Positive Percentage Research Articles

Analysis of clinically actionable alterations in baseline tumor versus plasma samples in participants of the TOPAZ-1 study of durvalumab plus gemcitabine and cisplatin in advanced biliary tract cancer.

625 Background: In the TOPAZ-1 study (NCT03875235), durvalumab + gemcitabine and cisplatin (D+GC) significantly improved overall survival (OS) versus placebo + GC (P+GC) in participants (pts) with advanced biliary tract cancer. Updated results have shown a clinically meaningful long-term OS benefit for D+GC versus P+GC at 3 years. This exploratory analysis tested circulating tumor DNA (ctDNA) in plasma samples for blood-based detection of clinically actionable alterations (CAAs) and investigated the potential of this method to guide treatment decisions. Methods: Baseline genomic alterations were retrospectively assessed in evaluable tumor (FMI biomarker evaluable population [BEP], n=441) and plasma samples (GH BEP, n=643) using FoundationOne (Foundation Medicine Inc., Cambridge, MA) and Guardant INFINITY (Guardant Health, Redwood City, CA) assays, respectively. Mutation prevalence and association with outcomes were compared in the FMI BEP and GH BEP. Positive and negative percent agreement of CAAs detected in tumor versus ctDNA were assessed in 419 pts with both tumor and plasma samples (FMI-GH BEP). Results: The FMI BEP and GH BEP represented 64% and 94% of the TOPAZ-1 final analysis set (685 pts), respectively. The relative prevalence and overall mutational landscape detected in plasma ctDNA was consistent with that observed by tumor profiling, with the notable exception that genes harboring complex alterations (e.g. gene amplification, rearrangements, or homozygous deletions) were less frequently detected in ctDNA (e.g. ERBB2 , FGFR2 , and CKDN2A/2B/MTAP ). The most common alterations (>15% prevalence in both BEPs) observed were in TP53 (49%/52%), KRAS (24%/17%), and ARID1A (21%/16%) (in the FMI/GH BEPs, respectively). The relative prevalence of alterations within geographic and anatomic subgroups was similar in tumor versus plasma for most CAAs. The overall percent agreement in CAAs was ≥93%, and negative percent agreement was ≥97%. However, positive percent agreement was notably low for ERBB2 amplification (52%) and FGFR2 fusions (47%). OS hazard ratios for D+GC versus P+GC in the GH BEP were <1 for both CAAs and wild-type, except for ERBB2 amplification, as previously reported in the FMI BEP. Conclusions: The overall concordance and relative prevalence of simple mutations (e.g. single nucleotide variants) were similar using the FoundationOne tumor assay and Guardant INFINITY ctDNA assay, suggesting that plasma ctDNA testing has potential utility in clinical practice. However, negative status by ctDNA for the complex alterations found in FGFR2 and ERBB2 would require further testing of tumors, based on their low detectability in plasma. Clinical trial information: NCT03875235 .

Concordance between human epidermal growth factor receptor 2 (HER2) status by immunohistochemistry (IHC) and next-generation sequencing (NGS) in tissue and blood samples from gastric and gastroesophageal junction cancers (GC/GEJC).

478 Background: An estimated 18% of patients (pts) with GC/GEJC have HER2-positive (HER2+; IHC 3+ or 2+/in situ hybridization–positive [ISH+]) tumors. Although IHC/ISH are the standard testing methods for determining HER2 positivity, NGS can identify HER2 ( ERBB2 ) amplification alongside other biomarkers. This study assessed the concordance of detecting HER2 amplification by tissue/plasma NGS with IHC/ISH HER2 status in pts with GC/GEJC. Methods: Pts were retrospectively identified in the Tempus database. The primary analysis assessed tissue and plasma NGS HER2 copy number (CN) concordance with a HER2+ (IHC 3+ or 2+/ISH+) or HER2− (IHC 0, 1+, or 2+/ISH−) status; NGS samples were collected in ≤30 days of the IHC sample collection date. HER2 amplification was defined as tissue CN ≥8 or plasma log2(CN) ≥0.5; additional amplifications were captured by manual inspection. A secondary analysis assessed the correlation between paired tissue and plasma HER2 log2(CN) in pts with plasma and tissue NGS samples collected ≤30 days apart. The secondary analysis included pts irrespective of IHC sample collection date. Results: Of 1578 pts with GC/GEJC and a curated IHC score evaluated in the primary analysis, 1507 and 305 pts underwent tissue and plasma NGS testing, respectively. Among pts with tissue and plasma NGS results, 902 (59%) and 201 (66%) had primary tumors of the stomach, respectively; all remaining pts had primary tumors of the cardia.According to IHC/ISH, 229 (15%) and 40 (13%) pts were HER2+ among those with tissue and plasma NGS testing, respectively. Of the pts identified as HER2 amplified by tissue and plasma NGS,95% and 92% were HER2+ by IHC/ISH, respectively. Positive percent agreement (PPA) of HER2 amplification by tissue and plasma NGS with IHC/ISH HER2+ status was 70% and 58%, respectively. Concordance rates by PPA, negative PA (NPA), and overall PA (OPA) are summarized in Table. In the secondary analysis, a positive correlation in NGS HER2 log2(CN) was observed in pts with paired tissue and plasma NGS (mutant variant allele frequency >5%; R=0.85; P<2.2e−16; n=307). Conclusions: Based on PPA, we observed moderate concordance between NGS HER2 amplification detection (plasma or tissue) and standard HER2 IHC assessment. NGS is not sufficient to detect all pts with HER2+ GC/GEJC. Further testing is warranted to determine if NGS testing could complement IHC/ISH in identifying pts who may benefit from HER2-directed therapies. IHC 3+ or 2+/ISH+ (HER2+), n IHC 0, 1+, or 2+/ISH− (HER2−), n Total Total 229 1120 Tissue HER2 amplified, n 160 8 168 Tissue HER2 not amplified, n 69 1112 1181 PPA: 69.9% NPA: 99.3% OPA: 94.3% IHC 3+ or 2+/ISH+ (HER2+), n IHC 0, 1+, or 2+/ISH − (HER2 − ), n Total Total 40 233 Plasma HER2 amplified, n 23 2 25 Plasma HER2 not amplified, n 17 231 248 PPA: 57.5% NPA: 99.1% OPA: 93.0%

Clinical relevance of tumor fraction assessment from circulating tumor DNA in metastatic colorectal cancer.

