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Background/Objectives : The International Organization for Standardization (ISO) 20776-2:2021, which replaces ISO 20776-2:2007, focuses solely on the performance of antimicrobial susceptibility testing (AST) assays, emphasizing the ISO 20776-1 broth microdilution method as the reference standard. Consequently, categorical agreement (CA) and associated errors should not be applied. We verified the Vitek Reveal AST assay according to both ISO 20776-2:2021 and ISO 20776-2:2007 criteria. Methods : Samples from 100 simulated and clinical Gram-negative (GN) positive blood cultures (PBCs) were tested at a large teaching hospital. The simulated GN-PBCs were obtained from a hospital collection of isolates selected to represent diverse antimicrobial resistance profiles. The Reveal assay results were compared with those from the reference assay, and the time to result (TTR) for the Reveal assay was calculated. Results : The essential agreement rates were 96.1% (816/849) for simulated and 98.8% (929/940) for clinical GN-PBC samples. The bias values were -3.1 for simulated and -11.0 for clinical samples. The CA rates were 97.7% (808/827) for simulated and 99.2% (924/931) for clinical samples. The mean TTR ± SD (hours) for resistant organisms was significantly lower (4.40 ± 1.15) than that for susceptible, increased exposure (5.52 ± 0.48) and susceptible (5.54 ± 0.49) organisms. Conclusions : Our findings reinforce the potential of the Reveal assay as a valuable tool and support its implementation in clinical microbiology laboratories.

The diagnosis of Candida bloodstream infection (BSI) may rely on a PCR-based analysis of a positive blood culture (PBC) obtained from the patient at the time of BSI. In this study, a yeast DNA extraction protocol for use on PBCs was developed and evaluated with the molecular mouse (MM) yeast blood (YBL) chip-based PCR assay, which allowed us to detect nine medically relevant Candida species. We studied 125 simulated or clinical PBCs for Candida species. A positive correlation between the DNA concentration and colony-forming unit count was found for simulated (Spearman's ρ = 0.58; p < 0.0001) and clinical (Spearman's ρ = 0.23, p = 0.09) PBCs. The extracted DNA yielded positive results with the MM YBL chip assay that agreed with the Candida species-level identification results for 63 (100%) of 63 isolates from simulated PBCs and 66 (99.5%) of 67 isolates from clinical PBCs. The false-negative result was for one C. tropicalis isolate that grew together with C. albicans in PBC. None of the 30 ( Candida )-negative clinical BCs included as negative controls yielded a positive result with the MM YBL chip assay. Our DNA extraction protocol for the Candida species couples efficiency and simplicity together. Nevertheless, further studies are needed before it can be adopted for use with the MM YBL chip assay.

This study aimed to assess the comparability of in vitro susceptibility testing methods to ceftazidime-avibactam (CZA) and ceftolozane-tazobactam (C/T). Meropenem-resistant and/or carbapenemase-producing clinical isolates of Enterobacterales ( Enterobacteriaceae ) and Pseudomonas aeruginosa were tested by both bioMérieux ETEST and VITEK-2 AST-N397 card and compared with a Micronaut AST-system broth microdilution (BMD) method. CZA and C/T MICs were interpreted using EUCAST breakpoints. Of the 153 Enterobacteriaceae isolates, 55.6% and 0.0% (VITEK 2) and 56.9% and 0.0% (ETEST and BMD) were susceptible to CZA and C/T, respectively. Of 52 P. aeruginosa isolates, 50.0% and 40.4% (VITEK 2, ETEST, and BMD) were susceptible to CZA and C/T, respectively. The essential agreement (EA) was 96.1% (197/205; VITEK 2 versus BMD) and 95.6% (196/205; ETEST versus BMD) for CZA testing, whereas EA was 98.0% (201/205; VITEK 2 versus BMD) and 96.6% (198/205; ETEST versus BMD) for C/T testing. The categorical agreement (CA) was 98.0% (201/205; VITEK 2 versus BMD) and 100% (ETEST versus BMD) for CZA testing, whereas CA was 100% (VITEK 2 versus BMD) and 100% (ETEST versus BMD) for C/T testing. Categorical errors regarded four Enterobacteriaceae isolates. VITEK 2 and ETEST yielded equivalent CZA and C/T susceptibility testing results, compared to the BMD method, in such a clinical context.

