared to the other groups (p < 0.001). CONCLUSIONS: Pre-analytical storage of tissue samples in thioglycolate broth did not improve the culture yield and did not detect additional cases of infection compared to the standard (pre-analytical storage in sterile containers). However, including a thioglycolate medium to the sampling algorithm reduced the rate of culture-negative infections and helped to identify additional organisms.

Slow clearance of histidine-rich protein-2 in Gabonese with uncomplicated malaria.

Malaria rapid diagnostic tests (RDTs), which detect Plasmodium falciparum (Pf)-specific histidine-rich protein-2 (HRP2), have increasing importance for the diagnosis and control of malaria, especially also in regions where routine diagnosis by microscopy is not available. HRP2-based RDTs have a similar sensitivity to expert microscopy, but their reported low specificity can lead to high false positivity rates, particularly in high-endemic areas. Despite the widespread use of RDTs, models investigating the dynamics of HRP2 clearance following Pf treatment focus rather on short-term clearance of the protein. The goal of this observational cohort study was to determine the long-term kinetic of HRP2-levels in peripheral blood after treatment of uncomplicated malaria cases with Pf mono-infection using a 3-day course of artesunate/amodiaquine. HRP2 levels were quantified at enrollment and on days 1, 2, 3, 5, 7, 12, 17, 22, and 28 post-treatment initiation. The findings reveal an unexpectedly prolonged clearance of HRP2 after parasite clearance from capillary blood. Terminal HRP2 half-life was estimated to be 9 days after parasite clearance using a pharmacokinetic two-compartmental elimination model. These results provide evidence that HRP2 clearance has generally been underestimated, as the antigen remains detectable in capillary blood for up to 28 days following successful treatment, influencing RDT-based assessment following a malaria treatment for weeks. A better understanding of the HRP2 clearance dynamics is critical for guiding the diagnosis of malaria when relying on RDTs. IMPORTANCE: Detecting Plasmodium falciparum, the parasite responsible for the severest form of malaria, typically involves microscopy, polymerase chain reaction (PCR), or rapid diagnostic tests (RDTs) targeting the histidine-rich protein 2 or 3 (HRP2/3). While microscopy and PCR quickly turn negative after the infection is cleared, HRP2 remains detectable for a prolonged period. The exact duration of HRP2 persistence had not been well defined. Our study in Gabon tracked HRP2 levels over 4 weeks, resulting in a new model for antigen clearance. We discovered that a two-compartment model accurately predicts HRP2 levels, revealing an initial rapid reduction followed by a much slower elimination phase that can take several weeks. These findings are crucial for interpreting RDT results, as lingering HRP2 can lead to false positives, impacting malaria diagnosis and treatment decisions.

Diagnostic accuracy of Savanna RVP4 (QuidelOrtho) for the detection of Influenza A virus, RSV, and SARS-CoV-2.

Seasonal increase of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus A/B (Flu A/B), and respiratory syncytial virus (RSV) require rapid diagnostic test methods for the management of respiratory tract infections. In this study, we compared the diagnostic accuracy of Savanna RVP4 (RVP4, QuidelOrtho) with Xpert Xpress Plus SARS-CoV-2/Flu/RSV (Xpert, Cepheid). Nasopharyngeal swabs from patients treated at a tertiary care hospital (Germany) were tested for SARS-CoV-2, Flu A/B, and RSV by RVP4 to assess diagnostic accuracy (reference standard: Xpert). The intra and inter assay precision of Ct-values was assessed by repeated test in triplicates (on day 1) and duplicates (days 2-3). All patients with a physician's order for a multiplex test for SARS-CoV-2, Flu, and RSV test were included. Duplicate swabs from the same patient, samples with a total volume ≤1 mL, or inappropriate shipment/storage were excluded. In total, 229 swabs were included between September 2023 and February 2024. The concordance between both tests was 96.5% (SARS-CoV-2), 98.7% (Flu A), and 99.6% (RSV). Flu B was not detected by both tests. The RVP4 test had a sensitivity of 85%-95% and a specificity of 100% for the detection of SARS-CoV-2, Flu A, and RSV. The intra and inter assay precision of Ct-values from RVP4 was 3% and 2% (SARS-CoV-2), 5% and 4% (Flu A), and 0% and 3% (RSV), respectively. The Savanna RVP4 has a favorable diagnostic accuracy for the detection of SARS-CoV-2, Flu A, and RSV. IMPORTANCE: We assessed the diagnostic accuracy of a new point-of-care test for the rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus A/B (Flu A/B), and respiratory syncytial virus (RSV). The new test has a concordance with the reference standard of 96.5% (SARS-CoV-2), 98.7% (Flu A), and 99.1% (RSV). The sensitivity of 85%-95% and specificity of 100% for the detection of SARS-CoV-2, Flu A, and RSV is comparable with similar nucleic acid amplification-based point of care tests but at lower costs.

