CLINICAL MICROBIOLOGY REVIEWS, Apr. 1990, p. 132-152 Vol. 3, No.

2
0893-8512/90/020132-21$02.00/0
Copyright © 1990, American Society for Microbiology

Immunoserology of Infectious Diseases

KAREN JAMES
Central DuPage Hospital, Winfield, Illinois 60190,t and Loyola University Medical Center,
Maywood, Illinois 60153

IMMUNE RESPONSE TO MICROORGANISMS ........................................................... 133

Immunity to Bacteria ........................................................... 133
Immunity to Viruses ........................................................... 133
Immunity to Fungi ........................................................... 133
TEST SYSTEMS FOR IMMUNODIAGNOSIS OF INFECTIOUS DISEASES ................................... 133
Nonspecific Indicators of Infectious Diseases ........................................................... 133
CRP ............................................................ 133
Endotoxin ........................................................... 134
TNF ........................................................... 134
Antibody Production and Purification ........................................................... 134
Polyclonal antibodies ........................................................... 134
Affinity-purified antibodies ........................................................... 135
Fractionated antibodies ........................................................... 135
MAbs ........................................................... 135
Soluble Antigen-Antibody Reactions ........................................................... 135
Double diffusion in agar (Ouchterlony reactions) ........................................................... 135
CIE ........................................................... 135
Particulate Antigen-Antibody Reactions ........................................................... 135
Hemagglutination assays ........................................................... 135
HI assays ........................................................... 136
Latex agglutination (LA) ............................................................ 136
Coagglutination ........................................................... 136
Lytic Assays ........................................................... 136
CF ........................................................... 136
Neutralization assays ........................................................... 136
Immunohistochemical Techniques ........................................................... 136
Direct IFAs ........................................................... 137
Indirect IFAs for total antibody ........................................................... 137
Indirect IFAs for IgM antibody ........................................................... 138
False-positive and false-negative IFAs for IgM antibody .......................................................... 138
Amplification IFAs ........................................................... 138
Immunoassay Techniques ............................................................ 139
Rapid EIAs to detect bacterial antigens ........................................................... 139
Solid-phase methods for detection of antibodies ............................................................ 139
IgM and IgG separation methods ........................................................... 140
Capture assays ........................................................... 140
SELECTION OF METHODS FOR CLINICAL LABORATORY USE ............................................. 141
Detection of Antibody to Verify Immunity ........................................................... 141
Preemployment screening ........................................................... 141
Prenatal screening ........................................................... 141
Pretransplant screening ........................................................... 143
Detection of Antibody to Diagnose Disease ........................................................... 143
Acute and convalescent specimens ........................................................... 143
IgM-specific assays ........................................................... 143
Method comparisons ........................................................... 144
Congenital infections ........................................................... 144
Transplantation and immunosuppression ........................................................... 144
Serologic tests for syphilis ............................................................ 145
Streptococcal antibodies ........................................................... 145
EBV antibodies ........................................................... 145
Legionella antibodies ........................................................... 146
Rickettsia antibodies ............................................................ 146
Lyme disease serology ........................................................... 146
Antibodies to other microorganisms ............................................................ 147

t Address for corrrespondence.

132
VOL. 3, 1990 IMMUNOSEROLOGY OF INFECTIOUS DISEASES 133

Other viral antibodies ............................................. 147

Detection of Soluble Antigen Correlates with Active Disease ............................................. 148
Neonatal bacterial infections .............................................148
Neonatal viral infections ............................................. 148
Group A streptococcal infections in children .............................................148
Other microbial antigens ............................................. 148
Other viruses ............................................. 149
CONCLUSIONS ............................................. 149
ACKNOWLEDGMENTS ............................................. 149
LITERATURE CITED ............................................. 149

