Negative Specimens Research Articles

SNaPshot minisequencing for SARS CoV 2 Variant Genotyping

Abstract Introduction/Objective The SNaPshot Multiplex System (Thermo Fisher) can investigate up to fifty SNP markers simultaneously by using PCR amplification and subsequent dideoxy single base extension of an unlabeled primer and capillary electrophoresis. After electrophoresis and fluorescence detection, the alleles of a single marker appear as different colored peaks at roughly the same size in the electropherogram plot. The size of the different allele peaks vary slightly due to differences in molecular weight of the dyes. We propose a new set of primers and probes to use this system to differentiate variants of SARs Cov 2 variants. Methods/Case Report SNAPSHOT analysis was performed using the primers designed internally and analysis was done of up to 7 mutation hot spots, which were 417, 4511, 478, 484, 501, 614, and 681. By implementing the algorithm of systems and methods herein with the traces depicted in Figures, true positive and true negative outcomes for the different COVID variants can be performed, based on the highlighted peaks, which are not necessarily the most intense peaks. 10 Positive controls achieved from in stro transcript plasmid was tested then 10 viral RNA obtained through BEI Resources, NIAID was tested after mixed into 30 pooled negative PCR-swab specimens. Genmapper was used for analysis of results. 40 Korean patient samples were tested as well. Results (if a Case Study enter NA) Both In vitro transcript and derived specimens of wild type, alpha, beta, delta, gamma, kappa, omicron, and pirola showed expected results in 78/80 of the specimens. For example,delta B.1.617.2 variant showed 1 = A (green) at Primer 452R-F, 2 = G (blue): Primer 484Q-F, and 3 = A (green) at Primer 614-F. Of note, hetero type was noted in a few of the patient strains but none of the BEI strains and this may be due to a transfromation of the virus and worth investigation. Conclusion The SNaPshot® Multiplex System is a accurate system that may be applied to multiple applications including viral strain anylsis and here we can show how it may be effectively used for SARS-Cov-2 gentotyping. Using this technique, it is possible to analyze more than 50 SNPs distributed throughout the genome in a single multiplex reaction, making it an advantage when compared with traditional sequencing reaction. This is a important study that shows that this technique may be used in multiple field applictions.

Development of an 18-color flow cytometry panel for B-lymphoblastic leukemia minimal residual disease detection

Abstract B-cell acute lymphoblastic leukemia (B-ALL) is characterized by a neoplastic proliferation of precursor B cells that have blast cytomorphology and immature immunophenotype. Following treatment of B-ALL, flow cytometry is utilized to detect minimal residual disease (MRD), which aids in monitoring therapeutic response and informing prognosis. In our institution, the BD FACSCanto cytometer is used in conjunction with two standardized 8-antibody EuroFlow panels to detect MRD in B-ALL patients. The objective of this study was to validate a new 18-color B-ALL MRD panel for bone marrow aspirate specimens to be run on a BD LSRFortessa cytometer, with the intention of improving sensitivity and consolidating the testing into one antibody panel. The proposed panel consisted of 22 antibodies, some of which were stacked within the same fluorescence channel, in addition to a viability stain. Twelve bone marrow aspirate specimens collected in 2023 (five positive and seven negative B-ALL MRD specimens) were stained with the new antibody panel and analyzed on the Fortessa cytometer. CD45 dim, CD19-positive blasts were analyzed using BD FACSDiva Software. For the five specimens previously reported as B-ALL MRD-positive using the EuroFlow panels, we were able to detect phenotypically identical B-ALL cells with the 18-color panel. A paired t-test done on the five MRD-positive specimens showed no significant difference in the percentage of residual B-ALL cells (out of total viable cells) measured by the 18-color panel when compared to the EuroFlow panels (EuroFlow/18-color: 0.0096%/0.0097%, 0.024%/0.012%, 0.94%/0.99%, 0.85%/0.85%, 0.022%/0.017%; p=0.58). All seven specimens that were previously reported as B-ALL MRD-negative using the EuroFlow panels were also negative for residual B-ALL cells when stained with the 18-color panel and analyzed on the Fortessa cytometer. The antibody cost per test of the EuroFlow panels ($20-$25, ~$44 for both panels) and new 18-color panel ($63) were calculated based on the unit cost of each antibody aliquot and the required volume of reagent per test. In summary, the new 18-color B-ALL MRD panel run on the BD LSRFortessa cytometer appears to provide similar results as the EuroFlow panels run on the BD FACSCanto cytometer. Although compared to the two EuroFlow panels, the single 18-color panel is more expensive per test in terms of antibodies, it may be preferable when there is scant tissue (such as cerebrospinal fluid) as there may not be enough material to run both EuroFlow panels.

