Advantage of precision metagenomics for urinary tract infection diagnostics

Frontiers in Cellular and Infection Microbiology, Год журнала: 2023, Номер 13

Опубликована: Июль 4, 2023

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 highly sensitive, its yield limited targeted microorganisms. Metagenomic next-generation sequencing (mNGS) hypothesis-free approach with potential of deciphering urobiome. However, clinically relevant information buried in enormous amount data.Precision metagenomics (PM) hybridization capture-based method enhanced discovery power better without diluting information. We collected 47 samples suspected parallel tested each sample by microbial culture, PCR, PM; then, we comparatively analyzed Next, phenotypically classified cumulative population using Explify® data analysis platform pathogenicity.Results revealed 100% positive predictive agreement (PPA) results, which identified only 13 different microorganisms, compared 19 62 organisms PM, respectively. All were into phenotypic groups (0-3) increasing pathogenic clinical relevance. This PM can simultaneously quantify classify readily through bioinformatic platforms like Explify®, essentially providing dissected quantitative results timely accurate empiric treatment.PM offers building effective models beyond usual care complex diseases. Future studies should assess impact PM-guided management on outcomes.

Язык: Английский

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Advancing Clinical Microbiology: Applications and Future of Next-Generation Sequencing

SAR Journal of Pathology and Microbiology, Год журнала: 2024, Номер 5(03), С. 107 - 111

Опубликована: Июнь 20, 2024

Next-generation sequencing (NGS) has reformed infectious disease management, including COVID-19. While real-time polymerase chain reaction (PCR) is widely used for rapid pathogen detection, it requires predefined targets. NGS offers an unbiased approach, detecting multiple pathogens simultaneously without prior knowledge. Despite its potential, implementation in clinical settings faces challenges like high costs and technical complexity. platforms Illumina, Ion Torrent, Nanopore provide high-throughput sequencing, identifying resistance markers. Applications include whole genome (WGS), metagenomic (mNGS), targeted (tNGS). Integrating with conventional methods could improve diagnostics, but current evidence mixed supporting widespread use.

Язык: Английский

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Navigating the New Norm: The FDA's Final Rule on Laboratory Developed Tests (LDTs) and Its Impact on Clinical Laboratory Operations

Clinical Microbiology Newsletter, Год журнала: 2024, Номер unknown

Опубликована: Сен. 1, 2024

Язык: Английский

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Enhancing Public Health Outcomes with AI-Powered Clinical Surveillance: Precise Detection of COVID-19 Variants using qPCR and Nanopore Sequencing

Journal of Infection and Public Health, Год журнала: 2025, Номер 18(3), С. 102663 - 102663

Опубликована: Янв. 10, 2025

Язык: Английский

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Streamlining Fungal Diagnostics: Evaluation of a Direct-to-PCR Extraction-Free Workflow for Candida Detection

Diagnostic Microbiology and Infectious Disease, Год журнала: 2025, Номер 113(2), С. 116923 - 116923

Опубликована: Май 22, 2025

Fungal infections are an escalating health threat, and delays from conventional nucleic acid extraction hinder timely diagnosis treatment. This study evaluated the diagnostic performance of a novel extraction-free technology, Direct-to-PCR (D2P; Scienetix, Tyler, TX, USA), for detection clinically significant Candida species (C. albicans, C. glabrata, auris, parapsilosis, tropicalis). D2P was compared against silica column-based (Qiagen) magnetic bead-based (KingFisher) methods, using microbial reference isolates, residual clinical specimens, limit-of-detection analyses. Diagnostic sensitivity specificity were comparable to approaches, with ranging 96.77 % 100 %. Concordance between methods high, Cohen's kappa coefficients (κ=0.93-1.00). Limit-of-detection analyses demonstrated strong analytical sensitivity, excellent linearity (R²=0.924-0.999) low replicate variability (coefficient variation 0.2-6.3 %). Statistical comparison cycle threshold values revealed no differences (p > 0.05), supporting equivalent recovery without need time-intensive steps. Despite these strengths, has limitations. The relatively small number specimens (n = 40) may restrict generalizability findings, further validation across broader patient populations, specimen types, settings is warranted. Nevertheless, offers streamlined, rapid workflow that reduces turnaround times enhances accessibility, particularly in resource-limited environments. Wider adoption PCR platforms such as could facilitate earlier invasive candidiasis, improve outcomes, mitigate healthcare-associated morbidity mortality attributable fungal infections.