IMMUNE RESPONSE TO MICROORGANISMS
Immunity to Bacteria
The immune response to extracellular bacteria must coun-
teract all of the mechanisms of invasion elicited by these
organisms (22, 26). The immune response includes antibod-
ies to capsular polysaccharides, to exotoxins (e.g., antistrep-
tolysin 0 [ASO]), and to extracellular enzymes (antihyalu-
ronidase). Antibodies to tetanus toxin or diphtheria toxin
can neutralize the effects of these toxins and prevent host
tissue destruction (16).
Complement activation promotes effective opsonization
with and without antibody (67). The membrane attack com-
plex of the terminal complement components are required to
lyse and eliminate certain gram-negative organisms (Neis-
seria spp.). For other gram-negative bacteria, a synergistic
destruction by complement in conjunction with lysozyme is
necessary (21). Complement activation is also necessary to
release chemotactic factors to attract phagocytic cells to the
site of infection.
Endotoxins elicited from certain gram-negative bacteria
can initiate the activation of the complement alternative
pathway in the absence of antibody. Endotoxin can also
degranulate neutrophils, enhance cytotoxicity, and prompt a
variety of other severe metabolic and potentially lethal
effects if gram-negative bacterial infections are not efficiently
treated (8).
Immunity to intracellular pathogens is primarily cellular
immunity, i.e., delayed T-cell hypersensitivity involving
lymphocytes, cytokines, and macrophages (48). There are
only two methods available to detect delayed T-cell hyper-
sensitivity: in vivo cutaneous injection of purified antigens
(skin or anergy testing) or in vitro lymphocyte transforma-
tion studies with purified antigens. Neither method is highly
reproducible and may be falsely negative due to the immu-
nosuppression experienced by individuals suffering from
invasion by intracellular pathogens. In many situations,
antibody is produced, but serves no demonstrable protective
mechanism. If antibody production is stimulated by the
intracellular pathogen, detection of that antibody and its
class specificity can be useful in diagnosing the invading
organism(s).
Immunity to Viruses
Antibody (immunoglobulin G [IgG] and IgM) capable of
binding directly to extracellular viruses may prevent viruses
from infecting other cells. If the virus has a viremic phase,
neutralizing antibodies may be produced. Two types of
neutralizing antibodies that can be demonstrated are com-
plement independent and complement facilitated. Antibod-
ies of the G, M, and A classes have been shown to neutralize
the infectivity of virtually all known viruses (21). Intercellu-
lar or vertically transmitted viruses or both would not be
subject to the neutralizing effects of antibodies.
Antibody can also diminish infectivity of viruses by pre-
venting attachment to the specific receptor or by introducing
conformational changes in the viral structure that promote
aggregation. Aggregation facilitates more effective elimina-
tion by antibody-mediated mechanisms such as opsonization
or complement activation or both. Arboviruses and hepatitis
B virus are examples of viruses that can be eliminated by
antibody-mediated events during their release into the blood-
stream or lymphatics.
In certain situations, antibody to viral proteins can be
detrimental to the host. For example, serum antibody to
respiratory syncytial virus (RSV), which is not protective
but was passively acquired across the placenta from the
mother, may produce an Arthus (immune complex) type of
hypersensitivity reaction in the lungs of infants (20). Similar
damaging effects of antiviral antibody have been described
with measles infections in infants.
The immune response to intercellularly or vertically trans-
mitted viruses involves cell-mediated cytotoxicity. Cyto-
toxic effector cells recognize the alterations to the membrane
antigens that are perturbed by viruses and either require
specific (T-cell) or nonspecific (natural killer cells or macro-
phages) cytolytic effector cells (88). Antibody-dependent
cell-mediated cytotoxicity has also been shown to be an
effector mechanism of antiviral cytotoxicity (85).
Immunity to Fungi
Immunity to fungi is primarily cell mediated. Detection of
specific IgM and IgG antibodies to certain fungi by immu-
noprecitin reactions can be helpful in establishing the diag-
nosis and following the course of the disease. However, the
antibodies do not play a protective role.
TEST SYSTEMS FOR IMMUNODIAGNOSIS OF
INFECTIOUS DISEASES
Nonspecific Indicators of Infectious Diseases
The local response to infection or tissue injury or both is
acute inflammation, resulting in vascular changes and at-
tracting leukocytes. During the first few days following
insult, systemic and metabolic changes occur which com-
prise the acute-phase response (57). Acute-phase proteins
include those that usually increase by 50% (complement
components and ferritin), alpha-1-antitrypsin, fibrinogen,
and haptoglobin which increase 200 to 400%, and C-reactive
protein (CRP) which can increase up to 1,000% in severe
tissue injury. CRP and complement components are the only
acute-phase proteins that have been shown to be directly
involved in the elimination of microorganisms.
CRP. CRP is the prototype acute-phase protein. CRP was
originally recognized for its ability to precipitate with the
C-polysaccharide fraction of pneumococcus (95). CRP did
134 JAMES
350 50
300
40
~~~~ 200 ~~ ~ ~ ~ ~ ~ 2
250 7 method for detecting trace quantities of endotoxin. LPS
O polyphemus, the horseshoe crab (88). The Limulus assay is
~~~~~~~~~~~~~~~~~~O
Days post antigen stimdation
ECRP ----- IgM IgG ESR
FIG. 1. Nonspecific and specific immune responses in relation to
time after antigenic stimulation. ESR, Erythrocyte sedimentation
rate.
not appear to be an antibody to pneumococcus since its level
decreased when patients recovered from the pneumonia.
CRP was present in sera from patients with other bacterial
illnesses, but not detectable in normal human sera.
CRP binds to phosphocholine and galactose residues,
ligands which are widely distributed among microbial prod-
ucts including fungi, parasites, lactobacilli, and streptococci
(67). When CRP binds to these surfaces, it activates com-
plement in much the same manner as antigen-antibody
activation of Clq (10). When CRP and C3 are bound to an
organism, phagocytosis is promoted by this opsonization
(23). Human CRP provides protection in vivo from a lethal
dose of Streptococcus pneumoniae in mice (99).
CRP is distinctive among human acute-phase proteins
because it is usually present in nanogram-per-milliliter con-
centrations, but can increase dramatically and rapidly to
hundreds of micrograms per milliliter within 3 days (9, 28).
The highest CRP levels are found in patients with bacterial
infections (>100 ,ug/ml), while moderate CRP elevations (10
to 100 ,ug/ml) are commonly found in chronic inflammatory
conditions such as autoimmune diseases, malignancies, al-
coholic hepatitis, congestive heart failure, and pregnancy
(68). When elevated CRP levels are found in patients with
chronic inflammation, superimposed bacterial infections
have been confirmed (68).
CRP elevates more rapidly and decreases sooner with
resolution of the infectious process than does the erythocyte
sedimentation rate, the classic nonspecific indicator of in-
flammation (Fig. 1). Measurement of spinal fluid CRP levels
has been shown to be sensitive and specific for differenti-
ating bacterial from viral meningitis, enabling efficient ther-
apeutic intervention (13, 72). Serum CRP levels have been
used to differentiate patients with bacteremia from those
with contaminated blood cultures (63), monitor treatment of
infective endocarditis (64), monitor spinal cord-injured pa-
tients for earlier detection of urinary tract infections (59),
differentiate pyelonephritis from cystitis in children (45) and
adults (68), differentiate bacterial pneumonia from acute
bronchitis (68), and monitor for postoperative infections (28)
and for other noninfectious disease applications (68, 73).
Endotoxin. Endotoxins are the lipopolysaccharide (LPS)
components of the outer membrane of gram-negative bacte-
ria. When released in vivo, endotoxin has toxic and pyro-
genic properties (65), including release of interleukin-1 and
tumor necrosis factor (TNF). Administration of endotoxin
can prompt severe metabolic and physiologic disturbances
CLIN. MICROBIOL. REV.
(including the production of TNF) referred to as septic or
gram-negative shock (65). Endotoxins are phagocytized and
detoxified by the liver; consequently, the concentration of
detectable LPS is high in portal blood, but often not detect-
able in the peripheral blood circulation (94).
The Limulus lysate assay is currently the only available
causes gelation of an extract from the lysate of Limulus
not specific for a particular microorganism, but detects LPS
from all gram-negative bacteria including Eschericia coli,
Neisseria meningitidis, and Haemophilus influenzae. Since
endotoxin is so rapidly cleared from peripheral blood, de-
tecting LPS in serum is unreliable, but detecting endotoxin
in cerebrospinal fluid (CSF) is a sensitive indicator of the
presence of gram-negative bacterial meningitis (88).
TNF. TNF is a cytotoxin which participates in the immune
response to microorganisms as well as effects the antitumor
activity for which it was named. TNF was first characterized
as an activity which appeared in murine serum after injection
with Mycobacterium bovis BCG (bacillus Calmette-Gudrin)
and LPS. When sera containing TNF were injected into
tumor-bearing mice, necrosis of the tumor was induced and
the tumor regressed (78). TNF can induce interleukin-1
production and can induce a factor that is directly cytotoxic
to malaria and other parasites (8).
Cachectin was first described when investigators were
searching for the mediators responsible for cachexia (wast-
ing) associated with parasitic infections (8). Subsequently,
cachectin and TNF were shown to have strong DNA se-
quence homologies and are now considered to be identical
cytotoxins.
When recombinant TNF is administered to mice, several
pathologic events occur, including severe metabolic acido-
sis, marked hemoconcentration, biphasic changes in blood
glucose concentrations, severe pulmonary leukostasis and
edema, hemorrhagic necrosis of adrenals and pancreas, and
tubular necrosis of the kidney (8). When TNF was neutral-
ized with (actively or passively acquired) antibodies, these
pathologic changes were prevented.
Recently, methods have become available to measure
TNF levels in human sera. Waage et al. (96) noted a
correlation between TNF levels and the degree of septic
shock and subsequent death in a series of patients with
meningococcal septicemia. Although TNF is a nonspecifi-
cally induced cytotoxin, detection of TNF may provide an
indicator of the extent of damage or the nature of the
infectious process or both.
Antibody Production and Purification
Polyclonal antibodies. Antisera used in many of the assay
systems to be described are prepared by hyperimmunizing
animals (rabbits or goats) with purified antigens emulsified in
adjuvants. After 6 to 8 weeks of injections, the animals are
bled and serum samples are removed and tested to ensure
the monospecificity of the resulting antisera for the antigen
injected. If contaminating antibodies or antibodies with
other specificities are present, they are removed by absorp-
tion with their corresponding antigens. The immunoglobulin
fraction of the animal sera is isolated by selective salt
precipitation (NH4SO4) or by ion-exchange chromatogra-
phy. Even a high-titered antibody represents only 10 to 15%
of the total IgG fraction of a polyclonal antibody. The
remaining IgG molecules are of undefined specificity, re-
flecting the myriad of antigens to which the animal has been
VOL. 3, 1990
exposed during its lifetime. It is necessary to characterize
painstakingly each polyclonal antibody to ensure that it is
detecting only the desired antigen. Still, the high percentage
(85 to 90%) of undesired antibodies increases the occurrence
of nonspecific binding of conjugated (but irrelevant) antibod-
ies in test systems.
Affinity-purified antibodies. To eliminate the antibodies of
undesired specificity, the IgG fraction of a polyclonal anti-
body can be purified by binding to and eluting from an
insolubilized form of the antigen. This is performed by
column chromatography, using a matrix to which the immu-
nizing antigen is coupled with a spacer molecule (an inert
molecule that serves as a carrier). The spacer is covalently
coupled to the antigen and to the insoluble matrix to pre-
serve optimally the relevant antigenic determinants. The
antibody is then applied to the column, using conditions
favorable for antigen-antibody reactions to occur (pH 7.5 to
8.5, physiologic ionic strength). IgG molecules of unrelated
specificity would pass through the column with the wash
buffer. Antigen-specific IgG molecules are then eluted off the
antigen matrix, using an elution buffer with a lower pH and
ionic strength. In this way, the functional integrity of the IgG
molecules is preserved, while the antibodies are gently
dissociated from the insolubilized antigen. The resulting
affinity-purified antibody is significantly higher (85 to 95%) in
specific antibody activity. This process decreases or entirely
eliminates any nonspecific binding due to extraneous anti-
bodies, resulting in a much better reagent for use with
immunohistochemical assays.
Polyclonal antibodies that have been affinity purified have
many uses in the clinical laboratory. Most antibodies to
human immunoglobulins, conjugated with fluorescent or
enzyme labels, are affinity-purified polyclonal antibodies.
Polyclonal antibodies bind to several antigenic determinants,
increasing the ability to detect their respective antigens.
Fractionated antibodies. Antibodies in antisera used to
detect antigens in cultured cell lines (especially herpesvi-
ruses) bind nonspecifically to the IgG Fc receptors of the
cells. One effective approach to decrease that nonspecific
binding significantly is to fractionate the IgG antisera to
eliminate the Fc regions of the IgG antibody molecules.
Under controlled conditions, the enzyme pepsin sequentially
cleaves the C-terminal end of the IgG molecule (the Fc
region), leaving the antibody-combining N terminals [the
F(ab')2 region]. The F(ab')2 fragments can still be effectively
conjugated, e.g., with fluorochromes or enzymes. Affinity-
purified antibodies can be pepsin digested, resulting in
affinity-purified F(ab')2 antisera.
MAbs. The production of monoclonal antibodies (MAbs)
was first described in 1975 by Kohler and Milstein (53). The
potential applications for clinical laboratory assays and in
immunotherapy were immediately recognized. MAbs are
prepared by hybridizing antibody-forming cells to continu-
ously replicating cell lines. Each antibody-forming cell,
programmed to produce antibody of a single (mono-) speci-
ficity with a single heavy-chain and single light-chain class,
can be cloned to replicate itself almost indefinitely. These
antibody-producing clones of cells (hybridomas) are used to
prepare virtually unlimited quantities of MAbs that are
chemically, physically, and immunologically completely ho-
mogeneous and definitively characterizable (93). Hybridoma
cells can be stored indefinitely, frozen in liquid nitrogen. As
needed, these hybridomas can be grown in large quantities in
tissue culture or propagated in syngeneic mice to form
ascites from which MAbs can be isolated.
The advantages of MAbs are obvious: the minimal purifi-
IMMUNOSEROLOGY OF INFECTIOUS DISEASES 135
cation required ensures optimal functional activity; charac-
terization of the specificity needs to be done only once
instead of each time an animal is immunized; MAbs gener-
ally do not bind to human IgG Fc receptors; and they
generally are unencumbered by nonspecificity or cross-
reactivity (93).
The disadvantages of MAbs are more subtle: a single
specificity does not enable effective cross-linking of antigens
with antibodies so one MAb cannot be used in agglutination
or precipitation reactions, although "cocktails" of several
MAbs can overcome that limitation; murine MAbs do not
efficiently bind Clq to activate complement so are not useful
in assays that rely on complement activation; the single
antigenic determinant to which a MAb binds may not be
expressed under certain conditions of antigen presentation
(e.g., viable versus fixed organisms). Nevertheless, an en-
tirely new field of study is available to use these reagents
effectively for clinically applicable testing systems.
Soluble Antigen-Antibody Reactions
Double diffusion in agar (Ouchterlony reactions). The classic
method for detecting antibody and evaluating its specificity
(identity, partial identity, or nonidentity) is Ouchterlony dou-
ble diffusion. Antigens can also be characterized by Ouchter-
lony diffusion when antibodies of known specificity are avail-
able. Since the procedure requires 18 to 24 h of diffusion for
the reactions to occur, this method is not as helpful in the
rapid diagnosis of acute infections as other methods discussed
below. The Ouchterlony reaction is primarily used to detect
antibodies in patients with suspected histoplasmosis, coccid-
iomycosis, or aspergillosis and to detect other fungal antigens
associated with hypersensitivity pneumonitis.
CIE. Counterimmunoelectrophoresis (CIE), one-dimen-
sional double electrophoresis, specifically directs the move-
ment of antigen and antibody toward each other in an
electric field. The buffer pH is selected to optimize the
electroendosmotic effects of antibody toward the cathode
(negative pole) while the antigen moves toward the anode
(positive pole). This electrophic movement rapidly (30 min)
concentrates the antigen and antibody in the zone between
the adjacent wells. CIE is approximately 10 times more
sensitive than double diffusion. This was the original method
used to detect hepatitis B surface antigen and antibody
known then as Australian antigen and antibody. CIE has also
been useful for rapid identification of antigens from bacteria
associated with meningitis, septicemia, disseminated intra-
vascular coagulation, pneumonia, and septic arthritis (88).
Depending on the sensitivity and specificity of the antibody
used, minimal detectable concentrations of bacterial antigen
range from 50 to 10 ng/ml (30). CIE has largely been replaced
with particulate antigen-antibody reactions for detecting
many bacterial antigens.
Particulate Antigen-Antibody Reactions
Hemagglutination assays. A variety of antigens can be
coupled to erythrocytes (RBCs) to provide the indicator
system to detect antibodies. Target antigens such as polysac-
charides readily adhere to RBCs, including antigens from E.
coli, N. meningitidis, and Toxoplasma sp. as well as purified
protein derivative from M. tuberculosis. Carbohydrate anti-
gens readily adhere to RBCs, but protein antigens require
pretreatment with tannic acid (producing "tanned" RBCs)
or with chromium chloride. Tanning the RBCs facilitates a
high-density coating which increases the sensitivity of the
test system. Subsequent Formalin or glutaraldehyde treat-
ment of tanned RBCs coated with either protein or carbo-
136 JAMES
hydrate allows long-term storage. Treated tanned RBCs
coated with protein antigens have been used for detection of
antibodies to toxins, e.g., diphtheria toxin or tetanus toxin
(16). Treponemal antibodies are detected by using trepone-
mal antigens adsorbed to tanned RBCs in the micro-hemag-
glutination assay for Treponema pallidum (MHA-TP) (98).
HI assays. Hemagglutination inhibition (HI) assays used in
infectious disease serology are based on the capacity of
certain viral antigens to agglutinate RBCs of selected species
spontaneously. Antibodies present in patient sera prevent
the spontaneous agglutination of the RBCs, thus resulting in
inhibition of agglutination, indicating a positive test for the
presence of antibody. HI was the original method used to
detect antibodies to rubella virus (76). HI tests cannot
distinguish between IgM and IgG classes of antibodies.
Immunoassay techniques which have replaced HI for detec-
tion of rubella virus antibodies will be reviewed below.
Antibodies to other less prevalent hemagglutinating viruses
are still detected by the HI method. These include influenza
viruses, arboviruses, reoviruses, and certain enteroviruses
(49).
Latex agglutination (LA). Latex particles are spheres of
polystyrene which readily bind IgG molecules by the Fc
region at pH 9.0 when a low-ionic-strength buffer is used.
When antibodies are bound by their Fc region, the antibody-
combining sites [F(ab) regions] remain exposed and are
capable of binding antigens. When the target antigens have
repetitive antigenic structures (e.g., polysaccharides), mul-
tivalent antibodies coupled to multiple latex particles can
bind antigen molecules and cross-link the latex particles,
resulting in agglutination. The latex particles serve as the
indicator system to detect the antigen-antibody reaction.
Theoretically, any antigen (but not hapten) to which
antibodies can be produced should be detectable by LA. LA
is particularly useful to detect bacterial polysaccharide anti-
gens in CSF or urine or both. LA has essentially replaced
CIE for the detection in CSF of capsular antigens of H.
influenzae type b, several N. meningiditis groups, and S.
pneumoniae for diagnosing bacterial meningitis and for
detection of Cryptococcus neoformans capsular antigens in
immunosuppressed patients. Group B streptococcal antigens
are detected by LA in urine or CSF from newborns.
Coagglutination. Staphylococcus aureus (Cowan strain)
contains protein A distributed evenly on the outermost layer
of the cell wall. Protein A binds the Fc region of IgG
subclasses 1, 2, and 4 (which constitute 95% of the total
IgG), analogous to the binding of IgG to latex particles. The
antibody-coated Staphylococcus becomes the indicator re-
agent to detect the presence of antigens corresponding to the
specificity of the coupled antibody. Coagglutination has been
used to detect the presence of bacterial antigens in CSF or
urine. Coagglutination is useful in the immunologic identifi-
cation of bacteria from culture, with commercially available
reagents for grouping streptococci and detecting Staphylo-
coccus aureus coagulase production (30).
Since protein A is an effective cross-linking reagent for
most IgG subclasses, any other preformed complexes con-
taining IgG and antigen would also cause agglutination of the
sensitized Staphylococcus reagent. Nonspecific agglutina-
tion is prevented by treating the body fluid to be tested with
soluble protein A to block binding of preformed complexes
or by heating the body fluid to 100'C to denature patient IgG
molecules before testing.
Coagglutination must be well controlled to detect nonspe-
cific agglutination in the body fluid, using unsensitized
Staphylococcus particles and particles sensitized with an
CLIN. MICROBIOL. REV.
irrelevant, but species-specific antibody. Coagglutination
reagents have a much shorter shelf life than latex reagents
due to deterioration of the bacteria upon storage. Many of
the clinical laboratory applications of coagglutination have
been essentially replaced by LA or immunoassay tech-
niques.
Lytic Assays
CF. Complement fixation (CF) is a two-step procedure
which uses complement to lyse indicator RBCs. The first
step involves reacting antigen with antibody in the presence
of complement in fluid phase. The complement used must be
from a standard source, e.g., rabbit serum which has been
appropriately collected and stored to preserve the hemolytic
activity. If corresponding antigens and antibodies are
present, the complement cascade will be activated through
the classical pathway. RBCs coated with RBC antibody (the
indicator system) are added to the first reaction mixture. If
complement had been activated (fixed) during the first incu-
bation, the indicator particles are not lysed. If the first step
did not contain either the specific antigen or antibody,
complement will bind to the antigen-antibody complex
present on the indicator particles, and lysis of the indicator
RBCs will occur.
CF is a semiquantitative method that can be used to detect
either antigens or antibodies if the corresponding specific
antibody or antigen is available. CF does not distinguish IgM
from IgG antibodies since both can fix complement. This
method is extremely sensitive and has broad applications for
infectious disease serology, but is rarely used outside of
reference laboratory situations because it is cumbersome
and complex (93).
CF may be the only method available for detecting anti-
body to less prevalent viruses (e.g., coxsackieviruses). An
advantage of the CF method is that, by keeping all other test
parameters unchanged, many antigens can be tested to
determine population exposure to rare organisms.
Neutralization assays. Beta-hemolytic group A strepto-
cocci produce a number of extracellular toxins that stimulate
the production of antibodies by infected patients (54). Sev-
eral of these toxins also function as hemolysins and lyse
RBCs. Specific antibodies neutralize the hemolysins and
inhibit RBC lysis. Analogous to the CF test, a positive test is
negative for hemolysis, and in a negative test hemolysis is
detectable. In the first step of the reaction, patient serum
(antibody) is incubated with the specific hemolysin being
assayed (antigen). In the second step, group 0 RBCs are
added. If the hemolysin has been neutralized during the first
incubation, the indicator particles will not lyse. If specific
antibody was not present to neutralize the hemolysin, the
RBCs will lyse. ASO is the most commonly used neutrali-
zation test to detect immunologic evidence of exposure to
streptococci.
Immunohistochemical Techniques
The most widely used immunohistochemical techniques
are immunofluorescence assays (IFAs). IFA uses tissue or
bacterial cells as the substrate (source of antigen) affixed to
a glass slide and a fluorochrome-conjugated (antibody) de-
tection system. IFA remains the "gold standard" for many
infectious disease serology test systems. The advantages of
IFA are that (i) the substrate can be visualized, ensuring
specificity of the reaction; (ii) it is significantly less cumber-
some than CF; (iii) it is highly reproducible when performed
VOL. 3, 1990 IMMUNOSEROLOGY OF INFECTIOUS DISEASES 137

