PathoDNA, an Advanced Diagnostic for Lyme Disease & Co-Infections Utilizing Next Generation DNA Sequencing with Greater Sensitivity and Selectivity than ELISA/Western Blot

The controversial subject of chronic Lyme disease has occupied medical dis-course for years while contributing to unprecedented patient suffering in the United States and abroad. A general misunderstanding of Lyme disease and overconfidence in the Center for Disease Control’s (CDC) recommended two-step test for Lyme disease has led to misdiagnosis and incorrect treatment over the years. This leads to increasing medical expenses and worse outcomes for patients. The two-step test, an ELISA immunoblot followed by a confirmatory Western blot, yields accuracy rates as low as 29% for acute Lyme disease and 75% for chronic Lyme disease. While these practices have been a staple of microbiology for decades, these accuracy rates are unaccepta-ble for diagnostic tests when better technology is available. PathoDNA, a Next-Generation DNA sequencing test for Lyme disease and other tick-borne pathogens, achieves accuracy rates of 98% for B. burgdorferi and 95% or greater for other common tick-borne pathogens with superior sensitivity and selectivity. PathoDNA is a Clinical Laboratory Improvement and Amendments (CLIA)-validated laboratory test that achieves these results utilizing Next Generational DNA Sequencing and a proprietary bioinformatics database. Thus, it allows for rapid results and specific identification of tick-borne illnesses. In this article, we will compare this promising technology against the existing standards for diagnosing and testing Lyme


Introduction
Every year, thousands of people across the United States go undiagnosed with infectious diseases. This is especially true of those suffering from Lyme disease and associated tick-borne infections. Unidentified infections result in prolonged stays in the hospital, more frequent readmission, and increased mortality and morbidity through delayed or inappropriate treatment [1] [2]. Unidentified infections have also been implicated as a potential cause of some cancers, autoimmune conditions, chronic fatigue syndrome, and many other acute and chronic disorders [3]- [9]. Correctly identifying an infection allows for the use of more closely targeted treatments and narrow-spectrum antibiotics. This is desirable because overuse of broad-spectrum antibiotics leads to the emergences of new, resistant strains of pathogens [10].
One of the most difficult to detect and diagnose infectious diseases that pose a threat to public health is Lyme disease. Lyme disease is the most common vector-borne disease in the United States, with the majority of cases occurring in the Northeast, mid-Atlantic, and upper Midwest regions [11]. Lyme disease is already a public health threat in both Europe and the USA and is currently poised to become the number one spreading, vector-borne epidemic in the world [12] [13]. Tick-borne illnesses increased from 48,600 cases in 2016 to 59,300 in 2017 [14]. A steady increase in the cases of reported Lyme disease has continued to be observed, which appear to be driven by both milder winter temperatures and extended spring and summer days, both of which benefit the primary vector for Lyme, Ixodes scapularis.
Across the United States, tick species capable of carrying Lyme disease are now found in nearly 50% of all counties [14]. In 2018, the Borrelia burgdorferi carrying Asian long-horned tick was newly identified in several US states [15].
The CDC has measured an estimated incidence rate of about 30,000 reported cases of Lyme disease each year between 2008 and 2015. This rate increases year after year and is triple the rate from 1992. The CDC acknowledges that Lyme disease is often misdiagnosed and is severely underreported in the United States [11]. Federal scientists have suggested that the actual incidence rate of Lyme disease is as much as ten times the number reported between 2008 and 2015 [11]. Thus, the actual incidence rate may exceed 300,000 cases per year [11].
A survey of seven of the largest commercial laboratories in the states (representing >76% of all Lyme disease tests performed in the same year) found that approximately 3.4 million Lyme disease tests were conducted on 2.4 million samples in 2008 at a cost to patients and health care providers of $492 million [16]; Two-tiered assays as mandated by the CDC accounted for 62% of all tests performed. This is actually higher when including tests performed individually (such as an initial ELISA upon arriving at a hospital), which were not included in this figure. The same researchers estimated that these reported tests corresponded to about 240,000 to 444,000 infected people for 2008; this aligns roughly with the estimate released by the CDC if only 10% of cases were reported to the official count. In New York, tick expansion coincided both spatially and temporally with warmer temperatures; the researchers there found that mild winter days were strongly predictive with summer encounters with I. scapularis [17]. Europe has reported a steady increase in Lyme infections for the past 20 years, with more than 360,000 patient cases recorded [18]. There is evidence suggesting that the infection rate is even higher in Europe than the reported number. Sykes et al.
