Serologic and Intradermal Tests for Parasitic Infections

Symposium on Parasitic Infections

Serologic and Intradermal Tests for Parasitic Infections

David A. Bruckner, Sc.D. *

Of the many thousands of known parasitic organisms, only a small number of them are pathogenic for man. Host parasite relationships have been of interest to scientists for years; however, only recently have studies focused on basic questions such as how parasites survive in an immune host and why the host is unable to protect itself against invading parasites. Parasitic organisms infecting man can be broken into two classes: 1. Those that multiply within the host-e.g., protozoan infections. 2. Those that mature within the host but never multiply-e.g., schistosomes and Ascaris. Protozoan pathogens multiply within the host and are somewhat analogous to infections caused by bacterial, fungal, or viral organisms. There is a continuous antigenic stimulation to the host's defense system as the infection progresses. In these instances there is a positive correlation between clinical symptoms and serologic tests for those organisms that have established themselves as pathogens. On the other hand, some helminthic organisms, as they migrate within the body, pass through a number of developmental stages without multiplying before becoming mature adults. With few exceptions, these infections have been difficult to reliably detect serologically, possibly because of limited antigenic stimulus to the host or failure to employ a relevant antigen in the test system used. The majority of parasitic antigens employed in assays are heterogeneous mixtures that are not well defined. The end results of the use of such antigen preparations may be cross-reactions or inadequate sensitivity. Even though parasites and their products are immunogenic for the host, the fact remains that the immune response does not protect the host effectively, and in some cases not at all. If immunity does develop, the response in many cases is usually species specific and often strain or stage specific. For years, the diagnosis of most parasitic infections has depended upon the direct demonstration of cysts, eggs, and larvae in tissue or fecal

*Assistant Professor, Department of Pathology, UCLA Medical Center, Los Angeles, California Pediatric Clinics of North America-Vol. 32, No.4, August 1985

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specimens or upon demonstration of the adult parasite itself. In many instances, serologic methods would be more practical for diagnosis, particularly where invasive techniques are required to demonstrate the parasite (Pneumocystis carinii). However, because of the complex life cycles of many of the parasitic organisms, few of these tests can be accurately used for their predictive values, even when the most advanced serologic technology is employed. One must also be aware that patients from endemic areas may have higher baseline titers than a patient from a nonendemic area where a low titer may be significant, thus indicating exposure to the organism rather than disease. Immunodiagnosis of parasitic infections commonly found in pediatric populations will be discussed in detail. PROTOZOAL INFECTIONS Toxoplasmosis

Toxoplasma gondii is a ubiquitous organism capable of infecting a wide range of hosts, including mammals, birds, and reptiles. Janku, an ophthalmologist, described the first recognized case of human toxoplasmosis in 1923. 59 The infection can be contracted by ingestion of oocysts from fecal material, ingestion of tissue cysts from raw or inadquately cooked meat, organ transplantation, transfusions, or by transplacental transmission. Human infection primarily occurs through the ingestion of raw or undercooked infected meat. Congenitally acquired toxoplasmosis can result in serious and debilitating sequelae; defects may be seen at birth, including stillbirths, or frequently the infant is asymptomatic at birth. However, in the latter cases, the sequelae of congenital infection may arise later in life, most commonly between the first and third decade. 45 The incidence of congenital toxoplasmosis is virtually unknown and may be greatly underreported. 45 The clinical severity of congenital toxoplasmosis varies considerably with the trimester of pregnancy when the maternal infection was acquired. 17. 45 Infections contracted during the third trimester result in a large number of congenital infections; however, the majority of the infants have no sign of disease. Although infections acquired by the mother in the first trimester result in a smaller number of infected infants, the disease is the most severe, including a number of stillbirths. 15 Recent evidence suggests that the majority of infants with subclinical infection at birth will develop signs or symptoms of disease (chorioretinitis) as they approach adulthood. 59 The infection or colonization by T. gondii appears to be quite common in most areas of the world, particularly where cats are found. In the United States, approximately 30 per cent of the adults have been exposed to toxoplasmosis; however, the vast majority of these adults did not have recognizable illness. 57. 58 The diagnosis of acute Toxoplasma infection can be established by isolation of tachyzoites from blood or body fluids; demonstration of tachyzoites in cytologic preparation or tissue sections; lymph node histology;

