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Copyright © 1990, American Society for Microbiology
IMMUNE RESPONSE TO MICROORGANISMS Antibody can also diminish infectivity of viruses by pre-
venting attachment to the specific receptor or by introducing
Immunity to Bacteria conformational changes in the viral structure that promote
The immune response to extracellular bacteria must coun- aggregation. Aggregation facilitates more effective elimina-
teract all of the mechanisms of invasion elicited by these tion by antibody-mediated mechanisms such as opsonization
organisms (22, 26). The immune response includes antibod- or complement activation or both. Arboviruses and hepatitis
ies to capsular polysaccharides, to exotoxins (e.g., antistrep- B virus are examples of viruses that can be eliminated by
tolysin 0 [ASO]), and to extracellular enzymes (antihyalu- antibody-mediated events during their release into the blood-
ronidase). Antibodies to tetanus toxin or diphtheria toxin stream or lymphatics.
can neutralize the effects of these toxins and prevent host In certain situations, antibody to viral proteins can be
tissue destruction (16). detrimental to the host. For example, serum antibody to
Complement activation promotes effective opsonization respiratory syncytial virus (RSV), which is not protective
with and without antibody (67). The membrane attack com- but was passively acquired across the placenta from the
plex of the terminal complement components are required to mother, may produce an Arthus (immune complex) type of
lyse and eliminate certain gram-negative organisms (Neis- hypersensitivity reaction in the lungs of infants (20). Similar
seria spp.). For other gram-negative bacteria, a synergistic damaging effects of antiviral antibody have been described
destruction by complement in conjunction with lysozyme is with measles infections in infants.
necessary (21). Complement activation is also necessary to The immune response to intercellularly or vertically trans-
release chemotactic factors to attract phagocytic cells to the mitted viruses involves cell-mediated cytotoxicity. Cyto-
site of infection. toxic effector cells recognize the alterations to the membrane
Endotoxins elicited from certain gram-negative bacteria antigens that are perturbed by viruses and either require
can initiate the activation of the complement alternative specific (T-cell) or nonspecific (natural killer cells or macro-
pathway in the absence of antibody. Endotoxin can also phages) cytolytic effector cells (88). Antibody-dependent
degranulate neutrophils, enhance cytotoxicity, and prompt a cell-mediated cytotoxicity has also been shown to be an
variety of other severe metabolic and potentially lethal effector mechanism of antiviral cytotoxicity (85).
effects if gram-negative bacterial infections are not efficiently
treated (8). Immunity to Fungi
Immunity to intracellular pathogens is primarily cellular
immunity, i.e., delayed T-cell hypersensitivity involving Immunity to fungi is primarily cell mediated. Detection of
lymphocytes, cytokines, and macrophages (48). There are specific IgM and IgG antibodies to certain fungi by immu-
only two methods available to detect delayed T-cell hyper- noprecitin reactions can be helpful in establishing the diag-
sensitivity: in vivo cutaneous injection of purified antigens nosis and following the course of the disease. However, the
(skin or anergy testing) or in vitro lymphocyte transforma- antibodies do not play a protective role.
tion studies with purified antigens. Neither method is highly
reproducible and may be falsely negative due to the immu- TEST SYSTEMS FOR IMMUNODIAGNOSIS OF
nosuppression experienced by individuals suffering from INFECTIOUS DISEASES
invasion by intracellular pathogens. In many situations,
antibody is produced, but serves no demonstrable protective Nonspecific Indicators of Infectious Diseases
mechanism. If antibody production is stimulated by the
intracellular pathogen, detection of that antibody and its The local response to infection or tissue injury or both is
class specificity can be useful in diagnosing the invading acute inflammation, resulting in vascular changes and at-
organism(s). tracting leukocytes. During the first few days following
insult, systemic and metabolic changes occur which com-
Immunity to Viruses prise the acute-phase response (57). Acute-phase proteins
Antibody (immunoglobulin G [IgG] and IgM) capable of include those that usually increase by 50% (complement
binding directly to extracellular viruses may prevent viruses components and ferritin), alpha-1-antitrypsin, fibrinogen,
from infecting other cells. If the virus has a viremic phase, and haptoglobin which increase 200 to 400%, and C-reactive
neutralizing antibodies may be produced. Two types of protein (CRP) which can increase up to 1,000% in severe
neutralizing antibodies that can be demonstrated are com- tissue injury. CRP and complement components are the only
plement independent and complement facilitated. Antibod- acute-phase proteins that have been shown to be directly
ies of the G, M, and A classes have been shown to neutralize involved in the elimination of microorganisms.
the infectivity of virtually all known viruses (21). Intercellu- CRP. CRP is the prototype acute-phase protein. CRP was
lar or vertically transmitted viruses or both would not be originally recognized for its ability to precipitate with the
subject to the neutralizing effects of antibodies. C-polysaccharide fraction of pneumococcus (95). CRP did
134 JAMES CLIN. MICROBIOL. REV.
exposed during its lifetime. It is necessary to characterize cation required ensures optimal functional activity; charac-
painstakingly each polyclonal antibody to ensure that it is terization of the specificity needs to be done only once
detecting only the desired antigen. Still, the high percentage instead of each time an animal is immunized; MAbs gener-
(85 to 90%) of undesired antibodies increases the occurrence ally do not bind to human IgG Fc receptors; and they
of nonspecific binding of conjugated (but irrelevant) antibod- generally are unencumbered by nonspecificity or cross-
ies in test systems. reactivity (93).
Affinity-purified antibodies. To eliminate the antibodies of The disadvantages of MAbs are more subtle: a single
undesired specificity, the IgG fraction of a polyclonal anti- specificity does not enable effective cross-linking of antigens
body can be purified by binding to and eluting from an with antibodies so one MAb cannot be used in agglutination
insolubilized form of the antigen. This is performed by or precipitation reactions, although "cocktails" of several
column chromatography, using a matrix to which the immu- MAbs can overcome that limitation; murine MAbs do not
nizing antigen is coupled with a spacer molecule (an inert efficiently bind Clq to activate complement so are not useful
molecule that serves as a carrier). The spacer is covalently in assays that rely on complement activation; the single
coupled to the antigen and to the insoluble matrix to pre- antigenic determinant to which a MAb binds may not be
serve optimally the relevant antigenic determinants. The expressed under certain conditions of antigen presentation
antibody is then applied to the column, using conditions (e.g., viable versus fixed organisms). Nevertheless, an en-
favorable for antigen-antibody reactions to occur (pH 7.5 to tirely new field of study is available to use these reagents
8.5, physiologic ionic strength). IgG molecules of unrelated effectively for clinically applicable testing systems.