found a much higher infection burden in Western Europe alone than the World Health Organization (WHO) figure would indicate 232,000 cases per year on average [19]. This result gives additional credence to the idea that the real infection rate in the US is much higher than the reported numbers [11]. A 2013 conference on Lyme disease released numbers from a self-reported survey which indicated a 0.3% infection rate across the US for the year 2012, which would amount to well over 900,000 people if it were applied to the entire country [20]. While the statistics from a self-reported survey should be taken with a grain of salt, it is an indicator that more honest and professional surveying of the actual disease rate is needed, given the wide discrepancy between reported infection rates for Lyme disease.
The increasing rates of Lyme disease seen in Figure 1 degrade public welfare and put individual lives in danger. It also places a burden on the health system, which is underreported because of commonly undiagnosed or misdiagnosed Lyme disease cases. An observational cohort conducted between 2000 and 2013 showed that incorrect diagnoses and unnecessary treatments were common for Lyme disease patients [21]. If the patient presents without a rash, the patient stands a 54% chance of being misdiagnosed [22]. When patients receive treatments that provide no benefit to them that places a burden on the healthcare system and contributes to inefficiencies of the system as a whole.
A 2015 study attempted to measure long-term issues Lyme patients faced and the increased cost of care associated with Lyme disease. Adrion et al. noted that despite standard treatment protocols that insist that Lyme disease treatment ends after about eight weeks, patients were returning to the doctor with persistent symptoms. This would then be followed by multiple additional rounds of testing and re-treatment. On average it is estimated that "people with Lyme disease cost the system $2968 more than matched controls, and they cost the health care system about $1 billion a year" [23]. However, Borrelia burgdorferi has been historically difficult to detect. This is because the organism contains many defensive countermeasures including interference with the active immune system. For example, Borrelia evades lysis through the complement immune response by expressing a factor H-binding protein identified by Kraiczy et al. [28]. Xu et al. write about the ability of Borrelia to change the expression of its outer surface proteins in order to evade the immune system during initial infection and then later during humoral response [29]. Lyme disease may also be confused for other conditions while making a diagnosis [30]. There have been cases where Lyme has been misdiagnosed as  [39].
In this article, the multiple problems with current standard testing regimen for Lyme disease will be examined in greater detail by the technology used to scan for the pathogen [40]. The primary concern over the standard diagnostic algorithm is that it relies on expression of relevant antibodies in the patient. This is unreliable in the case of early Lyme disease since it takes several weeks for an antibody response to develop, producing a high rate of false negatives [40] [41].
Additionally, in late Lyme disease, the immune system can become compromised which also limits the antibody reaction; in the late stage, two-tiered testing with ELISA and Western blot may miss as much as 44% of infected patients [42].
In addition to becoming immune-compromised, Borrelia antibodies frequently become bound up in circulating immune complexes leading to the inaccuracies measured in standard immunoassays [43]. Also, tests prepared in the US typically only attempt to detect a few prevalent species and are usually incapable of determining foreign infections from strains such as Borrelia miyamotoi [44] [

Western Blot
Western blot has been a staple technique in biochemistry since the 1980s [48]. It is a firmly established tool to detect and analyze proteins. And while as a forensic and diagnostic tool, Western blot is still considered a pillar of diagnostic science, it is also a 40-year-old technology. As newer and more advanced processes come along, there has always been resistance to the displacement of accepted methods.
In the face of that resistance, it can be useful to break down exactly what these older processes like Western blot and ELISA are, how they work, and their limitations.
Western blot primarily detects single proteins qualitatively, especially from complex mixtures of multiple proteins. The test is only semi-quantitative since the size and color of a protein band correlate roughly with the amount of protein present. While strong and faint bands are easy to distinguish, it is important to remember that smaller differences can be more difficult for an examiner to differentiate and introduce subjective interpretation into results. Western blot is more helpful for determining the presence or absence of a particular protein than obtaining more detailed information in practice. Liang et al. write that subjective interpretation of the banding patterns produced by Western blot can potentially introduce errors in reporting results [49].