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demonstration of cysts in tissues such as placenta, or in the fetus or neonate; or by serologic results. In older children or adults, the isolation of T. gondii from tissues may reflect only the presence of cysts. Rowever, the isolation of the organism from lymph node tissue may be due to the presence of tachyzoites, depending upon the clinical presentation. Because isolation of the parasite is not practical for many laboratories, most laboratories and physicians rely upon serologies for diagnosis. Properly interpreted serologic results provide excellent supportive evidence to the clinician. In toxoplasmosis, IgM antibodies are the first to be detected. The IgM titer will continue to rise during the first few weeks to a maximum and may remain positive for a year or longer. 57 In many cases, because of the steep rise in IgM antibody, a change in IgM titer may not be observed. When using current serologic techniques, the IgM antibody titer usually declines to undetectable levels by the fifth and sixth week postinfection. The IgG antibody titer rises shortly after the stimulation of IgM antibody. The IgG titer may continue to rise for as long as 2 months and remain elevated for years even though the clinical symptoms have disappeared. In addition to an antibody response of the IgM and IgG class, there is a rise in IgA titers. The IgA titers have not been useful for clinical diagnosis of toxoplasmosis. Serologic tests that have been employed for the diagnosis of toxoplasmosis are the Sabin-Feldman dye test, complement fixation (CF), indirect hemagglutination (IRA), indirect fluorescent antibody (IFA), and agglutination. ELISA methodology is just beginning to become commercially available, and agglutination tests using sensitized particles have not yet been well accepted. The most widely used tests are the Sabin-Feldman dye test, IRA, and IFA. Although the Sabin-Feldman dye test has been the method with which all other tests have been compared, a major drawback is that the test protocol requires live organisms obtained from the peritoneal exudate of mice. IRA titers generally lag behind the dye test and both remain positive for years, similar to the dye test. Because of the lag in titer rise, in some cases a month or more delay, the IRA has not been a satisfactory screening test. 3 , 27, 58 In addition, there have been cases of congenital toxoplasmosis in which the IRA test was negative; however, the dye test was positive. 45 The IF A method is the test of choice for most laboratories, because of its sensitivity, specificity, and general qualitative agreement with the dye test. False-positive IF A results have been noted to occur with some sera containing antinuclear antibodies. II Fluorescent conjugated antibody fractions to whole globulin (both anti-IgM and IgG), anti-IgG, and anti-IgM are commercially available. In the serologic diagnosis of congenital toxoplasmosis, the physician must know the limitations of the serologic test employed. Approximately 0.75 per cent of expecting mothers may contract toxoplasmosis during pregnancy. 57 Women who have a positive IgG titer before pregnancy have never been found to transmit Toxoplasma to the fetus regardless of the antibody titer. Women who have no demonstrable antibody must be considered at risk of infection. It has been recommended that seronegative

