Sequencing Rapidly Emerging for Epidemiology, Outbreak Control

Much as polymerase chain reaction (PCR) revolutionized microbiology 30 years ago, the application of next-generation sequencing (NGS) to the field of infectious disease will improve surveillance efforts, outbreak determination, and infection control activities, in addition to tailoring treatment decisions, thus improving both individual care and global antibiotic stewardship. Microbiologists and laboratorians will play a central role as NGS is employed for epidemiological, infection control, and patient care purposes. There is noticeable momentum toward the common use of NGS in molecular epidemiology by both public health agencies and hospital microbiology laboratories. The molecular data gleaned from these NGS-based surveillance efforts are informing the understanding of pathogen transmission, shifts in pathogen virulence or microbial sensitivity, and infection control practice. While use of NGS for individual patient diagnosis remains rare outside of translational research endeavors, experts surveyed by DTET believe that within the next five years whole genome sequencing (WGS) of pathogens will be well-integrated into routine public health surveillance and investigation of health care-associated outbreaks, particularly in large hospitals. "This is the year NGS and infectious disease jump to another level," George Weinstock, Ph.D., from the Jackson Laboratory for Genomic Medicine (Farmington, Conn.), tells DTET. "There are a number of examples in the literature where sequencing can detect one base differences in three million base genomes. This incredible sensitivity allows public health organizations [in multiple locations] to determine if clusters of E. coli from bad lettuce are the tip of the iceberg of a nationwide incident or coincidental local outbreaks."
NGS Improves Resolution Microbial genomics encompasses the full spectrum of investigation—from pathogen detection, identification, sensitivity testing, and incorporation of metadata into epidemiological tracking. Researchers say that more than 38,000 bacterial and 5,000 viral genomes have been sequenced to date and that pathogen characterization using NGS holds great potential to improve the resolution of the data over traditional microbiological techniques. While PCR is the most widely used molecular method in clinical microbiology, such targeted techniques lack the resolution necessary to determine chains of transmission, where single nucleotides may differentiate cases involved in an outbreak from others not directly involved. Additionally, NGS can provide a "universal" test that can answer questions not only regarding the identity of the pathogen, but also markers of virulence and antibiotic resistance in a single step. "As the genomes of bacteria and viruses are between one thousand to one million times smaller than the human genome they can be sequenced and analyzed more rapidly (in less than a day) and cheaply (for around £50 per genome) bringing the insights of pathogen genomics within reach of the budget and time frames in which clinical and public health microbiology services operate," writes Leila Lusheshi, Ph.D., from the health policy think tank PHG Foundation (United Kingdom) in a briefing note.
NGS Aids Public Health Policy Priorities Not only is evidence emerging that NGS is technically feasible for infectious disease identification purposes, but it plays a central role in dealing with top public health policy priorities, such as tackling emerging infections and antibiotic resistant bugs. Agencies such as the U.S. Centers for Disease Control and Prevention (CDC) and the U.S. Food and Drug Administration (FDA) are incorporating NGS into public health and food safety surveillance through their PulseNet and Genome Trakr networks. As the agencies build their NGS infrastructure both are utilizing a strategy that relies upon decentralized NGS testing in regional laboratory networks with centralized data management using cloud-based technology for cluster determination at the national level. The FDA's Genome Trakr Network entered into a $17 million contract with sequencer-maker Illumina (San Diego) back in 2012 to build NGS capacity for food safety testing at its 13 federal labs and 13 state health and university labs. The network is currently sequencing an average of over 800 isolates each month, the agency reports, using MiSeq sequencers. Results are transmitted to a genomic reference database housed at the U.S. Department of Health and Human Services' (HHS) National Center for Biotechnology Information. The real-time uploads enhance the agency's ability to identify potential outbreaks. While federal agencies are working on translating NGS-based surveillance into routine practice through a series of pilot programs, the President's Council of Advisors on Science and Technology issued a report Combating Antibiotic Resistance in September 2014 calling for coordinated efforts to further build national, genomic-based surveillance capacity to combat the threat of growing antibiotic resistant bacteria. In the report, the council calls for significant investment ($190 million annually) to strengthen national genomic-based pathogen surveillance through building state and local public health infrastructure. The council proposes a national laboratory network for pathogen surveillance (health care, agriculture, and environmental sampling), as well as heightened clinical laboratory capacity at 10 to 20 major health care facilities. The plan calls for ensuring laboratories would be able to receive specimens and relevant metadata; perform genomic analysis; rapidly return information to providers and to relevant public health entities for cluster identification; archive samples; and deposit genomic information and metadata in a publicly-accessible national database. The council estimates that the annual cost for the two laboratory components of the network is roughly $130 million ($80 million for the regional laboratories and $50 million for the hospital-based laboratories). The council says that by building sequencing capacity, an initial reference collection of antibiotic-resistant pathogens can be developed within three years. Additionally, the council calls for funds to foster the development of new computational methods and tools to aid genome assembly and comparison, metagenomic analysis and interpretation of epidemiologic data into reports for providers, and the development of surveillance and testing standards. "Ultimately, fully-automated sample handling and data analysis methods should allow the analysis of extremely large numbers of samples," the workgroup writes in the report. "Hospitals would be able to routinely monitor the many thousands of resistant isolates identified in their clinical practice. Surveillance efforts in other settings would become routine and could provide early warning signs about potential outbreaks, whatever their origin. Tracking patterns across facilities in the community would show patterns of spread to guide preventive interventions." Relatedly, HHS has identified the reduction of health care-acquired infections (HAI) as an agency priority goal. Diagnostics, including the potential use of NGS, are key to rapidly diagnosing infections, optimizing treatment as soon as possible, increasing the speed of an individual's recovery and thereby cutting their time in the hospital and associated health care costs. HAIs are receiving increased attention, in part because of payers' crackdown on reimbursements for care necessitated by preventable HAIs and hospital laboratories are looking for technologies that will enable a faster turnaround time for provision of antibiotic sensitivity results to a physician for a suspected HAI.
