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Volume: 1
Issue: 05
Date: 10-Mar-93


Table of Contents:

            Laboratory Diagnostic Tests for Lyme Disease
          by David W. Dorward, Ph. D.  dwd@rml.niaid.nih.gov
                         Senior Staff Fellow
                 Laboratory of Vectors and Pathogens
                   NIH/Rocky Mountain Laboratories
                         Hamilton, MT  59840


Newsletter:

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                     Volume 1 - Number 05 - 3/10/93


                       ***** SPECIAL ISSUE *****

I.   Introduction
II.  News from the wires
III. Jargon Index
IV.  How to Subscribe, Contribute and Get Back Issues



I. ***** INTRODUCTION *****

This issue of the LymeNet newsletter features a paper written by Dr. David
W. Dorward, Senior Staff Fellow at the NIH's Rocky Mountain Labs.  This
article was written for electronic publication in the Health Info-Com
Network Newsletter, a biweekly newsletter edited by Dr. David Dodell.  
My thanks to Dr. Dodell for the reprint permission.  More information
on the Heath Info-Com Newsletter can be found at the end of the article.


This article reviews the current state of Lyme testing.  Dr. Dorward looks
at the many different testing techniques, both existing and in development.
The pros and cons of each testing method are discussed, as well as
recent improvements.


-Marc.


II. ***** NEWS FROM THE WIRES ******

-----------------------------------------------------------------------------
Compilation Copyright 1993 by David Dodell,  D.M.D.  All  rights  Reserved.  
License  is  hereby  granted  to republish on electronic media for which no
fees are charged,  so long as the text of this copyright notice and license
are attached intact to any and all republished portion or portions.  
-----------------------------------------------------------------------------


                Laboratory Diagnostic Tests for Lyme Disease

             by David W. Dorward, Ph. D.  dwd@rml.niaid.nih.gov
                            Senior Staff Fellow
                    Laboratory of Vectors and Pathogens
                      NIH/Rocky Mountain Laboratories
                            Hamilton, MT  59840
                      _______________________________
                                INTRODUCTION


Lyme disease is an emerging disease, which has become the most prevalent
arthropod-borne disease in North America.  It is a protean disease, and can
cause a mild to severe, acute to chronic syndrome in humans and in wild and
domestic animals.


Despite considerable efforts to understand, recognize, diagnose, and treat
Lyme disease, many questions remain, particularly regarding methods used for
laboratory confirmation of infection.  In most cases, the results of
laboratory testing methods are consistent with clinical presentation of Lyme
disease.  However, when inconsistent, little confidence is placed on the
laboratory results because of the many published reports of relatively high
degrees of error among test systems.  In recent years several new testing
procedures have been proposed to address such problems.  This paper describes
the theoretical bases for current and "next generation" Lyme test systems and
discusses the benefits, potential problems, and recent improvements with each
type of system.  The emphasis of the descriptions and discussions of current
assays focuses on systems which have been developed and marketed by
manufacturers listed and which have been cleared for clinical use in the
United States by the U.S. Food and Drug Administration (FDA).  Readers should

note that many additional systems can be and are utilized by individual
clinical laboratories.  Furthermore, several additional companies supply test
systems which have previously received clearance by the FDA, after being
submitted for consideration by manufacturers listed herein.  Review of these
systems follows a brief general description of Lyme disease.


In just over a decade since the discovery of a spirochetal etiology for Lyme
disease, scientific interest in Lyme disease has led to a dramatic increase
in knowledge about the disease and it's agents, three members of Borrelia sp.
Originally isolated and identified by Willy Burgdorfer and co-workers (1),
these agents were initially named Borrelia burgdorferi.  More recently,
evidence of phylogenetic diversity among Lyme disease associated spirochetes
from the United States, Europe, and Asia, has led to acceptance of a proposal
for subdividing the agents into separate species, designated B.  burgdorferi,
B. garinii, and "group VS461" (2).


