COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme.

Laboratory tests for the accurate and rapid identification of SARS-CoV-2 variants can potentially guide the treatment of COVID-19 patients and inform infection control and public health surveillance efforts. Here, we present the development and validation of a rapid COVID-19 variant DETECTR assay incorporating loop-mediated isothermal amplification (LAMP) followed by CRISPR-Cas12 based identification of single nucleotide polymorphism (SNP) mutations in the SARS-CoV-2 spike (S) gene. This assay targets the L452R, E484K/Q/A, and N501Y mutations, at least one of which is found in nearly all major variants. In a comparison of three different Cas12 enzymes, only the newly identified enzyme CasDx1 was able to accurately identify all targeted SNP mutations. An analysis pipeline for CRISPR-based SNP identification from 261 clinical samples yielded a SNP concordance of 97.3% and agreement of 98.9% (258 of 261) for SARS-CoV-2 lineage classification, using SARS-CoV-2 whole-genome sequencing and/or real-time RT-PCR as test comparators. We also showed that detection of the single E484A mutation was necessary and sufficient to accurately identify Omicron from other major circulating variants in patient samples. These findings demonstrate the utility of CRISPR-based DETECTR as a faster and simpler diagnostic method compared with sequencing for SARS-CoV-2 variant identification in clinical and public health laboratories.

Metagenomic Next-Generation Sequencing for Identification and Quantitation of Transplant-Related DNA Viruses.

Infections with DNA viruses are frequent causes of morbidity and mortality in transplant recipients. This study describes the analytical and clinical performance characteristics of the Arc Bio Galileo Pathogen Solution, an all-inclusive metagenomic next-generation sequencing (mNGS) reagent and bioinformatics pipeline that allows the simultaneous quantitation of 10 transplant-related double-stranded DNA (dsDNA) viruses (adenovirus [ADV], BK virus [BKV], cytomegalovirus [CMV], Epstein-Barr virus [EBV], human herpesvirus 6A [HHV-6A], HHV-6B, herpes simplex virus 1 [HSV-1], HSV-2, JC virus [JCV], and varicella-zoster virus [VZV]). The mNGS 95% limit of detection ranged from 14 copies/ml (HHV-6) to 191 copies/ml (BKV), and the lower limit of quantitation ranged from 442 international units (IU)/ml (EBV) to 661 copies/ml (VZV). An evaluation of 50 residual plasma samples with at least one DNA virus detected in prior clinical testing showed a total percent agreement of mNGS and quantitative PCR (qPCR) of 89.2% (306/343), with a κ statistic of 0.725. The positive percent agreement was 84.9% (73/86), and the negative percent agreement was 90.7% (233/257). Furthermore, mNGS detected seven subsequently confirmed coinfections that were not initially requested by qPCR. Passing-Bablok regression revealed a regression line of y = 0.953x + 0.075 (95% confidence interval [CI] of the slope, 0.883 to 1.011; intercept, -0.100 to 0.299), and Bland-Altman analysis (mNGS - qPCR) showed a slight positive bias (0.28 log10 concentration; 95% limits of agreement, -0.62 to 1.18). In conclusion, the mNGS-based Galileo pipeline demonstrates analytical and clinical performance comparable to that of qPCR for transplant-related DNA viruses.

Development and clinical evaluation of a novel fully automated qualitative PCR assay for the diagnosis of anogenital herpes simplex virus infection.

Molecular detection of viral infections has the potential to improve microbial diagnostics, particularly with the emergence of rapid automated systems. We describe the design of the IDbox fully automated cassette-based system for nucleic acid extraction and real-time PCR amplification and perform a clinical evaluation for the diagnosis of genital herpes simplex infections. At optimal cutoff values determined by receiver-operator curves, the IDbox showed sensitivities of 94.9% (95% confidence interval [CI] 84.9-98.7%) and 97.0% (95% CI 88.5-99.5%) and specificities of 96.7% (95% CI 91.2-98.9%) and 97.3% (95% CI 91.9-99.3%) relative to herpes simplex virus culture and PCR, respectively. We discuss relevant design characteristics and approaches used for each step of the analytical process to enhance assay sensitivity and provide accurate results in the presence of potential cross-reactive organisms and interfering substances.