DOUBLE INDIRECT IFA

INDIRECT IFA
labeled rabbit
Labeled anti-goat Ig
goat antibody 2Goat antibody
Patient antibody Patient antibody

Antigens
.*.,.D....

ANTI-COMPLEMENT IFA AVIDIN-BIOTIN COMPLEX

C MATRIX ~ ~ D MATRIX:
FIG. 2. Amplification immunoassay systems. (A) Indirect IFA (shown for comparison) uses 'labeled goat anti-human immunoglobulin as
the indicator molecule to detect bound patient antibody. (B) Double-indirect IFA uses 'labeled rabbit anti-goat immunoglobubin to detect
unlabeled goat anti-human immunoglobulin which has bound to patient antibody. Fewer patient antibody molecules can be detected with
amplification techniques, thereby increasing sensitivity. (C) Anti-complement IFA uses 'labeled F(ab')2 goat anti-human immunoglobulin as
the indicator molecule to detect guinea pig 'complement bound to patient antibody. (D) Avidin-biotin complex reactions use 6biotinylated goat
anti-human immunoglobulin to bind to patient antibody. The indicator system is the 'labeled avidin which binds to the biotin.

by well-trained technologists; and (iv) in indirect IFA, the
same conjugate and dilution of patient sera can be used to
detect antibody to many different organisms or antigens.
The disadvantages of IFA are that it: (i) requires fresh or
frozen tissue or cells (processed tissue or smears fixed for
Grams stain are not usable); (ii) requires special equipment
and conditions (a fluorescence microscope and a dark room
for reading); (iii) is labor intensive, even when the substrate
for indirect IFAs can be purchased (reagent dilution, multi-
ple incubation and wash steps, and cover slips are required);
(iv) is subjective, requiring extensive training to read the
reactions and multiple controls to ensure test specificity and
sensitivity; and (v) has not been successfully automated.
Immunohistochemical assays have been developed that
use enzyme-conjugated antibodies (e.g., horseradish perox-
idase) with correponding substrates that yield different col-
ors when hydrolyzed to contrast with the colors of typical
histochemical strains. Immunoperoxidase techniques may
avoid the first two disadvantages of IFA, but the subjective
interpretation and labor intensiveness of immunoperoxidase
methods are even greater than with IFA. The systems
described below for direct and indirect IFA also apply to
immunoperoxidase techniques which have generally not
been used for microbiology applications.
Direct IFAs. Direct IFA is used to detect antigens or
organisms present in cells or tissues, using fluorochrome-
conjugated antisera (conjugate) specific for the antigen(s) in
question. Microbiologic applications of direct IFA include
detection of Chlamydia trachomatis elementary bodies in
columnar epithelial cells from the cervical canal, urethra,
eye, or rectum (2). Direct IFA is useful not only to detect the
microorganism, but also to evaluate whether the correct
and/or appropriate specimen was collected.
Treponema pallidum can be detected by direct IFA during
early stages of the disease when the organisms are concen-
trated in the chancre (primary) or in the mucocutaneous
lesions (secondary). Direct IFA, using conjugate to T. palli-
dum which has been absorbed with Reiter treponemes, has
essentially replaced dark-field examination as a diagnostic
test for early stages of syphilis before reaginic antibodies are
produced (4).
Direct IFA has been used successfully for detection of
Legionnella spp. (22) and for many viruses, including herpes
simplex virus (HSV) types 1 and 2, cytomegalovirus (CMV),
RSV, influenza virus types A and B, parainfluenza virus 1, 2,
and 3, varicella-zoster virus (VZV), and adenovirus (D.
Scholes, J. R. Daling, A. S. Stergachis, S. P. Wang, and J. T.
Grayston, Program Abstr. 28th Intersci. Conf. Antimicrob.
Agents Chemother., abstr. no. 1171, 1988). Enzyme-conju-
gated antibodies have also been used to detect these micro-
organisms in histologic tissue sections.
Indirect IFAs for total antibody. Indirect IFA is used to
detect antibodies in patient sera (Fig. 2A). Standardized
antigens (organisms or virus-infected cell cultures) are fixed
to glass slides. Patient serum is diluted, layered over the
substrate, and incubated to allow the antigen-antibody com-
plex to form. Unbound antibody is washed away, leaving
only bound antibody, which is then incubated with the
fluorescent conjugate. When antibodies are present in the
patient's serum, a second antigen-antibody reaction will take
place, with the conjugate becoming the third layer on the
slide.
138 JAMES CLIN. MICROBIOL. REV.

FALSE POSITIVE IgM ASSAY FALSE NEGATIVE IgM ASSAY

Labeled Anti-IgM

Patient antibodies

AnBige M
|B-; "if: 't' - . . -'. . '-"'.-' " .'-"'""-'...'.'..........
10
t
;
-

FIG. 3. Sources of error in specific IgM assays. (A) False-positive IgM assay due to patient RF binding to patient specific IgG. (B)
False-negative (or decreased intensity resulting in a borderline result) due to specific IgG competing with specific IgM for antigen-binding
sites.