Lyme spirochetes are transmitted to humans and other mammals by several
species of hard-shell ticks primarily those within the Ixodes ricinus
complex, comprising I. ricinus and I. persulcatus in Eurasia, and I. dammini,
I. scapularis, I. pacificus, and possibly I. cookei in North America.  There
is also evidence to suggest that Amblyomma americanum may also be a competent
vector in the southern United States.  The spirochetes gain entry to mammals
during feeding by these ticks.  Most human infections occur after bites by
the exceedingly tiny larval and nymphal stages of Ixodes sp.  ticks, which
emerge in late spring to early summer.  Hence, Lyme disease is most prevalent
in areas inhabited by these ticks, and occurs most frequently from May to
September.


Difficulties in diagnosis arise from several factors.  Such factors include
variability in the rate of onset and the types of symptoms exhibited, failure
of patients to see or remember tick bites, slow growth and low density of
infecting spirochetes, and differences in host responses to the infection.
Comprehensive reviews of these factors have previously been published (3, 4).
The U.S. Centers for Disease Control (CDC) has also released "case
definition" guidelines for Lyme disease.  Typically (in 50-80% of cases),
within several days of a tick bite, a characteristic and pathognomonic
"bulls-eye" rash, called erythema migrans, will encircle the site of the
bite.  Clinical isolation of spirochetes is often possible from skin biopsies
taken at or near the margins of erythema migrans lesions.  In cases in which
erythema migrans did not appear or was not noticed, isolation of spirochetes
is relatively rare (usually less than 10% of cases).  In those situations
diagnoses are based upon clinical observations, patient history, presence of

secondary symptoms such as arthritis, arthralgias, carditis, and neurological
manifestations, and results of clinical laboratory tests.  With few
exceptions, the laboratory tests involve determinations of diagnostic titers
of host antibodies directed against the Lyme agents, either by indirect
fluorescent assay (IFA), enzyme-linked immunoassay (EIA), or Western blot
analysis (WBA).  For reporting, the CDC case definition requires one or more
of the following indications: a) isolation of the causative spirochetes, b)
presence of erythema migrans, or c) a combination of "late" symptoms and
laboratory confirmation by serology as described above.


                    ___________________________________
                      LABORATORY DIAGNOSTIC STRATEGIES


Laboratory assays for the diagnosis of Lyme disease, which are in practice or
which have been proposed, can be grouped into the following categories:


1)  Isolation and culture of agents
2)  Histological examination of tissues
3)  Serology
4)  Antigen detection systems
5)  Nucleic acid detection systems


Most of the assays in the categories 3-5 are considered "rapid" tests, and
can be performed under normal circumstances in less than one day.  Such tests
are the focus of most research into Lyme diagnostics.  Furthermore, all
commercial test systems currently cleared by the FDA for clinical use in the
U.S. fall within the serological category.  Hence this discussion of rapid
test systems will concentrate on categories 3-5, with a brief description of
categories 1-2.


1)  Isolation

Attempts to isolate Lyme agents from patients are common, and certainly
conclusive when successful.  The ability to culture the spirochetes from
human patients is highly variable.  Furthermore, primary cultures generally
require 1-2 weeks or more before growth of the spirochetes is evident.  The
source of the sample factors into whether the spirochetes can be cultured.
Successful cultures have been obtained from ticks, skin biopsies, ear
punches, cerebrospinal fluid, blood, and synovial fluid (3, 4).  In animal
models, the spirochetes are reliably cultured from ticks, and from heart
muscle and the urinary bladder of mice (3, 4).  To help avoid contamination
of cultures, samples are handled carefully and antibiotics are usually added
to primary culture media.


