Effectiveness of Self-Collected, Ambient Temperature-Preserved Nasal Swabs Compared to Samples Collected by Trained Staff for Genotyping of Respiratory Viruses by Shotgun RNA Sequencing: Comparative Study.

BACKGROUND: The SARS-CoV-2 pandemic has underscored the need for field specimen collection and transport to diagnostic and public health laboratories. Self-collected nasal swabs transported without dependency on a cold chain have the potential to remove critical barriers to testing, expand testing capacity, and reduce opportunities for exposure of health professionals in the context of a pandemic. OBJECTIVE: We compared nasal swab collection by study participants from themselves and their children at home to collection by trained research staff. METHODS: Each adult participant collected 1 nasal swab, sampling both nares with the single swab, after which they collected 1 nasal swab from 1 child. After all the participant samples were collected for the household, the research staff member collected a separate single duplicate sample from each individual. Immediately after the sample collection, the adult participants completed a questionnaire about the acceptability of the sampling procedures. Swabs were placed in temperature-stable preservative and respiratory viruses were detected by shotgun RNA sequencing, enabling viral genome analysis. RESULTS: In total, 21 households participated in the study, each with 1 adult and 1 child, yielding 42 individuals with paired samples. Study participants reported that self-collection was acceptable. Agreement between identified respiratory viruses in both swabs by RNA sequencing demonstrated that adequate collection technique was achieved by brief instructions. CONCLUSIONS: Our results support the feasibility of a scalable and convenient means for the identification of respiratory viruses and implementation in pandemic preparedness for novel respiratory pathogens.

Metagenomic Next-Generation Sequencing for Pathogen Detection and Transcriptomic Analysis in Pediatric Central Nervous System Infections.

BACKGROUND: Pediatric central nervous system (CNS) infections are potentially life-threatening and may incur significant morbidity. Identifying a pathogen is important, both in terms of guiding therapeutic management and in characterizing prognosis. Usual care testing by culture and polymerase chain reaction is often unable to identify a pathogen. We examined the systematic application of metagenomic next-generation sequencing (mNGS) for detecting organisms and transcriptomic analysis of cerebrospinal fluid (CSF) in children with central nervous system (CNS) infections. METHODS: We conducted a prospective multisite study that aimed to enroll all children with a CSF pleocytosis and suspected CNS infection admitted to 1 of 3 tertiary pediatric hospitals during the study timeframe. After usual care testing had been performed, the remaining CSF was sent for mNGS and transcriptomic analysis. RESULTS: We screened 221 and enrolled 70 subjects over a 12-month recruitment period. A putative organism was isolated from CSF in 25 (35.7%) subjects by any diagnostic modality. Metagenomic next-generation sequencing of the CSF samples identified a pathogen in 20 (28.6%) subjects, which were also all identified by usual care testing. The median time to result was 38 hours. CONCLUSIONS: Metagenomic sequencing of CSF has the potential to rapidly identify pathogens in children with CNS infections.

A roadmap for the generation of benchmarking resources for antimicrobial resistance detection using next generation sequencing.

Next Generation Sequencing technologies significantly impact the field of Antimicrobial Resistance (AMR) detection and monitoring, with immediate uses in diagnosis and risk assessment. For this application and in general, considerable challenges remain in demonstrating sufficient trust to act upon the meaningful information produced from raw data, partly because of the reliance on bioinformatics pipelines, which can produce different results and therefore lead to different interpretations. With the constant evolution of the field, it is difficult to identify, harmonise and recommend specific methods for large-scale implementations over time. In this article, we propose to address this challenge through establishing a transparent, performance-based, evaluation approach to provide flexibility in the bioinformatics tools of choice, while demonstrating proficiency in meeting common performance standards. The approach is two-fold: first, a community-driven effort to establish and maintain "live" (dynamic) benchmarking platforms to provide relevant performance metrics, based on different use-cases, that would evolve together with the AMR field; second, agreed and defined datasets to allow the pipelines' implementation, validation, and quality-control over time. Following previous discussions on the main challenges linked to this approach, we provide concrete recommendations and future steps, related to different aspects of the design of benchmarks, such as the selection and the characteristics of the datasets (quality, choice of pathogens and resistances, etc.), the evaluation criteria of the pipelines, and the way these resources should be deployed in the community.

Metagenomic sequencing and evaluation of the host response in the pediatric aerodigestive population.

