Printer Friendly

Molecular diagnostics for infectious diseases.

Deoxyribonucleic acid (DNA) is a fundamental building block of life. It was first isolated in 1869 by Dr. Johannes Friedrick Miescher of Switzerland, a physician and biologist (Figure 1). Dr. Miescher suspected that it was involved in heredity. James Watson (an American biologist), Francis Crick (an English physicist and biochemist), and Maurice Wilkins (a New Zealand native, physicist, and molecular biologist) were awarded the Nobel Prize in 1962 for the discovery of the three-dimensional double helix structure of DNA. DNA consists of four different nucleotides: Adenine, Thymine, Guanine, and Cytosine (also A, T, G, and C, respectively). Nucleotides contain a nitrogenous base, a ribose sugar, and a phosphate. The nucleotides subunits bond to create a linear DNA sequence. When a complementary sequence is present a double-stranded spiral structure is formed that is commonly recognized as DNA molecules (Figure 2). These molecules encode the essential blueprint of life forms, and the specific sequence is uniquely different between individuals. Some viruses store genetic information as ribonucleic acid (RNA) instead of DNA. The RNA molecule is similar to DNA except that there is a substitution of the nucleotide base uracil for thymine, the ribose sugar differs, and it is usually single stranded. RNA is read by ribosomes in groups of three nucleotides at a time, called codons, and is translated into proteins. Proteins could be described as microscopic machines that perform the vast majority of chemical reactions and biological functions of cells.

The discovery of the double helix nature of DNA in the 1950s, the development of polymerase chain reaction in the 80s and 90s, and finally the automation of DNA sequencing set the current theoretical stage for modern molecular diagnostics. These technologies have now made it possible to diagnose nearly any infectious disease, map microbiomes, identify human genetic markers of disease, characterize human tumor types, and even provide information about the efficacy of potential therapies based on these results.

In clinical laboratory medicine, we use many molecular techniques and assays, but it is generally understood that when we discuss molecular diagnostics we are talking about human genomics or infectious diseases relying on DNA or RNA fingerprints for analysis. Below is a discussion of the core molecular technologies as well as examples of some tests ordered by physicians today and what clinicians may be ordering or employing soon.

All known life uses the same fundamental genetic system; and it offers the opportunity to identify and characterize all organisms, including viruses, bacteria, archaea, protozoans, fungi, algae, and all higher forms of life including humans, by a single core technology. Even organisms that utilize RNA as the main carrier of genetic information may be sequenced by using enzymes to convert the RNA into DNA by reverse transcription. Molecular-based technologies allow for the effective interrogation of genetic information and has gone through an evolution over the last 50 years. Relevant molecular technologies include simple Sanger sequencing by electrophoresis gels, Polymerase Chain Reaction (PCR), quantitative PCR, massively parallel multiplex PCR, and ultimately modern high throughput sequencing, also called Next Generation DNA sequencing.

Routine Molecular Diagnostics

A wide range of routinely ordered diagnostic tests are molecular assays, and many more are being adopted by the clinical laboratory. Ultimately, molecular diagnostics have cemented their role in mainstream medicine.

Human Genomics:

Down Syndrome results from a trisomy (three copies) of chromosome 21.

Cystic Fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator genes (CFTR.)

Coagulation genes play a role in appropriate blood clotting with Factor VII of relevance.

Oncogenes, such as mutations in the BCRA1/2 genes, result in an increased risk of developing breast cancer.

Human leukocyte antigen typing--The HLA gene, located on chromosome 6, is involved in transplant compatibility.

Pharmacogenomics uses genomic information to determine what drug to use for a condition.

Infectious Diseases:

Chlamydia species-a sexually transmitted disease

Neisseria gonorrhea--another sexually transmitted disease

Influenza--Determining the serotypes of these viruses is important for treatment and epidemiology.

Hepatitis typing--Typing of hepatitis virus is important for both treatment and projecting patient outcome.

HIV-Accurate viral load measurement is critical for monitoring therapy in patients with HIV and preventing the development of AIDS.

Mycobacteria species--Strain typing of tuberculosis is important for epidemiology and treatment.

Gastrointestinal Screens:

Biofire GI Panel--can simultaneously detect Campylobacter jejuni, Campylobacter coli, Campylobacter upsaliensis, toxin producing Clostridium difficile, Plesiomonas shigelloides, Salmonella species, Yersinia enterocolitica, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio cholera, a variety of pathogenic Shigella and Escherichia species, Adenovirus, Astrovirus, Norovirus, Rotavirus, Sapovirus, Cryptosporidium species, and Cyclospora cayetanensis.

