A reliable transcriptomic risk-score applicable to formalin-fixed paraffin-embedded biopsies improves outcome prediction in localized prostate cancer.

BACKGROUND: Clinical manifestation of prostate cancer (PCa) is highly variable. Aggressive tumors require radical treatment while clinically non-significant ones may be suitable for active surveillance. We previously developed the prognostic ProstaTrend RNA signature based on transcriptome-wide microarray and RNA-sequencing (RNA-Seq) analyses, primarily of prostatectomy specimens. An RNA-Seq study of formalin-fixed paraffin-embedded (FFPE) tumor biopsies has now allowed us to use this test as a basis for the development of a novel test that is applicable to FFPE biopsies as a tool for early routine PCa diagnostics. METHODS: All patients of the FFPE biopsy cohort were treated by radical prostatectomy and median follow-up for biochemical recurrence (BCR) was 9 years. Based on the transcriptome data of 176 FFPE biopsies, we filtered ProstaTrend for genes susceptible to FFPE-associated degradation via regression analysis. ProstaTrend was additionally restricted to genes with concordant prognostic effects in the RNA-Seq TCGA prostate adenocarcinoma (PRAD) cohort to ensure robust and broad applicability. The prognostic relevance of the refined Transcriptomic Risk Score (TRS) was analyzed by Kaplan-Meier curves and Cox-regression models in our FFPE-biopsy cohort and 9 other public datasets from PCa patients with BCR as primary endpoint. In addition, we developed a prostate single-cell atlas of 41 PCa patients from 5 publicly available studies to analyze gene expression of ProstaTrend genes in different cell compartments. RESULTS: Validation of the TRS using the original ProstaTrend signature in the cohort of FFPE biopsies revealed a relevant impact of FFPE-associated degradation on gene expression and consequently no significant association with prognosis (Cox-regression, p-value > 0.05) in FFPE tissue. However, the TRS based on the new version of the ProstaTrend-ffpe signature, which included 204 genes (of originally 1396 genes), was significantly associated with BCR in the FFPE biopsy cohort (Cox-regression p-value < 0.001) and retained prognostic relevance when adjusted for Gleason Grade Groups. We confirmed a significant association with BCR in 9 independent cohorts including 1109 patients. Comparison of the prognostic performance of the TRS with 17 other prognostically relevant PCa panels revealed that ProstaTrend-ffpe was among the best-ranked panels. We generated a PCa cell atlas to associate ProstaTrend genes with cell lineages or cell types. Tumor-specific luminal cells have a significantly higher TRS than normal luminal cells in all analyzed datasets. In addition, TRS of epithelial and luminal cells was correlated with increased Gleason score in 3 studies. CONCLUSIONS: We developed a prognostic gene-expression signature for PCa that can be applied to FFPE biopsies and may be suitable to support clinical decision-making.

Evaluation of the fully automated, sample-to-result Seegene STARlet-AIOS platform for detection of SARS-CoV-2, influenza virus A, influenza virus B, and RSV.

Early, accurate, and bulk detection of respiratory pathogens is essential for patient management and infection control. STARlet-All-in-One System (AIOS) (Seegene) is a new, fully automated, sample-to-result, molecular diagnostic platform. This study describes the first evaluation of STARlet-AIOS, by testing the Allplex™ SARS-CoV-2 (AS) and Allplex™ SARS-CoV-2/FluA/FluB/RSV combination (AC) assays in comparison to the SARS-CoV-2 assays used at our institute. Over a 3-week period, all naso-/oropharyngeal specimens tested for SARS-CoV-2 using either GeneXpert, Panther, or in-house developed test (LDT) were tested on the AIOS using the AS or AC assays. In addition, retrospective cohorts of specimens containing SARS-CoV-2, influenza virus A, influenza virus B, and RSV were tested. Discrepant results were re-tested with another assay used in this study. Hands-on time (HOT) and turn-around time (TAT) of the different systems were monitored and compared. A total of 738 specimens were tested on the AIOS using the AS assay. In addition, 210 specimens were tested using the AC assay. Overall agreement for SARS-CoV-2 detection was established as 98.5% and 95.2% for the AS and AC assay, respectively. Retrospective testing revealed high agreements for all targets, except for influenza virus A (agreement of 87.5%). HOT of the system was comparable to the HOT of GeneXpert and Panther and TAT comparable to Panther and LDT. The AIOS proved to be a robust sample-to-result system with low HOT and moderate TAT. This study showed reliable detection of SARS-CoV-2, influenza virus B, and RSV, whereas detection of influenza virus A using the AC assay appeared to be suboptimal.

