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Journal of Clinical Microbiology, December 1999, p. 3990-3996, Vol. 37, No. 12
Department of Parasitology, Tulane Regional
Primate Research Center, Tulane University Medical Center, Covington,
Louisiana 70433;1 Division of
Rheumatology, New England Medical Center, Tufts University School of
Medicine, Boston, Massachusetts 021112;
National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, Maryland 208923;
Division of Vector-Borne Infectious Diseases, Centers for
Disease Control and Prevention, Fort Collins, Colorado
805224; and Department of Microbiology
and Immunology, University of California at Los Angeles, Los
Angeles, California 900955
Received 30 June 1999/Returned for modification 12 August
1999/Accepted 19 August 1999
VlsE, the variable surface antigen of Borrelia
burgdorferi, contains an immunodominant conserved region named
IR6. In the present study, the diagnostic performance of a
peptide enzyme-linked immunosorbent assay (ELISA) based on a 26-mer
synthetic peptide (C6) with the IR6 sequence
was explored. Sensitivity was assessed with serum samples
(n = 210) collected from patients with clinically defined Lyme disease at the acute (early localized or early
disseminated disease), convalescent, or late disease phase. The
sensitivities for acute-, convalescent-, and late-phase specimens were
74% (29 of 39), 85 to 90% (34 of 40 to 35 of 39), and 100% (59 of
59), respectively. Serum specimens from early neuroborreliosis patients were 95% positive (19 of 20), and those from an additional group of
patients with posttreatment Lyme disease syndrome yielded a sensitivity
of 62% (8 of 13). To assess the specificity of the peptide ELISA, 77 serum samples from patients with other spirochetal or chronic
infections, autoimmune diseases, or neurologic diseases and 99 serum
specimens from hospitalized patients in an area where Lyme disease is
not endemic were examined. Only two potential false positives from the
hospitalized patients were found, and the overall specificity was 99%
(174 of 176). Precision, which was assessed with a panel of positive
and negative serum specimens arranged in blinded duplicates, was 100%.
Four serum samples with very high anti-OspA antibody titers obtained
from four monkeys given the OspA vaccine did not react with the
C6 peptide. This simple, sensitive, specific, and precise
ELISA may contribute to alleviate some of the remaining problems in
Lyme disease serodiagnosis. Because of its synthetic peptide base, it
will be inexpensive to manufacture. It also will be applicable to serum
specimens from OspA-vaccinated subjects.
In the United States, Lyme
borreliosis continues to be the most frequently reported
arthropod-borne infectious disease. Approaches taken towards Lyme
disease prevention and control include the recent development of
prophylactic vaccines (29, 34), one of which is already
commercially available (34), and the exertion of concerted
efforts to improve and standardize methods of serologic diagnosis
(7). Precise diagnosis of infection at an early phase is of
great importance in Lyme disease management, as the timely administration of appropriate antibiotics is usually curative (31). Because of the ambiguities that still bedevil
serodiagnosis of Lyme disease (1, 22, 28, 32, 35, 39),
patients with nonspecific clinical signs or symptoms of early infection (e.g., absence of erythema migrans, the skin rash that heralds infection) may remain undetected. Long courses of antibiotic therapy may be required if chronic infection ensues, sometimes with a prolonged
convalescence or an uncertain outcome (31, 33).
Over- and underdiagnosis (22, 28, 32, 35, 39), as well as
interlaboratory discrepancies (1), are current problems of
Lyme disease serodiagnosis. To improve specificity, a multiple-band set
of criteria was developed by Dressler et al. (10) and
Engstrom et al. (11) for a positive immunoblot test, and a
two-tiered approach composed of an initial enzyme-linked immunosorbent
assay (ELISA) of relatively high sensitivity but low specificity
followed by an immunoblot incorporating the Dressler (immunoglobulin G [IgG]) or Engstrom (IgM) band criteria was recommended by the Centers
for Disease Control and Prevention (CDC) (7). This approach
likely entails several advantages, such as enhanced specificity and the
opportunity to estimate duration of infection. However, the need to
include the Western blot (WB) technique will increase the cost of Lyme
disease diagnosis and possibly further enhance inter- and
intralaboratory discrepancies, as the test is itself more difficult to
perform than a standard ELISA and its outcome may depend on subjective
interpretation of the banding pattern.
