Tformation' concerning the availability

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1 A summary of information about serologic testing for parasitic diseases is presented. These diagnostic procedures are evaluated and conclusions about them are presented. EVALUATION OF ROUTINE SEROLOGIC TESTING FOR PARASITIC DISEASES Irving G. Kagan, Ph.D. THIRTEEN years ago, a summary of in- Tformation' concerning the availability and efficacy of serologic testing for the detection of parasitic diseases presented relatively few tests, and indicated many areas in need of further development. In the diagnostic serology laboratory at the Communicable Disease Center we have increased the number of routine tests for the diagnosis of a variety of parasitic infections, and have investigated a number of new tests and reagents. Some of our observations and conclusions concerning these diagnostic procedures will be discussed. In our evaluation studies we select one diagnostic test for use as a reference standard. For some infections we use more than one technic for reference. The sensitivity, specificity, and reproducibility of the reference test is carefully evaluated. To accomplish this a "battery" of approximately 200 sera from human cases is tested. These sera represent a variety of parasitic and nonparasitic diseases. In addition, sera received for routine diagnosis with known clinical histories are also titrated. Other technics and reagents are compared with the standard procedure. Trichinosis Parasitology diagnostic laboratories in the United States are probably more familiar with tests for trichinosis than any other parasitic disease. Complement-fixation (CF) tests have been used for many years, and in the past decade several kinds of flocculation tests that utilize antigen-coated-inert particles have been introduced. Reagents for some of these are now commercially available, making possible rapid diagnosis and the recognition of small outbreaks of the disease. The epidemiology of trichinosis in the United States has become closely associated with local outbreaks due to individual slaughtering of infected pigs. The incidence of infections in hogs has been greatly reduced since feeding of uncooked garbage was prohibited by law. Most of the parasitic infections found in infected pigs in the United States average less than one larva per gram body weight.2 An immunologic test applied to detection of infection in hogs must be sensitive enough to detect such light infections in the muscles and should be positive during the lifetime of the hog. For detecting human infections the most desirable serologic tests should only be positive during the acute stage of the disease. Such requirements complicate the evaluation and interpretation of trichinosis tests. In man the duration of a positive trichinosis CF test is believed to be one to two years,3 depending on the magni. VOL. 55, NO. 11. A.J.P.H.

2 SEROLOGIC TESTING FOR PARASITIC DISEASES tude of the initial antibody response. We once thought that the bentonite flocculation test (BFT) usually remained positive less than a year. Recently, we have had the opportunity to retest persons infected seven years previously and found some still had titers by flocculation tests. The duration of the antibody response will have to be reconsidered, but one must be careful in making judgments, especially when individuals continue to reside in an endemic area. We have evaluated in our laboratory the CF test, bentonite flocculation test (BFT), trichina-latex test, charcoalcholesterol-lecithin (C-L) flocculation, and the fluorescent antibody (FA) reaction. The BFT has been used as a standard procedure with two antigens, a somatic antigen prepared from larvae and a "metabolic" antigen containing the excretions of larvae after incubation for 48 hours in a simple medium. A comparison of these two antigens (Table 1) over a period of five years shows that for 3,262 sera submitted for diagnosis, 484 (15 per cent) gave positive reactions with the BFT. Of this number, 90 per cent (437) were positive with the somatic antigen and 97 per cent (468) with the metabolic antigen. The titers obtained with the metabolic antigen were higher in about half (49 per cent) and lower in 11 per cent of the sera. Evaluation of the specificity of the two antigens in the BFT (Table 2) showed no cross-reactions with the positive sera from other diseases in an evaluation battery. We also found4 that in large surveys of populations the test does not give a percentage of reactors comparable to the estimated infection rate in this country. On the basis of the results of animal experimentation, the BET has not been shown to be sensitive enough to detect very light infections in pigs.5 There has been no evidence that either of the two antigens is more sensitive in the detection of infection early in the disease. The fact that some sera are positive with one and negative with the other-usually only when titers are low-confirms our belief that probably no single crude antigen or single test will reveal all the positive reactions in sera submitted for parasitic serology. For this reason, we perform more than one test for most parasitic diseases. Other tests have been investigated and compared with the BET with particular interest in obtaining a more sensitive test, i.e., one that will detect early infections. BFT, latex-flocculation and FA tests have recently been used routinely with Table 1-Comparison of Somatic and Metabolic Antigens for the Diagnosis of Human Trichinosis Positive by Bentonite Flocculation Test Somatic Metabolic Year No. of Sera No. Antigen Antigen Total 3, NOVEMBER,