237 Background: Both tissue (TBx) and liquid (LBx) biopsies are indicated for detecting actionable alterations in metastatic colorectal cancer (mCRC). LBx is easier to obtain and often has faster turnaround time, but its informative results depend on the ctDNA tumor fraction (TF) content. We aimed to (1) evaluate the concordance between LBx and TBx in CRC; and (2) determine baseline lab and clinical factors associated with TF. Methods: CRC patients (pts) with both tissue and liquid Foundation Medicine comprehensive genomic profiling within an interval of up to 90 days between samples collection were analyzed. Positive percent agreement (PPA) and negative predictive value (NPV) for RAS (KRAS/NRAS) and BRAFV600E detection were calculated with tissue as reference. Clinical data was obtained by the US-wide de-identified Flatiron Health-Foundation Medicine real-world clinicogenomic CRC database, from ~280 cancer clinics (~800 sites of care) between 1/2011-12/2023. mCRC pts with LBx collected up to 60 days prior to therapy (Tx) start were analyzed for association between TF and baseline factors using logistic regression with TF≥1% as the binary outcome. Baseline factors include age, ECOG, race/ethnicity, line of therapy, practice type, metachronous vs. synchronous disease, tumor side, albumin, alkaline phosphatase (AP), serum creatinine, hemoglobin, lactate dehydrogenase (LDH), neutrophil-to-lymphocyte ratio, opioid and steroid pre-Tx. Foundation Medicine’s ctDNA TF is a composite algorithm prioritizing aneuploidy at higher levels to avoid germline signal and prioritizing variant allele frequency of canonical alterations at lower levels to maximize dynamic range. Results: A total of 402 pts had TBx and LBx (268 [67%] TF≥1% and 134 [33%] TF<1%). Without accounting for TF, the overall PPA for RAS and BRAFV600E was 77% and 83%, and the NPV was 65% and 99%. In LBx with TF≥1%, the PPA for RAS and BRAFV600E was 99% and 100%, and NPV was 98% and 100%. In contrast, for TF<1% samples, the PPA for RAS and BRAFV600E was 37% and 50%, and NPV was 57% and 97%. Of the 633 pts with LBx collected prior to Tx, 474 (75%) had TF≥1%. TF≥1% was independently associated with ECOG 1+ (odds ratio [OR]: 1.58, p=0.038), community oncology care site (OR: 2.58, p=0.002), elevated levels of AP (OR: 3.00, p<0.001) and LDH (OR: 3.97,p=0.002). TF≥1% was less likely to occur in right-side tumors (OR: 0.53, p=0.012) and metachronous disease (OR: 0.48, p<0.001). Conclusions: Approximately 70% of LBx samples from CRC pts have TF≥1%, with near-perfect concordance for RAS/BRAFV600E. For LBx with TF<1%, interpretation of negative results can be challenging due to low tumor shedding, especially for RAS mutations, where 43% may be false negatives and a subsequent TBx will provide more confidence in the negative results. Clinical factors such as right-sided tumors and metachronous disease may help anticipate low TF and guide decisions to pursue TBx.

Comparison of four commercial serological tests for the detection of Leishmania infantum antibodies in dogs.

Early detection and treatment of cases of Leishmania infantum infection are critical in controlling the spread of the disease in dogs. Several serological methods are available to support the diagnosis of canine leishmaniosis. The immunofluorescence antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) are the main tests used by clinicians. High antibody levels are associated with severe parasitism and disease and are diagnostic of clinical leishmaniosis. Conversely, the presence of low antibody levels is not necessarily indicative of disease and may be more difficult to detect by serological tests. The aim of this study was to evaluate and compare the diagnostic performance of four commercially available serological tests, including ELISAs (CIVTEST® CANIS LEISHMANIA short and standard protocols), LEISHMANIA-ELISA DOG®, ELISA/S7® and MegaFLUO®LEISH IFAT, for the detection of specific antibodies against L. infantum antigens in dogs in different states of infection. Canine sera samples from seropositive sick infected dogs (n = 75), seropositive apparently healthy dogs (n = 48), and seronegative apparently healthy dogs from a high endemic area (Cadiz, n = 40) and seronegative apparently healthy dogs from very low endemic area of L. infantum infection (Asturias, n = 40) were classified based on the results of an in-house UAB ELISA as a reference test. The positive percent of agreement (PPA) and negative percent of agreement (NPA) observed for each test were as follows: CIVTEST® standard (93.4 %, 100 %) on 202 samples tested, CIVTEST® short (84.4 %, 100 %) on 202 samples tested, LEISHMANIA-ELISA DOG® (81.8 %, 72.2 %) on 138 samples tested, ELISA/S7® (34.8 %, 45 %) on 195 samples tested and MegaFLUO®LEISH IFAT (100 %, 100 %) on 203 samples tested, respectively. The accuracy was as follows: CIVTEST® standard (0.96), CIVTEST® short (0.91), LEISHMANIA-ELISA DOG® (0.77), ELISA/S7® (0.39), and MegaFLUO®LEISH IFAT (1). The Cohen´s Kappa index (K) from best to worst was: MegaFLUO®LEISH IFAT (K = 1), CIVTEST® standard (K = 0.92), CIVTEST® short (K=0.81), LEISHMANIA-ELISA DOG® (K =0.54), and ELISA/S7® (K=-0.19). Finally, the area under the receiver operating characteristic curve (AUC-ROC), in the ELISAs test, ordered from the maximum to the minimum value was: CIVTEST® short (1), CIVTEST® standard (0.99), LEISHMANIA-ELISA DOG® (0.87), and S7® (0.37). In conclusion, this study demonstrated that the diagnostic performance of the commercially available ELISA tests against L. infantum antigen can vary widely. Moreover, it highlights the fact that CIVTEST® CANIS LEISHMANIA is a reliable test to support the diagnosis of canine leishmaniosis in clinical settings.

