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Dissatisfied With Lyme Disease Dx Methods, Researchers Emphasize Potential of New Technologies

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NEW YORK (360Dx) – Looking to overcome the limited capabilities of the current standard methods of detecting and diagnosing Lyme disease, researchers are hoping that new technologies, such as next-generation sequencing and mass spectrometry, can help provide better tools for clinicians.

Lyme disease is tricky to diagnose regardless of its stage of development in the human body, and traditional two-tiered testing, involving use of an enzyme immunoassay followed by a Western immunoblot, have unacceptably low levels of sensitivity. Hoping to change that outcome, a group of participants from the diagnostics industry and others with interest in Lyme disease testing met two years ago in the Cold Spring Harbor Laboratory (CSHL) Banbury Center in Huntington, New York, to assess current and potentially new laboratory tests for the diagnosis of Lyme disease.

As an outcome of the meeting and subsequent discussions among the meeting participants, the researchers jointly published two papers in the journal Clinical Infectious Diseases. One paper published at the end of last year describes the potential for commercial release of indirect serology tests, and a second published last Thursday describes alternate, direct testing methods that detect active infections including methods that identify protein antigens, use PCR-based diagnostics, or employ next-generation sequencing.        

Around 300,000 instances of Lyme disease are diagnosed in the US each year and more than 3 million diagnostic tests are conducted. All tests currently cleared for diagnostic use by the US Food and Drug Administration are serologic assays that follow the two-tier method.

One of the challenges with using these tests is that they have limited ability to confirm that a patient has an active infection, Steven Schutzer, a lead author on both studies and a physician-scientist at Rutgers New Jersey Medical School, said in an interview.

"Just as we all are aware of shortcomings of the current tests, we are also aware of newer technology being applied to other infectious diseases that could be applied to detecting Lyme disease," Schutzer said.

The researchers set out to assess the advantages and disadvantages of current tests, including assays cleared by the FDA and tests in development that could be applied to detect Lyme disease. "Most of the tests that are FDA cleared are antibody tests that are in use based on criteria established in 1994, and we know that those tests take quite a while before they show a positive result even if you know you have a tick bite," Schutzer said.

About 30 researchers attended the meeting at CSHL, which included those who are knowledgeable about Lyme disease and others who have experience with diagnostic testing outside of Lyme disease. The research papers that they produced are not intended to serve as a consensus statement about Lyme disease testing, and they are not intended to promote any single manufacturer's test, but they show what people interested in Lyme disease testing can do to improve upon current testing, Schutzer said.

Both indirect serology tests and direct tests — based on PCR or protein antigen detection or next-generation sequencing —have "their pros and cons," he said.

"The best solution for clinicians is probably a system that combines direct tests that quickly reveal an active infection and indirect tests for antibodies triggered by the immune system in response to the onset of Lyme disease a few weeks after infection," Schutzer said.

Both papers produced by the researchers as a result of the meeting and subsequent conversations emphasize unmet needs in the market for Lyme disease testing, Paul Auwerter, a clinician director in the division of infectious diseases at Johns Hopkins Medicine and president of the Infectious Disease Society of America, said in an interview.

"It would be very useful to have a test that can detect Lyme disease earlier than current serological, FDA-approved testing," said Auwerter, who is not an author on the studies. "It would also be useful to have a test that correlates with a microbiologic cure of Lyme disease — meaning that if people have ongoing symptoms, the test would assure us that the infection had been handled and the patient wouldn't require additional antibiotics."

The latter need — a test that tells a clinician whether the patient needs additional antibiotics — has implications for reducing overuse of antibiotics, and having a combination of the correct direct and indirect tests would take some of the guessing out of Lyme disease detection and treatment, he said.

The body's immune system plays an important role in the evolution of the disease as well. The immune system could clear an infection by itself, but clinicians can't be sure that this will happen, he said.

"We know from older studies that if you have untreated Lyme disease, half the people will develop Lyme arthritis weeks to months after acquiring the infection, and antibiotics are important to help people clear the infection," said Auwerter.

"Further, we know from careful studies of culture-proven Lyme disease that a quarter of people that present with a classic bulls-eye rash and have been diagnosed with early disease and treated have not healed well after three months," he said. "We believe that additional antibiotics don't help those people, but nonetheless practitioners feel compelled to prescribe antibiotics."

A testing approach for Lyme detection is needed that would enable clinicians "to be assured that their patients' infections had gone, and that they needed the body's immune system to settle. That is something all of us want to see," he said.

Current challenge

The challenge with current serology testing is that it depends on the generation of antibodies made by the body as markers of Lyme disease. "Antibodies can persist for years and even for decades after you have been successfully treated for an infection, just as you retain antibodies after an immunization, for example," Auwerter said.

