Pathogens In Clinical Samples Research Articles

Viral Metagenomics for the Identification of Emerging Infections in Clinical Samples with Inconclusive Dengue, Zika, and Chikungunya Viral Amplification.

Viral metagenomics is increasingly being used for the identification of emerging and re-emerging viral pathogens in clinical samples with unknown etiology. The objective of this study was to shield light on the metavirome composition in clinical samples obtained from patients with clinical history compatible with an arboviral infection, but that presented inconclusive results when tested using RT-qPCR. The inconclusive amplification results might be an indication of the presence of an emerging arboviral agent that is inefficiently amplified by conventional PCR techniques. A total of eight serum samples with inconclusive amplification results for the routinely tested arboviruses—dengue (DENV), Zika (ZIKV), and Chikungunya (CHIKV) obtained during DENV and CHIKV outbreaks registered in the state of Alagoas, Northeast Brazil between July and August 2021—were submitted to metagenomic next-generation sequencing assay using NextSeq 2000 and bioinformatic pipeline for viral discovery. The performed bioinformatic analysis revealed the presence of two arboviruses: DENV type 2 (DENV-2) and CHIKV with a high genome coverage. Further, the metavirome of those samples revealed the presence of multiple commensal viruses apparently without clinical significance. The phylogenetic analysis demonstrated that the DENV-2 genome belonged to the Asian/American genotype and clustered with other Brazilian strains. The identified CHIKV genome was taxonomically assigned as ECSA genotype, which is circulating in Brazil. Together, our results reinforce the utility of metagenomics as a valuable tool for viral identification in samples with inconclusive arboviral amplification. Viral metagenomics is one of the most potent methods for the identification of emerging arboviruses.

Molecular mechanisms of acquired antifungal drug resistance in principal fungal pathogens and EUCAST guidance for their laboratory detection and clinical implications.

The increasing incidence and changing epidemiology of invasive fungal infections continue to present many challenges to their effective management. The repertoire of antifungal drugs available for treatment is still limited although there are new antifungals on the horizon. Successful treatment of invasive mycoses is dependent on a mix of pathogen-, host- and antifungal drug-related factors. Laboratories need to be adept at detection of fungal pathogens in clinical samples in order to effectively guide treatment by identifying isolates with acquired drug resistance. While there are international guidelines on how to conduct in vitro antifungal susceptibility testing, these are not performed as widely as for bacterial pathogens. Furthermore, fungi generally are recovered in cultures more slowly than bacteria, and often cannot be cultured in the laboratory. Therefore, non-culture-based methods, including molecular tests, to detect fungi in clinical specimens are increasingly important in patient management and are becoming more reliable as technology improves. Molecular methods can also be used for detection of target gene mutations or other mechanisms that predict antifungal drug resistance. This review addresses acquired antifungal drug resistance in the principal human fungal pathogens and describes known resistance mechanisms and what in-house and commercial tools are available for their detection. It is emphasized that this approach should be complementary to culture-based susceptibility testing, given the range of mutations, resistance mechanisms and target genes that may be present in clinical isolates, but may not be included in current molecular assays.

Mitigating the Impact of Emerging Animal Infectious Disease Threats: First Emerging Animal Infectious Diseases Conference (EAIDC) Report.

From 29 November to 1 December 2021, an “emerging animal infectious disease conference (EAIDC)” was held at the Pennsylvania State University. This conference brought together distinguished thought leaders in animal health, veterinary diagnostics, epidemiology and disease surveillance, and agricultural economics. The conference’s primary objective was to review the lessons learned from past experiences in dealing with high-consequence animal infectious diseases to inform an action plan to prepare for future epizootics and panzootics. Invited speakers and panel members comprised world-leading experts in animal infectious diseases from federal state agencies, academia, professional societies, and the private sector. The conference concluded that the biosecurity of livestock operations is critical for minimizing the devastating impact of emerging animal infectious diseases. The panel also highlighted the need to develop and benchmark cutting-edge diagnostics for rapidly detecting pathogens in clinical samples and the environment. Developing next-generation pathogen agnostic diagnostics will help detect variants of known pathogens and unknown novel pathogens. The conference also highlighted the importance of the One Health approach in dealing with emerging animal and human infectious diseases. The recommendations of the conference may be used to inform policy discussions focused on developing strategies for monitoring and preventing emerging infectious disease threats to the livestock industry.

