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The standard-of-care (SOC) for genomic testing of myeloid cancers primarily relies on karyotyping/fluorescent in situ hybridization (FISH) (cytogenetic analysis) and targeted gene panels (usually ≤54 genes) that harbor hotspot pathogenic variants (molecular genetic analysis). 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 molecular etiology of many myeloid tumors. In this study, we evaluated the performance and clinical utility of combinatorial use of optical genome mapping (OGM) and a 523-gene next-generation sequencing (NGS) panel for comprehensive genomic profiling of 30 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 had an analytical concordance of 100% with karyotyping, FISH, and the 54-gene panel, respectively. Importantly, the IPSS-R cytogenetic risk group changed from very good/good to very poor in 22% of MDS (2/9) cases based on comprehensive profiling (karyotyping, FISH, and 54-gene panel vs. OGM and 523-gene panel), while additionally identifying six compound heterozygous events of potential clinical relevance in six cases (6/30, 20%). This cost-effective approach of using OGM and a 523-gene NGS panel for comprehensive genomic profiling of myeloid cancers demonstrated increased yield of actionable targets that can potentially result in improved clinical outcomes.

Effects of the combination therapy of anti-OX40 and anti-PD-1 Abs with tumor vaccine on spleen and tumor T-cell infiltration and clonality.

Combination therapies that depend on checkpoint inhibitor antibodies (Abs) such as for PD-1 or its ligand (PD-L1) together with immune stimulatory agonist Abs like anti-OX40 are being tested in the clinic to achieve improved antitumor effects. Here, we studied the potential therapeutic and immune effects of one such combination: Ab to PD-1 with agonist Ab to OX40/vaccine. We tested the antitumor effects of different treatment sequencing of this combination. We report that simultaneous addition of anti–PD-1 to anti-OX40 negated the antitumor effects of OX40 Ab. Antigen-specific CD8+ T-cell infiltration into the tumor was diminished, the resultant antitumor response weakened, and survival reduced. Although we observed an increase in IFNγ-producing E7-specifc CD8+ T cells in the spleens of mice treated with the combination of PD-1 blockade with anti-OX40/vaccine, these cells underwent apoptosis both in the periphery and the tumor. These results indicate that anti–PD-1 added at the initiation of therapy exhibits a detrimental effect on the positive outcome of anti-OX40 agonist Ab. These findings have important implications on the design of combination immunotherapy for cancer, demonstrating the need to test treatment combination and sequencing before moving to the clinic. Cancer Immunol Res; 5(9); 755–66. ©2017 AACR.

Supplementary Figure Legends

Response to vaccines in WT type and PI3K-delta KO mice

In vivo data from an experiment starting the therapy at a later date with larger starting tumor size

PI3K-delta inhibition preserves memory phenotype (all conetrations)

PI3K-delta KD in murine cells and PI3K-delta inhibition in human cell

PI3K-delta controls the differentiation of CD8 T-cells through the downstream Akt1 and Akt2

PI3K-delta inhibition preserves IL-2 production and does not affect cytotoxic cytokine secretion (all concentrations)

XLSX file - 43K, Supplementary Table 4 contains the top 100 down regulated genes that are differentially expressed in MB231-pCDH and MB231-GPR109A stable clones in the presence and absence of niacin.

XLSX file - 42K, Supplementary Table 3 contains the top 100 upregulated genes that are differentially expressed in MB231-pCDH and MB231-GPR109A stable clones in the presence and absence of niacin.

XLSX file - 27K, Supplementary Table 2 contains genes that are differentially expressed in MB231-pCDH and MB231-GPR109A stable clones without niacin treatment.