For general testing, fluorescein-conjugated anti-human
immunoglobulin is usually anti-IgG with reactivity to both
kappa and lambda light chains. This light chain reactivity
also detects antibodies of the IgA or IgM class or both,
which would be advantageous to detect all classes of anti-
body reactive with microorganisms.
The classic indirect IFA is the fluorescent treponemal
antibody absorption (FTA-ABS) test. The antigen, T. palli-
dum Nichols, is fixed to glass slides. Prior to incubation with
the antigen, the patient's serum is absorbed with the non-
pathogenic T. pallidum Reiter. This absorption step signifi-
cantly increases the specificity of the test (18); however,
false-positive reactions can still occur in patients with au-
toimmune diseases (55) and hypergammaglobulinemia and in
those with Lyme disease due to immunological cross-reac-
tivity with antigenic sites on the spirochetes (60).
Other commonly performed indirect IFAs include
TORCH titers for evaluating the immune status of pregnant
women. TORCH tests include toxoplasma (TO), rubella
virus (R), CMV (C), and HSV (H). These organisms cause
significant congenital infections resulting in stillbirths or a
spectrum of congenital diseases, although infections in
adults or children frequently are mild or even subclinical.
Although once ordered as a panel of tests, current preferred
practice is to perform only the specific test based on the
mother's clinical and exposure history.
Indirect IFAs for IgM antibody. If the test system is
designed to detect antibodies produced during acute infec-
tion, the conjugate must be specific for IgM heavy chains
with no light-chain or other heavy-chain reactivity. When
congenital infections are suspected as the cause of stillbirth
or abnormalities of a newborn, indirect IFAs for IgM anti-
body should be used to detect IgM antibodies produced by
neonates. Unlike maternal IgG, IgM does not cross the
placenta, so any specific IgM antibodies detected would
have been produced by the fetus in response to a congenital
infection. Healthy newborns have 5 to 15% of the adult level
of total IgM since the in utero environment is essentially
sterile. If an organism is transmitted to the fetus from the
mother during gestation, the fetus will develop its own IgM
response to the organism, which would be detectable in an
IgM-specific indirect IFA.
False-positive and false-negative IFAs for IgM antibody.
Indirect IFAs for IgM antibody are subject to false-positive
reactions attributable to the presence of rheumatoid factor
(RF) activity in the serum being tested (Fig. 3A). RF binds to
IgG when IgG is bound to antigen. The process of binding to
an antigen causes a confirmational change in IgG which
exposes new antigens on the Fc region of the IgG molecule.
The IgM RF binds to these newly exposed IgG determinants.
Unless separation methods are used, IgM RF cannot be
distinguished from organism-specific IgM. Distinguishing RF
from organism-specific IgM is particularly important in neo-
natal sera since a high percentage of congenitally infected
neonates have detectable RF (31). False-positive IgM assays
due to heterotypic antibody responses between herpesvi-
ruses may also be detected; i.e., patients infected with
Epstein-Barr virus (EBV) or VZV may demonstrate CMV
IgM antibodies without evidence of CMV infection (56).
IgM assays can also be subject to false-negative results if
IgG antibody inhibits or competes with IgM for binding sites
on the antigen (Fig. 3B). To avoid any possibility of false-
positive or false-negative reactions in most methods, IgG
should be separated from IgM before the assays are per-
formed. Separation methods will be discussed below.
The demand for assays that detect IgM antibodies to CMV
and HSV has recently increased due to infections in organ
transplant patients. The immunosuppressed state of trans-
plant patients increases their susceptibility to these oppor-
tunistic viruses since both humoral and cell-mediated immu-
nity are required for optimal viral immunity. Although
specific IgG antibody may be present, it may not be as
effective in controlling viral infections in patients whose
cell-mediated immunity has been abrogated to prevent trans-
plant rejection. The production of IgM antibodies is T
independent, i.e., does not require the presence or cooper-
ation of T cells (77). IgM antibodies are produced in immu-
nosuppressed patients and can be useful indicators of acute
infection.
Amplification IFAs. The sensitivity of IFAs is limited by
the level of fluorescence detectable by the human eye. The
optimal fluorescein/protein ratio of a polyclonal antibody
conjugate is 2.5 (34), i.e., two to three fluorescein molecules
for each immunoglobulin molecule, which is satisfactory for
detecting high-density antigens such as bacterial or proto-
zoan cell surface antigens. Directly conjugating antibodies at
a higher fluorescein/protein ratio results in significantly
increased nonspecific staining. Detection of low-density
antigens or using MAbs or both may require a higher
fluorescein/protein ratio to achieve desired sensitivity.
Indirect or double-indirect IFA has been used to amplify
the fluorescent signal and improve sensitivity. If the antigen
can be detected by direct IFA, indirect IFA increases the
test sensitivity. If the test antibody is difficult to detect by
indirect IFA, a double-indirect IFA would increase the
sensitivity of detection (Fig. 2B). For example, if organisms
were not visible in the T. pallidum direct IFA, the test could
be repeated with unconjugated rabbit antibody to the
VOL. 3, 1990
treponemes, any unbound antibody could be washed off, and
the bound rabbit antibody could be detected by using a
fluorochrome-conjugated goat anti-rabbit IgG as the conju-
gate. Each rabbit IgG molecule contains multiple antigenic
sites which would be recognized by the goat anti-rabbit IgG.
If four goat anti-rabbit IgG molecules bind to the rabbit IgG,
the fluorescent intensity would be magnified four times.
Additional controls would need to be included in each assay
to ensure that the extra step did not decrease the specificity
while increasing the sensitivity.
Complement-amplified IFA, also called anticomplement
IFA, has been used primarily for detection of herpesviruses
(74). Herpesvirus-infected tissue culture cells have an en-
hanced expression of IgG Fc receptors that nonspecifically
bind IgG antibody molecules.
Anticomplement IFA uses complement as the third of four
layers (Fig. 2C). Antigen is the substrate on a slide; patient's
serum is added analogously to performing an indirect IFA. A
source of active complement (e.g., guinea pig) is added.
While IgG molecules bound to Fc receptors cannot bind
Clq, IgG or IgM bound to antigen by the Fab region does
bind Clq and activates the classical complement pathway.
After unbound complement and other proteins are washed
away, an F(ab')2, fluorochrome-conjugated, anti-guinea pig
C3 is added (74). Anticomplement IFA eliminates the need
to control for nonspecific IgG binding since a complement
component is detected instead of IgG. Although not effi-
ciently performed in a clinical laboratory setting, this four-
step procedure may be the only method available for detect-
ing antibodies to certain herpesvirus antigens (e.g., the
nuclear antigen of EBV [EBNA]).
A very versatile amplification technique is the avidin-
biotin complex. Biotin covalently coupled to antibody is
used for the primary reagent. Conjugated avidin is the
second reagent (Fig. 2D). Avidin has a very high binding
affinity (1015 Kin) for biotin, and each biotin molecule will
bind four avidin molecules (3). Avidin can be saturated with
fluorescein molecules without loss of its ability to bind to
biotin. This results in very bright specific staining with
minimal nonspecific staining. Controls are few compared
with other amplification methods and include conjugated
avidin alone and an unrelated biotinylated antibody.
A distinct advantage of the avidin-biotin complex system
is that the same biotinylated antibody can be used with
several different conjugated avidins, i.e., fluorescein (green
fluorescence), phycoerythrin (red fluorescence), peroxidase
(light microscopy), or ferritin (for electron microscopy). The
avidin-biotin complex technique works well with MAbs
because biotinylation is fast (1 h), gentle (reaction at pH 7.0
to 8.5 at 22°C), and efficient (biotinylation of 95% of the
amino groups of an antibody molecule does not alter the
antigen-binding capacity of the antibody) (35). Biotinylation
reagents can be purchased commercially (Vector Laborato-
ries, Burlingame, Calif.), mixed with the desired antibody,
and used the same day. Avidin conjugated to any indicator
system feasible to use is commercially available. Avidin-
biotin complex has not realized its full potential in the field of
microbiology.
Immunoassay Techniques
Rapid EIAs to detect bacterial antigens. A plethora of
commercially available products has been released in the
past few years which use enzyme immunoassay (EIA) tech-
nology for detection of proteins and bacterial antigens. Most
of the early methods were qualitative assessments of the
IMMUNOSEROLOGY OF INFECTIOUS DISEASES 139
presence or absence of an antigen by a color change of liquid
in a test tube, of a matrix on a stick or a paddle, or of a
matrix on a plastic reaction vial (e.g., ICON; Hybritech Inc.,
San Diego, Calif.). Most of these methods were developed to
meet the demands for more rapid test results. The tech-
niques were reported to be simple enough to be performed
by nontechnical employees such as those in a doctor's office
setting. Although the price per test was high, the EIA
technology represented a cost savings to doctors' offices
because the results were available before the patient left the
office. This was particularly beneficial for rapid testing for
streptococcal pharyngitis since the physician could prescribe
or withhold antimicrobial agents based on the test results
rather than providing empirical treatment or telephone fol-
low-up with the patient or both. Clinical microbiology labo-
ratories have resisted accepting and applying this new tech-
nology because of a high percentage of false-negative results
(25). The compromise acceptable to many laboratories is to
take two throat swabs: if the rapid test is positive, discard
the second swab; if the test is negative, use the second swab
for culture.
A recently introduced technique uses liposomes, artificial
lipid spheres, to detect group A streptococcal antigens (BBL
Microbiology Systems, Cockeysville, Md.). In this test,
specific antibody is adsorbed to a porous matrix. The patient
specimen is added and any antigen present binds to the
antibodies. Liposomes containing a colored dye inside con-
centric lamillar layers and coated with antibodies to the same
antigen are then added. If no antigen was bound by the first
antibody, the liposomes flow through the porous matrix. If
liposomes are bound, the dye is released by a wash solution
which lyses the liposomes, depositing the dye on the matrix.
A recent entry into the solid-phase EIA bacterial antigen
marketplace is Chlamydia antigen detection (P. Coleman, V.
Varitek, T. Grier, J. Hansen, G. Kurpiewski, J. Safford, B.
Marchlewicz, and I. K. Mushahwar, Program Abstr. 28th
Intersci. Conf. Antimicrob. Agents Chemother., abstr. no.
1184, 1988). The IFA method done in many clinical micro-
biology laboratories depends on receiving a satisfactory
specimen. With the solid-phase EIA technology, the results
are either positive or negative (Eastman Kodak Clinical
Products, Rochester, N.Y.; Abbott Laboratories Diagnos-
tics Div., Abbott Park, Tll.). A negative EIA could mean
either "no chlamydia" or "unsatisfactory specimen." Re-
ports of negative results should clearly state both possibili-
ties and be confirmed by the IFA method, similar to per-
forming a streptococcal culture when the rapid antigen test is
negative.
Rapid detection EIA methods for other sexually transmit-
ted diseases may be available to doctors' offices and even to
the general public in the very near future. Under develop-
ment are tests for gonococcus, trichomonas, and human
immunodeficiency virus. Concerns of clinical microbiolo-
gists include the loss of epidemiologic tracking. Rapid anti-
gen EIA methods simply detect gonococcal antigens and do
not evaluate antimicrobial susceptibility. Therefore, they fail
to detect antimicrobial agent-resistant strains of N. gonor-
rhoeae. To respond to resulting problems, clinical microbi-
ologists should be aware of the availability of these test
systems, their level of sensitivity, and their degree of spec-
ificity.
Solid-phase methods for detection of antibodies. Enzyme-
linked immunosorbent assays (ELISAs) are a variation on
the coated-tube assay described above, but the roles of the
antibody and the antigen are reversed. Instead of an anti-
body being the solid-phase component, antigen is coupled to
140 JAMES
a surface. The second layer of the sandwich is antibody from
the patient serum. The third layer is an enzyme-conjugated
anti-human IgG or IgM. The indicator system is the color
change resulting from cleavage of the substrate by the
conjugated enzyme. The intensity of the color is directly
proportional to the amount of patient antibody bound to the
antigen.
Another interesting approach to EIA systems is FAST
ELISA (Falcon Assay Screening Test ELISA; Becton Dick-
inson Labware, Oxnard, Calif.), which uses coated polysty-
rene beads attached by a tine to the lid of a microdilution
tray (39). The advantage of the FAST system is that the same
dilution of patient sera, conjugated antibody, and enzyme
substrate could be used for multiple tests by simply using
beads coated with different antigens.
A manufacturer of automated microbiology equipment
(Vitek Systems, Hazelwood, Mo.) has developed an auto-
mated immunodiagnostic assay system (VIDAS) which uses
the solid-phase ELISA format to detect antigens and anti-
bodies directly from patient specimens in 1 to 2 h. The
solid-phase receptacle is a pipette tip-shaped device made of
polystyrene or polypropylene. Reagents are predispensed
into a cuvette strip which also includes containers of predis-
pensed wash solutions. A computer-controlled instrument
performs the tests in batch mode or by random access.
Antigen detection tests undergoing field trials include Chla-
mydia trachomatis, RSV, HSV, and Clostridium difficile
toxin. Antibody detection tests for human immunodeficiency
virus are also being tested (W. M. Janda, M. H. Graves, K.
Hoffman, L. M. Wilcoski, J. M. Stevens, and L. M. Gor-
niak, Abstr. Annu. Meet. Am. Soc. Microbiol. 1989, C-45, p.
401). The company has developed assays for tests that are
currently labor intensive or lengthy, have diagnostic utility,
and do not need antibiotic susceptibility testing. Plans for
future development include direct antigen detection of My-
cobacterium spp., Mycoplasma pneumoniae, and antibodies
to the other organisms of the TORCH panel.
IgM and IgG separation methods. To avoid false-positive
and false-negative results in specific IgM and IgG assays,
several methods are available to separate IgG from IgM in
serum. Physical separation based on the differential size of
these immunoglobulins can be achieved by molecular-
sieving column chromatography, but this is not practical for
clinical laboratory test systems. Sucrose gradient ultracen-
trifugation would also separate IgM from IgG based on size;
however, IgG-containing immune complexes sediment with
IgM (31). Most clinical laboratories do not have access to
ultracentrifugation equipment.
An efficient technique commonly used for IFA assays uses
miniature ion-exchange columns (46) which are commer-
cially available from at least two sources. IgG passes
through the column and IgM is retained. IgM is eluted from
the column by a buffer at a lower pH and higher ionic
strength than the application buffer. The volume of serum
used and the volume of eluting buffer are carefully controlled
to ensure that the final eluate is equivalent to a known
dilution of serum; therefore, the final results are semiquan-
titative as a titer.
In some methods, serum is absorbed with staphylococcal
protein A either by using the actual bacteria or by binding
protein A to an insoluble matrix used as an absorption
reagent. Protein A absorption removes IgG subclasses 1, 2,
and 4 (but not 3) from the serum. There is increasing
evidence that protein A also removes significant virus-
specific IgM activity (44).
To prevent interference by IgG, commercially available
CLIN. MICROBIOL. REV.
EIAs for IgM use either absorption with anti-human IgG (44)
or aggregated human gamma globulin (AHGG) (47). Anti-
IgG binds to IgG in the sample, removing the IgG reactivity
to the antigen; if no IgG is available to bind to the antigen,
RF would not be bound to IgG. AHGG neutralizes RF, but
does not eliminate the potential for false-negative results by
IgG competing with IgM for binding sites on the antigen.
One study that compared various methods of removing IgG
from patient sera found anti-IgG treatment superior to
AHGG for eliminating nonspecific IgM activities without
impairing specific activities (31), but this finding needs
confirmation. The simultaneous dilution of serum and ab-
sorption with either anti-IgG or AHGG, as used in several
commercial ETAs, is more efficient than ion-exchange sepa-
ration of IgM from IgG. Our experience with IgM EIAs from
three sources suggests that the quantity of anti-IgG used by
some manufacturers may not be adequate to remove the IgG
antibody from patients with hypergammaglobulinemia (K.
James, R. Van Enk, and K. Thompson, manuscript in
preparation).
Capture assays. Capture assays are another variation of
solid-phase technology that can be used to detect antibodies
or antigens. The capture assay for antigen detection uses
either polyclonal antibodies or MAbs attached to the solid
phase to bind (capture) the antigen being detected. The
enzyme-labeled antibody for detection can be either mono-
clonal or polyclonal. Polyclonal antibodies more reliably
detect different forms of the antigen, in contrast to MAbs
which bind to only a single epitope. There is some limited
evidence that capture assays are more sensitive when poly-
clonal antibodies that bind multiple antigenic epitopes are
used (M. D. Tolpin and M. A. Collins, Clin. Microbiol.
Newsl. 10:109-111, 1988).
In the capture assay for detecting IgM antibodies to
specific viral antigens, an animal antibody to the Fc region of
human IgM is attached to a solid-phase matrix (69). Poten-
tially, all of the IgM molecules in the patients' sera can be
bound to the anti-IgM antibody, regardless of their antigenic
specificity. The antigen to be bound by the IgM is conjugated
with an enzyme (or biotin when an amplification technique is
necessary) and incubated with the "captured" IgM. If IgM
specific for the enzyme-conjugated antigen is present, the
antigen is captured by the patient IgM, and enzyme substrate
will be cleaved. The color reaction is directly proportional to
the level of specific IgM antibody in the patients' sera.
The capture assay has wide potential for detecting IgM
antibodies to microorganisms. Microdilution trays can be
coated with anti-IgM and used for detecting antibodies with
many different antigenic specificities. A single dilution of
patient serum can be applied to the coated wells. Different
antigens can be enzyme conjugated (or biotinylated and used
with a single enzyme-conjugated avidin) and a single sub-
strate can be used to develop the color reaction. The capture
assay would be an efficient method to screen sera to detect
the presence or absence of several antibodies (e.g., evaluat-
ing congenital infections). Recently, a variation of the cap-
ture assay technique has been automated for high-volume
hepatitis tests (Abbott Laboratories Diagnostics Div.), in-
cluding hepatitis B surface antigen and antibody, hepatitis B
core antigen, and hepatitis A virus antibody (27).
The capture assay works well for detecting IgM antibod-
ies, but is not useful for specific IgG antibodies. During the
acute phase of infections, especially congenital viral infec-
tions, the majority of the IgM present is virus specific. In
contrast, virus-specific IgG would be <10% of the total IgG.
Consequently, an IgG capture assay would not be as sensi-
VOL. 3, 1990 IMMUNOSEROLOGY OF INFECTIOUS DISEASES 141

