Seroreactivity to the C6 Peptide in Borrelia miyamotoi Infections Occurring in the Northeastern United States Free

Abstract

Background

There are no US Food and Drug Administration (FDA)–approved diagnostic tests for Borrelia miyamotoi infection, an emerging tick-borne illness in the United States. The purpose of this study was to evaluate whether the FDA-approved C6 peptide enzyme-linked immunosorbent assay (ELISA) currently used to diagnose Lyme disease may potentially serve as a diagnostic test for B. miyamotoi infections.

Methods

Serum specimens from 30 patients from the northeastern United States with B. miyamotoi infection established by a polymerase chain reaction assay of a blood specimen were tested using the C6 ELISA. To reduce confounding with Borrelia burgdorferi coinfection, 6 sera were excluded: 3 from patients with a positive Western immunoblot for antibodies to B. burgdorferi and 3 from patients for whom immunoblot testing had not been performed.

Results

Twenty-two of 24 (91.7% [95% confidence interval, 73.0%–98.8%]) evaluable B. miyamotoi patients were C6 ELISA reactive, principally on a convalescent-phase serum specimen. C6 ELISA index values were often well above the positive cutoff value of 1.1, exceeding 4 in 11 of the 22 (50.0%) C6 ELISA-reactive patients.

Conclusions

Although previously regarded as a highly specific test for Lyme disease, the C6 ELISA is also regularly reactive on convalescent-phase serum samples of patients from the northeastern United States with B. miyamotoi infection.

Borrelia miyamotoi is the cause of an emerging tick-borne infection in the United States that is transmitted by the same Ixodes tick species that transmit Borrelia burgdorferi [1–6]. Borrelia miyamotoi, however, is a relapsing fever Borrelia species, not a Borrelia species that causes Lyme disease [7, 8]. Clinical features of B. miyamotoi infection in the United States, such as fever, headaches, leukopenia, and/or thrombocytopenia (cytopenia has been observed in >50% of cases), and elevated hepatic enzymes, are nonspecific precluding a purely clinical diagnosis [1, 2, 5]. Diagnosis can be established by detection of B. miyamotoi DNA in blood by polymerase chain reaction (PCR) or by detection of antibodies to glycerophosphodiester phosphodiesterase (GlpQ), a protein that is not present in B. burgdorferi [1, 2, 5, 8, 9]. However, there is no US Food and Drug Administration (FDA)–approved test kit for detection of B. miyamotoi infections and only a limited number of commercial laboratories offer testing for this microorganism.

The C6 peptide enzyme-linked immunosorbent assay (ELISA) is an FDA-approved first-tier serologic test for detection of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies to B. burgdorferi targeting a highly conserved 25 amino acid peptide derived from the sixth invariant region of the VlsE protein [10–12]. The commercially available C6 ELISA incorporates the C6 peptide found in the B31 strain of B. burgdorferi sensu stricto. A recent case report of an untreated patient from New York State with PCR-confirmed B. miyamotoi infection and no evidence of Lyme disease suggested that B. miyamotoi infection will potentially result in production of antibodies to the C6 peptide [13]. The purpose of this study was to determine if this finding could be confirmed in other US patients with documented B. miyamotoi infection.

METHODS

A convenience sample of serum specimens collected from 30 patients confirmed to have B. miyamotoi infection by whole-blood PCR at IMUGEN were tested for antibodies to the C6 peptide using the C6 Lyme ELISA kit (Immunetics, Boston, Massachusetts), in accordance with the manufacturer’s recommendations. PCR testing for B. miyamotoi and for B. burgdorferi was performed as previously described [1]. Sera were also tested for antibody to B. burgdorferi by an antibody capture enzyme immunoassay (EIA) for IgM and IgG isotypes and by IgM and IgG Western immunoblots, as previously described [1]. Immunoblots were interpreted according to Centers for Disease Control and Prevention (CDC) criteria [14], except that IgM Western immunoblots were assessed irrespective of the duration of the patient’s illness. Separate indirect EIAs to detect IgM and IgG antibodies to GlpQ were performed using recombinant GlpQ, as previously described [1]. Some of the sera studied were from a previously published case series of patients with B. miyamotoi infections [1]. The study was approved by the New England Institutional Review Board.