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women should be tested at 20 to 22 weeks and another test near or at term to detect a primary infection acquired during pregnancy. 45 This would allow time for a decision on therapy or therapeutic abortion for infections acquired early or appropriate management of the neonate. In many cases, because the diagnosis of acute toxoplasmosis must be based upon serologic tests and is frequently considered late in the patient's clinical course, the titers of the dye test or IF A test may have already reached their peak. Therefore, paired sera would be of little benefit; however, one must bear in mind that a single high titer does not indicate recent infection, since titers can remain high for years. In these situations, methods to detect IgM antibody have been quite useful in establishing a diagnosis. I, 37, 38 IgM antibody methods have been subject to both falsepositive and false-negative results owing to rheumatoid factor, blocking antibody, and the induction of fetal IgM antibodies against maternal globulin. 25, 32, 37, 44 Results from laboratories that employ test methods that do not try to eliminate the above sources of error may be subject to misinterpretation. The double-sandwich IgM test developed by Naot et al. 37 has eliminated many of these problems. In addition to this method, the ion exchange column chromatography methodology used by McGraw et al. 32 has successfully removed rheumatoid factor and blocking antibody from sera. The column method is simple and readily adaptable to routine clinical laboratory use. The serum collected from the column was relatively free of IgG; however, there is a final dilution of 1:10. Laboratories using fluorescent methods for diagnosis would not have to utilize other alternative tests for detection of IgM if the column method were used. IgM-IFA titers will rapidly rise to 1:160 or greater in acute acquired toxoplasmosis and rapidly decline within several months to 1:10 or less. As discussed earlier, the IgM titers may also remain positive in some patients for a prolonged period of time. In addition, IgM antibody may not be detected in 50 to 75 per cent of infants with congenital toxoplasmosis. 45 Generally, the demonstration of any IgM serum antibody to Toxoplasma is considered diagnostic; however, the use of cord serum is unreliable because of possible maternal antibody presence. Antibody response in patients who are immunodeficient and those with active ocular toxoplasmosis may not be demonstrable. Both mother and infant sera should be monitered at the same time. If both the infant and mother are positive, the infant's serum should be retested within 1 to 2 weeks. If IgM was maternally acquired, which occurs in less than 1 per cent of infants, there should be a marked fall in IgM antibody titer, for the half-life of IgM is approximately five days.45, 57 If the infant's IgM titer is stable or continues to rise, it is diagnostic of infection. Lack of an IgM titer in an infant Will not rule out the possibility of congenital infection. If there is suspicion of congenital toxoplasmosis in an infant with an absence of IgM antibodies to Toxoplasma, follow-up serologic testing may be the only means of diagnosis. In such infants, either IgG or IgM titers can be monitored over a period of time. Again, the physician must be aware of decreases in passively transferred maternal IgG in the infant. The half-life of maternal IgG is approximately 30 days. Because of the high avidity of IgG for antigenic binding sites, maternal IgG may

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prevent infant IgM from binding or the infant may respond by producing only IgG antibody. If the infant's serum contains only maternal IgG Toxoplasma antibody, the rate of decreasing titer should remain the same month after month. However, if the IgG titer remains constant or increases, this is evidence of infection. Therapy can alter the antibody response, particularly if instituted before the production of antibody in the infant. Depending upon when therapy was instituted, serologic diagnosis may be considerably delayed. The use of serologic tests on cerebrospinal fluid to establish central nervous system disease is not diagnostically useful, because of passive transfer of antibodies. 31 Methodology for the detection of Toxoplasma antigen in body fluids have been established; although it does lack sensitivity, it may be useful in detection of recently acquired acute and congenital toxoplasmosis. 4 Amebiasis The causative agent of amebiasis, Entamoeba histolytica, is cosmopolitan in distribution throughout the world and is a major infectious disease in developing countries. Although it is thought of mainly as a cause of diarrhea, the trophozoite stage can readily invade tissues, resulting in the development of extraintestinal infections. The diagnosis of amebic diarrhea is usually based upon finding either trophozoite or cyst stages in the stool. In disease caused by extraintestinal amebiasis, one mayor may not find diagnostic morphologic stages in the stool. The diagnosis of extraintestinal amebiasis is largely dependent upon serologic tests. The most commonly employed serologic tests are IHA, IFA, CF, ELISA, latex agglutination (LA), counterimmunoelectrophoresis (Cm), and immunodiffusion (ID). CF tests have not been widely adapted because of nonspecific and variable results. 35 IHA and LA appear to detect the same antibody. Although the IHA is one of the more sensitive tests, antibody titers persist for years, making interpretation difficult in certain patients. 20 The IHA test is quite practical for epidemiologic surveys. IF A and ID tests are not only sensitive tests but also have high positive predictive values. After successful therapy, titers in many of the patients decreased. This could allow physicians to monitor the therapeutic response of the patient. 2. 21,41 Although they are less demanding technically, cm and ID tests do not allow one to obtain a titer and therefore have limited usefulness. A major disadvantage for amebic serologic testing is the limited availability of commercial reagents. In newborns, maternal antibody disappeared within three months. 54 Persistence of antibody after this period of time may indicate active extraintestinal disease. Methods for the direct detection of antigen in feces and abscess fluids have been reported; however, commercial test kits are not available. 29 Giardiasis