Reverse-transcription PCR increases sensitivity of broad-range fungal detection in bronchoalveolar lavage fluid.

Fungal infection remains difficult to diagnose in the laboratory. Here, we have shown that detecting ribosomal RNA and DNA, rather than only ribosomal DNA, in a broad range fungal assay results in a significant enhancement in the ability to detect and identify fungal pathogens in clinical samples.

Performance of four commercial real-time PCR assays for the detection of bacterial enteric pathogens in clinical samples

ObjectivesMany laboratories use culture-independent diagnostic tests for bacterial gastroenteritis (i.e. real-time polymerase chain reaction, RT-PCR) instead of culture because of better sensitivity, automation, and faster turnaround times. To address some gaps in initial evaluations and lack of intraassay comparisons for many commercial RT-PCRs, this study compared the ability of four commercially available RT-PCR tests (Ridagene, Fast Track Diagnostics, BD Max, and Prodesse Progastro) to detect five major bacterial enteric pathogens: Campylobacter, Salmonella, Shiga-toxin producing Escherichia coli (STEC), Shigella, and Yersinia. MethodsClinical stool specimens and contrived samples comprising commonly circulating species, serotypes, biovars, and/or toxin subtypes were used for the comparison. ResultsConcordance rates for RT-PCR and culture using culture-positive and culture-negative clinical stools were >90% for Campylobacter (97.5-100%), Salmonella (97.5-100%), Shigella (100%), and STEC (90-100%). However, the agreement between RT-PCR and culture for Y. enteroccolitica ranged from 70-90%. For the contrived sample set, stx2f was detected by one of four assays. Of note, no assay could detect Yersinia non-enterocolitica and Campylobacter upsaliensis. ConclusionsDepending on the prevalence of certain stx sub-types, Yersinia species, and Campylobacter species in a laboratory's jurisdiction, without further improvement in culture-independent tests, culture methods remain critical for the detection of these pathogens.

Combining tag-specific primer extension and magneto-DNA system for Cas14a-based universal bacterial diagnostic platform

Nucleic acid-based diagnosis using CRISPR-Cas associated enzymes is essential for rapid infectious disease diagnosis and treatment strategies during a global pandemic. The obstacle has been blossomed CRIPSR-Cas based tools that can monitor wide range of pathogens in clinical samples with ultralow concentrations. Here, a universal nucleic acid magneto-DNA nanoparticle system was exploited for the detection of pathogenic bacteria, based on the collateral cleavage activity of CRISPR-Cas14a and tag-specific primer extension. In the system, the target nucleic acids were amplificated and be separated from mixtures by streptavidin-coated magnetic bead. The collateral cleavage activity of CRISPR-Cas14a can be activated via the tag sequence on the target product. Consequently, the fluorophore quencher reporter can be activated by CRISPR-Cas14a, leading to the increasing response. The exploited universal bacterial diagnostic can distinguish six different bacteria strains with 1 cfu/mL or 1 aM sensitivity, which may provide new strategies to construct fast, accurate, cost-effective and sensitive diagnostic tools in environments with limited resources.

A new quantitative 16S rRNA amplicon sequencing method

In recent years, 16S rRNA amplicon sequencing technology has been widely used to study human gut microbiota and to detect unknown pathogens in clinical samples. However, its resolution to bacterial population can only reach the relative abundance of genus level, and different factors affect the final bacterial profile, such as sample concentrations, PCR cycle numbers and amplification primers. In order to solve these problems, we developed a quantitative 16S rRNA amplicon sequencing method by combining random tag and internal marker method. The new methods improved the accuracy of human gut microbiota, reduced the impact of experimental operation on the results, and improved the comparability between sequencing and other molecular biological methods.

