Point of care diagnostics: Applying the science of genetic sequencing to serve the doctor’s need for rapid, accurate diagnosis of infectious disease

All living critters inhabiting this planet can be uniquely identified through analysis of their genetic sequences. The genes of all cells consist of DNA (made up of a large number of deoxyribonucleotides); some viruses however, use RNA (ribonucleic acid) as their genetic material. While the number of genes in each organism varies considerably, from prokaryotes with genomes of 2000 genes to humans with ~20,000 genes, each individual and each type of life evidences a unique genetic pattern. If you analyze genes from another human, your dog, your Aunt Millie’s parakeet, or something as small and simple as a virus or bacterium, they all contain unique features that can now be used for reliable identification. This facet of cellular biology and genetics, along with Akonni Biosystem’s latest microarray-based diagnostic technology, makes possible the accurate, timely, and low-cost detection of the presence, or absence, of a particular viral or bacterial infectious pathogen. The example below summarizes how Akonni’s diagnostic microarray uses pattern recognition of tuberculosis genetic signatures to differentiate a drug resistant strain from a drug susceptible strain.

Genetic analysis is also the key to identifying human predisposition to many diseases, including those for which environmental stresses contribute to susceptibility. For 23 years, The Human Genome Project has been focused on creating a comprehensive map of the entire human genome, in order to allow the identification of genetic anomalies in the human population. Genetic predisposition to a given disease in a patient, based on specific markers (gene variations, such as single nucleotide polymorphisms, insertions, deletions, etc.) for that disease, can be rapidly determined using the same Akonni technology depicted above.

The doctor’s dilemma: accurate, timely, and cost-effective diagnosis

From time immemorial, physicians have been hampered in their diagnosis of infectious disease by a lack of timely, accurate, and low-cost diagnostic tests - tests that will provide definitive answers immediately, while the patient is present in the examining room, or prior to their exhibiting acute and life-threatening symptoms. All too often a doctor must select a treatment protocol based on informed induction and probability, while waiting for test results that will not be available for days or weeks.

Current point-of-care testing modalities suffer from a number of shortfalls:

1. Lack of timeliness: The gold standard in FDA-approved tests is still mostly culture of the organism – growing the suspected pathogen in a Petri dish and examining it optically or chemically. Most cultures require 48 hours to 5 weeks to yield results.
2. Lack of accuracy or comprehensiveness: For a sore throat, a doctor will typically diagnose a patient by running a rapid strep test and sending a throat swab to a clinical lab for analysis, requiring 72 hours. The rapid strep test detects only one infectious disease. Unfortunately, it is not uncommon for the patient’s immune system to be doing battle with more than one pathogen, such as in the case when a patent has both a viral and a secondary bacterial infection. In the case of flu-like symptoms, there are over 50 highly infectious pathogens that can cause pneumonia, sepsis, or influenza, e.g., staph, strep, influenza, E. coli, common cold-rhinovirus, anthrax, Legionella, Chlamydia, tuberculosis, etc.). While waiting for the test results to be returned, more than likely the doctor has prescribed an antibiotic. If the doctor has misjudged the pathogen(s) causing the infection, the patient may well be taking the wrong medication. The scourge of new antibiotic-resistant bacterial strains has been greatly exacerbated by doctors having to prescribe without adequate factual data to make a definitive diagnosis.
3. High cost of diagnosis: Many of today’s more sophisticated tests cost $75 to several hundred dollars. Further, a battery of tests is typically run, costing several hundreds to thousands of dollars, on patients with infections that are difficult to diagnose, or on those whose immune systems are compromised, where multiple infections may be present. A significant component of high diagnostic costs is that most sophisticated tests require highly trained experts and very expensive medical equipment located in a central laboratory to conduct the tests.

The Akonni multi-array diagnostic tool (TruDiagnosis™) addresses the three principal shortfalls listed above by allowing the doctor to (i) screen a patient sample for multiple diseases, (ii) gain results within a typical patient visit, and (iii) do so at a modest cost utilizing fully-automated processes completed in the doctor’s office.