In the wake of the recent availability of the OspA (outer surface
protein A) vaccine, a new difficulty has been added to the field of
Lyme disease serodiagnosis, namely, the possible presence of anti-OspA
antibodies in patient serum. Whole-cell antigen-based tests will not be
useful in this context, as the antigen extracts currently used include OspA.
A procedure that was intrinsically unable to detect anti-OspA
antibodies and which retained, or improved upon, the simplicity and
sensitivity of the current ELISA and the specificity of a WB would, in
principle, circumvent the shortcomings of the two-tiered approach while
preserving some of its advantages. We recently identified, within the
variable (cassette) domain of VlsE, the variable surface antigen of
Borrelia burgdorferi (40), an invariable 26-amino-acid region, named IR6, which we determined to be
antigenically conserved among strains and species of the B. burgdorferi sensu lato complex and immunodominant in both human
and nonhuman primate hosts (17a). Based on these initial
results, we further investigated the sensitivity, specificity, and
precision of an ELISA based on a synthetic peptide (C6)
whose sequence is essentially that of IR6. Serial serum
samples from nonhuman primates that were tick inoculated with different
strains of B. burgdorferi were assessed to ascertain in
which phase of Lyme disease antibody to C6 first appeared
and the duration of its persistence in the serum. Sensitivity of the
C6 ELISA was assessed by using several serum panels with
specimens from patients with acute (early localized or early
disseminated phase) or late manifestations of Lyme disease or from
patients who were either convalescent or had posttreatment Lyme disease
syndrome. Specificity of the C6 ELISA was examined with a
panel of serum samples from patients living in a region where Lyme
disease is not endemic and with serum samples from an array of patients
with autoimmune or neurologic diseases, spirochetal diseases other than
Lyme borreliosis, or other chronic infections. Precision was assessed
with a subgroup of Lyme disease and non-Lyme disease serum specimens
arranged in blinded duplicates. Finally, absence of reactivity of the
C6 peptide with anti-OspA antibodies was assessed with
high-titer anti-OspA serum samples from rhesus monkeys. These animals
had been vaccinated with an OspA vaccine formulation and according to a
vaccine administration protocol which were similar to those employed in
human trials (36). Our results, related here, indicate that
the C6-peptide-based ELISA performs extremely well in terms
of simplicity, sensitivity, specificity, and precision and that it will
be utilizable with serum samples that contain anti-OspA antibodies.
Serum sources. (i) Monkey serum samples.
Serum specimens
from animals that were employed in previous studies were used (25,
26). For the serial study, samples were obtained from rhesus
monkeys (2- to 4-year-old Macaca mulatta animals) that had
been infected by the bite of Ixodes scapularis nymphal ticks
(nine animals) or by needle inoculation (one animal) as previously
described (25). Ticks feeding on the animals were themselves
infected with spirochetes of either the JD1 (25) or B31
(26) strain of B. burgdorferi sensu stricto. The
needle-inoculated animal received JD1 spirochetes. Blood specimens were
collected every 1 or 2 weeks postinoculation, and serum samples were
stored at (ii) Human serum samples.
Human serum samples were obtained
from several sources. To assess diagnostic sensitivity, three serum
panels from Lyme disease patients were used. One was from the CDC
(kindly provided by Martin Schriefer), a second panel was from patients
of the Tufts-New England Medical Center, and a third one was from the
National Institutes of Health (NIH). The CDC serum panel was composed
of 40 samples from patients who were in the convalescent phase of Lyme
disease. Twenty-seven of these samples were also culture confirmed. All
of the serum specimens in this panel were from patients whose case
satisfied the CDC Lyme disease case definition (6). In
addition, all of the samples had been tested at the CDC with
commercially available kits: ELISA was performed with Lyme Screen II
(bioMerieux, St. Louis, Mo.), and IgG and IgM WB was performed with
Marblot (MarDx, Carlsbad, Calif.). The panel from Tufts-New England
Medical Center was composed of 157 specimens, of which 39 were from
patients in the acute phase of Lyme disease. These patients were in the
early (localized or disseminated) phase of Lyme disease. Also in this
panel were 39 specimens from individuals in the convalescent phase
(obtained from the same patients whose sera had been obtained in the
acute phase), 20 from patients in the early disseminated phase who had
presented with signs and/or symptoms of early neuroborreliosis, and 59 from patients with late Lyme disease (49 with Lyme arthritis and 10 with late neuroborreliosis). All of the patients in the Tufts panel met
clinical criteria for Lyme disease diagnosis, as described previously
(10). The NIH panel was composed of 13 specimens obtained
from patients with posttreatment Lyme disease syndrome, defined as
persistent or intermittent symptoms for at least 6 months after
appropriate antibiotic therapy for Lyme disease. Usual symptoms include
widespread musculoskeletal pain and fatigue, memory and/or
concentration impairment, radicular pain, paresthesia, or dysesthesia.
