Test Turnaround Time Research Articles

Improving Turnaround Times for Routine Antimicrobial Sensitivity Testing Following European Committee on Antimicrobial Susceptibility Testing Methodology in Patients with Bacteraemia

Background/Objectives: Bacteraemia can be fatal without antibiotic intervention. Antibiotic Susceptibility Testing (AST) provides the necessary information for targeted antibiotic therapy; however, the traditional method using disc diffusion can take over two days from a positive blood culture. Inappropriate empiric therapy is associated with increased mortality and increased antibiotic resistance, highlighting the need for more rapid turnaround times for AST. By making changes to an established method, turnaround times can be reduced. Methods: Eighty-two patient positive blood culture samples were collected from January to April 2022, representing the range of common bacteria causing sepsis. This followed the normal methodology in the laboratory of inoculating agar from positive blood cultures in preparation for European Committee on Antimicrobial Susceptibility Testing (EUCAST) disc diffusion AST method. EUCAST methodology outlines that disc diffusion should be performed on isolates from an overnight culture of 16–24 h. This study looked at comparing disc diffusion results from cultures with 6 h of incubation to those with incubation times of 24 h, after organism identification by MALDI-ToF. Results from 6-h and 24-h cultures were compared by disc zone sizes and by interpreted susceptibility reading following EUCAST guidelines of sensitive, resistant, susceptible with increased exposure, or an area of technical uncertainty. Results: A total of 99.65% interpreted susceptibility readings matched across all organisms to all relevant antibiotics, with an average zone size difference of 1.08 mm between results from 6 h versus 24 h cultures. Conclusions: This method offers a non-automated way of using the traditional disc diffusion method, reducing turnaround times while still producing reliable and accurate results. This would mean validated ASTs can be set up in the same day as a blood culture flags positive rather than waiting for a longer culture. As this method is widely used within the laboratory already, it would mean that additional training is not required, as the process is the same, and only incubation time varies. This would positively impact patient outlook due to the shorter use of empiric therapy, and benefit antimicrobial stewardship (AMS).

Optimization of Turnaround Time and Insufficient Quantity Frequency for Molecular Testing on FFPE Material

Abstract Introduction/Objective With clinical decisions relying progressively more on molecular data extracted from formalin- fixed, paraffin-embedded (FFPE) tissue, it is pivotal to optimize the use of tissue for diagnostic and prognostic purposes. This optimization can be reached by identifying actionable instances that can decrease the turnaround time (TAT) and the rate of “quantity not sufficient” (QNS) results Methods/Case Report From January 2023 to December 2023, the total TAT from the time a molecular study is ordered to its delivery to the molecular lab at LL200A (Tomsich Laboratories), as well as the TAT from the time between the ordering pathologist circling the region of interest to its delivery to LL200A were broken down by subspecialty and site. In addition, the QNS results of Caris genomic profiling were evaluated considering subspecialty and site, amount of material present in the initial sample, and number of levels cut for other ancillary testing from the same material. Results (if a Case Study enter NA) 83% of subspecialties took longer than 48 hours to have material ordered for molecular studies delivered to LL200A. The median TAT for all specialties was 58 hours (average: 71.6 hours). When examining TAT from circling to LL200A, the median time was 19 hours (average: 28.4 hours). Regarding the QNS rate of the Caris assay, most QNS cases were gastrointestinal tract specimens (27%) or specimens obtained from either CT-guided core needle biopsies (30%) or endoscopic biopsies (32%). The longest dimension of QNS cases ranged from 7.5 cm to 0.1 cm (median: 1.3cm)—the average number of slides ordered before Caris ranged from 1 to 52 (median: 12). Conclusion The initial results pointed towards the time from circling to LL200A as one actionable item that could improve the TAT of molecular tests. Possible solutions include enacting a dedicated tumor circling service. In addition, to decrease the rate of Caris QNS results, we propose working together with proceduralists to increase biopsy material yield and to work with pathologists to preserve limited specimens for molecular studies.

Improving dihydropyrimidine dehydrogenase genotyping (DPYD) prior to initiation of fluoropyrimidine-based chemotherapy at a community-based hospital: A quality improvement initiative.

