Next-generation sequencing technologies are being rapidly adopted as a tool of choice for diagnos... more 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.
Rapid and reliable detection of rifampin (RIF) resistance is critical for the diagnosis and treat... more 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.
Familial Mediterranean fever (FMF) is a rare autoinflammatory disorder of the innate immune syste... more 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.
Background: With the rapid development of new advanced molecular detection methods, identificatio... more 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 ...
Whole-genome sequencing (WGS) of pathogens from pure culture provides unparalleled accuracy and c... more Whole-genome sequencing (WGS) of pathogens from pure culture provides unparalleled accuracy and comprehensive results at a cost that is advantageous compared to 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 NGS sequencing data for downstream bioinformatics analysis. A custom bioinformatics pipeline was created to optimize and remove non-E. coli reads, a...
We characterized a case of neonatal conjunctivitis in New York, USA, caused by Neisseria meningit... more 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.
Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, Jan 2, 2018
d The case fatality rate (CFR) from invasive meningococcal disease (IMD) in New York City (NYC) i... more d 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. We conducted a retrospective cohort study among 151 persons aged 15 years and older 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. Females had a higher CFR (23/62, 37%) following IMD than males (17/89, 19%) (adjusted relative risk [aRR], 2.1 [95% CI, 1.2 to 3.8]). Controlling for demographic and clinical factors, there was a significant interaction between sex and fatal outcomes related ...
We report a case of lymphadenitis caused by Mycobacterium orygis in an immunocompetent person in ... more 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. G enomic analysis has previously shown that the Mycobacterium tuberculosis (MTB) complex comprises >8 distinct subgroups: M. tuberculosis, M. africanum, M. canettii, M. bovis, M. caprae, M. pinnipedii, M. microti, and M. mungi (1). M. orygis was first characterized in Africa and South Asia in 2012 based on examination of 22 isolates selected for the similarity of their IS6110 restriction fragment length polymorphism patterns to previously described oryx bacilli (2). Eleven of these isolates were from animals (a cow, a rhesus monkey, and types of antelope including oryx), and 11 were from humans (9 from South Asia). On the basis of single-nucleotide polymorphism (SNP) and region of difference (RD) analysis, van Ingen et al. concluded that these mycobacteria belonged to a phylogenetically distinct lineage of the clonal MTB complex (2). M. orygis is also distinguished by a mutation in gene Rv2042c (2) and a G1113A mutation in the gyrB gene (3). We report a case of lymphadenitis caused by M. orygis in an immunocompetent person in Stony Brook, New York, USA. During July 2015, we diagnosed pneumonia in the upper lobe of the right lung in a woman, 71 years of age, who had a remote history of lymphoma. The condition was characterized by enlarged lymph nodes. The patient was born in Pakistan, moved to India at age 1, and emigrated to the United States ≈25 years before onset; her preimmigration TB skin test was <5 mm (bacillus Calmette-Guérin vaccinated), and chest radiograph results were negative. She drank unpasteurized milk while living in India.
Whole-genome sequencing (WGS) is a newer alternative for tuberculosis (TB) diagnostics and is cap... more 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 identifica...
Here, we report the release of a draft genome assembly of a Gram-positive cocciBranchiibiussp. NY... more Here, we report the release of a draft genome assembly of a Gram-positive cocciBranchiibiussp. NY16-3462-2 with a high-GC content, sequenced from a mixed clinical sample containingMycobacterium tuberculosis. This genome is the first publicly available sequence from a representative of the genusBranchiibius.
A Bordetella pertussis strain lacking 2 acellular vaccine immunogens, pertussis toxin and pertact... more 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.
In this study, a multicenter evaluation of the Life Technologies TaqMan(®) Array Card (TAC) with ... more 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.
The need for public health laboratories (PHLs) to prioritize resources has led to increased inter... more 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). We assessed multiple aspects of shared services including shipping, testing, reporting, and cost. Rhode Island State Health Laboratories shipped MTBC-positive specimens and isolates to Wadsworth Center. Average turnaround times were calculated and cost analysis was performed. Testing turnaround times were similar at both PHLs; however, the availability of conventional drug susceptibility testing (DST) results for Rhode Island primary specimens and isolates were extended by approximately four days of shipping time. An extended molecular testing panel was performed on eve...
Background: Mutations in the rpoB gene of Mycobacterium tuberculosis (Mtb) act as predictors of r... more Background: Mutations in the rpoB gene of Mycobacterium tuberculosis (Mtb) act as predictors of rifampin (RIF) resistance. There is a need for rapid identification procedures to screen for potential drug resistances to insure that proper drug therapy is being administered. Methods: A PyrosequencingTM protocol was developed to assess all possible mutations within the 81-bp core region of the rpoB gene. Two pyrosequencing primers were required to obtain a clear and accurate pyrogram within 2 hrs following PCR amplification. The sensitivity was determined and the pyrosequencing approach was evaluated in primary specimens positive for Mtb complex DNA by real-time PCR. Final results were compared with conventional RIF susceptibility testing results and/or DNA sequencing. Additionally, 60 clinical Mtb isolates with known mutations based on conventional DNA sequencing methodology were retested using this approach. Results: The assay sensitivity was determined to be 50 CFU per reaction. Ana...