Evaluation of the Seegene Allplex™ RV master assay for one-step simultaneous detection of eight respiratory viruses in nasopharyngeal specimens

BackgroundThe Seegene Allplex™ RV Master (RVM) assay is a one-step multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) system for detecting eight viral respiratory pathogens from nasopharyngeal swab, aspirate, and bronchoalveolar lavage specimens. The eight RVM targets are: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Influenza A (Flu A), Influenza B (Flu B), Human respiratory syncytial virus (RSV), adenovirus (AdV), rhinovirus (HRV), parainfluenza virus (PIV), and metapneumovirus (MPV). The assay is based on Seegene’s multiple detection temperature (MuDT) technology and provides cycle threshold (Ct) values for each of its viral targets upon PCR completion. ObjectiveWe aimed to evaluate the diagnostic performance of the RVM assay by calculating sensitivity, specificity, accuracy, Positive Predictive Value (PPV), Negative Predictive Value (NPV), Positive Percent Agreement (PPA), Negative Percent Agreement (NPA), and Overall Percent Agreement (OPA) compared to definite diagnosis and analogous reference assays. Study designDiagnostic sensitivity, specificity, accuracy, PPV, and NPV were calculated by comparing the results of the RVM assay to that of definite diagnosis assays; while PPA, NPA, and OPA were calculated by comparing results of the RVM assay to that of analogous reference products. Definite diagnosis and reference methods comprised whole genome sequencing and PCR genotyping, the Allplex™ SARS-CoV-2/FluA/FluB/RSV and Respiratory Panels 1, 2, and 3 assays (Seegene), and the Xpert® Xpress SARS-CoV-2/FluA/FluB/RSV Plus assay (Cepheid). Reproducibility of the RVM assay using fully-automated and semi-automated nucleic acid (NA) extraction workflows and as performed by independent operators was also assessed. In total, 249 positive respiratory specimens and at least 50 negative specimens for each target tested were used for this evaluation study. ResultsSensitivity, specificity, accuracy, PPV, NPV, PPA, NPA, and OPA ranged from 95.7 % to 100 % for detecting all eight targets tested on the RVM assay. Reproducibility PPA, NPA, and OPA between automated and semi-automated NA extraction workflows were all >97.9 %, while the reproducibility PPA, NPA and OPA between independent operators were all 100 %. ConclusionThese results demonstrate a high level of sensitivity, specificity, accuracy and diagnostic predictive value of the RVM assay and high agreement with comparable reference assays in identifying all eight of its targets. Taken together, our study underscores the diagnostic utility of the RVM assay in detecting eight viral respiratory pathogens.

B-263 Analytical Performance Evaluation of Eleven Drug of Abuse Assays on the Atellica CI Analyzer

Abstract Background The Atellica® CI Analyzer is an automated, high-throughput integrated chemistry and immunoassay analyzer utilizing both Atellica® CH and Atellica® IM Assays. This study evaluated the analytical performance of the Atellica CH Amphetamines (Amp), Barbiturates (Brb), Benzodiazepine (Bnz), Cannabinoids THC (Thc), Cocaine Metabolite (Coc), Ecstasy (Xtc), Methadone (Mdn), Opiates (Op), Oxycodone (OXY), Phencyclidine (Pcp), and Propoxyphene (Ppx) Assays on the Atellica CI Analyzer. Methods Precision and method comparison (MC) studies were used as performance indicators. Precision studies were performed according to CLSI EP05-A3 using quality control (QC) samples consisting of contrived human urine samples. One aliquot of each QC was tested in duplicate in two runs per day ≥2 hours apart on each analyzer for ≥ 20 days. Precision for each assay was evaluated with one reagent lot on one system. Method comparison studies were performed with three reagent lots according to CLSI EP12-A2. Individual native and contrived human urine samples were analyzed using the Atellica CH Amp, Barb, Bnz, Thc, Coc, Xtc, Mdn, Op, OXY, Pcp, and Ppx Assays on both the Atellica CH and Atellica CI Analyzers. The results were assessed based on analyte values used for distinguishing positive (value ≥cutoff) and negative (value <cutoff) specimens. Results As shown in table below, repeatability and within-lab %CVs based on manufacturer arbitrary units (mAU) were <0.8% and <3.2%, respectively or qualitative interpretation for each replicate remained unchanged for all 80 precision testing measurements (OXY, PcP). Qualitative accuracy assessed by concordance analysis demonstrated ≥95% agreement between the Atellica CI Analyzer and Atellica CH Analyzer. Conclusions Evaluation of the Atellica CH Amp, Barb, Bnz, Thc, Coc, Xtc, Mdn, Op, OXY, Pcp, and Ppx Assays using the Atellica CI Analyzer demonstrated acceptable precision and equivalent performance compared to the same assays on the Atellica CH Analyzer.

