Serologic Tests in Infectious Diseases: Clinical Utility and Interpretation

Symposium on Infectious Diseases

Serologic Tests in Infectious Diseases Clinical Utility and Interpretation

Allan J. Weinstein, M.D.,i.' and Stephen Farkas, M.D.**

Serodiagnosis is based on the principle that the reaction between an antibody and an antigen will result in a recordable event. Although techniques have become more refined over the years, the goals of serodiagnosis have remained unchanged: to identify an antigen or antibody in order to help determine the etiologic importance of a particular microorganism and to measure the immunologic response to the microorganism. The results of serologic tests alone are seldom sufficient to establish a diagnosis. Furthermore, a single antibody determination is of little value since it provides no indication of the timing of the infection. There must have been a four-fold increase in titer between the "acute" and "convalescent" samples in order to confirm the presence of an infection. The time interval between acute and convalescent determinations varies according to the organism being investigated. For any particular microorganism, a number of techniques are available to measure antibody activity. Each technique may assess the response to a different antigen, and each antibody may develop at a different time. The interpretation of a particular serodiagnostic method must be based both on its sensitivity and its specificity. Sensitivity is a measure of the ability of a method to be positive in persons known to have a disease, while specificity reflects the ability of the test to be negative in individuals known not to have the disease.

PROCEDURES In the past, most serodiagnostic procedures depended on direct methods which indicated the reaction between a known antibody and *Department of Infectious Diseases, The Cleveland Clinic Foundation; Clinical Assistant Professor of Medicine, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio **Special Fellow, Department of Infectious Diseases, The Cleveland Clinic Foundation, Cleveland, Ohio

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test antigen, or a known antigen and test antibody. One of these methods is the agglutination test. In this technique an insoluble antigen aggregates with an antibody. A constant concentration of test antigen is combined with serial dilutions of patient's serum; the antibody titer is equal to the highest dilution of serum that causes agglutination of the antigen. Antigen is sometimes present on the cell wall of the microorganism and the test measures antibody clumping of the organism. This technique is commonly modified by attaching the antigen to an agglutinable particle, such as a red blood cell pretreated with tannic acid (the hem agglutination test), or to inorganic particles, such as bentonite or polystyrene latex (the latex fixation test). Occasionally, the antigen itself produces hemagglutination, but this fails. to occur when antibody is present. The antibody titer at which "hemagglutination inhibition" occurs can then be determined. A technique similar to agglutination is flocculation, in which antibody causes droplets or suspended particles to be aggregated into relatively large masses. Complement fixation is an indirect method. One or more of the constituents of complement may be consumed during an antigenantibody reaction, and the complement-deficient mixture may then be unable to support a second antibody-antigen reaction; this is manifest by failure of the reaction to produce hemolysis of red blood cells. This technique has been adapted to a wide variety of soluble antigens, particulate antigens, and emulsions. A disadvantage of the complement fixation test is the presence of anticomplementary activity in serum. This occurs most commonly in narcotic addict.s. Neutralization procedures are based on the ability of antibodies to inactivate biologically active agents. Since neutralizing antibodies appear early in the course of viral infections, the usefulness of neutralization techniques in such infections is limited. By the time that a particular infection is suspected, the peak antibody response may already have occurred, and it may also be impossible to demonstrate a significant increase in antibody titer. Older neutralization techniques involved the administration of virus-antibody mixtures into animals or embryonated eggs, with death of the animal or embryo indicating the absence of neutralizing antibody. Recently, procedures performed in cell culture systems have been utilized. 50 These include: (1) measurements of the ability of an antibody to inhibit viral cytopathic effects in monolayers; (2) assessments of metabolic inhibition, in which cell cultures infected with a cytopathic virus are unable to generate enough acid to produce a color change in an indicator, but neutralizing antibodies allow the colorimetric process to proceed normally; and (3) plaque reduction assays, the most quantitative method, in which measurements are made of the serum's ability to decrease the number of plaques produced by a specific concentration of virus. Neutralization tests may also be performed with toxins. In the Schick test, minute amounts of diphtheria toxin are injected intradermally; in an immune individual, circulating antitoxin inhibits develop-

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ment of the local inflammatory response that would occur in a person not possessing antibodies to diphtheria toxin. The application of newer serodiagnostic techniques is best demonstrated by the modifications of the precipitin test: immunodiffusion, immunoelectrophoresis, and immunofluorescence. In the precipitin test, the interaction of antigen with antibody produces a visible precipitate. With immunodiffusion, multiple antigen-antibody systems can be identified on the basis of their ability to diffuse and precipitate in an agar gel medium. This technique is specific but may require 3 days to obtain optimal sensitivity. In immunoelectrophoresis antigens and antibodies are separated according to electrophoretic mobility; precipitation is then allowed to occur. Counterimmunoelectrophoresis (or immunoelectroosmophoresis) is preformed by permitting an electric current to be passed through the agar. This allows antibody and antigen to approach from opposite directions and produces rapid precipitation. Counterimmunoelectrophoresis is a sensitive technique. Many newer serologic techniques employ the principle of immunofluorescent staining. Antigen-antibody reactions are immunologic ally specific, and conjugation of an antigen or antibody to a fluorochrome such as fluorescein isothiocyanate does not alter either immunologic specificity or reactivity. The immunofluorescent methods in common use are the direct and indirect fluorescent antibody tests. The direct fluorescent antibody test is performed by placing a fluorescein-Iabelled antibody directly on the material which is to be examined (this may be a bacterial smear, a tissue secretion, or a cell culture). If the antigen is present, an antigen-antibody-fluorescein complex will be formed; this may then be observed microscopically utilizing an ultraviolet light source. The technique is specific, but lacks sensitivity. In the indirect fluorescent antibody test, known ahtigenic material is treated with test serum. If antibodies are present, they will be retained on the smear. Fluorescein-Iabelled anti-immunoglobulin antibodies that will react with antibodies in the test serum are then added to the mixture. The resultant antigen-test antibody-anti-immunoglobulin-fluorescein combination can be detected by ultraviolet illumination. This method may be modified by use of a labelled anticomplement antibody in place of antihuman immunoglobulin antibody. The indirect fluorescent antibody test is more sensitive than the direct method, but is less specific. Radioimmunoassays have been utilized to a limited extent in infectious diseases. 6o The radioimmunoassay involves competition between radioactively labelled antigen and various dilutions of unlabelled test antigen for a finite number of antibody binding sites. Since the amount of labelled antigen in the mixture is known, the amount of test antigen present may then be calculated. The most recently developed serodiagnostic method is the enzyme immunoassay, such as the enzyme-linked immunosorbent assay (ELISA).58 The principle of this technique is similar to that of indirect immunofluorescence. Anti-immunoglobulin antibodies are added to a