specificity would pass through the column with the wash
buffer. Antigen-specific IgG molecules are then eluted off the Soluble Antigen-Antibody Reactions
antigen matrix, using an elution buffer with a lower pH and Double diffusion in agar (Ouchterlony reactions). The classic
ionic strength. In this way, the functional integrity of the IgG method for detecting antibody and evaluating its specificity
molecules is preserved, while the antibodies are gently (identity, partial identity, or nonidentity) is Ouchterlony dou-
dissociated from the insolubilized antigen. The resulting ble diffusion. Antigens can also be characterized by Ouchter-
affinity-purified antibody is significantly higher (85 to 95%) in lony diffusion when antibodies of known specificity are avail-
specific antibody activity. This process decreases or entirely able. Since the procedure requires 18 to 24 h of diffusion for
eliminates any nonspecific binding due to extraneous anti- the reactions to occur, this method is not as helpful in the
bodies, resulting in a much better reagent for use with rapid diagnosis of acute infections as other methods discussed
immunohistochemical assays. below. The Ouchterlony reaction is primarily used to detect
Polyclonal antibodies that have been affinity purified have antibodies in patients with suspected histoplasmosis, coccid-
many uses in the clinical laboratory. Most antibodies to iomycosis, or aspergillosis and to detect other fungal antigens
human immunoglobulins, conjugated with fluorescent or associated with hypersensitivity pneumonitis.
enzyme labels, are affinity-purified polyclonal antibodies. CIE. Counterimmunoelectrophoresis (CIE), one-dimen-
Polyclonal antibodies bind to several antigenic determinants, sional double electrophoresis, specifically directs the move-
increasing the ability to detect their respective antigens. ment of antigen and antibody toward each other in an
Fractionated antibodies. Antibodies in antisera used to electric field. The buffer pH is selected to optimize the
detect antigens in cultured cell lines (especially herpesvi- electroendosmotic effects of antibody toward the cathode
ruses) bind nonspecifically to the IgG Fc receptors of the (negative pole) while the antigen moves toward the anode
cells. One effective approach to decrease that nonspecific (positive pole). This electrophic movement rapidly (30 min)
binding significantly is to fractionate the IgG antisera to concentrates the antigen and antibody in the zone between
eliminate the Fc regions of the IgG antibody molecules. the adjacent wells. CIE is approximately 10 times more
Under controlled conditions, the enzyme pepsin sequentially sensitive than double diffusion. This was the original method
cleaves the C-terminal end of the IgG molecule (the Fc used to detect hepatitis B surface antigen and antibody
region), leaving the antibody-combining N terminals [the known then as Australian antigen and antibody. CIE has also
F(ab')2 region]. The F(ab')2 fragments can still be effectively been useful for rapid identification of antigens from bacteria
conjugated, e.g., with fluorochromes or enzymes. Affinity- associated with meningitis, septicemia, disseminated intra-
purified antibodies can be pepsin digested, resulting in vascular coagulation, pneumonia, and septic arthritis (88).
affinity-purified F(ab')2 antisera. Depending on the sensitivity and specificity of the antibody
MAbs. The production of monoclonal antibodies (MAbs) used, minimal detectable concentrations of bacterial antigen
was first described in 1975 by Kohler and Milstein (53). The range from 50 to 10 ng/ml (30). CIE has largely been replaced
potential applications for clinical laboratory assays and in with particulate antigen-antibody reactions for detecting
immunotherapy were immediately recognized. MAbs are many bacterial antigens.
prepared by hybridizing antibody-forming cells to continu-
ously replicating cell lines. Each antibody-forming cell, Particulate Antigen-Antibody Reactions
programmed to produce antibody of a single (mono-) speci- Hemagglutination assays. A variety of antigens can be
ficity with a single heavy-chain and single light-chain class, coupled to erythrocytes (RBCs) to provide the indicator
can be cloned to replicate itself almost indefinitely. These system to detect antibodies. Target antigens such as polysac-
antibody-producing clones of cells (hybridomas) are used to charides readily adhere to RBCs, including antigens from E.
prepare virtually unlimited quantities of MAbs that are coli, N. meningitidis, and Toxoplasma sp. as well as purified
chemically, physically, and immunologically completely ho- protein derivative from M. tuberculosis. Carbohydrate anti-
mogeneous and definitively characterizable (93). Hybridoma gens readily adhere to RBCs, but protein antigens require
cells can be stored indefinitely, frozen in liquid nitrogen. As pretreatment with tannic acid (producing "tanned" RBCs)
needed, these hybridomas can be grown in large quantities in or with chromium chloride. Tanning the RBCs facilitates a
tissue culture or propagated in syngeneic mice to form high-density coating which increases the sensitivity of the
ascites from which MAbs can be isolated. test system. Subsequent Formalin or glutaraldehyde treat-
The advantages of MAbs are obvious: the minimal purifi- ment of tanned RBCs coated with either protein or carbo-
136 JAMES CLIN. MICROBIOL. REV.
hydrate allows long-term storage. Treated tanned RBCs irrelevant, but species-specific antibody. Coagglutination
coated with protein antigens have been used for detection of reagents have a much shorter shelf life than latex reagents
antibodies to toxins, e.g., diphtheria toxin or tetanus toxin due to deterioration of the bacteria upon storage. Many of
(16). Treponemal antibodies are detected by using trepone- the clinical laboratory applications of coagglutination have
mal antigens adsorbed to tanned RBCs in the micro-hemag- been essentially replaced by LA or immunoassay tech-
glutination assay for Treponema pallidum (MHA-TP) (98). niques.
HI assays. Hemagglutination inhibition (HI) assays used in
infectious disease serology are based on the capacity of Lytic Assays
certain viral antigens to agglutinate RBCs of selected species
spontaneously. Antibodies present in patient sera prevent CF. Complement fixation (CF) is a two-step procedure
the spontaneous agglutination of the RBCs, thus resulting in which uses complement to lyse indicator RBCs. The first
inhibition of agglutination, indicating a positive test for the step involves reacting antigen with antibody in the presence
presence of antibody. HI was the original method used to of complement in fluid phase. The complement used must be
detect antibodies to rubella virus (76). HI tests cannot from a standard source, e.g., rabbit serum which has been
distinguish between IgM and IgG classes of antibodies. appropriately collected and stored to preserve the hemolytic
Immunoassay techniques which have replaced HI for detec- activity. If corresponding antigens and antibodies are
tion of rubella virus antibodies will be reviewed below. present, the complement cascade will be activated through
Antibodies to other less prevalent hemagglutinating viruses the classical pathway. RBCs coated with RBC antibody (the
are still detected by the HI method. These include influenza indicator system) are added to the first reaction mixture. If
viruses, arboviruses, reoviruses, and certain enteroviruses complement had been activated (fixed) during the first incu-
(49). bation, the indicator particles are not lysed. If the first step
Latex agglutination (LA). Latex particles are spheres of did not contain either the specific antigen or antibody,
polystyrene which readily bind IgG molecules by the Fc complement will bind to the antigen-antibody complex
region at pH 9.0 when a low-ionic-strength buffer is used. present on the indicator particles, and lysis of the indicator
When antibodies are bound by their Fc region, the antibody- RBCs will occur.
