MultiGEN Diagnostics, Inc. : Frequently Asked Questions

Routine clinical ‘microbiology’ is in reality limited to mainly identifying bacteria (bacteriology), with a generally poor ability to detect the other important groups of disease causing organisms–viruses, parasites and fungi. As many infectious syndromes are caused by pathogens other than bacteria, clearly the term ‘microbiology laboratory’ is a misnomer. Rapid, specific and cost-effective laboratory diagnosis of most viral, parasitic and fungal diseases is just not a practical option with the methods in current use.

Bacteria are identified with present technology as a result of a complex mix of inputs from different sources: the shape, color and consistency of the colony; the shape, size, and color of the bacteria with artificial staining agents (e.g. Gram stain); the association with unusual phenomena (e.g. rupture of sheep red blood cells, or the production of gas) and sometimes specific antigen detection (antibody mediated latex flocculation and ELISA). Furthermore, some organisms are easily missed if they have unique growth requirements, and must be looked for specifically (e.g. Gonococcus, Listeria monocytogenes, and Mycobacteria). A ‘microbiology’ technologist must then judiciously weigh the significance of each of these many inputs and formulate an opinion as to the likely identity of the bacterium. Result reporting typically takes 3 days or more.

Viral infections are mainly identified indirectly by detecting a patient’s production of specific antibody (serology). But as antibodies are only produced a week or more after the start of an infection, results are delayed for a considerable period of time. In routine laboratories only a limited number of viral pathogens can be identified directly (usually with ELISA based methods), with positive results requiring a minimum concentration of the virus to be positive. A few viruses can be identified by detecting their unique DNA or RNA, but use of this method has been mainly limited to blood donation screening for HIV and Hepatitis viruses in specialized laboratories. Virus identification by culture takes far too long and is far too expensive for routine use, and is therefore used only by reference laboratories in unusual circumstances.

As currently practiced, the identification of parasites (parasitology) and fungi (mycology) is accomplished mainly by searching for the organism by direct microscopic examination of a sample. This method is often of questionable accuracy except in the most experienced hands.

If separate tests are performed for each of the different pathogens that can cause a particular patient’s infection, then the final cumulative price for all the results is quite high.

Furthermore, any laboratory report that takes a long time to produce is inevitably expensive, due to high labor costs, consumables, and the varied infrastructure required to process the tests (large incubators, microscopes, etc). The ‘microbiology’ sections of clinical labs are typically as large or larger than the chemistry section and this footprint too adds costs if full cost accounting is used. Relative to costing yardsticks applied to clinical chemistry and hematology, ‘microbiology’ therefore is an expensive section of the clinical laboratory, and given the limited test menus, delayed results, and cost it is fair to say ‘microbiology’ is relatively cost-ineffective as presently practiced.
Delayed results also may have a ripple effect, since delayed treatment can result in increased morbidity and death, and complications from unnecessary or inappropriate initial antibiotic therapy.

Patients present with syndromes, not diagnoses. For example, one of the causes of headaches is meningitis, which can itself be caused by a variety of specific bacteria, viruses, fungi or parasites. The final etiologic diagnosis therefore becomes bacterial meningitis or viral meningitis, etc each with different therapeutic options and differing prognoses. Likewise diarrhea with fever can be caused by pathogens from different phyla. An attending clinician, who may lack the knowledge to request searches for specific pathogens, should not be expected to ask the laboratory to identify all the potential pathogens by name and then have the laboratory search for each of them. It would be much more appropriate (and frankly a reflection of everyday reality in medical practice) for the clinician to request the "Meningitis Test" or the "Infectious Diarrhea Test" and leave it up to the laboratory to look for ALL the potential pathogens regardless of their phyla.

Unfortunately such comprehensive ‘Syndrome-Driven Panels’ are not presently available in ‘microbiology’ despite their longstanding availability in clinical chemistry (liver panels, renal panels, cholesterol/lipoproteins panels) and hematology (twelve or more parameter CBC profiles). Clearly, the present day ‘microbiology’ laboratory does not offer a logical approach to diagnosis that responds to clinical needs.

6.	Why have alternative DNA/RNA methods for microbial identification failed in large scale commercialization?

Any organism, whether a tree, a human or a bacterium, can be definitively identified by accurately detecting a short segment of its DNA or RNA (the so-called target) that is unique to that organism. This data base is readily available in the public domain, and the general method theoretically offers a truly microbiological diagnosis as to whether or not the target identified is from a bacterium, a virus, a parasite or a fungus. Unfortunately this promise has not been realized to date in other than relatively few applications, with the reasons centering mainly on concerns regarding accuracy, cost, and utility.