Clinical validation of plasma circulating-tumor DNA assay using highly sensitive Safe-SeqS technology for detecting RAS and BRAF V600E in metastatic colorectal cancer.

50 Background: We developed a circulating-tumor DNA (ctDNA) test assay for RAS/BRAF mutations using next-generation sequencing-based Safe-SeqS technology. The clinical performance of detecting RAS and BRAF mutations were evaluated versus approved liquid biopsy and tissue testing, respectively. Methods: Metastatic colorectal cancer (mCRC) patients (pts) were enrolled. The concordance of the RAS mutational status in ctDNA between Safe-SeqS assay and OncoBEAM RAS CRC kit, which is approved as a companion diagnostic in Japan, was evaluated using archival plasma samples. The primary endpoints were positive percent agreement (PPA) and negative percent agreement (NPA). The concordance of the BRAF V600E mutational status between plasma-Safe-SeqS assay and tissue-based testing using MEBGEN RASKET-B kit, which is approved as a companion diagnostic in Japan, was prospectively evaluated. The primary endpoints were sensitivity and specificity of Safe-SeqS assay compared to tissue-based testing. Multivariate analysis using LASSO regression model was performed to evaluate the discordance of BRAF V600E mutational status between Safe-SeqS assay and tissue-based testing. Results: In 125 eligible plasma samples, the PPA and NPA for RAS mutational status in ctDNA were 87.1% (95% confidence interval [CI]: 76.1–94.3) and 95.2% (95% CI: 86.7–99.0), respectively. On the other hand, in 103 eligible pts, the sensitivity and specificity of Safe-SeqS assay for BRAF V600E mutational status were 69.2% (95% CI: 48.2–85.7) and 98.7% (95% CI: 93.0–100.0), respectively. Multivariate analysis revealed the baseline longest diameter and the number of lesions in pts with peritoneal and/or lung metastasis were factors associated with discordance. The sensitivity for BRAF V600E mutations increased to 81.0% (95% CI: 58.1–94.6, fisher’s exact test p =0.02) when the 5 pts with lower tumor burden (peritoneal metastasis with the longest diameter <20 mm, and lung metastasis with the longest diameter <20 mm and <10 lesions, according to the previous report [1] ) were excluded. Conclusions: The clinical validity of the ctDNA assay for detecting RAS/BRAF V600E mutations using Safe-SeqS technology was confirmed in pts with mCRC. Careful attention should be paid for mCRC pts with only peritoneal and/or lung metastases having fewer metastases or smaller diameter lesions. 1. Kagawa Y, et al. Clin Cancer Res 2021.

Performance of a blood-based screening test for the early detection of colorectal cancer.

232 Background: While the clinical benefit of highly sensitive screening tools for colorectal cancer (CRC) has been established, adherence rates remain low. The convenience and familiarity of blood-based testing may reduce disease burden through greater access and compliance to regular and recommended asymptomatic screening. We report on preliminary findings from a minimally invasive blood-based early cancer detection (ECD) screening test for CRC. Methods: Plasma samples from healthy individuals and CRC patients sourced from biobanks were included. A targeted panel composed of differentially methylated genomic regions was developed. Using an independent cohort of plasma samples, a machine-learning model was trained to classify patient samples into cancer-free and CRC based on observed differential methylation patterns in circulating cell-free DNA (cfDNA). The assay performance was calculated based on concordance with clinical diagnosis (CRC, cancer-free). Results: The test set included 440 individuals: 127 with a confirmed CRC diagnosis (47% stage I/II, mean age 65±12 years, male 46%) and 313 without cancer (mean age 56±10 years, male 41%). Overall sensitivity was 95% (95% CI: 88-100%), and specificity was 91% (95% CI: 84-98%). Among patients with stage I disease, sensitivity was 92% overall, and 88% in screen-detected individuals. Sensitivity was 100% (n=14/14) in microsatellite-high tumors and 95% (n=107/113) in microsatellite-stable tumors. Multivariate analysis demonstrated associations between methylation signal and tumor size, cancer stage, and sex. Compared with a tumor-informed, variant-based circulating tumor DNA (ctDNA) test (Signatera), we observed a PPA (positive percent agreement) of 100%. Moreover, the differentially methylated allele fraction (DMAF) across target regions strongly correlated with variant allele frequencies (VAFs) from the tumor-informed test (Spearman’s correlation coefficient, 0.85). Conclusions: Using epigenetic markers specific for CRC, we developed a sensitive and specific blood-based test to detect cfDNA from early-stage and asymptomatic disease. The methylation signal strongly correlated with ctDNA VAFs from a clinically validated tumor-informed test and increased with cancer stage and disease burden. This test could serve as a means to increase patient adherence rates for CRC screening, thereby improving patient outcomes.

Analytical and Clinical Validation of the Plasma-Based Guardant360 CDx Test for Assessing HER2 (ERBB2) Mutation Status in Patients with Non-Small-Cell Lung Cancer for Treatment with Trastuzumab Deruxtecan in DESTINY-Lung01/02.

This study demonstrates the analytical and clinical validity of the approved (United States and Japan) plasma-based Guardant360 companion diagnostic (CDx) test for selecting patients with human epidermal growth factor receptor 2 (HER2 [ERBB2])-mutated (HER2m) non-small-cell lung cancer (NSCLC) for trastuzumab deruxtecan (T-DXd) treatment. Concordance between the Guardant360 CDx test and the plasma-based AVENIO ctDNA Expanded Kit Assay (AVENIO), as well as the tissue-based clinical trial assays (CTAs) was investigated. Clinical utility was assessed by comparing T-DXd clinical efficacy results of patients in DESTINY-Lung01/02 who tested positive for HER2 mutations using the Guardant360 CDx test to benchmark efficacy results from DESTINY-Lung01/02. Finally, concordance between the Guardant360 CDx test and the tissue-based Oncomine Dx Target (ODxT) test was explored. High concordance was observed between the Guardant360 CDx test versus AVENIO [positive percent agreement (PPA), 98.8%; negative percent agreement (NPA), 91.5%] and CTAs (DESTINY-Lung01 Cohort 2-PPA, 91.0%; NPA, 100%; DESTINY-Lung02 arm 1-PPA, 86.0%; NPA, 100%). Confirmed objective response rates were similar in patients with HER2m NSCLC identified by the Guardant360 CDx test and by CTAs. There was a high level of agreement between the Guardant360 CDx test and the ODxT test. The Guardant360 CDx test demonstrated analytical and clinical validity for identifying patients with HER2m NSCLC for T-DXd therapy; results support plasma-based testing when tissue-based testing is not feasible.

