European Journal of Clinical Microbiology & Infectious Diseases

BackgroundWhile the sensitivity of detection of pneumococcal carriage can be improved by testing respiratory tract samples with qPCR, concerns have been raised regarding the specificity of this approach. We therefore investigated the reliability of the widely-used lytA qPCR assay when applied to saliva samples from older adults in relation to a more specific qPCR assay (piaB). MethodsDuring the autumn/winter seasons of 2018/2019 and 2019/2020, saliva was collected at multiple timepoints from 103 healthy adults aged 21-40 (n=34) and [≥]64 (n=69) years. Following culture-enrichment, extracted DNA was tested using qPCR for piaB and lytA. By sequencing the variable region of rpsB (S2-typing), we identified the species of bacteria isolated from samples testing lytA-positive only. ResultsWhile 30/344 (8.7%) saliva samples (16.5% individuals) tested qPCR-positive for both piaB and lytA, 52 (15.1%) samples tested lytA-positive only. No samples tested piaB-positive only. Through extensive re-culture of the 32 lytA-positive samples collected in 2018/2019, we isolated 23 strains (from 8 samples, from 5 individuals) that were also qPCR-positive for only lytA. Sequencing determined that Streptococcus mitis and Streptococcus infantis were predominantly responsible for this lytA-positive qPCR signal. ConclusionsWe identified a comparatively large proportion of samples generating positive signals with the widely used lytA-qPCR and identified non-pneumococcal streptococcal species responsible for this signal. This highlights the importance of testing for the presence of multiple gene targets in tandem for reliable and specific detection of pneumococcus in respiratory tract samples.

The reliable detection of immunoglobulin G (IgG) or total antibodies directed against the novel severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is important for clinical diagnostics and epidemiological studies. Here, we compare the diagnostic accuracy of six commercially available SARS-CoV-2 IgG (Abbott SARS-CoV-2 IgG; Diasorin Liaison(R) SARS-CoV-2 S1/2 IgG; Epitope EDI Novel Coronavirus COVID-19 IgG ELISA Kit; Euroimmun Anti-SARS-CoV-2 ELISA (IgG); Mikrogen recomWell SARS-CoV-2 IgG) or total SARS-CoV-2 antibody assays (Roche Elecsys Anti-SARS-CoV-2). The test sensitivities were analyzed with a set of 34 sera obtained from 26 patients after PCR-confirmed SARS-CoV-2 infection and varied from 76.9% (Euroimmun) to 96.2% (Abbott). The majority of assay results were confirmed in a laboratory-developed plaque reduction neutralization test and by a SARS-CoV-2 IgG-specific line assay including measurement of generally low IgG avidities (Mikrogen recomLine Coronavirus IgG [Aviditat], prototype). Moreover, 100 stored sera collected during summer 2018 (N = 50) and winter season 2018/2019 (N = 50) were included to demonstrate test specificities. These varied from 96.0% (DiaSorin) to 100% (Epitope EDI). A subset of sera were retested with a lateral flow test (STANDARD Q COVID-19 IgM/IgG Duo) and a considerably lower sensitivity was noted. Overall, the diagnostic accuracy of the six SARS-CoV-2 IgG/total antibody assays was good and varied from 92.9% (Euroimmun) to 98.4% (Abbott). Due to the different specificities, results of commercially available SARS-CoV-2 antibody tests should be interpreted with caution. A high proportion of antibody-positive patient sera demonstrated neutralizing capacity against SARS-CoV-2.