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mixture of known antigen and test serum. The anti-globulin is conjugated with an enzyme such as alkaline phosphatase. An enzyme substrate is added, and the resultant enzymatic reaction, which may be measured spectrophotometrically, is proportional to the amount of antibody-fixed antigen. This method is very sensitive. 17

BACTERIAL INFECTIONS With the exception of the venereal diseases and bacterial meningitis, there have been few recent advances in the serodiagnosis of bacterial infections. This is probably a result of the fact that bacteria are usually identified rapidly and accurately by culture techniques. There are, however, certain bacterial diseases in which serologic techniques remain important. The anti-streptolysin 0 (ASO) titer has been utilized for many years in the diagnosis of acute rheumatic fever. The test is performed by adding streptolysin 0 to a patient's serum, combining this with red blood cells, and detennining whether hemolysis of the red blood cells occurs. This is a neutralization test. Antibodies to the 0 hemolysin (or streptolysin 0) appear approximately 7 days after acute streptococcal pharyngitis, attain peak levels 2 to 4 weeks later, and then may remain stable at high titers for weeks to months. A rising ASO titer suggests recent infection. A stable titer, despite its level, indicates only previous streptococcal exposure. Rising ASO titers are observed in 70 to 80 per cent of patients with streptococcal pharyngitis;59 this is much less common in individuals with streptococcal skin infection. Following epidemics, most children with streptococcal pharyngitis who have not received penicillin will demonstrate a significant ASO titer.33 In sporadic cases in children, only about one-half are positive. The ASO titer may be falsely positive in patients with liver disease. Antibody titers to another streptococcal enzyme, deoxyribonuclease B,35 may be elevated in streptococcal skin infections when the ASO titer is negative. The streptozyme test, which measures antibodies directed against five streptococcal enzymes - streptolysin 0, deoxyribonuclease B, hyaluronidase, streptokinase, and nicotinamide adenine dinucleotidase - is more sensitive than the ASO and may be useful in the diagnosis of post-streptococcal glomerulonephritis 2 and acute rheumatic fever3 4 and may be an important adjunct to, if not a replacement for, the ASO titer in the diagnosis of streptococcal infection, regardless of the site. Fluorescent antibodies directed against the C carbohydrate of Group A and other streptococci have been utilized to stain smears of pharyngeal exudate and early pharyngeal cultures. The results of these stains have been demonstrated to correlate well with subsequent culture results. 51 Pneumococci may be demonstrated in clinical specimens by the Quellung reaction, in which the pneumococcal capsule and typespecific antibody interact to produce "capsular swelling" which may be observed microscopically.

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Since counterimmunoelectrophoresis does not require the presence of living organisms, this technique may be useful in the assessment of patients who have received prior antibiotic therapy, in the evaluation of specimens which contain formalin or have been sent through the mail, and in other situations in which routine cultures may not be effective. When the efficacy of counterimmunoelectrophoresis has been compared to that of culture and direct microscopy in the diagnosis of bacterial meningitis,lO the routine bacterial culture has been found to be the most sensitive technique, detecting 85 per cent of cases. Counterimmunoelectrophoresis is positive in only 55 per cent of patients with bacterial meningitis, but the combination of counterimmunoelectrophoresis and direct microscopy is equivalent to culture alone, and results are available within 1 hour. Concentrated urine specimens 19 may also be investigated by counterimmunoelectrophoresis. The technique rarely produces falsepositive reactions. 10, 49. 53 Antisera currently employed in counterimmunoelectrophoresis include a polyvalent pneumococcal antiserum representing 83 pneumococcal serotypes, a polyvalent meningococcal antiserum, and Hemophilus injluenzae type B, and polyvalent he mophilus antisera. Counterimmunoelectrophoresis of serum and urine has been utilized in the detection of pneumococcal bacteremia in patients with pneumococcal pneumonia. 49 A counterimmunoelectrophoretic method which measures antibodies to teichoic acid, a major constituent of the cell wall of Staphylococcus aureus, has been employed in the diagnosis of severe staphylococcal infections, particularly bacterial endocarditis. Although the presence of teichoic acid antibodies distinguishes patients with staphylococcal endocarditis from those with endocarditis caused by other bacteria, individuals with staphylococcal bacteremia without endocarditis cannot be separated from those with bacterial endocarditis. 11 Measurement of teichoic acid antibodies may be useful in patients in whom blood cultures have been rendered sterile by previous antibiotic therapy, or when treatment must be instituted in seriously ill patients prior to the availability of blood culture results. Recently, a latex agglutination method has been employed to detect bacterial antigens in cerebrospinal fluid, blood, and concentrated urine. 32 This technique, which is rapid and specific, may prove to be more sensitive than counterimmunoelectrophoresis. The Widal reaction has been utilized in the diagnosis of salmonella infections. Agglutinins directed against somatic (0) and flagellar (H) antigens appear at the end of the first week of salmonella-induced enteric fever and reach maximum levels during the third week of infection. A four-fold or greater increase in the 0 agglutinin titer in nonimmunized individuals suggests, but does not prove, the presence of infection. The Widal reaction lacks both sensitivity and specificity. Since Salmonellatyphi, the species most commonly responsible for enteric fever, shares at least one, and more often two, 0 antigens with all other group D salmonellas, cross-reactivity is common. In addition,

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the bacterial antigens which have been utilized for the Widal test are poorly standardized. It has been suggested that a cholesterol flocculation test using the 0 antigen might be more specific and sensitive. 24 An agglutination test for Vi antigen has been employed to detect and monitor typhoid carriers, but its usefulness is limited by the observation that patients with enteric fever also develop antibodies to this antigen.41 The interpretation of agglutination titers for Brucella and Francisella tularensis is hindered by cross-reactivity both between these two microorganisms and with Vibrio choleraeY The definitive diagnosis of brucellosis and of plague is best made by isolation of the microorganism. Leptospiral serology is performed only in selected laboratories because live organisms must be used as antigens for the agglutination reaction. Recently an indirect hem agglutination test for leptospirosis has been developed. 41 An agglutination test for Listeria monocytogenes is available, but cross-reactivity with a staphylococcal aureus antigen is not uncommon.41

MYCOBACTERIAL INFECTIONS Several unsuccessful attempts have been made to develop serologic techniques for the diagnosis of infection due to Mycobacterium tuberculosis. 15

VENEREAL DISEASES Gonorrhea Isolation of Neisseria gonorrhoeae from the genitourinary tract or other sites is, at present, the only acceptable method by which to establish the diagnosis of gonococcal infection. Since cultures may occasionally be falsely negative and since mass culture programs are expensive, a reliable serologic test applicable to large populations would be useful. An indirect fluorescent antibody test,6 a latex agglutination test,18 and a microflocculation test 45 have been developed, but all have been associated with a high incidence of both false-positive and falsenegative results.