[Pitfalls in the diagnosis of house dust mite allergy]. / Tücken der Diagnostik der Hausstaubmilbenallergie.

House dust mite (HDM) is the most significant indoor allergen, responsible for not only many cases of rhinoconjunctivitis but also for many cases of bronchial asthma, rendering it of considerable socioeconomic relevance. Besides symptomatic treatment and avoidance measures, allergen immunotherapy (AIT) is crucial, as the only causal, disease-modifying therapeutic approach. However, high diagnostic certainty is essential for initiating AIT. The challenge in making a correct diagnosis lies in interpreting the demonstrated HDM sensitization regarding its clinical relevance (clinically silent sensitization vs. allergy). While the risk of allergy increases with the level of IgE titers against HDM extract, Der p 1, or Der p 2, as well as with the breadth of the molecular sensitization profile against HDM components (Der p 1, Der p 2, Der p 23), no threshold can be defined for the presence of allergy, nor can sensitization to a specific component be confidently considered allergy inducing. It should be noted that at least in Southern Bavaria, the prevalence of Der p 23 sensitization is too low to be considered a major allergen, and Der p 23 is not able to molecularly differentiate all HDM sensitizations when added to the two major allergens Der p 1 and Der p 2. Evidently, HDM possesses a diverse profile of allergens, with some relevant ones possibly yet to be described. Unfortunately, patient history does not provide a sufficient assessment of the clinical relevance of a demonstrated HDM sensitization, necessitating allergen provocation testing before initiating AIT with HDM, despite the relatively large effort involved.

Characterization and evaluation of gene fusions as a measure of genetic instability and disease prognosis in prostate cancer.

BACKGROUND: Prostate cancer (PCa) is one of the most prevalent cancers worldwide. The clinical manifestations and molecular characteristics of PCa are highly variable. Aggressive types require radical treatment, whereas indolent ones may be suitable for active surveillance or organ-preserving focal therapies. Patient stratification by clinical or pathological risk categories still lacks sufficient precision. Incorporating molecular biomarkers, such as transcriptome-wide expression signatures, improves patient stratification but so far excludes chromosomal rearrangements. In this study, we investigated gene fusions in PCa, characterized potential novel candidates, and explored their role as prognostic markers for PCa progression. METHODS: We analyzed 630 patients in four cohorts with varying traits regarding sequencing protocols, sample conservation, and PCa risk group. The datasets included transcriptome-wide expression and matched clinical follow-up data to detect and characterize gene fusions in PCa. With the fusion calling software Arriba, we computationally predicted gene fusions. Following detection, we annotated the gene fusions using published databases for gene fusions in cancer. To relate the occurrence of gene fusions to Gleason Grading Groups and disease prognosis, we performed survival analyses using the Kaplan-Meier estimator, log-rank test, and Cox regression. RESULTS: Our analyses identified two potential novel gene fusions, MBTTPS2,L0XNC01::SMS and AMACR::AMACR. These fusions were detected in all four studied cohorts, providing compelling evidence for the validity of these fusions and their relevance in PCa. We also found that the number of gene fusions detected in a patient sample was significantly associated with the time to biochemical recurrence in two of the four cohorts (log-rank test, p-value < 0.05 for both cohorts). This was also confirmed after adjusting the prognostic model for Gleason Grading Groups (Cox regression, p-values < 0.05). CONCLUSIONS: Our gene fusion characterization workflow revealed two potential novel fusions specific for PCa. We found evidence that the number of gene fusions was associated with the prognosis of PCa. However, as the quantitative correlations were only moderately strong, further validation and assessment of clinical value is required before potential application.

Clinical whole-genome sequencing in cancer diagnosis.

Characterizing the genomic landscape of cancers is a routine part of clinical care that began with the discovery of the Philadelphia chromosome and has since coevolved with genomic technologies. Genomic analysis of tumors at the nucleotide level using DNA sequencing has revolutionized the understanding of cancer biology and identified new molecular drivers of disease that have led to therapeutic advances and improved patient outcomes. However, the application of next-generation sequencing in the clinical laboratory has generally been limited until very recently to targeted analysis of selected genes. Recent technological innovations and reductions in sequencing costs are now able to deliver the long-promised goal of tumor whole-genome sequencing as a practical clinical assay.