Flexural strength and viscosity of dental luting composite reinforced with zirconia and alumina

Objectives Optimum flexural strength and viscosity are essential for longevity while efficiently handling dental luting composite. This study aims to investigate the effects of zirconia and alumina on the flexural strength (FS)and viscosity of dental cement made of silica rice husk. Materials and Methods Study groups with different percentages of weight (wt.): Group 1 (3 wt.% zirconia), Group 2 (3 wt.% alumina), and Group 3 (3 wt.% zirconia and 2 wt.% alumina), negative control specimen (0 wt.% zirconia/ alumina) and Rely-X U200 (3M ESPE). A universal testing machine and a rheometer were used to test FS and viscosity, respectively. One-way ANOVA and post-hoc Bonferonni test were used for data analyses. Results and Discussion FS and viscosity for Group 3 (3 wt.% zirconia and 2 wt.% alumina) were significantly higher compared to the negative control (p<0.05). Conclusion: Adding zirconia and alumina improved flexural strength without compromising the viscosity of dental luting composite. This experimental dental luting cement using hybrid fillers could be an alternative to resin luting cement. Bangladesh Journal of Medical Science Vol. 23 No. 04 October’24 Page : 1048-1053

B-189 Alinity m You-Create Lab-Developed Test for hepatitis delta virus

Abstract Background Lab developed tests (LDTs) play an important role in the realm of infectious diseases. Current molecular diagnostic platforms offer fully automated high-throughput performance capable of running LDTs. The Alinity m platform enables users to run LDTs concurrently with other FDA cleared or approved IVD assays with its Alinity m You-Create feature. Here, we investigated the performance of an LDT assay targeting hepatitis delta virus (HDV) using commercially available PCR reagents. Methods Alinity m You-Create LDT for HDV was evaluated using Applied Biosystems TaqMan Fast Virus 1-Step Multiplex Master Mix, No ROX, (ThermoFisher, Cat: 5555532). Sample extraction utilized Alinity m Sample Prep Kit 2. Alinity m You-Create HDV LDT analytical performance for sensitivity, linearity and precision was assessed using panels prepared by diluting the HDV 1st WHO international standard in negative plasma. Alinity m You-Create HDV LDT clinical performance was assessed by comparing 45 HDV positive samples previously tested on the m2000 HDV LDT. Assay specificity was assessed testing 97 HDV negative, HBV and/or HCV positive (by serology or nucleic acid amplification test). Results Alinity m You-Create HDV LDT had 100% detection as low as 2.5 IU/mL with SD ≤0.25 Log IU/mL for panels that were ≥2.5 IU/mL. Coefficient of correlation between Alinity m You-Create HDV LDT and m2000 HDV LDT was 0.969 and the mean bias was -0.16 Log IU/mL. All HDV negative specimens were undetected by Alinity m You-Create HDV LDT. Conclusions This study demonstrated successful implementation of an LDT for HDV on the Alinity m platform using the Alinity m You-Create feature using commercially available PCR reagents. The Alinity m You-Create feature for HDV has not been approved by FDA for use in the detection or diagnosis for HDV and its safety and effectiveness has not been established.

Spectrum of respiratory viruses identified from SARS-CoV-2-negative human respiratory tract specimens in Watansoppeng, Indonesia.

