Direct detection of drug-resistant using targeted next generation sequencing.

complex (MTBC) infections are treated with combinations of antibiotics; however, these regimens are not as efficacious against multidrug and extensively drug resistant MTBC. Phenotypic (growth-based) drug susceptibility testing on slow growing bacteria like MTBC requires many weeks to months to complete, whereas sequencing-based approaches can predict drug resistance (DR) with reduced turnaround time. We sought to develop a multiplexed, targeted next generation sequencing (tNGS) assay that can predict DR and can be performed directly on clinical respiratory specimens.

Characterization of sp. nov.: A novel nontuberculous mycobacteria isolated from a human wound infection.

Nontuberculous mycobacteria (NTM) are environmental bacteria commonly found in soil and water in almost every part of the world. While usually non-pathogenic, they can cause acute respiratory and cutaneous infections under certain circumstances or in patients with underlying medical conditions. Contrary to members of the complex, documented human-to-human transmissions of NTM have been rarely reported and most cases result from direct environmental exposure.

Novel Mycobacterium tuberculosis Complex Genotype Related to M. caprae.

We report the unusual genotypic characterization of a bacterium isolated from a clinical sample of a patient who grew up in Bangladesh and lives in the United States. Using whole-genome sequencing, we identified the bacterium as a member of the Mycobacterium tuberculosis complex (MTBC). Phylogenetic placement of this strain suggests a new MTBC genotype.

Laboratory diagnosis of bacterial meningitis by direct detection, serotyping and Next Generation Sequencing: How 10 years of testing in New York State has evolved to improve laboratory diagnosis and public health.

Since 2005, the Wadsworth Center (WC) has provided molecular testing on cerebrospinal fluid (CSF) and whole blood specimens in close collaboration with epidemiologists in New York State and New York City. In this study, we analyzed 10 years of data to demonstrate the significant value of utilizing molecular methods to assess patient specimens for etiologic agents of bacterial meningitis. A comprehensive molecular testing algorithm to detect and serotype/serogroup bacterial agents known to cause bacterial meningitis (Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae and Streptococcus agalactiae) has evolved, and retrospective specimen testing has been essential for each improvement.

Towards Unified Data Exchange Formats for Reporting Molecular Drug Susceptibility Testing.

Background: With the rapid development of new advanced molecular detection methods, identification of new genetic mutations conferring pathogen resistance to an ever-growing variety of antimicrobial substances will generate massive genomic datasets for public health and clinical laboratories. Keeping up with specialized standard coding for these immense datasets will be extremely challenging. This challenge prompted our effort to create a common molecular resistance Logical Observation Identifiers Names and Codes (LOINC) panel that can be used to report any identified antimicrobial resistance pattern.

Assessing Nanopore Sequencing for Clinical Diagnostics: a Comparison of Next-Generation Sequencing (NGS) Methods for Mycobacterium tuberculosis.

Next-generation sequencing technologies are being rapidly adopted as a tool of choice for diagnostic and outbreak investigation in public health laboratories. However, costs of operation and the need for specialized staff remain major hurdles for laboratories with limited resources for implementing these technologies. This project aimed to assess the feasibility of using Oxford Nanopore MinION whole-genome sequencing data of isolates for species identification, spoligotyping, detection of mutations associated with antimicrobial resistance (AMR) to accurately predict drug susceptibility profiles, and phylogenetic analysis to detect transmission between cases.

Low-Level Rifampin Resistance and Mutations in Mycobacterium tuberculosis: an Analysis of Whole-Genome Sequencing and Drug Susceptibility Test Data in New York.

Rapid and reliable detection of rifampin (RIF) resistance is critical for the diagnosis and treatment of drug-resistant and multidrug-resistant (MDR) tuberculosis. Discordant RIF phenotype/genotype susceptibility results remain a challenge due to the presence of mutations that do not confer high levels of RIF resistance, as have been exhibited in strains with mutations such as Ser450Leu. These strains, termed low-level RIF resistant, exhibit elevated RIF MICs compared to fully susceptible strains but remain phenotypically susceptible by mycobacterial growth indicator tube (MGIT) testing and have been associated with poor patient outcomes.