A simple and low-cost paper-based colorimetric method for detecting and distinguishing the GII.4 and GII.17 genotypes of norovirus

In modern times, viruses still threaten people's lives. Among them, norovirus was the main pathogenic factor in the cause of gastroenteritis and foodborne illness, of which the GII.4 and GII.17 genotypes are prevalent in China and most parts of the world. A simple and low-cost platform for rapid and accurate norovirus detection remains a major challenge. After the cell-free system and paper-based chromogenic system were optimized, a rapid and specific norovirus detection method was established based on norovirus-specific sequences in combination with toehold switch elements. The development of a visible color change during detection eliminates the need for any complicated instruments. We validated this strategy and its specificity in differentiating GII.4, GII.17, Zika virus, and human coronavirus HKU1. The results showed that the optimized detection system not only provided a simple and rapid detection method for the sufficient differentiation of the two norovirus genotypes but also showed high specificity and no cross-reactivity with other viruses. Using nucleic acid isothermal amplification, this assay showed a limit of detection of 0.5 pM for the GII.4 genotype and 2.6 fM for the GII.17 genotype in reactions that could be observed directly with the naked eye. Our results suggested that this paper-based colorimetric method could serve as a simple and low-cost visual detection method for pathogens in clinical samples, especially in remote or rural areas.

Antibody‐free bioimprint aided sandwich ELISA technique for cell recognition and rapid screening for bacteria

AbstractWe have developed and tested a novel ELISA‐like approach for bacterial detection based upon selective adhesion of targeted bacteria to microwells with prefabricated bacterial bioimprints. Bioimprints were produced from three bacterial species; Escherichia coli (Gram‐negative), Rhodococcus rhodochrous (Gram‐positive) and Sarcina aurantiaca (Gram‐negative), by using molding with curable silicone from dense layers of bacterial cells deposited on a glass substrate. We demonstrated that the surface functionalized whole cell bioimprints were able to selectively recognize and bind their own bacterial cell type. In order to detect target bacteria that are bound to the matching bioimprint, we also developed silica nanoparticles dual‐functionalized with (3‐glycidyloxypropyl)trimethoxysilane (GLYMO) coupled with 4‐hydroxyphenylboronic acid (4‐HPBA), SiO2NPs/GLYMO/4‐HPBA, which were further conjugated with horseradish peroxidase (HRP). Bacterial detection was demonstrated to work in the established ELISA‐like protocol using the colorimetric reaction of the conjugated HRP with 3,3′,5,5′‐tetramethylbenzidine (TMB). The bioimprints were used instead of capture antibodies and HRP‐coated dual functionalized silica nanoparticles instead of a secondary antibody with TMB as the enzyme‐converted reagent, producing a colored byproduct. This bacterial bioimprint‐based detection method does not rely on any antibodies, uses stable and inexpensive reagents, and could potentially find application for rapid diagnostics of bacterial pathogens in clinical samples at the point of care.

Challenges in Microfluidic and Point-of-Care Phenotypic Antimicrobial Resistance Tests

To combat the threat to public health of antimicrobial resistance, there is a need for faster, more portable diagnostic tools to aid in antibiotic selection. Current methods for determining antimicrobial resistance of pathogens in clinical samples take days to result and require high levels of user input. Microfluidics offers many potential benefits, reducing time to result, user input, and allowing point of care testing. This review focuses on the challenges of developing functional or phenotypic microfluidic antimicrobial susceptibility tests; such methods complement other vital tools such as nucleic acid detection. Some of the most important challenges identified here are not unique to microfluidics but apply to most antimicrobial susceptibility testing innovations and relate to the nature of the sample being tested. For many high priority samples, mixtures of bacteria, highly variable target cell density, and the sample matrix can all affect measurements, and miniaturization can create sensitivity problems if target bacteria are dilute. Recent advances including smartphone capability, new sensors, microscopy, and a resurgence in paper microfluidics offer important opportunities for microfluidic engineering to simplify functional and phenotypic antimicrobial susceptibility testing. But the complexity of most clinical samples remains one of the biggest barriers to rapid uptake of microfluidics for antimicrobial resistance testing.

Reporter Phage-Based Detection of Bacterial Pathogens: Design Guidelines and Recent Developments

Fast and reliable detection of bacterial pathogens in clinical samples, contaminated food products, and water supplies can drastically improve clinical outcomes and reduce the socio-economic impact of disease. As natural predators of bacteria, bacteriophages (phages) have evolved to bind their hosts with unparalleled specificity and to rapidly deliver and replicate their viral genome. Not surprisingly, phages and phage-encoded proteins have been used to develop a vast repertoire of diagnostic assays, many of which outperform conventional culture-based and molecular detection methods. While intact phages or phage-encoded affinity proteins can be used to capture bacteria, most phage-inspired detection systems harness viral genome delivery and amplification: to this end, suitable phages are genetically reprogrammed to deliver heterologous reporter genes, whose activity is typically detected through enzymatic substrate conversion to indicate the presence of a viable host cell. Infection with such engineered reporter phages typically leads to a rapid burst of reporter protein production that enables highly sensitive detection. In this review, we highlight recent advances in infection-based detection methods, present guidelines for reporter phage construction, outline technical aspects of reporter phage engineering, and discuss some of the advantages and pitfalls of phage-based pathogen detection. Recent improvements in reporter phage construction and engineering further substantiate the potential of these highly evolved nanomachines as rapid and inexpensive detection systems to replace or complement traditional diagnostic approaches.