2)  Histology

Histological examination of patient tissues and blood smears has been and
continues to be used in some laboratories as a diagnostic assay.
Histological examinations are usually coupled with immunological assays such
as fluorescence microscopy or immune histochemistry to enhance the
specificity of the assay.  Since Lyme disease is characterized by a low
density infection, examination of large numbers of sections is often required
before spirochetes are observed.  Similarly, unlike relapsing fever
borreliae, Lyme spirochetes are rarely observed in blood smears.  Thus,
although histological observation of Lyme spirochetes can successfully
confirm an infection, failure to observe the spirochetes does not necessarily
rule out Lyme disease.


3)  Serology

Serological assays for Lyme diagnosis include IFA, EIA, WBA, and spirochetal
neutralization assays.  Currently all commercial test systems which have
received clearance by the FDA are either IFA or EIA (Tables 1 and 2).  Tables
1 and 2 compare the formats, specificities, and scope of the assays which are
currently marketed and cleared.  The kits have been arranged alphabetically
by manufacturer.  As noted above, the tables do not list products which have
received clearance through one manufacturer but which are produced,
repackaged, or supplied under a different label.  The information shown was
provided by the manufacturers.


Among the fluorescent systems shown in table 1, most assays involve probing
fixed spirochetes with patient sera, labeling any immune complexes formed
with a fluorescent conjugate, and examining the preparation by fluorescent
microscopy.  Fluorescent "dot" and microtiter well assays utilizing whole
cell lysates have also been developed.  Approximately equal numbers of the
assays detect either IgG or IgM class antibodies in the sera, and most detect
both classes.


Table 2 shows that most enzyme-linked systems are also similar to each other.
Most have a microtiter well format to which whole cell antigens are
immobilized.  Dot assays and an automated system are also available.  Some
manufacturers have supplemented (or replaced) the whole cell antigens with
spirochetal membrane concentrates, the 41 kilodalton flagellar protein, or
with a recombinant 39 kilodalton antigen which is specific to Lyme
spirochetes.  As with fluorescent assays, approximately equal numbers of test
kits detect IgG or IgM antibodies, most detect both, and some also detect IgA
antibodies.



Table 1.  Fluorescent systems

                Format                Antigen         Detects
         ______________________     ____________     ___________
Mfg       Slide     Dot     Well     WC     Fixed     IgG     IgM
_________________________________________________________________
BE          +                                 +        +       +
DT          +                                 +        +       +
HB          +                                 +        +
           +                                 +                +
MD          +                                 +                +
           +                                 +        +
WB                    +               +                +       +
                     +               +                        +
                            +        +                +       +
           +                                 +        +       +
ZS          +                                 +        +       +
_________________________________________________________________


Abbreviations: Mfg-manufacturer; WC-Whole cell; BE-Bion
Enterprises; DT-Diagnostic Technology; HB-Hillcrest Biologicals; MD-
Mardx Diagnostics; WB-Whittaker Bioproducts; VS-Vitek Systems; ZS-Zeus
Scientific



Table 2.  Enzyme-linked systems

         Format         Antigen        Detects       Service
    ________________   ___________   _____________   Provided
Mfg  Well   Dot  Auto   WC  Peptide   IgG  IgM  IgA
______________________________________________________________
CB    +                 +              +    +
DX    +                 +     41k      +    +
     +                 +                   +
     +                 +     41k      +    +            +
     +                 +                   +            +
GB           +          +     41k      +
     +                 +     39k      +    +     +
     +                       39k      +    +     +
            +          +     39k      +    +     +
GL    +                 +                   +
HB    +                 +              +    +
     +                 +                   +
MD    +                 +              +    +
     +                 +                   +
     +                 +              +
MT           +          +              +    +
VS                +     +              +    +

WB    +                 +              +    +
     +                 +                   +
ZS    +                 +              +
     +                 +                   +
     +                 +              +    +
_________________________________________________________________
Abbreviations; Mfg-manufacturer; CB-Cambridge Biosciences; DX-
Diamedix; GB-General Biometrics; GL-Gull Laboratories; HB-Hillcrest
Biologicals; MD-Mardx Diagnostics; MT-Medical Diagnostic
Technology; VS-Vitek Systems; WB-Whittaker Bioproducts; ZS-Zeus
Scientific