We used nasopharyngeal swab samples of patients with a symptomatic (n = 82) or asymptomatic (n = 20) coronavirus disease 2019 (COVID-19) diagnosis to assess the ability of antigen detection tests to infer active (potentially transmissible) or inactive (potentially non-transmissible) infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using the subgenomic RNA (sgRNA) as an active replication marker of SARS-CoV-2, 48 (76.2%), 56 (88.9%), and 63 (100%) of 63 samples with sgRNA positive results tested positive with the SD BIOSENSOR STANDARD Q COVID-19 Ag (Standard Q), the SD BIOSENSOR STANDARD F COVID-19 Ag FIA (Standard F), or the Fujirebio LUMIPULSE G SARS-CoV-2 Ag (Lumipulse) assay, respectively. Conversely, 37 (94.9%), 29 (74.4%), and 7 (17.9%) of 39 samples with sgRNA negative results tested negative with Standard Q, Standard F, or Lumipulse, respectively. Stratifying results by the number of days of symptoms before testing revealed that most antigen positive/sgRNA positive results were among samples tested at 2-7 days regardless of the assay used. Conversely, most antigen negative/sgRNA negative results were among samples tested at 16-30 days only when Standard Q or Standard F were used. In conclusion, based on our findings, a negative antigen test, especially with the Lumipulse assay, or a positive antigen test, especially with the Standard F assay, may suggest, respectively, the absence or presence of replication-competent SARS-CoV-2.

To support the clinical laboratory diagnosis of Pneumocystis jirovecii ( PJ ) pneumonia (PCP), an invasive fungal infection mainly occurring in HIV-negative patients, in-house or commercial PJ -specific real-time quantitative PCR (qPCR) assays are todays' reliable options. The performance of these assays depends on the type of PJ gene (multi-copy mitochondrial versus single-copy nuclear) targeted by the assay. We described the development of a PJ -PCR assay targeting the dihydrofolate reductase (DHFR)-encoding gene. After delineating its analytical performance, the PJ -PCR assay was used to test bronchoalveolar lavage (BAL) fluid samples from 200 patients (only seven were HIV positive) with suspected PCP. Of 211 BAL fluid samples, 18 (8.5%) were positive and 193 (91.5%) were negative by PJ -PCR. Of 18 PJ -PCR-positive samples, 11 (61.1%) tested positive and seven (38.9%) tested negative with the immunofluorescence assay (IFA). All (100%) of the 193 PJ -PCR-negative samples were IFA negative. Based on IFA/PCR results, patients were, respectively, classified as having ( n = 18) and not having ( n = 182) proven ( PJ -PCR+/IFA+) or probable ( PJ -PCR+/IFA-) PCP. For 182 patients without PCP, alternative infectious or non-infectious etiologies were identified. Our PJ -PCR assay was at least equivalent to IFA, fostering studies aimed at defining a qPCR-based standard for PCP diagnosis in the future.

Molecular-based carbapenem resistance testing in Gram-negative bacterial bloodstream infections (BSIs) is currently limited because of the reliance on positive blood culture (BC) samples. The T2Resistance™ panel may now allow the detection of carbapenemase- and other β-lactamase encoding genes directly from blood samples. We detected carbapenem resistance genes in 11 (84.6%) of 13 samples from patients with BC-documented BSIs (10 caused by KPC-producing Klebsiella pneumoniae and 1 caused by VIM/CMY-producing Citrobacter freundii ). Two samples that tested negative for carbapenem resistance genes were from patients with BC-documented BSIs caused by KPC-producing K. pneumoniae who were receiving effective antibiotic therapy. In conclusion, our findings suggest that the T2Resistance™ panel can be a reliable tool for diagnosing carbapenem-resistant Gram-negative bacterial BSIs.

