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Conventional cytogenetic analysis of products of conception (POC) is of limited utility because of failed cultures, microbial and maternal cell contamination (MCC). Optical genome mapping (OGM) is an emerging technology that has the potential to replace conventional cytogenetic methods. The use of OGM precludes the requirement for culturing (and related microbial contamination). However, a high percentage of MCC impedes a definitive diagnosis, which can be addressed by an additional pre-analytical quality control step that includes histological assessment of H&E stained slides from FFPE tissue with macro-dissection for Chorionic villi to enrich fetal tissue component for Single nucleotide polymorphism (SNP) microarray analysis. An internal audit of POC cases subjected to karyotype-only analysis showed a low yield on clinically actionable information that contributed to patient care. To improve the diagnostic yield, an integrated workflow was devised that included MCC characterization of POC tissue, followed by OGM for MCC negative cases or SNPM with histological assessment for MCC positive cases. A result was obtained in 93% (29/31) cases with a diagnostic yield of 45.1% (14/31) with proposed workflow compared to 9.6% (3/31) and 6.4% (2/31) with routine workflow, respectively. The integrated workflow with these technologies demonstrates the clinical utility and higher diagnostic yield in evaluating POC specimens.

The standard-of-care (SOC) for genomic testing of myeloid cancers primarily relies on karyotyping and fluorescent in situ hydridization (FISH) (cytogenetic analysis) and targeted gene panels (≤54 genes) that harbor hotspot pathogenic variants (molecular genetic analysis). Both cytogenetic and molecular testing workup is necessary for the identification and detection of large structural variants (SVs) and small variants like single nucleotide variants (SNV) and indels, respectively. Despite this combinatorial approach, ∼50% of myeloid cancer genomes remain cytogenetically normal, and the limited sequencing variant profiles obtained from targeted panels are unable to resolve the genetic etiology of these myeloid tumors. In this study, we evaluated the performance and clinical utility of optical genome mapping (OGM) and a 523-gene next-generation sequencing (NGS) panel for comprehensive genomic profiling of 15 myeloid tumors and compared it to SOC cytogenetic methods (karyotyping and FISH) and a 54-gene NGS panel. OGM and the 523-gene NGS panel were found to have an analytical concordance of 100% with karyotyping, FISH, and the 54-gene panel, respectively. Additionally, OGM better characterized and resolved the structural variants previously reported by karyotyping in five cases, such as identifying the genomic content of marker and ring chromosomes. OGM also identified several additional translocations and eleven copy number variations (CNVs), of which the CNVs were validated/confirmed by the 523-gene panel. The 523-gene panel identified seven additional clinically relevant SNVs (two tier 1A variants and five tier 2C variants, as per the ACMG/AMP guidelines) in four cases. The simultaneous visualization of SVs and small NGS detected sequence variants (SNVs and small indels) from OGM and 523-gene NGS panel, respectively in the NxClinical software v6.1 identified two clinically relevant compound heterozygous events in two samples. This study demonstrates the higher sensitivity, resolution, accuracy, and ability to reveal cryptic and clinically relevant novel variants in myeloid cancers as compared to SOC methodologies. Our cost-effective approach of using OGM and a 523-gene NGS panel for comprehensive genomic profiling of myeloid cancers will not only increase the yield of actionable targets leading to improved clinical outcomes but also help resolve our ongoing conundrum of apparently genomically normal myeloid cancers by providing more answers.

