Mycobacterium tuberculosis complex whole-genome sequencing in New York State: Implementation of a reduced phenotypic drug susceptibility testing algorithm.

Whole-genome sequencing (WGS) can predict drug resistance and antimicrobial susceptibility in Mycobacterium tuberculosis complex (MTBC) and has shown promise in partially replacing culture-based phenotypic drug susceptibility testing (pDST). We performed a two-year side by side study comparing the prediction of drug resistance and antimicrobial susceptibility by WGS molecular DST (mDST) to pDST to determine resistance at the critical concentration by Mycobacterial Growth Indicator Tube (MGIT) and agar proportion testing. Negative predictive values of WGS results were consistently high for the first-line drugs: rifampin (99.9%), isoniazid (99.0%), pyrazinamide (98.5%), and ethambutol (99.8%); the rates of resistance to these drugs, among strains in our population, are 2.9%, 10.4%, 46.3%, and 2.3%, respectively. WGS results were available an average 8 days earlier than first-line MGIT pDST. Based on these findings, we implemented a new testing algorithm with an updated WGS workflow in which strains predicted pan-susceptible were no longer tested by pDST. This algorithm was applied to 1177 isolates between October 2018 and September 2020, eliminating pDST for 66.6% of samples and reducing pDST for an additional 22.0%. This algorithm change resulted in faster turnaround times and decreased cost while maintaining comprehensive antimicrobial susceptibility profiles of all culture-positive MTBC cases in New York.

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

Mycobacterium tuberculosis 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. A multiplex PCR was designed to amplify a group of thirteen full-length genes and promoter regions with mutations known to be involved in resistance to first- and second-line MTBC drugs. Long-read amplicon libraries were sequenced with Oxford Nanopore Technologies platforms and high-confidence resistance mutations were identified in real-time using an in-house developed bioinformatics pipeline. Sensitivity, specificity, reproducibility, and accuracy of the tNGS assay was assessed as part of a clinical validation study. In total, tNGS was performed on 72 primary specimens and 55 MTBC-positive cultures and results were compared to clinical whole genome sequencing (WGS) performed on paired patient cultures. Complete or partial susceptibility profiles were generated from 82% of smear positive primary specimens and the resistance mutations identified by tNGS were 100% concordant with WGS. In addition to performing tNGS on primary clinical samples, this assay can be used to sequence MTBC cultures mixed with other mycobacterial species that would not yield WGS results. The assay can be effectively implemented in a clinical/diagnostic laboratory with a two to three day turnaround time and, even if batched weekly, tNGS results are available on average 15 days earlier than culture-derived WGS results. This study demonstrates that tNGS can reliably predict MTBC drug resistance directly from clinical specimens or cultures and provide critical information in a timely manner for the appropriate treatment of patients with DR tuberculosis.

Characterization of Mycobacterium salfingeri 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 Mycobacterium tuberculosis complex, documented human-to-human transmissions of NTM have been rarely reported and most cases result from direct environmental exposure. Here we describe the identification of a new NTM species isolated from a hand laceration of a New York State patient after a fall. This new NTM forms rough, orange pigmented colonies and is naturally resistant to doxycycline and tobramycin. Whole genome analysis reveal no close relatives present in public databases, and our findings are in accordance with the recognition of a new taxonomic species of NTM. We propose the name Mycobacterium salfingeri sp. nov. for this new NTM representative. The type strain is 20-157661T (DSM = 113368T, BCCM = ITM 501207T).

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. Even though it had the same spoligotype as M. caprae strains, single-nucleotide polymorphism-based phylogenetic analysis placed the isolate as a sister lineage distinct from M. caprae, most closely related to 5 previously sequenced genomes isolated from primates and elephants in Asia. We propose a new animal-associated lineage, La4, within MTBC.

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. Over a ten-year span from 2010 to 2019 the WC received 831 specimens from 634 patients with suspected bacterial meningitis. Real-time PCR was positive for at least one of the agents in 223 (27%) specimens from 183 patients (29%). Of the 223 positives, 146 (66%) were further characterized by real-time PCR into serogroup/serotype. Additionally, examination of 131 paired specimens of CSF and whole blood from the same patients found better detection in CSF, but whole blood is a useful alternative for diagnosis when CSF is not available. For specimens initially PCR-negative, 16S rDNA Sanger sequencing was requested by the submitter for 146 cases resulting in the identification of bacterial agents in an additional 24 (16%) specimens. In a retrospective study, Next Generation Sequencing (NGS) was evaluated for the detection of pathogens in 53 previously tested PCR-negative CSF specimens and identified bacteria in 14 (26%) specimens. This molecular testing algorithm has provided clinicians a diagnosis when culture is negative with the potential to guide therapy. It has also aided public health in determining when antibiotic prophylaxis was needed, augmented surveillance data to yield a fuller picture of community prevalence, and highlighted gaps in the spectrum of agents that cause bacterial meningitis.