TABLE 1. Commercially available assays for TABLE 2. Commercially available assays for detecting
detecting VZV antibodies rubella virus antibodies
Method Antibodyb Company Method Antibodyb Company
Micro-ELISA IgG Diamedix Corp., Miami, Fla. Macro-ELISA IgG, IgM Abbott Laboratories Diagnostics
Div., Abbott Park, Ill.
IFA IgG Gull Labs, Salt Lake City, Utah
Agglutination Total Becton Dickinson Microbiology Sys-
Micro-ELISA Total, IgM Pharmacia ENI Diagnostics, tems, Cockeysville, Md.
IFA Total Fairfield, N.J.
Micro-ELISA Total Biotrol USA, Inc., West Chester, Pa.
Micro-ELISA IgG Sigma Diagnostics, St. Louis, Mo.
Micro-ELISA IgG, IgM Clinical Sciences, Inc., Whippany,
Agglutination Total Wampole Laboratories, Cranbury, N.J.
N.J.
Micro-ELISA IgG, IgM Diamedix Corp., Miami, Fla.
Micro-ELISA Total Whittaker Bioproducts, Walkers-
Stick-IFA Total ville, Md. Stick-ELISA Total General Biometrics, San Diego,
Calif.
IFA Total Zeus Scientific Inc., Raritan, N.J.
a Micro-ELISA, ELISA in a microdilution tray; agglutination, use of visible Micro-ELISA Total, IgM Labsystems, Inc., Research Triangle
particles (e.g. latex or coated RBCs); stick-IFA, use of a polystyrene stick
Park, N.C.
support.
b IgG antibody is heavy-chain (only) specific. Total antibody indicates Rapid-ELISA Total Murex Corp., Norcross, Ga.
antibody to IgG heavy and light chains (which would also react with light
chains of IgA or IgM). IgM antibody is heavy-chain specific. Rapid-ELISA Total Organon Teknika, Durham, N.C.
Stick-MIG Total Ortho Diagnostic Systems, Raritan,
tive for detecting specific IgG as are assay systems in which N.J.
specific antigen is coated to the matrix (80). Micro-ELISA Total, IgM Pharmacia ENI Diagnostics, Fair-
IFA Total field, N.J.
SELECTION OF METHODS FOR CLINICAL
LABORATORY USE Micro-ELISA IgG, IgM Sigma Diagnostics, St. Louis, Mo.
Agglutination Total Wampole Laboratories, Cranbury,
Detection of Antibody to Verify Immunity N.J.
Preemployment Screening. Prior to employment in the Micro-ELISA Total, IgM Whittaker Bioproducts, Walkers-
health care industry, many organizations require verification Stick-IFA Total, IgM ville, Md.
of immunity to rubella virus and VZV. Both of these viral
infections can be unwittingly spread to nonimmune patients a Macro-ELISA, ELISA in a tube; agglutination, use of visible particles
in the days between exposure to the viruses and eruption of (e.g., latex or coated RBCs); micro-ELISA, ELISA in a microdilution tray;
stick-ELISA, use of a polystyrene stick support; rapid-ELISA, use of a
the rash that would help diagnose these diseases. Infection polystyrene support with reaction membrane; stick-MIG, use of immunogold
with rubella virus or VZV could be fatal to immunosup- on a membrane support of a polystyrene stick; stick-IFA, use of a polystyrene
pressed patients, infants, and nonimmune adults. Test sys- stick support.
tems available for VZV are indirect IFA or EIA available b IgG antibody is heavy-chain (only) specific. Total antibody indicates
antibody to IgG heavy and light chains (which would also react with light
from a limited number of vendors (Table 1). chains of IgA or IgM). IgM antibody is heavy-chain specific.
Detection of immunity to rubella virus is the most com-
mon screening test performed in most laboratories. LA or
particle agglutination methods are available which detect an
antibody titer equivalent to .8 in the reference HI method immune status should be determined when specific risk
(75). This test requires no serum pretreatment and no factors are encountered, e.g., toxoplasma immunity when
elaborate equipment and can be performed in any clinical there is a new cat or a sick cat in the household, rubella virus
laboratory setting, including doctors' office laboratories immunity when there are school-age children, CMV immu-
(Becton Dickinson Microbiology Systems, Cockeysville, nity when there is exposure to an immunosuppressed patient
Md.). A variety of EIA methods are also available (Table 2). (e.g., transplantation, chemotherapy, other immunodefi-
If large volumes of tests for rubella virus immunity are ciency diseases), or herpesvirus immunity when there is
performed, a batch EIA method may be more efficient than exposure to genital or oral herpesvirus lesions.
the agglutination method. Pregnant women who are not immune to infection by an
Prenatal screening. Prenatal screening to determine im- organism to which they are at risk are advised to avoid
mune status is generally performed selectively based on past contact with and/or seek treatment immediately if they are
medical history and the risk factors for contracting infections knowingly exposed or symptoms develop. Unfortunately,
during pregnancy that could cause serious congenital anom- the organisms responsible for serious congenital defects may
alies. Rubella virus immune status should be determined for cause only subclinical disease in the pregnant woman with
women of child-bearing age (31). If the patient is not im- symptoms that are not pathognomonic and which may not be
mune, she should be vaccinated so that immunity can be differentiated from a mild viral illness.
established before pregnancy is considered. The TORCH The methods currently used for prenatal screening include
panel evaluates immunity to the other organisms responsible indirect IFA and EIA methods (Tables 2 to 5). Selection
for the most devastating congenital infections. The patient's criteria for a particular company's IFA product include (i)
142 JAMES CLIN. MICROBIOL. REV.

TABLE 3. Commercially available assays for TABLE 4. Commercially available assays for detecting
detecting CMV antibodies Toxoplasma antibodies
Method Antibodyb Company Method Antibodyb Company
Macro-ELISA Total, IgM Abbott Laboratories Diagnostics Macro-ELISA IgG, IgM Abbott Laboratories Diagnostics
Div., Abbott Park, Ill. Div., Abbott Park, Ill.
Agglutination Total Becton Dickinson Microbiology Agglutination Total Ampoor, Inc., Bridgeport, N.J.
Systems, Cockeysville, Md.
Micro-ELISA Total, IgM Biotrol USA, Inc., West Chester,
IFA IgG, IgM Bion Enterprises, Park Ridge, Pa.
Ill.
Micro-ELISA IgG, IgM Clinical Sciences, Inc., Whippany,
Micro-ELISA IgG, IgM Clinical Sciences, Inc., Whip- IFA Total, IgM N.J.
IFA Total, IgM pany, N.J.
IFA Total, IgM Diagnostic Technology, Inc.,
IFA Total, IgM Diagnostic Technology, Haup- Hauppauge, N.Y.
IgG, IgA pauge, N.Y.
Micro-ELISA IgG, IgM Diamedix Corp., Miami, Fla.
Micro-ELISA IgG, IgM Diamedix Corp., Miami, Fla.
Stick-ELISA Total General Biometrics, San Diego,
Stick-ELISA Total General Biometrics, San Diego, IFA IgG, IgM Calif.
IFA IgG, IgM Calif.
Rapid-ELISA IgG Gull Labs, Salt Lake City, Utah
Rapid-ELISA IgG Gull Labs, Salt Lake City, Utah IFA IgG, IgM
IFA IgG, IgM
Micro-ELISA Total, IgM Labsystems, Inc., Research
IFA Total, IgM Immuno Concepts Inc., Sacra- Triangle Park, N.C.
mento, Calif.
Rapid-ELISA Total Murex Corp., Norcross, Ga.
Micro-ELISA Total, IgM Labsystems, Inc., Research Tri-
angle Park, N.C. Rapid-ELISA Total Organon Teknika, Durham, N.C.
IFA IgG Ortho Diagnostic Systems, Rari- Micro-ELISA IgG, IgM Ortho Diagnostic Systems,
tan, N.J. IFA IgG Raritan, N.J.
Rapid-ELISA Total Organon Teknika, Durham, IFA Total, IgM Pharmacia ENI Diagnostics, Fair-
N.C. field, N.J.
Micro-ELISA Total, IgM Pharmacia ENI Diagnostics, Micro-ELISA IgG, IgM Sigma Diagnostics, St. Louis, Mo.
IFA Total Fairfield, N.J.
Rapid-ELISA Total Trend Scientific Inc., St. Paul,
Micro-ELISA IgG, IgM Sigma Diagnostics, St. Louis, Minn.
Mo.
Agglutination Total Wampole Laboratories, Cranbury,
Agglutination Total Wampole Laboratories, Cran- N.J.
bury, N.J.
Micro-ELISA Total, IgM Whittaker Bioproducts, Walkers-
Micro-ELISA Total, IgM Whittaker Bioproducts, Walk- Stick-IFA Total, IgM ville, Md.
Stick-IFA Total, IgM ersville, Md.
Micro-ELISA IgG Zeus Scientific Inc., Raritan, N.J.
Micro-ELISA IgG Zeus Scientific Inc., Raritan, IFA Total, IgM
IFA Total, IgM N.J. a Macro-ELISA, ELISA in a tube; agglutination, use of visible particles
a
Macro-ELISA, ELISA in a tube; agglutination, use of visible particles (e.g., latex or coated RBCs); micro-ELISA, ELISA in a microdilution tray;
(e.g., latex or coated RBCs); micro-ELISA, ELISA in a microdilution tray; stick-ELISA, use of a polystyrene stick support; rapid-ELISA, use of a
stick-ELISA, use of a polystyrene stick support; rapid-ELISA, use of a polystyrene support with reaction membrane; stick-IFA, use of a polystyrene
polystyrene support with reaction membrane; stick-IFA, use of a polystyrene stick support.
stick support. b IgG antibody is heavy-chain (only) specific. Total antibody indicates
b IgG antibody is heavy-chain (only) specific. Total antibody indicates antibody to IgG heavy and light chains (which would also react with light
antibody to IgG heavy and light chains (which would also react with light chains of IgA or IgM). IgM antibody is heavy-chain specific.
chains of IgA or IgM). IgM antibody is heavy-chain specific.

body is known (e.g., anti-DNA), insoluble antigen (e.g.,

preference for antigen expression; (ii) whether or not the
slide contains separate control wells; (iii) specificity and
strength of reactivity of the control antisera; and (iv) minimal
nonspecific binding of the fluorescent conjugate. False-
positive staining reactions resulting from antinuclear anti-
bodies or other autoantibodies must be differentiated from
true positive reactions. Strongly positive antinuclear anti-
bodies may mask the delectability of specific antibodies to
certain organisms. If the specificity of the antinuclear anti-
PM2-double-stranded DNA; Boehringer Mannheim Bio-
chemicals, Indianapolis, Ind.) can be used to absorb the
antinuclear antibody reactivity, leaving the specific viral
antibodies intact (unpublished observations).
Most companies that have developed EIAs for detecting
antibodies in patient sera supply the components of TORCH
assays separately. For these EIA methods, purified antigens
are attached to wells in microdilution trays or strips. For
evaluation of immunity, the enzyme-conjugated antibody is
VOL. 3, 1990 IMMUNOSEROLOGY OF INFECTIOUS DISEASES 143

