Some additional information from the blog Relative Risk, with my blue highlighting:Diagn Microbiol Infect Dis. 2012 Oct 10. pii: S0732-8893(12)00360-4. doi: 10.1016/j.diagmicrobio.2012.09.003. [Epub ahead of print]
Single-tier testing with the C6 peptide ELISA kit compared with two-tier testing for Lyme disease.
Wormser GP, Schriefer M, Aguero-Rosenfeld ME, Levin A, Steere AC, Nadelman RB, Nowakowski J, Marques A, Johnson BJ, Dumler JS.
Division of Infectious Diseases, New York Medical College, Valhalla, NY 10595, USA.
The 2-tier serologic testing protocol for Lyme disease has a number of shortcomings including low sensitivity in early disease; increased cost, time, and labor; and subjectivity in the interpretation of immunoblots.
The diagnostic accuracy of a single-tier commercial C6 ELISA kit was compared with 2-tier testing.
The C6 ELISA was significantly more sensitive than 2-tier testing with sensitivities of 66.5% (95% confidence interval [CI] 61.7-71.1) and 35.2% (95% CI 30.6-40.1), respectively (P < 0.001) in 403 sera from patients with erythema migrans. The C6 ELISA had sensitivity statistically comparable to 2-tier testing in sera from Lyme disease patients with early neurologic manifestations (88.6% versus 77.3%, P = 0.13) or arthritis (98.3% versus 95.6%, P = 0.38). The specificities of C6 ELISA and 2-tier testing in over 2200 blood donors, patients with other conditions, and Lyme disease vaccine recipients were found to be 98.9% and 99.5%, respectively (P < 0.05, 95% CI surrounding the 0.6 percentage point difference of 0.04 to 1.15).
Using a reference standard of 2-tier testing, the C6 ELISA as a single-step serodiagnostic test provided increased sensitivity in early Lyme disease with comparable sensitivity in later manifestations of Lyme disease. The C6 ELISA had slightly decreased specificity. Future studies should evaluate the performance of the C6 ELISA compared with 2-tier testing in routine clinical practice.
Copyright © 2012 Elsevier Inc. All rights reserved.
[PubMed - as supplied by publisher]
http://relative-risk.blogspot.com/2012/ ... stics.html
Just for reference purposes, here's an earlier article by Wormser et al about the C6 test:16 OCTOBER 2012
Wormser GP, Schriefer M, Aguero-Rosenfeld ME, Levin A, Steere AC, Nadelman RB, Nowakowski J, Marques A, Johnson BJ, Dumler JS. Single-tier testing with the C6 peptide ELISA kit compared with two-tier testing for Lyme disease. Diagn Microbiol Infect Dis. 2012 Oct 10.
The public health service recommendations in support of 2-tier testing (CDC, 1995) provide for the development of alternatives to 1 or both steps provided that equal or better performance is demonstrated by such alternative methods (Proceedings of the Second National Conference on Serologic Diagnosis of Lyme Disease, 1995). In principle, an ELISA of sufficiently high specificity and sensitivity would provide diagnostic information similar to existing 2-tier testing. A variety of recombinant and synthetic antigens have been evaluated in previous studies for use in serodiagnosis of Lyme disease. In particular, the VlsE protein and the highly conserved 25-amino acid peptide (“C6 peptide”) derived from the sixth invariant region of this protein have been shown to be both sensitive and specific antigens in the ELISA and immunoblot formats. The aim of the present study was to determine whether the C6 ELISA by itself is a suitable alternative to 2-tier testing by evaluating the comparative diagnostic accuracy in over 550 sera from well-characterized Lyme disease patients and in more than 2200 control sera.
The C6 Lyme ELISA kit (Immunetics, Boston, MA, USA), a test kit approved by the FDA for use as a first-tier assay, was modified for this study. Kits provided by the manufacturer for this study incorporated a simplified and comparable cut-off formula that was based on a negative calibrator serum. This modification yielded sensitivity and specificity statistically equivalent to what had been demonstrated with the original cut-off formula. Reproducibility testing of the C6 ELISA kit using the simplified cut-off yielded intra-assay and interassay coefficients of variation (CV) of 10% and 11.6%, respectively. Intra-assay and interassay CVs for the kit with the original cut-off were 9.5% and 14.3%, respectively. In all other respects, the modified and original kits were the same. The C6 peptide used as antigen in the kit is derived from the B. burgdorferi B31 strain sequence, which differs from the originally described IP90 sequence by 4 amino acids. The kit is formatted as an indirect ELISA in which both IgG and IgM antibodies to C6 peptide are detected by an enzyme conjugate. The C6 ELISA testing was performed at New York Medical College, the CDC, and at Immunetics.
The patient sera selected for this study were chosen to represent a broad range of clinical manifestations of Lyme disease from well-characterized patients. The sera comprised multiple reference panels from different sources that had been previously collected and frozen at −80 °C. The 569 Lyme disease sera studied were obtained from 528 patients. Erythema migrans was defined based on clinical diagnosis alone for 172 sera and on clinical diagnosis in conjunction with microbiologic confirmation of B. burgdorferi infection by either culture or polymerase chain reaction (PCR) for 231 sera.