Although no commercial WBA test systems have been cleared by the FDA, many
laboratories also screen sera by WBA.  With WBA, laboratories can determine
whether sera contain antibodies directed against a variety of different
spirochetal antigens.  Currently there is no clear consensus about which
antigens, when recognized, are most important for making diagnostic
conclusions for Lyme disease.  Binding of antibodies to several proteins have
been commonly observed in known Lyme sera.  These include (but are not
limited to) proteins at 92, 79, 60, 41, 39, 34, 31, and 22 kilodaltons (3,
4).  Proteins at 31 and 34 kilodaltons, designated outer surface protein A
(OspA) and OspB, respectively, are known to be plentiful in most Lyme
spirochete isolates.


Another, recently reported, serological approach involves testing sera for
the presence of neutralizing antibodies (5).  In this assay, sera are added
to a test strain culture of Borrelia burgdorferi, and the ability of serum
antibodies to kill the test strain is measured.


4)  Antigen detections

In contrast to serological systems, antigen detection systems screen for
antigenic products of the spirochete rather than the host's immune response
to the infection.  Therefore, antigen detection systems could be expected to
confirm both infections in patients with depressed immune systems and early
infections in patients who have not yet produced a detectable antibody
response.  It is presumed that clearance of such antigens from hosts would be
indicative of successful treatment and elimination of infecting spirochetes
from patients.  Furthermore, antigen detection systems can be utilized to
monitor the presence of Lyme spirochetes in ticks.  Two categories of antigen
detection systems have been developed and used in laboratories.  These are T-
cell (T-lymphocyte) proliferative assays and antibody-based antigen detection
systems.


T-cell proliferative assays are based upon the recognition of spirochetal
antigens by cloned, antigen-specific T-cells (6).  When such T-cells bind and
recognize Lyme spirochete products in a test sample, T-cell division is
induced.  This division can be measured by counting cells, or by measuring
the incorporation of labeled nutrients into the T-cells.  In theory, very few
molecules of the recognized antigens would be required to induce T-cell
proliferation.


Monoclonal or polyclonal antibodies directed against specific Lyme spirochete
antigens can also be used to detect antigens in clinical samples.  Initial
work found that detectable quantities of proteins including flagellin, OspA,
and OspB, were excreted in the urine of infected mammals (7).  Subsequent
work showed that the sensitivity of detection could be enhanced by
immobilizing, and effectively concentrating, the antigens onto an antibody-
coated surface before detection with a species-specific antibody probe.
Immobilization of antigens allows for removal of contaminants from a sample
preparation which might interfere with subsequent immunological binding
reactions in the assay (8).  Using such an "antigen capture" system, a
variety of different fluids and tissues from ticks and mammals can be
screened for involvement in the infection.  Mammalian fluids and tissues in
which antigens have been observed include: urine, blood, cerebrospinal fluid,
synovial fluid, tears, vitreous humor, breast milk, a variety of visceral

organs, and the brain.  Since such varied samples can contain spirochetal
antigens, it is presumed that other samples could also contain detectable
quantities of antigen.


When using antigen capture systems, occasionally intact spirochetes are
immobilized onto the antibody-activated substrate, whereas other
microorganisms are excluded from adhering to the same surface (8).  One
approach at exploiting this capture technique used capillary tubes, coated on
the inner surface with antibodies, to promote binding of spirochetes to the
glass (9).  Such selective adherence enabled recovery and isolation of Lyme
spirochetes from biologically diverse mixtures, such as contaminated
cultures, macerated ticks, and infected organs.  Therefore, use of antigen
capture systems may provide a means to facilitate isolating Lyme spirochetes
(and potentially many other types of pathogens) from infected hosts.