IntroductionRapid antigen detection tests (RDT) are suitable for large-scale testing for SARS-CoV-2 among the population and recent studies have shown that self-testing with RDT in the general population is feasible and yields acceptable sensitivities with high specificity. We aimed to determine the accuracy of two different RDTs, with two different sample collection methods for one of the RDTs among healthcare workers (HCW). Secondary objectives were to determine the accuracy of RDT using a viral load cut-off as proxy of infectiousness and to identify predictors for a false negative RDT. MethodsCenters that participated were secondary care hospitals, academic teaching hospitals, and long-term care facilities. All HCW that met inclusion criteria were asked to perform a RDT self-test next to a regular SARS-CoV-2 nucleic acid amplification test (NAAT). Three study groups were created. Study group 1; Veritor(tm) System, Becton Dickinson, Franklin Lakes, USA (BD-RDT) with combined oropharyngeal - mid-turbinate nasal sampling, group 2; BD-RDT with mid-turbinate nasal sampling only and group 3; SD Biosensor SARS-CoV-2 Rapid Antigen Test, Roche, Basel, Switzerland (Roche-RDT) with combined oropharyngeal - mid-turbinate nasal sampling. RDT accuracy was calculated using NAAT as reference standard. For samples processed in the cobas(R) 6800/8800 platform (Roche Diagnostics, Basel, Switzerland), established cycle threshold values (Ct-values) could be converted into viral loads. A viral load cut-off of [&ge;]5.2 log10 SARS-CoV-2 E gene copies/ml was used as proxy of infectiousness. Logistic regression analysis was performed to identify predictors for a false negative RDT. ResultsIn total, 7,196 HCW were included. Calculated sensitivities were 61.5% (95%CI 56.6%-66.3%), 50.3% (95%CI 42.8%-57.7%) and 74.2% (95%CI 66.4%-80.9%) for study groups 1, 2 and 3, respectively. After application of a viral load cut-off as a proxy for infectiousness for samples processed in the cobas(R) 6800/8800 platform sensitivities increased to 82.2% (95%CI 76.6-86.9%), 61.9% (95%CI 48.8%-73.9%) and 90.2% (95%CI 76.9%-97.3%) for group 1, group 2 and group 3, respectively. Multivariable regression analysis showed that use of Roche-RDT (p <0.01), combined oropharyngeal - mid-turbinate nasal sampling (p <0.05) and the presence of COVID-19 like symptoms at the time of testing (p <0.01) significantly reduced the likeliness of a false-negative RDT result. ConclusionSARS-CoV-2 RDT has proven able to identify infectious individuals, especially when upper respiratory specimen is collected through combined oropharyngeal - mid-turbinate sampling. Reliability of self-testing with RDT among HCW seems to depend on the type of RDT, the sampling method and the presence of COVID-19 like symptoms at the time of testing.

BackgroundPseudomonas aeruginosa (PA) is the most commonly detected pathogen in bronchiectasis. The clinical standard of care for pathogen detection is culture, which has low sensitivity. Early detection and improved monitoring could be used to enhance eradication and long-term suppressive treatments. MethodsA novel bacterial quantification assay (BQA) targeting ribosomal RNA for the detection of viable PA was developed. The BQA sensitivity and specificity in patient samples was assessed utilising culture (n=57); as well as BioFire pneumonia panel (n=111) and 16S rRNA gene sequencing (n=62). The potential clinical impact of the BQA in clinical settings was explored using two international patient cohorts. ResultsThe BQA detected PA in 100% of samples where PA was identified by other methods. The BQA quantification was positively correlated with quantitative culture (r=0.42; p<0.001) and BioFire (r=0.68; p<0.001). The BQA identified PA in an additional 23-44% of cases. To evaluate if this increased detection had clinical implications, we tested sputum samples considered to be PA negative by culture in patients who subsequently tested positive for PA. The BQA detected PA in 8/20 patient samples 8-484 days prior to primary detection by culture. In patients considered to have successful PA eradication by culture, the BQA detected PA in 13 samples from 27 patients who went on to relapse. ConclusionsThe BQA is a highly sensitive detection and quantification method for PA. The BQA demonstrated improved detection and treatment monitoring compared to culture, identifying patients who went on to either isolate a first PA or relapse.

