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.

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.

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 next-generation 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 post-vaccination 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 < 35 and for mNGS samples it was >40% target coverage, median depth of 1X and RPKM > 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.

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's 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<30 (~15 viral copies) and >200X 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.

Molecular detection of Oxyspirura larvae in arthropod intermediate hosts.

To determine potential intermediate hosts of Oxyspirura petrowi, a common nematode eyeworm of wild gallinaceous birds, various arthropod species including red harvester ants, beetles, wood cockroaches, crickets, grasshoppers, katydids, and desert termites were screened for the presence of O. petrowi using specific polymerase chain reaction (PCR) primers targeting the internal transcribed spacer 2 region (ITS2) of the eyeworm ribosomal deoxyribonucleic acid (rDNA). This is the first study to investigate the intermediate hosts of O. petrowi utilizing molecular techniques. We determined 38% (13/34) of the cockroaches, 27% (3/11) of the crickets, and 23% (68/289) of the grasshoppers which were positive for O. petrowi. Identifying potential intermediate hosts of O. petrowi is essential to better understanding the epizoology of the eyeworm's transmission mechanics and to controlling infections in wild gallinaceous birds.

Predictive Modeling for West Nile Virus and Mosquito Surveillance in Lubbock, Texas.

West Nile virus (WNV) was first detected in North America during 1999, and has since spread throughout the contiguous USA. West Nile virus causes West Nile fever and the more severe West Nile neuroinvasive disease. As part of a WNV vector surveillance program, we collected mosquitoes in Lubbock, Texas, using CO2-baited encephalitic vector survey (EVS) traps. During 219 wk from 2009 through 2017, EVS traps were operated for 1,748 trap nights, resulting in more than 101,000 mosquitoes captured. Weekly, selected female mosquito specimens were pooled by species and trap site, and screened for WNV using reverse transcription-polymerase chain reaction assay. Mosquitoes positive for WNV were detected during 16.9% (37/219) of the weeks. Using this information, we constructed a statistical model to predict the probability of detecting an infection within a mosquito pool as a factor of weather variables. The final model indicated that detection of WNV in mosquitoes was negatively associated with the week of year squared and average wind from 3 wk prior to sampling, and was positively associated with week of year, average visibility, average humidity from 2 wk prior to sampling, and average dew point from 4 wk prior to sampling. The model developed in this study may aid public health and vector control programs in swift and effective decision making relative to city-wide mosquito control efforts by predicting when the chances of mosquitoes having WNV are at their greatest.

Wnt signaling and podocyte dysfunction in diabetic nephropathy.

Nephropathy is a major microvascular complication of diabetes mellitus and often leads to terminal renal failure in addition to contributing significantly to cardiovascular morbidity and mortality. Despites continuous advances, the pathogenesis of diabetic nephropathy remains poorly understood. Recent studies have underscored the significance of structural and functional changes in podocytes in the development and progression of diabetic nephropathy. The role of podocytes in health and diabetic nephropathy and abnormalities including podocyte hypertrophy, effacement, and apoptosis, and a detailed discussion on the role played by the Wnt-ß-catenin signaling pathway in podocyte injury and dysfunction are the focus of this review. In addition, the role played by Wnt signaling in mediating the effects of known therapeutic strategies for diabetic nephropathy is also discussed.

EYEWORMS (OXYSPIRURA PETROWI) IN NORTHERN BOBWHITES (COLINUS VIRGINIANUS) FROM THE ROLLING PLAINS ECOREGION OF TEXAS AND OKLAHOMA, 2011-13.

The Northern Bobwhite ( Colinus virginianus ) has been steadily declining throughout much of its historic range for decades. The Rolling Plains ecoregion of Texas and western Oklahoma, historically rich with wild Northern Bobwhites and one of the last remaining quail strongholds, also has a declining population. During August and October in 2011-13, 348 Northern Bobwhites from the Rolling Plains were examined for eyeworms (Oxyspirura petrowi). Of these 348 Northern Bobwhites, 144 (41.4%) were infected with 1,018 total eyeworms. Eyeworm abundance (mean±SE) was 2.9±0.4 (range 0-64), with an intensity (mean±SE) of 7.1±0.6. Eyeworm prevalence was significantly higher in adult Northern Bobwhites (58.7%) than in juveniles (35.4%). Recent research suggests that eyeworms have the potential to cause cellular tissue damage to the eye, but it is unknown how these worms affect host survivability. This study further expands the regional distribution of O. petrowi in Northern Bobwhites in the Rolling Plains ecoregion and assesses the prevalence and abundance of infection across host age, host sex, and year. Further research is warranted on the life history of O. petrowi and assessing the impacts of eyeworms on their definitive host at individual and population levels.

A survey of neonicotinoid use and potential exposure to northern bobwhite (Colinus virginianus) and scaled quail (Callipepla squamata) in the Rolling Plains of Texas and Oklahoma.

Northern bobwhite (quail) (Colinus virginianus) and scaled quail (Callipepla squamata) populations have declined dramatically in the Rolling Plains ecoregion of Texas and Oklahoma (USA). There is rising concern about potential toxicity of neonicotinoids to birds. To investigate this concern, the authors examined crops of 81 northern bobwhite and 17 scaled quail to determine the presence or absence of seeds treated with 3 neonicotinoids (clothianidin, imidacloprid, and thiamethoxam). No treated seeds were found in the 98 crops examined. Liver samples from all 98 quail were collected and analyzed for neonicotinoid residues. Analysis revealed very low concentrations of neonicotinoids within the quail liver samples. The results suggest there is little to no risk of direct toxicity to quail from neonicotinoids. Environ Toxicol Chem 2016;35:1511-1515. © 2015 SETAC.