Implementation of a high-throughput whole genome sequencing approach with the goal of maximizing efficiency and cost effectiveness to improve public health.

This manuscript describes the development of a streamlined, cost-effective laboratory workflow to meet the demands of increased whole genome sequence (WGS) capacity while achieving mandated quality metrics. From 2020 to 2021, the Wadsworth Center Bacteriology Laboratory (WCBL) used a streamlined workflow to sequence 5,743 genomes that contributed sequence data to nine different projects. The combined use of the QIAcube HT, Illumina DNA Prep using quarter volume reactions, and the NextSeq allowed the WCBL to process all samples that required WGS while also achieving a median turn-around time of 7 days (range 4 to 10 days) and meeting minimum sequence quality requirements. Public Health Laboratories should consider implementing these methods to aid in meeting testing requirements within budgetary restrictions. IMPORTANCE: Public Health Laboratories that implement whole genome sequencing (WGS) technologies may struggle to find the balance between sample volume and cost effectiveness. We present a method that allows for sequencing of a variety of bacterial isolates in a cost-effective manner. This report provides specific strategies to implement high-volume WGS, including an innovative, low-cost solution utilizing a novel quarter volume sequencing library preparation. The methods described support the use of high-throughput DNA extraction and WGS within budgetary constraints, strengthening public health responses to outbreaks and disease surveillance.

Combining genomic and epidemiological data to compare the transmissibility of SARS-CoV-2 variants Alpha and Iota.

SARS-CoV-2 variants shaped the second year of the COVID-19 pandemic and the discourse around effective control measures. Evaluating the threat posed by a new variant is essential for adapting response efforts when community transmission is detected. In this study, we compare the dynamics of two variants, Alpha and Iota, by integrating genomic surveillance data to estimate the effective reproduction number (Rt) of the variants. We use Connecticut, United States, in which Alpha and Iota co-circulated in 2021. We find that the Rt of these variants were up to 50% larger than that of other variants. We then use phylogeography to show that while both variants were introduced into Connecticut at comparable frequencies, clades that resulted from introductions of Alpha were larger than those resulting from Iota introductions. By monitoring the dynamics of individual variants throughout our study period, we demonstrate the importance of routine surveillance in the response to COVID-19.

Combining genomic and epidemiological data to compare the transmissibility of SARS-CoV-2 lineages.

Emerging SARS-CoV-2 variants have shaped the second year of the COVID-19 pandemic and the public health discourse around effective control measures. Evaluating the public health threat posed by a new variant is essential for appropriately adapting response efforts when community transmission is detected. However, this assessment requires that a true comparison can be made between the new variant and its predecessors because factors other than the virus genotype may influence spread and transmission. In this study, we develop a framework that integrates genomic surveillance data to estimate the relative effective reproduction number (R t ) of co-circulating lineages. We use Connecticut, a state in the northeastern United States in which the SARS-CoV-2 variants B.1.1.7 and B.1.526 co-circulated in early 2021, as a case study for implementing this framework. We find that the R t of B.1.1.7 was 6-10% larger than that of B.1.526 in Connecticut in the midst of a COVID-19 vaccination campaign. To assess the generalizability of this framework, we apply it to genomic surveillance data from New York City and observe the same trend. Finally, we use discrete phylogeography to demonstrate that while both variants were introduced into Connecticut at comparable frequencies, clades that resulted from introductions of B.1.1.7 were larger than those resulting from B.1.526 introductions. Our framework, which uses open-source methods requiring minimal computational resources, may be used to monitor near real-time variant dynamics in a myriad of settings.

Early introductions and transmission of SARS-CoV-2 variant B.1.1.7 in the United States.

The emergence and spread of SARS-CoV-2 lineage B.1.1.7, first detected in the United Kingdom, has become a global public health concern because of its increased transmissibility. Over 2,500 COVID-19 cases associated with this variant have been detected in the United States (US) since December 2020, but the extent of establishment is relatively unknown. Using travel, genomic, and diagnostic data, we highlight that the primary ports of entry for B.1.1.7 in the US were in New York, California, and Florida. Furthermore, we found evidence for many independent B.1.1.7 establishments starting in early December 2020, followed by interstate spread by the end of the month. Finally, we project that B.1.1.7 will be the dominant lineage in many states by mid- to late March. Thus, genomic surveillance for B.1.1.7 and other variants urgently needs to be enhanced to better inform the public health response.

Early introductions and community transmission of SARS-CoV-2 variant B.1.1.7 in the United States.

The emergence and spread of SARS-CoV-2 lineage B.1.1.7, first detected in the United Kingdom, has become a global public health concern because of its increased transmissibility. Over 2500 COVID-19 cases associated with this variant have been detected in the US since December 2020, but the extent of establishment is relatively unknown. Using travel, genomic, and diagnostic data, we highlight the primary ports of entry for B.1.1.7 in the US and locations of possible underreporting of B.1.1.7 cases. Furthermore, we found evidence for many independent B.1.1.7 establishments starting in early December 2020, followed by interstate spread by the end of the month. Finally, we project that B.1.1.7 will be the dominant lineage in many states by mid to late March. Thus, genomic surveillance for B.1.1.7 and other variants urgently needs to be enhanced to better inform the public health response.