Multi-site blinded validation of a deep learning approach for clinical-grade MSI/dMMR detection in colorectal cancer from H&E-stained pathology images.

44 Background: Testing for microsatellite instability (MSI) or mismatch repair deficiency (dMMR) is part of the diagnosis and clinical management of patients with colorectal cancer (CRC). Healthcare services recommend MSI or dMMR testing for all CRC patients to guide therapeutic choices and assist in identifying Lynch Syndrome. However, in clinical practice, high costs and the demand for timely test results, combined with the rising prevalence of CRC and a shrinking pathology workforce, present a barrier to universal adoption. This highlights the need for rapid and affordable alternatives. PANProfiler CRC (PPC) is a deep learning-based solution for detecting MSI/dMMR in CRC tumors that only requires whole slide images (WSIs) of haematoxylin and eosin (H&E)-stained tissue to provide test results. Using only WSIs, PPC offers an efficient alternative to standard testing. This study evaluates PPC's performance in a multi-site blinded setting. Methods: Blinded validation was performed using 3246 WSIs of H&E-stained CRC specimens. PPC provided outputs as "Stable", "Unstable", or "Indeterminate", with "Unstable" indicating dMMR or MSI-High, and "Stable" indicating proficient mismatch repair or non-MSI-High. "Indeterminate" was returned when PPC did not have a definitive result. PPC was evaluated by comparison to standard MSI/dMMR tests. Validation data spanned three cohorts from two sites (Table). St James’s University Hospital (SJUH), Leeds, UK, supplied Cohorts 1 and 2; Cohort 3 was sourced from Wales Cancer Biobank (WCB), UK. Blinded analysis was performed at SJUH. Results: Results are given (Table). PPC demonstrated an overall percent agreement of 93.91%, a positive percent agreement of 92.17%, and a negative percent agreement of 94.15%, returning a definitive result for 88.05% of WSIs. Conclusions: This real-world, multi-site, blinded validation study demonstrates PPC’s remarkable performance, comparable to standard tests for detecting MSI/dMMR in CRC, with high test replacement rates. In the clinical setting, PPC could significantly accelerate testing and enable timely delivery of stratified treatment plans. This accurate and cost-effective diagnostic solution promises to revolutionize MSI/dMMR testing in CRC. Blinded validation results of PPC with confidence intervals (CI) at 95%. Site Cohort Sample Size (Unstable; Stable) Overall Percent Agreement % (CI) Positive Percent Agreement % (CI) Negative Percent Agreement %(CI) Test Replacement Rate % SJUH 1 488 (78; 410) 92.79 (89.86-95.08) 90.16 (79.81-96.30) 93.24(90.11-95.62) 85.25 SJUH 2 2704 (318; 2386) 94.31(93.31-95.21) 92.31(88.48-95.18) 94.57(93.52-95.50) 88.42 WCB 3 54 (11; 43) 84.31 (71.41-92.98) 100.00(71.51-100.00) 80.00(64.35-90.95) 94.44 All 3246 (407; 2839) 93.91 (92.97-94.76) 92.17(88.82-94.78) 94.15(93.16-95.04) 88.05

Concordance between PD-L1 assays 28-8 and SP263 and their respective scoring algorithms in procured upper gastrointestinal adenocarcinoma samples.

479 Background: PD-L1 is being studied as a predictive immunohistochemical (IHC) biomarker for gastroesophageal junction (GEJ), gastric (GC), and esophageal (EAC) adenocarcinomas, and anti-PD-(L)1 therapies are used to treat them. Here, we compare the VENTANA PD-L1 (SP263) IHC assay and its tumor area positivity (TAP) algorithm with the clinically validated Agilent PD-L1 IHC 28-8 pharmDX combined positive score (CPS) algorithm in GEJ, GC, and EAC samples. The degree of concordance between SP263 TAP ≥ 5% and 28-8 CPS ≥ 5 is important for interpreting real-world settings and emerging clinical trial data. Methods: GEJ, GC, and EAC tumor samples procured from company-sponsored clinical trials (NCT02545504, NCT02862535, NCT02864381) and external vendors with informed consent were analyzed 4 ways: SP263 TAP, SP263 CPS, 28-8 TAP, and 28-8 CPS. Readouts were evaluated by a single pathologist and were analyzed with Pearson correlation coefficients. Results: 286 patient samples were analyzed (GEJ, n = 106; GC, n = 88; EAC, n = 92). The agreement among the SP263 TAP ≥ 5% and 28-8 CPS ≥ 5 prevalence is shown in the Table. The overall correlation coefficient between 28-8 CPS and SP263 TAP was high ( R = 0.95; P < 2.2 × 10 −16 ). In addition, when applying the same scoring algorithm to both assays (SP263 TAP vs 28-8 TAP or SP263 CPS vs 28-8 CPS), the correlation coefficients remained high ( R = 0.97; P < 2.2 × 10 −16 ). When both algorithms were applied to the same IHC assay (SP263 TAP vs SP263 CPS or 28-8 TAP vs 28-8 CPS), the correlation coefficients were also high ( R = 0.98; P < 2.2 × 10 −16 ). Conclusions: In this controlled experiment the PD-L1 assays 28-8 and SP263 concordance was high. The scoring algorithms for GEJ, GC, and EAC samples were observed as highly correlated; minor differences were likely driven by the distinct PD-L1 monoclonal antibody clones utilized in the IHC assays. These data suggest that the 2 assays are comparable for evaluating PD-L1 expression at the TAP ≥ 5% and CPS ≥ 5 cutoffs. Clinical trial information: NCT02545504 , NCT02862535 , NCT02864381 . Concordance of SP263 TAP ≥ 5% with 28-8 CPS ≥ 5. Positive percentage agreement: 0.96Negative percentage agreement: 0.95Overall percentage agreement: 0.95 28-8 CPS ≥ 5: Positive 28-8 CPS ≥ 5: Negative SP263 TAP ≥ 5%: Positive 116 9 SP263 TAP ≥ 5%: Negative 5 156

Epidemiology of bacterial respiratory tract infections during the pre-pandemic, COVID-19 pandemic and post-pandemic era: A retrospective study of hospitalized adults in northern Greece between 2018 and 2023.