347 Background: Dihydropyrimidine dehydrogenase gene (DPYD) testing is crucial in preventing toxicities from fluoropyrimidine chemotherapy. Despite benefits and established guidelines, its adoption within healthcare organizations remains inconsistent and suboptimal. We implemented a quality improvement project to improve DPYD testing prior to fluoropyrimidine chemotherapy at a community-based hospital. We aimed to increase DPYD testing for patients receiving fluoropyrimidine-based chemotherapy from 65% to 95% between July 2023 to July 2024. Balancing measures included DPYD testing turnaround time. Methods: For baseline diagnostics, manual chart review was performed on 126 patients to identify frequency of DPYD testing, and turnaround time (TAT) between May 1, 2023 to August 4, 2023. Patients were excluded if they received fluoropyrimidine-based chemotherapy prior to May 1, 2023. A multidisciplinary team including medical oncology residents and staff, pharmacists, nurses, and clinical informatics specialists worked jointly to identify and address barriers to implementation. Barriers identified included lack of established workflows and awareness of the importance of testing. Diagnostic tools utilized included Ishikawa diagrams and process mapping. The team used Plan-Do-Study-Act (PDSA) cycles to address barriers. A p-chart was used to analyze the results. Results: The baseline DPYD testing rate was 65%. Barriers identified via multidisciplinary sessions included lack of established workflow, awareness and importance of testing. PDSA cycles included education sessions to breast medical oncologists and medical oncology nurses, and development of a best-practice advisory in the electronic medical record. The percentage of eligible patients receiving DPYD testing increased from 65% to 95.8% in the total population. For breast cancer patients, the rate increased from 16.7% to 100%. For gastrointestinal cancer patients, the rate increased from 56.1% to 94.2%. Sustainability was demonstrated two months post-intervention with TAT stable between 7-8 days. Conclusions: Through PDSA cycles, we improved the frequency of DPYD testing for patients receiving fluoropyrimidine chemotherapy from 65% to 95.8%. Future work will assess changes in chemotherapy prescribing behavior in the setting of variant DPYD results, and outcomes of patients with variant DPYD testing.

The American Cancer Society National Lung Cancer Roundtable strategic plan: Methods for improving turnaround time of comprehensive biomarker testing in non-small cell lung cancer.

Comprehensive biomarker testing for patients with non-small cell lung cancer is critical for selecting appropriate targeted therapy or immunotherapy. Ensuring timely ordering, processing, and reporting is key to optimizing patient outcomes. However, various factors can prevent or delay patients from being offered the option of treatment selection based on comprehensive biomarker testing. These factors include problems with access to testing, tissue adequacy, turnaround time, and health insurance coverage and billing practices. Turnaround time depends on several logistical and tissue handling factors, which involve institutional policies, processes, resources, testing methodology, and testing algorithms that vary across different practices. In this article, the authors identify key factors that prolong biomarker testing turnaround time, propose strategies to reduce it, and present a process map to aid physicians and key organizational stakeholders in improving testing efficiency.

Lean methodology in health care practice: an example of application for clinical laboratory urinalysis processing.

Improving quality and laboratory testing turnaround time (TAT) is a constant challenge for a clinical laboratory. The formulas that describe the best way to manage these goals are outlined in International Organization for Standardization standards. According to standards, improvement must be timely and continuous. Lean methodology is a tool to meet this requirement. One of the fundamental elements of Lean is a systematic approach to process improvement by removing waste to create value for the end-user (eg, patient) of the service. This methodology can be adapted in resource-limited settings. The aim of this study was to test the application of Lean methodology in urinalysis. Lean has various collections of tools and concepts. We applied the most useful for the clinical laboratory: Gemba walk, Takt time, cycle time, and value-stream mapping. Finally, we created and approved workplace standards to improve the performance of urinalysis. We compared the TATs of urinalysis tests before optimization, immediately after, and long after (~5 months). We found that TATs had significantly shortened. The TATs of emergency (STAT) urine tests immediately after optimization improved: automated microscopy to 16% (P =.194), fully automated test-strip to 23% (P = .0172), and standardized urine sediment examination to 20% (P =.0048). The TATs of routine urine tests also improved immediately after optimization: automated microscopy to 18% (P <.0001), fully automated test-strip to 11% (P =.0025), and standardized urine sediment examination to 18% (P =.0011). After 5 months of Lean application within the urinalysis laboratory, TATs of routine urine tests remained improved; however, the improvement of STAT urine test TATs dropped to approximately 4%. The application of the Lean methodology shows significant improvement in TATs of processes in our laboratory.