Next-generation sequencing technologies are being rapidly adopted as a tool of choice for diagnos... more 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.
Rapid and reliable detection of rifampin (RIF) resistance is critical for the diagnosis and treat... more 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.
Familial Mediterranean fever (FMF) is a rare autoinflammatory disorder of the innate immune syste... more 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.
Background: With the rapid development of new advanced molecular detection methods, identificatio... more 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 ...
Whole-genome sequencing (WGS) of pathogens from pure culture provides unparalleled accuracy and c... more Whole-genome sequencing (WGS) of pathogens from pure culture provides unparalleled accuracy and comprehensive results at a cost that is advantageous compared to 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 NGS sequencing data for downstream bioinformatics analysis. A custom bioinformatics pipeline was created to optimize and remove non-E. coli reads, a...
We characterized a case of neonatal conjunctivitis in New York, USA, caused by Neisseria meningit... more 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.
Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, Jan 2, 2018
d The case fatality rate (CFR) from invasive meningococcal disease (IMD) in New York City (NYC) i... more d 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. We conducted a retrospective cohort study among 151 persons aged 15 years and older 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. Females had a higher CFR (23/62, 37%) following IMD than males (17/89, 19%) (adjusted relative risk [aRR], 2.1 [95% CI, 1.2 to 3.8]). Controlling for demographic and clinical factors, there was a significant interaction between sex and fatal outcomes related ...
We report a case of lymphadenitis caused by Mycobacterium orygis in an immunocompetent person in ... more 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. G enomic analysis has previously shown that the Mycobacterium tuberculosis (MTB) complex comprises >8 distinct subgroups: M. tuberculosis, M. africanum, M. canettii, M. bovis, M. caprae, M. pinnipedii, M. microti, and M. mungi (1). M. orygis was first characterized in Africa and South Asia in 2012 based on examination of 22 isolates selected for the similarity of their IS6110 restriction fragment length polymorphism patterns to previously described oryx bacilli (2). Eleven of these isolates were from animals (a cow, a rhesus monkey, and types of antelope including oryx), and 11 were from humans (9 from South Asia). On the basis of single-nucleotide polymorphism (SNP) and region of difference (RD) analysis, van Ingen et al. concluded that these mycobacteria belonged to a phylogenetically distinct lineage of the clonal MTB complex (2). M. orygis is also distinguished by a mutation in gene Rv2042c (2) and a G1113A mutation in the gyrB gene (3). We report a case of lymphadenitis caused by M. orygis in an immunocompetent person in Stony Brook, New York, USA. During July 2015, we diagnosed pneumonia in the upper lobe of the right lung in a woman, 71 years of age, who had a remote history of lymphoma. The condition was characterized by enlarged lymph nodes. The patient was born in Pakistan, moved to India at age 1, and emigrated to the United States ≈25 years before onset; her preimmigration TB skin test was <5 mm (bacillus Calmette-Guérin vaccinated), and chest radiograph results were negative. She drank unpasteurized milk while living in India.
Whole-genome sequencing (WGS) is a newer alternative for tuberculosis (TB) diagnostics and is cap... more 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 identifica...
Here, we report the release of a draft genome assembly of a Gram-positive cocciBranchiibiussp. NY... more Here, we report the release of a draft genome assembly of a Gram-positive cocciBranchiibiussp. NY16-3462-2 with a high-GC content, sequenced from a mixed clinical sample containingMycobacterium tuberculosis. This genome is the first publicly available sequence from a representative of the genusBranchiibius.
A Bordetella pertussis strain lacking 2 acellular vaccine immunogens, pertussis toxin and pertact... more 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.
In this study, a multicenter evaluation of the Life Technologies TaqMan(®) Array Card (TAC) with ... more 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.
The need for public health laboratories (PHLs) to prioritize resources has led to increased inter... more 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). We assessed multiple aspects of shared services including shipping, testing, reporting, and cost. Rhode Island State Health Laboratories shipped MTBC-positive specimens and isolates to Wadsworth Center. Average turnaround times were calculated and cost analysis was performed. Testing turnaround times were similar at both PHLs; however, the availability of conventional drug susceptibility testing (DST) results for Rhode Island primary specimens and isolates were extended by approximately four days of shipping time. An extended molecular testing panel was performed on eve...
Background: Mutations in the rpoB gene of Mycobacterium tuberculosis (Mtb) act as predictors of r... more Background: Mutations in the rpoB gene of Mycobacterium tuberculosis (Mtb) act as predictors of rifampin (RIF) resistance. There is a need for rapid identification procedures to screen for potential drug resistances to insure that proper drug therapy is being administered. Methods: A PyrosequencingTM protocol was developed to assess all possible mutations within the 81-bp core region of the rpoB gene. Two pyrosequencing primers were required to obtain a clear and accurate pyrogram within 2 hrs following PCR amplification. The sensitivity was determined and the pyrosequencing approach was evaluated in primary specimens positive for Mtb complex DNA by real-time PCR. Final results were compared with conventional RIF susceptibility testing results and/or DNA sequencing. Additionally, 60 clinical Mtb isolates with known mutations based on conventional DNA sequencing methodology were retested using this approach. Results: The assay sensitivity was determined to be 50 CFU per reaction. Ana...
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