Infectious diseases probably impacted humans from the start. Despite the advances in treatments for many of these life-threatening illnesses, infectious diseases made up three of the top-10 killers in 2016, according to the World Health Organization. With sophisticated genomic technologies, such as the polymerase chain reaction (PCR) and next-generation sequencing (NGS), scientists continue to learn more about the transmission, mechanisms, and treatment of infectious diseases. With this class of diseases still killing countless people around the world, both basic and clinical research require even more advanced and unbiased ways to implement cutting-edge genomic techniques in combating infectious diseases.
“The spread of infectious disease is a serious public health issue that leads to millions of deaths each year,” says Anjali Shah, senior director of product management of clinical NGS at Thermo Fisher Scientific. “Infectious diseases are caused by pathogenic microorganisms, such as bacteria, viruses, parasites, or fungi, and can be spread directly or indirectly from one person to another.”
Scientists began studying infectious diseases with cultures and biochemical methods. For the latter, advances in instrumentation, such as mass spectrometry, helped scientists dig deeper into the ways that these diseases work and kill, but it was not enough. Many of today’s scientists made the “transition to molecular-based methods, which enable faster time to result and the ability to more comprehensively profile samples to detect not only pathogens, but also resistance factors to improve outcomes,” says Shah. “The ultimate goal of these emerging molecular techniques is to analyze primary samples, rather than cultured isolates, and to provide high sensitivity and the rapid turn-around time that is required for clinical research applications.”
Image: Many infectious diseases, such as the methicillin-resistant Staphylococcus aureus (MRSA, purple) shown here being ingested by a human neutrophil, endanger people around the world. Image courtesy of the National Institute of Allergy and Infectious Diseases, NIH.
Currently, there is no single method available that solves all of the major challenges in infectious disease research. As explained by experts from the European Centre for Disease Prevention and Control (ECDC), “Techniques used for studying infectious diseases vary based on the purpose of the study.” Consequently, ECDC prepared an Expert opinion on whole-genome sequencing for public-health surveillance.
Tools of the disease trade
Given the variety of infectious diseases, many tools can be used to study these illnesses. When asked which genomic technology is the most useful in studying infectious disease, Hayden Metsky, a computer science doctoral student at MIT, who uses computational biology and machine learning to study viral genomics, says, “I don’t think there’s any single technology that’s most useful.” That’s because all scientific methods come with pros and cons.
“For example, metagenomic sequencing provides a huge amount of data, allowing us to assemble complete genomes of pathogens,” Metsky explains, “but, depending on the goal, this can often be slow and expensive.” To just see if a sample contains some pathogen, simpler and less expensive options exist, such as real-time or quantitative PCR (qPCR). As an example, Shah says, “Use of Thermo Fisher Scientific genetic analysis platforms to address infectious disease and pathogenic outbreaks has been demonstrated through our genetic analysis technologies, such as real-time PCR, to understand diabetic foot ulcers, urinary tract infections, and bacterial vaginosis.”
The pathogen and the desired information can determine the best technique. As Metsky points out, “I work in a viral sequencing lab and a big challenge we face is that many viruses are present at low amounts in a sample, and therefore it can be difficult to detect and sequence their genomes directly from clinical samples.” For that, he says that techniques like hybridization capture can be useful. As Metsky notes, this process “can be used to enrich viral nucleic acid prior to sequencing.”
Shah adds that Thermo Fisher’s NGS technology has been used “to identify deadly strains of the Ebola virus in Sierra Leone.” In fact, Thermo Fisher makes specific NGS products for infectious disease research. The Ion AmpliSeq Pan Bacterial Research Panel, which Shah says, “contains hundreds of targets for 16S and species-specific identification and for antimicrobial resistance, can be utilized in medical centers seeking to improve outcomes for healthcare-acquired infections.” The Ion AmpliSeq Antimicrobial Resistance Research Panel, “assessing the presence or absence of 478 genes involved in antibiotic resistance, can be applied in environmental health agencies that want to understand antibiotic efficacy.”
Options in approaches
Digging deeper into nucleotide sequences can also be necessary. Pushpanathan Muthuirulan, a research associate in the department of human evolutionary biology at Harvard University, says, “deep sequencing technologies, such as microbiome analysis and dual RNA sequencing, are the most useful methods for studying infectious diseases.” He notes that microbiome analysis makes a good choice in searching for unknown organisms that cause a disease. “In microbiome analysis, profiling 16S ribosomal RNA gene, bacteria, and ITS1 fungal regions among healthy and diseased individuals would give better clues on the relevant pathogenic microbes—bacteria or fungi—responsible for diseases,” Muthuirulan explains. “In addition, this method also helps in evaluating the shift in microbial populations, or altered microbiome, which serves as diagnosis and prognosis indicators of diseases.”
Similarly, metagenome shotgun sequencing technologies can reveal the diversity, abundance, and function of microbes, including viruses, among healthy and diseased individuals. “Since, this method takes advantage of sheared DNA from environmental samples, it can capture all genes from all organisms in a given sample,” Muthuirulan adds.
To understand how a disease works or how the immune system fights it, Muthuirulan recommends dual RNA-sequencing. “This is the most powerful approach to measure the physiological state of either pathogen or eukaryotic cells under a given condition,” he explains. “This technology profiles gene expression simultaneously in a pathogen and its infected host at time of infection, which would enable discovery of novel potential targets for specific therapy.”
Image: Genomic techniques can be used to study infectious diseases. Image courtesy of Pushpanathan Muthuirulan.
In some infectious diseases, scientists deal with small amounts of virus in a sample. As an example, Metsky says, “During the 2015-16 Zika virus epidemic, we used NGS to sequence Zika virus genomes from throughout the Americas and, through a phylogenetic analysis of these genomes, traced how the virus spread.” The small amount of Zika virus in body fluids makes it very difficult to sequence. “Almost all of what we sequence is non-Zika material, like bacteria and human RNA, so finding Zika is like identifying a needle in a haystack.”
So, Metsky and his colleagues recovered the virus with two targeted-enrichment approaches: hybridization capture and amplicon sequencing. “Since viruses evolve quickly and they have plenty of diversity even over a short period of time, we could use the Zika virus genomes we assembled to estimate the dates at which the virus was introduced into different geographic regions in the Americas,” Metsky explains. “We found that Zika had been circulating undetected for months in each of these different regions, which stresses the importance of surveilling for viruses.
That surveillance will help people around the world, especially as populations fight diseases with no existing treatments. Covering the range of infectious diseases, though, will demand various genomic techniques and tweaks on many of them.
Hero image: Zika virus infecting brain cells. Credit: Kateryna Kon/Dreamstime.com