There are really only two generally accepted methods in molecular diagnostics: those based on DNA probes and those based on DNA sequencing (other novel approaches such as mass spectroscopy still have to be proven sufficiently accurate for clinical use).

In this method, a short string of nucleotides (a probe) is designed that is complementary to the short stretch of the DNA to be identified. This is based on the well-known binding reciprocity of the four standard DNA nucleotides: cytosine only binds to guanidine, and thymidine only binds to adenine (and vice versa). The specific probe could be thought of as a stereochemical mirror image of the DNA target, in very much the same way as an antibody is a mirror image of a special segment of an antigen.

In other words, probe-based technologies are very similar in general concept to Enzyme Linked ImmunoSorbent Assays (ELISA), and, not surprisingly, they share the same problems of nonspecific reactions. The reason for this is straightforward: a positive reaction is identified when the probe ‘sticks’ to the complementary DNA target, but a partial adhesion to a very short segment of DNA from a totally unrelated species with some similarity in its DNA to the intended target can produce a nonspecific false positive result. This is obviously unacceptable in clinical practice.

Probe-based assays also tend to be rather limited in the number of targets that can be accurately detected in the same test – typically up to three or four. This number of targets severely limits clinical applications as most ‘Syndrome-Driven Panels’ would require an ability to screen for a dozen or more pathogens to provide a diagnosis in 99% or so of cases.

The only other practical way to identify DNA or RNA depends on determining the precise nucleotide sequence (fingerprinting) of a short segment of the DNA or RNA that is known to uniquely identify that particular organism. This short DNA segment is called the Signature Sequence and is usually only 25 or so nucleotides long. In other words, it is not necessary to sequence the entire genome of an organism to conclusively prove its presence; a very short sequence will suffice.

DNA sequencing has been known for a couple of decades since being first described by Dr. Fred Sanger, and for which he received the Nobel Prize. It is generally accepted that DNA sequencing represents the ‘Gold Standard’ for microbiological identification in terms of specificity. Once a Signature Sequence is identified, it can only come from one specific organism eliminating the potential for false positives (other than through sample contamination).

Sequencing has an added advantage over DNA probes in that it can also identify mutations in the pathogen, and document evidence of genetic drift and shift. These factors are of course of particular importance with RNA viruses as they readily mutate (e.g., SARS, HIV, West Nile Virus etc.).

Unfortunately, traditional DNA sequencing, despite being the ‘Gold Standard’, cannot be used for routine diagnosis because it can only detect ONE signature sequence per test run. Any attempt at sequencing multiple targets simultaneously in the same test results in such overlap of the nucleotides during electrophoretic separation that no useful information can be obtained.

Traditional DNA and RNA sequencing has therefore been restricted to research applications or urgent investigations such as the recent SARS outbreak.

MultiGEN DNA Sequencing promises to transform present-day diagnostic capabilities to cost-effectively screen for and identify ANY pathogen that can cause a particular infectious disease syndrome with ‘Syndrome Driven Panels’. MultiGEN DNA Sequencing is a novel method of sequencing multiple DNA or RNA targets simultaneously in the same test. The test protocol is a modification of the well proven ‘Gold Standard’ Sanger method.

Molecular Epidemiology is a complex term describing very important ways of tracking the origin and spread of pathogens using DNA ‘fingerprinting’. The general method is called genotyping, and involves the comparison of the DNA fingerprints of the sub-types of a pathogen causing the same type of infection in different individuals. MultiGEN DNA sequencing is the only technology that can accurately identify, in the same test, not only the sub-type of the pathogen causing an infection but also unique mutations in its DNA . In a hospital setting this can enable the tracing of the source of a dangerous pathogen (e.g. MRSA and VRE) to a particular patient who can then be quarantined.

RNA viruses such as SARS-CoV and HIV regularly mutate as they are passed from host to host. In the case of SARS, the identification of the cumulative load of successive mutations allows a likely path of spread of the infection to be deduced, and a likely animal origin predicted so that counter-measures can be taken to control the source. Only MultiGEN sequencing has the ability to genotype accurately and cost-effectively, thereby providing information of great practical value for use in molecular epidemiology.