The COVID-19 pandemic has had impact on global healthcare and respiratory disease patterns. The aim was to evaluate the changes in positive respiratory cultures during the pandemic. A retrospective analysis of respiratory specimens in Papanikolaou Hospital during 2018-2023 was performed. In total, 18,852 samples (12,277 males, 6575 females) of respiratory samples were recorded. During 2018-2019, 684 BAL samples were received, of which 12.3 % were positive. Positive pharyngeal smears constituted 24.8 %, compared to 18.5 % in pandemic years, and 29.6 % in 2023. In PTC, pre-pandemic, positives were 12.3 %, while in 2021 the maximum percentage of positives (21.9 %) was observed. Positive levels remained high in 2023 (18.3 %). Concerning bronchial secretions, there was an increase (55.3 % in 2022 vs 47.0 % in 2019). The predominant bacteria were Acinetobacter spp, Klebsiella spp and Pseudomonas spp. Except for the bronchial secretions, the rest of the respiratory specimens do not exhibit any definite trends.

P-1376. Impact of COVID-19 and Mpox on Inpatient Screening of STIs

Abstract Background Sexually transmitted infections (STIs) continue to pose a considerable public health challenge, with a notable uptick in STI rates over the last few years. Access to care, both inpatient and outpatient, was disrupted during COVID-19 with a subsequent increase in STIs reported in many areas, followed by additional strain on services with the emergence of Mpox in Oregon. Abbreviations: sexually transmitted infection (STI) STIs include chlamydia, gonorrhea, syphilis, human immunodeficiency virus (HIV), and hepatitis C Methods We conducted a retrospective cohort of hospitalized patients at the Oregon Health & Science University (OHSU) Internal Medicine and Family Medicine services to evaluate STI screening and diagnoses (including chlamydia, gonorrhea, HIV, HCV and syphilis) across distinct periods: pre-COVID-19 pandemic (1/1/2018-2/29/2020), during the COVID-19 pandemic (3/1/2020-7/31/2022), and amidst the Mpox surge (8/1/2022-3/1/2023). We calculated the proportion screened, percent positivity by time and compared proportions between our pre-designated periods using the two-sample test of equal proportions. Results During the study period, 5802 patients were admitted to OHSU. Most were White (4705, 81.1%), non-Hispanic (5308, 91.5%) and Female sex (3600, 62.1%). In total, 2151 (37%) were screened for one or more STIs. HIV and syphilis were the most frequently screened STIs at 15.0% and 33.1%. The highest proportion of STI screenings were among those 25-44 years at 40.9% with the highest percent positivity in those 55-64 years at 34.0%. There was a statistically significant increase in proportion screened for all STIs (see Table 2), along with percent positivity for syphilis (P<0.01) between pre-COVID-19 and COVID-19 period, however this trend did not consistently continue after emergence of Mpox. Conclusion The proportion of inpatient STI screening is low for patients admitted to internal medicine and family medicine services with percent positivity by age ranging from 5.9-34.0%. We observed an overall increase in proportion screened during the COVID-19 period. Disclosures All Authors: No reported disclosures

P-360. Impact of a tiered masking intervention on healthcare worker illness and absenteeism during respiratory viral season

Abstract Background High community transmission of respiratory viruses can lead to employee illness, absenteeism, and the risk of spread to both patients and healthcare workers (HCWs). Masking in healthcare settings can mitigate these risks. Our hospital developed a policy to adjust the level of masking required based on current community transmission rates, intensifying or relaxing mandates as necessary. This study aims to evaluate the impact of our tiered masking policy on employee absenteeism. Weekly respiratory viral metrics and employee influenza-like illness for 2022 Methods Retrospective review of employee health data, public health respiratory virus surveillance metrics, and infection prevention masking requirements at a 151-bed safety-net hospital. Data from September to December 2022, when universal masking was in place, and September to December 2023, when our new masking policy was implemented was compared. In 2023, four masking levels were created: red always requires masks for HCWs and patients; orange requires them for HCWs; yellow requires masks for HCWs during patient contact; both orange and yellow recommends but doesn’t require masks for patients; green recommends but does not require masks for HCW and patients. Weekly public health respiratory surveillance data, masking requirements and employees reporting influenza-like illness (ILI) were tabulated and compared before and after our new masking policy. Weekly respiratory viral metrics and employee influenza-like illness for 2023 Results A total of 66 HCWs reported ILI during the 2022 respiratory viral season compared to 44 in 2023, a 33% reduction. Weekly respiratory viral metrics and HCW ILI is plotted in Figure 1 for 2022 and Figure 2 for 2023. When mask levels were raised to orange on October 6, HCW ILI slightly decreased. Masking changed to yellow on October 22 and correlated with a subsequent increase in ILI. Escalation to red on December 10 was followed by a decline in ILI. Conclusion A tiered masking policy in response to fluctuating community transmission appears to be an effective strategy in managing HCW absenteeism due to ILI. Despite higher percent positivity from multiple viruses, a 33% decrease in ILI occurred after implementation of the tiered masking policy, as compared the prior universal masking policy. A tiered masking policy based on percent positivity may be more effective than universal masking. Disclosures All Authors: No reported disclosures

P-2162. Utility of Metagenomic Sequencing of Microbial Plasma Cell-Free DNA with Different Testing Strategies