Case Studies of WGS for Outbreak Investigation "WGS is now poised to make an impact on hospital infection prevention and control, delivering cost-effective identification of routes of infection within a clinically relevant timeframe and allowing infection control teams to track, and even prevent, the spread of drug-resistant hospital pathogens," says Beryl Oppenheim, M.B.B.Ch., director of infection control at Queen Elizabeth Hospital. Molecular epidemiology, experts say, encompasses the process of identifying the genetic basis of disease (pathogen variants), transmission (including source and route), and informing prevention pathways supporting hospital infection control. NGS allows highly accurate, hypothesis-free analysis of multiple isolates for detection in a single test, replacing the need for multiple analyses to identify the organism, its resistance, and virulence. The resolution of NGS is so great, that it can distinguish pathogen strains that differ by as little as one single nucleotide polymorphism. This can speed investigation by public health officials and hospital infection control experts to take effective measures to contain and stop the spread of these pathogens. Real-life case studies are emerging, demonstrating the power of WGS in combination with traditional epidemiological tracking to control outbreaks. For 18 months, Queen Elizabeth Hospital (England) experienced a "protracted" outbreak of multi-drug resistant Acinetobacter baumannii (A. baumannii). WGS was used to determine the relationships between collected isolates. The outbreak involved both civilians and military casualties cared for in multiple wards. The outbreak was caused by A. baumannii pulsotype 27, as determined by pulse-field gel electrophoresis. Genomic DNA was extracted from 114 isolates beginning at week 40 of the outbreak. Genome sequences were determined from 102 isolates. Results revealed that 52 patients and 10 environmental isolates showed genomic similarity to the outbreak reference strain (eight or less single nucleotides differences). WGS determined 18 isolates were not part of the outbreak. Excluding isolates, the authors say, allowed efforts to focus on determining the connections between genetically-related cases, rather than trying to connect all cases. "By combining WGS and epidemiological data, we reconstructed potential transmission events that linked all but 10 of the patients and confirmed links between clinical and environmental isolates," writes co-author Beryl Oppenheim, in the case study published in November 2014 in Genome Medicine. "Identification of a contaminated bed and a burns theatre as sources of transmission led to enhanced environmental decontamination procedures."
Challenges to Adoption While there is mounting evidence that deploying NGS for pathogen characterization and outbreak detection is feasible, experts agree that in order for the technology to be incorporated into routine practice, some additional issues still need to be resolved. "While the sequencing technology and bioinformatic analysis underpinning the use of pathogen genomics in the management of infectious disease are relatively well established, the integration of these new techniques into the delivery of medical and public health microbiology services is only in its infancy," writes PHG Foundation. From a practical standpoint Oppenheim says that rapid declines in the cost of NGS and increasing speed to results coupled with significant reductions in the size of the equipment bode well for the future routine adoption of WGS in hospital infection control. But remaining challenges include ease-of-use issues, the skill-level needed of the laboratorians running sequencing tests, and the reproducibility of results all need to be addressed. Susan Knowles, senior market development manager for Illumina, tells DTET that one of the many reasons public health adoption has "taken off" in the United States is because of the lack of regulatory barriers, as opposed to use of the technology for clinical diagnostics, where FDA approval is needed. Hospitals, she says, are required to investigate potential outbreaks, but whether NGS is used is dictated by each hospital's infection control policy. Though, hospitals, she says, are increasingly aware of the high-quality, high-resolution data achieved with NGS. The PHG Foundation says that strategic planning and coordination will be necessary during this transition period to ready for widespread adoption. In addition to addressing systematic concerns (uniform protocols and analyses as well as standardized data sharing methods) and ease-of-use issues (low-cost data management and simplified bioinformatic interpretation and reporting), the economics of integrating the technology into medical care and public health systems must be evaluated. Experts predict that WGS will be adopted initially for priority applications with specific pathogens. Identifying for which pathogens diagnostic and epidemiologic gaps exist with current microbiological techniques will likely improve care and prevent infections, but developing scenarios for how, when and where it is appropriate to introduce pathogen genomics into microbiology services will ultimately require an economic evaluation of the benefits, expenses, and potential cost-savings. Technological advancements including culture-free sequencing directly from patient samples will further cut turnaround times, improving the actionability of the results from both a clinical care and public health perspective. Takeaway: NGS is now poised for meaningful adoption in genomic microbiology, for public health surveillance in the community and in health care settings. With improvements in ease-of-use, particularly for the analysis component, the high-resolution data provided by WGS will improve pathogen detection with a universal test, with results that can inform both case management and infection control efforts.