Syphilis The serologic tests currently utilized for the diagnosis of syphilis may be divided into nontreponemal and treponemal techniques. The former detect a globulin complex, reagin, that appears early in the course of syphilis. Reagin titers decline or disappear completely following treatment and may spontaneously fall over an extended period of time even in an untreated patient. Two methods are employed to docu-

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ment the presence of reagin. The first is a complement fixation procedure (the Wasserman reaction). The second is a flocculation test which measures the ability of heated serum reagin to aggregate a suspended cardiolipin lecithin antigen. This is the VDRL procedure. 28 Nontreponemal tests do not distinguish between reagin produced in syphilis and the abnormal globulin present in individuals with "biologic false-positivity." Biologic false-positivity occurs transiently in a number of febrile illnesses, and may be persistent in conditions such as leprosy, connective tissue disorders, sarcoidosis, and drug addiction. In addition to lack of specificity, nontreponemal tests are insensitive; 25 to 30 per cent of patients with primary, late, and latent syphilis have negative VDRL tests. The rapid plasma reagin (RPR) test has now replaced the VDRL in many centers. The VDRL antigen is employed in this method, but carbon particles are also added; these enable flocculation to be seen on a plastic card. The RPR is more convenient than the VDRL for use in mass testing; it has equivalent sensitivity and specificity.43 Nontreponemal tests permit results to be easily quantitated and can be utilized to assess the response to therapy. A majority of patients who have received adequate treatment for primary syphilis will become seronegative. Fewer revert to seronegativity after treatment for secondary syphilis. Many patients with late or latent syphilis are "serofast;" despite adequate therapy, a positive serologic test persists. A technique employed to measure specific treponemal antibodies is the fluorescent antibody test. This test has been modified to increase specificity by diluting test serum in a sorbent (the FTA-ABS test). Another specific technique, the Treponema pallidum immobilization test, has been used as a standard of specificity. It detects antibodies that decrease the mobility of live Treponema pallidum. Unlike the VDRL and RPR, the specific treponemal tests remain positive despite adequate antibiotic therapy, and, thus, are not useful in assessing the response to treatment. These techniques are, however, more sensitive than non-treponemal tests in early, late, and latent syphilis. At present, the VDRL and RPR are employed as screening tests for syphilis. If one of these is positive, the FTA-ABS is performed for confirmation. The diagnosis of neurosyphilis is based on the results of VDRL or RPR tests performed on cerebrospinal fluid. The usefulness of the FTA-ABS test of cerebrospinal fluid in neurosyphilis has not been established. 28 In the darkfield examination, material obtained from moist lesions or lymph nodes is evaluated by a fluorescent antibody technique. This method is useful in primary and, occasionally, in secondary syphilis. Since many laboratories are unable to perform this examination, the direct fluorescent antibody (DFATP) may be substituted. Unlike the darkfield examination, the DFATP employs antibodies specifically directed against Treponema pallidum and can be utilized to evaluate lesions of the oral. mucosa. In such a situation, the nonspecific antibodies used for darkfield examination may detect treponemas other

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than Treponema pallidum that are inhabitants of the normal oral microflora. The microhemagglutination test for Treponema pallidum CM HATP) is based on the ability of serum from patients with syphilis to agglutinate tanned red cells previously coated with Treponema pallidum antigen. The specificity of this technique must be further defined. 28

MYCOPLASMA PNEUMONIAE INFECTIONS Cold agglutinins are antibodies that actively agglutinate human blood group 0 cells at 4°C but not at 37°C. Approximately 50 per cent of patients with pneumonia due to Mycoplasma pneumoniae develop a significant cold agglutinin titers during the second to fourth week of illness; this usually disappears by the sixth to eighth week. Cold agglutinins are present in a number of conditions other than infections due to Mycoplasma pneumoniae. The lack of specificity and sensitivity and the late appearance of antibodies renders the cold agglutinin test of little value in the diagnosis of infection due to Mycoplasma pneumoniae. A metabolic inhibition test, a hem agglutination test, a complement fixation test, and a radioimmunoassay have been developed to demonstrate various mycoplasmal antibodies. 22 The complement fixation test is used most frequently in clinical practice. If paired serum specimens are obtained during the acute and convalescent phases of the illness, a four-fold or greater rise in titer is considered diagnostic of recent infection. When only serum obtained during convalescence is available, a high degree of suspicion of recent infection can be placed on sera with titers of 1:64 or greater. An indirect fluorescent antibody test has been developed to detect antibodies in the bronchial secretions of patients with Mycoplasma pneumoniae infections.3 This test is rapid 22 and is both sensitive and specific.

RICKETTSIAL INFECTIONS Antibodies develop relatively late in the course of Rocky Mountain spotted fever. Since antibiotic therapy must not be delayed, serologic techniques play a confirmatory rather than diagnostic role in this condition. Antibodies first appear between the eighth and twelfth day of illness and may be detected by the Weil-Felix method. In this technique, the serum of patients with Rocky Mountain spotted fever agglutinates a suspension of Proteus OX-19, which shares an antigen with Rickettsia rickettsii. This procedure has been largely replaced by a complement fixation test specific for the spotted fever group of infections, but early antibiotic therapy may suppress the complement fixation response. Both an indirect hem agglutination test and an indirect fluorescent

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antibody test may be superior to the complement fixation procedure in typhus and Rocky Mountain spotted fever. 42 The indirect fluorescent antibody test may be modified to detect IgM; this may be employed for the diagnosis of recent infection. All of the techniques are more useful in seroepidemiologic studies than in the diagnosis of acute infections.