Fracture-associated infection with Mycobacterium smegmatis in a 16-year old patient.

We present a patient who suffered an agricultural rollover trauma and developed a fracture-associated tissue infection caused by Mycobacterium smegmatis. Since cases are rare, treatment of infections with M. smegmatis requires an interprofessional approach and the combination of surgery and adjunctive antimicrobial treatment.

Integrated decentralized blood culture incubation: A step towards a 24/7 microbiology service?

To have an impact on the mortality of bloodstream infections, microbiological diagnostics of blood cultures (BC) should provide first results within 12 h. Here, we show how a decentralized BC incubation connected to the central BC incubators via a browser-based application significantly reduces turnaround times.

Bacterial vaginosis in pregnant women: A comparison of the Nugent Score with a multiplex PCR.

We evaluated the Nugent score against a multiplex real-time PCR (reference) for diagnosing bacterial vaginosis (BV) in 140 pregnant women. The Nugent score had a sensitivity of 60 %, a specificity of 81 % and a negative predictive value of 92 % - therefore a tool to rule out BV in pregnant women.

Temporal trends of skin and soft tissue infections caused by methicillin-resistant Staphylococcus aureus in Gabon.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of mortality due to bacterial antimicrobial resistance. While S. aureus is common in skin and soft tissue infections (SSTI) in Africa, data on MRSA rates are scarce and reports vary widely across the continent (5%-80%). In this study, we describe the proportion of MRSA causing SSTI in Lambaréné, Gabon, over an 11-year period. METHODS: We retrospectively analyzed data from 953 bacterial specimens collected from inpatients and outpatients with SSTI at the Albert Schweitzer Hospital, Lambaréné, Gabon, between 2009 and 2019. We determined temporal changes in the prevalence of MRSA and identified risk factors for SSTI with MRSA. RESULTS: 68% of all specimens with bacterial growth yielded S. aureus (n = 499/731), of which 7% (36/497) with antimicrobial susceptibility testing were identified as MRSA. Age above 18 years, admission to the surgical ward, and deep-seated infections were significantly associated with MRSA as the causative agent. After an initial decline from 7% in 2009, there was a marked increase in the proportion of MRSA among all S. aureus from SSTI from 3 to 20% between 2012 and 2019. The resistance rate to erythromycin was significantly higher in MRSA than in methicillin-susceptible S. aureus (73% vs. 10%), and clindamycin resistance was detected exclusively in MRSA isolates (8%). CONCLUSION: The increasing proportion of MRSA causing SSTI over the 11-year period contrasts with many European countries where MRSA is on decline. Continuous surveillance of MRSA lineages in the hospital and community along with antibiotic stewardship programs could address the increasing trend of MRSA.

Long-term immunity following yellow fever vaccination: a systematic review and meta-analysis.

BACKGROUND: Long-term immunity following yellow fever vaccination remains controversial. We aimed to summarise the literature regarding the long-term protection (≥10 years) conveyed by a single dose of yellow fever vaccination. METHODS: In this systematic review and meta-analysis, we searched 11 databases from database inception to Aug 24, 2023. We included cohort and cross-sectional studies reporting immunogenicity outcomes for children or adults who received a single dose of yellow fever vaccination 10 or more years ago. Case series and single case reports were excluded. Participants who received more than one dose of yellow fever vaccination before measurement of the outcome were excluded. Identified records were reviewed by two independent reviewers. The primary outcome of the meta-analysis was the pooled seroprotection rate. Risk of bias was assessed with the Risk Of Bias In Non-randomized Studies of Interventions tool, and the Joanna Briggs Institute tool for analytical cross-sectional studies. Studies of moderate or good quality that reported seroprotection were included for random-effects meta-analysis and stratified by endemicity and specific risk groups. The study was registered with PROSPERO, CRD42023384087. FINDINGS: Of the 7363 articles identified by our search, 39 were eligible for inclusion for systematic review. These studies comprised 2895 individuals vaccinated 10-60 years ago. 20 studies were included in the meta-analysis. Pooled seroprotection rates were 94% (95% CI 86-99) among healthy adults in a non-endemic setting (mostly travellers) and 76% (65-85) in an endemic setting (all Brazilian studies). The pooled seroprotection rate was 47% (35-60) in children (aged 9-23 months at time of vaccination) and 61% (38-82) in people living with HIV. Reported criteria for seroprotection were highly heterogeneous. INTERPRETATION: The gathered evidence suggests that a single dose of yellow fever vaccination provides lifelong protection in travellers. However, in people living with HIV and children (younger than 2 years), booster doses might still be required because lower proportions of vaccinees were seroprotected 10 or more years post-vaccination. Lower observed seroprotection rates among residents of endemic areas were partly explained by the use of a higher cutoff for seroprotection that was applied in Brazil. Studies from sub-Saharan Africa were scarce and of low quality; thus no conclusions could be drawn for this region. FUNDING: None.