Rapid Diagnosis of Meningococcal Meningitis in a Patient With Familial Mediterranean Fever by the FilmArray Meningitis/Encephalitis Panel: A Case Report.

Familial Mediterranean fever (FMF) is a rare autoinflammatory disorder of the innate immune system. Patients with innate immune system defects are at a high risk of meningococcal disease, although it is unclear if patients with FMF also have increased susceptibility to invasive infection. Herein, we present a diagnostically challenging case of a male adolescent with a past medical history of FMF stabilized on colchicine who presented with some of the early clinical features of community-acquired bacterial meningitis.

Carriage of Neisseria meningitidis in Men Who Have Sex With Men Presenting to Public Sexual Health Clinics, New York City.

Background: We conducted a Neisseria meningitidis (Nm) carriage study among men who have sex with men (MSM) to explore possible sexual transmission.

Methods: We paired information on patient characteristics with oropharyngeal, rectal, and urethral Nm culture results to assess associations with Nm carriage among 706 MSM at New York City sexual health clinics. The Nm isolates were characterized by whole genome sequencing.

Whole-Genome Single-Nucleotide Polymorphism (SNP) Analysis Applied Directly to Stool for Genotyping Shiga Toxin-Producing Escherichia coli: an Advanced Molecular Detection Method for Foodborne Disease Surveillance and Outbreak Tracking.

Whole-genome sequencing (WGS) of pathogens from pure culture provides unparalleled accuracy and comprehensive results at a cost that is advantageous compared with traditional diagnostic methods. Sequencing pathogens directly from a primary clinical specimen would help circumvent the need for culture and, in the process, substantially shorten the time to diagnosis and public health reporting. Unfortunately, this approach poses significant challenges because of the mixture of multiple sequences from a complex fecal biomass.

Neonatal Conjunctivitis Caused by Neisseria meningitidis US Urethritis Clade, New York, USA, August 2017.

We characterized a case of neonatal conjunctivitis in New York, USA, caused by Neisseria meningitidis by using whole-genome sequencing. The case was a rare occurrence, and the isolate obtained belonged to an emerging clade (N. meningitidis US nongroupable urethritis) associated with an increase in cases of urethritis since 2015.

Sex Difference in Meningococcal Disease Mortality, New York City, 2008-2016.

Background: The case fatality rate (CFR) from invasive meningococcal disease (IMD) in New York City (NYC) is greater than national figures, with higher rates among females than males across all age groups.

Methods: We conducted a retrospective cohort study among 151 persons aged ≥15 years diagnosed with IMD in NYC during 2008-2016 identified through communicable disease surveillance. We examined demographic, clinical, and community-level associations with death to confirm the elevated risk of mortality among female IMD patients after adjusting for confounders and to determine factors associated with female IMD mortality.

Mycobacterium orygis Lymphadenitis in New York, USA.

We report a case of lymphadenitis caused by Mycobacterium orygis in an immunocompetent person in Stony Brook, New York, USA. Initial real-time PCR assay failed to provide a final subspecies identification within the M. tuberculosis complex, but whole-genome sequencing characterized the isolate as M.

Comprehensive Whole-Genome Sequencing and Reporting of Drug Resistance Profiles on Clinical Cases of Mycobacterium tuberculosis in New York State.

Whole-genome sequencing (WGS) is a newer alternative for tuberculosis (TB) diagnostics and is capable of providing rapid drug resistance profiles while performing species identification and capturing the data necessary for genotyping. Our laboratory developed and validated a comprehensive and sensitive WGS assay to characterize and other complex (MTBC) strains, composed of a novel DNA extraction, optimized library preparation, paired-end WGS, and an in-house-developed bioinformatics pipeline. This new assay was assessed using 608 MTBC isolates, with 146 isolates during the validation portion of this study and 462 samples received prospectively.

Detection of Borrelia miyamotoi and other tick-borne pathogens in human clinical specimens and Ixodes scapularis ticks in New York State, 2012-2015.