5)  Nucleic acid detections

Like antigen detection systems, nucleic acid detection systems analyze
spirochetal bioproducts, rather than a host's immune response to infection.
Similarly, nucleic acid assays can also detect Lyme spirochetes in infected
ticks.  Currently, three types of nucleic acid detection systems have been
proposed as possible diagnostic procedures for Lyme disease.  These are DNA-
DNA hybridizations, polymerase chain reaction (PCR), and ligase chain
reaction (LCR).  Hybridization assays are based upon the ability of a labeled
fragment of DNA, specific to Lyme spirochetes, to locate and hybridize with
complementary DNA sequences in a test sample.  Chain reaction assays enable
the temperature-regulated amplification and detection of spirochetal DNA
molecules.  Both PCR and LCR reactions incorporate the following processes
a) melting of DNA strands, b) hybridization of short and species-specific
single-stranded DNA primer molecules to targeted sites on templet DNA
possibly present in test samples, and c) enzymatic activity by either heat

stable DNA polymerase (PCR) or DNA ligase (LCR).  Optimal temperatures for
each step are reached and maintained by instruments called thermocyclers.
Molecules amplified by PCR contain newly synthesized DNA which corresponds to
sequences which lie between the binding sites for the two primer molecules.
In contrast, molecules amplified by LCR result from ligation of the two
primer molecules which must bind to contiguous sites on a templet.


Direct DNA-DNA hybridization has been proposed and used to screen ticks for
the presence of Lyme spirochetes (10).  For effective detection of
spirochetal DNA by hybridization, numerous target DNA molecules must usually
be present in a test sample.  Because few if any intact spirochetes are
present in typical clinical samples, hybridization is not commonly employed
to directly test for infection of mammals.


Both PCR and LCR techniques for Lyme disease diagnosis are being developed
(10-15).  In theory, the presence of a single templet DNA molecule in a given
sample would be sufficient to allow confirmation of infection by these
techniques.  In practice the sensitivity can usually be extrapolated to
producing positive results with less than 10 templet DNA molecules in a
sample.  Several different studies have shown that amplification of Lyme
spirochetal DNA can be accomplished from ticks, and mammalian blood, urine,
and tissues.  Furthermore, several target sites which are apparently specific
for Lyme spirochetes have been identified (11, 12, 14, 15).  These include
sites within uncharacterized loci and within genes for Osp proteins,
flagellin, and ribosomal RNA.  Considerable interest and efforts are being
exerted toward developing amplification assays for confirming infections with
Lyme spirochetes, and many other microorganisms.


                  _______________________________________
                  PRACTICAL CONSIDERATIONS (PROS AND CONS)


1)  Isolation

As with any infection, the definitive confirmatory test is isolation of the
causative agent.  It is also desirable to have access to isolates from
patients for evaluating the antibiotic susceptibility and monitoring the
epidemiology of particular isolates or strains.  The low density infection
and slow growth rate, characteristic of Lyme spirochetes, can present several
difficulties in obtaining primary isolates.  Many studies have considered
methods by which to optimize the probability of obtaining spirochetal
cultures from patients (3, 4).  In general, the most reliable clinical
samples for culturing the spirochetes are skin biopsies taken from the
margins of erythema migrans lesions and from ear punches, and samples of
cerebrospinal fluid.  Although spirochetemia is known to occur, blood is not
a reliable sample for culture.  Techniques which have been developed for
capturing spirochetes onto antibody-activated surfaces prior to culturing,
may enable concentration of spirochetes from relatively large volume clinical

samples and may help reduce the potential for contamination of cultures.

2)  Histology

Histological examination of clinical samples can provide conclusive evidence
of infection when Lyme spirochetes are observed.  Coupling the microscopic
examination with an immunoassay such as immune fluorescence or immune
histochemistry, can enable an evaluation of whether structures resembling
spirochetes react with titrated antibodies specific for Lyme spirochetes.
However, since Lyme is characterized by a low density infection, thorough
histological examination of sectioned tissues can be laborious.  Moreover,
Lyme cannot be ruled out in patients with negative histology.  No recent
improvements for histological confirmation of Lyme disease have been proposed
or developed.



