Nikhil Shri Sahajpal

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.

Trigger finger is a common yet vastly understudied fibroproliferative hand pathology, severely affecting patients’ quality of life. Consistent trauma due to inadequate positioning within the afflicted finger’s tendon/pulley system leads to cellular dysregulation and eventual fibrosis. While the genetic characteristics of the fibrotic tissue in the trigger finger have been studied, the pathways that govern the initiation and propagation of fibrosis are still unknown. The complete gene expression profile of the trigger finger has never been explored. Our study has used the Nanostring nCounter gene expression assay to investigate the molecular signaling involved in trigger finger pathogenesis. We collected samples from patients undergoing trigger finger (n = 4) release surgery and compared the gene expression to carpal tunnel tissue (n = 4). Nanostring nCounter analysis identified 165 genes that were differentially regulated; 145 of these genes were upregulated, whereas 20 genes were downregulated. We found that several collagen genes were significantly upregulated, and a regulatory matrix metalloproteinase (MMP), MMP-3, was downregulated. Bioinformatic analysis revealed that several known signaling pathways were dysregulated, such as the TGF-β1 and Wnt signaling pathways. We also found several novel signaling pathways (e.g., PI3K, MAPK, JAK-STAT, and Notch) differentially regulated in trigger finger. The outcome of our study helps in understanding the molecular signaling pathway involved in the pathogenesis of the trigger finger.

Two serious public health challenges have emerged in the current COVID-19 pandemic namely, 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 is required to understand the transmission of the virus. To address these challenges, we evaluated 533 samples using a high-throughput next-generation sequencing (NGS) respiratory viral panel (RVP) that includes 40 viral pathogens. The performance metrics revealed a PPA, NPA, and accuracy of 95.98%, 85.96%, and 94.4%, respectively. The clade for pangolin lineage B that contains certain distant variants, including P4715L in ORF1ab, Q57H in ORF3a, and S84L in ORF8 covarying with the D614G spike protein mutation, were the most prevalent early in the pandemic in Georgia, USA. The isolates from the same county formed paraphyletic groups, indicating virus transmission between counties. The 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 and models that provide insights into viral transmission patterns and predict transmission/resurgence of regional outbreaks as well as providing 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.

Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer that accounts for almost 85% of lung cancer cases worldwide. Although recent advances in chemotherapy, radiotherapy, and immunotherapy have helped in the clinical management of these patients, the survival rate in advanced stages remains dismal. Furthermore, there is a critical lack of accurate prognostic and stratification markers for emerging immunotherapies. To harness immune response modalities for therapeutic benefits, a detailed understanding of the immune cells in the complex tumor microenvironment (TME) is required. Among the diverse immune cells, natural killer (NK cells) and dendritic cells (DCs) have generated tremendous interest in the scientific community. NK cells play a critical role in tumor immunosurveillance by directly killing malignant cells. DCs link innate and adaptive immune systems by cross-presenting the antigens to T cells. The presence of an immunosuppressive milieu in tumors can lead to inactivation and poor functioning of NK cells and DCs, which results in an adverse outcome for many cancer patients, including those with NSCLC. Recently, clinical intervention using modified NK cells and DCs have shown encouraging response in advanced NSCLC patients. Herein, we will discuss prognostic and predictive aspects of NK cells and DC cells with an emphasis on NSCLC. Additionally, the discussion will extend to potential strategies that seek to enhance the anti-tumor functionality of NK cells and DCs.

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.

Objectives: Limitations of widespread current COVID-19 diagnostic testing exist in both the pre-analytical and analytical stages. To alleviate these limitations, we developed a universal saliva processing protocol (SalivaSTAT) that would enable an extraction-free RT-PCR test using commercially available RT-PCR kits. Methods: We optimized saliva collection devices, heat-shock treatment, and homogenization. Saliva samples (879) previously tested using the FDA-EUA method were reevaluated with the optimized SalivaSTAT protocol using two widely available commercial RT-PCR kits. A five-sample pooling strategy was evaluated as per FDA guidelines. Results: Saliva collection (done without any media) showed performance comparable to that of the FDA-EUA method. The SalivaSTAT protocol was optimized by incubating saliva samples at 95 °C for 30-min and homogenization, followed by RT-PCR assay. The clinical sample evaluation of 630 saliva samples using the SalivaSTAT protocol with PerkinElmer (600-samples) and CDC (30-samples) RT-PCR assay achieved positive (PPA) and negative percent agreements (NPAs) of 95.0% and 100%, respectively. The LoD was established as ~60–180 copies/mL by absolute quantification. Furthermore, a five-sample-pooling evaluation using 250 saliva samples achieved a PPA and NPA of 92% and 100%, respectively. Conclusion: We have optimized an extraction-free RT-PCR assay for saliva samples that demonstrates comparable performance to FDA-EUA assay (Extraction and RT-PCR).