Role of Targeted Therapy and Immune Checkpoint Blockers in Advanced Non-Small Cell Lung Cancer: A Review.

Advanced non-small cell lung cancer (NSCLC) is a complex disease comprising molecularly distinct tumor types, each with a unique biology that is becoming increasingly better characterized. The aim of this review is to present an optimized treatment schema and the accompanying diagnostic testing approach for patients with advanced NSCLC. There are a number of therapies currently approved for patients with advanced NSCLC, including agents that target particular oncogenic drivers, as well as immune checkpoint blockers (ICBs) that elicit an antitumor response. Identification of genetic alterations (e.g., epidermal growth factor receptor, anaplastic lymphoma kinase, reactive oxygen species proto-oncogene 1, B-Raf proto-oncogene) or programmed cell death ligand-1 expression levels in NSCLC requires diligent molecular testing at initial diagnosis and, in some cases, at disease progression to ensure the most efficacious treatment is delivered. Accurate molecular diagnostic testing, along with the careful selection of currently approved targeted agents, ICBs, or systemic chemotherapy, provides therapy that is personalized according to patients' needs to achieve the best possible outcome. Enrollment in clinical trials that further the development of tailored therapies is highly recommended at all stages of treatment. IMPLICATIONS FOR PRACTICE: Targeted therapies and immune checkpoint blockers provide effective and tailored options for patients with non-small cell lung cancer. Careful molecular analysis of tumor samples is necessary to identify the genetic alterations that are present, to ensure that each patient receives the most efficacious treatment for their specific tumor type. Personalized therapy provides each patient with the best probability for prolonged survival. Enrolling patients in clinical trials should be the first consideration before making each treatment decision.

Evaluation of the GenoType MTBDRsl Version 2.0 Assay for Second-Line Drug Resistance Detection of Mycobacterium tuberculosis Isolates in South Africa.

Early detection of resistance to second-line antituberculosis drugs is important for the management of multidrug-resistant tuberculosis (MDR-TB). The GenoType MTBDRsl version 2.0 (VER 2.0) line probe assay has been redesigned for molecular detection of resistance-conferring mutations of fluoroquinolones (FLQ) (gyrA and gyrB genes) and second-line injectable drugs (SLID) (rrs and eis genes). The study evaluated the diagnostic performance of the GenoType MTBDRsl VER 2.0 assay for the detection of second-line drug resistance compared with phenotypic drug susceptibility testing (DST), using the Bactec MGIT 960 system on Mycobacterium tuberculosis complex isolates from South Africa. A total of 268 repository isolates collected between 2012 and 2014, which were rifampin monoresistant or MDR based on DST, were selected. MTBDRsl VER 2.0 testing was performed on these isolates and the results analyzed. The MTBDRsl VER 2.0 sensitivity and specificity indices for culture isolates were the following: FLQ, 100% (95% confidence interval [CI] 95.8 to 100%) and 98.9% (95% CI, 96.1 to 99.9%); SLID, 89.2% (95% CI, 79.1 to 95.6%) and 98.5% (95% CI, 95.7 to 99.7%). The sensitivity and specificity observed for individual SLID were the following: amikacin, 93.8% (95% CI, 79.2 to 99.2%) and 98.5% (95% CI, 95.5 to 99.7%); kanamycin, 89.2% (95% CI, 79.1 to 95.6%) and 98.5% (95% CI, 95.5 to 99.7%); and capreomycin, 86.2% (95% CI, 68.3 to 96.1%) and 95.9% (95% CI, 92.2 to 98.2%). An interoperator reproducibility of 100% and an overall interlaboratory performance of 93% to 96% were found. The overall improvement in sensitivity and specificity with excellent reproducibility makes the GenoType MTBDRsl VER 2.0 a highly suitable tool for rapid screening of clinical isolates for second-line drug resistance for use in high-burden TB/HIV settings.

Molecular-based diagnosis of respiratory allergic diseases in children from Curitiba, a city in Southern Brazil.