RESULTS

Serum samples were available from 30 patients with PCR-confirmed B. miyamotoi infection who were diagnosed between 2013 and 2016. Twenty-seven (90.0% [95% confidence interval {CI}, 74%–97%]) were C6 ELISA seroreactive on either an acute-phase sample (arbitrarily defined as a serum sample collected up to 6 days after the blood sample that confirmed the diagnosis of B. miyamotoi infection) or on a convalescent-phase sample (defined as a sample collected at ≥7 days after the diagnosis was established). Clinical histories were not available on these 30 subjects to determine if any had an erythema migrans skin lesion or another objective clinical manifestation of active Lyme disease. However, none of the 30 was PCR positive for B. burgdorferi DNA in blood. In addition, separate IgM and IgG whole cell sonicate–based EIAs for B. burgdorferi antibodies were performed for all study subjects and, in most cases, both IgM and IgG Western immunoblots were performed if either the C6 ELISA or a whole cell sonicate–based EIA was positive. Three of the 30 (10.0%) patients were found to have a positive IgM Western immunoblot for B. burgdorferi antibodies and were, therefore, excluded from further analyses; none was IgG Western immunoblot positive. One of these 3 patients was IgM Western immunoblot positive on the date of PCR testing for B. miyamotoi infection and 24 and 31 days later; one was negative on the date of PCR testing but positive 14 days later; and the third was positive at 20 days after the date of PCR testing on the only serum sample available for testing. In addition, 3 additional cases were excluded as IgM and IgG Western immunoblot test results were not both available for these patients.

Thus, 24 patients were regarded as unlikely to have concomitant Lyme disease and were considered evaluable to assess the performance of the C6 ELISA as a diagnostic test for identifying patients with B. miyamotoi infection (Table 1). Twenty-two (91.7% [95% CI, 73.0%–98.8%]) of the 24 cases were C6 ELISA seroreactive on either an acute or convalescent-phase serum specimen. C6 ELISA index values were often well above the positive cutoff value of 1.1, exceeding 4 in 11 (50.0%) of the 22 C6 ELISA-reactive patients. Eighteen acute-phase serum samples were available, and 3 (16.7% [95% CI, 5.0%–40.0%]) were C6 ELISA seroreactive. Seventeen of these serum specimens were obtained on the date of the PCR testing for B. miyamotoi, and the single additional specimen was collected 2 days following PCR testing. Of the 15 C6 ELISA negative acute-phase samples, 13 (86.7% [95% CI, 60%–98%]) became seroreactive on a convalescent-phase serum specimen collected between 7 and 130 days (median, 24 days) after the diagnosis of B. miyamotoi infection was established (Table 1). For 6 patients, only a convalescent-phase serum specimen was available. All 6 (100%) were C6 ELISA seroreactive. These samples were collected from 11 to 47 days after the date of the blood sample that confirmed the diagnosis of B. miyamotoi infection (Table 1).

Although all 24 patients were seroreactive for IgM antibody to B. burgdorferi by a whole cell sonicate EIA on either an acute or a convalescent-phase serum sample, none of the serum samples were seroreactive by EIA for IgG antibody to B. burgdorferi. Of the 15 C6 ELISA negative acute-phase serum specimens, 14 (93.3%) of these serum specimens were also negative for IgM antibodies to B. burgdorferi by EIA.

Twenty-two of the 24 (91.7%) patients were seropositive for IgM, IgG, or both IgM and IgG antibodies by EIA to GlpQ. However, of the 15 C6 ELISA-negative acute-phase specimens, all but 2 were also seronegative for GlpQ antibody. In total, all but 2 of the C6 ELISA-seroreactive sera were also GlpQ antibody positive.