Low-Level Rifampin Resistance and rpoB 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 rpoB 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. Here, we assess RIF resistance prediction by whole-genome sequencing (WGS) among a set of 1,779 prospectively tested strains by both prevalence of rpoB gene mutation and phenotype as part of routine clinical testing during a 2.5-year period. During this time, 139 strains were found to have nonsynonymous rpoB mutations, 53 of which were associated with RIF resistance, including both low-level and high-level resistance. Resistance to RIF (1.0 µg/ml in MGIT) was identified in 43 (81.1%) isolates. The remaining 10 (18.9%) strains were susceptible by MGIT but were confirmed to be low-level RIF resistant by MIC testing. Full rpoB gene sequencing overcame the limitations of critical concentration phenotyping, probe-based genotyping, and partial gene sequencing methods. Universal clinical WGS with concurrent phenotypic testing provided a more complete understanding of the prevalence and type of rpoB mutations and their association with RIF resistance in New York.

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. OBJECTIVE: To develop and utilize a common molecular resistance LOINC panel for molecular drug susceptibility testing (DST) data exchange in the U.S. National Tuberculosis Surveillance System using California Department of Public Health (CDPH) and New York State Department of Health as pilot sites. METHODS: We developed an interface and mapped incoming molecular DST data to the common molecular resistance LOINC panel using Health Level Seven (HL7) v2.5.1 Electronic Laboratory Reporting (ELR) message specifications through the Orion Health™ Rhapsody Integration Engine v6.3.1. RESULTS: Both pilot sites were able to process and upload/import the standardized HL7 v2.5.1 ELR messages into their respective systems; albeit CDPH identified areas for system improvements and has focused efforts to streamline the message importation process. Specifically, CDPH is enhancing their system to better capture parent-child elements and ensure that the data collected can be accessed seamlessly by the U.S. Centers for Disease Control and Prevention. DISCUSSION: The common molecular resistance LOINC panel is designed to be generalizable across other resistance genes and ideally also applicable to other disease domains. CONCLUSION: The study demonstrates that it is possible to exchange molecular DST data across the continuum of disparate healthcare information systems in integrated public health environments using the common molecular resistance LOINC panel.

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 Mycobacterium tuberculosis isolates for species identification, in silico spoligotyping, detection of mutations associated with antimicrobial resistance (AMR) to accurately predict drug susceptibility profiles, and phylogenetic analysis to detect transmission between cases. The results were compared prospectively in real time to those obtained with our current clinically validated Illumina MiSeq sequencing assay for M. tuberculosis and phenotypic drug susceptibility testing results when available. Our assessment of 431 sequenced samples over a 32-week period demonstrates that, when using the proper quality controls and thresholds, the MinION can achieve levels of genotyping analysis and phenotypic resistance predictions comparable to those of the Illumina MiSeq at a very competitive cost per sample. Our results indicate that nanopore sequencing can be a suitable alternative to, or complement, currently used sequencing platforms in a clinical setting and has the potential to be widely adopted in public health laboratories in the near future.

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. To our knowledge, this is the first case of meningococcal meningitis in a patient with FMF diagnosed with the FilmArray Meningitis/Encephalitis (ME) Panel. This case report demonstrates that rapid detection of Neisseria meningitidis by the ME Panel can aid in the early diagnosis and prompt treatment of patients with suspected meningitis and may be the only positive test in patients with early presentation and/or prior antimicrobial therapy.

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. RESULTS: Twenty-three percent (163 of 706) of MSM were Nm carriers. Oropharyngeal carriage was 22.6% (159 of 703), rectal 0.9% (6 of 695), and urethral 0.4% (3 of 696). Oropharyngeal carriage was associated with the following recent (past 30 days) exposures: 3 or more men kissed (adjusted relative risk [aRR], 1.38; 95% confidence interval [CI], 1.03-1.86), performing oral sex (aRR, 1.81; 95% CI, 1.04-3.18), and antibiotic use (aRR, 0.33; 95% CI, 0.19-0.57). Sixteen clonal complexes were identified; 27% belonged to invasive lineages. CONCLUSIONS: Our findings suggest that oral sex and the number of recent kissing partners contribute to Nm carriage in MSM.

