Erik Samayoa

Actionable Diagnosis of Neuroleptospirosis by Next-Generation Sequencing

A 14-year-old boy with severe combined immunodeficiency presented three times to a medical facility over a period of 4 months with fever and headache that progressed to hydrocephalus and status epilepticus necessitating a medically induced coma. Diagnostic workup including brain biopsy was unrevealing. Unbiased next-generation sequencing of the cerebrospinal fluid identified 475 of 3,063,784 sequence reads (0.016%) corresponding to leptospira infection. Clinical assays for leptospirosis were negative. Targeted antimicrobial agents were administered, and the patient was discharged home 32 days later with a status close to his premorbid condition. Polymerase-chain-reaction (PCR) and serologic testing at the Centers for Disease Control and Prevention (CDC) subsequently confirmed evidence of Leptospira santarosai infection.

Diagnosis of Neuroinvasive Astrovirus Infection in an Immunocompromised Adult With Encephalitis by Unbiased Next-Generation Sequencing

Metagenomic next-generation sequencing (NGS) was used to diagnose an unusual and fatal case of progressive encephalitis in an immunocompromised adult presenting at disease onset as bilateral hearing loss. The sequencing and confirmatory studies revealed neuroinvasive infection of the brain by an astrovirus belonging to a recently discovered VA/HMO clade.

A novel bocavirus in canine liver

BackgroundBocaviruses are classified as a genus within the Parvoviridae family of single-stranded DNA viruses and are pathogenic in some mammalian species. Two species have been previously reported in dogs, minute virus of canines (MVC), associated with neonatal diseases and fertility disorders; and Canine bocavirus (CBoV), associated with respiratory disease.FindingsIn this study using deep sequencing of enriched viral particles from the liver of a dog with severe hemorrhagic gastroenteritis, necrotizing vasculitis, granulomatous lymphadenitis and anuric renal failure, we identified and characterized a novel bocavirus we named Canine bocavirus 3 (CnBoV3). The three major ORFs of CnBoV3 (NS1, NP1 and VP1) shared less than 60% aa identity with those of other bocaviruses qualifying it as a novel species based on ICTV criteria. Inverse PCR showed the presence of concatemerized or circular forms of the genome in liver.ConclusionsWe genetically characterized a bocavirus in a dog liver that is highly distinct from prior canine bocaviruses found in respiratory and fecal samples. Its role in this animal’s complex disease remains to be determined.

Neurobrucellosis: Unexpected Answer From Metagenomic Next-Generation Sequencing

A diagnosis of brucellosis can be difficult because routine culture and serological methods exhibit variable sensitivity and specificity. We present the use of a metagenomic next- generation sequencing assay to diagnose a case of neurobrucellosis from cerebrospinal fluid, resulting in the institution of appropriate antibiotic treatment and a favorable clinical outcome.

Rosavirus: the prototype of a proposed new genus of the Picornaviridae family.

We describe a 8,724-nucleotide-long picornavirus genome encoding a single 2,470-aa polyprotein obtained from the feces of a wild mouse. Rosavirus is genetically closest to the double ORF Dicipivirus found in canine feces that is currently the only picornavirus with a second internal ribosome entry site (IRES). Of note, a section of rosavirus’ 5′UTR showed strong sequence and structural conservation with the type II IRES from the Parechovirus and Hungarovirus genera possibly reflecting exchange of genetic modules between genera. Based on genetic distance criteria rosavirus qualifies as prototype of a new genus of the Picornaviridae family.

Clinical Utility of Unbiased Metagenomic Next-Generation Sequencing in Diagnosis of Acute Infectious Diseases: A Prospective Case Series.

Background. Unbiased metagenomic next-generation sequencing (mNGS) allows the identification of any pathogen based on nucleic acid sequence. Pan-pathogen detection may constitute an ideal diagnostic test for acute infectious diseases (ID), often caused by a wide variety of pathogens. Methods. To evaluate mNGS utility for ID diagnosis, acutely ill patients were enrolled in a prospective, institutional review board-approved research study comparing mNGS with conventional testing. Patients were referred for mNGS testing by ID specialists. Inclusion criteria included (1) high clinical suspicion of infection but no diagnosis to date, (2) extensive negative workup (“last resort” testing), (3) confirmation of infection by an agent suspected based on symptoms and travel history for which testing was not readily available or (4) for which speciation or genomic characterization might be clinically useful. Clinical samples were sequenced by mNGS, and data were analyzed using the SURPI bioinformatics pathogen detection pipeline. Results were communicated to referring providers. Results. We enrolled 28 patients from 4 hospitals with clinical syndromes including neurological symptoms (n = 15), pneumonia (n = 2), influenza-like illness (4) or other (n = 7). (1) In patients with high suspicion of infection, credible infectious etiology was found in 4 of 9 patients by mNGS, including neuroleptospirosis and astrovirus encephalitis diagnoses impacting management. (2) “Last resort” mNGS testing was performed in 10 cases; mNGS as well as conventional testing revealed no infectious etiology in all 10. (3) mNGS confirmed suspected etiology in 4 of 4 patients, including Angiostrongylus cantonensis in a patient with eosinophilic meningitis. (4) In 4 cases, mNGS was used to confirm infection by genomic characterization. Leishmania infantum was speciated in an imported case of visceral leishmaniasis, Mycobacterium tuberculosis in a lung transplant patient, and influenza A(H1N1)pdm2009 in 2 previously vaccinated individuals. Conclusion. mNGS for pan-pathogen ID adds diagnostic value where infectious etiologies are suspected. In all 15 cases in which mNGS testing failed to detect a credible infectious etiology, conventional microbiological testing was also negative, suggesting that mNGS testing may be potentially useful as a “rule-out” assay to exclude infection. Disclosures. All authors: No reported disclosures.