DISCUSSION

The C6 ELISA has been considered highly specific for Lyme disease. In one study that tested >2200 control sera the specificity was 98.9% [10], and in another study that tested 1300 control sera the specificity was similar at 98.4% [11]. In most prior studies, no particular illness or infection besides Lyme disease was found to be associated with C6 ELISA positivity [10–12]. The current study challenges this understanding. In this evaluation of 24 patients with PCR-confirmed B. miyamotoi infections, 91.7% were found to be C6 ELISA seroreactive, all of whom were IgM and IgG Western immunoblot negative for antibodies to B. burgdorferi. A likely explanation for this is that the C6 peptide sequence of the B31 strain of B. burgdorferi, which is the C6 peptide sequence used in the C6 ELISA test kit, is significantly similar to the relapsing fever Borrelia variable large protein (Vlp) sequence, including Vlp 15/16 of B. miyamotoi, an observation first made by a Basic Local Alignment Search tool protein (BLASTp) search [13] and subsequently by direct sequencing data of a strain of B. miyamotoi from a tick found in Connecticut [15]. A recognized study limitation, however, is that despite using multiple laboratory approaches to detect B. burgdorferi coinfection, and despite excluding 6 patients from the analysis, it is, nevertheless, possible that coinfection may have been present in some of the remaining patients.

Of interest, the >90% rate of C6 ELISA seroreactivity in B. miyamotoi infections is higher than that found for patients with early localized Lyme disease, even using convalescent-phase serum samples, but approximately equivalent to that of patients with early disseminated Lyme disease [10]. It would be predicted based on a BLAST search that other types of relapsing fever Borrelia infections would also generate C6 ELISA reactivity and available, but limited data suggest that this is indeed true [13].

There has been increasing interest in finding an alternative 2-tier serologic testing algorithm for Lyme disease in which the second-tier IgM and IgG Western immunoblot tests are replaced by less technically complex, more objective, and less expensive alternative test methods [11, 16, 17]. One of the leading candidates to replace the second-tier Western immunoblots is the C6 ELISA. Multiple studies have confirmed that a whole sonicate–based EIA with reflex to the C6 ELISA as the second-tier test is more sensitive than conventional 2-tier testing for diagnosing early Lyme disease, with identical specificity. The specificity of this 2-tier algorithm was 99.5% in 2 separate studies [11, 17]. Our data, however, suggest that B. miyamotoi infections will regularly result in false positives using this 2-tier testing strategy, whereas this appears not to occur with conventional 2-tier testing with reflex to Western immunoblot testing [13]. However, more research is needed to determine whether a positive IgM Western immunoblot test result for Lyme disease (ie, antibody to B. burgdorferi) may on occasion occur solely as a result of infection with B. miyamotoi.

More data are also needed on the timing of C6 ELISA seroconversion in relapsing fever Borrelia infections to determine the most appropriate time point to obtain a convalescent-phase serum sample. It is clear that acute-phase testing is often negative for C6 ELISA seroreactivity, as it is for GlpQ seroreactivity. In addition, B. miyamotoi strains from different geographic locations may differ genetically [3, 8, 18], and thus whether the frequency of C6 ELISA reactivity would be as high in patients with this infection from other geographic areas remains to be determined. However, the patients studied in the current report should be representative of many other cases of B. miyamotoi infection occurring in the northeastern United States. Thus, the development of a positive C6 ELISA in a patient from this geographic area with tick exposure and an acute nonspecific febrile illness with leukopenia and/or thrombocytopenia, who is seronegative for Lyme disease by Western immunoblot testing, should be considered compatible with B. miyamotoi infection. Although nonspecific summertime febrile illnesses, occurring in the absence of the erythema migrans skin lesion, have also been attributed to Lyme disease, this is extremely rare in our experience and the presence of either leukopenia or thrombocytopenia would not be expected [19].

In conclusion, B. miyamotoi and other relapsing fever Borrelia infections may result in C6 ELISA seroreactivity, a fact that has not been appreciated to date. In the absence of the availability of other, more specific, FDA-approved diagnostic assays, C6 ELISA testing may be useful to diagnose B. miyamotoi and other relapsing fever borrelial infections, in addition to its role in the diagnosis of Lyme disease.

Notes

Acknowledgments. The authors thank Julia Singer, Artemio Zavala, Shana Warner, and Lisa Giarratano for their assistance.

Financial support. This study was internally funded by IMUGEN.

Potential conflicts of interest. P. J. M. is the paid medical director of IMUGEN. G. P. W. has received research grants from Immunetics, Institute for Systems Biology, Rarecyte, and Quidel Corporation; owns equity in Abbott; has been an expert witness in malpractice cases involving Lyme disease; and is an unpaid board member of the American Lyme Disease Foundation. K. E. W. and B. T. are employees of IMUGEN. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

References