Stopping antibacterial prophylaxis in pediatric allogeneic hematopoietic cell transplantation: An internal audit.

BACKGROUND: Antibacterial prophylaxis in children and adolescents undergoing allogeneic hematopoietic cell transplantation (HCT) is controversial and not recommended by international guidelines. We analyzed relevant posttransplant outcomes following discontinuation of antibacterial prophylaxis at a major European pediatric transplant center. METHODS: The single-center retrospective audit included all pediatric allogeneic HCT patients (pts) transplanted between 2011 and 2020 before (≤2014) and after (≥2015) stopping routine antibacterial prophylaxis with penicillin, metronidazole, and ciprofloxacin upon start of the conditioning regimen. The primary endpoint was overall survival until the first hospital discharge. Secondary endpoints included the occurrence of fever; bacterial infections; and cumulative days with antibacterial agents until discharge. RESULTS: A total of 257 HCT procedures were performed in 249 pts (median age: 10 years, range, 0.2-22.5) for leukemia/lymphoma (n = 150) and nonmalignant disorders (n = 107). Of these, 104 procedures were performed before (cohort 1) and 153 after (cohort 2) stopping prophylaxis. Overall survival until discharge was 90.4% in cohort 1 and 96.1% in cohort 2 (p = .06). No differences were observed in the occurrence of fever (92.3 vs. 94.1%; p = .57) and bacterial infections (34.6 vs. 25.5%; p = .11). The median number of days on antibacterial agents was significantly lower in cohort 2 (39 vs. 34; p = .002). Detection rates of resistant organisms were overall low. CONCLUSION: In this single-center audit, the stop of routine antibacterial prophylaxis had no effect on the occurrence of fever, bacterial infections, resistant organisms, and GVHD. Overall antibiotic use was significantly reduced, and survival was noninferior to the historical control cohort.

GEFAAR: a generic framework for the analysis of antimicrobial resistance providing statistics and cluster analyses.

Easy access to antimicrobial resistance data and meaningful visualization is essential to guide the empirical antimicrobial treatment and to promote the rational use of antimicrobial agents. Currently available solutions are commonly externally hosted, centralized systems. However, there is a need for close monitoring by local analysis tools. To fill this gap, we developed GEFAAR-a generic framework for the analysis of antimicrobial resistance data. Following the example of the German Robert Koch Institute (RKI), an interactive web-application is provided to determine basic pathogen and resistance statistics. In addition to the RKI's externally maintained database, our application provides a generic framework to import tabular data and to analyze them safely in a local environment. Moreover, our application offers an intuitive web-based user interface to visualize resistance trend analysis as well as advanced cluster analyses on species- or clinic/unit level to generate alerts of potential transmission events.

Pharyngeal Communities and Antimicrobial Resistance in Pangolins in Gabon.

Wildlife can be a reservoir and source of zoonotic pathogens for humans. For instance, pangolins were considered one of the potential animal reservoirs of SARS-CoV-2. The aim of this study was to assess the prevalence of antimicrobial-resistant species (e.g., extended-spectrum ß-lactamase [ESBL]-producing Enterobacterales) and Staphylococcus aureus-related complex and to describe the bacterial community in wild Gabonese pangolins. The pharyngeal colonization of pangolins sold in Gabon (n = 89, 2021 to 2022) was analyzed using culture media selective for ESBL-producing Enterobacterales, S. aureus-related complex, Gram-positive bacteria and nonfermenters. Phylogenetic analyses of ESBL-producing Enterobacterales was done using core-genome multilocus sequence typing (cgMLST) and compared with publicly available genomes. Patterns of cooccurring species were detected by network analysis. Of the 439 bacterial isolates, the majority of species belonged to the genus Pseudomonas (n = 170), followed by Stenotrophomonas (n = 113) and Achromobacter (n = 37). Three Klebsiella pneumoniae isolates and one Escherichia coli isolate were ESBL-producers, which clustered with human isolates from Nigeria (MLST sequence type 1788 [ST1788]) and Gabon (ST38), respectively. Network analysis revealed a frequent cooccurrence of Stenotrophomonas maltophilia with Pseudomonas putida and Pseudomonas aeruginosa. In conclusion, pangolins can be colonized with human-related ESBL-producing K. pneumoniae and E. coli. Unlike in other African wildlife, S. aureus-related complex was not detected in pangolins. IMPORTANCE There is an ongoing debate if pangolins are a relevant reservoir for viruses such as SARS-CoV-2. Here, we wanted to know if African pangolins are colonized with bacteria that are relevant for human health. A wildlife reservoir of antimicrobial resistance would be of medical relevance in regions were consumption of so-called bushmeat is common. In 89 pangolins, we found three ESBL-producing Klebsiella pneumoniae strains and one ESBL-producing Escherichia coli strains, which were closely related to isolates from humans in Africa. This points toward either a transmission between pangolins and humans or a common source from which both humans and pangolins became colonized.