FUNGAL INFECTIONS When systemic fungal infection is suspected, diagnosis is commonly based on clinical findings, cultures, and histopathology. Serologic procedures may occasionally be helpful. Complement fixation tests have been employed, but they lack sensitivity and specificity. Immunodiffusion, indirect fluorescent antibody tests, and other techniques are now being utilized. Histoplasmosis Since Histoplasma capsulatum may be difficult to isolate by standard culture techniques, serologic testing is an important element in diagnosis. Serodiagnostic studies may be performed on serum, cerebrospinal fluid, plasma, and peritoneal fluid. Two types of antigens are used: the histoplasmin mycelial antigen and the whole yeast antigen. Complement fixation, immunodiffusion, and latex agglutination methods are employed. The latter two techniques may be performed only with histoplasmin, while the complement fixation technique utilizes both antigens. In primary pulmonary histoplasmosis, complement-fixing antibodies to yeast antigen appear 10 to 21 days following exposure to the fungus, frequently by the time that symptoms have appeared. Histoplasmin antibodies are of greater importance in chronic infections, in which antibodies to yeast antigen may have decreased or may be absent.7 Although a complement-fixing antibody tit er of ~ 1:8 is presumed to indicate active infection, and most patients with histoplasmosis have titers higher than 1:16, some patients with severe infection may have lower titers. Several months after infection has subsided, antibody titers decline to 1:8 to 1:6. If the tit er remains elevated at 1:32 or greater, this may be an ominous prognostic sign. Complement fixing antibodies are nonspecific, and positive reactions may be observed in patients with blastomycosis and coccidioidomycosis. Skin testing may affect the results of serologic studies in histoplasmosis. A positive skin reaction develops one to three weeks following exposure to the fungus. Antibodies develop as the result of skin testing in 10 to 20 per cent of patients with positive reactions 4 and are directed primarily against the histoplasmin antigen rather than the yeast antigen. The antibody titer induced by skin testing usually is no higher than 1:32. Antibodies appear within 15 days and may remain elevated for as long as 6 .months.4 The skin test has very little utility in the diagnosis of acute histoplasmosis. When a histoplasmin complement

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fixation test is performed prior to skin testing, the tit er will be low or negative in acute disease. If the convalescent antibody titer is later found to be elevated, this could be the result either of skin testing or of active histoplasmosis. At present, the major indications for histoplasmosis skin testing are in the evaluation of cutaneous anergy and in the diagnosis of patients having cavitary lung disease with negative tuberculin skin tests. In the immunodiffusion test, the M band appears early in the course of illness, may persist after the patient has become asymptomatic, and may be induced by skin testing. The H band is usually associated with active infection and is not affected by the skin test. The latex agglutination test is a rapid, but insensitive, method for the diagnosis of histoplasmosis. 4 If the latex agglutination test is positive, a complement fixation test must be performed for confirmation. The combination of immunodiffusion and complement fixation represents the most accurate means for the diagnosis of acute histoplasmosis.41 In chronic pulmonary histoplasmosis, or in inactive disease, serologic tests are positive in fewer than half of the cases while skin tests are positive in 97 per cent of patients. 47

Coccidioidomycosis Although Coccidioides immitis can be cultured with relative ease,

skin tests and serologic techniques may be helpful in the diagnosis of coccidioidomycosis. The skin test becomes positive 1 to 3 weeks following exposure to the fungus. Antibody titers may be affected by skin testing. s Serologic studies may be performed on serum, plasma, ascitic fluid, pleural fluid, and joint fluid. The antigen utilized is coccidioidin, a culture filtrate of the mycelial phase of Coccidioides immitis. There is a cross-reactivity between coccidioidin, histoplasmin, and the antigen of Blastomyces dermatitidis. Four serodiagnostic techniques are currently employed for the diagnosis of coccidioidomycosis: tube precipitin, latex agglutination, immunodiffusion, and complement fixation. 4 The tube precipitin and latex agglutination tests are useful in acute illness and in acute exacerbations of chronic infections. Tube precipitins are highly specific, and can be detected in 80 per cent of patients within two weeks of the onset of symptoms; they are absent in 90 per cent after 6 months. 9 The latex agglutination is rapid and sensitive but lacks specificity. When positive, it must be confirmed by one of the other tests. The immunodiffusion test is sensitive but nonspecific. It becomes positive later than the latex agglutination and tube precipitin, and must be confirmed by the complement fixation test. The complement fixation test becomes positive 4 to 6 weeks following the onset of symptoms. The maximum response is present at 2 to 3 months. A complement-fixing antibody titer of 2:: 1:2 may indicate active disease. Higher titers are observed in severe infections, and 80 per cent of patients with disseminated coccidioidomycosis have antibody titers greater than 1: 16. 9 In disseminated coccidioidomycosis, the

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skin test is commonly negative at a time when the complement-fixing antibody titer is positive. When the skin test becomes positive and the complement fixation titer falls, this suggests a favorable prognosis. In coccidioidal meningitis, approximately 25 per cent of patients have negative complement fixation antibody tests in the cerebrospinal fluid despite seropositivity. 9 In coccidioidomycosis a combination of tests is superior to any single procedure. The combinations of immunodiffusion with latex agglutination and complement fixation with tube precipitin have been accurate in 85 to 93 per cent of patients later demonstrated to have coccidioidomycosis. 25 In the future, direct examination of specimens to demonstrate organisms by fluorescent antibody techniques may prove most effective in the diagnosis of coccidioidomycosis.

Blastomycosis A complement fixation test may be utilized for the diagnosis of blastomycosis, but the reactions are transient and inconsistent, there is a high incidence of cross-reactivity, and the sensitivity is poor.41 There is no antigen available for skin testing. In the future, agar gel double diffusion 5 and fluorescent antibody testing may be the most useful serodiagnostic techniques for blastomycosis.