IntroductionPneumocystis jirovecii (PJ) causes pneumonia primarily in immunocompromised individuals. Although direct immunofluorescence (IF) remains the diagnostic standard, PCR assays are increasingly used because of higher sensitivity and reduced observer dependency. This study evaluates the ELITe InGenius PJ PCR assay compared with IF for PJ detection in respiratory specimens. Material and MethodsRespiratory samples submitted for IF-based PJ testing at the University Hospital Zurich over a 19-month period were retrospectively analyzed using the ELITe InGenius PCR assay. Diagnostic accuracy was assessed using IF as the reference method, and performance was evaluated by receiver operating characteristic (ROC) analysis. ResultsA total of 222 samples from 213 patients were included (70 sputum, 152 bronchoalveolar lavage). PCR and IF results were concordant in 160 (72.1%) specimens, including 18 positives and 142 negatives. Sixty-two (27.9%) samples were discrepant: six IF-positive/PCR-negative and 56 IF-negative/PCR-positive. IF reexamination was possible for IF-positive/PCR-negative and IF-negative/PCR-high-positive samples (>100,000 copies/ml or Ct <29), resulting in revised IF findings in 3/3 and 2/8 cases, respectively. Three IF-negative/PCR-positive cases were clinically confirmed as true infections. ROC analysis identified an optimal PCR threshold of 6,233 copies/ml, yielding 70.8% sensitivity and 88.3% specificity, improving to 82.6% and 89.5% after reexamination. DiscussionThese findings support the higher sensitivity of PCR relative to IF for direct PJ detection. Clinical confirmation of IF-negative/PCR-positive cases and known limitations of IF underscore the diagnostic value of the ELITe InGenius assay. Its performance supports its use in routine diagnostics to improve early detection and guide timely treatment.

In children, the gold standard for the detection of pneumococcal carriage is conventional culture of a nasopharyngeal swab. Saliva, however, has a history as one of the most sensitive methods for surveillances on pneumococcal colonisation and has recently been shown to improve carriage detection in older age groups. Here, we compared the sensitivity of nasopharyngeal and saliva samples from PCV7-vaccinated 24-month-old children for pneumococcal carriage detection using conventional and molecular diagnostic methods. Nasopharyngeal and saliva samples were collected from 288 24-month-old children during the autumn/winter, 2012/2013. All samples were first processed by conventional diagnostic techniques. Next, DNA extracted from all plate growth was tested by qPCR for the presence of pneumococcal genes piaB and lytA and a subset of serotypes. By culture, 164/288 (57%) nasopharyngeal swabs tested positive for pneumococcus, but detection was not possible from saliva due to abundant polymicrobial growth on culture-plates. Molecular methods increased the number of children pneumococci-positive to 172/288 (60%) when testing culture-enriched saliva and to 212/288 (73%) when testing nasopharyngeal samples. Altogether, by molecular methods 239/288 (83%) infants were positive, with qPCR-based carriage detection of culture-enriched nasopharyngeal swabs significantly detecting more carriers compared to either conventional culture (p<0.001) or qPCR-detection of saliva (p<0.001). However, 27/240 (11%) carriers were positive only in saliva, significantly contributing to the overall number of carriers detected (p<0.01). While testing nasopharyngeal swabs with qPCR proved most sensitive for pneumococcal detection in infants, saliva sampling could be considered as complementary to provide additional information on carriage and serotypes which may not be detected in the nasopharynx and may be particularly useful in longitudinal studies, requiring repeated sampling events.

BackgroundPneumococcal pneumonia continues to be a significant global health burden, affecting both children and adults. Traditional diagnostic methods for sputum analysis remain challenging. The objective of this study was twofold: to develop a rapid and easy-to-perform assay for the identification of Streptococcus pneumoniae (Spn) directly in sputum specimens using fluorescence microscopy, and to characterize with high-resolution confocal microscopy the ultrastructure of pneumococci residing in human sputum. MethodsWe fluorescently labeled antibodies against the pneumococcal capsule (Spn-FLUO). The specificity and sensitivity of Spn-FLUO for detecting Spn was evaluated in vitro and in vivo using mouse models of carriage and disease, human nasopharyngeal specimens, and sputum from patients with pneumococcal pneumonia. Spn was confirmed in the specimens using culture and a species-specific qPCR assays. Confocal microscopy and Imaris software analysis were utilized to resolve the ultrastructure of pneumococci in human sputum. ResultsCompared with cultures and qPCR, Spn-FLUO demonstrated high sensitivity (78-96%) in nasopharyngeal samples from mice and humans. The limit of detection (LOD) in nasopharyngeal samples was [&ge;]1.6x10 GenEq/ml. The specificity in human nasopharyngeal specimens was 100%. In lung specimens from mice infected with pneumococci, Spn-FLUO reached 100% sensitivity with a LOD of [&ge;]1.39x10 GenEq/ml. In human sputum, the sensitivity for detecting Spn was 92.7% with a LOD of 3.6x103 GenEq/ml. Ultrastructural studies revealed that pneumococci are expectorated as large aggregates with a median size of 1336 {micro}m{superscript 2}. ConclusionsSpn-FLUO is a rapid and sensitive assay for detecting Spn in human sputum within 30 min. The study highlights that most pneumococci form aggregates in human sputum.