combining sites [F(ab) regions] remain exposed and are CF is a semiquantitative method that can be used to detect
capable of binding antigens. When the target antigens have either antigens or antibodies if the corresponding specific
repetitive antigenic structures (e.g., polysaccharides), mul- antibody or antigen is available. CF does not distinguish IgM
tivalent antibodies coupled to multiple latex particles can from IgG antibodies since both can fix complement. This
bind antigen molecules and cross-link the latex particles, method is extremely sensitive and has broad applications for
resulting in agglutination. The latex particles serve as the infectious disease serology, but is rarely used outside of
indicator system to detect the antigen-antibody reaction. reference laboratory situations because it is cumbersome
Theoretically, any antigen (but not hapten) to which and complex (93).
antibodies can be produced should be detectable by LA. LA CF may be the only method available for detecting anti-
is particularly useful to detect bacterial polysaccharide anti- body to less prevalent viruses (e.g., coxsackieviruses). An
gens in CSF or urine or both. LA has essentially replaced advantage of the CF method is that, by keeping all other test
CIE for the detection in CSF of capsular antigens of H. parameters unchanged, many antigens can be tested to
influenzae type b, several N. meningiditis groups, and S. determine population exposure to rare organisms.
pneumoniae for diagnosing bacterial meningitis and for Neutralization assays. Beta-hemolytic group A strepto-
detection of Cryptococcus neoformans capsular antigens in cocci produce a number of extracellular toxins that stimulate
immunosuppressed patients. Group B streptococcal antigens the production of antibodies by infected patients (54). Sev-
are detected by LA in urine or CSF from newborns. eral of these toxins also function as hemolysins and lyse
Coagglutination. Staphylococcus aureus (Cowan strain) RBCs. Specific antibodies neutralize the hemolysins and
contains protein A distributed evenly on the outermost layer inhibit RBC lysis. Analogous to the CF test, a positive test is
of the cell wall. Protein A binds the Fc region of IgG negative for hemolysis, and in a negative test hemolysis is
subclasses 1, 2, and 4 (which constitute 95% of the total detectable. In the first step of the reaction, patient serum
IgG), analogous to the binding of IgG to latex particles. The (antibody) is incubated with the specific hemolysin being
antibody-coated Staphylococcus becomes the indicator re- assayed (antigen). In the second step, group 0 RBCs are
agent to detect the presence of antigens corresponding to the added. If the hemolysin has been neutralized during the first
specificity of the coupled antibody. Coagglutination has been incubation, the indicator particles will not lyse. If specific
used to detect the presence of bacterial antigens in CSF or antibody was not present to neutralize the hemolysin, the
urine. Coagglutination is useful in the immunologic identifi- RBCs will lyse. ASO is the most commonly used neutrali-
cation of bacteria from culture, with commercially available zation test to detect immunologic evidence of exposure to
reagents for grouping streptococci and detecting Staphylo- streptococci.
coccus aureus coagulase production (30).
Since protein A is an effective cross-linking reagent for
most IgG subclasses, any other preformed complexes con- Immunohistochemical Techniques
taining IgG and antigen would also cause agglutination of the The most widely used immunohistochemical techniques
sensitized Staphylococcus reagent. Nonspecific agglutina- are immunofluorescence assays (IFAs). IFA uses tissue or
tion is prevented by treating the body fluid to be tested with bacterial cells as the substrate (source of antigen) affixed to
soluble protein A to block binding of preformed complexes a glass slide and a fluorochrome-conjugated (antibody) de-
or by heating the body fluid to 100'C to denature patient IgG tection system. IFA remains the "gold standard" for many
molecules before testing. infectious disease serology test systems. The advantages of
Coagglutination must be well controlled to detect nonspe- IFA are that (i) the substrate can be visualized, ensuring
cific agglutination in the body fluid, using unsensitized specificity of the reaction; (ii) it is significantly less cumber-
Staphylococcus particles and particles sensitized with an some than CF; (iii) it is highly reproducible when performed
VOL. 3, 1990 IMMUNOSEROLOGY OF INFECTIOUS DISEASES 137
Antigens
.*.,.D....
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 microorganism, but also to evaluate whether the correct
same conjugate and dilution of patient sera can be used to and/or appropriate specimen was collected.
detect antibody to many different organisms or antigens. Treponema pallidum can be detected by direct IFA during
The disadvantages of IFA are that it: (i) requires fresh or early stages of the disease when the organisms are concen-
frozen tissue or cells (processed tissue or smears fixed for trated in the chancre (primary) or in the mucocutaneous
Grams stain are not usable); (ii) requires special equipment lesions (secondary). Direct IFA, using conjugate to T. palli-
and conditions (a fluorescence microscope and a dark room dum which has been absorbed with Reiter treponemes, has
for reading); (iii) is labor intensive, even when the substrate essentially replaced dark-field examination as a diagnostic
for indirect IFAs can be purchased (reagent dilution, multi- test for early stages of syphilis before reaginic antibodies are
ple incubation and wash steps, and cover slips are required); produced (4).
(iv) is subjective, requiring extensive training to read the Direct IFA has been used successfully for detection of
reactions and multiple controls to ensure test specificity and Legionnella spp. (22) and for many viruses, including herpes
sensitivity; and (v) has not been successfully automated. simplex virus (HSV) types 1 and 2, cytomegalovirus (CMV),
Immunohistochemical assays have been developed that RSV, influenza virus types A and B, parainfluenza virus 1, 2,
use enzyme-conjugated antibodies (e.g., horseradish perox- and 3, varicella-zoster virus (VZV), and adenovirus (D.
idase) with correponding substrates that yield different col- Scholes, J. R. Daling, A. S. Stergachis, S. P. Wang, and J. T.
ors when hydrolyzed to contrast with the colors of typical Grayston, Program Abstr. 28th Intersci. Conf. Antimicrob.
histochemical strains. Immunoperoxidase techniques may Agents Chemother., abstr. no. 1171, 1988). Enzyme-conju-
avoid the first two disadvantages of IFA, but the subjective gated antibodies have also been used to detect these micro-
interpretation and labor intensiveness of immunoperoxidase organisms in histologic tissue sections.
methods are even greater than with IFA. The systems Indirect IFAs for total antibody. Indirect IFA is used to
described below for direct and indirect IFA also apply to detect antibodies in patient sera (Fig. 2A). Standardized
immunoperoxidase techniques which have generally not antigens (organisms or virus-infected cell cultures) are fixed
been used for microbiology applications. to glass slides. Patient serum is diluted, layered over the
Direct IFAs. Direct IFA is used to detect antigens or substrate, and incubated to allow the antigen-antibody com-
organisms present in cells or tissues, using fluorochrome- plex to form. Unbound antibody is washed away, leaving
conjugated antisera (conjugate) specific for the antigen(s) in only bound antibody, which is then incubated with the
question. Microbiologic applications of direct IFA include fluorescent conjugate. When antibodies are present in the
detection of Chlamydia trachomatis elementary bodies in patient's serum, a second antigen-antibody reaction will take
columnar epithelial cells from the cervical canal, urethra, place, with the conjugate becoming the third layer on the
eye, or rectum (2). Direct IFA is useful not only to detect the slide.