Giardia lamblia is a cause of chronic diarrhea and malabsorption, especially in children. Even though the organism is one of the most easily

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Table 1. Serologic Tests Performed in the Parasitic Diseases Division, Center for Disease Control, for the Diagnosis of Parasitic Diseases* DISEASE

Amebiasis Babesiosis Chagas' disease Cysticercosis Echinococcosis Filariasis Leishmaniasis Malaria Paragonimiasis Pneumocystosis Schistosomiasis Strongyloidiasis Toxocariasis Toxoplasmosis Trichinellosis

TESTS

IHA IIF CF, IHA IHA IHA IHA DAT IIF CF IIF IIF IHA ELISA IIF, IIF-IgM BFT

DIAGNOSTIC TITERS

1:256 1:16 1:8, 2: 1:128 2: 1:128 2: 1:256 2: 1:128 2: 1:64 2: 1:64 2: 1:8 2: 1:16 Positive 2: 1:64 2: 1:32 2: 1:256, 2: 1:16 2: 1:5 2:

2: 2:

*Courtesy of Kenneth W. Walls, Center for Disease Control, Atlanta, Georgia. IHA = Indirect Hemagglutination CF = Complement Fixation DAT = Direct Agglutination IIF = Indirect Immunofluorescence BFT = Bentonite Flocculation ELISA = Enzyme-Linked Immunosorbent Assay

recognized parasites when detected in stool, variability in the concentration of organisms in the stool makes this infection difficult to diagnose. Serum antibodies to C. lamblia have been detected by IF A and ELISA methodology. 51, 55, 60 These techniques have most been reliable in the diagnosis of symptomatic patients, whereas predictive values were not reliable with asymptomatic patients (carriers). After successful therapy, antibodies could be detected from two weeks to 15 months. Antigen detection in stool and duodenal fluids have been reported using CIE and IFA methodology. 13, 46 These tests do appear to be sensitive and reliable and offer an alternative method for diagnosis of an infection that is sometimes difficult to detect. Commercial test kits for antibody or anLgen detection are not available. Pneumocystis

Pneumocystis carinii pneumonia is an opportunistic infection of increasing frequency in adult and pediatric populations. Because diagnosis usually requires invasive techniques (i.e., open lung biopsy or bronchial biospy), serologic methodology for diagnosis has been evaluated. Serologic methods have included CF, IFA, and LA tests. 7, 33, 39 None of these tests have been successfully utilized in routine clinical diagnosis because of a lack of specificity or sensitivity. Because a large portion of the general population is positive for Pneumocystis antibody, the detection of specific antibody in selected patients may be of little value. 24 These serologic tests primarily employ cyst antigens. If methods could be developed where

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African Trypanosomiasis Amebiasis Ancylostomiasis 0 Ascariasis Chagas Clonorchiasis Cysticercosis Echinococcosis Fascioliasis Filariasis Giardiasis Leishmaniasis Malaria Paragonimiasis Pneumocystosis Schistosomiasis Strongyloidiasis Toxocariasis 0 Toxoplasmosis Trichinellosis

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Figure 1. Serodiagnostic tests for parasitic diseases. (Courtesy of Dr. Irving Kagan, Center for Disease Control, Atlanta, Georgia.)

one would detect antibody against trophozoite antigens, it is possible that test specificity and sensitivity could be improved. A method to detect antigenemia has also been developed. 43 However, in addition to antigen being detected in a significant number of patients with Pneurrwcystis pneumonia, it was also detected in a large number of patients with no pneumonia and patients with pneumonia due to other etiologies. 34 Chagas' Disease Trypanosoma cruzi infects millions of people in Central and South America and is a significant public health problem there. Definitive