Blood-culture bottles (BCBs) are widely used to improve the diagnosis of orthopedic device-related infections. Data is scarce on the growth of Cutibacterium acnes and its genotypes in BCBs under real-life clinical conditions. We studied 39 cases of revision arthroplasty for which at least one intraoperative sample yielded a pure C. acnes culture from anaerobic BCBs (BD Bactec Lytic/10 Anaerobic/F [Lytic Ana]) and/or solid media. Genotyping of C. acnes isolates from the 39 cases allowed: i) the identification of 49 non-redundant isolates belonging to four clonal complexes (CCs): CC18, CC28, CC36, and CC53 and ii) the determination of infectant and contaminant strains. Under real-life clinical conditions, Lytic Ana alone was more often positive for contaminants than infectant strains (18/36 [50%] versus 2/13 [15.4%]; p = 0.047). The time to detection (TTD) values in Lytic Ana were shorter for CC53 than other CCs (mean [SD] TTD: 77 [15] versus 165 [71] hours; p = 0.02). CC53 was confirmed to grow faster than other CCs by studying an enlarged panel of 70 genotyped C. acnes strains inoculated in vitro into Lytic Ana vials (mean [SD] TTD: 73 [13] versus 122 [50] hours; p < 0.001). The use of Lytic Ana BCBs in orthopedics increases the recovery rate of C. acnes but leads to the isolation of proportionally more contaminants than true infectant strains. TTD values are much shorter for CC53 strains, irrespective of their being infectant or contaminant. TTD does not solely reflect the bacterial load of samples but also clonal complex-related traits.

IntroductionSeveral viral respiratory infections - notably influenza - are associated with secondary bacterial infection and additional pathology. The extent to which this applies for COVID-19 is unknown. Accordingly, we aimed to define the bacteria causing secondary pneumonias in COVID-19 ICU patients using the FilmArray Pneumonia Panel, and to determine this tests potential in COVID-19 management. MethodsCOVID-19 ICU patients with clinically-suspected secondary infection at 5 UK hospitals were tested with the FilmArray at point of care. We collected patient demographic data and compared FilmArray results with routine culture. ResultsWe report results of 110 FilmArray tests on 94 patients (16 had 2 tests): 69 patients (73%) were male, the median age was 59 yrs; 92 were ventilated. Median hospital stay before testing was 14 days (range 1-38). Fifty-nine (54%) tests were positive, with 141 bacteria detected. Most were Enterobacterales (n=55, including Klebsiella spp. [n= 35]) or Staphylococcus aureus (n=13), as is typical of hospital and ventilator pneumonia. Community pathogens, including Haemophilus influenzae (n=8) and Streptococcus pneumoniae (n=1), were rarer. FilmArray detected one additional virus (Rhinovirus/Enterovirus) and no atypical bacteria. Fewer samples (28 % vs. 54%) were positive by routine culture, and fewer species were reported per sample; Klebsiella species remained the most prevalent pathogens. ConclusionFilmArray had a higher diagnostic yield than culture for ICU COVID-19 patients with suspected secondary pneumonias. The bacteria found mostly were Enterobacterales, S. aureus and P. aeruginosa, as in typical HAP/VAP, but with Klebsiella spp. more prominent. We found almost no viral co-infection. Turnaround from sample to results is around 1h 15 min compared with the usual 72h for culture, giving prescribers earlier data to inform antimicrobial decisions.