Molecular typing and whole genome next generation sequencing of human adenovirus 8 strains recovered from four 2012 outbreaks of keratoconjunctivitis in New York State.

Ocular infections caused by human adenovirus (HAdV) are highly contagious. The most severe are usually caused by members of species HAdV-D (types HAdV8, 19, 37, 53, 54, and 56) and can manifest as epidemic keratoconjunctivitis (EKC), often resulting in prolonged impairment of vision. During the early months of 2012, EKC outbreaks occurred in neonatal intensive care units (NICUs) in 3 hospitals in New York State (New York and Suffolk Counties). A total of 32 neonates were affected. For 14 of them, HAdV8 was laboratory-confirmed as the causative agent. Nine healthcare workers were also affected with 3 laboratory-confirmed, HAdV-positive EKC. A fourth EKC outbreak was documented among patients attending a private ophthalmology practice in Ulster County involving a total of 35 cases. Epidemiological linkage between the neonatal intensive care unit outbreaks was demonstrated by molecular typing of virus isolates with restriction enzyme analysis and next generation whole genome sequencing. The strain isolated from the ophthalmology clinic was easily distinguishable from the others by restriction enzyme analysis.

Oseltamivir-zanamivir combination therapy suppresses drug-resistant H1N1 influenza A viruses in the hollow fiber infection model (HFIM) system.

Drug-resistant influenza is a significant threat to global public health. Until new antiviral agents with novel mechanisms of action become available, there is a pressing need for alternative treatment strategies with available influenza antivirals. Our aims were to evaluate the antiviral activity of two neuraminidase inhibitors (oseltamivir and zanamivir) as combination therapy against H1N1 influenza A viruses, as these agents bind to the neuraminidase active site differently: oseltamivir requires a conformational change for binding whereas zanamivir does not. We performed pharmacodynamic studies in the hollow fiber infection model (HFIM) system with oseltamivir (75mg Q12h, t1/2: 8h) and zanamivir (600mg Q12h, t1/2: 2.5h), given as mono- or combination therapy, against viruses with varying susceptibilities to oseltamivir and zanamivir. Each antiviral suppressed the replication of influenza strains which were resistant to the other neuraminidase inhibitor, showing each drug does not engender cross-resistance to the other compound. Oseltamivir/zanamivir combination therapy was as effective at suppressing oseltamivir- and zanamivir-resistant influenza viruses and the combination regimen inhibited viral replication at a level that was similar to the most effective monotherapy arm. However, combination therapy offered a clear benefit by preventing the emergence and spread of drug-resistant viruses. These findings demonstrate that combination therapy with two agents that target the same viral protein through distinctly different binding interactions is a feasible strategy to combat resistance emergence. This is a novel finding that may be applicable to other viral and non-viral diseases for which different classes of agents do not exist.

Detection and Genetic Characterization of Adenovirus Type 14 Strain in Students with Influenza-Like Illness, New York, USA, 2014-2015.

During the 2014-15 influenza season, 13/168 respiratory samples from students with influenza-like illness (ILI) at a college in New York, USA, were positive for human adenovirus (HAdV); 4/13 samples were positive for HAdV-B14p1. During influenza season, HAdV should be included in the differential diagnostic panel used to determine the etiology of ILI.

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.

Next generation sequencing for whole genome analysis and surveillance of influenza A viruses.

BACKGROUND: The Wadsworth Center, New York State Department of Health (NYSDOH), conducts routine diagnosis and surveillance of influenza viruses. Whole genome sequencing (WGS) with next generation sequencing (NGS) was initiated to provide more rapid, detailed, thorough, and accurate analysis. OBJECTIVES: To optimize and implement a method for routine WGS of influenza A viruses. To use WGS to monitor influenza A viruses for reassortment, mutations associated with antiviral resistance and antigenicity changes, as well as those potentially affecting virulence and tropism. STUDY DESIGN: Multiple extraction and amplification methods were investigated and optimized for the production of template to be used for NGS. Additionally, software options were considered for data analysis. Initial WGS influenza projects have included the comparison of mixed population sequence data obtained with NGS, Sanger dideoxy sequencing, and pyrosequencing, the comparison of sequences obtained from paired primary/cultured samples, the analysis of sequence changes over several influenza seasons, and phylogenetic analysis. RESULTS: Procedures were optimized for extraction and amplification such that WGS could be successfully performed on both cultured isolates and primary specimens. Data is presented on 15 A/H1pdm09 and 44 A/H3N2 samples. Analysis of influenza A viruses identified and confirmed variant and mixed populations affecting antigenicity and antiviral susceptibility in both primary specimens and cultured isolates. CONCLUSIONS: An influenza A whole genome PCR method has been optimized for the reliable production of template for NGS. The WGS method has been successfully implemented for enhanced comprehensive surveillance and the generation of detailed clinical data on drug resistance and virulence. Data obtained with this method will also aid in future vaccine selection.