138 JAMES CLIN. MICROBIOL. REV.
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 The IgM RF binds to these newly exposed IgG determinants.
immunoglobulin is usually anti-IgG with reactivity to both Unless separation methods are used, IgM RF cannot be
kappa and lambda light chains. This light chain reactivity distinguished from organism-specific IgM. Distinguishing RF
also detects antibodies of the IgA or IgM class or both, from organism-specific IgM is particularly important in neo-
which would be advantageous to detect all classes of anti- natal sera since a high percentage of congenitally infected
body reactive with microorganisms. neonates have detectable RF (31). False-positive IgM assays
The classic indirect IFA is the fluorescent treponemal due to heterotypic antibody responses between herpesvi-
antibody absorption (FTA-ABS) test. The antigen, T. palli- ruses may also be detected; i.e., patients infected with
dum Nichols, is fixed to glass slides. Prior to incubation with Epstein-Barr virus (EBV) or VZV may demonstrate CMV
the antigen, the patient's serum is absorbed with the non- IgM antibodies without evidence of CMV infection (56).
pathogenic T. pallidum Reiter. This absorption step signifi- IgM assays can also be subject to false-negative results if
cantly increases the specificity of the test (18); however, IgG antibody inhibits or competes with IgM for binding sites
false-positive reactions can still occur in patients with au- on the antigen (Fig. 3B). To avoid any possibility of false-
toimmune diseases (55) and hypergammaglobulinemia and in positive or false-negative reactions in most methods, IgG
those with Lyme disease due to immunological cross-reac- should be separated from IgM before the assays are per-
tivity with antigenic sites on the spirochetes (60). formed. Separation methods will be discussed below.
Other commonly performed indirect IFAs include The demand for assays that detect IgM antibodies to CMV
TORCH titers for evaluating the immune status of pregnant and HSV has recently increased due to infections in organ
women. TORCH tests include toxoplasma (TO), rubella transplant patients. The immunosuppressed state of trans-
virus (R), CMV (C), and HSV (H). These organisms cause plant patients increases their susceptibility to these oppor-
significant congenital infections resulting in stillbirths or a tunistic viruses since both humoral and cell-mediated immu-
spectrum of congenital diseases, although infections in nity are required for optimal viral immunity. Although
adults or children frequently are mild or even subclinical. specific IgG antibody may be present, it may not be as
Although once ordered as a panel of tests, current preferred effective in controlling viral infections in patients whose
practice is to perform only the specific test based on the cell-mediated immunity has been abrogated to prevent trans-
mother's clinical and exposure history. plant rejection. The production of IgM antibodies is T
Indirect IFAs for IgM antibody. If the test system is independent, i.e., does not require the presence or cooper-
designed to detect antibodies produced during acute infec- ation of T cells (77). IgM antibodies are produced in immu-
tion, the conjugate must be specific for IgM heavy chains nosuppressed patients and can be useful indicators of acute
with no light-chain or other heavy-chain reactivity. When infection.
congenital infections are suspected as the cause of stillbirth Amplification IFAs. The sensitivity of IFAs is limited by
or abnormalities of a newborn, indirect IFAs for IgM anti- the level of fluorescence detectable by the human eye. The
body should be used to detect IgM antibodies produced by optimal fluorescein/protein ratio of a polyclonal antibody
neonates. Unlike maternal IgG, IgM does not cross the conjugate is 2.5 (34), i.e., two to three fluorescein molecules
placenta, so any specific IgM antibodies detected would for each immunoglobulin molecule, which is satisfactory for
have been produced by the fetus in response to a congenital detecting high-density antigens such as bacterial or proto-
infection. Healthy newborns have 5 to 15% of the adult level zoan cell surface antigens. Directly conjugating antibodies at
of total IgM since the in utero environment is essentially a higher fluorescein/protein ratio results in significantly
sterile. If an organism is transmitted to the fetus from the increased nonspecific staining. Detection of low-density
mother during gestation, the fetus will develop its own IgM antigens or using MAbs or both may require a higher
response to the organism, which would be detectable in an fluorescein/protein ratio to achieve desired sensitivity.
IgM-specific indirect IFA. Indirect or double-indirect IFA has been used to amplify
False-positive and false-negative IFAs for IgM antibody. the fluorescent signal and improve sensitivity. If the antigen
Indirect IFAs for IgM antibody are subject to false-positive can be detected by direct IFA, indirect IFA increases the
reactions attributable to the presence of rheumatoid factor test sensitivity. If the test antibody is difficult to detect by
(RF) activity in the serum being tested (Fig. 3A). RF binds to indirect IFA, a double-indirect IFA would increase the
IgG when IgG is bound to antigen. The process of binding to sensitivity of detection (Fig. 2B). For example, if organisms
an antigen causes a confirmational change in IgG which were not visible in the T. pallidum direct IFA, the test could
exposes new antigens on the Fc region of the IgG molecule. be repeated with unconjugated rabbit antibody to the
VOL. 3, 1990 IMMUNOSEROLOGY OF INFECTIOUS DISEASES 139
treponemes, any unbound antibody could be washed off, and presence or absence of an antigen by a color change of liquid
the bound rabbit antibody could be detected by using a in a test tube, of a matrix on a stick or a paddle, or of a
fluorochrome-conjugated goat anti-rabbit IgG as the conju- matrix on a plastic reaction vial (e.g., ICON; Hybritech Inc.,
gate. Each rabbit IgG molecule contains multiple antigenic San Diego, Calif.). Most of these methods were developed to
sites which would be recognized by the goat anti-rabbit IgG. meet the demands for more rapid test results. The tech-
If four goat anti-rabbit IgG molecules bind to the rabbit IgG, niques were reported to be simple enough to be performed
the fluorescent intensity would be magnified four times. by nontechnical employees such as those in a doctor's office
Additional controls would need to be included in each assay setting. Although the price per test was high, the EIA
to ensure that the extra step did not decrease the specificity technology represented a cost savings to doctors' offices
while increasing the sensitivity. because the results were available before the patient left the
Complement-amplified IFA, also called anticomplement office. This was particularly beneficial for rapid testing for
IFA, has been used primarily for detection of herpesviruses streptococcal pharyngitis since the physician could prescribe
(74). Herpesvirus-infected tissue culture cells have an en- or withhold antimicrobial agents based on the test results
hanced expression of IgG Fc receptors that nonspecifically rather than providing empirical treatment or telephone fol-
bind IgG antibody molecules. low-up with the patient or both. Clinical microbiology labo-
Anticomplement IFA uses complement as the third of four ratories have resisted accepting and applying this new tech-
layers (Fig. 2C). Antigen is the substrate on a slide; patient's nology because of a high percentage of false-negative results
serum is added analogously to performing an indirect IFA. A (25). The compromise acceptable to many laboratories is to
source of active complement (e.g., guinea pig) is added. take two throat swabs: if the rapid test is positive, discard
While IgG molecules bound to Fc receptors cannot bind the second swab; if the test is negative, use the second swab
Clq, IgG or IgM bound to antigen by the Fab region does for culture.