A quantitative reverse transcription-PCR assay for rapid, automated analysis of breast cancer sentinel lymph nodes.

We have previously reported that a quantitative reverse transcription (QRT)-PCR assay accurately analyzes sentinel lymph nodes (SLNs) from breast cancer patients. The aim of this study was to assess a completely automated, cartridge-based version of the assay for accuracy, predictive value, and reproducibility. The triplex (two markers + control) QRT-PCR assay was incorporated into a single-use cartridge for point-of-care use on the GeneXpert system. Three academic centers participated equally. Twenty-nine positive lymph nodes and 30 negative lymph nodes were analyzed to establish classification rules. SLNs from 120 patients were subsequently analyzed by QRT-PCR and histology (including immunohistochemistry), and the predetermined decision rules were used to classify the SLNs; 112 SLN specimens produced an informative result by both QRT-PCR and histology. By histological analysis, 21 SLNs were positive and 91 SLNs were negative for metastasis. QRT-PCR characterization produced a classification with 100% sensitivity, 97.8% specificity, and 98.2% accuracy compared with histology (91.3% positive predictive value and 100% negative predictive value). Interlaboratory reproducibility analyses demonstrated that a 95% prediction interval for a new measurement (DeltaCt) ranged between 0.403 and 0.956. This fully automated QRT-PCR assay accurately characterizes breast cancer SLNs for the presence of metastasis. Furthermore, the assay is not dependent on subjective interpretation, is reproducible across three clinical environments, and is rapid enough to allow intraoperative decision making.

A rapid, fully automated, molecular-based assay accurately analyzes sentinel lymph nodes for the presence of metastatic breast cancer.

OBJECTIVE: To develop a fully automated, rapid, molecular-based assay that accurately and objectively evaluates sentinel lymph nodes (SLN) from breast cancer patients. SUMMARY BACKGROUND DATA: Intraoperative analysis for the presence of metastatic cancer in SLNs from breast cancer patients lacks sensitivity. Even with immunohistochemical staining (IHC) and time-consuming review, alarming discordance in the interpretation of SLN has been observed. METHOD: A total of 43 potential markers were evaluated for the ability to accurately characterize lymph node specimens from breast cancer patients as compared with complete histologic analysis including IHC. Selected markers then underwent external validation on 90 independent SLN specimens using rapid, multiplex quantitative reverse transcription-polymerase chain reaction (QRT-PCR) assays. Finally, 18 SLNs were analyzed using a completely automated RNA isolation, reverse transcription, and quantitative PCR instrument (GeneXpert). RESULTS: : Following analysis of potential markers, promising markers were evaluated to establish relative level of expression cutoff values that maximized classification accuracy. A validation set of 90 SLNs from breast cancer patients was prospectively characterized using 4 markers individually or in combinations, and the results compared with histologic analysis. A 2-marker assay was found to be 97.8% accurate (94% sensitive, 100% specific) compared with histologic analysis. The fully automated GeneXpert instrument produced comparable and reproducible results in less than 35 minutes. CONCLUSIONS: A rapid, fully automated QRT-PCR assay definitively characterizes breast cancer SLN with accuracy equal to conventional pathology. This approach is superior to intraoperative SLN analysis and can provide standardized, objective results to assist in pathologic diagnosis.

Technology for automated, rapid, and quantitative PCR or reverse transcription-PCR clinical testing.