3)  Serology

As noted above, all FDA-cleared and commercially available Lyme test systems
are serological.  In addition many public, private, and educational
laboratories, have developed and use their own serological assays for Lyme
disease.  Serological assays are advantageous in that they are readily
available; they generally produce rapid results; and guidelines for their use
and interpretation are available from manufacturers, published literature,
and governmental agencies such as the CDC and state health departments.


Numerous studies have evaluated commercially available Lyme test kits.  Among
these studies there is virtually no consensus on the sensitivity and
specificity, hence the accuracy and utility of the kits is brought into
question.  In perhaps the most comprehensive comparison of kits marketed in
the U.S., the CDC evaluated the performance of 20 kits using a set of 53
human sera from patients who were clinically defined as positive or negative
for Lyme disease.  Figures 1 and 2 show the results of comparisons between
the seven most accurate kits and the clinical definition of the samples (Fig.
1), and the degree of consistency obtained by each kit when the sera were
tested at three different reference laboratories (Fig. 2).



Figure 1.  Sensitivity and specificity of Lyme test kits, based on clinical
definition.
___________________________________________________________________
Percent:     20        40        60        80        100
___________________________________________________________________
1  :NNNNNNNNNNNNNN
  :CCCCCCCCCCCCCCCCCCCCCCCCCCCCC
2  :NNNNNNNNNNNNNNNNNNNNNNNNN
  :CCCCCCCCC
3  :NNNNNNNNNNNNNNNNNNNNNNNNNN
  :CCCCCCCCCC
4  :NNNNNNNNNNNNNNN
  :CCCCCCCCCCCCCCCCCCCCCCC
5  :NNNNNNNNNNNNNNNNNNNNNNNNNNNNN
  :CCCCCC
6  :NNNNNNNNNNNNNNNNNNN
  :CCCCCCCCCCCCCCCCCCCC
7  :NNNNNNNNNNNNNNNNNNNNNNNNN
  :CCCCCCCCCC
WB :NNNNNNNNN
  :CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC
__________________________________________________________________
Abbreviations: N-sensitivity; C-specificity; WB-western blot
Source: U.S. Public Health Service, Centers for Disease Control



Figure 2.  Average sensitivity and specificity of Lyme test kits
analyzed at three independent laboratories.
__________________________________________________________________
Percent:    20       40        60        80       100
__________________________________________________________________
1 :NNNNNNNNNNNNNNNNNNNN
 :CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC
2 :NNNNNNNNNNNNNNNNNNNNNNNNNNN
 :CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC
3 :NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
 :CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC
4 :NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
 :CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC
5 :NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
 :CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC
6 :NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
 :CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC
7 :NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
 :CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC
___________________________________________________________________
Abbreviations: N-sensitivity; C-specificity;
Source: U. S. Public Health Service, Centers for Disease Control


Such results indicated that a significant degree of variability could be
expected when these kits were used to screen patient sera for evidence of
infection with Lyme spirochetes.  Similar independent evaluations of test
systems, performed at many different locations, have been consistent with
these results.  Taken together, such findings have led physicians to place
limited confidence on the results of commercial test systems.  Several
factors could contribute to the variability and error rate associated with
these systems.  These include the inherent variability of patient humoral
responses, the stage of infection at the time of serum sampling, the
antigenic similarity of related commensal and pathogenic spirochetes (which
can lead to false-positive reactions), and the antigenic variability of Lyme
spirochetes within and between different geographic locations (which can
result in false-negative reactions).  Recently, purified spirochetal antigens
or recombinant antigens have been included in some kits in an attempt to

enhance the accuracy of the kits.  However, evidence is mounting that a
significant proportion of Lyme cases, particularly congenital cases, are
seronegative.  Thus, considerable effort has gone into development of
alternative testing protocols.


