0 Package Insert excerpt: Athena Multi-Lyte Borrelia VlsE-1/pepC10 Plus Test System

The following excerpt contains only the intended use, significance, background, and cross reactivity portions of the package insert.

For your review of the remaining sections and more microbio prep info, the complete package insert is here: http://www.zeusscientific.com/fileadmin/media/pdfs/inserts/athena/autoimmune/R2455E.pdf

Borrelia VlsE-1/pepC10 Plus Test System
A Multiplexed, Microparticle-Based Immunoassay for IgG Antibodies
to VlsE-1 and IgM antibodies to pepC10 antigens.
Product Number: A90151

INTENDED USE

The Zeus Scientific, Inc AtheNA Multi-Lyte Borrelia VlsE-1/ pepC10 Plus Test System is a multiplexed sandwich assay for the qualitative detection of IgG class antibody to recombinant VlsE-1 and the IgM class of antibody to synthetic pepC10 in human serum.

The AtheNA Multi-lyte Borrelia VlsE-1/pepC10 Plus Test System is intended for use in testing human serum samples which have been found equivocal or positive by alternate serological procedures to provide supportive evidence of infection by Borrelia burgdorferi.

This kit is for in vitro diagnostic use only.

Assay performance characteristics have not been established for immunocompromised or immunosuppressed patients, cord blood, neonatal specimens, or infants.

SIGNIFICANCE AND BACKGROUND

Borrelia burgdorferi is a spirochete that causes Lyme disease. The organism is transmitted by ticks of the genus Ixodes. In endemic areas, these ticks are commonly found on vegetation and animals such as deer, mice, dogs, horses, and birds. B. burgdorferi infection shares features with other spirochetal infections (diseases caused by three genera in humans: Treponema, Borrelia, and Leptospira). Skin is the portal of entry for B. burgdorferi and the tick bite often causes a characteristic rash called erythema migrans (EM). EM develops around the tick bite in 60% to 80% of patients. Spirochetemia occurs early with wide spread dissemination through tissue and body fluids. Lyme disease occurs in stages, often with intervening latent periods and with different clinical manifestations.

In Lyme disease there are generally three stages of disease often with overlapping symptoms. Symptoms vary according to the sites affected by the infection such as joints, skin, central nervous system, heart, eye, bone, spleen, and kidney. Late disease is most often associated with arthritis or CNS syndromes. Asymptomatic subclinical infection is possible and infection may not become clinically evident until the later stages.

Patients with early infection produce IgM antibodies during the first few weeks after onset of EM and produce IgG antibodies more slowly (1). Although IgM only may be detected during the first month after onset of illness, the majority of patients develop IgG antibodies within one month. Both IgG and IgM antibodies can remain detectable for years. Isolation of B. burgdorferi from skin biopsy, blood, and spinal fluid has been reported (2). However, these direct culture detection methods may not be practical in the large scale diagnosis of Lyme borreliosis.

Serological testing methods for antibodies to B. burgdorferi include indirect fluorescent antibody (IFA) staining, immunoblotting, and enzyme immunoassay (EIA).

B. burgdorferi is antigenically complex with strains that vary considerably. Early antibody responses often are to flagellin which has cross reactive components. Patients in early stages of infection may not produce detectable levels of antibody. Also, early antibiotic therapy after EM may diminish or abrogate good antibody response. Some patients may never generate detectable antibody levels.

Thus, serological tests for antibodies to B. burgdorferi are known to have low sensitivity and specificity and because of such inaccuracy, these tests cannot be relied upon for establishing a diagnosis of Lyme disease (3,4).

In 1994, the Second National Conference on Serological diagnosis of Lyme disease recommended a two-step testing system toward standardizing laboratory serologic testing for B. burgdorferi.

Because EIA and IFA methods were not sufficiently specific to support clinical diagnosis, it was recommended that positive or equivocal results from a sensitive EIA or IFA (first step) should be further tested, or supplemented, by using a standardized Western Blot method (second step) for detecting antibodies to B. burgdorferi (Western Blot assays for antibodies to B. burgdorferi are supplemental rather than confirmatory because their specificity is less than optimal, particularly for detecting IgM). Two-step positive results provide supportive evidence of exposure to B. burgdorferi, which could support a clinical diagnosis of Lyme disease but should not be used as a sole criterion for diagnosis.