Global medical associations (ACOG, ISUOG, ACMG) recommend diagnostic prenatal testing for the detection and prevention of genetic disorders. Historically, cytogenetic methods such as karyotype analysis, fluorescent in situ hybridization (FISH) and chromosomal microarray (CMA) are utilized worldwide to diagnose common syndromes. However, the limitations of each of these methods, either performed in tandem or simultaneously, demonstrates the need of a revolutionary technology that can alleviate the need for multiple technologies. Optical genome mapping (OGM) is a novel method that fills this void by being able to detect all classes of structural variations (SVs), including copy number variations (CNVs). OGM is being adopted by laboratories as a tool for both postnatal constitutional genetic disorders and hematological malignancies. This commentary highlights the potential for OGM to become a standard of care in prenatal genetic testing based on its capability to comprehensively identify large balanced and unbalanced SVs (currently the strength of karyotyping and metaphase FISH), CNVs (by CMA), repeat contraction disorders (by Southern blotting) and multiple repeat expansion disorders (by PCR-based methods or Southern blotting). Next-generation sequencing (NGS) methods are excellent at detecting sequence variants, but they are unable to accurately resolve repeat regions of the genome, which limits their ability to detect all classes of SVs. Notably, multiple molecular methods are used to identify repeat expansion and contraction disorders in routine clinical laboratories around the world. With non-invasive prenatal testing (NIPT) becoming the standard of care screening assay for all global pregnancies, we anticipate that OGM can provide a high-resolution, cytogenomic assay to be employed following a positive NIPT screen or for high-risk pregnancies with an abnormal ultrasound. Accurate detection of all types of genetic disorders by OGM, such as liveborn aneuploidies, sex chromosome anomalies, microdeletion/microduplication syndromes, repeat expansion/contraction disorders is key to reducing the global burden of genetic disorders.

Lung cancer is one of the leading causes of death worldwide. Cell death pathways such as autophagy, apoptosis, and necrosis can provide useful clinical and immunological insights that can assist in the design of personalized therapeutics. In this study, variations in the expression of genes involved in cell death pathways and resulting infiltration of immune cells were explored in lung adenocarcinoma (The Cancer Genome Atlas: TCGA, lung adenocarcinoma (LUAD), 510 patients). Firstly, genes involved in autophagy (n = 34 genes), apoptosis (n = 66 genes), and necrosis (n = 32 genes) were analyzed to assess the prognostic significance in lung cancer. The significant genes were used to develop the cell death index (CDI) of 21 genes which clustered patients based on high risk (high CDI) and low risk (low CDI). The survival analysis using the Kaplan–Meier curve differentiated patients based on overall survival (40.4 months vs. 76.2 months), progression-free survival (26.2 months vs. 48.6 months), and disease-free survival (62.2 months vs. 158.2 months) (Log-rank test, p < 0.01). Cox proportional hazard model significantly associated patients in high CDI group with a higher risk of mortality (Hazard Ratio: H.R 1.75, 95% CI: 1.28–2.45, p < 0.001). Differential gene expression analysis using principal component analysis (PCA) identified genes with the highest fold change forming distinct clusters. To analyze the immune parameters in two risk groups, cytokines expression (n = 265 genes) analysis revealed the highest association of IL-15RA and IL 15 (> 1.5-fold, p < 0.01) with the high-risk group. The microenvironment cell-population (MCP)-counter algorithm identified the higher infiltration of CD8+ T cells, macrophages, and lower infiltration of neutrophils with the high-risk group. Interestingly, this group also showed a higher expression of immune checkpoint molecules CD-274 (PD-L1), CTLA-4, and T cell exhaustion genes (HAVCR2, TIGIT, LAG3, PDCD1, CXCL13, and LYN) (p < 0.01). Furthermore, functional enrichment analysis identified significant perturbations in immune pathways in the higher risk group. This study highlights the presence of an immunocompromised microenvironment indicated by the higher infiltration of cytotoxic T cells along with the presence of checkpoint molecules and T cell exhaustion genes. These patients at higher risk might be more suitable to benefit from PD-L1 blockade or other checkpoint blockade immunotherapies.

Background: The 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. Methods: The 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. Results: In 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. Conclusion: This 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. Summary: SalivaAll 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.