BACKGROUNDAutomated antimicrobial susceptibility testing (AST) systems are crucial for accurate, timely detection of drug-resistant microbial isolates. This meta-analysis assessed the performance of the BD Phoenix ("Phoenix", BD Diagnostic Solutions), Vitek(R) 2 ("Vitek 2", bioMerieux), and DxM MicroScan WalkAway ("MicroScan", Beckman Coulter, Inc.) AST systems relative to common reference methodology. METHODSA systematic literature search in Ovid (MEDLINE and Embase) yielded 275 unique (not duplicated) records, with 44 additional records retrieved from handsearching; 39 studies met inclusion criteria. Categorical agreement (CA), essential agreement (EA), very major errors (VMEs), and major errors (MEs) for the three instruments were compared to a common reference method. Ratios of proportions were analyzed using random-effect meta-regression. RESULTSThe instruments did not differ significantly in CA, EA, or ME. Vitek 2 showed a higher overall VME rate than Phoenix ([~]44% higher; Vitek 2-to-Phoenix ratio = 1.44; p=0.062 [approaching significance]) and MicroScan (74% higher; ratio = 1.74; p=0.045). No appreciable difference was observed for VME between Phoenix and MicroScan. Subgroup analyses should be interpreted cautiously due to limited overall significance indicating varying performance across systems. Vitek 2 generally had higher relative VMEs for gram-negative organisms and lower relative VMEs for gram-positive organisms, whereas Phoenix showed the opposite pattern. MicroScan had relatively low VMEs when stratified by Clinical and Laboratory Standards Institute (CLSI) criteria; no differences in VMEs were observed using European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. CONCLUSIONAlthough some VME differences were noted, overall performance of the three systems was comparable. Organism- and drug-specific VME patterns--and updates to CLSI criteria over time--highlight the importance of continued monitoring of current breakpoints for all three instruments.

SynopsisO_ST_ABSObjectivesC_ST_ABSTo describe the genotypic epidemiological distribution and the antibiotic resistance mechanisms of recent carbapenem-resistant Acinetobacter baumannii (CRAb) strains recovered from clinical samples in Belgium. MethodsA total of 40 clinical isolates of CRAb collected by the national reference center from 19 acute-care hospitals through national microbiological surveillance in 2014 and 2017 were analysed in this study. The isolates were tested for antimicrobial susceptibility by broth microdilution and determined for carbapenemase-encoding genes by multiplex PCR targeting major carbapenemases families. Isolates were subjected to whole-genome sequencing (WGS) with Illumina technology and the complete chromosomal sequences were de novo assembled. Genome analysis was performed to identify intrinsic and acquired resistance determinants and to characterize clonal lineage according to the sequence type (ST). ResultsAll 40 isolates were resistant to carbapenems and exhibited extensively drug-resistant phenotype with blaOXA-23 (n=29) being the most abundant detected acquired AMR gene with 38 isolates encoding at least two different types of OXA enzymes. The majority of the isolates were globally disseminated clones of ST2 (n=25) while less frequent sequence types such as ST636 (n=6), ST1 (n=3), ST85 (n=2) and per one isolate from ST604, ST215, ST158 and ST78 were also detected. ConclusionsWe have detected extensively drug-resistant globally occurring clones of A. baumannii ST1 and ST2 throughout Belgium as well as other sporadic ST including ST636 causing local outbreaks. Our results show the presence of high-risk clones of A. baumannii with common travel importation and the crucial need of constant surveillance.