The beginning of the symptoms coincides with, or occurs within 6 months
of, the initial B. burgdorferi infection. Symptoms are
significant enough to interfere with daily life activities, and other
causes have been excluded. All of these samples met CDC criteria for seropositivity (7).
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Sensitive and Specific Serodiagnosis of Lyme
Disease by Enzyme-Linked Immunosorbent Assay with a Peptide Based
on an Immunodominant Conserved Region of Borrelia
burgdorferi VlsE
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
20°C until being tested. To assess cross-reactivity of
the C6 peptide with anti-OspA antibodies, serum samples
from animals that had been administered an OspA vaccine were utilized
(26). Specimens were obtained from four rhesus macaques
before and after they were vaccinated with recombinant lipidated OspA
adsorbed onto aluminum hydroxide. The vaccine formulation and
administration protocol, which also had been used in human trials
(36), have been described previously (26).
Definitions. Sensitivity was defined as true positives/(true positives plus false negatives), specificity was defined as true negatives/(true negatives plus false positives), and precision was defined as the frequency of obtaining the same result on duplicate analysis of a set of positive and negative specimens. Early localized, early disseminated, and late Lyme disease were defined as described in reference 31.
Peptide synthesis and conjugation to biotin. A 26-amino-acid peptide (C6) was prepared by using the fluorenylmethoxycarbonyl synthesis protocol (4). A cysteine residue was included at the N terminus and used as a biotinylation site. Biotinylation was performed by the N-succinimidyl maleimide carboxylate method. The maleimide reagent was from Molecular Probes (Eugene, Oreg.), and the protocol suggested by the manufacturer was followed. The sequence of the peptide (C6) used in this study is CMKKDDQIAAAMVLRGMAKDGQFALK. Its immunologic properties have been described elsewhere (17a).
Peptide ELISA. Ninety-six-well ELISA plates (Corning Inc., Corning, N.Y.) were coated with 100 µl of streptavidin (4 µg/ml), (Pierce Chemical Company, Rockford, Ill.) per well in coating buffer (0.1 M carbonate buffer, pH 9.2) and incubated at 4°C overnight. After two 3-min washes with 200 µl of PBST (10 mM sodium phosphate, 150 mM NaCl, and 0.1% Tween 20, pH 7.4) per well at 200 rpm in a rotatory shaker (orbital shaker; Lab-Line Instruments Inc., Melrose Park, Ill.), 200 µl of biotinylated peptide (5 µg/ml) dissolved in blocking buffer (PBST supplemented with 5% nonfat dry milk [Carnation; Nestlé Food Company, Glendale, Calif.]) was applied to each well. The plate was shaken at 150 rpm for 2 h at room temperature (RT). After three washes with PBST as described above, 50 µl of serum (monkey or human) diluted 1:200 with blocking buffer was added to each well. The plate was incubated with shaking at 150 rpm for 1 h at RT and then washed three times with PBST as before. Each well then received 100 µl of goat anti-monkey IgG (0.2 µg/ml) or goat anti-monkey IgM (0.5 µg/ml) (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) goat anti-human IgG (0.1 µg/ml) (Pierce), or goat anti-human IgM (0.5 µg/ml) (Sigma), each conjugated to horseradish peroxidase and dissolved in blocking buffer. Incubation was at RT for an additional 1 h with shaking at 150 rpm. After four washes with PBST for 3, 4, 5, and 6 min, respectively, 100 µl of a solution composed of the chromogen 3,3',5,5'-tetramethylbenzidine at 0.2 mg/ml and 0.01% hydrogen peroxide in the buffer supplied by the manufacturer (Kirkegaard & Perry) was added, and color was allowed to develop for 10 min. The enzyme reaction was stopped by addition of 100 µl of 1 M H3PO4. The optical density (OD) was measured at 450 nm with an ELISA plate spectrophotometer model SLT Spectra (SLT Lab Instruments, Salzburg, Austria). The cutoff OD value was defined as the mean OD plus 3 standard deviations (SDs) for 97 serum samples collected from patients of a hospital in Louisiana (where Lyme disease is not endemic). All of the samples were assessed blindly, in duplicate, at least twice. Mean OD values from duplicate determinations are reported. OD values of individual samples never varied more than 5%.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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IgG and IgM antibody responses to C6 in infected rhesus monkeys. Serum samples serially collected from 10 monkeys that had been inoculated with either the B31 or JD1 strain of B. burgdorferi were tested by ELISA for antibody responses to C6. IgG antibody was detectable in seven animals as early as 3 weeks postinoculation. At week 5 postinoculation, nine animals had responded, and all had responded at week 6 (Fig. 1A). Antibody to C6 persisted at high levels in all animals during the entire study period, which was between 25 and 160 weeks postinoculation. The results for 3 of the 10 animals studied are shown in Fig. 1B. IgM anti-C6 antibody responses were detectable in only some of the animals and did not appear earlier than the corresponding IgG responses (not shown).