BACKGROUND: PCR-based assays can improve clinical care, but they remain technically demanding and labor-intensive. We describe a new instrument, the GeneXpert, that performs automated nucleic acid isolation, reverse transcription, and fluorescence-based quantitative PCR in approximately 35 min. METHODS: Yield and integrity of RNA isolated on the GeneXpert were compared with Qiagen-based extraction for parallel samples (5-microm frozen tissue sections). The reproducibility of automated RNA isolation, reverse transcription, and quantitative PCR was determined by replicate (n = 10) analysis of 10 tissues, using duplex (target and endogenous control) reverse transcription-PCR reactions for two gene combinations. The GeneXpert was then used to perform rapid analysis of lymph nodes from melanoma, breast cancer, and lung cancer patients and analysis of melanoma metastatic to the lung, primary lung adenocarcinoma, and healthy lung tissue. RESULTS: On the GeneXpert, RNA was recovered in slightly over 6 min, and the yield was approximately 70% of that from parallel Qiagen reactions. The RNA integrity was comparable to that of Qiagen-isolated RNA as determined by gel electrophoresis. For the melanoma samples, the 95% prediction interval for the deltaCt for a new measurement was +/-1.54 cycles, and for breast cancer samples, the interval for a newly observed deltaCt was +/-1.40 cycles. GeneXpert assays successfully detected the presence of metastatic melanoma, breast cancer, and lung cancer in lymph nodes and also differentiated among metastatic melanoma, lung adenocarcinoma, and healthy lung. CONCLUSIONS: RNA yield and integrity on the GeneXpert are comparable to benchtop methods. Reproducibility of the GeneXpert data is similar to that seen with manual methods in our hands but may need improvement for some applications. The GeneXpert can perform RNA isolation, reverse transcription, and quantitative PCR in approximately 35 min and could therefore be used for intraoperative testing when applicable.

Molecular staging of cervical lymph nodes in squamous cell carcinoma of the head and neck.

Clinical staging of cervical lymph nodes from patients with squamous cell carcinoma of the head and neck (SCCHN) has only 50% accuracy compared with definitive pathologic assessment. Consequently, both clinically positive and clinically negative patients frequently undergo neck dissections that may not be necessary. To address this potential overtreatment, sentinel lymph node (SLN) biopsy is currently being evaluated to provide better staging of the neck. However, to fully realize the potential improvement in patient care afforded by the SLN procedure, a rapid and accurate SLN analysis is necessary. We used quantitative reverse transcription-PCR (QRT-PCR) to screen 40 potential markers for their ability to detect SCCHN metastases to cervical lymph nodes. Seven markers were identified with good characteristics for identifying metastatic disease, and these were validated using a set of 26 primary tumors, 19 histologically positive lymph nodes, and 21 benign nodes from patients without cancer. Four markers discriminated between positive and benign nodes with accuracy >97% but only one marker, pemphigus vulgaris antigen (PVA), discriminated with 100% accuracy in both the observed data and a statistical bootstrap analysis. A rapid QRT-PCR assay for PVA was then developed and incorporated into a prototype instrument capable of performing fully automated RNA isolation and QRT-PCR. The automated analysis with PVA provided perfect discrimination between histologically positive and benign lymph nodes and correctly identified two lymph nodes with micrometastatic tumor deposits. These assays were completed (from tissue to result) in approximately 30 minutes, thus demonstrating the feasibility of intraoperative staging of SCCHN SLNs by QRT-PCR.

Human genomic DNA analysis using a semi-automated sample preparation, amplification, and electrophoresis separation platform.

The growing importance of analyzing the human genome to detect hereditary and infectious diseases associated with specific DNA sequences has motivated us to develop automated devices to integrate sample preparation, real-time PCR, and microchannel electrophoresis (MCE). In this report, we present results from an optimized compact system capable of processing a raw sample of blood, extracting the DNA, and performing a multiplexed PCR reaction. Finally, an innovative electrophoretic separation was performed on the post-PCR products using a unique MCE system. The sample preparation system extracted and lysed white blood cells (WBC) from whole blood, producing DNA of sufficient quantity and quality for a polymerase chain reaction (PCR). Separation of multiple amplicons was achieved in a microfabricated channel 30 microm x 100 microm in cross section and 85 mm in length filled with a replaceable methyl cellulose matrix operated under denaturing conditions at 50 degrees C. By incorporating fluorescent-labeled primers in the PCR, the amplicons were identified by a two-color (multiplexed) fluorescence detection system. Two base-pair resolution of single-stranded DNA (PCR products) was achieved. We believe that this integrated system provides a unique solution for DNA analysis.