OBJECTIVES: To assess the diagnostic utility of metagenomic sequencing in pediatric aerodigestive clinic patients being evaluated for chronic aspiration. We hypothesize that using a metagenomics platform will aid in the identification of microbes not found on standard culture. STUDY DESIGN AND METHODS: Twenty-four children referred to an aerodigestive clinic were enrolled in a prospective, single-site, cross-sectional cohort study. At the time of clinical evaluation under anesthesia, two samples were obtained: an upper airway sample and a sample from bronchoalveolar lavage (BAL). Samples were sent for routine culture and analyzed using Explify® Respiratory, a CLIA Laboratory Developed Test which identifies respiratory commensals and pathogens through RNA and DNA sequencing. Since RNA was sequenced in the course of the metagenomic analysis to identify organisms (RNA viruses and bacteria), the sequencing approach also captured host derived messenger RNA during sample analysis. This incidentally obtained host transcriptomic data were analyzed to evaluate the host immune response. The results of these studies were correlated with the clinical presentation of the research subjects. RESULTS: In 10 patients, organisms primarily associated with oral flora were identified in the BAL. Standard culture was negative in three patients where clinical metagenomics led to a result with potential clinical significance. Transcriptomic data correlated with the presence or absence of dysphagia as identified on prior videofluoroscopic evaluation of swallowing. CONCLUSIONS: Clinical metagenomics allows for simultaneous analysis of the microbiota and the host immune response from BAL samples. As the technologies in this field continue to advance, such testing may improve the diagnostic evaluation of patients with suspected chronic aspiration.

Added Diagnostic Utility of Clinical Metagenomics for the Diagnosis of Pneumonia in Immunocompromised Adults.

BACKGROUND: In the evaluation of community-acquired pneumonia, 30% to 60% of cases remain undiagnosed, despite extensive conventional microbiologic testing (CMT). Clinical metagenomics (CM) is an unbiased pathogen detection method that can increase diagnostic yield. RESEARCH QUESTION: Does adding clinical metagenomics to conventional microbiologic testing improve the diagnostic yield for pneumonia in immunocompromised adults? STUDY DESIGN AND METHODS: We performed a noninterventional prospective study of immunocompromised adults with pneumonia who underwent bronchoscopy and BAL over 2 years. CMT was performed per standard of care. A commercial CM test was performed on residual BAL fluid. Final microbiologic diagnoses were based on CMT vs CMT + CM. Final clinical diagnoses for CMT and CMT + CM were made based on laboratory results in conjunction with clinical and radiologic findings. Hypothetical impact of CMT + CM on management and antimicrobial stewardship was also assessed. RESULTS: A total of 30 immunocompromised adult patients (31 episodes of pneumonia) were included. Final microbiologic diagnoses were made in 11 cases (35%) with the use of CMT and in 18 cases (58%) with the use of CMT + CM. Bacterial pneumonia was diagnosed in five cases (16%) by CMT and in 13 cases (42%) by CMT + CM; fungal pneumonia was diagnosed in six cases (19%) by CMT and in seven cases (23%) by CMT + CM, and viral pneumonia was diagnosed in two cases (6%) by CMT and in five cases (16%) by CMT + CM. The hypothetical impact of CMT + CM on management was deemed probable in one case, possible in eight cases, and unlikely in two cases, whereas the impact on antimicrobial stewardship was possible in 13 cases and unlikely in seven cases. Final clinical diagnoses were made in 20 of 31 cases (65%) based on CMT and in 23 of 31 cases (74%) based on CMT + CM. INTERPRETATION: CMT + CM increased diagnostic yield in immunocompromised adults with pneumonia from 35% to 58%, mostly by the detection of additional bacterial causes but was less useful for fungal pneumonia.

Contact Transmission of Vaccinia to an Infant Diagnosed by Viral Culture and Metagenomic Sequencing.

Targeted molecular diagnostic tests and accurate immunoassays have transformed the landscape of clinical virology, calling into question the usefulness of traditional viral culture. Here we present a case where viral culture, followed by metagenomic sequencing, was central to the diagnosis of an unexpected viral infection, with significant clinical and public health implications.

Microbiome Diagnostics.