Aspergillosis Aspergillus species are easily identified by stain and culture techniques. However, because these fungi are ubiquitous, it is often difficult to establish a diagnosis of aspergillosis since the organisms are frequently isolated from healthy individuals as well as from those with infection. Alternative methods for the diagnosis of aspergillosis have been developed. The agar gel diffusion test demonstrates serum precipitins, and is a useful technique in certain forms of aspergillosis. Precipitins are detected in virtually 100 per cent of patients with aspergillomas and in 70 per cent of those with allergic pulmonary aspergillosis. Precipitins are rarely present in normal individuals. However, approximately 40 per cent of patients with pulmonary tuberculosis have these antibodies. 39 There are no useful serologic procedures in invasive aspergillosis. A complement fixation test is available, but is less specific than agar gel diffusion. Counterimmunoelectrophoresis has been employed to increase the speed and sensitivity of precipitin testing. An indirect fluorescent antibody procedure is currently being evaluated. 41

Cryptococcosis Because India ink examination of the cerebrospinal fluid is insufficiently sensitive, and culture of Cryptococcus neoformans may be difficult, reliable sero-diagnostic techniques for cryptococcal meningitis have become particularly important. Initial attempts to demonstrate cryptococcal antibodies were unsuccessful. This failure was due probably to neutralization of antibody by the presence of abundant cryptococcal capsular antigen. 21 Methods to detect cryptococcal antigen rather than antibody were then developed.

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The latex agglutination test for cryptococcal antigen is rapid and accurate. 21 In cryptococcal meningitis, the cerebrospinal fluid antigen tit er decreases with successful administration of amphotericin B. Cryptococcal antigen may also be detected in the serum of patients with cryptococcal meningitis, although the titers are lower than in the cerebrospinal fluid. The latex agglutination test for cryptococcal antigen is specific. However, since there may be false-positive reactions in individuals with rheumatoid arthritis and chronic lymphocytic leukemia, a rheumatoid factor test must be performed in all patients in whom cryptococcal antigen has been demonstrated. False-negative latex agglutination reactions occur in 5 to 15 per cent of patients with cryptococcal meningitis. 21 A fluorescent antibody test may prove useful in cryptococcosis in the future. Cryptococcal antibody tests may occasionally be useful when the cryptococcal antibody test is negative, but the appearance of antibody usually is indicative of improvement; this commonly occurs at a time when antigen titers are declining. 1

Candidiasis Candida species are frequently responsible for infections in patients with compromised host defenses. Systemic candidal infection may be curable if recognized early, but the diagnosis of candidiasis is difficult to establish. It has been suggested 48, 56, 61 that precipitins directed against cytoplasmic candidal antigens may be useful indicators of disseminated candidal infection. The precipitin reaction is very specific,48 but lacks sensitivity, and is negative in 25 to 40 per cent of patients with disseminated candidal infection. Candidal agglutinating antibodies may be detected in systemic candidal infection. However, such antibodies may also be present in patients with superficial candidal infections and in healthy individuals. 4 When both agglutinins and precipitins are present, this may be more indicative of serious infection than the presence either of precipitins or agglutinins alone.16 The germ tube dispersion test (the inhibition of the clumping of Candida albicans filaments by an IgG antibody) may be the most specific test in disseminated candidal infection. 40 A fluorescent antibody test for candidiasis is currently being evaluated. Skin tests are of no value in candidiasis, since 80 per cent of normal individuals may have skin reactions to candida antigen. Other Fungal Infections There are no serodiagnostic techniques presently available for the diagnosis of actinomycosis. A fluorescent antibody procedure is being developed to identify certain actinomycetes in cultures and in smears of tissue and exudate. 20 Patients with paracoccidioidomycosis (South American blastomycosis), develop precipitating antibodies early in the course of illness. These may be detected by an immunodiffusion technique. They are

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specific and have prognostic importance. Complement-fixing antibodies appear later in paracoccidioidomycosis and persist longer than precipitating antibodies. Higher complement fixation antibody titers are present in disseminated infection; falling titers indicate a response to therapy. A tube agglutination test and a more sensitive and specific latex agglutination procedure are available for the diagnosis of sporotrichosis.41 A fluorescent antibody test is being developed to identify Sporotrichum schenckii in clinical materials and in cultures.

VIRAL INFECTIONS With refinements in neutralization tests, the development of fluorescent antibody techniques, and the prospect of effective viral chemotherapy, viral serodiagnosis may assume a more important role in the future.

Hepatitis Hemagglutination and hemagglutination inhibition techniques were formerly employed to measure hepatitis B surface antigen (HBsAg). These methods, and the complement fixation test, were specific but lacked sensitivity. The need for greater sensitivity led to the development of the radioimmunoassay for HBsAg.60 Although employed in the evaluation of patients with acute and chronic hepatitis, HBsAg testing is used most commonly in the investigation of potential blood donors. Radioimmunoassays detect two to ten times as many HBsAg-positive individuals as did the earlier techniques. However, it has been estimated that approximately two thirds of cases of transfusion-associated hepatitis are transmitted by blood that is HBsAg-negative, either because of insensitivity of serodiagnostic techniques or because another agent has been responsible for the infection.60 Hepatitis B core antibody (HBcAb), detectable by a complement fixation test, persists in chronic carriers of HBsAg. HBcAb normally disappears after recovery from hepatitis B.23 An inability to purify sufficient quantities of antigen has delayed the development of serodiagnostic techniques for hepatitis A.60

Infectious Mononucleosis Patients with infectious mononucleosis develop elevated titers of heterophile antibodies which agglutinate sheep red blood cells. However, similar antibodies are present in other infections, in serum sickness, and in normal individuals (Forssman antibodies). The differential heterophile test utilized for the diagnosis of infectious mononucleosis is based on two specific properties of the sheep red cell agglutinates in this infection: (1) the antibodies are not completely removed by absorption with guinea pig kidney antigen, and (2) the antibodies are readily absorbed by beef red· blood cells. The agglutinating

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antibodies found in serum sickness are removed both by guinea pig kidney and by beef red blood cells. 13 Although an enzyme test and a beef hemolysin test have been utilized in the diagnosis of infectious mononucleosis, the most important recent development has been the introduction of the "mono spot" test. A 4 per cent saline suspension of formalinized horse red blood cells serves as the antigen. One drop of the antigen is added to serum. Coarse granulation indicates a positive reaction. This method is rapid, specific, and sensitive. 12, 52 Patients with infectious mononucleosis develop heterophile agglutinins and positive "mono spot" tests at similar times: approximately 40 per cent have positive tests during the first week of illness. and 80 per cent by the third week. 12 Under usual circumstances the "mono spot" test is sufficient to establish the diagnosis of infectious mononucleosis. However, if the clinical findings are atypical, the differential heterophile absorption test must also be performed. Occasionally, a patient with typical clinical findings of infectious mononucleosis will demonstrate a negative heterophile agglutinin test. In such a situation confirmation of the diagnosis may be possible by performance of specific serologic studies for the etiologic agent of infectious mononucleosis, Epstein-Barr virus. Alternatively, the illness may be due to another agent, cytomegalovirus; this may also be confirmed serologically.