Borrelia miyamotoi (Bm) is a recently emerging bacterial agent transmitted by several species of ixodid ticks. Diagnosis of Bm infection can be challenging, as the organism is not easily cultivable. We have developed and validated a multiplex real-time PCR to simultaneously identify Bm infection and the agents causing human granulocytic anaplasmosis and human monocytic ehrlichiosis, Anaplasma phagocytophilum and Ehrlichia chaffeensis, respectively.

Draft Genome Sequence of Branchiibius sp. NY16-3462-2, Isolated from a Mixed Clinical Sample.

Here, we report the release of a draft genome assembly of a Gram-positive cocci Branchiibius sp. NY16-3462-2 with a high-GC content, sequenced from a mixed clinical sample containing Mycobacterium tuberculosis This genome is the first publicly available sequence from a representative of the genus Branchiibius.

Bordetella pertussis Strain Lacking Pertactin and Pertussis Toxin.

A Bordetella pertussis strain lacking 2 acellular vaccine immunogens, pertussis toxin and pertactin, was isolated from an unvaccinated infant in New York State in 2013. Comparison with a French strain that was pertussis toxin-deficient, pertactin wild-type showed that the strains carry the same 28-kb deletion in similar genomes.

Comparative analytical evaluation of the respiratory TaqMan Array Card with real-time PCR and commercial multi-pathogen assays.

In this study, a multicenter evaluation of the Life Technologies TaqMan(®) Array Card (TAC) with 21 custom viral and bacterial respiratory assays was performed on the Applied Biosystems ViiA™ 7 Real-Time PCR System. The goal of the study was to demonstrate the analytical performance of this platform when compared to identical individual pathogen specific laboratory developed tests (LDTs) designed at the Centers for Disease Control and Prevention (CDC), equivalent LDTs provided by state public health laboratories, or to three different commercial multi-respiratory panels. CDC and Association of Public Health Laboratories (APHL) LDTs had similar analytical sensitivities for viral pathogens, while several of the bacterial pathogen APHL LDTs demonstrated sensitivities one log higher than the corresponding CDC LDT.

A Model of Shared Mycobacteriology Testing Services: Lessons Learned.

Objective: The need for public health laboratories (PHLs) to prioritize resources has led to increased interest in sharing diagnostic services. To address this concept for tuberculosis (TB) testing, the New York State Department of Health Wadsworth Center and the Rhode Island State Health Laboratories assessed the feasibility of shared services for the detection and characterization of Mycobacterium tuberculosis complex (MTBC).

Methods: We assessed multiple aspects of shared services including shipping, testing, reporting, and cost.

Direct molecular testing to assess the incidence of meningococcal and other bacterial causes of meningitis among persons reported with unspecified bacterial meningitis.

Confirmed and probable cases of invasive Neisseria meningitidis (Nm) infection are reportable in New York City. We conducted a study to identify Nm among culture-negative reports of bacterial and viral meningitis. During the study period, 262 reports of suspected meningitis were eligible.

Detection of Mycobacterium avium complex DNA directly in clinical respiratory specimens: opportunities for improved turn-around time and cost savings.

We developed, evaluated, and implemented a Taqman multiplex real-time polymerase chain reaction (PCR) assay for the detection of Mycobacterium avium complex (MAC), targeting the 16S-23S rRNA internal transcribed spacer, which we have combined with an existing Mycobacterium tuberculosis complex assay for use directly in clinical respiratory specimens. Evaluation of the performance of this assay for MAC detection included 464 clinical respiratory specimens tested prospectively. This real-time PCR assay was found overall to have a sensitivity of 71.

Postmortem diagnosis of invasive meningococcal disease.

We diagnosed invasive meningococcal disease by using immunohistochemical staining of embalmed tissue and PCR of vitreous humor from 2 men in New York City. Because vitreous humor is less subject than other body fluids to putrefaction, it is a good material for postmortem analysis.

Pertactin-negative variants of Bordetella pertussis in New York State: a retrospective analysis, 2004-2013.

The first report of pertactin-negative variants of Bordetella pertussis in the United States has raised questions about the role of acellular pertussis vaccines in the recent increase of pertussis cases. Our laboratory utilized a sequence-based method to identify mutations in the pertactin gene responsible for these variants and assessed vaccination status from the associated cases.