Engineered Mycobacteriophage TM4::GeNL Rapidly Determines Bedaquiline, Pretomanid, Linezolid, Rifampicin, and Clofazimine Sensitivity in Mycobacterium tuberculosis Clinical Isolates.

Background: Drug-resistant tuberculosis is a growing public health threat, and early characterization of the resistance phenotype is essential for guiding treatment and mitigating the high mortality associated with the disease. However, the slow growth rate of Mycobacterium tuberculosis, the causative agent of tuberculosis, necessitates several weeks for conventional culture-dependent drug susceptibility testing (DST). In addition, there are no widely available molecular diagnostic assays for evaluating resistance to newer tuberculosis drugs or drugs with complex resistance mechanisms.

Molecular Epidemiology of Isoniazid-resistant in Port-au-Prince, Haiti.

Background: Isoniazid-resistant, rifampin-susceptible tuberculosis (Hr-TB) is associated with poor treatment outcomes and higher rates of acquisition of further drug resistance during treatment. Due to a lack of widespread diagnostics, Hr-TB is frequently undetected and its epidemiology is incompletely understood.

Methods: We studied the molecular epidemiology of Hr-TB among all patients diagnosed with culture-positive pulmonary tuberculosis between January 1 and June 30, 2017, at an urban referral tuberculosis clinic in Port-au-Prince, Haiti.

Differential rates of Mycobacterium tuberculosis transmission associate with host-pathogen sympatry.

Several human-adapted Mycobacterium tuberculosis complex (Mtbc) lineages exhibit a restricted geographical distribution globally. These lineages are hypothesized to transmit more effectively among sympatric hosts, that is, those that share the same geographical area, though this is yet to be confirmed while controlling for exposure, social networks and disease risk after exposure. Using pathogen genomic and contact tracing data from 2,279 tuberculosis cases linked to 12,749 contacts from three low-incidence cities, we show that geographically restricted Mtbc lineages were less transmissible than lineages that have a widespread global distribution.

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.

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.

Revised Interpretation of the Hain Lifescience GenoType MTBC To Differentiate and Members of the Mycobacterium tuberculosis Complex.

Using 894 phylogenetically diverse genomes of the complex (MTBC), we simulated the ability of the Hain Lifescience GenoType MTBC assay to differentiate the causative agents of tuberculosis. Here, we propose a revised interpretation of this assay to reflect its strengths (e.g.

Using Reduced Inoculum Densities of in MGIT Pyrazinamide Susceptibility Testing to Prevent False-Resistant Results and Improve Accuracy: A Multicenter Evaluation.

The primary platform used for pyrazinamide (PZA) susceptibility testing of is the MGIT culture system (Becton Dickinson). Since false-resistant results have been associated with the use of this system, we conducted a multicenter evaluation to determine the effect of using a reduced cell density inoculum on the rate of false resistance. Two reduced inoculum densities were compared with that prescribed by the manufacturer (designated as "BD" method).

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.

Performance of the G4 Xpert MTB/RIF assay for the detection of Mycobacterium tuberculosis and rifampin resistance: a retrospective case-control study of analytical and clinical samples from high- and low-tuberculosis prevalence settings.

Background: The Xpert MTB/RIF (Xpert) assay is a rapid PCR-based assay for the detection of Mycobacterium tuberculosis complex DNA (MTBc) and mutations associated with rifampin resistance (RIF). An updated version introduced in 2011, the G4 Xpert, included modifications to probe B and updated analytic software.

Methods: An analytical study was performed to assess Xpert detection of mutations associated with rifampin resistance in rifampin-susceptible and -resistant isolates.

Within Host Evolution Selects for a Dominant Genotype of Mycobacterium tuberculosis while T Cells Increase Pathogen Genetic Diversity.

Molecular epidemiological assessments, drug treatment optimization, and development of immunological interventions all depend on understanding pathogen adaptation and genetic variation, which differ for specific pathogens. Mycobacterium tuberculosis is an exceptionally successful human pathogen, yet beyond knowledge that this bacterium has low overall genomic variation but acquires drug resistance mutations, little is known of the factors that drive its population genomic characteristics. Here, we compared the genetic diversity of the bacteria that established infection to the bacterial populations obtained from infected tissues during murine M.

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.

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.

Rapid prediction of inducible clarithromycin resistance in Mycobacterium abscessus.

We have developed a single tube TaqMan(®) real-time PCR assay that differentiates the full-length and truncated erm(41) gene to predict inducible resistance to clarithromycin in Mycobacterium abscessus. A study of 87 clinical isolates found this assay to be 90.8% concordant to conventional drug susceptibility testing results for the prediction of inducible clarithromycin drug resistance.

Multilocus sequence typing of Mycobacterium xenopi.

Mycobacterium xenopi is an opportunistic mycobacterial pathogen of increasing clinical importance. Surveillance of M. xenopi is hampered by the absence of tools for genotyping and molecular epidemiology.

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.

Correlation between genotypic and phenotypic testing for resistance to rifampin in Mycobacterium tuberculosis clinical isolates in Haiti: investigation of cases with discrepant susceptibility results.

The World Health Organization has recommended use of molecular-based tests MTBDRplus and GeneXpert MTB/RIF to diagnose multidrug-resistant tuberculosis in developing and high-burden countries. Both tests are based on detection of mutations in the Rifampin (RIF) Resistance-Determining Region of DNA-dependent RNA Polymerase gene (rpoB). Such mutations are found in 95-98% of Mycobacterium tuberculosis strains determined to be RIF-resistant by the "gold standard" culture-based drug susceptibility testing (DST).

Use of Luminex MagPlex magnetic microspheres for high-throughput spoligotyping of Mycobacterium tuberculosis isolates in Port-au-Prince, Haiti.

Genotyping of Mycobacterium tuberculosis strains became indispensable for understanding tuberculosis transmission dynamics and designing measures to combat the disease. Unfortunately, typing involves sophisticated laboratory analysis, is expensive, and requires a high level of technical expertise, which limited its use in the resource-poor countries where the majority of tuberculosis cases occur. Spoligotyping is a PCR-based M.

Outbreak of Mycobacterium chelonae infection associated with tattoo ink.

Background: In January 2012, on the basis of an initial report from a dermatologist, we began to investigate an outbreak of tattoo-associated Mycobacterium chelonae skin and soft-tissue infections in Rochester, New York. The main goals were to identify the extent, cause, and form of transmission of the outbreak and to prevent further cases of infection.

Methods: We analyzed data from structured interviews with the patients, histopathological testing of skin-biopsy specimens, acid-fast bacilli smears, and microbial cultures and antimicrobial susceptibility testing.

Multidrug-resistant tuberculosis in Port-au-Prince, Haiti.

Objective: To determine the prevalence of multidrug-resistant tuberculosis (MDR-TB) among patients with new smear-positive pulmonary TB in Port-au-Prince, Haiti.

Methods: Sputum samples were cultured from 1 006 patients newly diagnosed with TB in 2008. The core region of the rpoB gene that is associated with resistance to rifampin was sequenced.