4)  Antigen detection systems

Both antibody based and T-cell based antigen detection systems have been
developed, but none has been cleared and marketed for clinical use.  These
systems offer a potential advantage over serological assays in that
development of an immune response to the infection is not necessary for
detection of spirochetal antigens in a sample.  Such tests can also
effectively screen ticks, and a variety of mammalian fluids and tissues for
the presence of Lyme antigens.  Like serological tests, the antibody-based
antigen detection systems are also rapid.  Limitations of antigen detection
systems include the possibility that antigens may be excreted or remain in
hosts for extended periods after effective treatment.  Furthermore, these
systems are vulnerable to antigenic variation among different strains and
species of Lyme spirochetes, particularly when based upon recognition of
individual epitopes.  One recent improvement to antigenic detection systems
is the use of a capture step which allows for immobilization and
concentration of antigens from relatively large volumes of samples prior to

testing, and can also allow for capturing intact spirochetes for subsequent
aseptic isolation and culture.


5)  Nucleic acid detection systems

Systems for detection of spirochetal nucleic acids by hybridization, PCR, and
LCR, also offer the potential advantage of being independent of the host
immune response, and being applicable to screening ticks.  For practical
reasons discussed above, screening of samples for Lyme spirochetes by
hybridization techniques are usually limited to ticks and to cultured
spirochetes.  Detections by PCR and LCR can be sufficiently sensitive to be
used successfully on clinical samples.  Furthermore, it is likely that
thermocyclers will increasingly become common equipment for clinical
laboratories as enzyme chain reaction diagnostic protocols are established
for more pathogens.  Potential problems with diagnostic use of PCR and LCR in
Lyme cases include the vulnerability of the technique to mutation and genetic
sequence diversity within the templet DNA target site, presence of enzyme
inhibitors in clinical samples, and the potential for chance contamination of
clinical samples by templet DNA molecules.  Enzyme inhibitors can include a

wide variety of agents such as salts, proteases, detergents, chelating
agents, and solvents.  If necessary, these can be removed before
amplification by common DNA extraction techniques.  Genetic stability of
target sequences has been addressed by developing primer sets which bind to
evolutionarily-conserved, yet species-specific sequences within the genes for
ribosomal RNA.  Furthermore, use of ultraviolet radiation to covalently
cross-link any potentially contaminating DNA in vessels and reagents, and on
equipment used in the PCR and LCR protocols, has helped reduce false-positive
amplifications.  Finally, the potential for contamination can be monitored by
performing all steps in the amplification protocols with mixtures to which no
known source of templet DNA has been added.


                    ___________________________________
                                  SUMMARY


In summary, variability and uncertainty with currently available serological
techniques for rapid laboratory confirmation of Lyme disease limit the
utility of such tests.  Such uncertainty compounds the difficulty of
physicians to either confirm or rule out Lyme disease in patients with
symptoms and histories consistent with infection by Lyme spirochetes.
Questions surrounding the ability to accurately diagnose Lyme disease have
been cited as reasons behind delayed or inappropriate treatment of patients,
and in disallowance of insurance coverage for treatment.  During the last few
years, several new approaches for confirming infections have been
investigated and developed.  These include detection of spirochetal antigens
by T-cell proliferative assays, antibody-based capture and detection assays,
and detection of spirochetal nucleic acids by enzyme chain reactions.
Continued development and evaluation of these techniques may provide
effective tools to facilitate monitoring infections by Lyme spirochetes in
arthropod and mammalian hosts.