TABLE 5. Commercially available assays for anti-IgG with kappa and lambda light-chain reactivity. The
detecting HSV antibodies results can be expressed as an index relative to a negative
control or converted to standard international units, if estab-
Method' Antibodyb Company lished. Reference ranges have been established to correlate
HSV-1 and -2 to immune status as indicated by the reference methods (HI
combined for rubella virus; IFA for toxoplasma, CMV, and HSV).
IFA Total Clinical Sciences, Inc., Since the antigens have been extracted from organisms
Whippany, N.J. growing in tissue culture, ETAs should be evaluated for the
same factors that cause false-positive reactions in indirect
Micro-ELISA IgG, IgM Diamedix Corp., Miami, Fla. IFAs. These include nuclear and cytoplasmic autoantibodies
or cross-reactions with antibodies to other organisms in the
Stick-ELISA Total General Biometrics, San Diego, same genus or class. When EIAs are being considered to
Calif. replace indirect IFAs, they must be extensively validated to
determine the specificity and sensitivity of the proposed
IFA Total, IgM Gull Labs, Salt Lake City, Utah method compared with the existing method. There are
Macro-ELISA Total Kallestad Diagnostics, Austin, Tex.
significant differences in specificity and sensitivity between
commercially available EIAs for Toxoplasma spp. and CMV
Rapid-ELISA Total Organon Teknika, Durham, N.C. (James et al., in preparation). A definite need exists for
extensive evaluation of antigen preparations and antibody
Micro-ELISA Total, IgM Pharmacia ENI Diagnostics, Fair- cross-reactivities before currently available EIA methods
IFA Total field, N.J. are adopted for routine clinical use.
Pretransplant screening. Transplant patients are particu-
Micro-ELISA Total, IgM Whittaker Bioproducts, Walkers- larly prone to developing CMV infections which can be
Stick-IFA Total, IgM ville, Md. accidentally transmitted with donor organs, bone marrow, or
blood transfusions, since the virus resides in leukocytes (90).
IFA Total, IgM Zeus Scientific Inc., Raritan, N.J. CMV infections in these patients may be due to primary
infection, reactivation of latent virus, or reinfection with
HSV-1 and -2 as exogenous virus. The immune status of transplant recipients
separate and donors must be determined prior to the transplantation
assays procedure so that special precautions can be taken with
Bion Enterprises, Park Ridge, Ill.
CMV seronegative patients. If possible, CMV-negative pa-
IFA IgG, IgM tients are transplanted with tissues from CMV-negative
Micro-ELISA IgG, IgM Clinical Sciences, Inc., donors. Blood products to be transfused to CMV-negative
IFA Total Whippany, N.J. transplant patients should also be CMV seronegative (73).
Sensitive and specific rapid agglutination methods that are
IFA Total Diagnostic Technology, Inc., well suited for screening blood donor sera are commercially
Hauppauge, N.Y. available to determine CMV seroreactivity or immunity
(Table 3) (50).
HSV-1 and -2
separate but Detection of Antibody to Diagnose Disease
on same
slide Acute and convalescent specimens. Serologic studies con-
IFA IgG, IgM General Biometrics, San Diego, tribute significantly to the diagnosis of viral infections in
Calif. patients. Methods manuals specify that "serologic confirma-
IFA IgG Ortho Diagnostic Systems, Rari-
tion of a suspected viral etiology of an illness optimally
tan, N.J. requires two serum specimens, the first obtained as soon as
possible after the onset of the illness and another obtained 1
Rapid-ELISA Total Organon Teknika, Durham, N.C. to 2 weeks later" (40). These paired samples are termed
acute and convalescent specimens. For accurate test inter-
Micro-ELISA Total Pharmacia ENI Diagnostics, Fair- pretation, these specimens should be assayed simulta-
IFA Total, IgM field, N.J. neously, using the same method in the same assay. A
fourfold or greater rise in titer in the convalescent specimen
Micro-ELISA IgG, IgM Sigma Diagnostics, St. Louis, Mo. compared with the acute specimen is considered diagnostic
of an active infection at the time the acute specimen was
Micro-ELISA Total Whittaker Bioproducts, Walkers- taken (40). The disadvantage of requiring acute and conva-
ville, Md. lescent specimens is the delay in diagnosis and treatment,
which interferes with patient compliance for medications
Micro-ELISA IgG Zeus Scientific Inc., Raritan, N.J. and subsequent follow-up.
IFA Total, IgM (HSV-2 only) IgM-specific assays. New technologies being developed
I
Macro-ELISA, ELISA in a tube; agglutination, use of visible particles will result in changes in the definitions of significance limits
(e.g., latex or coated RBCs); micro-ELISA, ELISA in a microdilution tray; of diagnostic tests. Johnson and Nakamura have advocated
stick-ELISA, use of a polystyrene stick support; rapid-ELISA, use of a
polystyrene support with reaction membrane; stick-IFA, use of a polystyrene
that a single specimen tested for both IgG- and IgM-specific
stick support. antibodies to the suspected organisms is sufficient for eval-
b IgG antibody is heavy-chain (only) specific. Total antibody indicates uating patient serum to diagnose disease (46). If neither IgG
antibody to IgG heavy and light chains (which would also react with light nor IgM antibodies to the organism are detected, the patient
chains of IgA or IgM). IgM antibody is heavy-chain specific. has not been exposed or testing was performed too early. If
144 JAMES
700
0*
600
0 00
:I 500
- 400
U 0@0
8 300
cc 200 0~~
I dation, blindness, and other severe problems. A high (68%)
100 .L MI IU
0
0
.5.~~~~-
100 200 300 400
EIA Unis
FIG. 4. Comparison of the detection of Toxoplasma antibodies
by indirectIFA (Gull Laboratories, Salt Lake City, Utah) and by
micro-ELISA (EIA) (Diamedix Corp., Miami, Fla.). SpecificIgM
(U) and total IgG (0) antibody heavy- and light-chain reactivity are
included to illustrate the approximately linear relationship between
generally lower-titerIgM antibodies and higher-titer IgG antibodies.
IgM antibodies are detectable, the patient has been exposed
and is mounting an immune response regardless of the
presence of IgG antibodies. IfIgM antibodies are not de-
tected but IgG antibodies are present, the patient has immu-
nity to the specific organism, but not necessarily to a current
infection (unless tested prior to developing an IgM re-
sponse). Using a single specimen for diagnosis of acute
infection is feasible if the assay systems used are specific and
sensitive and the specimen is obtained at a time whenIgM
antibodies are detectable. Most manufacturers have IgM-
specific assays as well as assays that detect total antibody or
IgG (Table 1 to 5; see also Tables 8 and 9).
Method comparisons. The transition from CF to IFA to
detect viral antibodies was made without a need to reeducate
the medical staff regarding significance of results since both
methods reported titers. A change from IFA titer to EIA
units requires additional effort by the laboratory, perhaps
including providing a conversion table or reporting both sets
of results for a period of time. Each organism and each
antibody tested must be treated separately. A representative
comparison of these two methods is illustrated in Fig. 4 and
a conversion format is presented in Table 6.
Congenital infections. If the mother has not been screened
for antibodies prenatally, testing maternal serum after deliv-
ery of a possibly infected infant is useless. Unless maternal
symptoms can be documented within the previous 4 weeks,
TABLE 6. Sample conversion table to compare ELISA
units with IFA titersa
Equivalent
ELISA units titer
<45 ............................................... <20
50 ............................................... 20
100 ....................................... 40
150 ....................................... 80
200 ............................................... 160
250 ............................................... 320
300 ....................................... 640
>300 ....................................... -1,280
a Data illustrated in Fig. 4 are summarized in table format as an example.
When converting from IFA to ELISA, a conversion table is helpful to educate
clinicians in the significance of ELISA units.
CLIN. MICROBIOL. REV.
maternalIgM antibody titers are not helpful since anIgM
response peaks at 7 days postinfection and may no longer be
detectable by 28 days. Evaluating the mother for IgG anti-
bodies is noncontributory because IgG antibodies are detect-
able between 7 and 14 days postinfection and can remain
positive for life.
The severity of congenital infections is related to the time
of exposure to the organism relative to the stage of fetal
development. Congenital infections can result in stillbirths,
death in the neonatal period, hydrocephalus, mental retar-
rubella had no abnor-
percentage of infants with congenitalclinically
mal findings at birth but developed
ease in the first 5 years (39).
apparent dis-
If the mother demonstrates immunity to TORCH organ-
isms, her IgG antibodies (which cross the placenta)during may
protect her Iffetus if she is exposed to the organism
pregnancy. the mother is nottheimmune, viruses and Toxo-
plasma gondiithe(which cross placenta) can cause fetal
harm before mother can develop protective IgG antibod-
ies. The fetus eventually responds to the infection by pro-
ducingIgM, but the response is much slower than in an
adult. This lag time allows the infecting organism to repro-
duce unchecked.
Methods for diagnosing congenital infections include cul-
ture of fetal tissue or placenta, demonstration of character-
istic lesions within affected tissues, or detection of neonatal
IgM-specific antibodies. Culture may take 2 days to 4 weeks
to demonstrate virus, histologic changes may not be detect-
able, or tissue may not be feasibly obtained from an infant.
Thus,IgM antibody detection would be the method of choiceas
in order to initiate appropriate therapy (if available)
IgM levels are generally
quickly as possible. Quantitativebut would not be as useful
elevated in congenital infections,
as detecting specific IgM antibodies. A useful congenital
infection profile would include quantitative IgM, CRP, and
IgM-specific antibodies to the TORCH profile. Stagno et al.
advocate screening for intrauterine infections with an RF
assay which was found to be as sensitive as and more
specific thanin quantitating IgM levels for detecting congenital
infections a large series of neonates (89).
In congenital infections, maternal IgG specific for the
the neonatal
organism crosses the placenta and is present in the antigens
serum. Because the maternal IgG binds to
present in the test system, a false-negative reaction could
result by inhibiting neonatal specific IgM from binding. A
false-positive result could occur if neonatal IgM RF is
3A).
present to bind to the complexed maternal IgG (Fig.
Neonatal IgM specific for the organism cannot be distin-
guished from neonatal IgM RF. RF would be prevented the
from
neonatal
binding if the maternal IgG was removed from
serum prior to testing. The methods described previously to
separate IgG from IgM would also separate maternal IgG
from neonatal IgM.
Transplantation and immunosuppression. With the intro-
duction of cyclosporine, which selectively suppresses cell-
mediated immunity (6), there has been a dramatic increase in
organ and bone marrow transplantation procedures per-
formed. These immunosuppressed patients are at risk for
opportunistic infections. Bacterial infections can be treated
with antimicrobial agents; however, differentiating viral in-
fections from symptoms of chronic allograft rejection may be
difficult. Transplant recipients who are seronegative for
CMV or HSV or both prior to transplant are particularly at
risk. An IgM assay for CMV that can be performed on a
daily basis is necessary for clinical evaluation of patients
VOL. 3, 1990
both pre- and posttransplant. If IgM antibodies are undetect-
able prior to transplant or prior to appearance of symptoms,
detection of IgM antibodies would confirm a currently active
viral infection. Although specific IgM generally does not
persist for longer than 4 to 8 weeks, IgM antibodies to
viruses (especially CMV) have been reported to be detect-
able for months or years after a documented infection due to
the continued intracellular presence of the virus (42). Detec-
tion of CMV IgM is convenient for diagnosing CMV infec-
tions in transplant patients; however, there is conflicting
evidence about whether the detection of CMV viremia is a
better indicator of clinically significant infection (J. Nelson,
F. Strie, C. Benson, J. Pottage, and H. Kessler, Abstr. Clin.
Res. 464A, 1988; G. A. Storch, E. D. Spitzer, L. Marsano,
M. W. Flye, D. W. Hanto, P. R. Murray, and R. P. Perillo,
Program Abstr. 28th Intersci. Conf. Antimicrob. Agents
Chemother., abstr no. 816, 1988).
The absence of specific IgM may be misleading. Timing of
specimen collection is critical to its detection. If collection is
too early, IgM may not be present in detectable levels (Fig.
1). If the specimen is obtained too late, IgM may have
disappeared. Conversely, with some viral infections, specific
IgM antibody can be detected from 12 to 18 months after the
acute onset of the disease (81). Consequently, IgG and IgM
assays should be performed simultaneously to evaluate a
patient with an acute infectious process. Both acute and
convalescent specimens should be collected if there is any
ambiguity about interpretation of results from the acute
specimen. When transplant patients are being evaluated for
immune status, both IgG- and IgM-specific tests should be
performed to establish base-line levels by which to compare
posttransplant results.
Serologic tests for syphilis. Nontreponemal tests for syph-
ilis are the most common applications of screening for
disease by the detection of antibodies. The rapid plasma
reagin (RPR) and the Venereal Disease Research Laboratory
(VDRL) tests detect Wasserman or reaginic antilipid anti-
bodies. Reaginic antibodies are produced in response to
lipoidal material released from damaged host cells early in
infection as well as to lipid present on the cell surface of the
treponeme (62). Although very sensitive, the screening tests
are not specific for T. pallidum infections. Reactive results
must be confirmed by specific treponemal tests, e.g., FTA-
ABS or MHA-TP.
Premarital and prenatal serologic tests for syphilis are
required in most states to detect syphilis during the repro-
ductive years. Syphilis can be effectively treated with peni-
cillin or other antimicrobial agents if detected in early stages.
Prenatal screening is intended to quell congenital transmis-
sion of the disease. Biologic false-positive nontreponemal
serologic tests for syphilis are commonly observed in pa-
tients with autoimmune diseases (55). Biologic false-positive
RPRs range from a low titer (undiluted) to a high titer of 32;
some, but not all, biologic false-positive tests are associated
with cardiolipin antibodies. The highly specific but less
sensitive treponemal test(s) should be performed to confirm
a reactive RPR prior to instituting therapy. An RPR titer can
be used to monitor the effectiveness of treatment since the
nontreponemal antibodies decrease with treatment and dis-
appear altogether with time if treatment was effective. The
FTA-ABS or MHA-TP assays may, however, remain reac-
tive for years after the spirochete has been successfully
eradicated.
Streptococcal antibodies. In spite of readily available anti-
microbial agents, group A streptococci remain significant
pathogens whose immunologic sequelae play an important
IMMUNOSEROLOGY OF INFECTIOUS DISEASES 145
role in human disease. A positive ASO titer is useful in
establishing the diagnosis of acute rheumatic fever or acute
poststreptococcal glomerulonephritis which may occur
weeks or months after resolution of a streptococcal infection
(51). ASO is produced by most strains of group A strepto-
cocci and elicits a strong antibody response during 80% of
pharyngeal streptococcal infections, but in only 25% of
streptococcal pyoderma skin infections (E. M. Ayoub, Clin.
Immunol. Newsl. 3:107-109, 1982). An LA kit is available
for ASO testing (Behring Diagnostics, Inc., Somerville,
N.J.) which yields results equivalent to those by the neutral-
ization assay but much more efficiently (unpublished obser-
vations).
Anti-DNase B is the assay most frequently used to confirm
streptococcal sequela which can result from skin infections.
DNase B is produced by most strains of group A strepto-
cocci, but the antibody response appears later than the
response to streptolysin 0 and lasts longer. Consequently,
anti-DNase is more valuable than ASO in detecting strepto-
coccal antibodies in Sydenham's chorea because of the long
latent period between streptococcal infection and develop-
ment of chorea (51). Antihyaluronidase titers are useful to
detect antibodies to streptococcal hyaluronidase following
pyoderma. In patients with suspected streptococcal se-
quelae, a negative ASO titer would be the indication to
perform anti-DNase or antihyaluronidase or both.
A commercially available product, Streptozyme (Wam-
pole Laboratories, Cranbury, N.J.), consists of particles
coated with a mixture of various intracellular and extracel-
lular antigens produced by group A beta-hemolytic strepto-
cocci. Streptozyme may be an effective screening test for
streptococcal antibodies other than ASO. A positive Strep-
tozyme with a negative ASO test indicates an antibody
response to another extracellular streptococcal toxin. The
manufacturers of this product have not revealed the specific
content of antigens, toxins, or hemolysins used in the
coating process, and lot-to-lot variations in reactivity with a
panel of sera have been reported (33). We have observed
negative Streptozyme tests in patients with positive ASO
titers and hypothesize that insufficient streptolysin 0 antigen
was present on the particles to cross-link with antibody and
agglutinate the coated particles. For that reason, we use
Streptozyme as a follow-up to a negative ASO test. If the
ASO test and Streptozyme test are both negative in a patient
with a clinical presentation consistent with poststreptococ-
cal sequelae, we recommend the anti-DNase and antihyalu-
ronidase tests (41).
Detection of high titers of one or more of these antibodies
or a rise in titer over time or both confirm the diagnosis of
streptococcal infection. If acute poststreptococcal glomeru-
lonephritis is suspected, obtaining C3 and C4 levels may be
helpful in making the diagnosis. Acute poststreptococcal
glomeralonephritis provides the activating surface necessary
for alternative pathway activation, significantly decreasing
C3 levels, while C4 remains at normal levels.
EBV antibodies. EBV is the primary cause of infectious
mononucleosis (IM), also contributes to Burkitt's lymphoma
and nasopharyngeal carcinoma, and has been erroneously
implicated in chronic fatigue syndrome (70). Because the
virus is so ubiquitous and most healthy adults in the United
States have antibody to EBV, the appropriate screening test
forIM is still the classic heterophile. Although 30% of EBV
IM cases may be heterophile negative at the time of presen-
tation, in 85 to 90% of IM cases, Paul-Bunnel heterophile
tests are positive by week 2 of illness and no further testing
is necessary (58). The classic sheep cell heterophile with
146 JAMES CLIN. MICROBIOL. REV.