Development and clinical evaluation of a novel fully automated qualitative PCR assay for the diagnosis of anogenital herpes simplex virus infection

Molecular detection of viral infections has the potential to improve microbial diagnostics, particularly with the emergence of rapid automated systems. We describe the design of the IDbox fully automated cassette-based system for nucleic acid extraction and real-time PCR amplification and perform a clinical evaluation for the diagnosis of genital herpes simplex infections. At optimal cutoff values determined by receiver-operator curves, the IDbox showed sensitivities of 94.9% (95% confidence interval [CI] 84.9-98.7%) and 97.0% (95% CI 88.5-99.5%) and specificities of 96.7% (95% CI 91.2-98.9%) and 97.3% (95% CI 91.9-99.3%) relative to herpes simplex virus culture and PCR, respectively. We discuss relevant design characteristics and approaches used for each step of the analytical process to enhance assay sensitivity and provide accurate results in the presence of potential cross-reactive organisms and interfering substances.

No viral association found in a set of differentiated vulvar intraepithelial neoplasia cases by human papillomavirus and pan-viral microarray testing

Vulvar Intraepithelial Neoplasia (VIN) is the precursor lesion of Vulvar Squamous Cell Carcinoma (VSCC), and the differentiated type (dVIN) is more frequently observed in relation to VSCC. In contrast to usual-type VIN (uVIN), which is related to infection by human papillomavirus (HPV), a germline mutation in the p53 gene is thought to be associated with ~90% of dVIN cases. To date, no infectious agent has been identified in association with dVIN, and studies investigating this possibility have been hindered by the difficulty in accurately diagnosing dVIN from small biopsies. Here, we used immunostaining for p16ink4a, a biomarker for HPV infection, to study 14 uVIN high-grade VIN and 14 dVIN cases, and to select 10 dVIN cases to broadly screen for all known viruses using a pan-viral microarray platform (ViroChip). All of the uVIN tissue samples, including 8 warty and 6 basaloid cases, showed positivity with the p16ink4a immunostain. The staining pattern was full-thickness for all except two cases in which positive staining was localized in the lower 1/3 of the epidermis. In contrast, immunostaining for p16ink4a was negative in all dVIN cases. ViroChip analysis of 10 pure dVIN samples confirmed the absence of human papillomavirus subtypes or any other virus with the exception of a single sample that showed a weak microarray signature to a porcine herpesvirus. Follow-up PCR testing of the sample was negative for herpesvirus, and in-depth metagenomic next-generation sequencing revealed only sequences corresponding to non-pathogenic viral flora and bacterial contamination. In this study, we demonstrated lack of a virus association in 10 dVIN cases. Alternative pathways for carcinogenesis such as the p53 mutation should be considered for investigation of potential treatment options in dVIN.

Laboratory Validation of a Clinical Metagenomic Sequencing Assay for Pathogen Detection in Cerebrospinal Fluid

Metagenomic next-generation sequencing (mNGS) for pan-pathogen detection has been successfully tested in proof-of-concept case studies in patients with acute illness of unknown etiology, but to date has been largely confined to research settings. Here we developed and validated an mNGS assay for diagnosis of infectious causes of meningitis and encephalitis from cerebrospinal fluid (CSF) in a licensed clinical laboratory. A clinical bioinformatics pipeline, SURPI+, was developed to rapidly analyze mNGS data, automatically report detected pathogens, and provide a graphical user interface for evaluating and interpreting results. We established quality metrics, threshold values, and limits of detection of between 0.16 – 313 genomic copies or colony forming units per milliliter for each representative organism type. Gross hemolysis and excess host nucleic acid reduced assay sensitivity; however, a spiked phage used as an internal control was a reliable indicator of sensitivity loss. Diagnostic test accuracy was evaluated by blinded mNGS testing of 95 patient samples, revealing 73% sensitivity and 99% specificity compared to original clinical test results, with 81% positive percent agreement and 99% negative percent agreement after discrepancy analysis. Subsequent mNGS challenge testing of 20 positive CSF samples prospectively collected from a cohort of pediatric patients hospitalized with meningitis, myelitis, and/or encephalitis showed 92% sensitivity and 96% specificity relative to conventional microbiological testing of CSF in identifying the causative pathogen. These results demonstrate the analytic performance of a laboratory-validated mNGS assay for pan-pathogen detection, to be used clinically for diagnosis of neurological infections from CSF.