Advantage of precision metagenomics for urinary tract infection diagnostics

Background: Urinary tract infections (UTIs) remain a diagnostic challenge and often promote antibiotic overuse. Despite urine culture being the gold standard for UTI diagnosis, some uropathogens may lead to false-negative or inconclusive results. Although PCR testing is fast and highly sensitive, its diagnostic yield is limited to targeted microorganisms. Metagenomic next-generation sequencing (mNGS) is a hypothesis-free approach with potential of deciphering the urobiome. However, clinically relevant information is often buried in the enormous amount of sequencing data. Methods: Precision metagenomics (PM) is a hybridization capture-based method with potential of enhanced discovery power and better diagnostic yield without diluting clinically relevant information. We collected 47 urine samples of clinically suspected UTI and in parallel tested each sample by microbial culture, PCR, and PM; then, we comparatively analyzed the results. Next, we phenotypically classified the cumulative microbial population using the Explify® data analysis platform for potential pathogenicity. Results: Results revealed 100% positive predictive agreement (PPA) with culture results, which identified only 13 different microorganisms, compared to 19 and 62 organisms identified by PCR and PM, respectively. All identified organisms were classified into phenotypic groups (0–3) with increasing pathogenic potential and clinical relevance. This PM can simultaneously quantify and phenotypically classify the organisms readily through bioinformatic platforms like Explify®, essentially providing dissected and quantitative results for timely and accurate empiric UTI treatment. Conclusion: PM offers potential for building effective diagnostic models beyond usual care testing in complex UTI diseases. Future studies should assess the impact of PM-guided UTI management on clinical outcomes

COVIDSeq as Laboratory Developed Test (LDT) for Diagnosis of SARS-CoV-2 Variants of Concern (VOC)

Rapid classification and detection of SARS-CoV-2 variants have been critical in comprehending the virus\u27s transmission dynamics. Clinical manifestation of the infection is influenced by comorbidities such as age, immune status, diabetes, and the infecting variant. Thus, clinical management may differ for new variants. For example, some monoclonal antibody treatments are variant-specific. Yet, a U.S. Food and Drug Administration (FDA)-approved test for detecting the SARS-CoV-2 variant is unavailable. A laboratory-developed test (LDT) remains a viable option for reporting the infecting variant for clinical intervention or epidemiological purposes. Accordingly, we have validated the Illumina COVIDSeq assay as an LDT according to the guidelines prescribed by the College of American Pathologists (CAP) and Clinical Laboratory Improvement Amendments (CLIA). The limit of detection (LOD) of this test is Ct(~15 viral copies) and \u3e200X genomic coverage, and the test is 100% specific in the detection of existing variants. The test demonstrated 100% precision in inter-day, intra-day, and intra-laboratory reproducibility studies. It is also 100% accurate, defined by reference strain testing and split sample testing with other CLIA laboratories. Advanta Genetics LDT COVIDSeq has been reviewed by CAP inspectors and is under review by FDA for Emergency Use Authorization

Optimization of the Illumina COVIDSeq™ protocol for decentralized, cost-effective genomic surveillance

A decentralized surveillance system to identify local outbreaks and monitor SARS-CoV-2 Variants of Concern is one of the primary strategies for the pandemic’s containment. Although nextgeneration sequencing (NGS) is a gold standard for genomic surveillance and variant discovery, the technology is still cost-prohibitive for decentralized sequencing, particularly in small independent labs with limited resources. We have optimized the Illumina COVIDSeq™ protocol for the Illumina MiniSeq instrument to reduce cost without compromising accuracy. We slashed the library preparation cost by half by using 50% of recommended reagents at each step and normalizing the libraries before pooling to achieve uniform coverage. Reagent-only cost (~ $43.27/sample) for SARS-CoV-2 variant analysis with this normalized input protocol on MiniSeq instruments is comparable to what is achieved on high throughput instruments such as NextSeq and NovaSeq. Using this modified protocol, we tested 153 clinical samples, and 90% of genomic coverage was achieved for 142/153 samples analyzed in this study. The lineage was correctly assigned to all samples (152/153) except for one. This modified protocol can help laboratories with constrained resources to contribute in decentralized COVID-19 surveillance in the postvaccination era