This qualitative testing ability suits the needs of many doctors in making certain kinds of diagnoses. However, Western blot meets challenges in circumstances where there are weak or muddled protein signals which can make or break an early diagnosis. In the case of Lyme disease, Western blot is less than ideal for making an early diagnosis because the IgM response, the key factor for which Western blot is looking for, is insensitive, nonspecific, or both in the first weeks following infection [50]. It is not until late Lyme disease that Western blot can make a concrete diagnosis.
In the case of Human Immunodeficiency Virus (HIV), Western blot is being phased out for use in detection and diagnosis entirely. Once the gold standard, Western blot is being gradually removed from the recommended HIV diagnostic algorithm due to its inability to detect early HIV infection, the length of time the testing requires, and a tendency to misclassify HIV-2 infections as HIV-1 [51].
Interestingly, diseases such as Lyme, syphilis and lupus can cause false positives in the ELISA test for HIV infection [52]. The similarity here between HIV and Lyme is striking because advanced Lyme disease can also result in an immune-compromised state [53].
In North America, the species, Borrelia burgdorferi is associated with Lyme disease [54], and Western blot tests produced in the US reflect that. Outside North America, however, the situation can be very different. For example, in

Elisa
Enzyme-linked immunosorbent assay, or ELISA, has been around since first described by Engvall  When there are problems with ELISA, many of them come down to the immobilization technique. For instance, in direct ELISA, when extracted serum is dried to the bottom or sides of a testing well, there will usually be many other proteins aside from the one of interest in the mix and in unknown quantities.
There may be only a tiny portion of protein exposed on the surface where antibodies can react. This can cause weak enzyme signals and equivocal results.
Sandwich ELISA is an attempt to solve this by immobilizing capture antibodies instead so that they can pull target antigens out of the solution, but this technique has its issues, often needing separate validation due to the risk of false positives [63].
In the case of Lyme disease, the sensitivity of ELISA can vary substantially,  [68]. The tests were retrospective, allowing the researchers to categorize the children into groups of "Definite," "Possible," and "non-Lyme" in terms of their Lyme disease status; another group of children not exposed to Lyme disease was used as a control group. The PCR tests identified only a small portion of the definite Lyme cases as positive for Lyme disease (5%) while changing one child from the "Possible" group to "Definite" and another child from the "non-Lyme" group to "Definite" [68]. The conclusion that Skogman and her colleagues drew from the results was that these two PCR tests were better suited to a complementary role in Lyme disease testing due to its very low sensitivity but could still remain useful for its ability to catch certain cases that would otherwise not be classified as a Lyme infection.
One explanation for this stunning lack of sensitivity for PCR tests designed specifically for Lyme disease is that PCR is reliant on the choice of primers/probes for the test. An incorrect or suboptimally designed set of primers would result in failed PCR reactions. Another possible explanation is the suspected low population of spirochetes in cerebral spinal fluid which Skogman was using for her study [68]. Other studies offer a range of sensitivities for Lyme PCR detection depending on the origin of the source tissue. Dessau et al. report a median of 69% sensitivity with the best results for biopsies of erythema migrans and the worst for cerebrospinal fluid with sensitivity approaching 40% [69]. Under certain circumstances, sensitivity is reported to be higher [70] [71], however, the wide range of reported sensitivity for this test is a major concern when trying to make a firm diagnosis. PCR is undoubtedly an invaluable technology for a large number of procedures dealing with DNA, but as a diagnostic test, there are certainly better options such as Next-Generation DNA Sequencing. There are two major approaches to shotgun sequencing, metagenomics, which examines present DNA from a sample, and metatranscriptomics, which sequences the mRNAs present in a sample. PathoDNA takes a metagenomic approach to classifying DNA. Figure 2 illustrates the 4 main steps in the PathoDNA diagnostic process. The test was designed to process either a blood or a urine sample from a patient. The inclusion of the ability to test urine for spirochetes and coinfections is important since there is data suggesting that these organisms reside in greater concentrations inside the urine and bladder tissue of the host [78]. DNA is extracted and purified from the patient sample and then sent through a DNA amplification process. AmpliSeq primers are used following the AmpliSeq Library protocol according to CLN-00001-SOP [79]. During this step, the samples are normalized using the AmpliSeq Equalizer Kit before being pooled for emulsion PCR. The PCR product is loaded onto enriched beads before being placed onto a chip for DNA sequencing. Sequencing is performed on the Ion Torrent S5 sequencer [79]. Specialized software is used to trim and filter sequences as they are generated because the shotgun sequencing process produces many small sequences of DNA that need to be identified and sorted into a library for analysis. It is important to remove reads from the sequence output that map to the known human genome, leaving only non-human DNA for data processing. DNA sequencing by the shotgun process requires that reads be pooled according to probable species and counted. Software counts the number of reads generated by the sequencing process and uses this information to assign probability thresholds for matching sets of amplicons. The number of reads of a particular sequence that is generated by the sequencing process corresponds to the quantity of DNA that was present in the original sample [79].