Short-chain fatty acids, generated in colon by bacterial fermentation of dietary fiber, protect against colorectal cancer and inflammatory bowel disease. Among these bacterial metabolites, butyrate is biologically most relevant. GPR109A is a G-protein–coupled receptor for nicotinate but recognizes butyrate with low affinity. Millimolar concentrations of butyrate are needed to activate the receptor. Although concentrations of butyrate in colonic lumen are sufficient to activate the receptor maximally, there have been no reports on the expression/function of GPR109A in this tissue. Here we show that GPR109A is expressed in the lumen-facing apical membrane of colonic and intestinal epithelial cells and that the receptor recognizes butyrate as a ligand. The expression of GPR109A is silenced in colon cancer in humans, in a mouse model of intestinal/colon cancer, and in colon cancer cell lines. The tumor-associated silencing of GPR109A involves DNA methylation directly or indirectly. Reexpression of GPR109A in colon cancer cells induces apoptosis, but only in the presence of its ligands butyrate and nicotinate. Butyrate is an inhibitor of histone deacetylases, but apoptosis induced by activation of GPR109A with its ligands in colon cancer cells does not involve inhibition of histone deacetylation. The primary changes in this apoptotic process include down-regulation of Bcl-2, Bcl-xL, and cyclin D1 and up-regulation of death receptor pathway. In addition, GPR109A/butyrate suppresses nuclear factor-κB activation in normal and cancer colon cell lines as well as in normal mouse colon. These studies show that GPR109A mediates the tumor-suppressive effects of the bacterial fermentation product butyrate in colon. [Cancer Res 2009;69(7):2826–32]

PDF file - 548K, Supplementary Table S1- Primers used for the RT-PCR assays. Figure S1. DNA methylation inhibitor re-activates GPR109A expression in human breast cancer cells. Figure S2. Stable expression of GPR109A in human breast cancer cell lines. Figure S3. GPR109A activation in breast cancer cells inhibits anti-apoptotic genes and induces pro-apoptotic genes. Figure S4. GPR109A activation in breast cancer cells regulates genes involved in various cellular processes. Figure S5. GPR109A binding is inhibited by components in the media. A, MB231-pCDH and MB231-GPR109A stable cell lines were subjected to radio-labeled nicotinate (NA) binding with binding buffer and L15 media.

Supplementary Figure Legends 1-5 from GPR109A Is a G-protein–Coupled Receptor for the Bacterial Fermentation Product Butyrate and Functions as a Tumor Suppressor in Colon

Supplementary Figure S3 from SLC5A8 Triggers Tumor Cell Apoptosis through Pyruvate-Dependent Inhibition of Histone Deacetylases

The clinical performance of saliva compared with nasopharyngeal swabs (NPSs) has shown conflicting results in healthcare and community settings. In the present study, a total of 429 matched NPS and saliva sample pairs, collected in either healthcare or community setting, were evaluated. Phase-1 (protocol U) tested 240 matched NPS and saliva sample pairs; phase 2 (SalivaAll protocol) tested 189 matched NPS and saliva sample pairs, with an additional sample homogenization step before RNA extraction. A total of 85 saliva samples were evaluated with both protocols. In phase-1, 28.3% (68/240) samples tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from saliva, NPS, or both. The detection rate from saliva was lower compared with that from NPS samples (50.0% versus 89.7%). In phase-2, 50.2% (95/189) samples tested positive for SARS-CoV-2 from saliva, NPS, or both. The detection rate from saliva was higher compared with that from NPS samples (97.8% versus 78.9%). Of the 85 saliva samples evaluated with both protocols, the detection rate was 100% for samples tested with SalivaAll, and 36.7% with protocol U. The limit of detection with SalivaAll protocol was 20 to 60 copies/mL. The pooled testing approach demonstrated a 95% positive and 100% negative percentage agreement. This protocol for saliva samples results in higher sensitivity compared with NPS samples and breaks the barrier to using pooled saliva for SARS-CoV-2 testing.

Conventional cytogenetic analysis of products of conception (POC) is of limited utility because of failed cultures, as well as 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 formalin-fixed paraffin embedded (FFPE) tissue with macro-dissection for chorionic villi to enrich fetal tissue component for single nucleotide polymorphism microarray (SNPM) analysis. 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) of cases with a diagnostic yield of 45.1% (14/31) with the 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.

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.