Evaluation of Turnaround Times for Antigen Testing in Hospitalized Patients With Histoplasmosis and Blastomycosis.

Review of histoplasmosis and blastomycosis antigen testing for 39 patients hospitalized with these diseases found that there were significantly longer turnaround times between the time of specimen collection and receipt of positive test results among those patients who had worse outcomes.

Impact of a University-Led COVID-19 Case Investigation and Contact Tracing Program.

The coronavirus disease 2019 (COVID-19) pandemic devastated societies and economies worldwide. Given the major disruptions to higher education, reflection on university responses to the COVID-19 pandemic may provide insights for future outbreaks. Here, we describe the epidemiology of COVID-19 on the Emory University campus during the 2020-2021 academic year and provide an evaluation of the performance of a university-led program with the purpose of describing the effectiveness of efforts to augment the public health authority's case investigation and contact tracing efforts during a public health emergency. Evaluation of a case investigation and contact tracing program regarding operations, timeliness, and performance. We analyzed quality metrics to determine the proportion of cases and contacts interviewed and the time to completion of each step from case diagnosis to testing of contacts. During the 2020-2021 academic year, 1267 COVID-19 cases among Emory students, faculty, and staff were confirmed by polymerase chain reaction, with 1132 reported close contacts. Among cases, the median test turnaround time was 1 day (interquartile range: 1, 2). Among both cases and close contacts, 98% were successfully interviewed. The team called a majority of cases on the same day as their test result was reported to the program (87%; n =1052). Almost all (98%; n =1247) cases completed isolation or were advised to isolate during the review period. Close to half (46%; n =513) of contacts interviewed began quarantine before their interview. Among close contacts interviewed, 13% (n=145) subsequently converted to an index case. The impact and performance of Emory's program may provide useful and actionable data for future university-led infectious disease outbreak response programs. The program structure, performance metrics, and information collected via interviews provide practical implications and an organized structure to guide other programs during future outbreaks.

Application of cell-free DNA fungal polymerase chain reaction for invasive fungal disease evaluation in pediatric oncology and stem cell transplant patients.

Molecular diagnostics may enable early, noninvasive detection of invasive fungal disease (IFD) in immunocompromised patients. Cell-free deoxyribonucleic acid (cfDNA) fungal polymerase chain reaction (PCR) assays were recently incorporated into institutional prolonged febrile neutropenia pathways. We aimed to evaluate the performance of plasma cfDNA PCR panels (mold and Candida panels) in pediatric oncology and hematopoietic stem cell transplant (HSCT) patients with clinical concern for IFD. This single-center, observational study assessed plasma cfDNA fungal PCR performance for noninvasive IFD detection in hospitalized pediatric oncology and HSCT patients. The primary outcome was IFD diagnosis per published consensus definitions within 1month. Positive and negative agreement between plasma cfDNA fungal PCR and consensus definitions were calculated. We also described test turnaround time and patient survival. From October 2021 to 2022, 54 patients underwent 60 evaluations with 11 proven/probable IFD cases. Comparing plasma cfDNA fungal PCRs to consensus definitions for proven/probable IFD, there was 73% positive agreement and 96% negative agreement. Two proven/probable cases with negative PCRs were caused by organisms not included on either panel. Median time to cfDNA fungal PCR result was 35hours (interquartile range: 19-69) in eight proven/probable cases detected by cfDNA fungal PCR. There were 17 deaths among 54 patients, and IFD contributed to 45% of deaths in patients with proven/probable IFD. Plasma cfDNA fungal PCRs detected relevant molds or yeast in most cases classified as proven/probable IFD. However, this targeted approach missed some cases. More studies are required to determine optimal utilization of molecular diagnostics in pediatric patients.

Cross-sectional evaluation of online direct-to-public calprotectin testing

Objective Why about a quarter of patients with inflammatory bowel disease (IBD) suffer symptoms for more than a year before their diagnosis made is unclear. Low public awareness, embarrassment and...