Metagenomics is often used to understand the functional component of a community of organisms (i.e., an infection). This can be inferred by comparison Figure 2. The four main steps of PathoDNA, starting with blood or a urine sample. PCR composes the second step where the DNA is amplified. Next-generation DNA sequencing is the third step where all of the DNA that was amplified in step 2 is sequenced.
Step 4 compares the results of the DNA sequencing against a proprietary database containing known sequences for Borrelia and other tick-related infectious organisms. Lyme and other tickborne infections. Below, Figure 3 illustrates a list of organisms detected by PathoDNA that cause Lyme disease or its associated coinfections.  formed by a single laboratory to be a "Laboratory Developed Test" (LDT). These are also sometimes referred to as "in-house" or "homebrew" tests. LDT's can be FDA-approved or CLIA validated, though due to poor enforcement, sometimes LDT's are neither of these.
So-called "alternative tests" have been available to patients willing to go outside the medical insurance system for many years, but it is frequently difficult for the average person to distinguish between science-backed medicine and pseu- Even when an independently developed test is not FDA-approved, the laboratory must still demonstrate the competency of both the staff and equipment present in the lab, evidence supporting the efficacy of the test, and that results are not falsified in any way. With these safeguards in place, the FDA has made the wise decision to allow LDT's in the marketplace to fill unique health care niches better and prevent delays in medical treatment. More patients would be better informed to learn that even when a lab test is not FDA-approved, it is still subject to oversight by the FDA to ensure its safety and validity. In fact, the FDA is still updating its draft guidances on LDT's which were last done in 2017 [82]. The FDA explicitly features a class system for laboratory tests and medical de- vices ranging from potentially dangerous and highly regulated tests to minimally dangerous tests which general controls can monitor. The process for obtaining FDA approval, however, is long and onerous for most companies, often requiring an enormous financial cost and taking years to complete. This prevents many ideas for tests, especially ones that are not optimally marketable, from making it to the clinical trial stage [83]. Typically, only commercially manufactured tests, sold as kits to other laboratories, are FDA-approved. This is why the FDA allows LDT's to exist, because there are many niches, especially for uncommon diseases, where a laboratory test might be desirable, yet it would not make commercial sense to seek an FDA approval. In this case, LDT's can still seek a CLIA validation to assure patients and the doctors who use them that the test has been reviewed for veracity and clinical soundness. These distinguishing qualities benefit a patient seeking better health care and avoiding fake products and quackery that are presented as illegitimate substitutes for scientifically backed tests. By achieving CLIA validation, the PathoDNA diagnostic test demonstrated its clinical validity, a scientific measure of its reliability, reproducibility and validity.
The final report submitted for validation reported 98.8% for the detection of B. burgdorferi in blood and urine samples and 95% accuracy for the detection of other tick-borne pathogens [79]. Test validation was measured with several variables: • Accuracy of Detecting Single Spike-in of Pathogen DNA in Human Urine. In each of these categories, PathoDNA achieved passing acceptance criteria for CLIA validation. In fact, 98.8% accuracy for B. burgdorferi exceeds the threshold by a large margin. This accuracy level is needed and should be expected for a medical diagnostic test rather than the low accuracies of the ELISA and Western blot tests quoted earlier in this article.