The phenomenon in Lyme disease can confuse clinicians seeking a diagnosis. A positive serology test for Lyme could indicate the presence of antibodies from previous years, or it could represent a new flare up of the condition. But the clinician, without a means of detecting an active infection, doesn't know for sure that a new outbreak has occurred.

The cause of Lyme disease, Borrelia burgdorferi, was discovered in 1983, and just over a decade later researchers established the two-tiered testing protocol for serodiagnosis involving an enzyme immunoassay, or indirect detection of a fluorescence antibody, followed by immunoglobulin M and immunoglobulin G Western immunoblots if the enzyme immunoassay is reactive.

These assays were prepared from whole-cell cultured B. burgdorferi, lacking key in vivo expressed antigens and expressing antigens that can bind non-Borrelia antibodies, the researchers said. Further, low sensitivity in early infection, technical complexity, and subjective interpretation when scored by visual examination have hampered getting an accurate diagnosis, particular for the Western immunoblot test.

Although that two-tiered approach has remained a benchmark for evaluating new methods or approaches, next-generation serologic assays prepared with recombinant proteins or synthetic peptides and alternative testing protocols can now overcome or circumvent many of these past drawbacks, the researchers said.

They noted in their Clinical Infectious Diseases papers that multiplexed serologic assays using synthetic peptides or chimeric or recombinant proteins, or both, are being developed using simpler and more flexible platforms than immunoblots that use beads and microchips. Further, these multiplex serologic assays provide detailed information about the targets of antibody response and are being designed to include antigens expressed in vivo.

Recent studies suggest that single multiplex or two-tiered strategies involving two different EIAs perform as well or better than two-tiered testing with EIA and Western blots.

Detection assays that directly target a pathogen-causing disease have effectively been applied to other emerging infections and show promise in direct detection of B. burgdorferi infections, Schutzer said.

Culture of the pathogen, microbial nucleic acid, or protein may be detected in skin biopsies of erythema migrans, a circular rash that often appears in the early stages of Lyme disease before the development of a positive serology. However, culture requires specialized media and B. burgdorferi grows slowly, requiring weeks before it comes detectable, Schutzer said.

Among other performance characteristics, a direct test for B. burgdorferi needs to be highly sensitive and specific soon after a tick bite or infection, or before the time of symptom onset; highly sensitive and specific in the later stage of disease when extracuteneous infection has been established; and would need to have a turnaround time of less than 24 hours.

Direct nucleic acid, protein detection, and next-generation sequencing would complement improved indirect serological tests and enable more comprehensive diagnosis of Lyme disease, Schutzer said.

Development of long-read sequencing technology by Pacific Biosciences and use of high-throughput sequencing technologies with very low sequencing error rates, including on the Illumina Novae or MiSeq platforms, has allowed resolution of the repeated sequences that are the hallmark of Borrelia genomes, the researchers said. Additionally, next-gen sequencing could potentially be used as a direct-detection method in clinical diagnostics, the researchers said.

Antigen-capture assays provide another means of direct detection. Bacteria often shed or secrete antigens that can be detected in body fluids, but earlier attempts to create antigen-capture assays may have been hindered by lack of data for optimal selection of targets and antibodies. Advances in proteomics, specimen processing, and mass spectrometry are making identification of pathogen-specific antigens possible and represent a potential path forward, the researchers said.

For infectious agents that are difficult to visualize or cultivate, nucleic acid amplification tests can identify specific pathogen DNA. However, in Lyme disease, the infectious agent resides in very low numbers in tissue outside the trademark Lyme disease ring and body fluids. Meanwhile, standard PCR methods, as applied to B. burgdorferi detection, have suffered from poor sensitivity, especially in blood and cerebrospinal fluid, the researchers said.

Lyme disease has very low microbe numbers in the majority of clinical samples, which also hinders molecular detection. However, with enhancement and enrichment methods, detection of B. burgdorferi in the blood is becoming increasingly possible, the researchers noted. Strategies in which DNA is exponentially amplified by isothermal amplification has been shown to increase the yield of B. burgdorferi by at least twohundred fold.

A recent clearance is reflective of the trend toward antibody tests. In August, the FDA cleared and granted a CLIA waiver for a test from Quidel to detect antibodies to the bacterial pathogen that causes Lyme disease.

But, obtaining the FDA nod for tests that detect active Lyme infections can be challenging, Auwerter noted, not only because of the expense of conducting clinical trials but also because of challenges associated with obtaining samples representative of active infections.

In the meantime, technologies to improve sensitivity are also on the agency's radar. In July, Ceres Nanosciences received FDA Breakthrough Device designation for its Nanotrap system, which consists of hydrogel nanoparticles functionalized with internal affinity baits to enrich target analytes for downstream analysis, thereby improving the sensitivity of the ultimate clinical detection method — for instance, ELISA or mass spectrometry.