bind Clq and activates the classical complement pathway. A recently introduced technique uses liposomes, artificial
After unbound complement and other proteins are washed lipid spheres, to detect group A streptococcal antigens (BBL
away, an F(ab')2, fluorochrome-conjugated, anti-guinea pig Microbiology Systems, Cockeysville, Md.). In this test,
C3 is added (74). Anticomplement IFA eliminates the need specific antibody is adsorbed to a porous matrix. The patient
to control for nonspecific IgG binding since a complement specimen is added and any antigen present binds to the
component is detected instead of IgG. Although not effi- antibodies. Liposomes containing a colored dye inside con-
ciently performed in a clinical laboratory setting, this four- centric lamillar layers and coated with antibodies to the same
step procedure may be the only method available for detect- antigen are then added. If no antigen was bound by the first
ing antibodies to certain herpesvirus antigens (e.g., the antibody, the liposomes flow through the porous matrix. If
nuclear antigen of EBV [EBNA]). liposomes are bound, the dye is released by a wash solution
A very versatile amplification technique is the avidin- which lyses the liposomes, depositing the dye on the matrix.
biotin complex. Biotin covalently coupled to antibody is A recent entry into the solid-phase EIA bacterial antigen
used for the primary reagent. Conjugated avidin is the marketplace is Chlamydia antigen detection (P. Coleman, V.
second reagent (Fig. 2D). Avidin has a very high binding Varitek, T. Grier, J. Hansen, G. Kurpiewski, J. Safford, B.
affinity (1015 Kin) for biotin, and each biotin molecule will Marchlewicz, and I. K. Mushahwar, Program Abstr. 28th
bind four avidin molecules (3). Avidin can be saturated with Intersci. Conf. Antimicrob. Agents Chemother., abstr. no.
fluorescein molecules without loss of its ability to bind to 1184, 1988). The IFA method done in many clinical micro-
biotin. This results in very bright specific staining with biology laboratories depends on receiving a satisfactory
minimal nonspecific staining. Controls are few compared specimen. With the solid-phase EIA technology, the results
with other amplification methods and include conjugated are either positive or negative (Eastman Kodak Clinical
avidin alone and an unrelated biotinylated antibody. Products, Rochester, N.Y.; Abbott Laboratories Diagnos-
A distinct advantage of the avidin-biotin complex system tics Div., Abbott Park, Tll.). A negative EIA could mean
is that the same biotinylated antibody can be used with either "no chlamydia" or "unsatisfactory specimen." Re-
several different conjugated avidins, i.e., fluorescein (green ports of negative results should clearly state both possibili-
fluorescence), phycoerythrin (red fluorescence), peroxidase ties and be confirmed by the IFA method, similar to per-
(light microscopy), or ferritin (for electron microscopy). The forming a streptococcal culture when the rapid antigen test is
avidin-biotin complex technique works well with MAbs negative.
because biotinylation is fast (1 h), gentle (reaction at pH 7.0 Rapid detection EIA methods for other sexually transmit-
to 8.5 at 22°C), and efficient (biotinylation of 95% of the ted diseases may be available to doctors' offices and even to
amino groups of an antibody molecule does not alter the the general public in the very near future. Under develop-
antigen-binding capacity of the antibody) (35). Biotinylation ment are tests for gonococcus, trichomonas, and human
reagents can be purchased commercially (Vector Laborato- immunodeficiency virus. Concerns of clinical microbiolo-
ries, Burlingame, Calif.), mixed with the desired antibody, gists include the loss of epidemiologic tracking. Rapid anti-
and used the same day. Avidin conjugated to any indicator gen EIA methods simply detect gonococcal antigens and do
system feasible to use is commercially available. Avidin- not evaluate antimicrobial susceptibility. Therefore, they fail
biotin complex has not realized its full potential in the field of to detect antimicrobial agent-resistant strains of N. gonor-
microbiology. rhoeae. To respond to resulting problems, clinical microbi-
ologists should be aware of the availability of these test
Immunoassay Techniques systems, their level of sensitivity, and their degree of spec-
ificity.
Rapid EIAs to detect bacterial antigens. A plethora of Solid-phase methods for detection of antibodies. Enzyme-
commercially available products has been released in the linked immunosorbent assays (ELISAs) are a variation on
past few years which use enzyme immunoassay (EIA) tech- the coated-tube assay described above, but the roles of the
nology for detection of proteins and bacterial antigens. Most antibody and the antigen are reversed. Instead of an anti-
of the early methods were qualitative assessments of the body being the solid-phase component, antigen is coupled to
140 JAMES CLIN. MICROBIOL. REV.
a surface. The second layer of the sandwich is antibody from EIAs for IgM use either absorption with anti-human IgG (44)
the patient serum. The third layer is an enzyme-conjugated or aggregated human gamma globulin (AHGG) (47). Anti-
anti-human IgG or IgM. The indicator system is the color IgG binds to IgG in the sample, removing the IgG reactivity
change resulting from cleavage of the substrate by the to the antigen; if no IgG is available to bind to the antigen,
conjugated enzyme. The intensity of the color is directly RF would not be bound to IgG. AHGG neutralizes RF, but
proportional to the amount of patient antibody bound to the does not eliminate the potential for false-negative results by
antigen. IgG competing with IgM for binding sites on the antigen.
Another interesting approach to EIA systems is FAST One study that compared various methods of removing IgG
ELISA (Falcon Assay Screening Test ELISA; Becton Dick- from patient sera found anti-IgG treatment superior to
inson Labware, Oxnard, Calif.), which uses coated polysty- AHGG for eliminating nonspecific IgM activities without
rene beads attached by a tine to the lid of a microdilution impairing specific activities (31), but this finding needs
tray (39). The advantage of the FAST system is that the same confirmation. The simultaneous dilution of serum and ab-
dilution of patient sera, conjugated antibody, and enzyme sorption with either anti-IgG or AHGG, as used in several
substrate could be used for multiple tests by simply using commercial ETAs, is more efficient than ion-exchange sepa-
beads coated with different antigens. ration of IgM from IgG. Our experience with IgM EIAs from
A manufacturer of automated microbiology equipment three sources suggests that the quantity of anti-IgG used by
(Vitek Systems, Hazelwood, Mo.) has developed an auto- some manufacturers may not be adequate to remove the IgG
mated immunodiagnostic assay system (VIDAS) which uses antibody from patients with hypergammaglobulinemia (K.
the solid-phase ELISA format to detect antigens and anti- James, R. Van Enk, and K. Thompson, manuscript in
bodies directly from patient specimens in 1 to 2 h. The preparation).
solid-phase receptacle is a pipette tip-shaped device made of Capture assays. Capture assays are another variation of
polystyrene or polypropylene. Reagents are predispensed solid-phase technology that can be used to detect antibodies
into a cuvette strip which also includes containers of predis- or antigens. The capture assay for antigen detection uses
pensed wash solutions. A computer-controlled instrument either polyclonal antibodies or MAbs attached to the solid
performs the tests in batch mode or by random access. phase to bind (capture) the antigen being detected. The
Antigen detection tests undergoing field trials include Chla- enzyme-labeled antibody for detection can be either mono-
mydia trachomatis, RSV, HSV, and Clostridium difficile clonal or polyclonal. Polyclonal antibodies more reliably
toxin. Antibody detection tests for human immunodeficiency detect different forms of the antigen, in contrast to MAbs
virus are also being tested (W. M. Janda, M. H. Graves, K. which bind to only a single epitope. There is some limited
Hoffman, L. M. Wilcoski, J. M. Stevens, and L. M. Gor- evidence that capture assays are more sensitive when poly-
niak, Abstr. Annu. Meet. Am. Soc. Microbiol. 1989, C-45, p. clonal antibodies that bind multiple antigenic epitopes are
401). The company has developed assays for tests that are used (M. D. Tolpin and M. A. Collins, Clin. Microbiol.