BACKGROUND: During the past decade, breakthroughs in sequencing technology and computational biology have provided the basis for studies of the myriad ways in which microbial communities ("microbiota") in and on the human body influence human health and disease. In almost every medical specialty, there is now a growing interest in accurate and replicable profiling of the microbiota for use in diagnostic and therapeutic application. CONTENT: This review provides an overview of approaches, challenges, and considerations for diagnostic applications borrowing from other areas of molecular diagnostics, including clinical metagenomics. Methodological considerations and evolving approaches for microbiota profiling from mitochondrially encoded 16S rRNA-based amplicon sequencing to metagenomics and metatranscriptomics are discussed. To improve replicability, at least the most vulnerable steps in testing workflows will need to be standardized and continuous efforts needed to define QC standards. Challenges such as purity of reagents and consumables, improvement of reference databases, and availability of diagnostic-grade data analysis solutions will require joint efforts across disciplines and with manufacturers. SUMMARY: The body of literature supporting important links between the microbiota at different anatomic sites with human health and disease is expanding rapidly and therapeutic manipulation of the intestinal microbiota is becoming routine. The next decade will likely see implementation of microbiome diagnostics in diagnostic laboratories to fully capitalize on technological and scientific advances and apply them in routine medical practice.

Clinical Validation of the Aptima Bacterial Vaginosis and Aptima Candida/Trichomonas Vaginitis Assays: Results from a Prospective Multicenter Clinical Study.

Infectious vaginitis due to bacterial vaginosis (BV), vulvovaginal candidiasis (VVC), and Trichomonas vaginalis accounts for a significant proportion of all gynecologic visits in the United States. A prospective multicenter clinical study was conducted to validate the performance of two new in vitro diagnostic transcription-mediated amplification nucleic acid amplification tests (NAATs) for diagnosis of BV, VVC, and trichomoniasis. Patient- and clinician-collected vaginal-swab samples obtained from women with symptoms of vaginitis were tested with the Aptima BV and Aptima Candida/Trichomonas vaginitis (CV/TV) assays. The results were compared to Nugent (plus Amsel for intermediate Nugent) scores for BV, Candida cultures and DNA sequencing for VVC, and a composite of NAAT and culture for T. vaginalis The prevalences of infection were similar for clinician- and patient-collected samples: 49% for BV, 29% for VVC due to the Candida species group, 4% for VVC due to Candida glabrata, and 10% for T. vaginalis Sensitivity and specificity estimates for the investigational tests in clinician-collected samples were 95.0% and 89.6%, respectively, for BV; 91.7% and 94.9% for the Candida species group; 84.7% and 99.1% for C. glabrata; and 96.5% and 95.1% for T. vaginalis Sensitivities and specificities were similar in patient-collected samples. In a secondary analysis, clinicians' diagnoses, in-clinic assessments, and investigational-assay results were compared to gold standard reference methods. Overall, the investigational assays had higher sensitivity and specificity than clinicians' diagnoses and in-clinic assessments, indicating that the investigational assays were more predictive of infection than traditional diagnostic methods. These results provide clinical-efficacy evidence for two in vitro diagnostic NAATs that can detect the main causes of vaginitis.

Clinical Performance of SurePath™ Preservative Compared to PreservCyt® with Cobas® and Hybrid Capture® 2 HPV Tests in a Colposcopy Population.

BACKGROUND: Until recently, no HPV test had been US FDA-approved for SurePath preservative. Clinical performance remains incompletely understood. The clinical performances of the Cobas HPV Test (Cobas) and Hybrid Capture 2 High-Risk HPV DNA Test (HC2) with PreservCyt and SurePath preservatives were compared. METHODS: Cervical cytology samples were collected in both preservatives in random order from women age 21+ (n = 244) referred for colposcopy. Before cytology processing and pelleting, SurePath samples were tested by the Cobas test with and without buffered SDS heat pretreatment. SurePath pellets were tested by the HC2 test and by the Cobas test (with pretreatment). Performance characteristics were calculated in relation to cases of cervical in-traepithelial neoplasia grade 2 or higher (CIN2+) as the clinical target outcome. All HPV-positive samples were also genotyped with the Linear Array test. RESULTS: CIN2+ was detected in 42 patients (17.2%). For both HPV tests, there was a trend towards higher positivity and sensitivity for SurePath compared to PreservCyt preservative. The Cobas test had higher sensitivity than HC2 and the HC2 test had higher specificity than Cobas. Pretreated SurePath samples produced results similar to untreated ones, despite a two-fold dilution during pretreatment [sensitivity %: 95.1 (82.2 - 99.2) vs. 94.3 (79.5 - 99.0); specificity %: 33.0 (26.6 - 40.1) vs. 33.0 (26.4 - 40.3)]. CONCLUSIONS: There was good agreement between the preservatives and HPV tests in detecting HPV and between the Cobas and Linear Array tests for genotyping HR-HPV. These trends were not statistically significant due to the limited number of CIN2+ cases. However, these data may help in evaluations of preservative selection for colposcopy samples. Pre-treatment for Cobas testing eliminated invalid results due to clots. The Cobas test has been FDA-approved for use with heat pretreated SurePath samples.