Respiratory infections account for millions of hospital admissions worldwide. The aetiology of respiratory infections can be attributed to a diverse range of pathogens including viruses, bacteria and fungi. SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2)-negative specimens from Wattansoppeng city, South Sulawesi, were analysed to study the spectrum of respiratory viruses. Samples were screened for influenza virus, enterovirus, Paramyxoviridae, Nipah virus, Coronaviridae and Pneumoviridae. Of 210 specimens, 19 were positive for respiratory syncytial virus (RSV)-A, RSV-B, human parainfluenza virus type 1 (HPIV-1), HPIV-2, human rhinovirus (HRV)-A, HRV-B, HRV-C, human metapneumovirus (HMPV), influenza A virus (IAV) and coxsackievirus A6 (CV-A6). Influenza virus was of seasonal H3N2 subtype. The HMPVs were of genotypes B1 and A2a, while one RSV-A was of the ON-1 genotype. The viruses mostly affected children with unknown severity.

Massilia shenzhen sp. nov., isolated from blood of one premature infant, causing sepsis

This study explores a premature infant with respiratory failure and pneumonia, suggestive of neonatal sepsis. Despite initially negative clinical specimens, blood testing revealed a pathogen. MALDI-TOF-MS and physiological tests initially failed to identify it accurately. Subsequent analysis of the 16S rRNA gene, housekeeping genes, and whole genome sequencing placed it in the genus Massilia. Average Nucleotide Identities (ANIs) indicated 88.47 % similarity with the type strain of Massilia norwichensis. Detailed characterization showed it as Gram-negative, aerobic, flagellated, measuring 0.45–0.55 × 1.75–2.40 μm. Major fatty acids included C16:0, C16:1ω7c, C18:1ω7c, and cyclo-C17:0. Minimum inhibitory concentrations to ceftazidime, penicillin, and meropenem were <0.032 μg/mL, ≤0.75 μg/mL, and <0.002 μg/mL respectively. Phylogenetic analysis, fatty acid composition, and physiological parameters confirmed it as Massilia shenzhen sp. nov., with strain GZ0329T. Given limited research on Massilia drug resistance, ceftazidime and imipenem show promise in treating Massilia infections.

Adequacy Assessment in Lymph Node Aspirates: An Exploratory Cytomorphologic Analysis of Negative Cervical Node Aspirates of Head and Neck Carcinomas

Introduction: Fine-needle aspiration cytology (FNAC) of lymph node is sensitive for detection of metastatic carcinoma but not without a significant false-negative rate. This study reviews clinicocytological features of negative node aspirates to identify predictive factors for establishing adequacy criteria. Methods: Negative FNAC specimens matched with neck dissection from a primary diagnosis of head and neck squamous cell, or undifferentiated (nasopharyngeal) carcinoma were reviewed for clinical and cytological parameters including lymphoid, inflammatory, and background components. Results: Slides from 86 lymph node aspirates including 50 positive for metastasis on follow-up were retrieved. Higher total lymphocyte count, lymphoid fragment count, germinal center fragment count, undifferentiated histology, presence of histiocytes and absence of blood were associated with a true negative cytologic diagnosis (p < 0.05), but not node size or location (p > 0.05). Undifferentiated histology, small lymphoid and germinal center fragments were independent factors indicative of a true negative diagnosis (p < 0.05). Large lymphoid fragments (p = 0.052) demonstrated a trend. Assessment of lymphoid components over five hotspots high-power fields (HPFs) was more robust in predictive value than only one hotspot. Receiver operating characteristic curve identified >10 small lymphoid, >20 large lymphoid and >2 germinal center fragment per five HPFs as optimal adequacy thresholds. Stricter total lymphocyte count cutoff accompanies increase of diagnostic accuracy, up to 0.67 for ≥5 HPFs with >500 lymphocytes. Conclusion: Total counts of lymphoid and germinal center fragments from multiple HPFs are useful in adequacy assessment of lymph node aspirates and improve diagnostic performance of FNAC in exclusion of metastatic carcinoma.