Rubella During maternal rubella, IgM antibodies develop first, but are present only transiently. IgG antibodies appear later and cross the placenta during the second and third trimesters of pregnancy. The rubella-infected fetus produces IgM antibodies late in gestation, and at birth neonatal blood may contain both maternal IgG and IgM produced by the child. Maternal IgG is lost during the early postpartum period; IgM may persist at high levels into the second year of life. IgG production in the infant begins during the first year. 37 Serologic tests utilized in the diagnosis of rubella include hemagglutination inhibition, complement fixation, and neutralization. 37 Although the hem agglutination test does not distinguish between IgM and IgG antibodies, the absence of hemagglutination inhibition antibodies in fetal cord blood makes rubella very unlikely. The presence of hemagglutination inibition antibodies at birth followed by their decline during the first 6 months of life suggests passive transfer of maternal antibodies rather than intrapartum infection. In congenital rubella there is persistence or elevation of hemagglutination inhibition antibody titers during the first year of life. Immunofluorescent techniques 57 can be utilized to distinguish IgM from IgG. The presence of IgM in cord blood strongly suggests congenital rubella infection. Serologic testing is employed in the evaluation of pregnant women who have been exposed to rubella, in the detection of susceptible women for the purposes of vaccination, and in the assessment of the response to vaccine. A hemagglutination inhibition antibody titer of

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~1:8 within 10 days of exposure to rubella indicates susceptibility; a higher titer implies immunity. Recent infection may be documented by the presence of a four-fold increase in hemagglutination inhibition antibody titer in the convalescent serum of a susceptible individual. 27 Reinfection occurs when exposure to rubella virus produces a recrudescence of infection in an individual who had previously been immune either as the result of primary infection or of immunization. A diagnosis of reinfection is established when there has been a four-fold increase in the preexisting antibody titer. Reinfection is less frequent in those with natural immunity than in those who have been vaccinated, and probably does not occur in individuals with hem agglutination inhibition antibody titers greater than 1 :64. 27 Viremia develops only rarely in reinfection, and transplacental transmission of virus is unlikely. Recently, it has been suggested that a hemolysis-in-gel procedure may be the most sensitive and reproducible method for detecting rubella immunity. 55

Rubeola The exanthem of rubeola appears on approximately the fourteenth day after exposure to the virus, and antibodies develop shortly thereafter. Neutralizing, complement-fixing, and hemagglutination inhibition antibodies are present. Hemagglutination inhibition antibodies persist for years and are reliable indicators of immunity. 37 Subacute sclerosing panencephalitis is believed to be a recrudescence of measles and is a progressive and degenerative central nervous system disease of childhood and adolescence which is invariably fatal. Measles antibodies are present in all patients with subacute sclerosing panencephalitis, including those without a history of measles infection or vaccination. IgM antibodies are present in the serum in this condition, and may persist until death.37 In acute measles IgM antibodies usually disappear within two months. IgM and IgG antibodies may be present in the cerebrospinal fluid of patients with subacute sclerosing panencephalitis. PARASITIC INFECTIONS Because the etiologic agent is difficult to isolate, serologic testing may be the only practical means of diagnosis in certain parasitic diseases. Effective methods for antigen isolation and improved testing techniques have recently become available. Toxoplasmosis Toxoplasma gondii is a ubiquitous microorganism. It can produce a condition which resembles infectious mononucleosis manifest by lymphadenopathy, fever, malaise, and other constitutional symptoms. Toxoplasma gondii may also be responsible for disseminated disease, chorioretinitis, and congenital infection. Isolation of the microorgan-

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ism is expensive and difficult, and diagnosis frequently depends upon serologic tests. The most reliable techniques are the Sabin-Feldman dye test, the complement fixation test, the indirect fluorescent antibody test, and the indirect hemagglutination test. 38 The Sabin-Feldman procedure is based on the observation that Toxoplasma gondii is stained by methylene blue dye, except when the organism has previously been exposed to anti-toxoplasmic antibodies. Although the test is specific, its cost and the potential danger involved with the use of living organisms has led to the development of alternative methods of diagnosis. The complement fixation test is useful in the diagnosis of acquired toxoplasmosis. The indirect hemagglutination test is specific but time-consuming. 44 The indirect fluorescent antibody test is both specific and sensitive. IgM fluorescent antibodies appear in the first week and reach a peak within a month. A high tit er (> 1 :80) or a two-fold increase in titer establishes the diagnosis of recent infection. When toxoplasmosis develops in an immunocompromised individual, Sabin-Feldman and indirect fluorescent antibody titers are usually greater than 1: 1000. 44 The presence of antibodies in a baby's serum may be due either to infection or to the passive transfer of maternal antibodies. Congenital infection of the newborn is manifest by a persistent or increasing antibody titer two to three months following birth or by high titers at birth in a child with clinical evidence of toxoplasmosis. An indirect fluorescent antibody test measuring IgM is most specific in congenital toxoplasmosis. 46 The indirect hemagglutination and complement fixation tests are often falsely negative in this condition. 38 Serodiagnostic techniques are least valuable in ocular toxoplasmosis, although testing of aqueous humor may be useful in establishing the diagnosis of toxoplasma chorioretinitis. 14

Amebiasis Examination of the stool is the most effective method available for the diagnosis of intestinal amebiasis, but this is sometimes technically difficult, and amoebae are frequently absent from the stool in hepatic amebiasis. Serologic tests have, therefore, become important in the diagnosis of infections caused by Entamoeba histolytica. Gel diffusion precipitation and indirect hem agglutination techniques are utilized. Antibodies may be detected in as many as 95 per cent of patients with hepatic abscess and 85 per cent with intestinal amebiasis. 38 Cellular acetate diffusion, a modification of the gel diffusion precipitation test, is employed for rapid diagnosis in life-threatening situations. 54 Echinococcosis Serologic methods are particularly important in the diagnosis of echinococcal infections. The most effective techniques are the indirect hemagglutination and the Casoni skin test. Both are sensitive, but the indirect hemagglutination test is more specific and is positive in approximately 90 per cent of patients with hepatic cysts and 30 per cent of those with cysts in the lungs. 31