Various antigens have been tested in recent years to improve in assisting in the diagnosis of Lyme disease. One such attempt has been the use of a two tier algorithm to test for IgG antibodies towards VlsE1 and IgM antibodies for pepC10 antigen. Serodiagnosis of Lyme disease using the two-tier approach has greater percent sensitivity than individual EIA’s (5). Using this paradigm the manufacturer has developed a two tier assay to perform IgG detection towards VlsE1 and IgM detection towards pepC10 antigen.

(Kit Component, Assay info., etc. to be found at above PDF link for interested parties.)

CROSS REACTIVITY AND INTERFERING SUBSTANCES

Cross Reactivity Studies were performed at two sites to assess cross reactivity with the Athena Multi-Lyte Borrelia VlsE-1/pepC10 Plus test system using sera that were sero-positive to EBV VCA IgG, EBV VCA IgM, RF, ANA, Syphilis, CMV IgG, CMV IgM, Rubella, VZV IgM and Toxoplasma.

Site one was Zeus Scientific’s manufacturing facility and site two was a state Department of Public Health (DOH) located in the northeast. ELISA, IFA and micro-particle immunoassay test systems manufactured by various companies for commercial distribution were used to determine the sero-positivity of the samples. Ten samples minimally for each possible crossreactant were tested.

The cross reactivity data has been summarized in the following table.

In total, 180 samples were tested for possible cross reactivity with 10 analytes.

AtheNA Multi-Lyte Borrelia VlsE-1/pepC10 Plus Cross Reactivity Study
Possible Positive Results/
Cross-Reactants Number Tested
EBV VCA IgG 0 / 21
EBV VCA IgM 4 / 10*
ANA 2 / 20
Syphilis 0 / 22
CMV IgG 0 / 21
CMV IgM 0 / 10
Rubella IgG 1 / 21
Toxo IgG 3 / 24
VZV IgM 0 / 10
RF 0 / 21
*Please note that one out of the four samples that had a positive AtheNA Score was also positive for B.burgdorferi by the two tier.


REFERENCES:
1. Steere AC, et al: J. Infect. Dis. 154:295-300, 1986.
2. Rosenfeld MEA:Serodiagnosis of Lyme disease. J. Clin. Microbiol. 31:3090-3095, 1993.
3. Steere AC, et al:The Spirochetal Etiology of Lyme Disease. N. Engl. J. Med. 308:733-740, 1983.
4. Bakken LL, Callister SM, Wand PJ, and Schell RF:Interlaboratory Comparison of Test Results for Detection of Lyme Disease by 516 Patients in the Wisconsin State Laboratory of Hygiene/College of American Pathologists Proficiency Testing Program. J. Clin. Microbiol. 35:537-543, 1997.
5. Bacon R M, et al: J. Infect. Dis. 187:1187-99, 2003
6. U.S. Department of Health and Human Services. Public Health Service. Centers for Disease Control and Prevention and  National Institutes of Health.  U.S. Government Printing Office, Washington D.C., 4th Ed., 1999.
7. U.S. Department of Labor, Occupational Safety and Health Administration; Occupational Exposure to Bloodborne Pathogens, Final Rule. Fed.Register 56:64175-64182, 1991.
8. Protection of Laboratory Workers from Instrument Biohazards and Infectious Disease Transmitted by Blood, Body Fluids and  Tissues; Approved Guideline.  NCCLS/CLSI Document M29, Vol.17 (12), 1997.
9. CLSI. User Protocol for Evaluation of Qualitative Test Performance: Approved Guideline. CLSI document EP-12-A (ISBN 1-56238-468-6). CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2002.
10. CLSI EP7-A2. Interference Testing in Clinical Chemistry; Approved Guideline, 2nd Ed. (2005).

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Okay, where is my bottle of scotch? I think I need it about now...

I'm going to take a break from posting about vlsE for a bit, unless something more compelling comes up about it.

I have mixed feelings about this, by the way - I don't like reading the significance and background on this insert when I know what the public statements have been about persisting Lyme disease - yet at the same time, if this test really is more accurate in catching early cases and people get early treatment because of it, I can't complain about that. We need more accurate tests for early Lyme disease detection and diagnosis.