Half of oncologists fail to use ordered NGS results to guide their first-line treatment decision in advanced NSCLC: A retrospective study in a community-based integrated healthcare system

PurposeNext generation sequencing (NGS) testing is used to identify driver mutation(s) in non-small cell lung cancer (NSCLC) that are amenable to targeted therapy, resulting in superior outcomes and improved tolerability. We characterized how clinicians in a large integrated healthcare system utilized NGS testing to inform first line treatment decisions in patients with stage IV NSCLC shortly after diagnosis. MethodsWe conducted a cross-sectional study of 964 patients within an integrated healthcare system, Kaiser Permanente Southern California (KPSC), who were diagnosed with stage IV NSCLC and completed NGS testing (Strata Oncology) between May 2019 to June 2021. Treatment start dates were used to divide patients into those who started treatment before or after NGS results, or those who did not receive treatment after NGS results. Patients harboring alterations in seven genes (EGFR, ALK, ROS-1, BRAF, KRAS, RET, and MET) were considered candidates for targeted first line therapy. ResultsFirst line treatment was initiated in half (52 %; n = 284) of all treated patients prior to NGS results. Just under half (48 %; n = 137) of these patients were found to have a targetable mutation by NGS, of whom 59 % received first line chemotherapy and/or immunotherapy, rather than targeted therapy. Nearly 27 % of the sample never received treatment, of which 31 % had a targetable mutation, and may have been candidates for targeted therapy. Not undergoing first line treatment was correlated with older age, higher comorbidity index, smoking history, and the lack of an identifiable driver mutation. ConclusionNGS tests results were not exclusively used to inform first line treatment decisions in most patients with stage IV NSCLC, and most patients with a targetable mutation were not treated with targeted therapy. Possible explanations include lengthy turnaround times for NGS testing and the availability of timelier but less accurate single gene testing.

Reducing turnaround time for routine outpatient biochemical tests through Lean Six Sigma: A case study in China.

Routine clinical biochemistry tests are crucial for clinical diagnostics and play a key role in enhancing outpatient turnover efficiency and patient satisfaction. This study aimed to implement Lean Six Sigma in the biochemistry laboratory of a hospital in China to improve efficiency and quality by reducing turnaround time. The study was conducted from January to December 2023, using the DMAIC (Define, Measure, Analyze, Improve, Control) framework, and employed tools such as the voice of the customer, Value Stream Mapping, '5 whys' technique, Nominal Group Technique, and Pareto chart. The turnaround time for outpatient routine clinical biochemistry tests was reduced from 139 min to 58 min (p < 0.05), effectively increasing both patient and physician satisfaction. Lean Six Sigma aimed to reduce the turnaround time for biochemical tests have significant advantages. This study confirms the effectiveness of Lean Six Sigma in a Chinese clinical laboratory setting and provides guidance for optimizing efficiency in global clinical laboratories with limited implementation experience, constrained technical and equipment resources, and high demand for medical diagnostics.

‘Plasma first’ approach for detecting epidermal growth factor receptor mutation in advanced non-small cell lung carcinoma

IntroductionThe treatment approach for recently diagnosed advanced non-small cell lung cancer (NSCLC) with EGFR mutations primarily relies on confirming the tissue diagnosis as non-squamous NSCLC. This routine clinical practice of tissue diagnosis imposes several barriers and delays in turnaround time (TAT) for biomarker testing, significantly delaying the time to treatment. The objective of this study is to investigate the ‘plasma first’ approach for detection of EGFR mutation in advanced stage treatment naïve NSCLC patients.MethodsWe prospectively collected blood samples of treatment naïve patients with clinical and radiological suspicion of advanced stage NSCLC prior to obtaining tissue biopsy. Plasma cfDNA was tested for EGFR mutation using two different methods. We compared the sensitivity and TAT of liquid biopsy with tissue biopsy.ResultsIn total, we analyzed plasma cell-free DNA (cfDNA) of 236 patients suspected of having advanced NSCLC for EGFR mutations. We observed a notably shorter turnaround time (TAT) of 3 days, which was significantly quicker compared to the 12-day TAT for tissue biopsy (p < 0.05). The ddPCR method had a sensitivity of 82.8%, which was higher than 66.34% sensitivity of ARMS-PCR. The current study also highlights that there is no significant difference in the clinical outcome of the patients whether treated based on liquid biopsy only or tissue biopsy (median progression-free survival of 11.56 vs. 11.9 months; p = 0.94).ConclusionsUtilizing a ‘plasma first’ strategy, given its shorter turnaround time, strong positive concordance and comparable outcomes to tissue biopsy, emerges as a highly specific and reliable method for detecting EGFR mutations in advanced-stage NSCLC.