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) is an infectious virus that causes coronavirus disease 2019 (COVID-19) transmitted mainly through droplets and aerosol affecting the respiratory tract and lungs. Little is known regarding why some individuals are more susceptible than others and develop severe symptoms. In this study, we analyzed the nasopharyngeal microbiota profile of aged patients with COVID-19 (asymptomatic vs. symptomatic) vs. healthy individuals. We examined the nasopharynx swab of 84 aged-matched patients, out of which 27 were negative asymptomatic (NegA), 30 were positive asymptomatic (PA), and 27 patients were positive symptomatic (PSY). Our analysis revealed the presence of abundant Cyanobacterial taxa at phylum level in PA (p-value = 0.0016) and PSY (p-value = 0.00038) patients along with an upward trend in the population of Litoricola, Amylibacter, Balneola, and Aeromonas at the genus level. Furthermore, to know the relationship between the nasal microbiota composition and severity of COVID-19, we compared PA and PSY groups. Our data show that the nasal microbiota of PSY patients was significantly enriched with the signatures of two bacterial taxa: Cutibacterium (p-value = 0.045) and Lentimonas (p-value = 0.007). Furthermore, we also found a significantly lower abundance of five bacterial taxa, namely: Prevotellaceae (p-value = 7 × 10−6), Luminiphilus (p-value = 0.027), Flectobacillus (p-value = 0.027), Comamonas (p-value = 0.048), and Jannaschia (p-value = 0.012) in PSY patients. The dysbiosis of the nasal microbiota in COVID-19 positive patients might have a role in contributing to the severity of COVID-19. The findings of our study show that there is a strong correlation between the composition of the nasal microbiota and COVID-19 severity. Further studies are needed to validate our finding in large-scale samples and to correlate immune response (cytokine Strome) and nasal microbiota to identify underlying mechanisms and develop therapeutic strategies against COVID-19.

We describe the clinical validation of a targeted DNA and RNA-based next-generation sequencing (NGS) assay at two clinical molecular diagnostic laboratories. This assay employs simultaneous DNA and RNA analysis of all coding exons to detect small variants (single-nucleotide variants, insertions, and deletions) in 148 genes, amplifications in 59 genes, and fusions and splice variants in 55 genes. During independent validations at two sites, 234 individual specimens were tested, including clinical formalin-fixed, paraffin-embedded (FFPE) tumor specimens, reference material, and cell lines. Samples were prepared using the Illumina TruSight Tumor 170 (TST170) kit, sequenced with Illumina sequencers, and the data were analyzed using the TST170 App. At both sites, TST170 had ≥98% success for ≥250× depth for ≥95% of covered positions. Variant calling was accurate and reproducible at allele frequencies ≥5%. Limit of detection studies determined that inputs of ≥50 ng of DNA (with ≥3.3 ng/μl) and ≥50 ng RNA (minimum of 7 copies/ng) were optimal for high analytical sensitivity. The TST170 assay results were highly concordant with prior results using different methods across all variant categories. Optimization of nucleic acid extraction and DNA shearing, and quality control following library preparation is recommended to maximize assay success rates. In summary, we describe the validation of comprehensive and simultaneous DNA and RNA-based NGS testing using TST170 at two clinical sites.

This review discusses the current testing methodologies for COVID-19 diagnosis and explores next-generation sequencing (NGS) technology for the detection of SARS-CoV-2 and monitoring phylogenetic evolution in the current COVID-19 pandemic. The review addresses the development, fundamentals, assay quality control and bioinformatics processing of the NGS data. This article provides a comprehensive review of the obstacles and opportunities facing the application of NGS technologies for the diagnosis, surveillance, and study of SARS-CoV-2 and other infectious diseases. Further, we have contemplated the opportunities and challenges inherent in the adoption of NGS technology as a diagnostic test with real-world examples of its utility in the fight against COVID-19.

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.