A novel high-performance rapid screening test for the detection of total HTLV-I and HTLV-II antibodies in HTLV-I/II infected patients

Rapid diagnosis of human T-cell lymphotropic virus (HTLV) type-I and -II infections are essential for timely and cost-effective disease interventions. MP Diagnostics ASSURE HTLV-I/II Rapid Test was developed for the rapid detection of anti-HTLV-I/II antibodies in patients’ serum, plasma, and whole blood specimens. ASSURE HTLV-I/II Rapid Test employed MP Biomedicals’ proprietary HTLV-I/II Trifusion recombinant antigen conjugated with gold nanoparticles and HTLV-I / HTLV-II recombinant antigens immobilized on the nitrocellulose membrane to detect total HTLV-I and HTLV-II antibodies. The overall performance of the ASSURE HTLV-I/II Rapid Test was found to be 99.42% sensitivity (95% Confidence Interval, 98.32–99.88%) and 100% specificity (95% Confidence Interval, 99.58–100.00%) in the tested clinical samples, including a total of 518 HTLV-I/II positive specimens (396 HTLV-I infection, 97 HTLV-II infection and 25 HTLV-I/II dual infection) and 872 HTLV negative clinical specimens consisting of 691 healthy donor samples, 116 potentially cross-reactive samples, and 65 samples with interfering substances. The ASSURE HTLV-I/II Rapid Test can effectively be deployed as a screening tool in any prevalence studies, blood banks or organ transplant centres.

Cushioning performance of origami negative poisson's ratio honeycomb steel structure

The honeycomb structure with a negative Poisson's ratio, inspired by the Miura origami unit, exhibits three-dimensional negative Poisson's ratio characteristics and effective energy dissipation performance. This unique property provides significant potential in the field of protection applications. This study aims to comprehensively understand the impact of auxetic and origami structures on the cushioning performance of honeycomb structures. For this purpose, 316 L stainless steel specimens were fabricated using 3D printing technology and subjected to drop hammer impact tests, and the results were verified by finite element simulation. This paper focuses on assessing the deformation mode and energy dissipation performance of origami auxetic honeycomb structures with varying width-to-thickness ratios and folding angles under different impact energy levels. The results indicate that the negative Poisson's ratio origami specimens exhibit higher plateau stress and specific energy absorption compared to their positive and zero Poisson's ratio origami counterparts with the same geometry. In addition, a smaller width-to-thickness ratio and higher input impact energy enhance the cushioning performance of honeycomb structures.

A Cross-Sectional Study of Measles-Specific Antibody Levels in Australian Blood Donors-Implications for Measles Post-Elimination Countries.

Passive immunisation with normal human immunoglobulin (NHIG) is recommended as post-exposure prophylaxis (PEP) for higher-risk measles contacts where vaccination is contraindicated. However, the concentration of measles-specific antibodies in NHIG depends on antibody levels within pooled donor plasma. There are concerns that measles immunity in the Australian population may be declining over time and that blood donors' levels will progressively decrease, impacting levels required to produce effective NHIG for measles PEP. A cross-sectional study of Australian plasmapheresis donors was performed using an age-stratified, random sample of recovered serum specimens, collected between October and November 2019 (n = 1199). Measles-specific IgG antibodies were quantified by ELISA (Enzygnost anti-measles virus IgG, Siemens), and negative and equivocal specimens (n = 149) also underwent plaque reduction neutralisation testing (PRNT). Mean antibody levels (optical density values) progressively decreased from older to younger birth cohorts, from 2.09 [±0.09, 95% CI] to 0.58 [±0.04, 95% CI] in donors born in 1940-1959 and 1990-2001, respectively (p < 0.0001). This study shows that mean measles-specific IgG levels are significantly lower in younger Australian donors. While current NHIG selection policies target older donors, as younger birth cohorts become an increasingly larger proportion of contributing donors, measles-specific antibody concentrations of NHIG will progressively reduce. We therefore recommend monitoring measles-specific antibody levels in future donors and NHIG products in Australia and other countries that eliminated measles before the birth of their youngest blood donors.

BD SurePath Direct to Slide (DTS) cervical cytology: Migrating the benefits of liquid-based cytology to low-resource settings.

The benefits of liquid-based cytology (LBC) in routine cervical cancer screening are often associated with the availability of instrumented platforms and economic considerations. A low-cost alternative to LBC in low-volume settings remains an unmet need. A multisite evaluation of the BD SurePath (SurePath) LBC Direct to Slide (DTS) method was conducted. The DTS preparations were evaluated across 3 sites. Cytology features for DTS preparation included predetermined thresholds for total cellularity, cell distribution, cellular preservation, and stain quality. Rare event detection was evaluated using SiHa cells spiked into pools from negative cytology specimens. Concordance between Bethesda classification results was evaluated for SurePath LBC and DTS methods using routinely collected SurePath specimens in a split-sample study design. The DTS specimens met criteria for total cellularity, cell distribution, cellular preservation, and stain quality in more than 98% of all cases. Rare event detection was observed with an average detection of 5 SiHa cells per 2mL of specimen. Concordant cervical cytology classifications were observed between SurePath LBC and DTS methods. The results demonstrate that the DTS process is suitable for routine cervical cytology evaluation. The procedure is reproducible and detected abnormal cervical cells in concordance with standard SurePath LBC preparation.