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diagnosis depends upon demonstration of the parasite in blood or tissues. However, examination of blood is of value only during the initial acute disease or during ' chronic exacerbation. Serologic tests employed for the diagnosis of Chagas' disease include CF (Guerreiro-Machado), IFA, IHA, ELISA, and agglutination. 9, 18,56 Crossreactions with low titers have been noted with leishmaniasis, infectious mononucleosis, lepromatous leprosy, and pemphigus foliaceus. An IgM-IFA test has been developed for use in acute and congenital disease diagnosis. lO, 23, 53 In the diagnosis of congenital disease, one must differentiate between maternal and fetal antibodies. Maternally derived antibody in the fetus should rapidly decrease in titer.9 Lack of an IgM titer should not rule out the possibility of congenital infection, because some infants will not produce IgM antibody. 23, 53 Because of the presence of maternal antibody in infants with whom there is strong suspicion of congenital disease, follow-up serologies should be done. Detection of circulating antigen has been accomplished in recently acquired infections. 6 The usefulness of this new diagnostic approach needs to be further explored. A summary of serologic tests and diagnostic titers for protozoan infections can be found in Table 1 and Figure 1. HELMINTH INFECTIONS Toxocariasis

Toxocara spp. are nonhumari ascarids that are capable of undergoing limited development in the human host. Infections with the organism have been classified into two categories: visceral larva migrans (VLM) is a systemic infection occurring in patients with a mean age of 4.6 ± 3.6 years. 22 Ocular larva migrans (OLM) also occurs in children; however, concurrent VLM and OLM in the same patient is rare. Eosinophilia, which is pronounced in VLM, is almost absent in OLM. The average age of patients with OLM is higher than patients with VLM.48, 49, 62 The diagnosis of toxocariasis can be very difficult, and only recently has a reliable immunodiagnostic test been developed. 14 Previous serologic tests had employed adult or larval stage somatic extracts and whole or sectioned larvae. The ELISA test uses third stage larva secretory antigen and is superior to all other serodiagnostic tests. The test is highly specific and does not cross-react With sera obtained from patients infected with other commonly occurring human parasites. Even though ELISA is an extremely sensitive test, the diagnostic predictive value for VLM is significantly different from that for OLM infections. 48 A titer of 2=1:32 is considered diagnostic for VLM, whereas a titer of 2=1:8 is considered diagnostic for OLM. This lower predictive titer for OLM raises the possibility of a false-positive diagnosis in a patient who has an asymptomatic Toxocara infection and ocular disease due to other etiology. Vitreous antibody can also be measured in addition to serum antibody. Titers in the vitreous are equal to or greater than the serum titer.

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Measurement of ocular fluid antibody should increase the specificity of the ELISA test, yielding a better definitive diagnosis for the patient. Hydatid Disease Serum antibodies to Echinococcus have been detected by IRA, IFA, ELISA, CF, LA, immunoelectrophoresis, and immuodiffusion. 26 , 47, 50, 52 Cysts in the liver will produce a greater serologic response in the patient than will hyaline or lung cysts. As many as 50 per cent of the patients with lung cysts will be serologically negative. Serologic cross-reactions have been noted in low titers in patients with other parasitic diseases, liver cirrhosis, and collagen diseases. 26 A test using a selected antigen called arc-5 has been developed for the specific diagnosis of Echinococcus infections. 11, 12 Reaction with arc-5 antigen will also occur in cysticercosis patients. Because antibody to arc-5 antigen may be absent in some patients, it is recommended that the serum be tested with one of the more sensitive tests, such as IRA or IF A, in combination with tests for arc-5 antigen. False-positive reactions to cysticercosis should be easily differentiated on a clinical basis. Cysticercosis Ruman cysticercosis caused by Taenia solium is a public health problem of considerable importance in developing nations. Serologic tests that have been used for diagnostic purposes include CF, IRA, IFA, ELISA, agar diffusion, and radioimmunoassay. 16, 19, 20, 26, 36 Immunologic diagnosis has been disappointing because a significant number of infected patients fail to respond to the test antigens used. Immunologic reactions in patients with ventricular cysts and meningitis are greater than those with parenchymal cysts. A comparison of serologic tests is quite difficult because of variations in methodology. IRA test has had better acceptance as a diagnostic tool; however, the test appears to be more sensitive with serum than with spinal fluid. Cross-reactions have been noted in patients infected with coenurus, Echinococcus spp., and T. saginata. A summary of serologic tests and diagnostic titers for helminthic infections can be found in Table 1 and Figure 1.