Hospital mortality and length of stay associated with Enterobacterales positive blood cultures: a multicenter analysis.

Delayed time to antimicrobial susceptibility results can impact patients' outcomes. Our study evaluated the impact of susceptibility turnaround time (TAT) and inadequate empiric antibacterial therapy (IET) in patients with bloodstream infections (BSI) caused by Enterobacterales (ENT) species on in-hospital mortality and length of stay (LOS). This retrospective, multicenter investigation which included 29,570 blood ENT-positive admissions across 161 US healthcare facilities evaluated the association between antimicrobial susceptibility testing (AST) TAT, carbapenem susceptibility, and empiric therapy on post-BSI in-hospital mortality and LOS following an ENT BSI event in adult patients. After adjusting for outcomes covariates, post-BSI in-hospital mortality was significantly higher for patients in the IET vs adequate empiric therapy (AET) group [odds ratio (OR): 1.61 (95% CI: 1.32, 1.98); P < 0.0001], and when AST TAT was >63 h [OR:1.48 (95% CI: 1.16, 1.90); P = 0.0017]. Patients with carbapenem non-susceptible (carb-NS) ENT BSI had significantly higher LOS (16.6 days, 95% CI: 15.6, 17.8) compared to carbapenem susceptible (carb-S, 12.2 days, 95% CI: 11.8, 12.6), (P < 0.0001). Extended AST TAT was significantly associated with longer LOS for TAT of 57-65 h and >65 h (P = 0.005 and P< 0.0001, respectively) compared to TAT ≤42 h (reference). Inadequate empiric therapy (IET), carb-NS, and delayed AST TAT are significantly associated with adverse hospital outcomes in ENT BSI. Workflows that accelerate AST TAT for ENT BSIs and facilitate timely and adequate therapy may reduce post-BSI in-hospital mortality rate and LOS.IMPORTANCEFor patients diagnosed with bloodstream infections (BSI) caused by Enterobacterales (ENT), delayed time to antimicrobial susceptibility (AST) results can significantly impact in-hospital mortality and hospital length of stay. However, this relationship between time elapsed from blood culture collection to AST results has only been assessed, to date, in a limited number of publications. Our study focuses on this important gap using retrospective data from 29,570 blood ENT-positive admissions across 161 healthcare facilities in the US as we believe that a thorough understanding of the dynamic between AST turnaround time, adequacy of empiric therapy, post-BSI event mortality, and hospital length of stay will help guide effective clinical management and optimize outcomes of patients with ENT infections.

Impact of point-of-care gonorrhea and chlamydia testing in the emergency department on reducing overtreatment rates

BackgroundChlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infections continue to increase in the United States. Advancement in technology with point-of-care (POC) testing can improve the overall treatment of sexually transmitted infections (STI) in the emergency department (ED) by shortening the time to test result and administration of accurate treatment. The purpose of this study was to assess if the POC test reduced the rate of overtreatment for CT and/or NG compared to the standard-of-care (SOC) test. MethodsThis retrospective cohort study included adult patients tested for CT and NG at two urban EDs between August 2020 and October 2022. This cohort excluded hospital admissions, elopement, pregnancy, rectal and oral samples, victims of sexual assault, and diagnoses for which antimicrobial treatment overlapped that of CT/NG. The primary outcome assessed overtreatment, defined as receiving treatment in the ED or a prescription prior to discharge for patients who tested negative for CT and/or NG. Secondary outcomes included undertreatment rates, overtreatment rates in select populations, test turnaround time, and ED length of stay (LOS). ResultsOf 327 patients screened, 97 patients were included in the SOC group and 100 in POC. Overtreatment for CT was provided in zero POC patients and 29 (29.9%) SOC patients (p < 0.001). NG was overtreated in 1 (1%) POC and 23 (23.7%) SOC (p < 0.001). POC was associated with undertreatment of CT and/or NG in two patients, compared to four patients tested with SOC. Overall, treatment was deemed inappropriate for 5 (5%) of those tested with POC, compared to 35 (36%) tested with SOC (p < 0.001). There was no difference in ED LOS (2.7 vs 3.01 h, p = 0.41). ConclusionsPOC testing facilitated the return of results prior to patients being discharged from the ED. Compared to standard testing, POC improved appropriateness of CT and NG treatment by reducing the rates of overtreatment.