Oxidative damage has been identified as a major causative factor in degenerative diseases of the retina, retinal pigment epithelial (RPE) cells are at high risk. Hence, identifying novel strategies for increasing the antioxidant capacity of RPE cells, the purpose of this study, is important. Specifically, we evaluated the influence of selenium in the form of selenomethionine (Se-Met) in cultured RPE cells on system xc- expression and functional activity and on cellular levels of glutathione, a major cellular antioxidant. ARPE-19 and mouse RPE cells were cultured with and without selenomethionine (Se-Met), the principal form of selenium in the diet. Promoter activity assay, uptake assay, RT-PCR, northern and western blots, and immunofluorescence were used to analyze the expression of xc-, Nrf2, and its target genes. Se-Met activated Nrf2 and induced the expression and function of xc- in RPE. Other target genes of Nrf2 were also induced. System xc- consists of two subunits, and Se-Met induced the subunit responsible for transport activity (SLC7A11). Selenocysteine also induced xc- but with less potency. The effect of Se-met on xc- was associated with an increase in maximal velocity and an increase in substrate affinity. Se-Met increased the cellular levels of glutathione in the control, an oxidatively stressed RPE. The Se-Met effect was selective, under identical conditions, taurine transport was not affected and Na+-coupled glutamate transport was inhibited. This study demonstrates that Se-Met enhances the antioxidant capacity of RPE by inducing the transporter xc- with a consequent increase in glutathione.

RT-PCR-based assays for the detection of SARS-CoV-2 have played an essential role in the current COVID-19 pandemic. However, the sample collection and test reagents are in short supply, primarily due to supply chain issues. Thus, to eliminate testing constraints, we have optimized three key process variables: RNA extraction and RT-PCR reactions, different sample types and media to facilitate SARS-CoV-2 testing. By performing various validation and bridging studies, we have shown that various sample types such as nasopharyngeal swab, bronchioalveolar lavage and saliva, collected using conventional nasopharyngeal swabs, ESwab or 3D-printed swabs and, preserved in viral transport media, universal transport media, 0.9% sodium chloride or Amies media are compatible with RT-PCR assay for COVID-19. Besides, the reduction of PCR reagents by up to fourfold also produces reliable results.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing has lagged in many countries because of test kit shortages and analytical process bottlenecks. This study investigated the feasibility and accuracy of a sample pooling approach for wide-scale population screening for coronavirus disease 2019. A total of 940 nasopharyngeal swab samples (934 negative and 6 positive) previously tested for SARS-CoV-2 were deidentified and assigned random numbers for analysis, and 94 pools of 10 samples each were generated. Automated RNA extraction, followed by RT-PCR, was performed in a 96-well plate. Positive pools were identified, and the individual samples were reanalyzed. Of the 94 pools/wells, four were positive [Ct values: N (22.7 to 28.3), ORF1ab (23.3 to 27.2), and internal control (34.4 to 35.4)]. The 40 samples comprising the four pools were identified and reanalyzed individually; six samples were positive, with Ct values of N gene, ORF1ab, and internal control comparable to their respective wells. Additional experiments were performed on samples with high Ct values, and overall results showed 91.6% positive and 100% negative agreement compared with individual testing approach. Thus, 940 samples were tested in 148 reactions compared with 940 reactions in routine screening. The sample pooling strategy may help catch up with testing needs and minimal turnaround times and facilitate enormous savings on laboratory supplies, extraction, and PCR kits currently in short supply.

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 availability of large quantities and diverse of live foods organisms such as microalgae, rotifer, Artemia, cladocerans, copepods, polychaetes etc., to meet the different stages of fry production is need of the hour. Live feeds such as microalgae, rotifers, Artemia, cladocerans and copepods play vital role as initial feed for the tiny finfish and shellfish larvae, fingerlings and juveniles during hatchery rearing. Some appreciable works are available on microalgae culture in India especially in laboratory level including Gopinathan who made attempt on the culture of phytoflagellates. A few attempts have been made by some previous workers on cladocerans culture in laboratory level in India. Although the polychaetes are good alternate feed for larvae and brooders of both edible and ornamental fish and shrimp, unfortunately even till today the polychaetes culture is not started in India.

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.