Digital Image Comparison of Cellular Yield in Bronchial Brushing: Pre- and Post-Biopsy Lavage Cytology

Introduction: Bronchoscopy is a useful diagnostic tool capable of performing core biopsy, forceps biopsy, bronchoalveolar lavage, and bronchial brushing. This study compares the cellularity of bronchial cytology including pre- and post-biopsy lavage by digital image analysis, aiming to increase diagnostic and tumor yield by optimizing the sequence and combination of bronchial biopsy and cytology. Methods: Alveolar macrophage, bronchial epithelium, and tumor cell cellularity from liquid-based cytology preparations of bronchial brushing and pre-biopsy and post-biopsy bronchoalveolar lavage were annotated on digitized whole-slide images and compared. Secondary analysis on the relationship of tumor cell and non-lesional cell yield was performed. Results: Overall, 118 cytology specimens from 43 patients were retrieved in total. Bronchial epithelium count was higher in pre-biopsy than post-biopsy lavage (p < 0.01) but not for alveolar macrophages nor tumor cell (p > 0.05). Tumor cell count was higher for bronchial brushing cytology samples than lavage (p = 0.018). The alveolar macrophage count was higher in post-biopsy lavage than bronchial brushing (p = 0.033); otherwise, brushing showed consistently higher bronchial epithelium and tumor cell counts. There were 33 false negative (tumor cell absent) specimens, and the combination of bronchial brushing and pre-biopsy lavage yielded the lowest false negative cases. Correlation between bronchial epithelium and alveolar macrophage counts with tumor cell count was weak (correlation coefficient = −0.168–0.203) except for post-biopsy lavage (correlation coefficient = 0.412–0.479, p < 0.05). Conclusion: Bronchial brushing yields a greater amount of tumor cell than lavage, and timing lavage before or after core biopsy does not affect tumor cell yield. Combining bronchial brushing and pre-biopsy lavage results in the lowest false negative rate.

Enhancing cancer detection through AI-driven high content analysis of circulating tumor cells.

3054 Background: The advancements in automated High Content Analysis (HCA) for Circulating Tumor Cells (CTCs) detection necessitate the integration of high-throughput screening (HTS) and Artificial Intelligence (AI) to improve cancer diagnosis efficiency and accuracy. Traditional approaches to cancer detection have faced limitations in speed, cost, and analysis depth, driving the need for innovative solutions in assay development and execution. Methods: We obtained peripheral blood from 150 asymptomatic individuals and 63 newly diagnosed cases of solid organ cancers including Lung, Breast, Head and Neck, Colorectal, Pancreas, Esophagus, Gynecologic cancers. We used differential apoptosis based negative enrichment technique and HCS fluorescent imaging for identification of CTCs and CTC like immune cells from the relevant blood samples. We devised AutoML algorithms which were trained on a cohort of 4,135 cropped region of interest (ROI) images of true CTCs and CTC-mimicking immune cells. These images were annotated with the coordinates of the top-left and bottom-right corners of rectangles to specify ROIs. This method employs a combination of automated sample processing, integration with the Laboratory Information Management System (LIMS) integration, digital filtration, and decision matrix algorithms for sample classification and smooth data flow. The image quality criteria were established prior to dataset analysis. The algorithms underwent training, testing, and validation based on expert recommendations, with dataset splitting of 80% for training, 10% for testing, and 10% for validation phases. Results: The automated deep learning model achieved remarkable performance metrics, presenting an accuracy of 96.66% (95% CI, 96.07%-97.19%), a specificity of 95.34% (95% CI, 94.37%-96.18%), and a sensitivity of 98.14% (95% CI, 97.44%-98.69%) in detecting true CTCs. Additional analysis included the area under the curve and confusion matrix evaluations, indicating the model's robustness in distinguishing between true positive and true negative specimens. This efficiency and precision in data handling, processing, and analysis demonstrate significant advancements in HCA screening, offering enhanced assay development, execution flexibility, and the facilitation of detailed cell-by-cell analysis for cancer detection from a large set of Peripheral Blood Mononuclear Cells (PBMCs) from samples obtained for screening or diagnosis. Conclusions: This study successfully evaluated a deep learning model for the detection of circulating tumor cells (CTCs) in peripheral blood mononuclear cells (PBMCs) using image data from HTS platform. The model achieved superior performance, demonstrating its potential for cancer detection from blood samples.