Accurate Detection of HPV Integration Sites in Cervical Cancer Samples Using the Nanopore MinION Sequencer Without Error Correction.

During the carcinogenesis of cervical cancer, the DNA of human papillomavirus (HPV) is frequently integrated into the human genome, which might be a biomarker for the early diagnosis of cervical cancer. Although the detection sensitivity of virus infection status increased significantly through the Illumina sequencing platform, there were still disadvantages remain for further improvement, including the detection accuracy and the complex integrated genome structure identification, etc. Nanopore sequencing has been proven to be a fast yet accurate technique of detecting pathogens in clinical samples with significant longer sequencing length. However, the identification of virus integration sites, especially HPV integration sites was seldom carried out by using nanopore platform. In this study, we evaluated the feasibility of identifying HPV integration sites by nanopore sequencer. Specifically, we re-sequenced the integration sites of a previously published sample by both nanopore and Illumina sequencing. After analyzing the results, three points of conclusions were drawn: first, 13 out of 19 previously published integration sites were found from all three datasets (i.e., nanopore, Illumina, and the published data), indicating a high overlap rate and comparability among the three platforms; second, our pipeline of nanopore and Illumina data identified 66 unique integration sites compared with previous published paper with 13 of them being verified by Sanger sequencing, indicating the higher integration sites detection sensitivity of our results compared with published data; third, we established a pipeline which could be used in HPV integration site detection by nanopore sequencing data without doing error correction analysis. In summary, a new nanopore data analysis method was tested and proved to be reliable in integration sites detection compared with methods of existing Illumina data analysis pipeline with less sequencing data required. It provides a solid evidence and tool to support the potential application of nanopore in virus status identification.

Molecular identification of Cryptococcus gattii from cerebrospinal fluid using single-cell sequencing: A case study

A 31-year-old man presented with cryptococcal meningitis (CM) without typical clinical characteristics, but with abnormal walking, difficult leg lifting and frequent falling. He was admitted to Peking Union Medical College Hospital. After multiple tests failed to identify the pathogen, single-cell sequencing (scS) was used to test the cerebrospinal fluid (CSF). Comparing the sequence obtained from single-cell sequencing with the reference database, it was found that the infection was caused by Cryptococcus gattii sensu stricto (AFLP4/VGI genotype). Cryptococcus is difficult to cultivate from complex body fluids. The etiological agent of this patient was identified and the patient was treated. This is the first case in which scS was used to detect and identify fungal pathogen after conventional testing failed to identify the cause of the disease. This report demonstrates that the scS approach can be used to generate fungal genome sequences directly from the CSF of a CM patient. The scS technology could become a powerful tool to precise detect microscopically visible but uncultured pathogens in clinical samples.

Chimeric Phage Nanoparticles for Rapid Characterization of Bacterial Pathogens: Detection in Complex Biological Samples and Determination of Antibiotic Sensitivity.

Rapid, specific, and sensitive detection of pathogenic bacteria in drink, food, and clinical samples is an important goal for public health. In addition, rapid characterization of antibiotic susceptibility could inform clinical choices and improve antibiotic stewardship. We previously reported a straightforward, inexpensive strategy to detect Gram-negative bacterial pathogens, including Pseudomonas aeruginosa, Vibrio cholerae, and Escherichia coli, taking advantage of the high affinity and specificity of phages for their bacterial hosts. Chimeric phages targeted different bacterial pathogens, and thiolation of the phages induced aggregation of gold nanoparticles (AuNPs), leading to a visible colorimetric response in the presence of at least ∼100 cells of the target bacteria. Here, we apply this strategy to complex biological samples (milk, urine, and swabs from a porcine ex vivo model of P. aeruginosa infection). We also show that this assay can be used to identify the antibiotic susceptibility profile based on detection of bacterial growth in the presence of different antibiotics. The prospect for using phage-conjugated AuNPs to detect bacterial pathogens in clinical samples and guide antibiotic choice is discussed.