BACKGROUND: Prevalence of respiratory allergic diseases has increased worldwide. Identification of the aeroallergens involved in allergenic sensitisation is important for diagnosis, treatment and prevention. OBJECTIVE: To verify the molecular pattern of sensitisation to aeroallergens in patients with allergic respiratory diseases using microarray technique for specific IgE antibody detection. METHODS: Cross-sectional study of 101 children with allergic rhinitis was followed in an outpatient clinic. All patients had positive skin prick tests (SPT) to at least one of the following antigens: Dermatophagoides pteronyssinus, Blomia tropicalis, Blattella germanica, Lolium multiflorum, and dog and cat epithelium. Serum specific IgE antibodies (sIgE) to mites, animal epithelia, fungi, cockroach and pollens components were determined by ImmunoCAP ISAC. RESULTS: sIgE to group 1 and 2 mite allergens showed higher positive rates: Der p 1 (74.2%), Der p 2 (73.3%), Der f 1 (74.2%), Der f 2 (72.3%). sIgE to animal epithelia were less frequent, Can f 1, Can f 2, Can f 3 in 4.9%, 2.9%, 1.9% respectively and Fel d 1, Fel d 2, Fel d 4 in 16.8%, 0.9% and 1.9%. respectively. Sensitisation to fungi and cockroach were rare, except for Bla g 7, to which 16.8% were positive. There was no significant recognition for tree pollens group. For grass, sIgE were detected to Cyn d 1 in 16.8%, Phl p 1 and Phl p 4 in 14.8% and 12.9%, respectively. CONCLUSION: Knowing that the pattern of allergic sensitisation varies according to environment and population, our results reinforce the need for local studies, using molecular-based diagnosis.

Molecular Testing for Treatment of Metastatic Non-Small Cell Lung Cancer: How to Implement Evidence-Based Recommendations.

The recent discovery of relevant biomarkers has reshaped our approach to therapy selection for patients with non-small cell lung cancer. The unprecedented outcomes demonstrated with tyrosine kinase inhibitors in molecularly defined cohorts of patients has underscored the importance of genetic profiling in this disease. Despite published guidelines on biomarker testing, successful tumor genotyping faces significant hurdles at both academic and community-based practices. Oncologists are now faced with interpreting large-scale genomic data from multiple tumor types, possibly making it difficult to stay current with practice standards in lung cancer. In addition, physicians' lack of time, resources, and face-to-face opportunities can interfere with the multidisciplinary approach that is essential to delivery of care. Finally, several challenges exist in optimizing the amount and quality of tissue for molecular testing. Recognizing the importance of biomarker testing, a series of advisory boards were recently convened to address these hurdles and clarify best practices. We reviewed these challenges and established recommendations to help optimize tissue acquisition, processing, and testing within the framework of a multidisciplinary approach.

Automated processing of fluorescence in-situ hybridization slides for HER2 testing in breast and gastro-esophageal carcinomas.

BACKGROUND: HER2 fluorescence in-situ hybridization (FISH) is used in breast and gastro-esophageal carcinoma for determining HER2 gene amplification and patients' eligibility for HER2 targeted therapeutics. Traditional manual processing of the FISH slides is labor intensive because of multiple steps that require hands on manipulation of the slides and specifically timed intervals between steps. This highly manual processing also introduces inter-run and inter-operator variability that may affect the quality of the FISH result. Therefore, we sought to incorporate an automated processing instrument into our FISH workflow. METHODS: Twenty-six cases including breast (20) and gastro-esophageal (6) cancer comprising 23 biopsies and three excision specimens were tested for HER2 FISH (Pathvysion, Abbott) using the Thermobrite Elite (TBE) system (Leica). Up to 12 slides can be run simultaneously. All cases were previously tested by the Pathvysion HER2 FISH assay with manual preparation. Twenty cells were counted by two observers for each case; five cases were tested on three separate runs by different operators to evaluate the precision and inter-operator variability. RESULTS: There was 100% concordance in the scoring between the manual and TBE methods as well as among the five cases that were tested on three runs. Only one case failed due to poor probe hybridization. In total, seven cases were positive for HER2 amplification (HER2:CEP17 ratio >2.2) and the remaining 19 were negative (HER2:CEP17 ratio <1.8) utilizing the 2007 ASCO/CAP scoring criteria. Due to the automated denaturation and hybridization, for each run, there was a reduction in labor of 3.5h which could then be dedicated to other lab functions. CONCLUSION: The TBE is a walk away pre- and post-hybridization system that automates FISH slide processing, improves work flow and consistency and saves approximately 3.5h of technologist time. The instrument has a small footprint thus occupying minimal counter space. TBE processed slides performed exceptionally well in comparison to the manual technique with no disagreement in HER2 amplification status.