Background: SARS-CoV-2 antigen detection has currently expanded the testing capacity for COVID-19, which yet relies on the SARS-CoV-2 RNA RT-PCR amplification., Objectives: To report on a COVID-19 testing algorithm from a tertiary care hospital emergency department (ED) that combines both antigen (performed on the ED) and RT-PCR (performed outside the ED) testing., Methods: Between December 2020 and January 2021, in a priori designated, spatially separated COVID-19 or non-COVID-19 ED areas, respectively, symptomatic or asymptomatic patients received SARS-CoV-2 antigen testing on nasopharyngeal swab samples. Antigen results were promptly accessible to guide subsequent, outside performed confirmatory (RT-PCR) testing., Results: Overall, 1083 (100%) of 1083 samples in the COVID-19 area and 1815 (49.4%) of 3670 samples in the non-COVID-19 area had antigen results that required confirmation by RT-PCR. Antigen positivity rates were 12.4% (134/1083) and 3.7% (66/1815), respectively. Compared to RT-PCR testing results, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of antigen testing were, respectively, 68.0%, 98.3%, 88.8%, and 94.1% in the COVID-19 area, and 41.9%, 97.3%, 27.3%, and 98.6% in non-COVID-19 area. Practically, RT-PCR tests were avoided in 50.6% (1855/3670) of non-COVID-19 area samples (all antigen negative) from patients who, otherwise, would have needed antigen result confirmation., Conclusions: Our algorithm had value to preserve RT-PCR from avoidable usage and, importantly, to save time, which translated into a timely RT-PCR result availability in the COVID-19 area.

The aim of this study was to characterize COVID-19 (SARS-CoV-2-infected) patients who develop bloodstream infection (BSI) and to assess risk factors associated with in-hospital mortality. We conducted a retrospective observational study of adult patients admitted for ≥48 h to a large Central Italy hospital for COVID-19 (1 March to 31 May 2020) who had or had not survived at discharge. We included only patients having blood cultures drawn or other inclusion criteria satisfied. Kaplan-Meier survival or Cox regression analyses were performed of 293 COVID-19 patients studied, 46 patients (15.7%) had a hospital-acquired clinically relevant BSI secondary to SARS-CoV-2 infection, accounting for 58 episodes (49 monomicrobial and 9 polymicrobial) in total. Twelve episodes (20.7%) occurred at day 3 of hospital admission. Sixty-nine species were isolated, including Staphylococcus aureus (32.8%), Enterobacterales (20.7%), Enterococcus faecalis (17.2%), Candida (13.8%) and Pseudomonas aeruginosa (10.3%). Of 69 isolates, 27 (39.1%) were multidrug-resistant organisms. Twelve (54.5%) of 22 patients for whom empirical antimicrobial therapy was inappropriate were infected by a multidrug-resistant organism. Of 46 patients, 26 (56.5%) survived and 20 (43.5%) died. Exploring variables for association with in-hospital mortality identified > 75-year age (HR 2.97, 95% CI 1.15-7.68, p = 0.02), septic shock (HR 6.55, 95% CI 2.36-18.23, p < 0.001) and BSI onset ≤ 3 days (HR 4.68, 95% CI 1.40-15.63, p = 0.01) as risk factors independently associated with death. In our hospital, mortality among COVID-19 patients with BSI was high. While continued vigilance against these infections is essential, identification of risk factors for mortality may help to reduce fatal outcomes in patients with COVID-19.

Diagnostic methods based on SARS-CoV-2 antigens detection are a promising alternative to SARS-CoV-2 RNA amplification. We evaluated the automated chemiluminescence-based Lumipulse ® G SARS-CoV-2 Ag assay on saliva samples, using Simplexa™ COVID-19 Direct assay as a reference test. Analytical performance was established on a pool of healthy donors' saliva samples spiked with the 2019-nCoV/Italy-INMI1 isolate, whereas clinical performance was assessed on fresh saliva specimens collected from hospitalized patients with suspect or confirmed COVID-19 diagnosis. The limit of detection (LOD) was 0.65 Log TCID50/mL, corresponding to 18,197 copies/mL of SARS-CoV-2 RNA. Antigen concentrations and SARS-CoV-2 RNA were highly correlated (r = 0.99; p < 0.0001). Substantial agreement (80.3%) and significant correlation (r = -0.675; p = 0.0006) were observed between Lumipulse ® G assay results and Ct values on clinical samples, with 52.4% sensitivity and specificity 94.1%. Sensitivity exceeded 90.0% when calculated on samples with Ct < 25, and specificity was 100% when excluding samples from recovered patients with previous COVID-19 diagnosis. Overall, chemiluminescence-based antigen assay may be reliably applied to saliva samples to identify individuals with high viral loads, more likely to transmit the virus. However, the low positive predictive value in a context of low SARS-CoV-2 prevalence underscores the need for confirmatory testing in SARS-CoV-2 antigen-positive cases.