BackgroundIn the current phase of COVID-19 pandemic, we are facing two serious public health challenges that include deficits in SARS-CoV-2 variant monitoring, and neglect of other co-circulating respiratory viruses. Additionally, accurate assessment of the evolution, extent and dynamics of the outbreak are required to understand the transmission of the virus amongst seemingly unrelated cases and provide critical epidemiological information. To address these challenges, we evaluated a new high-throughput next-generation sequencing (NGS), respiratory viral panel (RVP) that includes 40 viral pathogens with the aim of analyzing viral subtypes, mutational variants of SARS-CoV-2, model to understand the spread of the virus in the state of Georgia, USA, and to assess other circulating viruses in the same population.MethodsThis study evaluated a total of 522 samples that included 483 patient samples and 42 synthetic positive control material. The performance metrics were calculated for both clinical and reference control samples by comparing detection results with the RT-PCR assay. The limit of detection (LoD) studies were conducted as per the FDA guidelines. Inference and visualization of the phylogeny of the SARS-CoV-2 sequences were performed through the Nextstrain Command-Line Interface (CLI) tool, utilizing the associated augur and auspice toolkits.ResultsThe performance metrics calculated using both the clinical samples and the reference controls revealed a PPA, NPA and accuracy of 95.98%, 85.96% and 94.4%, respectively. The LoD was determined to be 10 copies/ml with all 25 replicates detected across two different runs. The clade for pangolin lineage B that contains certain distant variants, including P4715L in ORF1ab, Q57H in ORF 3a and, S84L in ORF8 covarying with the D614G spike protein mutation were the most prevalent, early in the pandemic, in Georgia, USA. In our analysis, isolates from the same county formed paraphyletic groups, which indicated virus transmission between counties.ConclusionThe study demonstrates the clinical and public health utility of the NGS-RVP to identify novel variants that can provide actionable information to prevent or mitigate emerging viral threats, models that provide insights into viral transmission patterns and predict transmission/ resurgence of regional outbreaks and provide critical information on co-circulating respiratory viruses that might be independent factors contributing to the global disease burden.

BackgroundThe COVID-19 pandemic has resulted in a significant diversion of human and material resources to COVID-19 diagnostics, to the extent that testing of viral pathogens normally contributing to seasonal respiratory tract infections have been markedly neglected. The global health burden due to influenza viruses and co-infection in COVID-19 patients remains undocumented but clearly pose serious public health consequences. To address these clinical and technical challenges, we have optimized and validated a highly sensitive RT-PCR based multiplex assay for the detection of SARS-CoV-2, Influenza A and B viruses in a single test.MethodsThis study evaluated clinical specimens (n=1411) that included 1019 saliva and 392 nasopharyngeal swab (NPS) samples collected in either healthcare or community setting. Samples were tested using two assays: FDA-EUA approved SARS-CoV-2 assay that targets N and ORF1ab gene, and the PKamp RT-PCR based assay that targets SARS-CoV-2, Influenza viruses A and B. The limit of detection (LoD) studies was conducted as per the FDA guidelines using SARS-CoV-2 and Influenza A and B reference control materials.ResultsOf the 1019 saliva samples, 17.0% (174/1019) tested positive for SARS-CoV-2 using either assay. The detection rate for SARS-CoV-2 was higher with our multiplex assay compared to SARS-specific assay [91.9% (160/174) vs. 87.9% (153/174)], respectively. Of the 392 NPS samples, 10.4% (41/392) tested positive for SARS-CoV-2 using either assay. The detection rate for SARS-CoV-2 was higher with our multiplex assay compared to SARS-specific assay [97.5% (40/41) vs. 92.1% (39/41)], respectively. The Ct values for SARS-CoV-2 were comparable between the two assays, whereas the Ct values of the housekeeping gene was significantly lower with multiplex assay compared to SARS-specific assay. The LoD was established as 60 copies/ml for SARS-CoV-2 and 180 copies/ml for Influenza A and B viruses for both saliva and NPS samples.ConclusionThis study presents clinical validation of a multiplex PCR assay for testing SARS-CoV-2, Influenza A and B viruses, using NPS and saliva samples, and demonstrates the feasibility of implementing the assay without disrupting the existing laboratory workflow. This novel assay uses the same instruments, sample types, supplies, and laboratory personnel as needed for the testing of SARS-CoV-2 virus.