Biofire Respiratory Panel--can simultaneously detect Adenovirus, Coronaviruses, Metapneumovirus, Rhinovirus, Enterovirus, Influenza viruses, Parainfluenza viruses, Respiratory Syncytial Virus, Bordatella perstussis, Chlamydophila pneumoniae, and Mycoplasma pneumoniae.

Biofire Blood Culture ID Panel--can simultaneously detect Enterococcus species, Listeria monocytogenes, Staphylococcus species, Staphylococcus aureus, Streptococcus species, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Acinetobacter baumannii, Haemophilus influenza, Neisseria meningib'des, Psuedomonas aeruginosa, Enterobacteriaceae species, Enterobacter cloacae complex, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus species, Serratia marcescens, Candida albicans, Candida glabrate, Candida krusei, Candida parapsilosis, and Candida tropicalis.

Biofire Meningitis/Encephalitis Panel--can simultaneously detect Escherichia coli, Haemophilus influenza, Listeria monocytogenes, Neisseria meningitidis, Streptococcus agalactiae, Streptococcus pneumoniae, Cryptococcus neoformans, Cryptoccous gattii, Cytomegalovirus, Enterovirus, Herpes simplex viruses, Parechovirus, Varicella zoster virus.

DNA Extraction, Replication, and Sequencing

Extraction and Purification Technologies

One major obstacle for molecular methodologies begins with the appropriate DNA extraction or purification from the primary biological source. A case in point being our own laboratory, whereby adequate and comprehensive DNA yield for analysis for eukaryotes required an investment of a year of research and development. To further complicate subsequent workflows is how different tissue types require different extraction methodologies. In general, biological material is lysed by detergents allowing for subsequent separation of DNA from all the remaining cellular debris. The lysis times, temperatures, and concentrations of reagents can be critical for optimum extraction. Once extraction is complete the purity, quality, and concentration of the DNA may be determined for further downstream applications and analysis.

Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) is a technique that allows for selective amplification of DNA sequences. This technology was first developed by Dr. Kary Mullis, another Nobel Prize winner, in 1983 while working at the Cetus Corporation. The core technological breakthrough enabling PCR is the DNA polymerase isolated from Thermophilus aquotica found in a geothermal pool at Yellowstone National Park. This polymerase enzyme was found to be stable at high temperatures. By combining this enzyme with a process of thermal cycling (or rapidly changing the temperature of an amplification reaction) a new technology was developed that allowed for rapid and selective replication of DNA fragments without the aid of living cells (Figure 3).

Early Sequencing-Gels and Sanger Sequencing

The Nobel Prize winner Frederick Sanger and his team at Applied Biosystems developed one of the first DNA sequencing methods in 1977. Sanger sequencing is inexpensive, accurate, and is still in use today with minor modifications. This technology uses the incorporation of chain terminating nucleotides called di-deoxynucleoside triphosphates (ddNTPs) which randomly terminate DNA strand elongation by DNA polymerase during replication. The terminating ddNTPs are radioactively or fluorescently labeled. Four different amplification reactions corresponding to each of the ddNTPs are separated by gel electrophoresis. The resulting bands are then read on X-ray film (in the case of radioactively labeled ddNTPs) or by a fluorescence detector (in the case of fluorescently tagged ddNTPs) allowing the sequence to be derived by reading the terminating fragment sizes from shortest to longest (Figure 4).

Quantitative, Real Time, or qPCR

Quantitative PCR monitors the amplification of the specific DNA during the polymerase chain reaction extension time. (Figure 5) The main purpose of this technology is using fluorescence to detect the amplification of a target sequence in "real time" allowing us the ability to quantitate the amount of that specific sequence present in the sample.

Multiplex PCR

This is a technique first developed in the late 1980s to amplify multiple target sequences simultaneously. Many of the commercial wide array assays for infectious disease rely on this technique. The 'Biofire' system using the PCR multiplex 'Film Array' technology is an example of this.

High Throughput or Next-Generation DNA Sequencing

Next-Generation DNA Sequencing (NGS) relies on newer devices that take the PCR product and then directly sequences through three primary methods. These instruments sequence the DNA (determine the order of the nucleotide bases). The core technology for sequencing may rely on semiconductor electronic chip technology, fluidic interferometry, or fluorescent tag detection. The three main manufacturers of instruments include Pacific Biosciences, Illumina, and the Ion Torrent by Life Technologies/ThermoFisher. The most rapid DNA sequencer, the Ion Torrent, was originally developed by a group of Stanford researchers using a pH sensitive semiconductor-based technology, Illumina sequencers are arguably the industry standard due to their high quality, but slower sequencing methodology by reversible terminator fluorescence detection. The Pacific Biosciences sequencers operate using the single molecule, real time sequencing which is quite different than the clonal detection methods used by Illumina and Ion Torrent. Overall, these devices can provide rapid results in days to hours. Other emerging methods, such as Oxford Nanpore and Genia, have the potential to sequence the same samples in a matter of minutes in the not too distant future.