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Sensitivity of the C6 ELISA.
Forty serum samples
obtained by the CDC from patients in the convalescent phase of Lyme
borreliosis were assessed with the C6 ELISA. Thirty-four
were positive (Table 1; Fig.
2A), thus yielding a sensitivity of
detection of 85%. An assessment of the same samples performed at the
CDC with commercially available assay kits yielded sensitivities of
80% (32 of 40) with a conventional ELISA and 75% (30 of 40) with the
combination of both IgG and IgM immunoblots.
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Specificity of the C6 ELISA.
The C6
ELISA yielded a specificity of 100% (77 of 77) when serum samples from
patients with other chronic infections or autoimmune diseases were
tested (Table 3; Fig. 2B). This panel
included 9 specimens from relapsing fever patients, 22 specimens from
syphilis patients (10 from NIH and 12 from UCLA), and 46 specimens from patients with either multiple sclerosis (n = 10),
positive anticardiolipin antibody (n = 10), positive
rheumatoid factor (n = 10), positive antinuclear
antibody (n = 10), Guillain-Barré syndrome
(n = 1), or mycobacterial infection (n = 5). Only two potential false-positive results were obtained when a
group of 99 human serum samples randomly collected at a local hospital
in Louisiana were tested (Table 3; Fig. 2B). Lyme disease is not
endemic in Louisiana, but the identities of the patients and their
clinical histories are unknown.
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Precision of the C6 ELISA. To assess precision, a panel of serum specimens composed of samples from patients with posttreatment Lyme disease syndrome (n = 7), early disseminated Lyme disease (n = 1), mycobacterial infection (n = 5), positive antinuclear antibody (n = 5), positive rheumatoid factor (n = 5), or positive rapid plasma reagin (n = 5) was set up in blinded duplicates and assessed with the C6 ELISA. Precision was 100%. Results for any pair of duplicates derived from true-positive specimens were both positive, and, in addition, their OD values differed by less than 10%. Similarly, duplicates derived from true-negative samples were both negative in that both OD values were below the cutoff line.