Abstract Background While promising, optimal use criteria for metagenomic next-generation sequencing of microbial cell-free DNA (mcfDNA) to diagnose infectious diseases (ID) are not yet established. We examined this question by comparing the real-world clinical impact of mcfDNA testing on ID management in two academic medical systems with similar patient case mix but differing test ordering practices. Both institutions used the Karius assay (Redwood City, CA). Intermountain Health (IH) used an expedited pathway for specimen shipping and restricted test ordering to ID physicians with suggested use criteria. By contrast, at University of Utah Health (UU), testing occurred without systemic education and lab processing was slower. Demographics Methods We conducted a retrospective study of all mcfDNA testing at IH and UU from April 2022 – June 2023. We compared indications, incidence (per ID consult), and turn-around-time (TAT) for testing at each institution. Clinical impact of mcfDNA testing was evaluated for each institution using published criteria (Table 2). Clinical Impact Criteria UU, University of Utah; IH, Intermountain Health; mcfDNA, metagenomic next generation sequencing of plasma microbial cell-free DNA. Criteria adapted from Hogan CA, Yang S, Garner OB, et al. Clinical Impact of Metagenomic Next-Generation Sequencing of Plasma Cell-Free DNA for the Diagnosis of Infectious Diseases: A Multicenter Retrospective Cohort Study. Clin Infect Dis. 2021;72(2):239-245. doi:10.1093/cid/ciaa035 Results During the study period 123 tests were ordered (106 at IH and 17 at UU) (Table 1). Most were obtained via ID consult (118/122). Testing incidence at IH was 3.24/100 ID consults and at UU was 0.3/100 ID consults. At least one organism was identified in 44% (47/106) of tests at IH and 70% (12/17) at UU (p=0.06). Median TAT was 2 days at IH vs 8 days at UU (p < 0.05, Figure 1). Clinical impact varied by criterion between institutions (Table 2). Overall, mcfDNA testing had no discernible clinical impact in 64% (68/106) of IH cases and 76% (13/17) UU cases, (p= 0.41). A positive change in management resulted after 14% (15/106) of tests at IH and 23% (4/17) of tests at UU, p=0.46. Ordering and clinical impact varied by clinical indication (Table 3). Tests by clinical indication UU, University of Utah; IH, Intermountain Health Conclusion At UU, test ordering was more selective but yielded higher test positivity. At IH, where ordering practices were slightly more liberal, a clinical diagnosis was more often confirmed by mcfDNA results. Despite differences in testing availability and strategy, positive impact percentages were similar between institutions. At IH, mcfDNA results positively changed management for more patients, at the cost of more tests sent overall. Further research is needed to further clarify best mcfDNA testing strategies. Turnaround time UU, University of Utah; IH, Intermountain Health Disclosures Bert K. Lopansri, MD, D(ABMM), FIDSA, Cepheid: Grant/Research Support|Seegene: Advisor/Consultant

P-2081. Prototype Assay for Detection of Carbapenem-Resistant Acinetobacter baumannii Complex (CRABC)

Abstract Background Carbapenem-resistant Acinetobacter baumannii complex (CRABC) is recognized as an urgent/critical antimicrobial resistance (AMR) threat, causing hospital-acquired and ventilator-associated pneumonia (HAP/VAP) and bacteremia. Due to limited treatment options, there is an urgent need for novel targeted antibiotics against CRABC. As these drugs become available, their timely and appropriate use will require the rapid identification of patients with CRABC infection. We evaluated the performance of a prototype assay for the rapid detection of CRABC directly from bronchoalveolar lavage (BAL), sputum/endotracheal aspirate (S/ETA), and positive blood culture (PBC) samples. Methods A prototype assay was designed for the qualitative detection of A. baumannii complex (ABC) harboring the most prevalent associated carbapenemase genes (blaOXA-23, blaOXA-24, blaOXA-58, and/or blaNDM). The prototype assay was developed for use on the high-throughput cobas® 5800/6800/8800 Systems with a defined oligo pool and positive control. A total of 452 BAL, 681 S/ETA, and 392 PBC samples (including prospective, archived, and contrived specimens) were tested by the prototype assay and compared with routine microbiology for ABC detection and the Unyvero Hospitalized Pneumonia or BCU panel for carbapenemase detection. Discrepant resolution was performed using the BioFire® FilmArray® Pneumonia or BCID Panel and/or the Streck ARM-D® Kit, OXA assay, depending on the type of discrepancy. Results After resolution of discrepant results, the overall percentage agreement, positive percent agreement, and negative percent agreement between the prototype assay and the comparator methods (culture/nucleic acid amplification tests [NAATs]) was 97.1%, 98.5%, and 96.5%, respectively, for BAL; 99.3%, 100%, and 99.1%, respectively, for S/ETA; and 99.2%, 100%, and 99.1%, respectively, for PBC. Conclusion This prototype assay exhibited a high percentage agreement for the detection of CRABC in BAL, S/ETA, and PBC samples when compared to culture/NAATs. A future assay based on this prototype could be evaluated in a clinical trial to establish its utility in facilitating timely and appropriate antibiotic selection for patients with HAP/VAP or bacteremia caused by CRABC. Disclosures Brian Lee, MD, GE Healthcare: Stocks/Bonds (Public Company)|Invitae: Stocks/Bonds (Public Company)|Merck: Stocks/Bonds (Public Company)|OPKO Health: Stocks/Bonds (Public Company)|Roche Diagnostics Solutions: Employment|Roche Diagnostics Solutions: Stocks/Bonds (Public Company)|Spero Therapeutics: Stocks/Bonds (Public Company) Natacha Martins Sorenson, PhD, Roche Diagnostics Solutions: Employment|Roche Diagnostics Solutions: Stocks/Bonds (Public Company) Hai Nguyen, n/a, Roche Diagnostics Solutions: Employment Arrash Moghaddasi, n/a, Roche Diagnostics Solutions: Employment Aishwarya Sathish, n/a, Roche Diagnostics Solutions: Employment Patrick Lin, n/a, Roche Diagnostics Solutions: Employment Kyle C. Cady, PhD, Roche Diagnostics Solutions: Employment|Roche Diagnostics Solutions: Stocks/Bonds (Public Company)