3 695 sera received for diagnosis during 1963 and In this group, 111 were positive by the BFT, 133 by the FA, and 102 by a CDC latex antigen. Examination of the results obtained with the evaluation battery (Table 2) leads us to conclude at this time that the latex test, using our experimental antigens, is not as reactive as the others. The FA test was the most reactive. Until further evaluation of the FA test is available, we cannot determine whether or not the additional reactions obtained by FA are specific, i.e., represent low antibody levels or are cross-reactions with other antibodies. Testing of swine sera with the same types of antigens and procedures shows both the BFT and the latex to be less sensitive than the Suessenguth-Kline (SK) test, charcoal, and FA tests. The New York State type of latex antigen was superior to our own latex antigen. Recent studies of the C-L test for trichinosis6 have shown this procedure to have great promise as a field diagnostic and laboratory technic. Further evaluation studies are in progress. Hydatid Disease Although the number of indigenous infections with Echinococcus granulosus is low in the United States, we have a continual demand for testing sera from naturalized Americans who have been exposed to or have acquired hydatid infections in endemic areas. About 300 sera are examined in our laboratory each year. Although the CF test was originally used, the standard methods chosen for ease of performance as well as accuracy are the hemagglutination (HA) test and the BFT. Titers of over 1:400 in the HA test and 1:5 or higher in the BFT are reported as positive specific diagnostic tests. However, infections with lung cysts frequently fail to produce measurable levels of antibody. When testing general populations, 0) 02 0S 0 C._ Cl._._ 4-0) 5- v- 0 0) A ;0._ CQ 0 C.) W. U.) 0U) co; co * a N CD C._ C cu - Cl X C.)a Ht*u co E0 U.) H U.) 2. o o 0 0 o0h oo 00 + o Cl Cl Cl Cl Cl o co c C- c4 cq o o 0 0 oo Cl Cl Cl lclc._ CN (N CN CNN N Ns N N NN o o o ~ o o Cl CO C'O co 0%N -, Lo 0 LO o sp Cl o o % ooci CZo Id i N N N NS N N CO CO Cl Cl Cl Cl *_ - 4- \0 C Cl Cl Cl Cl Cl Cl Cl o Cl Cl E - E- -E-- C.._ c; c 0_ U C C ou C U I td) C. C. C. C. C. a- a-.5 a-.5 a- HHHHHH=r.0 S U)1 U. -U AU z VOL. 55, NO. 11. A.J.P.H.