BackgroundThe adoption of saliva as a specimen type for SARS-CoV-2 mass surveillance can significantly increase population compliance with decreased exposure risk for healthcare workers. However, studies evaluating the clinical performance of saliva compared to nasopharyngeal swab (NPS) samples have demonstrated conflicting results regardless of the collection being in healthcare or community settings. Further, pooled testing with saliva remains a challenge owing to the ambiguous sensitivity, limit of detection (LoD), and processing challenges. To overcome these limitations, SalivaAll protocol was developed and validated as a cost-effective measure that must be used on saliva collected in health care or community settings with pooling utility for SARS-CoV-2 mass surveillance.MethodsThe study evaluated 429 matched NPS and saliva samples collected from 344 individuals in either healthcare or community setting. In phase I (protocol U), 240 matched NPS, and saliva samples were tested for SARS-CoV-2 detection by RT-PCR. In phase II (SalivaAll protocol), 189 matched NPS and saliva samples were tested, with an additional sample homogenization step for saliva before RNA extraction, followed by RT-PCR. Eighty-five saliva samples were evaluated with both protocols (U and SalivaAll). Subsequently, adopting SalivaAll protocol, a five-sample pooling strategy was evaluated for saliva samples based on FDA recommendations.ResultsIn phase I, 28.3% (68/240) samples tested positive for SARS-CoV-2 from either saliva, NPS, or both. The detection rate was lower in saliva compared to NPS samples (50.0% vs. 89.7%). In phase II, 50.2% (95/189) samples tested positive for SARS-CoV-2 from either saliva, NPS, or both. The detection rate for SARS-CoV-2 was higher in saliva compared to NPS testing (97.8% vs. 78.9%). Of the 85 saliva samples evaluated by both protocols, 57.6% (49) tested positive for SARS-CoV-2 with either protocol U, SalivaAll, or both. The detection rate was 100% for samples tested with SalivaAll, whereas it was 36.7% with protocol U. Also, the LoD with SalivaAll protocol was 20 copies/ml. The pooled testing approach demonstrated a 95% positive and 100% negative percent agreement.ConclusionThis single-site study demonstrated the variability of results reported in the literature for saliva samples, and found that the discrepancies are explained by processing challenges associated with saliva samples. We have optimized a protocol for saliva samples that results in higher sensitivity compared to NPS samples and also breaks the barrier to using pooled saliva testing for SARS-CoV-2.SummarySalivaAll is a very sensitive (LoD 20 copies/ml) cost-effective test validated on saliva collected in health care and community settings with pooling utility and submitted for FDA Emergency Use Authorization.

The current gold-standard molecular diagnosis for COVID-19 is based on a multi-step assay involving RNA-extraction and RT-PCR analysis for the detection of SARS-CoV-2. RNA-extraction step has been a major rate-limiting step in implementing high-throughput screening for COVID-19 during this pandemic. Moreover, clinical laboratories are facing several challenges that include cost, reagents, instrumentation, turn-around time, trained personnel, and supply-chain constraints to efficiently implement and sustain testing. Cognizant of these limitations, we evaluated the extraction-free methods described in the literature and have developed an innovative, simplified and easy protocol employing limited reagents to extract RNA for subsequent RT-PCR analysis. Nasopharyngeal-swab samples were subjected to the following individual conditions: 65°C for 15 minutes; 80°C for 5 minutes; 90°C for 5 minutes or 80°C for 1 minute, and processed for direct RT-PCR. These groups were also compared with a supplemental protocol adding isopropanol-ethanol-water elution steps followed by RT-PCR assay. The direct RT-PCR assay did not detect SARS-CoV-2 within the various temperature incubation only groups, whereas, the 90°C for 5 minutes-isopropanol-ethanol-water method was found to be comparable to the FDA-EUA method. Evaluation of the performance metrics for 100 clinical samples demonstrated a sensitivity of 94.2% and a specificity of 100%. The limit of detection was ascertained to be ∼40 copies/ml by absolute-quantification. The protocol presented for this assay employs limited reagents and yields results with high sensitivity. Additionally, it presents a simplified methodology that would be easier to implement in laboratories in limited resource countries in order to meet the high current COVID-19 testing needs.