RespiDisk: a point-of-care platform for fully automated detection of respiratory tract infection pathogens in clinical samples.

We present the RespiDisk enabling the fully automated and multiplex point-of-care (POC) detection of (currently) up to 19 respiratory tract infection (RTI) pathogens from a single sample based on reverse transcriptase polymerase chain reaction (RT-PCR). RespiDisk comprises a RTI-specific implementation of the centrifugal microfluidic LabDisk platform and combines new and existing advanced unit operations for liquid control, thereby automating all assay steps only by a spinning frequency and temperature protocol in combination with the use of a permanent magnet for in situ bead handing. The capabilities of the system were demonstrated with 36 tested quality samples mimicking clinical conditions (clinical and/or cultured material suspended in transport medium or synthetic bronchoalveolar lavage (BAL)) from past external quality assessment (EQA) panels covering 13 of the 19 integrated RTI detection assays. In total, 36 samples × 19 assays/sample resulting in 684 assays were performed with the RespiDisk, and its analytical performance was in full agreement with the routine clinical workflow serving as reference. A strong feature of the platform is its universality since its components allow the simultaneous detection of a broad panel of bacteria and viruses in a single run, thereby enabling the differentiation between antibiotic-treatable diseases. Furthermore, the full integration of all necessary biochemical components enables a reduction of the hands-on time from manual to automated sample-to-answer analysis to about 5 min. The study was performed on an air-heated LabDisk Player instrument with a time-to-result of 200 min.

Graphene Quantum Dot-Based Electrochemical Immunosensors for Biomedical Applications.

In the area of biomedicine, research for designing electrochemical sensors has evolved over the past decade, since it is crucial to selectively quantify biomarkers or pathogens in clinical samples for the efficacious diagnosis and/or treatment of various diseases. To fulfil the demand of rapid, specific, economic, and easy detection of such biomolecules in ultralow amounts, numerous nanomaterials have been explored to effectively enhance the sensitivity, selectivity, and reproducibility of immunosensors. Graphene quantum dots (GQDs) have garnered tremendous attention in immunosensor development, owing to their special attributes such as large surface area, excellent biocompatibility, quantum confinement, edge effects, and abundant sites for chemical modification. Besides these distinct features, GQDs acquire peroxidase (POD)-mimicking electro-catalytic activity, and hence, they can replace horseradish peroxidase (HRP)-based systems to conduct facile, quick, and inexpensive label-free immunoassays. The chief motive of this review article is to summarize and focus on the recent advances in GQD-based electrochemical immunosensors for the early and rapid detection of cancer, cardiovascular disorders, and pathogenic diseases. Moreover, the underlying principles of electrochemical immunosensing techniques are also highlighted. These GQD immunosensors are ubiquitous in biomedical diagnosis and conducive for miniaturization, encouraging low-cost disease diagnostics in developing nations using point-of-care testing (POCT) and similar allusive techniques.

Development of a multiplex PCR assay for the simultaneous and rapid detection of six pathogenic bacteria in poultry

Escherichia coli, Pasteurella multocida, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella spp. and Staphylococcus aureus are six bacterial pathogens of avian. However, these pathogens may cause many similar pathological changes, resulting in clinical isolates that are difficult to quickly and simultaneously detect and identify. Here, a multiplex polymerase chain reaction (m-PCR) assay is reported to rapidly identify targets genes (phoA, KMT1, ureR, toxA, invA, and nuc) of these six pathogens in clinical samples. Six pairs of specific primers were designed. The optimal reaction conditions, specificity, and sensitivity of the m-PCR assay were investigated. The results showed that betaine remarkably improved amplification of the target genes. Specific test results showed that all six pathogens were detected by the proposed m-PCR protocol without cross-amplification with viruses or parasites. Sensitivity test results showed that the m-PCR system could amplify the six target genes from bacterial genomes or cultures with template amounts of 500 pg or 2.8–8.6 × 103 colony forming units, respectively. Furthermore, the six bacterial pathogens isolated from the infected tissue samples were successfully identified. The proposed m-PCR assay is a useful tool to monitor and diagnose bacterial infection in birds with high specificity, sensitivity and throughput.