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The complement fixation test for echinococcosis is insensitive, but is useful in the postoperative evaluation of echinococcal infections.30 While other serologic tests remain positive postoperativelY,36 the complement fixation test becomes negative following surgery.36 A significant decline in complement fixing antibody titer indicates surgical cure; an unchanged or rising titer suggests persistence or recurrence of infection. Fluorescent antibody testing is available and is a sensitive technique. 30

Trichinosis The diagnosis of trichinosis is usually based upon a history of pork ingestion, findings of muscle tenderness and eosinophilia, and recovery of the parasite from a muscle biopsy specimen. Because the latex agglutination and complement fixation tests are frequently negative early in the course of trichinosis, these techniques are not useful in diagnosis since immediate therapy may be required in severe infections. The major utility of serologic tests in trichinosis is to confirm a diagnosis that has been established by other means. The latex agglutination test 26 is sensitive. However, false-positive tests may occur in patients with tuberculosis, polyarteritis nodosa, and infectious mononucleosis. 29 REFERENCES 1. Baum, G. L., and Schwarz, J.: Diagnosis and treatment of systemic mycoses. MED. CLIN. N. AMER. 58:661·681, 1974. 2. Bergner-Rabinowitz, S., Ofek, 1., Fleiderman, S., et al.: Evaluation ofstreptozyme and antistreptolysin 0 tests in streptococcal pyodermal nephritis. Appl. Microbiol., 26:5658, 1973. 3. Biberfeld, G., and Sterner, G.: Antibodies in bronchial secretions following natural infection with Mycoplasma pneumoniae. Acta Pathol. Microbiol. Scand., 79B:599-605, 1971. 4. Buechner, H. A., Seabury, J. H., Campbell, C. C., et al.: The current status ofserologic, immunologic, and skin tests in the diagnosis of pulmonary mycoses. Chest, 63:259270,1973. 5. Busey, J. F., and Hinton, P. F.: Precipitins in blastomycosis. Amer. Rev. Resp. Dis., 95:112-113,1967. 6. Caloenescu, M., Clecner, B., Petrow, S., et al.: Immunofluorescent antibody test for diagnosis of gonorrhoeae. J. Clin. Microbiol., 1: 143-146, 1975. 7. Campbell, C. C.: The accuracy of serologic methods in diseases. Ann. N. Y. Acad. Sci., 89: 163-177, 1960. 8. Campbell, C. C.: Use and interpretation of serologic and skin tests in the respiratory mycoses: Current considerations. Dis. Chest, 54 (Suppl. 1):305-310, 1968. 9. Chick, E. W., Baum, G. L., Furcolow, M. L., et al.: Scientific assembly statement. The use of skin tests and serologic tests in histoplasmosis, coccidioidomycosis, and blastomycosis, 1973. Am. Rev. Resp. Dis., 108:156-159, 1973. 10. Colding, H., and Lind, 1.: Counterimmunoelectrophoresis in the diagnosis of bacterial meningitis. J. Clin. Microbiol., 5:405-409, 1977. 11. Crowder, J. G., and White, A.: Teichoic acid antibodies in staphylococcal and nonstaphylococcal endocarditis. Ann. Intern. Med., 77:87-90, 1972. 12. Dann, T. C.: A new test for the detection of infectious mononucleosis. Brit. J. Clin. Pract., 21 :511-512, 1967. 13. Davidsohn, I.: Serologic diagnosis of infectious mononucleosis. J.A.M.A., 108:289295,1937. 14. Desmonts, G.: Definitive serological diagnosis of ocular toxoplasmosis. Arch. Ophthalmol., 76:839-851, 1966. 15. Diena, B. B.: Serology in tuberculosis and the bentonite flocculation test. Can. Med. Assoc. J., 99:763-764, 1968.