Role of the clinical pathology laboratory in the evaluation of endometrial carcinomas for Lynch syndrome.

Molecular diagnostic testing of endometrial carcinomas in the pathology laboratory has recently emerged as a key component of the clinical evaluation of Lynch syndrome in many centers. Testing modalities involve immunohistochemical and PCR-based analyses. This article outlines the routine application of these analyses, provides a practical guide for troubleshooting some of the common technical issues related to their performance, and reviews common pitfalls in their interpretation. Discrepancies between tissue testing and genetic testing results are discussed in the context of the current understanding of endometrial cancer biology. The merits of universal versus targeted tissue testing based on clinical patient history and histological tumor appearance are also addressed.

Pathogen-Agnostic Advanced Molecular Diagnostic Testing for Difficult-to-Diagnose Clinical Syndromes-Results of an Emerging Infections Network Survey of Frontline US Infectious Disease Clinicians, May 2023.

During routine clinical practice, infectious disease physicians encounter patients with difficult-to-diagnose clinical syndromes and may order advanced molecular testing to detect pathogens. These tests may identify potential infectious causes for illness and allow clinicians to adapt treatments or stop unnecessary antimicrobials. Cases of pathogen-agnostic disease testing also provide an important window into known, emerging, and reemerging pathogens and may be leveraged as part of national sentinel surveillance. A survey of Emerging Infections Network members, a group of infectious disease providers in North America, was conducted in May 2023. The objective of the survey was to gain insight into how and when infectious disease physicians use advanced molecular testing for patients with difficult-to-diagnose infectious diseases, as well as to explore the usefulness of advanced molecular testing and barriers to use. Overall, 643 providers answered at least some of the survey questions; 478 (74%) of those who completed the survey had ordered advanced molecular testing in the last two years, and formed the basis for this study. Respondents indicated that they most often ordered broad-range 16S rRNA gene sequencing, followed by metagenomic next-generation sequencing and whole genome sequencing; and commented that in clinical practice, some, but not all tests were useful. Many physicians also noted several barriers to use, including a lack of national guidelines and cost, while others commented that whole genome sequencing had potential for use in outbreak surveillance. Improving frontline physician access, availability, affordability, and developing clear national guidelines for interpretation and use of advanced molecular testing could potentially support clinical practice and public health surveillance.

An algorithm for the characterization of influenza A viruses from various host species and environments.

Due to the extensive host range of influenza A viruses, it is difficult to determine the best diagnostic algorithm to efficiently screen samples from a variety of host species for influenza A viruses. While there are some influenza diagnostic algorithms that are specific to host species, to our knowledge, no single algorithm exists for the characterization of influenza A viruses across multiple host species. In this paper, we propose an algorithm that can serve as a guide for screening human, animal, and environmental samples for influenza A viruses of high human and animal health importance.

CTNS Mutations Causing Autosomal Recessive Cystinosis in a Subset of Iranian Population: Report of Two New Variants.

Background: Nephropathic cystinosis (NC) is an uncommon autosomal recessive disease with abnormality in lysosomal storage that appearances in patients with mutations in the CTNS gene encoding a lysosomal transporter cystinosin. Disrupted function of this transporter is followed by accumulation of cysteine crystals in cells of many various organs. This study aimed to investigate the mutations of the CTNS gene in 20 Iranian patients suffering from NC. Materials and Methods: Twenty Iranian cystinosis patients referring to Imam Hossein Hospital of Isfahan were employed in this case-series study. After extraction of genomic DNA, the promoter and entire coding regions of CTNS were analysed using sanger sequencing in all patients. Gap-Polymerase Chain Reaction was used to detect 57 kb deletion in the CTNS gene. In silico study was performed to analyse variants. Results: The large deletion was not seen in any NC patients. Molecular analysis which conducted to screen the CTNS gene of patients, identified eight different mutations, including two new mutations, c.971_972insC and c.956_956delA, which have not been reported before, and c.681G>A mutation, which was identified as a frequently founded mutation in the Middle East and was observed in 35% of patients. In this study, five other mutations including c.1015G>A, c.922G>A, c.323_323delA, c.433C>T, and c.18_21delGACT were also observed, which have been reported in previous studies. Conclusion: The mutational spectrum in the Iranian patients is the same as previously reported mutations except that two new mutations were found. The present findings will present suggestions for regular molecular diagnosis of cystinosis in Iran.