                     __________________________________
                              ACKNOWLEDGMENTS


Special thanks go to Ronald Harbeck, Leonard Mayer, and Roxanne Shively for
critical review of this article, and to Betty Kester for help in preparing
the article.
                     __________________________________


                                 REFERENCES

1.   Burgdorfer, W., et al. 1982.  "Lyme disease--a tick-borne
    spirochetosis?"  Science 216:1317-1319.
2.   Baranton, G., et al. 1992  "Delineation of Borrelia
    burgdorferi sensu stricto, Borrelia garinii sp. nov., and
    group VS461 associated with Lyme borreliosis."  Int. J. Syst.
    Bacteriol.  42:378-383.
3.   Steere, A. C. 1989.  "Lyme disease."  New. Engl. J. Med.
    321:586-596.
4.   Szczepanski, A., and J. L. Benach.  1991.  "Lyme borreliosis:
    host responses to Borrelia burgdorferi."  Microbiol. Rev.
    55:21-34.
5.   Callister, S. M., et al. 1991.  "Lyme disease assay which
    detects killed Borrelia burgdorferi."  J. Clin. Microbiol.
    29:1773-1776.
6.   Dressler, F. el al. 1991.  "The T-cell proliferative assay in
    the diagnosis of Lyme disease."  Ann. Int. Med.  115:533-539.
7.   Hyde, F. W., et al. 1989.  "Detection of antigens in urine of
    mice and humans infected with Borrelia burgdorferi, etiologic
    agent of Lyme disease."  J. Clin. Microbiol.  27:58-61.
8.   Dorward, D. W., et al.  1991.  "Immune capture and detection

    of extracellular Borrelia burgdorferi antigens in fluids and
    tissues of ticks, mice, dogs, and humans."  J. Clin.
    Microbiol.  29:1162-1171.
9.   Dorward, D. W., and C. F. Garon.  Unpublished data.
10.  Schwartz, J., et al.  1991.  "Determination of Borrelia
    burgdorferi infection rates in Ixodes dammini ticks by three
    methods."  Abstracts of the General Meeting of the American
    Society for Microbiology  91:80.
11.  Rosa, P. A., and T. G. Schwan.  1989.  "A specific and
    sensitive assay for the Lyme disease spirochete Borrelia
    burgdorferi using the polymerase chain reaction."  J. Infect.
    Dis.  160:1018-1029.
12.  Persing, D. H., et al.  1990.  "Detection of Borrelia
    burgdorferi DNA in museum specimens of Ixodes dammini ticks."
    Science  249:1420-1423.
13.  Goodman, J. L., et al.  1991.  "Molecular detection of
    persistent Borrelia burgdorferi infection in the urine of
    patients with active Lyme disease."  Infect. Immun.  59:269-
    278.
14.  Hu, H., et al.  1991.  "Detection of Borrelia burgdorferi by

    ligase chain reaction."  Abstracts of the General Meeting of
    the American Society for Microbiology.  91:79.
15.  Marconi, R. T., and C. F. Garon.  1992.  "Development of
    polymerase chain reaction primer sets for diagnosis of Lyme
    disease and for species-specific identification of Lyme
    disease isolates by 16S rRNA signature nucleotide analysis."
    J. Clin. Microbiol.  30:2830-2834


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III. ***** JARGON INDEX *****

Bb - Borrelia burgdorferi - The scientific name for the LD bacterium.
CDC - Centers for Disease Control - Federal agency in charge of tracking
     diseases and programs to prevent them.
CNS - Central Nervous System.
ELISA - Enzyme-linked Immunosorbent Assays - Common antibody test
EM - Erythema Migrans - The name of the "bull's eye" rash that appears in
    ~60% of the patients early in the infection.
IFA - Indirect Fluorescent Antibody - Common antibody test.
LD - Common abbreviation for Lyme Disease.
NIH - National Institutes of Health - Federal agency that conducts medical
     research and issues grants to research interests.
PCR - Polymerase Chain Reaction - A new test that detects the DNA sequence
     of the microbe in question.  Currently being tested for use in
     detecting LD, TB, and AIDS.
Spirochete - The LD bacterium.  It's given this name due to it's spiral
     shape.
Western Blot - A more precise antibody test.



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