currently labor intensive or lengthy, have diagnostic utility, Newsl. 10:109-111, 1988).
and do not need antibiotic susceptibility testing. Plans for In the capture assay for detecting IgM antibodies to
future development include direct antigen detection of My- specific viral antigens, an animal antibody to the Fc region of
cobacterium spp., Mycoplasma pneumoniae, and antibodies human IgM is attached to a solid-phase matrix (69). Poten-
to the other organisms of the TORCH panel. tially, all of the IgM molecules in the patients' sera can be
IgM and IgG separation methods. To avoid false-positive bound to the anti-IgM antibody, regardless of their antigenic
and false-negative results in specific IgM and IgG assays, specificity. The antigen to be bound by the IgM is conjugated
several methods are available to separate IgG from IgM in with an enzyme (or biotin when an amplification technique is
serum. Physical separation based on the differential size of necessary) and incubated with the "captured" IgM. If IgM
these immunoglobulins can be achieved by molecular- specific for the enzyme-conjugated antigen is present, the
sieving column chromatography, but this is not practical for antigen is captured by the patient IgM, and enzyme substrate
clinical laboratory test systems. Sucrose gradient ultracen- will be cleaved. The color reaction is directly proportional to
trifugation would also separate IgM from IgG based on size; the level of specific IgM antibody in the patients' sera.
however, IgG-containing immune complexes sediment with The capture assay has wide potential for detecting IgM
IgM (31). Most clinical laboratories do not have access to antibodies to microorganisms. Microdilution trays can be
ultracentrifugation equipment. coated with anti-IgM and used for detecting antibodies with
An efficient technique commonly used for IFA assays uses many different antigenic specificities. A single dilution of
miniature ion-exchange columns (46) which are commer- patient serum can be applied to the coated wells. Different
cially available from at least two sources. IgG passes antigens can be enzyme conjugated (or biotinylated and used
through the column and IgM is retained. IgM is eluted from with a single enzyme-conjugated avidin) and a single sub-
the column by a buffer at a lower pH and higher ionic strate can be used to develop the color reaction. The capture
strength than the application buffer. The volume of serum assay would be an efficient method to screen sera to detect
used and the volume of eluting buffer are carefully controlled the presence or absence of several antibodies (e.g., evaluat-
to ensure that the final eluate is equivalent to a known ing congenital infections). Recently, a variation of the cap-
dilution of serum; therefore, the final results are semiquan- ture assay technique has been automated for high-volume
titative as a titer. hepatitis tests (Abbott Laboratories Diagnostics Div.), in-
In some methods, serum is absorbed with staphylococcal cluding hepatitis B surface antigen and antibody, hepatitis B
protein A either by using the actual bacteria or by binding core antigen, and hepatitis A virus antibody (27).
protein A to an insoluble matrix used as an absorption The capture assay works well for detecting IgM antibod-
reagent. Protein A absorption removes IgG subclasses 1, 2, ies, but is not useful for specific IgG antibodies. During the
and 4 (but not 3) from the serum. There is increasing acute phase of infections, especially congenital viral infec-
evidence that protein A also removes significant virus- tions, the majority of the IgM present is virus specific. In
specific IgM activity (44). contrast, virus-specific IgG would be <10% of the total IgG.
To prevent interference by IgG, commercially available 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.
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 CLIN. MICROBIOL. REV.
both pre- and posttransplant. If IgM antibodies are undetect- role in human disease. A positive ASO titer is useful in
able prior to transplant or prior to appearance of symptoms, establishing the diagnosis of acute rheumatic fever or acute
detection of IgM antibodies would confirm a currently active poststreptococcal glomerulonephritis which may occur
viral infection. Although specific IgM generally does not weeks or months after resolution of a streptococcal infection
persist for longer than 4 to 8 weeks, IgM antibodies to (51). ASO is produced by most strains of group A strepto-
viruses (especially CMV) have been reported to be detect- cocci and elicits a strong antibody response during 80% of
able for months or years after a documented infection due to pharyngeal streptococcal infections, but in only 25% of
the continued intracellular presence of the virus (42). Detec- streptococcal pyoderma skin infections (E. M. Ayoub, Clin.
tion of CMV IgM is convenient for diagnosing CMV infec- Immunol. Newsl. 3:107-109, 1982). An LA kit is available
tions in transplant patients; however, there is conflicting for ASO testing (Behring Diagnostics, Inc., Somerville,
evidence about whether the detection of CMV viremia is a N.J.) which yields results equivalent to those by the neutral-
better indicator of clinically significant infection (J. Nelson, ization assay but much more efficiently (unpublished obser-
F. Strie, C. Benson, J. Pottage, and H. Kessler, Abstr. Clin. vations).
Res. 464A, 1988; G. A. Storch, E. D. Spitzer, L. Marsano, Anti-DNase B is the assay most frequently used to confirm
M. W. Flye, D. W. Hanto, P. R. Murray, and R. P. Perillo, streptococcal sequela which can result from skin infections.
Program Abstr. 28th Intersci. Conf. Antimicrob. Agents DNase B is produced by most strains of group A strepto-
Chemother., abstr no. 816, 1988). cocci, but the antibody response appears later than the
The absence of specific IgM may be misleading. Timing of response to streptolysin 0 and lasts longer. Consequently,
specimen collection is critical to its detection. If collection is anti-DNase is more valuable than ASO in detecting strepto-
too early, IgM may not be present in detectable levels (Fig. coccal antibodies in Sydenham's chorea because of the long
1). If the specimen is obtained too late, IgM may have latent period between streptococcal infection and develop-
disappeared. Conversely, with some viral infections, specific ment of chorea (51). Antihyaluronidase titers are useful to
IgM antibody can be detected from 12 to 18 months after the detect antibodies to streptococcal hyaluronidase following
acute onset of the disease (81). Consequently, IgG and IgM pyoderma. In patients with suspected streptococcal se-
assays should be performed simultaneously to evaluate a quelae, a negative ASO titer would be the indication to
patient with an acute infectious process. Both acute and perform anti-DNase or antihyaluronidase or both.