In this evaluation of the performance of serologic methods for detection of antibodies to B. burgdorferi, the C6 ELISA as a standalone test had significantly greater sensitivity than either the WCS ELISA-based 2-tier testing method (66.5% versus 35.2%, P < 0.001) or the C6 ELISA-based 2-tier testing method (66.5% versus 34.5%, P < 0.001) for patients with erythema migrans.
In contrast, on sera from the groups of patients with neurologic or rheumatologic manifestations of Lyme disease, each of the 3 testing methods had high sensitivity without a statistically significant difference (P < 0.05). The specificity of either of the 2-tier testing methods on more than 2200 control sera, however, exceeded that of the C6 ELISA alone by 0.6 percentage points (99.5% versus 98.9%, 95% CI surrounding the difference of 0.04 to 1.15), and this difference was significant (P < 0.05). The 0.4–0.7 percentage point lower specificity values observed for these serologic methods in the endemic compared with the nonendemic blood donor group might be explained by the inclusion of individuals with prior exposure to B. burgdorferi in the endemic group, although this hypothesis cannot be evaluated due to the lack of information on donor histories. Our overall results for specificity are quite comparable to that for the C6 ELISA in another published study that involved a sizeable but smaller number of control serum samples (present study 98.9% [2216/2241] versus 98.4% [1279/1300], P = 0.22). The authors of that report (Branda et al., 2011) observed that a novel 2-tier strategy using a WCS ELISA followed by the C6 ELISA would have increased the specificity to 99.5%, a figure that was identical to the conventional WCS ELISA–immunoblot-based 2-tier testing. A reanalysis of our study data with such an algorithm yielded the same 99.5% specificity value.
Interestingly, however, in the group of individuals with other disease conditions as well as in the group of LymeRx® vaccine recipients, which collectively might be more representative of the target population for testing than blood donors, the specificity of this 2-tier ELISA algorithm was identical to that of the C6 ELISA alone. Serial 2-step algorithms generically offer improved specificity at the cost of some loss of sensitivity; in the present study, the serial ELISA algorithm would result in a loss of about 6% in sensitivity for erythema migrans patients, and a similar decrease was observed by Branda et al. (2011). A cost–benefit analysis of this tradeoff may indicate its utility relative to other algorithms.
Overall, our results indicate that the C6 ELISA should be preferred over 2-tier testing in patients with early infection, for example, in patients with a skin lesion(s) for whom a clinical diagnosis is uncertain.
Given the ease of performance, objectivity and greater simplicity of single-tier testing using an ELISA, the C6 ELISA may be preferred over 2-tier testing in patients with early neurologic Lyme disease and other manifestations of early disseminated Lyme disease.
An advantage of 2-tier testing over the C6 ELISA is its higher specificity. The high specificity of 2-tier testing found in this study, however, is not consistent with observations in clinical practice where overreading of weak bands on the IgM immunoblot in particular has served to reduce specificity and stimulate interest in alternative testing strategies to avoid the IgM immunoblot entirely. Another potential advantage of 2-tier testing over C6 ELISA is that the former provides information on the presence of an expanded IgG response specifically, a serologic prerequisite for the diagnosis of late Lyme disease. Thus, for the diagnosis of late Lyme disease, it may be advantageous to perform an IgG immunoblot to supplement the C6 or another ELISA.
The full article is here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2773679/Clin Infect Dis. 2008 Oct 1;47(7):910-4.
Effect of Borrelia burgdorferi genotype on the sensitivity of C6 and 2-tier testing in North American patients with culture-confirmed Lyme disease.
Wormser GP, Liveris D, Hanincová K, Brisson D, Ludin S, Stracuzzi VJ, Embers ME, Philipp MT, Levin A, Aguero-Rosenfeld M, Schwartz I.
Division of Infectious Diseases, Department of Medicine, New York Medical College, Valhalla, NY 10595, USA.
A potential concern with any serologic test to detect antibodies to Borrelia burgdorferi is whether the epitopes incorporated in the test provide sufficient cross-reactivity to detect infection with all of the pathogenic strains of the species. This is a particular concern for the C6 test, which is based on reactivity to a single peptide.
C6 testing and 2-tier testing were performed on acute-phase serum samples obtained from >158 patients with erythema migrans for whom the genotype of the borrelial isolate was defined on the basis of an analysis of the 16S-23S ribosomal DNA spacer region and/or on the genetic variation of the outer surface protein C gene (ospC). The sonicated whole cell-based enzyme-linked immunosorbent assay, the immunoblots used in the 2-tier testing, and the C6 assay all used antigens from B. burgdorferi sensu stricto strain B31.
The sensitivity of C6 testing (69.5%) was greater than that of 2-tier testing (38.9%) (P<.001); the difference in sensitivity, however, was statistically significant only for patients infected with 2 of the 3 ribosomal spacer type-defined genotypes. The lower sensitivity of 2-tier testing was attributable to the low sensitivity of the immunoblot tests, rather than the first-tier enzyme-linked immunosorbent assay. There was also a trend for the sensitivity of 2-tier testing to vary according to the ospC genotype for the 14 genotypes represented in the study (P=.07); this relationship was not observed with C6 testing.