This accuracy is gained in large part by applying a specialized reference library for the pathogens that cause Lyme disease along with a list of organisms commonly associated with tick-borne disease. The reference library is a database of known DNA sequences used during the shotgun DNA sequencing process after raw sequencing collection and data processing.
Lastly, PathoDNA was designed to accept either a blood or urine sample from a patient, with separate protocols for extracting DNA from either blood or urine [79]. This is important because pathogens can hide in different excreta from the body. An organism present in the blood might not necessarily be detectable in the urine and vice versa. In "Modern Methods of Pathogen Detection" the authors write that direct analysis is often made difficult by low pathogen counts and high organic and inorganic contents in the sample (such as heme in blood) [78]. The author also mentions that NGS represents the next step towards using genome sequencing as a diagnostic tool due to the impressive speed and quantity of sequence data produced by methods such as IonTorrent (which PathoDNA relies upon), although NGS systems are still not being used for routine diagnostics. In the case of urine, small molecules can become enriched to higher content than found in the plasma due to the filtration process of the blood by the kidneys. In this way, urine can represent an ultrafiltrate of the plasma [78]. Some species of intracellular bacteria can be determined more easily from the urine than from blood, such as Mycobacterium. Figure 4 shows that it was far easier for researchers to isolate spirochetes from the bladder tissue than from blood samples. In this way, a comprehensive detection system should examine multiple kinds of sample specimens for a more comprehensive analysis of the patient and a better chance of making an accurate diagnosis. Figure 4. A graph indicating the relative distribution of spirochetes in the body by tissue type. Researchers were far more likely to isolate spirochetes from the bladder than the blood in murine model. These results strongly indicate that a urine test will be more likely to uncover spirochetes in the body than a blood test.

Discussion
In this article, we have taken a closer look at the state of technology used to detect and diagnose Lyme disease and other tick-borne ailments. PathoDNA was developed as a diagnostic test for doctors to address the tremendous need for sound and accurate diagnostic testing in light of the spreading areas where Lyme disease impacts the country. This test uses newer and proven Next-Generation DNA Sequencing technology which offers much greater accuracy for detecting B. burgdorferi and other parasites while maintaining high selectivity. This is a step forward past the CDC-recommended two-tier process for diagnosing Lyme disease utilizing ELISA and Western blot.
We briefly covered how ELISA and Western blot technologies are currently used for the diagnosis of Lyme disease. Both technologies were defined between forty and fifty years ago. The technologies are relatively low cost due to their ubiquitous adoption by the scientific and medical community and existing infrastructure; however, better conclusions can be drawn from newer technology, which is far more sensitive, a feature needed with low-population infections such as Lyme. Even with the combination of both technologies in a two-tier format, ELISA-Western-blot achieves only a 52% sensitivity for early Lyme disease, well below the FDA's standard for diagnostic tests of at least 95% [56]. This level of sensitivity means that half of the tests performed come up as a false negative. While two-tier testing eventually increases sensitivity the longer the patient has already been infected, catching Lyme early on and beginning appropriate treatment is critical to affecting a permanent recovery and preventing the development of chronic Lyme disease syndrome. The worst-case scenario here is that a person receives the FDA-mandated version of the test, falsely believes it to be negative and does nothing for the latent Lyme infection, which can cause years of debilitating symptoms ranging from widespread aches and pains and chronic fatigue to neurological deterioration. Unfortunately, this is exactly what is happening and in significant numbers with as many as 300,000 new cases of Lyme each year and all signs indicating that this number will rise in the future [11]. The following Figure 5 illustrates the major difference in accuracy between the two-step, CDC-recommended test, and newer technology like PathoDNA.