Molecular Detection and Typing of Treponema pallidum in Non-Ocular Samples from Patients with Ocular Syphilis.

INTRODUCTION: Ocular syphilis is a rare but potentially sight-threatening manifestation of infection with the spirochete Treponema pallidum subspecies pallidum. Molecular strain typing of clinical specimens obtained from patients with syphilis can provide useful epidemiological and clinical information. In this study, we assess the utility of non-ocular clinical samples in strain typing for patients with diagnosed ocular syphilis. METHODS: We collected samples of excess blood, serum, and cerebrospinal fluid (CSF) from 6 patients with ocular syphilis treated in 2013-2016. DNA was extracted, purified, and then analyzed using an enhanced molecular typing method including sequence analysis of tp0548, number of repeats in the arp gene, and restriction fragment length polymorphism of the tpr gene. RESULTS: Molecular strain typing based on tp0548 gene sequence analysis revealed two cases of type F and two cases of type G in 3 of 6 (50%) cases with CSF samples, 1 of which was obtained after starting antibiotics. In a patient with 2 distinct episodes, the same tp0548 type (type G) was identified in both episodes using different sample types (CSF, whole blood). Serum samples were available in 6 cases, but none were successfully typed with any of the methods. Amplification of the tpr and arp genes was unsuccessful in all cases. Overall, strain types were identified in 4 of the 7 episodes. CONCLUSION: Treponema pallidum strain types F and G were detected in CSF or whole blood in 4 of 7 episodes in this series. We demonstrate moderate sensitivity of strain typing in ocular syphilis using non-ocular clinical specimens.

Molecular diagnostics of infectious disease: Detection and characterization of microbial agents in cytology samples.

BACKGROUND: Cytology samples are widely used to diagnose various infectious diseases by detection and identification of causative infectious agents, including bacteria, fungi, and viruses. The role of cytopathology in infectious disease has expanded tremendously in the past decades with the advances in molecular techniques. Molecular diagnostic methods, compared to conventional methods, have shown improved patient outcome, reduction in cost, and shortened hospital stay times. The aim of this article is to review molecular testing in cytology samples for diagnosis of infectious diseases. METHODS: The literature search for molecular testing in common cytology samples for diagnosis of infectious diseases was performed. The findings of the studies were summarized. The common cytology samples included in this article were gynecologic specimens, cerebrospinal fluid, bronchoalveolar lavage, and urine samples. CONCLUSIONS: There are a number of molecular diagnostic tests that are available to be used in common cytology samples to detect infectious agents. Each test has its own advantages and limitations. It is our hope that upon reading this review article, the readers will have better understanding of molecular diagnostic testing of infectious diseases utilizing commonly sampled cytology specimens in daily practice.

An Old Acquaintance: Could Adenoviruses Be Our Next Pandemic Threat?

Human adenoviruses (HAdV) are one of the most important pathogens detected in acute respiratory diseases in pediatrics and immunocompromised patients. In 1953, Wallace Rowe described it for the first time in oropharyngeal lymphatic tissue. To date, more than 110 types of HAdV have been described, with different cellular tropisms. They can cause respiratory and gastrointestinal symptoms, even urinary tract inflammation, although most infections are asymptomatic. However, there is a population at risk that can develop serious and even lethal conditions. These viruses have a double-stranded DNA genome, 25-48 kbp, 90 nm in diameter, without a mantle, are stable in the environment, and resistant to fat-soluble detergents. Currently the diagnosis is made with lateral flow immunochromatography or molecular biology through a polymerase chain reaction. This review aimed to highlight the HAdV variability and the pandemic potential that a HAdV3 and 7 recombinant could have considering the aggressive outbreaks produced in health facilities. Herein, we described the characteristics of HAdV, from the infection to treatment, vaccine development, and the evaluation of the social determinants of health associated with HAdV, suggesting the necessary measures for future sanitary control to prevent disasters such as the SARS-CoV-2 pandemic, with an emphasis on the use of recombinant AdV vaccines to control other potential pandemics.

Examining the clinical impact of rapid multiplex polymerase chain reaction-based diagnostic testing for bloodstream infections in a national cohort of the Veterans Health Administration.