378. Visby Medical Women’s Sexual Health Test: An Over-the-Counter Molecular Diagnostics Solution to STI Testing *The Visby Medical Women’s Sexual Health test has not been cleared or approved by the FDA for in vitro diagnostic use

Abstract Background Visby Medical has developed a palm-sized, single-use, PCR platform that combines sample preparation, nucleic acid amplification and detection into an integrated device (Figure 1) that delivers results within 30 minutes. This platform was used to develop the Women’s Sexual Health Test for the detection of DNA from pathogens Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis. Testing instructions were designed for lay users. A smartphone application was developed to guide users through the testing process with videos, including digital interpretation of the results using the smartphone camera.Table 1:The data summary table below shows the aggregate results from testing conducted by lay users across all thirteen sites to assess the performance of the Visby test relative to the comparator tests.PPA=Positive Percent Agreement; NPA=Negative Percent Agreement.TP=true positive; FP=false positive; TN=true negative; FN=false negative. Methods Performance characteristics of an investigational use only (IUO) version of the test were evaluated using clinical and analytical studies. The clinical study was carried out at 13 geographically diverse sites. A total of 2203 female subjects (14+ years old) were included in the performance evaluation. Subjects followed the printed and/or the digital instructions to self-collect a vaginal swab, place it in the Visby collection media, conduct the test, and interpret the results. Three additional swabs were collected from each subject for comparator testing. The analytical studies included limit of detection (LoD), inclusivity, cross reactivity, interference and flex studies. Human factors usability studies were also conducted.Figure 1:Visby Medical Women’s Sexual Health Test Results The Visby Medical Women’s Sexual Health Test* had a sensitivity of at least 95.0% and a specificity of at least 98.0% (see Table 1) for each of the three target organisms. Human Factors usability studies substantiated that a lay user could successfully perform the testing, and the analytical studies demonstrated that the test is sensitive (LoD, inclusivity), specific (cross-reactivity), and robust (interference and flex testing). Conclusion The performance characteristics of the Visby Medical Women’s Sexual Health Test when used by lay users in an at-home setting was equivalent to the results from high complexity assays, conducted in centralized laboratories by trained laboratory professionals. This test could potentially be the first PCR test cleared for at-home use with the ability to offer highly accurate testing for sexually transmitted infections (STIs) and remove barriers to promptly and accurately treating STIs. Disclosures Jennifer Albrecht, PhD, Visby Medical, Inc: Employee Brittany Owings, n/a, Visby Medical: Employee Paul Dentinger, PhD, Visby Medical: Stocks/Bonds (Private Company) Gary Schoolnik, MD, Visby Medical: I am the Chief Medical Officer of Visby Medical|Visby Medical: Stocks/Bonds (Private Company) Beth Lingenfelter, n/a, Visby Medical: Employee

P-2167. "Clinical Utility of Cell-free Next-generation Sequencing, Single Center Experience"

Abstract Background Timely diagnosis of infectious diseases is crucial, but it can be challenging. The literature about impact of Karius in clinical care is still insufficient. Overall, Karius has been very effective on selected patients but the clinical impact is difficult to assess due to absence of gold standard comparator. In addition, a negative result doesn’t always mean a “true negative” but means that the organism’s DNA wasn’t detected. Also, Identification of non-relevant results has been reported. Methods This is a retrospective cohort study of patients who received a Karius testing between January 2018 until March 2023 at Lifespan institution in Rhode Island. Clinical and demographic data was abstracted from the electronic medical record. Quality checks were applied to the data to remove duplicates and errors. Descriptive statistics were reported using Microsoft excel. Clinical impact was abritrated after review of each case by three independent investigators. We predefined clinical impact categories criteria based on criteria from previous studies. Each case was reviewed by each investigator independently and disparities were solved via consensus and discussions. inter-rater reliability was assessed using Cohen's Kappa coefficient. Results Overall, 65 patient had Karius testing and included 35 male and 30 female. 30 patients were immunocompromised and 16 of them had hematologic malignancy. Mean age was 46.7 and the study included 12 pediatric and 53 adult patient. The most common infectious syndrome was Pneumonia/pulmonary followed by osteoarticular and endovascular. Management was changed in 11/65 patients. The Karius testing had positive impact in 12 patients, negative impact in 1 patient and no impact in 52 patients. Positive percent agreement with conventional testing was 0.49 and negative percent agreement was 0.83 Conclusion Cell-free next-generation sequencing has a valuable role in diagnosis and management of infections in challenging cases especially in patients with high pretest probability. Further research is needed to define the population who would benefit most from it. Disclosures All Authors: No reported disclosures

Glucometer versus analyzer: comparable results with negligible clinical risk

This study assessed the reliability of Roche Accu-Chek Inform II glucometers in a real-world setting. A retrospective analysis was conducted on 6,695 paired results. Capillary samples were tested using Roche Accu-Chek Inform II glucometers, while venous samples were analyzed using Roche Cobas c503/702 analyzers. Compliance was assessed using modified criteria based on the ISO 15197 guideline and the CLSI EP09-A3 guideline using Passing-Bablok regression analysis, Bland-Altman plots, and Surveillance Error Grid (SEG) analysis. The overall compliance of glucometer results within ±15% or 0.83 mmol/L (15 mg/dL) of the reference method was 81.5%, below the acceptance criterion of 94.6%. SEG analysis showed that 90.3% of the paired results fell within the No-risk zone, with less than 0.001% in the moderate/lower-risk zone. The Emergency Department results indicated 87.8% overall compliance and 92.2% of pairs falling in the No-risk zone. Based on the regression analysis, the glucometer results showed a positive constant bias of nearly 0.33 mmol/L (6 mg/dL). The Bland-Altman plots showed a positive mean difference of 0.43 mmol/L for results ≤5.55 mmol/L (≤100 mg/dL) and a positive mean percentage difference of 3.77% for results >5.55 mmol/L (>100 mg/dL), within the permissible deviation. The compliance values ranged from 76.0% to 90.3% for clinical concentration groups, with the highest compliance found between >16.65–22.20 mmol/L (>300–400 mg/dL). The Accu-Chek Inform II glucometers demonstrated in real-world reliability, with most results falling within acceptable risk categories. However, compliance still needs improvement, so manufacturers should assess opportunities for advancement.