INTRADERMAL TESTS In the absence of reliable diagnostic tests, skin tests have been used as valuable adjuncts to provide indirect evidence of infection. The majority of skin tests have been used primarily for research and epidemiologic purposes. Some of the most widely used skin tests are the Casoni and Montenegro tests. In many cases, antigens employed are difficult to obtain and are not commercially available. Skin tests have not been well standardized and in many instances lack both specificity and sensitivity.

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Casoni Test Until recently, the Casoni test was the only means of diagnosing exposure to hydatid disease. This intradermal test has many advantages because of its simplicity; however, its nonspecificity has been the major limitation of the test. 28,61 Low diagnostic sensitivity was noted with patients who had intact and hyaline types of cysts. An immune response was detected more frequently in patients with liver cysts than in those with lung cysts. False-positive reactions have been reported in patients with other parasitic diseases and nonparasitic diseases. In addition to false-positive reactions, the Casoni antigen may also sensitize the patient, leading to antibody production. Montenegro Test The Montenegro test utilizes formalinized promastigotes of any species of Leishmania to evoke a delayed hypersensitivity reaction in infected patients. 30, 42 In some areas, it is the method of choice for diagnosis because of its simplicity. Patients infected with Leishmania donovani (kala-azar) will have a negative Montenegro test during active disease. The test will become positive after successful treatment or when there is a spontaneous cure. Similar problems have occurred with the Montenegro test as mentioned above for the Casoni test. The skin test may induce antibody production and also cause anaphylactic reactions.

SUMMARY For years, the diagnosis of most parasitic diseases has depended upon the direct demonstration of the parasite or its cysts, eggs, or larvae in specimens. In some infections, direct demonstration of the causative agent or its stages is almost impossible. In such cases, indirect techniques, such as serologic methods, have been found to be more practical and sensitive than are direct methods. Most serologic methods have been devised to detect antibodies. Tests for the detection of antigen are just beginning to be utilized; however, the practicality and under what situations antigen detection tests can be used await further testing. The majority of the serum antibody tests employ a heterogeneous mixture of antigens. Antigens derived from whole adult or larval stages usually result in tests with poor specificity and/or sensitivity. The necessity for better purified and standardized antigens cannot be over emphasized. Although there have been many major advances in the serodiagnosis of parasitic infections, a major drawback to routine use of parasitic serologies is the lack of commercially available reliable test kits. For the diagnosis of most parasitic diseases, one must rely upon speciality laboratories or public health laboratories. Before any laboratory begins to offer parasitic test serologies, they should contact their local or state public health laboratory or the Parasitic Serologic Section of the Centers for Disease Control to

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determine the pros and cons of these tests. Information of this type should be used to inform the physician of the limitations of the test in the differential diagnosis.