Akonni Biosystems’ microarray: origins and technology

Origins of the Technology. Akonni’s microarray technology was invented in 1988 by Dr. Andrei Mirzabekov, the Director of the Engelhardt Institute of Molecular Biology in Moscow, Russia. The Engelhardt (EIMB)/ Argonne portfolio defines one of three pioneering platforms in the field of microarrays:

• Steve Fodar (Affymax/ Affymetrix)
• Ed Southern (Isis/ Oxford Gene Technologies, OGT)
• Andrei Mirzabekov (EIMB/ Argonne)

These are all recognized as patent-protected platforms that established themselves as founding microarray technologies in the late 80s and early 90s.

In 1995, the U.S. government brought Mirzabekov and his team to this country, where he continued his work on the microarray at Argonne National Laboratory, near Chicago. Since 1995, the technology has been developed under federal programs funded by Department of Defense agencies such as DARPA and DTRA, the Department of Energy, the National Institutes of Health, the Centers for Disease Control, and the National Science Foundation. The government and private investment in this technology already exceeded $22 million by the time Akonni Biosystems gained its exclusive license to it in 2002. Since then, the Federal government has continued to invest heavily in its development, and Akonni has greatly advanced the technology, making important new discoveries that will allow this point-of-care testing to be affordable and become the "gold standard" in diagnosis of infectious and genetic disease.

The Akonni technology in brief. A microarray is an ordered pattern (an array) of tiny drop-like biosensors anchored on a piece of plastic or glass. Each biosensor can identify a different disease marker (which is a unique molecular feature that can be used to differentiate one disease from another). A single sample can be screened for hundreds of disease markers.

Akonni uses precision robotics to place an array of micro-gel-drops on a plastic or glass microfluidic slide. Each gel-drop is 100 microns in diameter by 20 microns in height (100 microns is about the width of a human hair); each gel-drop is about 0.2 nanoliters in volume. Akonni manufactures arrays carrying from 10 to 1000 gel-drops. Each gel-drop behaves as a “micro-test tube”, and each test tube can be tailored to carry out many diagnostic biochemical tests. The individual micro-test-tubes are packed into a 1 cm2 area, such that a small amount of clinical sample (i.e., blood or sputum) can be analyzed for hundreds to thousands of infectious disease markers simultaneously, without interfering with each other. The microfluidic device automates all of the processes necessary to achieve an accurate result. The following figure is a flow chart of the components of the TruDiagnosisTM system for both nucleic acid and antibody-based TruArrayTM tests. Also depicted is a plot of multiple disease identifiers, produced by Akonni’s Respiratory TruArrayTM panel test, which is capable of screening for over 3925 strains and species of infectious diseases.

Utilizing TruDiagnosis™ in medical practice

The doctor, nurse, or allied health professional first takes a patient sample – sputum, buccal swab, blood, etc., depending on the suspected infectious disease. The sample is introduced into a prepared syringe, which is then used to inject the sample into the test cartridge (microarray). The microarray is placed on the reader/controller loading platform and the “start” button depressed. All further processing is automated. Depending on the battery of tests being run, test results are available within 10 to 30 minutes.

Infectious disease tests under development

The following Akonni infectious disease tests are currently being utilized by the Federal government for medical surveillance and research purposes. Today, tests are available for research use only (RUO), see Table below. Application for FDA approval will be initiated shortly. There are more than two dozen additional infectious disease tests currently under development.

An additional test application under development is a comprehensive panel test for nosocomial infections – infections patents acquire during hospitalization. The incidence of nosocomial infections has been increasing; the CDC estimates that 2 million patients are affected per annum and 99,000 died last year as a result. Akonni is combining tests for the most common nosocomial infections into one low-cost test, so that it will be economical for a hospital to monitor all patients at intake and periodically throughout their stay. Given the timeliness of the test results, those patients harboring infections can then be immediately quarantined and given additional appropriate therapy.

Supplementing clinical judgment with molecular evidence

In summary, from a small non-invasive patient sample, Akonni’s technology enables a doctor to test a patient for a panel of common infectious diseases within a 30-minute appointment and at a fraction of the cost of today’s most common tests. The doctor will have an unprecedented amount of information to aid diagnosis; further, the doctor can be better informed as to alternative FDA-approved medications designed to treat the disease. Genetic analysis is finally beginning to dramatically improve the practice of medicine to the benefit of both the patient and the doctor, while at the same time lowering the cost of care.