BackgroundO_ST_ABSAbstractC_ST_ABSReliable SARS-CoV-2 serological assays are required for diagnosing infections, for the serosurveillance of past exposures and for assessing the response to future vaccines. In this study, the analytical and clinical performances of a chemiluminescent immunoassays for SARS-CoV-2 IgM and IgG detection (Mindray CL-1200i), targeting Nucleocapsid (N) and receptor binding domain (RBD) portion of the Spike protein, were evaluated. MethodsPrecision and linearity were evaluated using standardized procedures. A total of 157 leftover serum samples from 81 hospitalized confirmed COVID-19 patients (38 with moderate and 43 with severe disease) and 76 SARS-CoV-2 negative subjects (44 healthcare workers, 20 individuals with rheumatic disorders, 12 pregnant women) were included in the study. In an additional series of 44 SARS-CoV-2 positive, IgM and IgG time kinetics were also evaluated in a time-period of 38 days. ResultsPrecision was below or equal to 4% for both IgM and IgG, in all the studied levels, whilst a slightly significant deviation from linearity was observed for both assays in the range of values covering the manufacturers cut-off. Considering a time frame [&ge;] 12 days post symptom onset, sensitivity and specificity for IgM were 92.3% (95%CI:79.1%-98.4%) and 92.1% (95%CI:83.6%-97.0%). In the same time frame, sensitivity and specificity for IgG were 100% (95%CI:91.0%-100%) and 93.4% (95%CI:85.3%-97.8%). The assays agreement was 73.9% (Cohens kappa of 0.373). Time kinetics showed a substantial overlapping of IgM and IgG response, the latter values being elevated up to 38 days from symptoms onset. ConclusionsAnalytical imprecision is satisfactory as well as the linearity, particularly when taking into account the fact that both assays are claimed to be qualitative. Diagnostic sensitivity of IgG was excellent, especially considering specimens collected [&ge;]12 days post symptom onset. Time kinetics suggest that IgM and IgG are detectable early in the course of infection, but the role of SARS-CoV-2 antibodies in clinical practice still requires further evaluations.

The aim of this study was to determine the performance of both cefotaxime and ceftazidime containing agars on the specificity and sensitivity for chromosomal AmpC-hyperproducing and plasmid AmpC harboring Escherichia coli compared to ESBL-producing E. coli and E. coli without ESBL, pAmpC or cAmpC hyperproduction. Second, we evaluated the influence of adding cefoxitin to these agars for detection of both chromosomal AmpC-hyperproducing and plasmid AmpC harboring E. coli.\n\nFour different homemade screening agars with cefotaxime (1mg/L), ceftazidime (1mg/L), cefotaxime (1mg/L) with cefoxitin (8mg/L), and ceftazidime (1mg/L) with cefoxitin (8mg/L) were compared to each other for the identification of AmpC producing E. coli. A total of 40 isolates with plasmid encoded AmpC {beta}-lactamases, 40 isolates with alterations in the promoter/attenuator region of the AmpC gene leading to hyperproduction of the {beta}-lactamase, 40 isolates with ESBL genes and 39 isolates lacking both a AmpC and ESBL genotype were used to test the four agars.\n\nThe sensitivity and specificity were 100% (95% confidence interval (95% CI) 96.1% to 100%) and 48.1% (95% CI 38.6%-60.2%), respectively, for the cefotaxime agar; 100% (95% CI 96.1% to 100%) and 49.41% (95% CI 39.8%-61.4%), respectively, for the ceftazidime agar; 96.3% (95% CI 89.1% to 99.2%) and 77.2% (95% CI 66.7%-85.2%) respectively, for the cefotaxime with cefoxitin agar; 98.8% (95% CI) 92.6% to 99.6%) and 81.0% (95% CI 70.9%-88.3%) respectively, for the ceftazidime agar with cefoxitin. The main reason for false-positive results were ESBL-harboring strains that grew on various agars; therefore, the specificity of each agar reported here was influenced mainly by the proportion of ESBL isolates tested. In conclusion addition of cefoxitin to cefotaxime and ceftazidime containing agars had little influence on sensitivity, but increased specificity for the detection of AmpC in E. coli.

ObjectivesDiagnosis of community-acquired Legionnaires disease (CALD) relies on microbiological testing. Routine testing in hospitalised CAP patients has low positivity rates. We externally validated a Legionella prediction score, assessed its applicability in routine care, and explored potential updates. MethodsWe analysed data from 196 CALD patients from 20 Swiss hospitals and 196 Legionella-negative CAP controls matched by date of diagnosis ({+/-}14 days; August 2022-March 2024). We assessed the availability of the original score predictors (fever, no/dry cough, hyponatremia, elevated CRP, elevated LDH, low platelet count) in routine care and the original scores discriminative performance. The dataset was split into development and validation cohorts to evaluate whether simplifying modifications improved predictive performance. ResultsThe original score showed 91% (95% CI: 86-96%) sensitivity and 35% (95% CI: 28-42%) specificity at a cut-off [&ge;]2; LDH was infrequently measured, and platelet count was a poor predictor. The simplified SwissLEGIO score (fever >38{degrees}C, sodium <133 mmol/L, CRP >180 mg/L, no/dry cough, prior {beta}-lactam therapy) maintained high sensitivity (88-92%) and showed improved specificity (46-58%) at cut-off [&ge;]2. ConclusionThe SwissLEGIO score is an easy-to-apply screening tool to rule out CALD in hospitalised CAP patients with scores <2 and may reduce testing by 36-52% at a CALD prevalence of 4%.