Lack of sensitivity of the C6 test because of strain diversity seems less likely to be a limitation of this serologic test, compared with 2-tier testing in North American patients with early Lyme disease.
[PubMed - indexed for MEDLINE]
Free PMC Article
Here's the link for the Immunetics C6 peptide test kit:
Serum specimens from patients with Lyme disease
Serum specimens that had been stored at −80°C were tested using the C6 and 2-tier methods as part of a performance comparison. The serum samples were originally obtained from patients with erythema migrans whose clinical diagnosis was confirmed by the growth of B. burgdorferi from culture of a skin biopsy or blood sample, with use of methods reported elsewhere . Serum specimens were obtained before antibiotic treatment was initiated. The patients who provided the serum samples were subjects in approved research studies at New York Medical College (Valhalla). The patients resided in Westchester County, New York, or in the immediately surrounding geographic areas. Information on duration of illness and number of erythema migrans skin lesions was obtained from study records.
A limitation of our study is that the patients were infected in a single location in New York State or in the immediately surrounding geographic areas; thus, the results may not be generalizable to infections acquired in other locations. Another limitation may be related to the choice of the MarDx/Trinity Biotech immunoblot kits as the second-tier test. This may have contributed to the reduced sensitivity observed in this study, because 1 comparative study has shown that the IgM kit is less sensitive than some other immunoblot assays .
The IR6 peptide in some VlsE proteins of strains of other species of B. burgdorferi sensu lato, such as Borrelia afzelii and B. garinii, may vary by up to 5 amino acids, compared with the IR6 region of the VlsE protein of B. burgdorferi B31 . In North America, only strains of B. burgdorferi are known to cause Lyme disease, and variations in the IR6 region among North American isolates of B. burgdorferi have not been directly examined with a large number of isolates. Lack of sensitivity of the commercially available C6 test attributable to strain diversity, although a theoretical concern, has not yet been demonstrated and, on the basis of our findings in New York State, seems unlikely to be a limitation of this serologic test.
C6 (B. burgdorferi) Lyme ELISA
For in vitro diagnostic use
Catalog # DK-E352-096
Lyme disease can be difficult to diagnose. Traditional Lyme ELISA tests are short on specificity and can generate over 20 percent false positive results. This uncertain performance can result in time-consuming, expensive Western Blot confirmatory tests. The C6 Lyme ELISA from Immunetics sets a new standard in Lyme test accuracy, and delivers more reliable results sooner and more economically than any other ELISA. It demonstrates 99 percent specificity in an average population – equivalent to the specificity of Western Blot testing and a 10-fold improvement over Whole Cell Sonicate ELISAs. Additionally, the C6 Lyme ELISA is a far more sensitive assay – showing twice the sensitivity of the standard Western Blot in patients with symptoms of acute or early Lyme disease. These features mean that more patients are correctly detected as positive for the disease. From early onset to late stage disseminated infection, C6 Lyme ELISA demonstrates superior results.
Accurate: Western Blot accuracy in an ELISA
Sensitive: More sensitive detection of antibodies in early and late Lyme disease than competing kits
Specific: Eliminates vast majority of other ELISA's false positives at the screening step
Broadly applicable: Detects infection with all strains of Borrelia which cause Lyme disease, including European strains B. garinii and B. afzelii.
Convenient: One hour turnaround time, breakaway strips for any number of samples, ready-to-use reagents and controls included
Cost-effective: Greatly reduces the number of samples sent for expensive and lengthy confirmatory testing
Immunetics has pioneered the use of the C6 peptide, a 26-amino acid sequence within the Borrelia membrane protein VlsE. This is the first truly specific and sensitive marker for infection with the Borrelia burgdorferi spirochete. This peptide has been shown to represent the immunodominant portion of VlsE, containing substantially all of the antigenic reactivity of the whole protein. The C6 peptide sequence is highly antigenic, generating an immune response in nearly all human cases soon after infection, and is specific to Borrelia strains causing Lyme disease. It is not found in other infectious organisms, eliminating cross-reactivity at the source.
Immunetics’ C6 ELISA uses a synthetic version of the C6 peptide antigen*. This precisely defined antigen makes possible a highly sensitive, specific and reproducible ELISA for infection with the B. burgdorferi spirochete. Current ELISA tests do not match the specificity or sensitivity of Immunetics’ C6 ELISA because they are based on Whole Cell Sonicates (WCS) containing multiple antigens, leading to cross-reactivity and inaccurate or ambiguous results.
Along with superior performance, Immunetics’ C6 Lyme ELISA offers a rapid and easy-to-use protocol. Turnaround time is approximately one hour, and tests can be run on automated ELISA systems or manually. The C6 Lyme ELISA kit is approved for in vitro diagnostic use.
U.S. Patents 6,475,492, 6,719,983, and 6,740,744 and International Patents
FDA approved and CE marked
C6 test kits (Cat # DK-E352-096) include:
Coated microwell plate with 96 breakaway wells
Ready to use reagents – everything needed to perform the assay
Positive and negative controls
One-hour protocol instructions