Nonreactivity of the C6 peptide with anti-OspA antibodies. To ascertain whether high-titer anti-OspA antibodies could cross-react with the C6 peptide, serum samples from four rhesus macaques that had been vaccinated with an OspA vaccine formulation also used in humans (26, 36) were tested with the C6 ELISA. The anti-OspA antibody geometric mean titer in these four serum samples was 1:0.9 × 106, as measured by an OspA ELISA (24). No antibody to C6 was detected in any of the four specimens.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Current problems of Lyme disease serodiagnosis include over- and underdiagnosis (22, 28, 32, 35, 39) as well as interlaboratory discrepancies (1). A late or feeble antibody response to B. burgdorferi antigens may lead to serologic underdiagnosis, whereas antigenic determinants shared by proteins of B. burgdorferi and other bacteria are likely a contributing factor to overdiagnosis. The 10 or more antigen bands that can be detected on spirochete whole-cell lysate immunoblots when such blots are reacted with Lyme disease patient serum (10, 19) include homologues of the omnipresent bacterial heat shock proteins (74, 66, and 58 kDa) (16, 18); flagellin (41 kDa), which is shared with other spirochetes such as Borrelia hermsii (3), one of the relapsing fever agents, and T. pallidum (19), the cause of syphilis; and a 60-kDa antigen which is expressed by a wide range of bacteria (16). Antiflagellin serum antibody can be detected in 87% of Lyme disease patients, 75% of syphilis patients, and 43% of normal humans (19). The frequencies of detection of antibody to the 60-kDa antigen are 58, 42.9, and 16.6% in borreliosis, syphilis, and normal human serum, respectively (19). Therefore, ELISAs based on detection of antibody to B. burgdorferi whole-cell extracts are not expected to be highly specific and may thus lead to overdiagnosis. In fact, in a recent interlaboratory comparison of results of tests for detection of Lyme disease, in which the methodology used was based on enzyme immunoassay, 516 laboratories each received 28 serum samples from Lyme disease patients and 22 samples from individuals without previous or current Lyme disease. False-positive test results approached 55% with some serum samples from healthy donors (1). A serum sample with anti-T. pallidum antibodies was reported as positive by 70% of the participants (1). Also, in a survey of normal human serum samples, most samples had antibodies to at least one B. burdorferi immunoblot band (12), 64% of serum samples from tick-borne relapsing fever patients were falsely diagnosed as positive by a whole-antigen ELISA (20), and human serum samples collected in a tropical country where Lyme disease is not endemic yielded 98% false-positive results by ELISA and 57% by immunoblotting (5).
The two-tiered ELISA-WB approach that was recently recommended by the
CDC (7), although more costly, is almost certain to improve
diagnostic specificity (e.g., none of the bands recommended by Dressler
et al. [10] are heat shock proteins). However,
interlaboratory, and even intralaboratory, variation may persist
because of two types of possible shortcomings of the whole-cell antigen
immunoblot procedure: first, the procedure is technically
involved
more so than the ELISAand its results are largely
qualitative and dependent on subjective interpretation; second, the
composition of the whole-cell antigen extract is ill defined. This is
so in part because some B. burgdorferi antigens are
polymorphic and thus their apparent molecular masses vary from strain
to strain (2, 37, 38). Conversely, different antigens may
have the same apparent molecular mass, e.g., OspC (outer surface
protein C) and decorin binding protein (22 kDa) (15) BmpA
(Borrelia membrane protein A) (39 kDa) (30), or
flagellin (41 kDa) (3) and VlsE (39 to 45 kDa) (40). In addition, expression of several B. burgdorferi antigens is dependent on the spirochetal growth phase
and on the length of time spent in continuous culture by a given
spirochetal isolate. Thus, spirochetes harvested in mid-log phase
during growth in vitro do not express an array of antigens that are
detectable only in late log or stationary growth phase (17,
27), and the expression of several antigens is lost after
repeated serial passage of cultured spirochetes (21, 23).
Hence, the whole-cell antigen extract composition will depend on how
long a particular isolate has been maintained in continuous culture and
on whether cells have been harvested at an early or late growth phase.
A procedure with the simplicity of an ELISA and based on synthetic antigens would in principle circumvent the technical complexity and potential antigen variability associated with immunoblot diagnosis. Synthetic peptides used as antigens have the added advantage of avoiding contamination with low-level impurities derived from Escherichia coli or other cloning vectors required to produce recombinant proteins. Such impurities could decrease the specificity of the assay if they were cross-reactive with B. burgdorferi antigens. Indeed, the specificity of diagnostic ELISAs for Lyme disease can be significantly improved by adsorption of the serum samples with soluble E. coli protein (13, 14). The C6 ELISA utilizes a chemically synthesized 26-mer peptide as the diagnostic antigen, thus permitting the uniformity of antigen preparations at relatively low costs and minimizing spurious cross-reactivity.