Serologic responses to the MVA-based JYNNEOS mpox vaccine in a cohort of participants from the District of Columbia (D.C.)

We assessed early antibody responses after two doses of JYNNEOS (IMVANEX) mpox vaccine in the District of Columbia (D.C.) in persons at high risk for mpox without characteristic lesions or rash. Participants with PCR mpox negative specimens (oral swab, blood, and/or rectal swab) on the day of receipt of the first vaccine dose and who provided a baseline (day 0) serum sample and at least one serum sample at ∼28, ∼42–56 days, or 180 days post vaccination were included in this analysis. Orthopoxvirus (OPXV)-specific IgG and IgM ELISAs and neutralizing antibody titers were performed, and longitudinal serologic responses were examined. Based on participants’ IgG and IgM antibody levels at baseline, they were categorized as naïve or non-naïve. Linear mixed effects regression models were conducted to determine if IgG antibody response over time varied by age, sex, HIV status, and route of administration for both naïve and non-naïve participants. Among both naïve and non-naïve participants IgG seropositivity rates increased until day 42–56, with 89.4 % of naïve and 92.1 % of non-naïve participants having detectable IgG antibodies. The proportion of naive participants with detectable IgG antibodies declined by day 180 (67.7 %) but remained high among non-naïve participants (94.4 %). Neutralizing antibody titers displayed a similar pattern, increasing initially post vaccination but declining by day 180 among naïve participants. There were no significant serologic response differences by age, sex, or HIV status. Serologic response did vary by route of vaccine administration, with those receiving a combination of intradermal and subcutaneous doses displaying significantly higher IgG values than those receiving both doses intradermally. These analyses provide initial insights into the immunogenicity of a two-dose JYNNEOS PEP regimen in individuals at high risk of mpox exposure in the United States.

Evaluation of different genomic regions of rotavirus B and rotavirus C for development of real-time RT‒PCR assays

BackgroundThe causative agents of diarrhea, rotavirus B (RVB) and rotavirus C (RVC) are common in adults and patients of all age groups, respectively. Due to the Rotavirus A (RVA) vaccination program, a significant decrease in the number of gastroenteritis cases has been observed globally. The replacement of RVA infections with RVB, RVC, or other related serogroups is suspected due to the possibility of reducing natural selective constraints due to RVA infections. The data available on RVB and RVC incidence are scant due to the lack of cheap and rapid commercial diagnostic assays and the focus on RVA infections. The present study aimed to develop real-time RT‒PCR assays using the data from all genomic RNA segments of human RVB and RVC strains available in the Gene Bank.ResultsAmong the 11 gene segments, NSP3 and NSP5 of RVB and the VP6 gene of RVC were found to be suitable for real-time RT‒PCR (qRT‒PCR) assays. Fecal specimens collected from diarrheal patients were tested simultaneously for the presence of RVB (n = 192) and RVC (n = 188) using the respective conventional RT‒PCR and newly developed qRT‒PCR assays. All RVB- and RVC-positive specimens were reactive in their respective qRT‒PCR assays and had Ct values ranging between 23.69 and 41.97 and 11.49 and 36.05, respectively. All known positive and negative specimens for other viral agents were nonreactive, and comparative analysis showed 100% concordance with conventional RT‒PCR assays.ConclusionsThe suitability of the NSP5 gene of RVB and the VP6 gene of RVC was verified via qRT‒PCR assays, which showed 100% sensitivity and specificity. The rapid qRT‒PCR assays developed will be useful diagnostic tools, especially during diarrheal outbreaks for testing non-RVA rotaviral agents and reducing the unnecessary use of antibiotics.