Rapid and Highly Sensitive DNA Flow Technology Platform to Detect Tick-Borne Bacterial Pathogens in Clinical Samples.

Zoonotic diseases represent a significant public health concern worldwide due to the emergence/re-emergence of vector-borne diseases in the last decade. Ticks are the most important vectors in the northern hemisphere and can transmit diseases such as Lyme disease, human granulocytic anaplasmosis, and spotted fever rickettsioses, among others. Therefore, there is a growing need to develop better and faster diagnostic tools that can detect zoonotic human pathogens in clinical samples. In this study, we present the results for a new kit tick-borne bacteria flow chip (TBFC), which allows the simultaneous screening of seven different bacterial pathogens in human samples using a DNA flow technology platform (hybriSpot system). The analytical sensitivity and specificity of the TBFC were calculated spiking bacterial DNA in human DNA samples, and the results were compared with an in-house single PCR-reverse line blot (RLB) routinely used for diagnosis at the National Center for Microbiology in Spain. The analytical sensitivity and specificity of the TBFC were almost identical to the PCR-RLBs used in diagnosis. In addition, samples from patients (n = 212) with a wide range of clinical signs/symptoms consistent with multisystem disorders suggestive of a tick-borne infection were tested using the TBFC, and the results were compared with those obtained by PCR-RLB. The concordance of both methods using patient samples was 97.2%. The TBFC kit is a rapid new and cost-efficient diagnostic molecular tool capable of detecting tick-borne pathogens in clinical samples.

Evaluation of the Vibrant DNA microarray for the high-throughput multiplex detection of enteric pathogens in clinical samples

ABSTRACTBackgroundRapid detection of a wide range of etiologic agents is essential for appropriate treatment and control of gastrointestinal (GI) infections. A variety of microbial species including bacteria, viruses, parasites, and fungi have been recognized as diarrheagenic enteric pathogens. However, multiplex testing of various targets in a single reaction needs further improvement because of its limitation in species and throughput.ResultsThis study aims at developing and evaluating a DNA microarray-based qualitative multiplexed polymerase chain reaction (PCR) assay, Vibrant GI pathogen panel (GPP), for simultaneous detection of 27 enteric GI pathogenic targets (16 bacteria, 5 viruses, 4 parasites, and 2 fungi) directly from stool specimens. Limits of detection ranged from 102 to 104 cells/mL for bacteria, 102 to 103 cells/mL for parasites, 102 to 103 RNA copies/mL for viruses, and 102 to 103 cells/mL for fungi. Performance characteristics were determined using 27 Quantitative Genomic DNAs, 212 spiked stool specimens, 1067 clinical and archived stool specimens. Overall sensitivity was 95.9% (95% CI 92.4–98.1) and specificity was 100% (95% CI 99.9–100). Polymicrobial detections contained either two or three organisms was 20.2% (35/173) of positive clinical specimens and 3.3% (35/1055) of all clinical specimens.ConclusionThe Vibrant GPP is a comprehensive, high-throughput, and rapid DNA microarray to provide etiologic diagnosis of GI infections in the laboratory setting.

A cell-free DNA metagenomic sequencing assay that integrates the host injury response to infection

High-throughput metagenomic sequencing offers an unbiased approach to identify pathogens in clinical samples. Conventional metagenomic sequencing, however, does not integrate information about the host, which is often critical to distinguish infection from infectious disease, and to assess the severity of disease. Here, we explore the utility of high-throughput sequencing of cell-free DNA (cfDNA) after bisulfite conversion to map the tissue and cell types of origin of host-derived cfDNA, and to profile the bacterial and viral metagenome. We applied this assay to 51 urinary cfDNA isolates collected from a cohort of kidney transplant recipients with and without bacterial and viral infection of the urinary tract. We find that the cell and tissue types of origin of urinary cfDNA can be derived from its genome-wide profile of methylation marks, and strongly depend on infection status. We find evidence of kidney and bladder tissue damage due to viral and bacterial infection, respectively, and of the recruitment of neutrophils to the urinary tract during infection. Through direct comparison to conventional metagenomic sequencing as well as clinical tests of infection, we find this assay accurately captures the bacterial and viral composition of the sample. The assay presented here is straightforward to implement, offers a systems view into bacterial and viral infections of the urinary tract, and can find future use as a tool for the differential diagnosis of infection.