The sensitivity of the C6 ELISA ranged from 62 to 100%, depending on when in the course of disease the serum samples were collected and on the clinical definition of the patients' signs and symptoms (Table 1). Based on the results of our longitudinal analysis of the anti-C6 antibody response in infected rhesus monkeys (Fig. 1A), it is unlikely that such a response may be detectable with good sensitivity earlier than 1 to 2 weeks postinfection. However, by week 5 postinfection, 90% of the animals (9 of 10) had responded. This result is consistent with that obtained with the acute-phase serum panel (panel 2 [Tufts]), which showed that 80% or more of the patients whose serum had been collected 3 or more weeks after disease onset (5 or more weeks after infection?) were positive by the C6 ELISA (Fig. 3). With the CDC serum panel of 40 specimens from convalescent patients, the sensitivity of the C6 ELISA (85%) was slightly higher than that of the conventional ELISA (80%) and higher than the combination of IgG and IgM WB (75%). A lower sensitivity (74%) was obtained with serum specimens collected during the acute phase of Lyme disease (74%) (39 samples from Tufts-New England Medical Center). However, only three of these patients remained negative in the convalescent phase (Table 1). The patient with the fourth negative sample among the convalescent-phase samples had been positive during the acute phase. We explored the possibility of making an earlier diagnosis by testing IgM responses to C6 both in monkeys and in humans. In either case, no IgM antibody response was seen in the absence of an IgG response, no matter how early after infection or disease onset the serum samples had been collected.
Overall, and as expected from the results obtained with rhesus monkeys as infection progressed in these animals (Fig. 1), the sensitivity of anti-C6 antibody detection was higher in humans with later forms of Lyme disease. For patients in the early disseminated phase (neuroborreliosis), sensitivity was 95%, and for patients with late Lyme disease it was 100% (Table 1, panel 2). The exception was the samples from patients with posttreatment Lyme disease syndrome, which were only 62% positive (Table 1, panel 3). Patients in this group had a history of Lyme disease, and despite having received antibiotic therapy, they had persistent Lyme disease symptoms. The etiology of these symptoms is currently under investigation at the NIH.
The C6 ELISA is highly specific (Table 3; Fig. 2B). It could discriminate between Lyme borreliosis and infections with spirochetes of different species but of the same (B. hermsii) or different (T. pallidum) genera. Moreover, none of the samples from patients with autoimmune diseases, Mycobacterium infections, or diseases that often need to be differentially diagnosed with respect to Lyme disease, such as multiple sclerosis or Guillain-Barré syndrome, were positive with the C6 test. Only 2 of 99 serum samples from a local hospital in Louisiana, where Lyme disease is not endemic, yielded a positive C6 ELISA result. Given that the samples were collected randomly from unknown patients, it is impossible to assess whether these two patients could have been exposed to B. burgdorferi. The specificity results obtained are supported by our finding that, except for the Vls cassette of B. burgdorferi, no other sequences homologous to C6 could be identified (by using the BLAST search algorithm) in the National Center for Biotechnology Information protein sequence database. High diagnostic specificity is extremely critical to improve the positive predictive value of a test, especially when the incidence of a disease is very low. The incidence of Lyme disease in most areas of endemicity is less than 0.01% (8, 28); the highest incidence in the United States is 0.09% in Connecticut (8).
The cassette portion of VlsE contains six invariable regions (IRs) interspersed with an equal number of variable regions (40). The latter, by their very nature, have no diagnostic value. The additional five IRs are not as conserved as IR6 (17a). Nonetheless, we assessed whether the remaining IRs (IR1 to IR5) could contribute to improve the diagnostic performance of C6, whose sequence is based on that of IR6. No antibody responses to peptides reproducing the sequences of IR1 to IR5 were detected in humans or monkeys, infected with B. burgdorferi, in the absence of a response to C6 (17b). On this basis, we concluded that no improvement would be accrued from incorporating any of these peptides into the C6 ELISA. We did not test the diagnostic potential of other conserved regions of VlsE, which are located outside the cassette region (40). The C6 ELISA was very precise. This suggests that intralaboratory variations with this test may not be a problem. As yet, the assay has not been tested by other laboratories to evaluate interlaboratory performance.
As expected, monkey serum samples which contained high-titer anti-OspA antibody did not react with C6. Our test, therefore, will be suitable in the OspA vaccine era. Moreover, because of its simplicity and high sensitivity, specificity, and precision, it may contribute to alleviate some of the remaining problems in Lyme disease serodiagnosis.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health grants AI35027 and RR00164 and by a grant from SmithKline Beecham Biologicals.
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FOOTNOTES |
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* Corresponding author. Mailing address: Tulane Regional Primate Research Center, Tulane University Medical Center, 18703 Three Rivers Rd., Covington, LA 70433. Phone: (504) 871-6221. Fax: (504) 871-6390. E-mail: philipp{at}tpc.tulane.edu.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Clinical Microbiology, December 1999, p. 3990-3996, Vol. 37, No. 12
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