Comparison of AccuPower Diarrhea V1&V2 RT-PCR to a Chromatographic Immunoassay for Detecting Viral Pathogens from Human Diarrheal Stool Specimens

Viruses are a frequent cause of self-limited diarrhea, with more severe outcomes in immunocompromised patients. This study aimed to compare the performance of Real-Time RT-PCR to chromatographic immunoassays (CIAs) for detecting the major gastrointestinal viruses in human stool. This study was conducted at the University Hospital of Split, Croatia, from October 2023 to May 2024. Stool samples were simultaneously analyzed with CIA (Acro Biotech Rotavirus and Adenovirus Combo Rapid Test Cassette, USA and JusChek Norovirus Rapid Test Cassette, China) and Real-Time RT-PCR (AccuPower Diarrhea V1&V2 Real-Time RT-PCR, Bioneer, Republic of Korea), according to the manufacturers’ instructions. Positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement (OPA) were calculated. For norovirus, CIA had a low PPA (25%), indicating that it missed 75% of norovirus-positive cases identified by RT-PCR. Adenovirus detection by CIA showed poor agreement with RT-PCR (PPA 0%; NPA 100%). Rotavirus detection presented a relatively better performance with CIA (PPA 90.9% and OPA 84.13%). However, the presence of false positives (15.8%) highlights the need for confirmatory RT-PCR testing. One specimen was sapovirus-RT-PCR-positive, marking the first documented case from human specimens in Croatia. Although CIA provided rapid results, limitations regarding reliability highlight the value of RT-PCR, particularly in the case of ambiguous clinical cases with negative antigenic test results and newly emerged viruses. A two-step diagnostic approach, with initial CIA screening followed by confirmatory RT-PCR, could balance cost-effectiveness with diagnostic accuracy.

Coproantigen detection and molecular identification of Cryptosporidium species among newborn and adult farm animals

Cryptosporidium sp. is an obligatory intracellular apicomplexan protozoan parasite that causes a disease called cryptosporidiosis with substantial veterinary and medical importance. Therefore, this study aimed to evaluate an early diagnosis of cryptosporidiosis using the anti-Cryptosporidium parvum oocyst immunoglobulin IgG polyclonal antibodies (anti-C. parvum IgG PAbs)-based sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of Cryptosporidium oocyst antigens in fecal samples of farm animals in Egypt. Further molecular identification and sequencing were performed for the detected isolates. Eight hundred and twenty fecal samples of farm animals; 102 buffalo calves, 120 cattle calves, 100 lambs and 98 goat kids, 80 buffaloes, 60 cattle, 160 sheep and 100 goats, collected from different small-scale farms and local holders were examined for cryptosporidiosis by Modified Ziehl-Neelsen (MZN) technique. The percentage of positivity was 45.1%, 50%, 20%, 18.4%, 31.25%, 38.3%, 18.8%, and 11% in buffalo calves, cattle calves, lambs, goat kids, adult buffaloes, adult cattle, sheep, and goats, respectively. Molecular identification of Cryptosporidium samples was performed based on COWP gene, revealing the isolates: GenBank: OQ121955.1, OR029973.1 and PP316107.1 which were identical to the C. parvum and GenBank: PP316108.1 and OR029972.1 which were identical to C. hominis and C. andersoni, respectively. Then, C. parvum oocysts were used for preparation of antigens and rabbit immunization. Anti-C. parvum IgG PAbs were purified and characterized by SDS-PAGE and then labeled with horseradish peroxidase (HRP). Anti-C. parvum IgG PAbs in-house sandwich ELISA was prepared, then tested this ELISA on 820 samples and compared results with MZN microscopical examination and a commercial sandwich ELISA kit. In this study, in-house sandwich ELISA scored higher sensitivity of 98%, 100% specificity, validity 99% and relative agreement 98.6% than (92%, 90%, 91% and 91.4%) of MZN and (96%, 95%, 95.5% and 95.7%) of coproantigen commercial sandwich ELISA kit, respectively. Moreover, we used PCR to evaluate the positivity of in-house sandwich ELISA results, and the total PCR positive samples were 263 out of 268 sandwich ELISA positive samples (98.13%). In conclusion, the prepared Anti-C. parvum IgG PAbs based sandwich ELISA offered a simple and accurate diagnostic method for cryptosporidiosis in the fecal samples of different species of farm animals in Egypt with high sensitivity (98%) and specificity (100%). Further studies on this Anti-C. parvum IgG PAbs may help also in the protection against cryptosporidiosis.

Clinical Evaluation of a Rapid Reciprocal-Flow PCR Assay and Real-Time PCR Assay with Quenching Probe for Detection of Mycobacterium tuberculosis Complex.

This study investigated the diagnostic efficiencies of two assays for the detection of Mycobacterium tuberculosis complex: (1) the reciprocal-flow real-time polymerase chain reaction (PCR)-based GeneSoC assay and (2) the real-time PCR based GENECUBE MTB assay with quenching probe. These assays were performed for stored clinical samples and results were compared with the confirmed results based on culture and COBAS TaqMan MTB assay. A total of 53 samples (20 confirmed positives and 33 confirmed negatives) were included in the performance analysis. The GeneSoC assay showed concordance in all 53 samples, regardless of specimen type, while the GENECUBE MTB assay showed concordance in 19 of the 20 confirmed positive samples and all 33 confirmed negative samples. The overall agreement was 100.0% for the GeneSoC assay and 98.1% for the GENECUBE MTB assay. Positive and negative percent agreements were 100.0% each for the GeneSoC assay and 95.0% and 100.0%, respectively, for the GENECUBE MTB assay. Both the GeneSoC and GENECUBE MTB assays exhibited excellent performance in detecting M. tuberculosis complex. The GeneSoC assay is useful for independent assays of individual samples, whereas the GENECUBE MTB assay is suitable for batch assays of multiple samples.