Cost-effectiveness of large-panel next-generation sequencing in guiding first-line treatment decisions for patients with nonsquamous advanced non-small cell lung cancer.

Clinical practice guidelines recommend broad-panel genomic profiling to identify actionable genomic alterations for patients with advanced non-small cell lung cancer (aNSCLC). To assess the cost-effectiveness of large-panel next-generation sequencing (LP-NGS) compared with current empirical single-gene test (SGT) patterns to inform first-line treatment decisions for patients with aNSCLC from a US commercial payer perspective, accounting for the effect of testing turnaround time and time to treatment initiation. We developed a discrete-event simulation model to estimate the impact of LP-NGS vs SGT for patients with nonsquamous aNSCLC. Discrete events and timing included testing patterns, receipt of the initial test result, treatment initiation (targeted vs nontargeted therapies), switching, retesting, rebiopsies, clinical trial participation, progression on therapy, and death. LP-NGS and SGT cohorts each comprised 100,000 adults with aNSCLC simulated over a 5-year postdiagnosis period, assumed to have the same distribution of genomic alterations. The model predicted the proportion of patients receiving appropriate first-line therapy according to clinical practice guidelines. Economic outcomes included expected life-years gained, quality-adjusted life-years, and the total costs of care over 5 years. Sensitivity and scenario analyses explored the robustness of the base-case model results. In the base-case model, LP-NGS was likely to identify more alterations than SGT. Total 5-year costs per patient were $539,658 for LP-NGS and $544,550 for SGT (net difference, $4,892 lower costs per patient for LP-NGS), which is likely to be cost-effective 95.1% of the time. The most influential model parameters on the 5-year total costs of care were preprogression nondrug medical costs on nontargeted therapy, NGS turnaround time, and clinical trial participation. This study suggests that LP-NGS to guide first-line treatment decisions is clinically more appropriate (more likely to identify alterations and subsequently allocate patients to clinically appropriate therapy) and provides a dominant cost-effectiveness treatment strategy over 5 years for patients with newly diagnosed aNSCLC in the United States.

A case for implementing an HSV1/2, VZV, and syphilis lesion panel in Manitoba, Canada.

Syphilis, caused by Treponema pallidum subsp. pallidum (TPA), is becoming a significant public health concern, with rising incidence in Manitoba exceeding the national average. The province has also seen a demographic shift leading to women representing 51.9% of cases in 2021, leading to the re-emergence of congenital syphilis. Given the similarities in lesion appearance between TPA and other pathogens such as herpesviruses, accurate diagnosis is crucial for effective management and prevention. In order to address the potential for missed TPA cases, we conducted a quality assurance study from June 2021 to March 2023, screening over 5,000 mucocutaneous lesion swabs for TPA, initially submitted for herpes simplex virus (HSV) and varicella zoster virus (VZV) testing. Positivity rates were 13% for HSV1, 13% for HSV2, 6.7% for VZV, and 6.6% for TPA. Turnaround times (TAT) for TPA testing, as a send-out to the reference laboratory, averaged 17.8 days. Of the TPA-positive specimens, 36% did not have a corresponding TPA PCR test ordered, and 19% did not have accompanying syphilis serology within 30 days of collection. Creation of a multiplex lesion panel identified high sensitivity and specificity for HSV1, HSV2, VZV, and TPA, with robust reproducibility across multiple runs. Incorporation of TPA into a lesion panel improved the TAT to 4 days. Our findings emphasize the need for improved testing strategies to combat the syphilis epidemic and enhance public health outcomes.IMPORTANCESyphilis resurgence has become a significant global public health concern. In particular, the Canadian Prairies have been struggling with high incidence since 2016, exceeding the national Canadian average. We undertook a quality assurance study that highlighted significant gaps in diagnosis of acute syphilis, which led to the development of a highly sensitive and specific multiplex lesion assay for the dual detection of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella zoster virus (VZV), and syphilis.

Torquetenovirus Viremia Quantification Using Real-Time PCR Developed on a Fully Automated, Random-Access Platform.