Fecal Host Transcriptomics for Non-Invasive Human Mucosal Immune Profiling: Proof of Concept in Clostridium Difficile Infection.

Background: Host factors play an important role in pathogenesis and disease outcome in Clostridium difficile infection (CDI), and characterization of these responses could uncover potential host biomarkers to complement existing microbe-based diagnostics. Methods: We extracted RNA from fecal samples of patients with CDI and profiled human mRNA using amplicon-based next-generation sequencing (NGS). We compared the fecal host mRNA transcript expression profiles of patients with CDI to controls with non-CDI diarrhea. Results: We found that the ratio of human actin gamma 1 (ACTG1) to 16S ribosomal RNA (rRNA) was highly correlated with NGS quality as measured by percentage of reads on target. Patients with CDI could be differentiated from those with non-CDI diarrhea based on their fecal mRNA expression profiles using principal component analysis. Among the most differentially expressed genes were ones related to immune response (IL23A, IL34) and actin-cytoskeleton function (TNNT1, MYL4, SMTN, MYBPC3, all adjusted P-values < 1 × 10-3). Conclusions: In this proof-of-concept study, we used host fecal transcriptomics for non-invasive profiling of the mucosal immune response in CDI. We identified differentially expressed genes with biological plausibility based on animal and cell culture models. This demonstrates the potential of fecal transcriptomics to uncover host-based biomarkers for enteric infections.

Gestational Psittacosis Resulting in Neonatal Death Identified by Next-Generation RNA Sequencing of Postmortem, Formalin-Fixed Lung Tissue.

Psittacosis is a rare zoonosis that can cause severe disease and adverse outcomes during pregnancy. We identified a previously elusive case of psittacosis causing premature delivery and infant death by next-generation RNA sequencing of postmortem tissues. Hypothesis-free pathogen detection in postmortem specimens can increase the yield of epidemiologic and cause-of-death studies.

Human Bocavirus Capsid Messenger RNA Detection in Children With Pneumonia.

Background: The role of human bocavirus (HBoV) in respiratory illness is uncertain. HBoV genomic DNA is frequently detected in both ill and healthy children. We hypothesized that spliced viral capsid messenger RNA (mRNA) produced during active replication might be a better marker for acute infection. Methods: As part of the Etiology of Pneumonia in the Community (EPIC) study, children aged <18 years who were hospitalized with community-acquired pneumonia (CAP) and children asymptomatic at the time of elective outpatient surgery (controls) were enrolled. Nasopharyngeal/oropharyngeal specimens were tested for HBoV mRNA and genomic DNA by quantitative polymerase chain reaction. Results: HBoV DNA was detected in 10.4% of 1295 patients with CAP and 7.5% of 721 controls (odds ratio [OR], 1.4 [95% confidence interval {CI}, 1.0-2.0]); HBoV mRNA was detected in 2.1% and 0.4%, respectively (OR, 5.1 [95% CI, 1.6-26]). When adjusted for age, enrollment month, and detection of other respiratory viruses, HBoV mRNA detection (adjusted OR, 7.6 [95% CI, 1.5-38.4]) but not DNA (adjusted OR, 1.2 [95% CI, .6-2.4]) was associated with CAP. Among children with no other pathogens detected, HBoV mRNA (OR, 9.6 [95% CI, 1.9-82]) was strongly associated with CAP. Conclusions: Detection of HBoV mRNA but not DNA was associated with CAP, supporting a pathogenic role for HBoV in CAP. HBoV mRNA could be a useful target for diagnostic testing.

Viral Pathogen Detection by Metagenomics and Pan-Viral Group Polymerase Chain Reaction in Children With Pneumonia Lacking Identifiable Etiology.