STUDY OBJECTIVE: Bloodstream infections (BSIs) are a significant cause of mortality. Use of a rapid multiplex polymerase chain reaction-based blood culture identification panel (BCID) may improve antimicrobial utilization and clinical outcomes by shortening the time to appropriate therapy and de-escalating antibiotics among patients on overly broad-spectrum empiric therapy. The effect of BCID on clinical outcomes across varying institutional antimicrobial stewardship program (ASP) practices is unclear. This study evaluated clinical outcomes associated with the "real-world" implementation of BCID in a national health system with varying ASP practices. DESIGN: National, multicenter, retrospective, pre-post quasi-experimental study of hospitalized patients admitted from 2015 to 2020 to VHA facilities, which introduced the BCID for ≥1 year. SETTING: United States Veterans Health Administration (VHA) hospitals with BCID. PATIENTS: Hospitalized VHA patients with ≥1 blood culture positive for bacteria featured on the BCID panel. INTERVENTION: Comparison of outcomes between the pre- and post-BCID implementation groups. MEASUREMENTS: Outcomes evaluated included early antimicrobial de-escalation within 48 h, defined as reduction in antimicrobial spectrum scores, time to appropriate therapy, and 30-day mortality. MAIN RESULTS: A total of 4138 patients (pre-BCID, n = 2100; post-BCID, n = 2038) met the study criteria. Implementation of BCID was associated with significant improvements in early antimicrobial de-escalation (34.6%: pre-BCID vs. 38.1%: post-BCID; p = 0.022), which persisted after adjusting for other covariates (adjusted risk ratio [aRR], 1.11; 95% confidence interval [CI], 1.02-1.20; p = 0.011). Median time to appropriate therapy was shorter in the post-BCID implementation group relative to the pre-BCID group (9 h: pre-BCID vs. 8 h: post-BCID, respectively, p = 0.005), and a greater percentage of patients received early appropriate antimicrobial therapy within 48 h in the post-BCID implementation group (91.7%: pre-BCID vs. 93.8%: post-BCID; p = 0.008). In the multivariable regression analysis, BCID implementation was significantly associated with a higher likelihood of appropriate therapy within 48 h (aRR, 1.02; 95% CI, 1.01-1.08; p = 0.020). There was no difference in 30-day mortality between groups overall (12.6% pre-BCID vs. 11.2% post-BCID; p = 0.211). CONCLUSIONS: In a "real-world" clinical setting, the implementation of BCID was associated with clinical improvements in antimicrobial utilization. The BCID platform may serve as a useful adjunct for BSI management in facilities with ASP.

Bacteroides meningitis in a healthy child: A case report and review of the literature.

A 7-year-old male presented with meningitis. CSF gram stain showed gram negative rods, but the organism failed to grow on culture. 16 s rRNA sequencing identified the organism as Bacteroides fragilis. The patient fully recovered with antibiotic therapy targeting that organism.

Pathological criteria for multiplex gene-panel testing using next-generation sequencing in non-small cell lung cancer.

BACKGROUND: Multiplex gene-panel tests have recently been developed, including the Oncomine Dx Target Test multi-CDx system (ODxTT), and are commonly used to determine the adaptation of molecular-targeting drugs in non-small cell lung cancer. However, in actual clinical settings, we obtain false results owing to the small biopsy samples. We aimed to optimize tissue preparation methods to improve the success rate. PATIENTS AND METHODS: We investigated 88 biopsy samples. The area and nucleated cell count in the first cut section were quantified using a morphometric software. Pathological parameters, including "total tissue area" and "total nucleated cell count," were calculated by multiplying the total number of slides submitted to ODxTT. Optimal cutoff values to obtain the best success rate were also determined. Additionally, we morphometrically measured actual tumor cell proportions and attempted to determine the lower limit possible to detect mutations. RESULTS: Optimal cutoff values for "total nucleated cell count" and "total tissue area" were 132,885 and 32.94 mm2, respectively. The actual tumor cell proportions ranged from 4.6 to 97.7%. Even in cases with actual tumor cell proportions of less than 20% (ranging from 4.6 to 19.7%), there was no false negative. CONCLUSION: Thus, we proposed the pathological criteria for accurate ODxTT. Our result suggested that tumor cell proportions of less than 20% (around 5%) could be applicable for ODxTT. We hope that our results will help pathologists to choose between the multi-plex test (ODxTT) or single-plex test in routine diagnostics.