Quantification of Torquetenovirus (TTV) viremia is becoming important for evaluating the status of the immune system in solid organ transplant recipients, monitoring the appearance of post-transplant complications, and controlling the efficacy of maintenance immunosuppressive therapy. Thus, diagnostic approaches able to scale up TTV quantification are needed. Here, we report on the development and validation of a real-time PCR assay for TTV quantification on the Hologic Panther Fusion® System by utilizing its open-access channel. The manual real-time PCR previously developed in our laboratories was optimized to detect TTV DNA on the Hologic Panther Fusion® System. The assay was validated using clinical samples. The automated TTV assay has a limit of detection of 1.6 log copies per ml of serum. Using 112 samples previously tested via manual real-time PCR, the concordance in TTV detection was 93% between the assays. When the TTV levels were compared, the overall agreement between the methods, as assessed using Passing-Bablok linear regression and Bland-Altman analyses, was excellent. In summary, we validated a highly sensitive and accurate method for the diagnostic use of TTV quantification on a fully automated Hologic Panther Fusion® System. This will greatly improve the turnaround time for TTV testing and better support the laboratory diagnosis of this new viral biomarker.

Evaluation of Whole Genome Sequencing-Based Predictions of Antimicrobial Resistance to TB First Line Agents: A Lesson from 5 Years of Data.

Phenotypic susceptibility testing of the Mycobacterium tuberculosis complex (MTBC) isolate requires culture growth, which can delay rapid detection of resistant cases. Whole genome sequencing (WGS) and data analysis pipelines can assist in predicting resistance to antimicrobials used in the treatment of tuberculosis (TB). This study compared phenotypic susceptibility testing results and WGS-based predictions of antimicrobial resistance (AMR) to four first-line antimicrobials-isoniazid, rifampin, ethambutol, and pyrazinamide-for MTBC isolates tested between the years 2018-2022. For this 5-year retrospective analysis, the WGS sensitivity for predicting resistance for isoniazid, rifampin, ethambutol, and pyrazinamide using Mykrobe was 86.7%, 100.0%, 100.0%, and 47.8%, respectively, and the specificity was 99.4%, 99.5%, 98.7%, and 99.9%, respectively. The predictive values improved slightly using Mykrobe corrections applied using TB Profiler, i.e., the WGS sensitivity for isoniazid, rifampin, ethambutol, and pyrazinamide was 92.31%, 100%, 100%, and 57.78%, respectively, and the specificity was 99.63%. 99.45%, 98.93%, and 99.93%, respectively. The utilization of WGS-based testing addresses concerns regarding test turnaround time and enables analysis for MTBC member identification, antimicrobial resistance prediction, detection of mixed cultures, and strain genotyping, all through a single laboratory test. WGS enables rapid resistance detection compared to traditional phenotypic susceptibility testing methods using the WHO TB mutation catalog, providing an insight into lesser-known mutations, which should be added to prediction databases as high-confidence mutations are recognized. The WGS-based methods can support TB elimination efforts in Canada and globally by ensuring the early start of appropriate treatment, rapidly limiting the spread of TB outbreaks.

PIK3CA testing and treatment patterns among patients with metastatic breast cancer in US community clinical practice.