TABLE 7. Commercially available assays detect the viral capsid antigen. IgM-EBV-viral capsid anti-
for heterophile antibodies gen-specific serologic tests should be performed to diagnose
Differential primary infections in young children and atypical disease in
Indicator particles absorption Company adults (70, 86). Since CMV can present with symptoms
similar to EBV, if EBV-IgM tests are negative, CMV-TgM
Horse RBC GPK only Access Medical Sys- should be evaluated. In cases of CMV negative- and EBV-
tems, Branford, viral capsid antigen-negative IM, or to detect antibody levels
Conn. from patients with EBV-induced malignancies, or to assess
Horse RBC (1 reagent) Blocked Ampoor, Inc., reactivation of EBV in immunocompromised patients, it
stroma Bridgeport, N.J. may be useful to evaluate the presence of antibodies to the
Horse RBC (3 reagent) GPK/BRBC EBV early antigen, restricted or diffuse (86).
LegioneUa antibodies. Antibodies to Legionella spp. may
Latex (IM-Latex) No Microscan, Div. of provide current as well as retrospective evidence of an
Horse RBC (IM-RBC) No Baxter, Bellevue, infection with this organism (100). IgM antibodies can be
Wash. detected by IFA as early as 1 week after onset of symptoms
Medical Technology
of a suspected Legionella pneumonia (100). Total or IgG
Macro-ELISAb No antibodies are detectable by EIA or IFA within 3 to 6 weeks
Corp., Somerset,
N.J. (1). Since this organism is endemic in certain areas of the
country, a single positive IgG result may be inconclusive. In
Horse RBC GPK/BRBC Organon Teknika, the absence of direct fluorescent detection of the organism
(Monosticon) Durham, N.C. from clinical specimens or an IgM antibody titer of >256,
acute and convalescent specimens should be collected to
Horse RBC (Monospot) GPK/BRBC Ortho Diagnostics, detect a significant increase in antibody titer to diagnose
Systems, Raritan, legionellosis reliably.
N.J. Rickettsia antibodies. Rickettsial organisms are hazardous
Ventrex Laborato- to culture and difficult to detect with light microscopy.
Macro-ELISA Noc
ries, Portland, Consequently, serologic tests are frequently the most reli-
Maine able method of detecting these diseases (71). The Weil-Felix
test, which uses Proteus vulgaris strains to detect cross-
Latex (Monolatex) No Wampole Laborato- reacting rickettsial antibodies, is rarely used now. The most
Latex (Monodiff) HK/BRBC ries, Cranbury, standard and reliable test appears to be the indirect IFA
Horse RBC (Monotest) No N.J. (Hillcrest Laboratories, Division of MRL, Cypress, Calif.),
Horse RBC (Monosure) HK/BRBC available to detect antibodies to at least nine different
Horse RBC (Monotest No rickettsial species (11). An LA test (New York State Health
FTB) Lab, New York, N.Y.) is available (38), and an EIA method
a GPK, Guinea pig kidney; BRBC, beef RBCs; HK, horse kidney. has been reported (11).
b Macro-ELISA, ELISA in a test tube.
I
Product uses a highly purified antigen that does not bind the Forssman
Lyme disease serology. It has long been suspected that
some chronic inflammatory diseases may have an infectious
antibody.
disease etiology. Lyme disease, the first syndrome con-
firmed to be associated with a tick-borne etiologic agent,
Borrelia burgdorferi, was described in 1981 (5). Lyme dis-
differential absorptions has been replaced by a variety of ease was first described as an outbreak of arthritis among
slide agglutination tests that use latex particles or treated children in Lyme, Conn., in 1975 (92). The manifestations of
RBCs (Table 7). When differently absorbed, these rapid tests the disease reflect the patient's immune response to the
are reliable for diagnosing most cases of classic IM. Clinical organism. In early Lyme disease, symptoms include a dis-
microbiologists should be aware that many commercially tinctive skin lesion, erythema chronicum migrans (ECM),
available assays (Table 7) for detecting heterophile antibod- followed by intermittent attacks of arthritis in the large
ies do not use an absorption reagent(s). False-positive reac- joints. In later stages of the disease, neurologic abnormali-
tions due to Forssman antibodies could result in misdiagno- ties and cardiac symptoms may be confused with symptoms
sis (58). of other diseases (91). Certain patients diagnosed with mul-
Recently, a rapid EIA which separately detects IgM and tiple sclerosis have demonstrated high titers of antibodies to
IgG antibodies to the EBV-associated nuclear antigen B. burgdorferi, but recent reports conclude that this organ-
(EBNA-1) synthetic peptide 62 has been developed (Ortho ism is probably not the cause of their multiple sclerosis (15,
Diagnostic Systems, Raritan, N.J.). Detection of IgM anti- 83).
bodies to EBNA is reported to be more sensitive than Diagnostic criteria include the classic ECM reaction to the
heterophile antibodies for the detection of IM (M. Levin, G. initial tick bite, although this distinctive skin lesion is ex-
Rhodes, C. Haberzetl, J. Geltosky, C. Wren, C. Sumaya, M. pressed in less than half of the cases. For the majority of
Weinstein, and Monolert Study Group, Progr. Abstr. 28th patients, serologic diagnosis must prevail since the organism
Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. is not easily isolated or cultivated in the clinical laboratory
813, 1988). EBNA IgG antibody is absent during a primary (79). Indirect IFAs are commercially available (Table 9),
acute infection, can be detected weeks to months postinfec- some of which can differentiate between IgG and IgM
tion, and persists for life. Detection of IgM-specific antibod- antibodies. ETAs have been introduced recently by several
ies to EBNA would diagnose reinfection in normal individ- commercial companies (Table 9).
uals, but immunologically compromised persons often lack Significant problems are still associated with Lyme dis-
antibody to EBNA (70). ease serology. Many patients have detectable cross-reactive
Indirect IFA and EIA systems (Table 8) are available to antibodies (IgM>IgG) in the absence of characteristic symp-
VOL. 3, 1990 IMMUNOSEROLOGY OF INFECTIOUS DISEASES 147

TABLE 8. Commercially available assays for EBV antibodies

Method Antigen expressed' Antibodyc Company
IFA VCA IgG, IgM Bion Enterprises, Park Ridge, Ill.
Micro-ELISA VCA IgG, IgM Clinical Sciences, Inc., Whippany, N.J.
EBNA IgG, IgM
Stick-ELISA VCA Total General Biometrics, Inc., San Diego, Calif.
IFA VCA IgG, IgM Gull Labs, Salt Lake City, Utah
EA IgG
EBNA Total (ACIFA)
IFA VCA Total, IgM Immuno Concepts Inc., Sacramento, Calif.
IFA VCA Total, IgM Organon Teknika, Durham, N.C.
EBNA Total (ACIFA)
Stick-ELISA EBNA IgM vs IgG Ortho Diagnostic Systems, Raritan, N.J.
Micro-ELISA VCA (80%) Total, IgM Pharmacia ENI Diagnostics, Fairfield, N.J.
IFA VCA Total
Micro-ELISA VCA Total, IgM Whittaker Bioproducts, Walkersville, Md.
Stick-IFA VCA Total
IFA VCA Total, IgM Zeus Scientific Inc., Raritan, N.J.
EA Total
EBNA Total (ACIFA), IgM
a Micro-ELISA, ELISA in a microdilution tray; stick-ELISA, use of a polystyrene stick support; stick-IFA, use of a polystyrene stick support.
b VCA, Viral capsid antigen; EBNA, Epstein-Barr nuclear antigen; EA, early antigen.
c IgG antibody is heavy-chain (only) specific. Total antibody indicates antibody to IgG heavy and light chains (which would also react with light chains of IgA
or IgM). IgM antibody is heavy-chain specific. ACIFA is an anti-complement immunofluorescence assay, as described in the text.