Deciphering microbiota of acute upper respiratory infections: A comparative analysis of PCR and mNGS methods for lower respiratory trafficking potential

Although it is clinically important for acute respiratory tract (co)infections to have a rapid and accurate diagnosis, it is critical that respiratory medicine understands the advantages of current laboratory methods. In this study, we tested nasopharyngeal samples (n = 29) with a commercially available PCR assay and compared the results with those of a hybridization-capture-based mNGS workflow. Detection criteria for positive PCR samples was Ct \u3c 35 and for mNGS samples it was \u3e40% target coverage, median depth of 1X and RPKM \u3e 10. A high degree of concordance (98.33% PPA and 100% NPA) was recorded. However, mNGS yielded positively 29 additional microorganisms (23 bacteria, 4 viruses, and 2 fungi) beyond PCR. We then characterized the microorganisms of each method into three phenotypic categories using the IDbyDNA Explify® Platform (Illumina® Inc, San Diego, CA, USA) for consideration of infectivity and trafficking potential to the lower respiratory region. The findings are significant for providing a comprehensive yet clinically relevant microbiology profile of acute upper respiratory infection, especially important in immunocompromised or immunocompetent with comorbidity respiratory cases or where traditional syndromic approaches fail to identify pathogenicity. Accordingly, this technology can be used to supplement current syndrome-based tests, and data can quickly and effectively be phenotypically characterized for trafficking potential, clinical (co)infection, and comorbid consideration—with promise to reduce morbidity and mortality

Investigation of the Enhancement of Neuroprotective Efficacy of Water Soluble Ashwagandha Extract Alone and in Combination with Ubisol- Q10 for Parkinson’s Disease

Parkinson’s disease (PD) is the second most common chronic and progressive neurodegenerative disease and the most common movement disorder. PD is characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc), leading to symptoms such as tremors, bradykinesia, postural instability, and rigidity. Biochemical pathologies of PD include oxidative stress, autophagy and mitochondrial dysfunction, and neuroinflammation. Current treatments for PD provide only symptomatic relief and fail to halt progression of neurodegeneration. Prolonged treatment can result in adverse motor/behavioural side effects. Previous studies have shown that supplementary antioxidants such as coenzyme Q10 have neuroprotective abilities and can target mechanisms of oxidative stress and mitochondrial and autophagic dysfunction, however, it has limited bioavailability since it is lipophilic.A water-soluble formulation of CoQ10 (Ubisol-Q10) was created to increase bioavailability. Ubisol-Q10 was shown to protect cultured neurons from paraquat (PQ) (an environmental toxin known to cause PD) toxicity and DA neurons in PQ induced rodent models of PD. Ethanolic ashwagandha has also been shown to provide neuroprotective, anti-inflammatory, and even neuro-regenerative properties in PQ induced rodent models of PD. Similar to CoQ10, ashwagandha extract phytochemicals are lipophilic, resulting in poor bioavailability. The same technology used in Ubisol-Q10 was used to enhance solubility of ashwagandha extract. Previously, we investigated the efficacy of combined Ubisol-Q10 and ethanolic ashwagandha treatments and we have begun to test the enhanced efficacy of the combination of both water-soluble formulations. Both physiological and behavioral components of PD progression in response to treatment in rats will be studied in this project. We anticipate that this combinatorial treatment will halt the progression by reducing oxidative stress, stabilizing mitochondrial function and activating autophagy mechanisms

SARS-CoV-2 Next Generation Sequencing (NGS) data from clinical isolates from the East Texas Region of the United States