e13059 Background: PIK3CA mutations are present in ~35-40% of HR+/HER2- breast cancer patients. Targeted treatments are approved and in development for patients with advanced disease, and clinical guidelines recommend biomarker testing. This retrospective cohort study characterized PIK3CA mutation testing and treatment patterns among HR+/HER2- metastatic breast cancer (mBC) patients in United States (US) community clinical practices. Methods: We used the nationwide Flatiron Health electronic health record-derived de-identified database to select patients diagnosed with HR+/HER2- mBC from January 2011 to May 2023. We described baseline characteristics of patients tested and not tested for PIK3CA mutation. We captured rates of PIK3CA mutation testing (anytime and ≤1 year) prior to starting first-line (1L), second-line (2L), and third-line (3L) treatment; test and sample types; secular trends over time; and test turnaround time (TAT). Treatment patterns for patients with a PIK3CA-mutated tumor were also described from 2019 onwards. Results: Among 14,246 HR+/HER2- mBC patients treated at community sites, 4,537 had a PIK3CA mutation test result during the study period. 25% of patients had more than one PIK3CA mutation test. Test rates increased from 2011 to 2023 for all patients starting 1L, 2L, or 3L during the study period (Table). In 2023, the percentage of patients tested anytime prior to 1L, 2L, and 3L start dates were 11% (37 of 324 patients), 52% (140 of 271 patients), and 65% (133 of 204 patients), respectively. Among tested patients, those who were tested between 1L and 2L increased from 32% in 2018 to 45% in 2023. Median time from mBC diagnosis date to first test was variable across years: 8, 10, and 3 months in 2018, 2020, and 2022, respectively. During the study period, most PIK3CA mutation tests were next-generation sequencing (NGS) (88%) and used tumor tissue (72%). Median TAT decreased from 12 days in 2018 to 7 days in 2023. Among patients with a PIK3CA-mutated tumor in 2019 and onwards who received treatment, 36% of patients received alpelisib-containing regimens. Conclusions: PIK3CA mutation testing in HR+/HER2- mBC patients has increased over time in community clinical practice, and timing has shifted to earlier lines of therapy among tested patients. However, overall testing and subsequent treatment with pathway-directed therapy are still low despite clinical guidelines, which may reflect potential gaps in access to PIK3CA mutation testing and the continued unmet need regarding available targeted agents within the current treatment landscape. [Table: see text]

Implementation of a comprehensive genomic profiling (CGP) panel for precision oncology at a cancer center in Brazil.

e23520 Background: Assessing molecular alterations in solid tumors is essential for providing the best care in precision oncology. CGP tests utilize next-generation sequencing (NGS) to detect clinically relevant alterations and molecular signatures that are increasingly utilized in cancer careand can be performed in-house, which decreases test turnaround time (TAT) and costs. A paucity of studies have documented the experience of in-house testing at academic centers in Brazil. Methods: The TruSight Oncology (TSO) 500 assay/TSO 500 HRD are research use only (RUO) CGP assays that were validated for the detection of small variants (SVs), copy number variants (CNVs), microsatellite instability (MSI), tumor mutation burden (TMB) and genomic instability score (GIS) in DNA and gene fusions/splice variants in RNA in the NextSeq 500 system. Secondary analysis was performed with Illumina Connected Analytics. Tertiary analysis was performed with Illumina Connected Insights (ICI) and Sophia DDM. TMB ≥ 10mut/Mb was defined as TMB-High and GIS ≥ 42 was homologous recombination deficient (HRD). Results: We performed CGP on 40 FFPE samples (16 with HRD) with variable quality and block age most of which had known alterations detected by other genomics/molecular tests: 17 TNBC (16 paired DNA and RNA, 1 DNA only), 18 lung cancer (13 paired DNA and RNA, 5 DNA only), 1 endometrium cancer (DNA only, POLE mutated, 4 replicates), 1 colorectal cancer (DNA only, MSI unstable) and 11 sarcomas (RNA only). 84%, 84%, 90% and 89% of samples with DNA integrity number (DIN) &gt; 2.5 were qualified for analysis of SVs/TMB, MSI, CNVs, and GIS, respectively, while for samples with DIN &lt; 2.5 these values felt to 47%, 74%, 68% and 43%, respectively. RNA isolated from blocks dated up to 5 y.o. were qualified in 90% of cases while from older blocks only 6%. The 4 replicate (single sample) analysis showed 82% and 83% overall variant concordance in the ICI and Sophia DDM software, respectively. All previously known oncogenic alterations (SVs ≥5% VAF, fusions/splice, MSI, TMB) were detected (qualified samples) and with similar performance in ICI and Sophia, respectively: concordance 0.91/p &lt; 0.0001 vs 0.94/p &lt; 0.0001; sensitivity 100% vs 100%; specificity 98% vs 100%. TMB had 100% concordance with previous TMB results. Of note, 73% (8/11) TNBC samples analyzed for GIS were HRD and 75% (6/8) explained by BRCA1 germline mutation (25%) and BRCA1or RAD51C somatic promoter methylation (75%). Conclusions: This study demonstrates the utility of CGP and HRD testing in identifying multiple molecular alterations in FFPE tissue samples with high sensitivity and specificity. Pre-analytical variables directly impact data generation. BRCA1/RAD51C promoter methylation showed to be a potential biomarker for selecting metastatic TNBC patients for PARP inhibitor therapies. Offering CGP can potentially bring precision oncology to more patients with cancer in Brazil.