convalescent specimens should be collected if there is any A commercially available product, Streptozyme (Wam-
ambiguity about interpretation of results from the acute pole Laboratories, Cranbury, N.J.), consists of particles
specimen. When transplant patients are being evaluated for coated with a mixture of various intracellular and extracel-
immune status, both IgG- and IgM-specific tests should be lular antigens produced by group A beta-hemolytic strepto-
performed to establish base-line levels by which to compare cocci. Streptozyme may be an effective screening test for
posttransplant results. streptococcal antibodies other than ASO. A positive Strep-
Serologic tests for syphilis. Nontreponemal tests for syph- tozyme with a negative ASO test indicates an antibody
ilis are the most common applications of screening for response to another extracellular streptococcal toxin. The
disease by the detection of antibodies. The rapid plasma manufacturers of this product have not revealed the specific
reagin (RPR) and the Venereal Disease Research Laboratory content of antigens, toxins, or hemolysins used in the
(VDRL) tests detect Wasserman or reaginic antilipid anti- coating process, and lot-to-lot variations in reactivity with a
bodies. Reaginic antibodies are produced in response to panel of sera have been reported (33). We have observed
lipoidal material released from damaged host cells early in negative Streptozyme tests in patients with positive ASO
infection as well as to lipid present on the cell surface of the titers and hypothesize that insufficient streptolysin 0 antigen
treponeme (62). Although very sensitive, the screening tests was present on the particles to cross-link with antibody and
are not specific for T. pallidum infections. Reactive results agglutinate the coated particles. For that reason, we use
must be confirmed by specific treponemal tests, e.g., FTA- Streptozyme as a follow-up to a negative ASO test. If the
ABS or MHA-TP. ASO test and Streptozyme test are both negative in a patient
Premarital and prenatal serologic tests for syphilis are with a clinical presentation consistent with poststreptococ-
required in most states to detect syphilis during the repro- cal sequelae, we recommend the anti-DNase and antihyalu-
ductive years. Syphilis can be effectively treated with peni- ronidase tests (41).
cillin or other antimicrobial agents if detected in early stages. Detection of high titers of one or more of these antibodies
Prenatal screening is intended to quell congenital transmis- or a rise in titer over time or both confirm the diagnosis of
sion of the disease. Biologic false-positive nontreponemal streptococcal infection. If acute poststreptococcal glomeru-
serologic tests for syphilis are commonly observed in pa- lonephritis is suspected, obtaining C3 and C4 levels may be
tients with autoimmune diseases (55). Biologic false-positive helpful in making the diagnosis. Acute poststreptococcal
RPRs range from a low titer (undiluted) to a high titer of 32; glomeralonephritis provides the activating surface necessary
some, but not all, biologic false-positive tests are associated for alternative pathway activation, significantly decreasing
with cardiolipin antibodies. The highly specific but less C3 levels, while C4 remains at normal levels.
sensitive treponemal test(s) should be performed to confirm EBV antibodies. EBV is the primary cause of infectious
a reactive RPR prior to instituting therapy. An RPR titer can mononucleosis (IM), also contributes to Burkitt's lymphoma
be used to monitor the effectiveness of treatment since the and nasopharyngeal carcinoma, and has been erroneously
nontreponemal antibodies decrease with treatment and dis- implicated in chronic fatigue syndrome (70). Because the
appear altogether with time if treatment was effective. The virus is so ubiquitous and most healthy adults in the United
FTA-ABS or MHA-TP assays may, however, remain reac- States have antibody to EBV, the appropriate screening test
tive for years after the spirochete has been successfully forIM is still the classic heterophile. Although 30% of EBV
eradicated. IM cases may be heterophile negative at the time of presen-
Streptococcal antibodies. In spite of readily available anti- tation, in 85 to 90% of IM cases, Paul-Bunnel heterophile
microbial agents, group A streptococci remain significant tests are positive by week 2 of illness and no further testing
pathogens whose immunologic sequelae play an important 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
toms or documented exposure to the spirochete. A properly Antibodies to Campylobacter pylon may be useful in the
performed negative test rules out Lyme disease, but a differential diagnosis of gastritis and peptic ulcer disease
positive test should be interpreted cautiously. A rise in titer (S. P. Holland, M. F. Go, B. I. Hirshowitz, W. J. Koops-
after acute onset of symptoms would be diagnostic. The man, and S. M. Michalek, Abstr. Annu. Meet. Am. Soc.
difficult diagnoses are in patients with chronic progressive Microbiol. 1988, Abstr. E85, p. 123); patients with gastric
symptoms that could be those of Lyme disease. Cross- ulcer had elevated levels of Campylobacter pylori-specific
reactivity can occur with antibodies to other spirochetes; IgM antibodies.
e.g., Treponema pallidum, which can result in a biologic Until recently, detecting high titers of cold agglutinins was
false-positive FTA-ABS (60). The RPR or VDRL test or considered an appropriate screening test for Mycoplasma
both, however, would not be falsely reactive. Although not pneumoniae, although this test was positive in only 50% of
yet commercially available, a Western blot (immunoblot) documented cases. IgG and IgM indirect IFAs have been
assay to confirm questionable antibody reactions is being developed (7, 87) and are now available commercially (Zeus
used at major medical centers located in endemic areas (12). Scientific Inc., Raritan, N.J.). Recently, an LA method
As with serologic tests for syphilis, there is a need for two (Access Medical Systems, Bradford, Conn.) has been intro-
tests for Lyme disease antibodies: (i) one that uses washed duced as an effective screening test for antibodies to Myco-
whole organisms or sonicated B. burgdorferi as a screening plasma pneumoniae (D. L. Smalley and C. E. Dunn, Abstr.
test (analogous to RPR or VDRL); and (ii) an absorption test Annu. Meet. Am. Soc. Microbiol. 1988, V12, p. 419).
(analogous to FTA-ABS) or Western blot for confirmation. Other viral antibodies. A number of other viral antibodies
Antibodies to other microorganisms. Chlamydial antibodies have diagnostic or prognostic significance. IgM antibodies to
can be found in a high proportion of individuals who are not hepatitis B core antigen are diagnostic, as are IgM antibodies
currently infected, thus limiting the usefulness of serologic to hepatitis A virus. An abundance of literature is already
methods (2). An ELISA which detects IgM antibodies has available about interpreting hepatitis antigen and antibody
been reported to be more useful than the detection of tests (14, 82). Diagnostic testing for detecting antibodies to
chlamydial antigen for diagnosing chlamydial pneumonitis in and antigens of human immunodeficiency virus are used to
infants (61). A recent report indicates that an IFA antibody determine exposure to this virus. These tests have recently
titer to Chlamydia trachomatis correlates with the incidence been extensively reviewed (43). EIA tests for human T-
of pelvic inflammatory disease in women of childbearing age lymphotrophic virus type antibodies are used to screen the
(Scholes et al., 28th ICAAC). These data support the role of blood supply to minimize the possiblity of transfusion-
prior chlamydial infection in pelvic inflammatory disease, acquired exposure to this virus which has been etiologically
but this finding remains to be confirmed. Recently, a test associated with adult T-cell leukemia or lymphoma (66).
system to detect chlamydial antibodies has become commer- Other, less prevalent viral antibodies can be detected by CF
cially available (Labsystems, Inc., Research Triangle Park, methods at reference laboratories such as state department
N.C.). 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 IMMUNOSEROLOGY OF INFECTIOUS DISEASES 149
TABLE 10. Commercially available assays for RSV antigens techniques or both, have potential for standardizing the
Method Company laboratory diagnosis of infectious diseases. These new tech-
nologies will permit and promote sharing of resources.