The SARS-CoV-2 virus has evolved throughout the pandemic and is likely to continue evolving into new variants. Some of these variants may affect functional properties, including infectivity, interactions with host immunity, and disease severity. And compromised vaccine efficacy is an emerging concern with every new viral variant. Next-generation sequencing (NGS) has emerged as the tool of choice for discovering new variants and understanding the transmission dynamics of SARS-CoV-2. Deciphering the SARS-CoV-2 genome has enabled epidemiological survivance and forecast of altered etiologically. Clinical presentations of the infection are influenced by comorbidities such as age, immune status, diabetes, and the infecting variant. Thus, clinical management and vaccine efficacy may differ for new variants. For example, some monoclonal antibody treatments are variant-specific, and some vaccines are less efficacious against the omicron and delta variants of SARS-CoV-2. Consequently, determining the local outbreaks and monitoring SARS-CoV-2 Variants of Concern (VOC) is one of the primary strategies for the pandemic's containment. Although next-generation sequencing (NGS) is a gold standard for genomic surveillance and variant discovery, the assays are not approved for variant diagnosis for clinical decision-making. Advanta Genetics, Texas, USA, optimized Illumina COVID-seq protocol to reduce cost without compromising accuracy and validated the Illumina COVID-Seq assay as a Laboratory Developed Test (LDT) according to the guidelines prescribed by the College of American Pathologists (CAP) and Clinical Laboratory Improvement Amendments (CLIA). The whole genome of the virus was sequenced in (n = 161) samples from the East Texas region using the Illumina MiniSeq® instrument and analyzed by using Illumina baseSpace (https://basespace.illumina.com) bioinformatics pipeline. Briefly, the library was prepared by using Illumina COVIDSeq research use only (RUO) kit, and the individual libraries were normalized using the DNA concentration measured by Qubit Flex Fluorometer, and the pooled libraries were sequenced on Illumina MiniSeq® Instrument. Illumina baseSpace application was used for sequencing QC, FASTQ generation, genome assembly, and identification of SARS-CoV-2 variants. This whole genome shotgun project (n = 161) has been deposited at GISAID

Two Ports Suture Less Laparoscopic Appendectomy using the new Enseal Device and Ultra Grasper Safe and Feasible

This operation was performed by me (1) in pre-conference surgical workshop day 2 at Minimal Invasive Surgery Centre, Liaquat University of Medicine and Health Sciences (LUMHS) on 20th November 2019. A 22 years old female resident of Hyderabad presented the Out Patients Department with complain of right iliac fossa pain associated with vomiting for the past 4 days. Physical examination was significant for tenderness and rebound tenderness in RIF region, Rovsing’s sign was positive. She was diagnosed as a case of acute appendicitis. After obtaining an informed consent she underwent 2-ports laparoscopic appendectomy under general anaesthesia. In this case we did not use any sutures or clips. Mesoappendix was divided and appendicectomy performed with Enseal device. Haemostasis was secured and wound closed with Vicryl 2.0. Patient made uneventful recovery and was discharged on the first postoperative day. We would therefore like to conclude that the suture-less 2-ports appendectomy was considered safe, reliable, and more minimal invasive, with decreased hospitalization duration and decreased risk of postoperative complications

Safety and efficacy of percutaneous coronary intervention versus coronary artery bypass graft in patients with STEMI and unprotected left main stem disease: a systematic review & meta-analysis

Introduction: Owing to its large area of supply, left main coronary artery disease (LMCAD) has the highest mortality rate among coronary artery lesions, resulting in debate about its optimal revascularization technique. This meta-analysis compares percutaneous coronary intervention (PCI) versus coronary artery bypass grafting (CABG) for the treatment of LMCAD. Method: MEDLINE, TRIP, and Cochrane Central databases were queried from their inception until 25 April 2021, to determine MACCE (major adverse cardiac and cardiovascular events), all-cause mortality, repeat revascularization, myocardial infarction (MI) and stroke rates post-revascularization for different follow-ups. 7 RCTs and 50 observational studies having 56,701 patients were included. A random-effects model was used with effect sizes calculated as odds ratios (odds ratio, OR). Results: In the short term (1 year), PCI had significantly higher repeat revascularizations (OR = 3.58, 95% CI 2.47-5.20; p intermediate term (2-5 years), PCI had significantly higher rates of repeat revascularizations (OR = 3.47, 95% CI 2.72-4.44; p long term (≥5 years), while in the very long term (≥10 years), PCI had significantly lower all-cause mortalities (OR = 0.77, 95% CI 0.61-0.96; p = 0.02). Conclusion: PCI was safer than CABG for patients with stroke for most follow-ups, while CABG was associated with lower repeat revascularizations. However, further research is required to determine PCI's safety over CABG for reducing post-surgery MI.</p