Monday, August 17, 2015

A biosignature of early Lyme disease

Laboratory testing for Lyme disease involves two-tier antibody testing with sera from patients suspected of having the disease.  The first step is usually an ELISA with a cell lysate of Borrelia burgdorferi as antigen.  If the ELISA results are positive or borderline, a Western blot is done to confirm that the patient has Lyme disease.  Direct detection of Borrelia burgdorferi by culture would be the preferred laboratory test, but it takes too long for the spirochete to grow.  Culture of patient specimens is done only for research studies.

Source: CDC
In general, the problem with antibody testing for infectious diseases is that it takes time for the immune system to generate antibody against the pathogen.  Therefore, patients in the early stages of infection may test negative.  False-negative tests may delay appropriate treatment until the illness worsens.  For these reasons, scientists have been trying to come up with better laboratory tests for infections whose diagnosis relies on detecting antibody against the infectious agent.

One new approach being developed for a few pathogens involves measuring the amounts of each of the thousands of small molecules found in the sera of infected patients.  This is done by liquid chromatography/mass spectrometry (LC-MS), which accurately and precisely measures the size of small molecules, even in complex substances like serum.  The assumption is that the composition of small molecules (the so-called "metabolome") starts to change in a predictable manner as soon as someone is infected.  The metabolome changes because tissues react to the pathogen by generating inflammatory molecules that leak into the bloodstream.  Another critical assumption is that the changes that occur in the metabolome are unique to each pathogen.  Analysis of the patient's metabolome may therefore allow clinicians to quick diagnose any infectious disease whose metabolome has been characterized.

A recent CDC study revealed the metabolome of early Lyme disease.  The investigators obtained sera from 89 patients who had early-stage Lyme disease.  All had erythema migrans (EM), the rash characteristic of Lyme disease.  Most were also culture or PCR positive for Borrelia burgdorferi.  The patient sera were compared with sera from 50 healthy individuals by LC-MS.

After two runs of LC-MS with each sample, the researchers identified a set of 95 small molecules whose levels consistently differed between patients with early Lyme disease and healthy individuals.  Statistical modeling of the data allowed the investigators to refine the biosignature to a set of 44 molecules that identified Lyme disease in the 139 (89 + 50) subjects with the highest sensitivity and specificity.

To better gauge the performance of the biosignature in identifying those with early Lyme disease, the investigators conducted LC-MS on sera from another group of 91 patients shown to have early Lyme disease by the same criteria as the first set of patients.  Control sera came from 108 healthy individuals.  Another set of control sera was obtained from 101 patients with other diseases that could be confused with Lyme disease clinically, serologically, or microbiologically: syphilis, severe periodontitis, infectious mononucleosis, and fibromyalgia.  All patient and control sera were also tested by the standard two-tier antibody test.

The sensitivity of LC-MS testing turned out to be much higher than that of two-tier testing: 88% vs. 44%.  The specificity of LC-MS was 94% with healthy sera and 95% when sera from patients with other diseases were tested.  These values were not significantly different from the specificities of 100% and 95% achieved with two-tier testing.

These results show the promise of using the metabolic biosignature to help diagnose early Lyme disease.  However, note that all patient sera used to uncover the biosignature and assess its performance came from individuals with EM.  In practice, a clinical diagnosis involving the classic bulls-eye EM with a patient history suggestive of Lyme disease does not require confirmation by laboratory testing.  Patients without EM are more likely to need laboratory testing.  According to the CDC, 20-40% of Lyme disease patients do not have EM.

To get an idea of how well the biosignature performs on patients without EM, the investigators obtained sera drawn from 22 cases with early Lyme disease who tested positive with the C6 ELISA, a newer antibody test.  The antigen for the C6 ELISA is a highly-conserved peptide from the B. burgdorferi surface protein VlsE.  Eight of the 22 patients did not have EM.  The EM status was unknown in another eight patients.  The remaining six patients had EM.  Unfortunately, the results for each subgroup were not presented by the authors, so we don't have a firm answer about the performance of LC-MS testing on patients without EM.  What we can say is that even though more than a third of the 22 patients did not have EM, the sensitivity of LC-MS testing remained high at 86%.  In contrast, the sensitivity of two-tier testing with this group was only 41%, even though the investigators stacked the deck by using the C6 ELISA as the first tier with this group.  Future testing of the biosignature should include a larger number of sera from EM-negative patients in the early stages of Lyme disease.

As discussed in the paper, sera from patients with skin conditions that could be confused with EM (e.g., STARI, cellulitis) should be examined in future studies to make sure that the early Lyme biosignature can be used to rule out those conditions.  The authors recommend that sera from patients with neurologic, cardiac, and arthritic forms of Lyme disease also be examined to see if biosignatures specific for these more serious forms of Lyme disease could be identified.


Molins CR, Ashton LV, Wormser GP, Hess AM, Delorey MJ, Mahapatra S, Schriefer ME, & Belisle JT (2015). Development of a metabolic biosignature for detection of early Lyme disease. Clinical Infectious Diseases, 60 (12), 1767-1775 PMID: 25761869