Multiplex PCR panels have been used for the diagnosis of viral respiratory infections in the last years. While the types of manufacturer validated and thus officially approved materials are usually limited, the tested materials in the clinical routine or studies often vary, which presents a challenge in light of the new EU-IVDR guideline. Aim of our present study was to evaluate if testing of lower respiratory tract (LRT) or saline gargle specimens (SGS) provided an advantage compared to the testing of nasopharyngeal swabs (NPS) and if the testing of pleural effusions (PE) provided any advantage compared to the testing of LRT samples. We included 367 NPS vs LRT cases, of which 202 (55%) were negative in both samples, 108 (29%) were positive in both samples, 28 (8%) had a positive NPS and a negative LRT and 29 (8%) had a negative NPS but a positive LRT, with no significant differences between immunocompetent and immunosuppressed cases. We included 46 NPS vs SGS cases, of which 18 (39%) were negative in both samples, 18 (39%) were positive in both samples, 4 (9%) had a positive NPS and a negative SGS and 6 (13%) had a negative NPS but a positive SGS. Out of the 82 tested PE samples, only one (1%) was positive for Influenza B RNA (detected in the PE but not LTR), while for 5 positive LTR samples no viral genome could be detected in the PE. The samples were tested with the FTD respiratory viral panel for common respiratory viruses. Testing of a lower respiratory tract sample after a negative upper respiratory tract sample may have an incremental diagnostic value. Gargle and nasopharyngeal swab samples seem to have a comparable diagnostic performance, while pleural effusion is a substandard material for the diagnosis of common respiratory virus infections.

Empirical antibiotic selection often fails to be optimal targeted in the era of increasingly common resistant organisms. We prospectively evaluated the usefulness or rapid AST for optimal antibiotic selection by infectious disease (ID) physicians in patients with bacteremia of Gram-positive organisms. QMAC-dRAST results led to optimal antibiotic treatment in 33 (89.2%) of the 37 cases receiving non-optimal targeted antibiotics. Optimal targeted treatments based on QMAC-dRAST results were possible in 133 (97.1%) of the 137 cases. In conclusion, the introduction of rapid phenotypic AST can help increase the selection of optimal targeted antibiotics during the early period of bacteremia.

Nasopharyngeal swabs (NPS) collected by trained healthcare professionals are the preferred specimen for SARS-CoV-2 testing. Self-collected specimens might decrease patient discomfort, conserve healthcare resources, and be preferred by patients. During August - November 2020, 1,806 adults undergoing SARS-CoV-2 testing in Denver, Colorado and Atlanta, Georgia, provided self-collected anterior nares swabs (ANS) and saliva specimens before NPS collection. Compared to NPS, sensitivity for SARS-CoV-2 detection by rRT-PCR appeared higher for saliva than for ANS (85% versus 80% in Denver; 67% versus 58% in Atlanta) and higher among participants reporting current symptoms (94% and 87% in Denver; 72% and 62% in Atlanta, for saliva and ANS, respectively) than among those reporting no symptoms (29% and 50% in Denver; 50% and 44% in Atlanta, for saliva and ANS, respectively). Compared to ANS, saliva was more challenging to collect and process. Self-collected saliva and ANS are less sensitive than NPS for SARS-CoV-2 detection; however, they offer practical advantages and might be most useful for currently symptomatic patients.