Identification of a novel intertypic recombinant species D human adenovirus in a pediatric stem cell transplant recipient.

BACKGROUND: Human adenoviruses (HAdV) are known opportunistic pathogens in hematopoietic stem cell transplant (SCT) recipients. The detection of HAdV infection in children after SCT has been implicated as a determinant of poor outcome but specific associations between HAdV species or individual HAdV types and disease are poorly understood. OBJECTIVES: Characterization of a HAdV-D strain isolated from multiple clinical specimens of an 11-year-old female recipient of a matched unrelated donor peripheral SCT for T-cell lymphoma and case report. STUDY DESIGN: Archived HAdV PCR-positive plasma, urine, and stool specimens were processed for virus isolation and detailed molecular typing. Complete genomic sequencing was carried out on 2 isolates. RESULTS: The patient tested positive for HAdV DNA by real-time PCR of a stool specimen at 44 days after initiation of a SCT conditioning regimen. In the subsequent 3 months, HAdV was detected in plasma, urine and stool specimens in association with symptoms of gastroenteritis and hemorrhagic cystitis. A novel HAdV-D with a HAdV20-like hexon gene was isolated from both urine and stool specimens. All isolates yielded identical restriction profiles with endonucleases BamHI, BglII, BstEII, HindIII, PstI and SmaI. Analysis of 2 complete genomic sequences further identified the virus as a novel intertypic recombinant HAdV-D (P20/H20/F42) closely related to HAdV42. CONCLUSIONS: This case highlights the identification of a previously unknown HAdV-D from an immunocompromised host. In this patient, the course of adenovirus infection is compatible with reactivation of a latent virus or a primary opportunistic infection. Adenoviremia in this patient resolved without definitive adenovirus-directed antiviral therapy.

Rapid whole-genome sequencing for surveillance of Salmonella enterica serovar enteritidis.

For Salmonella enterica serovar Enteritidis, 85% of isolates can be classified into 5 pulsed-field gel electrophoresis (PFGE) types. However, PFGE has limited discriminatory power for outbreak detection. Although whole-genome sequencing has been found to improve discrimination of outbreak clusters, whether this procedure can be used in real-time in a public health laboratory is not known. Therefore, we conducted a retrospective and prospective analysis. The retrospective study investigated isolates from 1 confirmed outbreak. Additional cases could be attributed to the outbreak strain on the basis of whole-genome data. The prospective study included 58 isolates obtained in 2012, including isolates from 1 epidemiologically defined outbreak. Whole-genome sequencing identified additional isolates that could be attributed to the outbreak, but which differed from the outbreak-associated PFGE type. Additional putative outbreak clusters were detected in the retrospective and prospective analyses. This study demonstrates the practicality of implementing this approach for outbreak surveillance in a state public health laboratory.

Influenza antiviral resistance testing in new york and wisconsin, 2006 to 2008: methodology and surveillance data.

The need for effective influenza antiviral susceptibility surveillance methods has increased due to the emergence of near-universal adamantane resistance in influenza A/H3N2 viruses during the 2005-2006 season and the appearance of oseltamivir resistance in the influenza A/H1N1 virus subtype during the 2007-2008 season. The two classes of influenza antivirals, the neuraminidase inhibitors (NAIs) and the adamantanes, are well characterized, as are many mutations that can confer resistance to these drugs. Adamantane resistance is imparted mainly by a S31N mutation in the matrix gene, while NAI resistance can result from a number of mutations in the neuraminidase gene. During the 2007-2008 season, a neuraminidase mutation (H274Y) conferring resistance to the NAI oseltamivir emerged worldwide in the A/H1N1 virus subtype. Surveillance methodology and data from New York (NY) and Wisconsin (WI) for the 2006-2007 and 2007-2008 influenza seasons are presented. We used an existing pyrosequencing method (R. A. Bright et al., Lancet 366:1175-1181, 2005) and a modified version of this method for detection of adamantane resistance mutations. For NAI resistance mutation detection, we used a mutation-specific pyrosequencing technique and developed a neuraminidase gene dideoxy sequencing method. Adamantane resistance in the A/H3N2 virus samples was 100% for 2007-2008, similar to the 99.8% resistance nationwide as reported by the CDC. Adamantane resistance was found in only 1.2% of NY and WI A/H1N1 virus samples, compared to that found in 10.8% of samples tested nationwide as reported by the CDC. Influenza A/H1N1 virus H274Y mutants were found in 11.1% of NY samples for 2007-2008, a level comparable to the 10.9% nationwide level reported by the CDC; in contrast, mutants were found in 17.4% of WI samples. These results indicate the need for regional influenza antiviral surveillance.