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. The aim of this project was to develop a proof of concept protocol for the sequencing and genotyping of Shiga toxin-producing Escherichia coli (STEC) directly from stool specimens. We have developed an enrichment protocol that reliably achieves a substantially higher DNA yield belonging to E. coli, which provides adequate next-generation sequencing (NGS) data for downstream bioinformatics analysis. A custom bioinformatics pipeline was created to optimize and remove non-E. coli reads, assess the STEC versus commensal E. coli population in the samples, and build consensus sequences based on population allele frequency distributions. Side-by-side analysis of WGS from paired STEC isolates and matched primary stool specimens reveal that this method can reliably be implemented for many clinical specimens to directly genotype STEC and accurately identify clusters of disease outbreak when no STEC isolate is available for testing.

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.

Lymphogranuloma Venereum: An Increasingly Common Anorectal Infection Among Men Who Have Sex With Men Attending New York City Sexual Health Clinics.

Using Chlamydia trachomatis anorectal specimens routinely tested for lymphogranuloma venereum (LGV) (2008-2011) and samples of archived specimens tested for LGV (2012-2015), we observed increased LGV positivity among men who have sex with men attending NYC Sexual Health Clinics. Using clinical data, we determined predictors of anorectal LGV that may guide clinical management.

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. Relative risks of death were estimated using multivariable log-linear Poisson regression with a robust error variance. Results: Females had a higher CFR (n = 23/62; 37%) following IMD than males (n = 17/89; 19%) (adjusted relative risk [aRR], 2.1; 95% confidence interval [CI], 1.2-3.8). Controlling for demographic and clinical factors, there was a significant interaction between sex and fatal outcomes related to meningitis: the relative risk of death for females with meningitis was 13.7 (95% CI, 3.2-58.1) compared with males. In the model restricted to females, altered mental status (aRR, 7.5; 95% CI, 2.9-19.6) was significantly associated with an increased risk of death. Conclusions: Female mortality from IMD was significantly increased compared with males, controlling for other predictors of mortality. Sex-based differences in recognition and treatment need to be evaluated in cases of meningococcal disease. Our study highlights the importance of analyzing routine surveillance data to identify and address disparities in disease incidence and outcomes.

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. orygis.

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 Mycobacterium tuberculosis and other M. tuberculosis 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. In February 2016, this assay was implemented to test all clinical cases of MTBC in New York State, including isolates and early positive Bactec mycobacterial growth indicator tube (MGIT) 960 cultures from primary specimens. Since the inception of the assay, we have assessed the accuracy of identification of MTBC strains to the species level, concordance with culture-based drug susceptibility testing (DST), and turnaround time. Species identification by WGS was determined to be 99% accurate. Concordance between drug resistance profiles generated by WGS and culture-based DST methods was 96% for eight drugs, with an average resistance-predictive value of 93% and susceptible-predictive value of 96%. This single comprehensive WGS assay has replaced seven molecular assays and has resulted in resistance profiles being reported to physicians an average of 9 days sooner than with culture-based DST for first-line drugs and 32 days sooner for second-line drugs.

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. The assay is 100% specific; highly sensitive, detecting 11 gene copies of Bm DNA in both whole blood and cerebral spinal fluid; and provides rapid results in less than two hours. A retrospective study of 796 clinical specimens collected between the years 2012 and 2014 and a prospective study of 366 clinical specimens were performed utilizing this novel assay to evaluate the frequency of Bm infection in New York State (NYS). Eight clinical specimens (1%) were found to be positive for Bm, 216 were positive for A. phagocytophilum, and 10 were positive for E. chaffeensis. Additionally, we tested 411 I. scapularis ticks collected in NYS during 2013 and 2014 in a separate multiplex real-time PCR to determine the prevalence of Bm, A. phagocytophilum, Borrelia burgdorferi s.s., and Borrelia species. Our results indicated rates of 1.5%, 27%, 19.7%, and 8.8% respectively. The ability to monitor both the frequency and geographic distribution of Bm cases and the prevalence and geographic distribution of Bm in ticks will help create a better understanding of this emerging tick-borne pathogen.

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. When compared to CDC LDTs, TAC assays were generally one to two logs less sensitive depending on the site performing the analysis. Finally, TAC assays were generally more sensitive than their counterparts in three different commercial multi-respiratory panels. TAC technology allows users to spot customized assays and design TAC layout, simplify assay setup, conserve specimen, dramatically reduce contamination potential, and as demonstrated in this study, analyze multiple samples in parallel with good reproducibility between instruments and operators.