Zoonotic sources and the spread of antimicrobial resistance from the perspective of low and middle-income countries.

BACKGROUND: Antimicrobial resistance is an increasing challenge in low and middle-income countries as it is widespread in these countries and is linked to an increased mortality. Apart from human and environmental factors, animal-related drivers of antimicrobial resistance in low- and middle-income countries have special features that differ from high-income countries. The aim of this narrative review is to address the zoonotic sources and the spread of antimicrobial resistance from the perspective of low- and middle-income countries. MAIN BODY: Contamination with extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli is highest in poultry (Africa: 8.9-60%, Asia: 53-93%) and there is a risk to import ESBL-producing E. coli through poultry meat in Africa. In aquacultures, the proportion of ESBL-producers among E. coli can be high (27%) but the overall low quality of published studies limit the general conclusion on the impact of aquacultures on human health. ESBL-producing E. coli colonization of wildlife is 1-9% in bats or 2.5-63% birds. Since most of them are migratory animals, they can disperse antimicrobial resistant bacteria over large distances. So-called 'filth flies' are a relevant vector not only of enteric pathogens but also of antimicrobial resistant bacteria in settings where sanitary systems are poor. In Africa, up to 72.5% of 'filth flies' are colonized with ESBL-producing E. coli, mostly conferred by CTX-M (24.4-100%). While methicillin-resistant Staphylococcus aureus plays a minor role in livestock in Africa, it is frequently found in South America in poultry (27%) or pork (37.5-56.5%) but less common in Asia (poultry: 3%, pork: 1-16%). CONCLUSIONS: Interventions to contain the spread of AMR should be tailored to the needs of low- and middle-income countries. These comprise capacity building of diagnostic facilities, surveillance, infection prevention and control in small-scale farming.

Impact of the COVID-19 Pandemic on the Management of Staphylococcus aureus Bloodstream Infections in a Tertiary Care Hospital.

Staphylococcus aureus bacteremia (SAB) is associated with a high mortality rate. The clinical outcome of SAB patients highly depends on early diagnosis, adequate antibiotic therapy and source control. In the context of the COVID-19 pandemic, the health care system faced additional organizational challenges and the question arose whether structured screening and triaging for COVID-19 and shifting resources influence the management of SAB. Patients (n = 115) with SAB were enrolled in a retrospective comparative study with historical controls (March 2019-February 2021). The quality of SAB therapy was assessed with a point score, which included correct choice of antibiotic, adequate dosage of antibiotic, sufficient duration of therapy, early start of therapy after receipt of findings, focus search and taking control blood cultures 3-4 days after starting adequate antibiotic therapy. The quality of treatment before and after the onset of the COVID-19 pandemic were compared. No significant differences in the total score points were found between the pre-COVID-19 and COVID-19 cohort. All quality indicators, except the correct duration of antibiotic therapy, showed no significant differences in both cohorts. Furthermore, there were no significant differences in the outcome between both cohorts. The treatment quality of SAB therapy was comparable before and during the COVID-19 pandemic.

Research article network analysis of polymicrobial chronic wound infections in Masanga, Sierra Leone.

BACKGROUND: Chronic wounds are frequently colonized or infected with multiple bacterial or fungal species, which can both promote or inhibit each other. Network analyses are helpful to understand the interplay of these species in polymicrobial infections. Our aim was to analyse the network of bacterial and fungal species in chronic wounds. METHODS: Swabs (n = 163) from chronic wound infections (Masanga, Sierra Leone, 2019-2020) were screened for bacterial and fungal species using non-selective agars. Some of these wounds were suspected but not confirmed Buruli ulcer. Species identification was done with MALDI-TOF mass spectrometry. Network analysis was performed to investigate co-occurrence of different species within one patient. All species with n ≥ 10 isolates were taken into account. RESULTS: Of the 163 patients, 156 had a positive wound culture (median of three different species per patient; range 1-7). Pseudomonas aeruginosa (n = 75) was the dominating species with frequent co-detections of Klebsiella pneumoniae (21 cases; OR = 1.36, 95%CI: 0.63-2.96, p = 0.47), Staphylococcus aureus (14 cases; OR = 1.06, 95%CI: 0.44-2.55, p = 1) and Proteus mirabilis (13 cases; OR = 0.84, 95%CI: 0.35-1.99, p = 0.69). CONCLUSION: The culturome of chronic wounds in Sierra Leonean patients is highly diverse and characterized by the co-occurrence of P. aeruginosa, K. pneumoniae and S. aureus.