Unfortunately, there is a divergence of opinion on how to best address the problem of Lyme disease among medical professionals. This can be seen with Igenex, which is continued to be lauded as a leader of Lyme disease detection. In truth, they only use a more liberal definition of the same test prescribed by the CDC to return a positive result for Lyme [59]. Their tests suffer from the same inherent flaws of technology as the conventional test and are more likely to return a false positive [60]. That is not to say that the CDC is entirely blameless either. For decades, the organization has sat on its hands, recommending and requiring doctors to use a procedure with unacceptably low sensitivity rates by its own standards for other diseases. This may have been fine in the early to mid-90s when no other options were realistically available [84], but miniaturization and automation have now put DNA sequencing technology within the grasp  of nearly everyone in the country. Issuing guidance for doctors for an inadequate test actively harms tens of thousands of people each year. Health insurance companies base what they will cover on issuances made by the CDC. As long as two-tier testing for Lyme disease is standard in the US, other, better forms of diagnostics will not be covered, which effectively prevents access to adequate care for millions of people in this country. Unfortunately, the CDC has a history of silence concerning past mistakes, and remedies may come years or decades after the fact if they come at all [85].
It is fortunate then that visionary groups like Envita Medical Centers are willing to explore a niche for advanced genetic testing where there is a real health need that is not being met. The leaders at Envita felt it was necessary to step forward to meet the popular demand from both practitioners and patients to fulfill the need for better diagnostic tests. New, better technology should be embraced readily and rapidly for areas where they offer tangible improvements, and existing techniques are either subpar or problematic. Envita's vision is to provide a diagnostic service designed to help patients and doctors, improving health outcomes while lowering medical costs. Better diagnostic technology is key to improving both the patient's and the doctor's experience when dealing with a potential case of Lyme disease as well as associated coinfections. The truth is that Next-Generation Sequencing has already demonstrated both efficiency and reliability for the detection of infectious organisms in many scientific applications [75] [86]- [93]. Shotgun sequencing is a widely used technique for the detection of pathogens [94]. Successful metagenomic profiling critically relies on a good DNA reference library for the organisms being identified. Pa-thoDNA is the first test of its kind with a custom reference library to enable bio- informatics responsible for its remarkably high levels of accuracy and selectivity. Data from over 1000 Lyme disease patients who came to Envita during the past decade was collected to form the base of this incredible, novel reference library that allows PathoDNA to function. The detection of Borrelia and other spirochetes is an application for NGS which ought to receive more widespread attention not simply because it is a superior technology, but because the existing technologies of ELISA and Western blot are inadequate to the current needs of hundreds of thousands of people infected each year in the US alone.
The result of that research is an incredible difference in accuracy between ELISA-Western blot and PathoDNA (Next-Generation DNA Sequencing): 29% -40% for ELISA-Western blot and >95% for PathoDNA. In the case of B. burgdorferi specifically, PathoDNA was shown to be 98.8% accurate! PathoDNA meets and exceeds the CDC recommended accuracy threshold of 95% or greater for a diagnostic test, while the two-tier process mandated by the CDC itself does not. This is an ironic result, but the CDC is not wrong to suggest that a high level of accuracy should be expected for medical diagnostic tests since the wellbeing of hundreds of thousands of human lives often depends on sound and accurate test results. Below, Figure 6 offers a concise comparison of features between ELISA/Western blot and PathoDNA.
PathoDNA is also a broader test than ELISA-Western blot, which only looks for antibodies for a narrow range of organisms, namely specific species of Borrelia. PathoDNA utilizes a reference library of DNA containing information for 11 different species of pathogen that cause Lyme disease or are common coinfections that coincide with Chronic Lyme disease Syndrome. PathoDNA achieves many times greater accuracy and a broader scan for other types of Lyme disease fashion. All these qualities make PathoDNA a vastly more useful test for Lyme disease and its coinfections than the CDC recommended two-tier test.
In conclusion, the state of Lyme disease detection and diagnosis recommended by the CDC has existed for far too long and is now actively harming the population in the United States and abroad by allowing for increasing rates of Lyme to go undetected and untreated. The two-tier process of ELISA and Western blot is not sensitive or accurate enough even by the CDC's criteria and ought to be replaced by a new, high-tech detection platform. With over 95% accuracy, the Next-generation DNA sequencing platform "PathoDNA" utilizing the Ion Torrent S5 instrument may be the solution we need to counter the growing rates of untreated and undetected Lyme disease in this country and elsewhere.