toms or documented exposure to the spirochete. A properly
performed negative test rules out Lyme disease, but a
positive test should be interpreted cautiously. A rise in titer
after acute onset of symptoms would be diagnostic. The
difficult diagnoses are in patients with chronic progressive
symptoms that could be those of Lyme disease. Cross-
reactivity can occur with antibodies to other spirochetes;
e.g., Treponema pallidum, which can result in a biologic
false-positive FTA-ABS (60). The RPR or VDRL test or
both, however, would not be falsely reactive. Although not
yet commercially available, a Western blot (immunoblot)
assay to confirm questionable antibody reactions is being
used at major medical centers located in endemic areas (12).
As with serologic tests for syphilis, there is a need for two
tests for Lyme disease antibodies: (i) one that uses washed
whole organisms or sonicated B. burgdorferi as a screening
test (analogous to RPR or VDRL); and (ii) an absorption test
(analogous to FTA-ABS) or Western blot for confirmation.
Antibodies to other microorganisms. Chlamydial antibodies
can be found in a high proportion of individuals who are not
currently infected, thus limiting the usefulness of serologic
methods (2). An ELISA which detects IgM antibodies has
been reported to be more useful than the detection of
chlamydial antigen for diagnosing chlamydial pneumonitis in
infants (61). A recent report indicates that an IFA antibody
titer to Chlamydia trachomatis correlates with the incidence
of pelvic inflammatory disease in women of childbearing age
(Scholes et al., 28th ICAAC). These data support the role of
prior chlamydial infection in pelvic inflammatory disease,
but this finding remains to be confirmed. Recently, a test
system to detect chlamydial antibodies has become commer-
cially available (Labsystems, Inc., Research Triangle Park,
N.C.).
Antibodies to Campylobacter pylon may be useful in the
differential diagnosis of gastritis and peptic ulcer disease
(S. P. Holland, M. F. Go, B. I. Hirshowitz, W. J. Koops-
man, and S. M. Michalek, Abstr. Annu. Meet. Am. Soc.
Microbiol. 1988, Abstr. E85, p. 123); patients with gastric
ulcer had elevated levels of Campylobacter pylori-specific
IgM antibodies.
Until recently, detecting high titers of cold agglutinins was
considered an appropriate screening test for Mycoplasma
pneumoniae, although this test was positive in only 50% of
documented cases. IgG and IgM indirect IFAs have been
developed (7, 87) and are now available commercially (Zeus
Scientific Inc., Raritan, N.J.). Recently, an LA method
(Access Medical Systems, Bradford, Conn.) has been intro-
duced as an effective screening test for antibodies to Myco-
plasma pneumoniae (D. L. Smalley and C. E. Dunn, Abstr.
Annu. Meet. Am. Soc. Microbiol. 1988, V12, p. 419).
Other viral antibodies. A number of other viral antibodies
have diagnostic or prognostic significance. IgM antibodies to
hepatitis B core antigen are diagnostic, as are IgM antibodies
to hepatitis A virus. An abundance of literature is already
available about interpreting hepatitis antigen and antibody
tests (14, 82). Diagnostic testing for detecting antibodies to
and antigens of human immunodeficiency virus are used to
determine exposure to this virus. These tests have recently
been extensively reviewed (43). EIA tests for human T-
lymphotrophic virus type antibodies are used to screen the
blood supply to minimize the possiblity of transfusion-
acquired exposure to this virus which has been etiologically
associated with adult T-cell leukemia or lymphoma (66).
Other, less prevalent viral antibodies can be detected by CF
methods at reference laboratories such as state department
of health laboratories or the Centers for Disease Control.
148 JAMES CLIN. MICROBIOL. REV.

TABLE 9. Commercially available assays for Lyme immune system develops sufficiently to combat infectious
disease antibodies processes. Neonates are markedly susceptible to group B
Method Antibodyb Company beta-hemolytic streptococci during the first 2 months of life
(17). Group B streptococcal antigen can be rapidly detected
Rapid-ELISA Total Access Medical Systems, in urine, CSF, and other body fluids. In the absence of
Branford, Conn. detectable group B streptococcal antigens, the differential
diagnosis of neonatal sepsis would include the other organ-
Micro-ELISA Total, IgG, Cambridge BioScience Corp., isms to which newborn infants are particularly susceptible,
IgM Worcester, Mass. e.g., Listeria monocytogenes and E. coli. Neonates lack
IFA Total, IgM Diagnostic Technology, transplacentally acquired antibodies from the mother in
Hauppauge, N.Y. virtually every instance of serious disease caused by these
microbes.
Micro-ELISA Total Diamedix Corp., Miami, Fla. Infants 3 months to 2 years old are susceptible to menin-
gitis caused by three groups of bacteria that possess polysac-
Stick-ELISA Total General Biometrics, San charide capsules: H. influenzae type B, S. pneumoniae, and
Diego, Calif. N. meningitidis. LA methods may improve the efficiency of
Micro-ELISA Total, IgG, MarDx Diagnostics, Scotch diagnosis and initiation of appropriate therapy in bacterial
IgM Plains, N.J. meningitis of infants, especially in partially treated, culture-
IFA IgG, IgM negative patients.
Neonatal viral infections. Viral infection of infants can
Micro-ELISA Total Sigma Diagnostics, St. Louis, occur during delivery by exposure to HSV or CMV in the
Mo. birth canal of asymptomatic women. If HSV is known or
suspected in the mother, a Caesarean section could be
Micro-ELISA Total Whittaker Bioproducts, performed to avoid subjecting the infant to the virus during
Stick-IFA Total Walkersville, Md. birth (R. A. Greene, P. A. Kasila, A. E. Rugg, T. A. Travis,
Micro-ELISA Total, IgG, Zeus Scientific, Raritan, N.J.
and E. Joachim, Abstr. Annu. Meet. Am. Soc. Microbiol.
IgM 1988, S27, p. 319). Most CMV cervical infections are asymp-
IFA Total, IgG, tomatic, however. To facilitate detecting cervical infections,
IgM there is a need for rapid antigen assays for HSV and CMV
that could be performed upon admission for a vaginal
Micro-F.ELISA Total 3M Diagnostic Systems, delivery. Under those conditions, physicians may choose to
Santa Clara, Calif. perform a Caesarean section to spare the infant exposure to
a Rapid-ELISA, use of a polystyrene support with reaction membrane; HSV from cervical secretions. Nursing babies can acquire
micro-ELISA, ELISA in a microdilutin tray; stick-ELISA, use of a polysty- CMV infection from maternal milk or colostrum (19); moth-
rene stick support; micro-F.ELISA, fluorescence ELISA in a microdilution ers with documented infections could choose not to nurse
tray.
b IgG antibody is heavy-chain (only) specific. Total antibody indicates
their infants.
antibody to IgG heavy and light chains (which would also react with light Group A streptococcal infections in children. Rapid antigen
chains of IgA or IgM). IgM antibody is heavy-chain specific. detection methods have been available for group A strepto-
coccal antigens for over 5 years. The antigens must be
released from the bacteria and inflammatory cell debris that
Detection of Soluble Antigen Correlates with Active Disease typically surround the organisms when swabbed from the
Soluble bacterial capsular polysaccharide and cell wall throats of patients presenting with pharyngitis. The antigen
antigens can be detected by a variety of methods including can then be detected by particle agglutination or, more
double immunodiffusion, CIE, particle agglutination tech- commonly, by an EIA technique. The specificity of a posi-
niques, and ElAs. Body fluids such as serum, CSF, and tive reaction is very good, but a negative test in a patient
urine are commonly assayed. Urine is a particularly effective with suppurative pharyngitis requires retesting by a throat
specimen because it can be collected noninvasively and culture.
because the kidneys concentrate the urine, thereby increas- Other microbial antigens. Immunoassay methods are avail-
ing the antigen level (L. J. LaScolea, Clin. Microbiol. able to detect Candida antigens in serum (Ramco Laborato-
Newsl. 10:21-23, 1988). Direct antigen detection is advanta- ries, Houston, Tex.), but interpretation of results must
geous because it does not depend on the presence of viable consider the patient history and culture findings (29). Toxo-
organisms as do culture techniques. Previous antimicrobial plasma gondii antigens can also be detected by EIA in sera
treatment that could inhibit growth of an organism in culture and CSF of congenitally infected infants, immunocompro-
will not affect rapid methods to detect antigens. Conse- mised patients, and patients with recently acquired infec-
quently, rapid antigen testing may facilitate administration of tions; the antigen is absent in chronic infections (S. L.
appropriate antimicrobial therapy and may be the only Josephson, Clin. Immunol. Newsl. 9:165-168, 1987). Direct
means of establishing the source of an infection in a partially IFA can detect small numbers of Cryptosporidium oocysts in
treated patient (30). Direct antigen detection methods cannot feces of patients with minimal symptoms (32). LA methods
be used to evaluate therapy since the antigens can persist for are available to detect Cryptococcus neoformans antigen in
long periods of time even after appropriate therapy (42, 54; CSF (DuPont Medical Products, Wilmington, Del.) and
LaScolea, Clin. Microbiol. Newsl. 10:21-23, 1988). Legionella spp. in urine (54). B. burgdorferi antigen detec-
Neonatal bacterial infections. Unless a congenital infection tion in urine has also recently been reported as detectable by
has been transmitted to the fetus from the mother during Western blot and by an ELISA method (42). The clinical
gestation, the in utero environment is essentially sterile. relevance of antigenuria for detection and/or monitoring of
Neonates, especially premature infants, are susceptible to Lyme disease remains to be determined. Although detection
infections for the first few months of life until their own of microbial antigens in urine may be useful to diagnose
VOL. 3, 1990
TABLE 10. Commercially available assays for RSV antigens
Method Company
Macro-ELISA Abbott Laboratories Diagnostic Div.,
Abbott Park, Ill.
IFA Bartels Div. of Baxter, Bellevue, Wash.
DFA
Rapid-ELISA Becton Dickinson Microbiology Systems,
Cockeysville, Md.
IFA Difco Laboratories, Detroit, Mich.
Macro-ELISA Kallestad Diagnostics, Austin, Tex.
Micro-ELISA Ortho Diagnostic Systems, Raritan, N.J.
IFA
a Macro-ELISA, ELISA in a tube; IFA, indirect IFA; DFA, direct immu-
nofluorescence assay; micro-ELISA, ELISA in a microdilution tray; rapid-
ELISA, use of a polystyrene support with reaction membrane.
newly acquired infections, this method will not be helpful to
monitor patients, since antigenuria in most systems tested
can persist for months to years after resolution of the
infectious process.
Pneumococcal C-polysaccharide detection in sputum and
other body fluids has been helpful in confirming the diagnosis
of pneumococcal pneumonia (A. J. Parkinson, M. Davidson,
and M. Rabiego, Progr. Abstr. 28th Intersci. Conf. Antimi-
crob. Agents Chemother., abstr. no. 473, 1988). The EIA
method was found to be more sensitive than culture to detect
S. pneumoniae in patients who had received antimicrobial
agents.
Other viruses. Many lower-respiratory-tract virus infec-
tions in infants and children are due to RSV. Several EIAs
for RSV are available which appear to be more sensitive than
culture for detecting RSV (Table 10). Rapid diagnosis of
RSV among hospitalized children may minimize the occur-
rence of nosocomial infections and facilitate initiation of
appropriate antiviral therapy instead of antimicrobial agents
(52, 97). At least one of these kits (Abbott Diagnostics) is
considered to be appropriate for the physician office labora-
tory (C. G. Wren, B. J. Bate, B. A. Masters, and B. A.
Lauer, Program Abstr. 28th Intersci. Conf. Antimicrob.
Agents Chemother., abstr. no. 208, 1988).
Direct IFA methods are available for detecting HSV and
VZV in vesicular lesions (81). CMV detection by direct IFA
has been reported for lung biopsies (36) and bronchoalveolar
savage cells (24). A modification of direct IFA for detection
of CMV uses short-term (24-h) culture in shell vials followed
by direct IFA detection of the CMV. This method signifi-
cantly decreases the turnaround time for confirmation of
CMV in patients with low levels of organisms (B. A. Forbes
and N. L. Dock, Clin. Microbiol. Newsl. 10:17-21, 1988).
CONCLUSIONS
Automation of infectious disease serology is on the hori-
zon. Techniques for detecting organism-specific antigens
and antibodies have been described and are in process of
development for use in clinical laboratories. A difficult
aspect of this emerging technology will be redefining the
"gold standards." The standards that have been used for
many years may no longer apply to these emerging technol-
ogies as diagnostic criteria become more sophisticated.
The new methods of EIAs, using MAbs or amplification
IMMUNOSEROLOGY OF INFECTIOUS DISEASES 149
techniques or both, have potential for standardizing the
laboratory diagnosis of infectious diseases. These new tech-
nologies will permit and promote sharing of resources.
Consensus conferences will be held to define better the
relationships between test results in various laboratories as
they relate to documented infectious diseases.
Current definitions of infectious diseases can be somewhat
vague and depend on culturing the organism, detecting
antigens from the organisms, detecting IgM antibodies to the
organisms, or detecting an increase in antibody titer after the
acute phase of the infection has passed. Commercially
available reagents have been approved by the Food and
Drug Administration for use in the clinical laboratory, but
consistent methods (e.g., proficiency testing, clinical corre-
lations, etc.) to validate the results have not yet been
established. Each laboratory must validate manufacturer's
claims for their products, not just accept the claims and
report results obtained from performing kit assays. With
increasing availability of specific (but expensive and poten-
tially toxic) antiviral drugs, it will be critical to accurately
identify viral diseases early in the disease process so di-
rected therapy can be instituted.
Much has been accomplished, but much remains to be
discovered, defined, and developed to bring the level of
automation necessary for efficient diagnosis and treatment of
infectious diseases. The 1990s will bring many changes that
will dramatically affect the operations of the clinical micro-
biology laboratory in this era of medical cost containment
and shortages of qualified personnel to perform these assays.
ACKNOWLEDGMENTS
I acknowledge the artistic assistance of Jeffrey James in providing
Fig. 2 and 3. The preliminary review of the manuscript by my
colleagues Robert Chase, Barbara Harrer Lewis, Ed Nowakowski,
and Kenneth Thompson is acknowledged and sincerely appreciated.
The critical review of John B. Carter contributed significantly to the
final report.
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