Macro-ELISA Abbott Laboratories Diagnostic Div., Consensus conferences will be held to define better the
Abbott Park, Ill. relationships between test results in various laboratories as
they relate to documented infectious diseases.
IFA Bartels Div. of Baxter, Bellevue, Wash. Current definitions of infectious diseases can be somewhat
DFA vague and depend on culturing the organism, detecting
Rapid-ELISA Becton Dickinson Microbiology Systems, antigens from the organisms, detecting IgM antibodies to the
Cockeysville, Md. organisms, or detecting an increase in antibody titer after the
acute phase of the infection has passed. Commercially
IFA Difco Laboratories, Detroit, Mich. available reagents have been approved by the Food and
Drug Administration for use in the clinical laboratory, but
Macro-ELISA Kallestad Diagnostics, Austin, Tex. consistent methods (e.g., proficiency testing, clinical corre-
lations, etc.) to validate the results have not yet been
Micro-ELISA Ortho Diagnostic Systems, Raritan, N.J. established. Each laboratory must validate manufacturer's
IFA claims for their products, not just accept the claims and
a Macro-ELISA, ELISA in a tube; IFA, indirect IFA; DFA, direct immu- report results obtained from performing kit assays. With
nofluorescence assay; micro-ELISA, ELISA in a microdilution tray; rapid- increasing availability of specific (but expensive and poten-
ELISA, use of a polystyrene support with reaction membrane. tially toxic) antiviral drugs, it will be critical to accurately
identify viral diseases early in the disease process so di-
newly acquired infections, this method will not be helpful to rected therapy can be instituted.
monitor patients, since antigenuria in most systems tested Much has been accomplished, but much remains to be
can persist for months to years after resolution of the discovered, defined, and developed to bring the level of
infectious process. automation necessary for efficient diagnosis and treatment of
Pneumococcal C-polysaccharide detection in sputum and infectious diseases. The 1990s will bring many changes that
other body fluids has been helpful in confirming the diagnosis will dramatically affect the operations of the clinical micro-
of pneumococcal pneumonia (A. J. Parkinson, M. Davidson, biology laboratory in this era of medical cost containment
and M. Rabiego, Progr. Abstr. 28th Intersci. Conf. Antimi- and shortages of qualified personnel to perform these assays.
crob. Agents Chemother., abstr. no. 473, 1988). The EIA
method was found to be more sensitive than culture to detect ACKNOWLEDGMENTS
S. pneumoniae in patients who had received antimicrobial I acknowledge the artistic assistance of Jeffrey James in providing
agents. Fig. 2 and 3. The preliminary review of the manuscript by my
Other viruses. Many lower-respiratory-tract virus infec- colleagues Robert Chase, Barbara Harrer Lewis, Ed Nowakowski,
tions in infants and children are due to RSV. Several EIAs and Kenneth Thompson is acknowledged and sincerely appreciated.
for RSV are available which appear to be more sensitive than The critical review of John B. Carter contributed significantly to the
culture for detecting RSV (Table 10). Rapid diagnosis of final report.
RSV among hospitalized children may minimize the occur- LITERATURE CITED
rence of nosocomial infections and facilitate initiation of
appropriate antiviral therapy instead of antimicrobial agents 1. Barka, N., J. P. Tomasi, and S. Stadtsbaeder. 1986. ELISA
(52, 97). At least one of these kits (Abbott Diagnostics) is using whole Legionella pneumophila cell as antigen: compar-
ison between monovalent and polyvalent antigens for the
considered to be appropriate for the physician office labora- serodiagnosis of human legionellosis. J. Immunol. Methods
tory (C. G. Wren, B. J. Bate, B. A. Masters, and B. A. 93:77-81.
Lauer, Program Abstr. 28th Intersci. Conf. Antimicrob. 2. Barnes, R. C. 1989. Laboratory diagnosis of human chlamydial
Agents Chemother., abstr. no. 208, 1988). infections. Clin. Microbiol. Rev. 2:119-136.
Direct IFA methods are available for detecting HSV and 3. Berman, J. W., and R. S. Basch. 1980. Amplification of the
VZV in vesicular lesions (81). CMV detection by direct IFA biotin-avidin immunofluorescence technique. J. Immunol.
has been reported for lung biopsies (36) and bronchoalveolar Methods 36:335-338.
4. Bradford, L. L., and S. A. Larsen. 1985. Serological tests for
savage cells (24). A modification of direct IFA for detection syphilis, p. 910-920. In E. H. Lennette, A. Balows, W. J.
of CMV uses short-term (24-h) culture in shell vials followed Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical
by direct IFA detection of the CMV. This method signifi- microbiology, 4th ed. American Society for Microbiology,
cantly decreases the turnaround time for confirmation of Washington, D.C.
CMV in patients with low levels of organisms (B. A. Forbes 5. Burgdorfer, W. A., A. G. Barbour, S. F. Hayes, J. L. Benach,
and N. L. Dock, Clin. Microbiol. Newsl. 10:17-21, 1988). E. Grunwaldt, and J. P. Davis. 1982. Lyme disease-a tick-
borne spirochetosis? Science 216:1317-1319.
CONCLUSIONS 6. Canadian Multicentre Transplant Study Group. 1986. A ran-
domized clinical trial of cyclosporine in cadaveric renal trans-
Automation of infectious disease serology is on the hori- plantation. N. Engl. J. Med. 314:1219-1225.
zon. Techniques for detecting organism-specific antigens 7. Carter, J. B., and S. L. Carter. 1983. Acute-phase, indirect
and antibodies have been described and are in process of fluorescent antibody procedure for diagnosis of Mycoplasma
development for use in clinical laboratories. A difficult pneumoniae infection. Ann. Clin. Lab. Sci. 13:150-155.
8. Cerami, A., and B. Beutler. 1988. The role of cachectin/TNF in
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"gold standards." The standards that have been used for 9. Claus, D. R., A. P. Osmand, and H. Gewurz. 1976. Radioim-
many years may no longer apply to these emerging technol- munoassay of human C-reactive protein and levels in normal
ogies as diagnostic criteria become more sophisticated. sera. J. Lab. Clin. Med. 87:120-128.
The new methods of EIAs, using MAbs or amplification 10. Claus, D. R., J. Siegel, K. Petras, A. P. Osmand, and H.
150 JAMES CLIN. MICROBIOL. REV.
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11. Clements, M. L., J. S. Dumler, and P. Fiset. 1983. Serodiagno- 31. Fung, J. C., and R. C. Tilton. 1985. TORCH serologies and
sis of Rocky Mountain spotted fever: comparison of IgM and specific IgM antibody determination in acquired and congenital
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12. Coleman, J. L., and J. L. Benach. 1987. Isolation of antigenic 32. Garcia, L. S., T. C. Brewer, and D. A. Bruckner. 1987.
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group A streptococci directly from throat swabs. J. Clin. cytomegalovirus immunoglobulin M activities in the enzyme-
Microbiol. 25:504-508. linked immunosorbent assay by using anti-human immuno-
26. Farmer, S. G. 1985. Immunology of bacterial infections, p. globulin G. J. Clin. Microbiol. 23:576-581.
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