Язык: Английский

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Evaluation of direct-to-PCR (D2P) method for molecular diagnosis of infectious diseases

Experimental and Molecular Pathology, Год журнала: 2025, Номер 143, С. 104972 - 104972

Опубликована: Май 26, 2025

This study evaluates the performance of Direct-to-PCR (D2P) method as a streamlined, extraction-independent alternative to conventional nucleic acid extraction techniques for diagnosing urinary tract infections, sexually transmitted and respiratory infections. The D2P approach employs proprietary antimicrobial peptide-based lysis buffers tailored bacterial, fungal, viral targets, enabling direct amplification from clinical contrived specimens without column- or bead-based purification. Comparative analyses were conducted against silica column-based (QIAGEN) magnetic (KingFisher) methods using both microbial reference isolates 116 residual samples. Results demonstrate that yields comparable sensitivity specificity workflows across diverse panel pathogens-including Gram-negative Gram-positive bacteria, Candida species, ssRNA viruses (e.g., CoV-229E, Parainfluenza Virus 1 2), dsDNA HSV, HAdV). Notably, outperformed QIAGEN KingFisher in extracting acids auris, multidrug-resistant fungal pathogen. Limit detection efficiency remained within acceptable ranges all platforms, with R2 values between 0.92 0.99, slopes consistent MIQE standards. protocol reduced total sample processing time ∼120 min ∼45 min, minimized hands-on steps, demonstrated effective turbid hemolyzed samples-making it suitable high-throughput resource-limited settings. However, limitations observed samples high PCR-inhibitor content low target yield, broader validation additional matrices is recommended. These findings support reliable, efficient, scalable molecular diagnostic broad utility. Integration into could enhance access rapid, cost-effective pathogen centralized laboratories decentralized point-of-care environments.

Язык: Английский

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Deciphering Microbiota of Acute Upper Respiratory Infections: A Comparative Analysis of PCR and mNGS Methods for Lower Respiratory Trafficking Potential

Advances in respiratory medicine, Год журнала: 2023, Номер 91(1), С. 49 - 65

Опубликована: Фев. 2, 2023

Although it is clinically important for acute respiratory tract (co)infections to have a rapid and accurate diagnosis, critical that medicine understands the advantages of current laboratory methods. In this study, we tested nasopharyngeal samples (n = 29) with commercially available PCR assay compared results those hybridization-capture-based mNGS workflow. Detection criteria positive was Ct < 35 >40% target coverage, median depth 1X RPKM > 10. A high degree concordance (98.33% PPA 100% NPA) recorded. However, yielded positively 29 additional microorganisms (23 bacteria, 4 viruses, 2 fungi) beyond PCR. We then characterized each method into three phenotypic categories using IDbyDNA Explify® Platform (Illumina® Inc, San Diego, CA, USA) consideration infectivity trafficking potential lower region. The findings are significant providing comprehensive yet relevant microbiology profile upper infection, especially in immunocompromised or immunocompetent comorbidity cases where traditional syndromic approaches fail identify pathogenicity. Accordingly, technology can be used supplement syndrome-based tests, data quickly effectively phenotypically potential, clinical (co)infection, comorbid consideration-with promise reduce morbidity mortality.

Язык: Английский

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

Data in Brief, Год журнала: 2023, Номер 49, С. 109312 - 109312

Опубликована: Июнь 16, 2023

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

Язык: Английский

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

Archives of Clinical and Biomedical Research, Год журнала: 2022, Номер 06(06)

Опубликована: Янв. 1, 2022

Rapid classification and detection of SARS-CoV-2 variants have been critical in comprehending the virus's transmission dynamics. Clinical manifestation infection is influenced by comorbidities such as age, immune status, diabetes, infecting variant. Thus, clinical management may differ for new variants. For example, some monoclonal antibody treatments are variant-specific. Yet, a U.S. Food Drug Administration (FDA)-approved test detecting variant unavailable. A laboratory-developed (LDT) remains viable option reporting intervention or epidemiological purposes. Accordingly, we validated Illumina COVIDSeq assay an LDT according to guidelines prescribed College American Pathologists (CAP) Laboratory Improvement Amendments (CLIA). The limit (LOD) this Ct<30 (~15 viral copies) >200X genomic coverage, 100% specific existing demonstrated precision inter-day, intra-day, intra-laboratory reproducibility studies. It also accurate, defined reference strain testing split sample with other CLIA laboratories. Advanta Genetics has reviewed CAP inspectors under review FDA Emergency Use Authorization.

Язык: Английский

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

Practical Laboratory Medicine, Год журнала: 2023, Номер 34, С. e00311 - e00311

Опубликована: Фев. 1, 2023

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

Язык: Английский

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