Characterization of the unique oral microbiome of children harboring Helicobacter pylori in the oral cavity

ABSTRACT Objective Helicobacter pylori infection is acquired in childhood via the oral cavity, although its relationship with the characteristics of the oral microbiome has not been elucidated. In this study, we performed comprehensive analysis of the oral microbiome in children and adults with or without H. pylori in the oral cavity. Methods Bacterial DNA was extracted from 41 adult and 21 child saliva specimens, and H. pylori was detected using PCR. 16S rRNA gene amplification was performed for next-generation sequencing. Bioinformatic analyses were conducted using Quantitative Insights into Microbial Ecology 2 (QIIME 2). Results Faith’s phylogenetic diversity analysis showed a significant difference between H. pylori-negative adult and child specimens in terms of α-diversity (p < 0.05), while no significant difference was observed between H. pylori-positive adult and child specimens. There was also a significant difference in β-diversity between H. pylori-positive and negative child specimens (p < 0.05). Taxonomic analysis at the genus level revealed that Porphyromonas was the only bacterium that was significantly more abundant in both H. pylori-positive adults and children than in corresponding negative specimens (p < 0.01 and p < 0.05, respectively). Conclusion These results suggest unique oral microbiome characteristics in children with H. pylori infection in the oral cavity.

Molecular Diagnosis of Human Monkeypox Virus during 2022-23 Outbreak: Preliminary Evaluation of Novel Real-Time Qualitative PCR Assays.

In 2022-23, the human monkeypox virus (MPXV) caused a global outbreak in several non-endemic countries. Here, we evaluated the diagnostic performance of four real-time qualitative PCR assays for the laboratory diagnosis of mpox (monkeypox) monkeypox disease. From July to August 2022, 27 positive and 10 negative specimens (lesion, crust and exudate swabs) were tested in the laboratory of the Hygiene Unit of the San Martino Hospital (Genoa, Italy) by using home-made real-time PCR to detect MPXV generic G2R_G DNA. According to the manufacturer's instructions, we also retrospectively analyzed these specimens using RealCycler MONK-UX/-GX (Progenie Molecular), STANDARD M10 MPX/OPX (SD Biosensor), Novaplex MPXV (Seegene Inc.) and RealStar Orthopoxvirus PCR Kit 1.0 (Altona Diagnostics) assays, recognized as research-use-only tests. The diagnostic accuracy and sensitivity of these assays ranged from 97.3% (95% CI: 86.2-99.5%) to 100% (95% CI: 90.6-100%) and 96.3% (95% CI: 81.72-99.34%) to 100% (95% CI: 72.2-100%), respectively. The RealCycler MONK-UX and STANDARD M10 MPX/OPX did not detect one positive sample with a cycle threshold of 36. The overall specificity was 100% (95% CI: 72.2-100%), and Cohen's Kappa values ranged from 1 (95% CI: 0.67-1) to 0.93 (95% CI: 0.61-1). As they are highly accurate, reliable and user-friendly, these tests should be recommended for the routine or rapid laboratory discrimination of mpox from other rash illnesses.

Strengthening Bordetella pertussis genomic surveillance by direct sequencing of residual positive specimens.

Whole-genome sequencing (WGS) of microbial pathogens recovered from patients with infectious disease facilitates high-resolution strain characterization and molecular epidemiology. However, increasing reliance on culture-independent methods to diagnose infectious diseases has resulted in few isolates available for WGS. Here, we report a novel culture-independent approach to genome characterization of Bordetella pertussis, the causative agent of pertussis and a paradigm for insufficient genomic surveillance due to limited culture of clinical isolates. Sequencing libraries constructed directly from residual pertussis-positive diagnostic nasopharyngeal specimens were hybridized with biotinylated RNA "baits" targeting B. pertussis fragments within complex mixtures that contained high concentrations of host and microbial background DNA. Recovery of B. pertussis genome sequence data was evaluated with mock and pooled negative clinical specimens spiked with reducing concentrations of either purified DNA or inactivated cells. Targeted enrichment increased the yield of B. pertussis sequencing reads up to 90% while simultaneously decreasing host reads to less than 10%. Filtered sequencing reads provided sufficient genome coverage to perform characterization via whole-genome single nucleotide polymorphisms and whole-genome multilocus sequencing typing. Moreover, these data were concordant with sequenced isolates recovered from the same specimens such that phylogenetic reconstructions from either consistently clustered the same putatively linked cases. The optimized protocol is suitable for nasopharyngeal specimens with diagnostic IS481 Ct < 35 and >10 ng DNA. Routine implementation of these methods could strengthen surveillance and study of pertussis resurgence by capturing additional cases with genomic characterization.