Partial ORF1ab Gene Target Failure with Omicron BA.2.12.1.

Mutations in the genome of SARS-CoV-2 can affect the performance of molecular diagnostic assays. In some cases, such as S-gene target failure, the impact can serve as a unique indicator of a particular SARS-CoV-2 variant and provide a method for rapid detection. Here, we describe partial ORF1ab gene target failure (pOGTF) on the cobas SARS-CoV-2 assays, defined by a ≥2-thermocycle delay in detection of the ORF1ab gene compared to that of the E-gene. We demonstrate that pOGTF is 98.6% sensitive and 99.9% specific for SARS-CoV-2 lineage BA.2.12.1, an emerging variant in the United States with spike L452Q and S704L mutations that may affect transmission, infectivity, and/or immune evasion. Increasing rates of pOGTF closely mirrored rates of BA.2.12.1 sequences uploaded to public databases, and, importantly, increasing local rates of pOGTF also mirrored increasing overall test positivity. Use of pOGTF as a proxy for BA.2.12.1 provides faster tracking of the variant than whole-genome sequencing and can benefit laboratories without sequencing capabilities.

Clinical Evaluation of a Real-Time PCR Assay for Simultaneous Detection of Helicobacter pylori and Genotypic Markers of Clarithromycin Resistance Directly from Stool.

Helicobacter pylori infection is mainly diagnosed noninvasively, with susceptibility testing traditionally requiring endoscopy. Treatment is empirical, with clarithromycin-based triple therapy recommended where resistance rates are below 15%. Rising rates of clarithromycin resistance, resulting in high clarithromycin-based therapy failure rates, are seen worldwide, but U.S. data are limited. We developed a real-time PCR assay for simultaneous detection of H. pylori and genotypic markers of clarithromycin resistance directly from stool specimens. The assay was validated by testing 524 stool samples using an H. pylori stool antigen test as the reference method for detection accuracy and Sanger sequencing to confirm genotypic susceptibility results. A separate set of 223 antigen-positive stool samples was tested and retrospective medical record review conducted to define clinical utility. PCR resulted in 88.6% and 92.8% sensitivity in the validation and clinical study sets, respectively. Sequencing confirmed correct detection of clarithromycin resistance-associated mutations in all positive validation samples. The PCR-predicted clarithromycin resistance rate was 39% in the clinical data set overall and 31% in treatment-naive patients; the clarithromycin-based triple therapy eradication rate in treatment-naive patients was 62%. The clarithromycin-based triple therapy success was lower when resistance was predicted by PCR (41%) than when no resistance was predicted (70%; P = 0.03). PCR results were positive in 98% of antigen-positive stools from patients tested for eradication. The described PCR assay can accurately and noninvasively diagnose H. pylori, provide genotypic susceptibility, and test for eradication. Our findings support the need for susceptibility-guided therapy in our region if a clarithromycin-based regimen is considered.

SARS-CoV-2 coinfection with additional respiratory virus does not predict severe disease: a retrospective cohort study.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) claimed over 4 million lives by July 2021 and continues to pose a serious public health threat. OBJECTIVES: Our retrospective study utilized respiratory pathogen panel (RPP) results in patients with SARS-CoV-2 to determine if coinfection (i.e. SARS-CoV-2 positivity with an additional respiratory virus) was associated with more severe presentation and outcomes. METHODS: All patients with negative influenza/respiratory syncytial virus testing who underwent RPP testing within 7 days of a positive SARS-CoV-2 test at a large, academic medical centre in New York were examined. Patients positive for SARS-CoV-2 with a negative RPP were compared with patients positive for SARS-CoV-2 and positive for a virus by RPP in terms of biomarkers, oxygen requirements and severe COVID-19 outcome, as defined by mechanical ventilation or death within 30 days. RESULTS: Of the 306 SARS-CoV-2-positive patients with RPP testing, 14 (4.6%) were positive for a non-influenza virus (coinfected). Compared with the coinfected group, patients positive for SARS-CoV-2 with a negative RPP had higher inflammatory markers and were significantly more likely to be admitted (P = 0.01). Severe COVID-19 outcome occurred in 111 (36.3%) patients in the SARS-CoV-2-only group and 3 (21.4%) patients in the coinfected group (P = 0.24). CONCLUSIONS: Patients infected with SARS-CoV-2 along with a non-influenza respiratory virus had less severe disease on presentation and were more likely to be admitted-but did not have more severe outcomes-than those infected with SARS-CoV-2 alone.

Performance Characteristics of a Targeted Sequencing Platform for Simultaneous Detection of Single Nucleotide Variants, Insertions/Deletions, Copy Number Alterations, and Gene Fusions in Cancer Genome.

CONTEXT.­: An increasing number of molecular laboratories are implementing next-generation sequencing platforms to identify clinically actionable and relevant genomic alterations for precision oncology. OBJECTIVE.­: To describe the validation studies as per New York State-Department of Health (NYS-DOH) guidelines for the Oncomine Comprehensive Panel v2, which was originally tailored to the National Cancer Institute Molecular Analysis for Therapy Choice (NCI-MATCH) trial. DESIGN.­: Accuracy, precision, and reproducibility were investigated by using 130 DNA and 18 RNA samples from cytology cell blocks; formalin-fixed, paraffin-embedded tissues; and frozen samples. Analytic sensitivity and specificity were tested by using ATCC and HapMap cell lines. RESULTS.­: High accuracy and precision/reproducibility were observed for single nucleotide variants and insertion/deletions. We also share our experience in the detection of gene fusions and copy number alterations from an amplicon-based sequencing platform. After sequencing analysis, variant annotation and report generation were performed by using the institutional knowledgebase. CONCLUSIONS.­: This study serves as an example for validating a comprehensive targeted next-generation sequencing assay with both DNASeq and RNASeq components for NYS-DOH.

Subareolar Sclerosing Ductal Hyperplasia.

Subareolar sclerosing duct hyperplasia (SSDH) remains to be fully characterized nearly 20 years after initial description. Thirty-five SSDH cases diagnosed over a 16-year period (January 2000 to December 2015) were reviewed. All patients were female (mean age = 59 years, range = 18-80) who had presented with a unilateral solitary lesion (left 22, right 13) with a mean size of 1.3 cm (range = 0.4-3.0 cm), and showed florid and papillary epithelial hyperplasia with dense sclerosis without involvement of nipple or areolar epidermis. Significant lesions concurrent within SSDH included low-grade adenosquamous carcinoma (n = 1), ductal carcinoma in situ (DCIS; n = 1), lobular carcinoma in situ (LCIS; n = 1), and atypical ductal hyperplasia (ADH; n = 13). No case of SSDH recurred in a mean follow-up of 44 months (range = 6-189). Subsequent significant lesions occurred in 6 patients: DCIS (n = 3; ipsilateral 2, contralateral 1), ipsilateral ADH (n = 2), and ipsilateral atypical lobular hyperplasia (n = 1). Long-term follow-up for patients with SSDH is indicated as DCIS can occur subsequently in either breast.