REFERENCES 1. Alford, C. A., Jr., Stagno, S., and Reynolds, D. W.: Congenital toxoplasmosis: Clinical laboratory and therapeutic considerations, with special reference to subclinical disease. Bull. N.Y. Acad. Med., 50:160-181, 1974. 2. Ambroise-Thomas, P., and Truong, T. K.: Fluorescent antibody test in amebiasis. Am. J. Trop. Med. Hyg., 21:907-912, 1972. 3. Ambroise-Thomas, P., Simon, J., and Bayard, M.: Indirect hemagglutination using whole mixed antigen for checking toxoplasmosis immunity and for serodiagnosis of human toxoplasmosis, compared with immunofluorescence. Biomedicine, 29:245-248, 1978. 4. Araujo, F. G., and Remington, J. S.: Antigenemia in recently acquired acute toxoplasmosis. J. Infect. Dis., 141:144-150, 1980. 5. Araugo, F. G., Barnett, E. V., Gentry, L. 0., et al.: False positive anti-toxoplasma fluorescent-antibody tests in patients with anti-nuclear antibodies. Appl. Microbio!., 22:270-275, 1971. 6. Araujo, F. G., Chiari, E. and Dias, J. C. P.: Demonstration of Trypanosoma cruzi antigen in serum from patients with Chagas' disease. Lancet, 1 :246--249, 1981. 7. Benaz, P. J.: A propos du diagnostic immunologique des affections a Pneumocystis carinii, Le test d' agglutination au latex. Bull. Soc. Patho!. Exot., 66:32-42, 1973. 8. Biglan, A. W., Glickman, L. T., and Lokes, L. A.: Serum and vitreous Toxocara antibody in nematode ophthalmitis. Am. J. Ophthalmo!., 88:898-901, 1979. 9. Bittencourt, A. L.: Congenital Chagas' disease. Am. J. Dis. Child., 130:97-103, 1976. 10. Camargo, M. E., and AmatoNeto, V.: Anti T. cruzi antibodies as a serological evidence of recent infection. Rev. Inst. Med. Trop. Sao Paulo, 16:200-202, 1974. 11. Capron, A. L., Yarzabal, L. A., Vernes, A., et al.: Le diagnostic immunologique de l'echinococcose humaine. Patho!. Bio!., 18:357-365, 1970. 12. Coltorti, E. A., and Varela-Diaz, V. M.: Detection of antibodies against Echinococcus granulosus arc 5 antigens by double diffusion test. Trans. R. Soc. Trop. Med. Hyg., 72:226--229, 1978. 13. Craft, J. C., and Nelson, J. D.: Diagnosis of giardiasis by counterimmunoelectrophoresis offeces. J. Infect. Dis., 145:499-504, 1982. 14. Cypress, R. H., Karol, M. H., Zician, J. L., et al.: Larva specific antibodies in patients with visceral larva migrans. J. Infect. Dis., 135:633-640, 1977. 15. Desmonts, G., and Couvier, J.: Congenital toxoplasmosis: A prospective study of 378 pregnancies. N. Eng!. J. Med., 290:1110-1116, 1974. 16. Espinoza, B., Flisser, A., Plancarte, A., et al.: Immunodiagnosis of human cysticercosis: ELISA and immunoelectrophoresis. In Flisser, A., Willms, K., Laclette, J. P., et al. (eds.): Cysticercosis: Present state of knowledge and perspectives. New York, Academic Press, 1980, pp. 163-170. 17. Feign, R. D., and Cherry, J. D.: Textbook of Pediatric Infectious Disease. Philadelphia, W. B. Saunders Co., 1981. 18. Fife, E. H., and Muschel, L. A.: Fluorescent antibody technique for serodiagnosis of Trypansosoma cruzi infection. Proc. Soc. Exp. Bio!. Med., 101 :540-543, 1959. 19. Flisser, A., Perez-Montfort, R., and Larralde, C.: The immunology of human and animal cysticercosis: A review. Bul!. WHO, 57:839--856, 1979. 20. Flisser, A., Woodhouse, E., and Larralde, C.: Human cysticercosis: Antigen, antibodies and non-responders. Clin. Exp. Immuno!., 39:27-37, 1980. 21. Garcia, L. S., Bruckner, D. A., Brewer, T. C., et aI.: Comparison of indirect fluorescentantibody amoebic serology with counterimmunoelectrophoresis and indirect hemagglutination amoebic serologies. J. Clin. Microbio!., 15:603-605, 1982. 22. GlickJ!lan, L. T., Schantz, P. M., and Cypress, R. H.: Epidemiological characteristics and clinical findings in patients with serologically proven toxocariasis. Trans. R. Soc. Trop. Med. Hyg., 73:254-258, 1979.

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Department of Pathology UCLA Medical Center Los Angeles, California 90024