Development and Validation of Three Automated High-Throughput Molecular Tests to Detect Monkeypox Virus Infections.

BACKGROUND: The 2022 outbreak of the clade IIb monkeypox virus and subsequent global spread lead to an urgent need for the development of high-throughput, sensitive, and reproducible diagnostic tests. METHODS: We developed 3 assays to detect monkeypox virus, 2 (MPXV+ and MPXV) for m2000 RealTime and 1 (MPXV) for Alinity m platforms. Dual targets in E9L and B6R (MPXV+) and J2L and B7R (MPXV) increased mutation resistance. In silico prediction indicates MPXV+ cross-reactivity with orthopox viruses and specific monkeypox virus detection with MPXV. RESULTS: m2000 RealTime MPXV+ and MPXV assay sensitivity was determined to be 3.2 plaque-forming units/mL using a reference virus culture diluted into universal transport medium (UTM). Alinity m MPXV lower limit of detection was 200 copies/mL using monkeypox virus plasmids in pooled UTM matrix. m2000 RealTime MPXV+ and MPXV assays were validated with lesion swabs in UTM and 1:1 saliva to UTM mixtures. Commercially available and remnant clinical lesion specimens in UTM were tested with RealTime MPXV+, RealTime MPXV and Alinity m MPXV assays and demonstrated high agreement to known mpox (MPX)-positive specimens. CONCLUSIONS: RealTime MPXV+, RealTime MPXV, and Alinity MPXV are high throughput and sensitive assays used for the detection of monkeypox virus. These assays maybe useful during MPX outbreaks.

Development of the RealTime SARS-CoV-2 quantitative Laboratory Developed Test and correlation with viral culture as a measure of infectivity.

While diagnosis of COVID-19 relies on qualitative molecular testing for the absence or presence of SARS-CoV-2 RNA, quantitative viral load determination for SARS-CoV-2 has many potential applications in antiviral therapy and vaccine trials as well as implications for public health and quarantine guidance. To date, no quantitative SARS-CoV-2 viral load tests have been authorized for clinical use by the FDA. In this study, we modified the FDA emergency use authorized qualitative RealTime SARS-CoV-2 assay into a quantitative SARS-CoV-2 Laboratory Developed Test (LDT) using newly developed Abbott SARS-CoV-2 calibration standards. Both analytical and clinical performance of this SARS-CoV-2 quantitative LDT was evaluated using nasopharyngeal swabs (NPS). We further assessed the correlation between Ct and the ability to culture virus on Vero CCL81 cells. The SARS-CoV-2 quantitative LDT demonstrated high linearity with R2 value of 0.992, high inter- and intra-assay reproducibility across the dynamic range (SDs ± 0.08-0.14 log10 copies/mL for inter-assay reproducibility and ± 0.09 to 0.19 log10 copies/mL for intra-assay reproducibility). Lower limit of detection was determined as 1.90 log10 copies/mL. The highest Ct at which CPE was detected ranged between 28.21-28.49, corresponding to approximately 4.2 log10 copies/mL. Quantitative tests, validated against viral culture capacity, may allow more accurate identification of individuals with and without infectious viral shedding from the respiratory tract.

Multicenter clinical evaluation of alinity m HCV assay performance.

BACKGROUND: Nucleic acid testing is essential for the detection and quantification of HCV RNA in the diagnosis of HCV infection and treatment monitoring. The Alinity m HCV assay was recently developed by Abbott Molecular for rapid detection and quantification of HCV RNA on the fully automated, continuous, random-access Alinity m analyzer. OBJECTIVES: Our study assessed the performance of the new Alinity m HCV assay for detection and quantification of HCV RNA in a large series of patient samples of various genotypes. This international, multicentric study evaluated the linearity, precision, and reproducibility of the Alinity m HCV assay and its performance in comparison to three other HCV assays currently used in clinical practice. RESULTS: The Alinity m HCV assay demonstrated high linearity (correlation coefficient r = 1.00), precision (coefficients of variation [CV] 6.6-13.5 %) and reproducibility (CV 1.7-4.3 % across three control lots). At a concentration near the lower limit of detection, the Alinity m HCV assay exhibited >98 % detectability. The Alinity m HCV assay showed excellent correlation with comparator HCV assays in serum (n = 406) and plasma (n = 1401) samples (correlation coefficients ≥0.96, bias 0.01 to 0.14 Log10 IU/mL). More than 95 % of the quantified results with the Alinity m HCV assay were less than mean bias ± 1.96 SD different from those of the comparator assays. CONCLUSIONS: The newly developed Alinity m HCV assay is sensitive, reproducible, and accurately quantifies HCV RNA levels in serum and plasma samples from patients with chronic HCV infection, with no impact of HCV genotype on assay performance.

Lack of a Prognostic Impact of the MyD88 L265P Mutation for Diffuse Large B Cell Lymphoma Patients Undergoing Autologous Stem Cell Transplantation.