The cefazolin inoculum effect (CzIE) has been associated with poor clinical outcomes in patients with MSSA infections. We aimed to investigate the point prevalence of the CzIE among nasal colonizing MSSA isolates from ICU patients in a multicenter study in Colombia (2019-2023). Patients underwent nasal swabs to assess for S. aureus colonization on admission to the ICU and some individuals had follow-up swabs. We performed cefazolin MIC by broth-microdilution using standard and high-inoculum and developed a modified nitrocefin-based rapid test to detect the CzIE. Whole genome sequencing was carried out to characterize BlaZ types and allotypes, phylogenomics and Agr-typing. All swabs were subjected to 16S-rRNA metabarcoding sequencing to evaluate microbiome characteristics associated with the CzIE. A total of 352 patients were included; 46/352 (13%) patients were colonized with S. aureus; 22% (10/46) and 78% (36/46) with MRSA and MSSA, respectively. Among 36 patients that contributed with 43 MSSA colonizing isolates, 21/36 (58%) had MSSA exhibiting the CzIE. BlaZ type A and BlaZ-2 were the predominant type and allotype in 56% and 52%, respectively. MSSA belonging to CC30 were highly associated with the CzIE and SNP analyses supported transmission of MSSA exhibiting the CzIE among some patients of the same unit. The modified nitrocefin rapid test had 100%, 94.4% and 97.7% sensitivity, specificity and accuracy, respectively. We found a high prevalence point prevalence of the CzIE in MSSA colonizing the nares of critically-ill patients in Colombia. A modified rapid test was highly accurate in detecting the CzIE in this patient population.

Streptococcus pneumoniae is the leading cause of empyema and pneumonia in children, and monitoring of effectiveness of polyvalent pneumococcal vaccines has been essential for controlling invasive pneumococcal disease (IPD) in children and elderly adults. Conventional serotyping of pneumococci has relied on Quellung reaction following laboratory culture, however more recently whole genome sequencing (WGS) has been implemented in many reference laboratories to enhance traditional typing. Pleural fluid samples from cases with empyema are often culture negative, limiting the utility of WGS and requiring polymerase chain reaction (PCR) or 16S rRNA sequencing to detect S. pneumoniae. These molecular methods have limited sensitivity and capacity to characterise pneumococcus in clinical samples, especially in specimens with a low pathogen abundance. This study applied capture-based enrichment (tNGS) to identify and characterise S. pneumoniae directly from pleural fluid samples. A total of 51 pleural fluid samples were subjected to tNGS with a custom probe panel, for 39 known positive fluids collected from IPD cases between 2018-2025 in New South Wales, Australia. tNGS results were benchmarked against molecular-based serotyping. Our tNGS achieved 100% sensitivity and specificity in detecting S. pneumoniae. Serotyping results were concordant with PCR and 95% (37/39) of S. pneumoniae PCR positive pleural fluid cases could be serotyped using tNGS. Standard molecular methods however could only determine serotype in 56% (22/39) of samples. This tNGS enabled 39% improvement in ability to directly identify and serotype IPD-associated serotypes of S. pneumoniae in difficult-to-culture pleural fluids can significantly enhance laboratory surveillance of IPD as well as our understanding of vaccine effectiveness.

Penicillin susceptible Staphylococcus aureus (PSSA) may occasionally be encountered as a cause of complicated S. aureus infection, such as endocarditis or bloodstream infections. Clinicians may choose to treat these patients with penicillin over a semi-synthetic penicillin derivative, such as flucloxacillin or oxacillin, due to a favourable Pk/Pd profile. In this study, we prospectively evaluated the penicillin disc (1-IU) method for detection of blaZ, with interpretation of the penicillin edge according to EUCAST recommendations. 472 PSSA isolates were collected between September 2014 to December 2015 from three clinical microbiology laboratories in Queensland, Australia. Initial antimicrobial susceptibility testing was performed by the Vitek 2 system. Real-time PCR for blaZ was performed following phenotypic testing with the 1-IU penicillin disc and the PCR used as the gold standard for detection of penicillinase. The prevalence of blaZ amongst the isolates was 7%. The sensitivity, specificity, positive predictive value and negative predictive value of the penicillin disc method was 97%, 95%, 61% and 100% when compared to blaZ PCR. In summary, the penicillin disc zone size and edge interpretation is a reliable method for detection of blaZ in S. aureus isolates that otherwise test susceptible to penicillin by Vitek 2 AST.