In the case of microbial diagnostics, the resulting sequences are then compared to a database. One of the most comprehensive and more frequently updated organism databases is the "NT" database maintained by NCBI with over 36,000,000 million entries. Using BLAST (Basic Local Alignment Search Tool) the sequences may be compared to all of the entries in the "NT" database. The nature of the matches to the sequenced DNA allows you to identify your microbe. Interestingly, this unique approach allows for the identification of new or novel organisms as matches to the database might be close matches (likely to be the same organism as listed in the database) or more divergent matches (likely to be a novel organism). This has clear implications when searching for the causality of disease, outbreaks of new diseases, and personalized medicine.

NGS has already replaced quantitative PCR and multiplex PCR in our laboratory because of its ease of use and accuracy. Improved scope of testing yields fewer false positive results particularly when combining both prokaryotic and eukaryotic searches by NGS. NGS will replace most other technologies for infectious disease detection in the next 5 to 7 years and will be widely available in hospitals, larger clinics, and reference laboratories.

Why Use NGS Over Other Techniques?

Quantitative-PCR and heavily multiplexed PCR are affordable and useful when ruling out specific organisms. Additionally, these technologies are common among many clinical laboratories. Assays dependent on these technologies are also relatively quick. NGS has the advantage of simultaneously detecting and characterizing nearly all organisms even if they are novel or previously undiscovered. These systems are rapidly becoming automated and have the capability to analyze multiple samples simultaneously. While currently expensive compared to quantitative PCR and multiplex PCR, the cost of operation is decreasing and will soon reach a similar level to these legacy methods. Think of using NGS when no organisms are found in an obvious infectious disease case and when you suspect something new. In addition, there are few laboratory tests for many of the fungi or protozoans; and, thus, they are not even available for routine PCR except in research laboratories.

The Future of Molecular Diagnostics

The future for molecular diagnostics will include comprehensive and sophisticated DNA-based analysis using sophisticated bioinformatics, reporting, and full automation. Once these systems become widespread the cost per test will reduce dramatically. In the more distant future it is not unreasonable to speculate that we will be able to identify deleterious gene mutations and repair them by slicing out defective genomic segments and replacement with corrected genomic material. This technology is still in its infancy but holds great promise, especially correcting infectious disease predispositions, compensating for genetic deficiencies, and selectively treating cancers.

Selected and Cited References:

(1.) Espy MJ, et al. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev. 2006;19(1): p. 165-256.

(2.) Rao A, Fader B, Hocker K. Molecular detection and survellance of healthcare-associated infecbons. In Molecular Diagnostics: Techniques and Applications for the Clinical Laboratory., W. Grody, et al. Editors. Boston, MA: Academic Press, Inc.; 2010: 327-346.

(3.) Dahm, R. Discovering DNA: Friedrich Miescher and the early years of nucleic acid research. Hum Genet. 2008;122(6): 565-81.

(4.) Franca LT, Carrilho E, Kist TB. A review of DNA sequencing techniques. Q Rev Biophys. 2002;35(2):169-200.

(5.) Ellis JE, et al. Rapid infectious disease identification by next-generation DNA sequencing. J Microbiol Methods. 2016.

(6.) Ramawat K. Molecular Biology and Biotechnology. 2014: S CHAND.

(7.) Coleman W, Tsongalis G. Diagnostic Molecular Pathology: A Guide to Applied Molecular Testing. 1st ed. Academic Press; 2016.

by Stephen Fry, MD

Declarations: Dr. Fry is the owner of Fry Laboratories, LLC, a clinical research and development laboratory which provides NGS services to clinics and hospitals worldwide.


Please note: Some tables or figures were omitted from this article.
COPYRIGHT 2017 The Townsend Letter Group
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2017 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Fry, Stephen
Publication:Townsend Letter
Article Type:Report
Date:Jan 1, 2017
Previous Article:Health and disease by iridology examination.
Next Article:Thirty-seven years of practice in electrodermal screening (EAV).

Terms of use | Privacy policy | Copyright © 2021 Farlex, Inc. | Feedback | For webmasters |