Cell-of-origin determination has emerged as an important prognostic factor for patients initially diagnosed with diffuse large B cell lymphoma (DLBCL). Specifically, the nongerminal center B cell-like (non-GCB) subtype, composed predominantly of the activated B cell-like (ABC) molecular subtype, has been shown to portend poor prognosis because of its more aggressive nature and resistance to standard cyclophosphamide, hydroxydaunorubicin, oncovin, prednisone (CHOP)-like chemotherapy compared with the GCB subtype. The recurrent MyD88 L265P mutation, present in 29% of ABC DLBCL, was reported as an independent poor prognostic factor for patients with newly diagnosed DLBCL. For patients whose disease relapses or is refractory to first-line chemotherapy, high-dose chemotherapy with autologous stem cell transplantation (ASCT) is frequently offered as salvage therapy. However, the impact of MyD88 mutation status on post-ASCT outcome has not been reported. Here, we retrospectively analyzed, with up to 20 years of follow-up, 165 patients who underwent ASCT for relapsed/refractory DLBCL at our institution. We found that MyD88 mutation status did not correlate with overall survival (OS), post-ASCT OS, or progression-free survival (PFS). Patients with non-GCB subtype had significantly worse OS from initial diagnosis and after ASCT. Notably, high International Prognostic Index score was predictive of poor pre- and post-transplant PFS and post-transplant OS.

Evaluation of the Abbott RealTime HCV genotype II plus RUO (PLUS) assay with reference to core and NS5B sequencing.

BACKGROUND: HCV genotyping remains a critical tool for guiding initiation of therapy and selecting the most appropriate treatment regimen. Current commercial genotyping assays may have difficulty identifying 1a, 1b and genotype 6. OBJECTIVE: To evaluate the concordance for identifying 1a, 1b, and genotype 6 between two methods: the PLUS assay and core/NS5B sequencing. STUDY DESIGN: This study included 236 plasma and serum samples previously genotyped by core/NS5B sequencing. Of these, 25 samples were also previously tested by the Abbott RealTime HCV GT II Research Use Only (RUO) assay and yielded ambiguous results. The remaining 211 samples were routine genotype 1 (n=169) and genotype 6 (n=42). Genotypes obtained from sequence data were determined using a laboratory-developed HCV sequence analysis tool and the NCBI non-redundant database. RESULTS: Agreement between the PLUS assay and core/NS5B sequencing for genotype 1 samples was 95.8% (162/169), with 96% (127/132) and 95% (35/37) agreement for 1a and 1b samples respectively. PLUS results agreed with core/NS5B sequencing for 83% (35/42) of unselected genotype 6 samples, with the remaining seven "not detected" by the PLUS assay. Among the 25 samples with ambiguous GT II results, 15 were concordant by PLUS and core/NS5B sequencing, nine were not detected by PLUS, and one sample had an internal control failure. CONCLUSIONS: The PLUS assay is an automated method that identifies 1a, 1b and genotype 6 with good agreement with gold-standard core/NS5B sequencing and can aid in the resolution of certain genotype samples with ambiguous GT II results.

Performance attributes of the LCx HCV RNA quantitative assay.

The LCx HCV RNA quantitative assay (Abbott Laboratories, North Chicago, IL) is designed to use competitive reverse transcriptase-polymerase chain reaction (RT-PCR) and microparticle enzyme immunoassay (MEIA), in combination with a modified Qiagen sample preparation method, to measure the level of hepatitis C virus (HCV) in human plasma and serum. The assay provides quantitative results in international units (IU) of HCV RNA/ml, in copies of HCV RNA/ml, or their log (base 10) equivalents. A conversion study determined that 1IU equals 4.3 copies. The LCx HCV assay detected HCV RNA transcripts representative of genotypes 1-6 with near equal efficiency. The assay did not cross-react with high concentrations of 21 potentially cross-reactive microorganisms or with 100 HCV-negative specimens. The lower limit of detection was demonstrated to be 23IU/ml. The LCx assay had similar sensitivity to the Roche Amplicor HCV (version 2.0) qualitative assay when used to test panels containing 6, 12, 23, and 47IU/ml. The assay linear range was shown to extend from 23 to 2.3millionIU/ml. The intra-assay standard deviation (S.D.) was < or =0.066 logIU/ml for the four HCV positive samples tested, while for the same samples the observed inter-assay S.D. was < or =0.075 logIU/ml. The overall mean assay quantitation value for seven HCV-positive WHO-standardized Acrometrix NAP linearity panel members was within 0.06 logIU/ml of the mean assigned value. The assay was demonstrated to correlate acceptably against the Roche Amplicor HCV monitor test (version 2.0). These data suggest that the assay is standardized appropriately against the WHO standard across its linear range and can be used for quantitation of HCV. In addition, with a sensitivity of 23IU/ml, the assay can be used to determine if post-therapy viral clearance has occurred.