Background: Community-acquired pneumonia (CAP) is a leading cause of pediatric hospitalization. Pathogen identification fails in approximately 20% of children but is critical for optimal treatment and prevention of hospital-acquired infections. We used two broad-spectrum detection strategies to identify pathogens in test-negative children with CAP and asymptomatic controls. Methods: Nasopharyngeal/oropharyngeal (NP/OP) swabs from 70 children <5 years with CAP of unknown etiology and 90 asymptomatic controls were tested by next-generation sequencing (RNA-seq) and pan viral group (PVG) PCR for 19 viral families. Association of viruses with CAP was assessed by adjusted odds ratios (aOR) and 95% confidence intervals controlling for season and age group. Results: RNA-seq/PVG PCR detected previously missed, putative pathogens in 34% of patients. Putative viral pathogens included human parainfluenza virus 4 (aOR 9.3, P = .12), human bocavirus (aOR 9.1, P < .01), Coxsackieviruses (aOR 5.1, P = .09), rhinovirus A (aOR 3.5, P = .34), and rhinovirus C (aOR 2.9, P = .57). RNA-seq was more sensitive for RNA viruses whereas PVG PCR detected more DNA viruses. Conclusions: RNA-seq and PVG PCR identified additional viruses, some known to be pathogenic, in NP/OP specimens from one-third of children hospitalized with CAP without a previously identified etiology. Both broad-range methods could be useful tools in future epidemiologic and diagnostic studies.

Development of a real-time PCR assay for the direct detection of Candida species causing Vulvovaginal candidiasis.

Identification of Candida species by traditional methods can be time-consuming and have limited analytical sensitivity. We developed a multiplex real-time PCR assay for detection and differentiation of Candida species causing vulvovaginal candidiasis (VVC). Overall, this PCR assay is a powerful diagnostic tool offering superior accuracy, sensitivity, and specificity.

Validation of Metagenomic Next-Generation Sequencing Tests for Universal Pathogen Detection.

CONTEXT: - Metagenomic sequencing can be used for detection of any pathogens using unbiased, shotgun next-generation sequencing (NGS), without the need for sequence-specific amplification. Proof-of-concept has been demonstrated in infectious disease outbreaks of unknown causes and in patients with suspected infections but negative results for conventional tests. Metagenomic NGS tests hold great promise to improve infectious disease diagnostics, especially in immunocompromised and critically ill patients. OBJECTIVE: - To discuss challenges and provide example solutions for validating metagenomic pathogen detection tests in clinical laboratories. A summary of current regulatory requirements, largely based on prior guidance for NGS testing in constitutional genetics and oncology, is provided. DATA SOURCES: - Examples from 2 separate validation studies are provided for steps from assay design, and validation of wet bench and bioinformatics protocols, to quality control and assurance. CONCLUSIONS: - Although laboratory and data analysis workflows are still complex, metagenomic NGS tests for infectious diseases are increasingly being validated in clinical laboratories. Many parallels exist to NGS tests in other fields. Nevertheless, specimen preparation, rapidly evolving data analysis algorithms, and incomplete reference sequence databases are idiosyncratic to the field of microbiology and often overlooked.

Taxonomer: an interactive metagenomics analysis portal for universal pathogen detection and host mRNA expression profiling.

BACKGROUND: High-throughput sequencing enables unbiased profiling of microbial communities, universal pathogen detection, and host response to infectious diseases. However, computation times and algorithmic inaccuracies have hindered adoption. RESULTS: We present Taxonomer, an ultrafast, web-tool for comprehensive metagenomics data analysis and interactive results visualization. Taxonomer is unique in providing integrated nucleotide and protein-based classification and simultaneous host messenger RNA (mRNA) transcript profiling. Using real-world case-studies, we show that Taxonomer detects previously unrecognized infections and reveals antiviral host mRNA expression profiles. To facilitate data-sharing across geographic distances in outbreak settings, Taxonomer is publicly available through a web-based user interface. CONCLUSIONS: Taxonomer enables rapid, accurate, and interactive analyses of metagenomics data on personal computers and mobile devices.

Expanded Geographic Distribution and Clinical Characteristics of Ehrlichia ewingii Infections, United States.

Ehrlichiosis is a bacterial zoonosis, spread through the bites of infected ticks, that is most commonly caused in the United States by infection with the bacterium Ehrlichia chaffeensis. We retrospectively reviewed samples from an 18-month study of ehrlichiosis in the United States and found that E. ewingii was present in 10 (9.2%) of 109 case-patients with ehrlichiosis, a higher rate of infection with this species than had previously been reported. Two patients resided in New Jersey and Indiana, where cases have not been reported. All patients with available case histories recovered. Our study suggests a higher prevalence and wider geographic distribution of E. ewingii in the United States than previous reports have indicated.