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16. Dolan, C. T., and Stried, R P.: Serologic diagnosis of yeast infections. Amer. J. Clin. Pathol., 59:49-55. 1973. 17. Draper, C. C.: Immunodiagnosis of tropical infections. Editorial. J. Trop. Med. Hyg., 79:23-24,1976. 18. Dyckman, J. D., Wende, R D., and Williams, RP.: Evaluation of the gonosticon dri dot test in females with a low incidence of gonorrhea. Appl. Microbiol., 238:431-434, 1974. 19. Feigin, RD., Wong, M., Shackelford, P. G., et al.: Countercurrent immunoelectrophoresis of urine as well as of CSF and blood for diagnosis of bacterial meningitis. J. Pediat., 89:773-775, 1976. 20. Gereneser, M. A., and Slack, J. M.: Isolation and characterization of Actinomyces propionicus. J. Bacteriol., 84: 109-115, 1967. 21. Gordon, M. A., and Vedder, D. K.: Serologic tests in diagnosis and prognosis of cryptococcosis. J.A.M.A., 197:961-967,1966. 22. Honda, H.: Studies on the infection of Mycoplasma pneumoniae. I. Application of immunofluorescent antibody method to serodiagnosis of primary atypical pneumonia. Kumamoto Med. J., 23:92-102, 1970. 23. Hoofuagle, J. H., Gerety, R J., and Barker, L. F.: Antibody to hepatitis-B virus core in man. Lancet, 2:869-873, 1973. 24. Hunter, C. A., and Burdorff, R: Serologic tests for typhoid fever. Amer. J. Clin. Path., 37:162-167, 1962. 25. Huppert, M., Peterson, E. T., Sun, S. H., et al.: Evaluation of a latex particle agglutination test for coccidioidomycosis. Amer. J. Clin. Path.,49:96-102, 1968. 26. Innella, F., and Redner, W. J.: Latex-agglutination serologic test for trichinosis. Preliminary report. J.A.M.A., 171 :885-887, 1959. 27. Iwakata, S., Rhodes, A. H., and Labzoffsky, N. A.: Laboratory diagnosis of rubella virus infections. Can. Med. Assoc. J., 108:894-895, 1973. 28. Jaffe, H. W.: The laboratory diagnosis of syphilis: New concepts. Ann. Intern. Med., 83:846-850, 1975. 29. Kagan, I. G.: Trichinosis: a review of biologic, serologic, and immunologic aspects. J. Infect. Dis., 107:65-93, 1960. 30. Kagan, I. G.: A review of serological tests for the diagnosis of hydatid disease. Bull. WHO, 39:25-37, 1968. 31. Kagan, I. G., Osimani, J. J., Varela, J. C., et al.: Evaluation of intradermal and serologic tests for diagnosis of hydatid disease. Amer. J. Trop. Med. Hyg., 15:172-179, 1966. 32. Kaldor, J., Asznowicz, R, and Buist, D. G. P.: Latex agglutination in diagnosis of bacterial infections, with special reference to patients with meningitis and septicemia. Amer. J. Clin. Path., 8:284-289,1977. 33. Kaplan, E. L: Unresolved problems in diagnosis and epidemiology of streptococcal infection. In Wannamaker, L. W., and Matsen, J. M., eds.: Streptococci and Streptococcal Diseases - Recognition, Understanding, and Management. New York, Academic Press, 1972, pp. 557-570. 34. Kaplan, 0., Halfon, S. T., Ever-Hadani, P., et al.: Sensitivity of serological tests and the diagnosis of streptococcal sore throat in children. Hlth. Sci. Lab., 11 :178-181, 1974. 35. Klein, G. C., Baker, C. N., Addison, B. V., et al.: Micro test for streptococcal antideoxyribonuclease B. Appl. Microbiol., 18:204-206, 1969. 36. Lass, N., Laver, Z., and Lengy, J.: The immunodiagnosis of hydatid disease: postoperative evaluation of the skin test and four serological tests. Ann. Allergy, 31 :430436, 1973. 37. Meyer, H. M., Jr., Parkman, P. D., and Hopps, H. E.: The clinical application oflaboratory diagnostic procedures for rubella and measles (Rubeola). Amer. J. Clin. Path., 57:803-813, 1972. 38. Miller, L. H., and Brown, H. W.: The serologic diagnosis of parasitic infections in medical practice. Ann. Intern. Med., 71 :983-992, 1969. 39. Muchmore, H. G., McKown, B. A., and Mohr, J. A.: Aspergillus precipitins in hospitalized and non-hospitalized subjects. Bacteriological Proceedings. Abstracts of the 71st Annual Meeting of the American Society for Microbiology, 1971, p. 120. 40. Oblack, D., Schwarz, J., and Holder, I. A.: Comparative evaluation of the candida agglutinin test, precipitin test, and germ tube dispersion test in the diagnosis of candidiasis. J. Clin. Microbiol., 3:175-179, 1976. 41. Palmer, D. F.: Immunological diagnosis of bacterial and fungal diseases. In Prier, J. E., Bartola, J. T., Friedman, H., eds.: Modern Methods in Medical Microbiology. Baltimore, University Park Press, 1976, pp. 87-98. 42. Philip, RN., Casper, E. A., MacCormack, J. N., et al.: A comparison of serologic methods for diagnosis of Rocky Mountain spotted fever. Amer. J. Epidemiol., 105:5667, 1977.

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43. Portnoy, J., Brewer, J. H., and Harris, A.: Rapid plasma reagin card test for syphilis and other treponematoses. Pub. Hlth. Rep., 77:645-652, 1962. 44. Prakash, 0., and Vinayak, V. K: Serological diagnosis of parasitic diseases. Indian J. Pediatr., 37:395-408, 1970. 45. Reising, G.: Microflocculation assay for gonococcal antibody. AppI. MicrobioI., 21 :852853, 1971. 46. Remington, J. S., and Miller, M. J.: 19S and 17S anti-toxoplasma antibodies in diagnosis of acute congenital and acquired toxoplasmosis. Proc. Soc. Exper. BioI. Med., 121 :357-361, 1966. 47. Richert, J. H., and Campbell, C. C.: The significance of skin and serologic tests in the diagnosis of pulmonary residuals of histoplasmosis. A review of 123 cases. Amer. Rev. Resp. Dis., 86:381-384, 1962. 48. Rosner, F., Gabriel, F. D., Taschdjian, C. L., et al.: Serologic diagnosis of systemic candidiasis in patients with acute leukemia. Amer. J. Med., 51 :54-62, 1971. 49. Rytel, M. W.: Counterimmunoelectrophoresis in diagnosis of infectious disease. Hosp. Pract., 10:75-82, 1975. 50. Schmidt, N. J., and Lennette, E. H.: Advances in the serodiagnosis of viral infections. Prog. Med. ViroI., 15:244-308, 1973. 51. Sherris, J. C.: Some recent advances in diagnostic medical bacteriology. Ann. Rev. MicrobioI., 17:565-592, 1963. 52. Sigler, A. T.: Comparison of screening tests for infectious mononucleosis in children. Johns Hopkins Med. J., 126:312-319, 1970. 53. Sillanpaa, M., Vaha-Eskeli, E., and Willman, K: Immunoelectroosmophoresis (IEOP) for detection of bacterial antigens in cerebrospinal fluid. Scand. J. Infect. Dis., 7: 113115,1975. 54. Sodeman, W. A., Jr., and Dowda, M. C.: Rapid serological methods for the demonstration of entamoeba histolytic a activity. Gastroenterology, 65 :604-607, 1973. 55. Strannegard, 0., Grillner, L., and Lindberg, I. M.: Hemolysis-in-gel test for the demonstration of antibodies to rubella virus. J. Clin. MicrobioI., 1 :491-494, 1975. 56. Taschdjian, C. L., Kozinn, P. J., Cuesta, M. B., et al.: Serodiagnosis ofcandidal infections. Amer. J. Clin. Path., 57:195-205, 1972. 57. Vesikari, T., and Vaheri, A.: Rubella: A method for rapid diagnosis of a recent infection by demonstration of the IgM antibodies. Brit. Med. J., 1 :221-223, 1968. 58. Voller, A., Bidwell, D. E., and Bartlett, A.: Enzyme immunoassays in diagnostic medicine. Theory and practice. Bull. WHO, 53:55-65,1976. 59. Vosti, K L: Streptoccoccal diseases. In Hoeprich, P. D., ed.: Infectious Diseases. Hagerstown, Harper & Row, 2nd ed., 1977, pp. 235-246. 60. Walsh, J. H.: Serology and epidemiology. West. J. Med., 123:206-210, 1975. 61. Wise, G. J., Ray, B., and Kozinn, P. J.: The serodiagnosis of significant genitourinary candidiasis. J. UroI., 107: 1043-1046, 1972. The Cleveland Clinic 9500 Euclid Avenue Cleveland, Ohio 44106