4 SEROLOGIC TESTING FOR PARASITIC DISEASES Table 3-Sensitivity and Specificity of the Bentonite Flocculation and Hemagglutination Tests with an Evaluation Battery for Hydatid Disease Anti- Normal Anti-Tst Anti-Egt Filaria Anti- Anti- Anti- Anti- Tests Sera Sera Sera Sera Asc/Tox. Schisto. Protozoal Bact. HA 0/25* 1/29 20/20 2/32 1/20 0/24 1/21 0/49 BFT 0/30 3/33 18/19 1/23 2/21 0/28 2/18 0/39 * Number positive/number tested t Trichinella spiralis + Echinococcus granulosus a fairly large number of low-level reactions is obtained. Although the number of such reactions was reduced when sheep hydatid fluid was substituted for pig hydatid fluid as antigen, they have not been eliminated. Reactions with the 203 sera in the evaluation battery by the BFT and hemagglutination test are shown in Table 3. None of the crossreactions in the BFT showed titers above 1:10, while the hydatid cases are usually of very high titer. An echinococcus-latex test has the same level of sensitivity and specificity as the BFT. The latex antigen is difficult to standardize, but it has proved to be useful.7 In a recent evaluation of clinical sera from proven cases of hydatid disease, the levels of sensitivity and spe- cificity obtained for the HA, flocculation and latex tests are shown in Table 4. Note the higher specificity obtained with liver hydatid cases and low reactivity with lung cases. Schistosomiasis Serologic tests for schistosomiasis are numerous, and occasionally a single serum does not react with all procedures employed. The C-L test described by Anderson,8 using extracts of cercariae as antigen, has been recently chosen as the standard procedure in our laboratory. Neither the hemagglutination test nor the circumoval precipitin (COP) reactions are as sensitive or as reproducible. The FA test for schistosomiasis is Table 4-Serologic Results with Sera from Individuals Infected with Echinococcus granulosus Serologic Tests Cases Hemagglu- Floccutination lation Latex Echinococcus granulosus Liver 32/39* 32/39 32/39 Lung 6/18 9/18 8/18 Controls Normal 0/22 0/22 1/22 Miscellaneous diseases 0/13 1/13 1/12 Cancer 0/16 2/15 1/16 Taenia saginata 0/16 2/16 2/16 * Number positive/number tested NOVEMBER,

5 Table 5-Evaluation of the Fluorescent Antibody Test for Schistosomiasis Serologic Results Sera No. No. Positive/Tested % Positive Schistosomiasis 18/25 72 Trichinosis 24/31 77 Filariasis 4/23 17 Visceral larva migrans 2/22 9 Venereal disease 1/20 5 Echinococcosis 0/28 0 Kala azar 0/7 0 Chagas' disease 0/4 0 Tuberculosis 0/4 0 Virus disease 0/20 0 Toxoplasmosis 0/10 0 Normal 0/25 0 useful with certain types of sera. There are many more positive reactions with the FA test using cercariae as antigen. When 749 routine diagnostic sera were tested by two methods, 215 were positive by FA and 124 by C-L. When the FA test was evaluated on over 1,000 sera, the following conclusions were reached: The test is highly reproducible and a sensitive procedure. The specificity, however, is low with sera from individuals living in some areas of the world and shows many false-positive reactions. Further evaluation of the FA test as an epidemiologic tool is needed. Sera from cases of trichinosis are usually positive with schistosome antigens. Results obtained with the evaluation battery are shown in Table 5. Ascaris and Toxocara Serology-The Diagnosis of Visceral Larva Migrans (VLM) HA tests and BFT with crude somatic antigens prepared from Ascaris lumbricoides and Toxocara sp. worms are used routinely in our laboratory for the diagnosis of VLM. The results are often difficult to interpret and evaluate. Clin ical confirmation is usually not available. Results of the evaluation of the BFT with crude Ascaris and Toxocara antigens and our evaluation battery of sera are shown in Table 6. Agreement between the results of HA and the BET is poor, and there is evidence of considerable cross-reacting with antisera from nonrelated infections. However, the possibility is always present that previous infection with Ascaris may leave detectable antibodies for a long period of time. This may account for some of the reactions in the evaluation battery, since the individuals who provided the sera live in hyperendemic areas for A. lumbricoides. Of a variety of antigens evaluated, metabolic antigens made from hatched eggs gave the highest titers with experimentally infected animal sera. The production of such antigen is difficult, but not impossible. FA tests are in early stages of evaluation. The development of more specific tests for the diagnosis of Toxocara infection in children is needed. In spite of a great deal of research in this area, specific antigens or direct specific serologic procedures are still not available. Ascaris antibody can be differentiated from Toxocara antibody by absorption methods.9 Filariasis The BFT and HA tests using Dirofilaria immitis (Di) antigens are performed in our laboratory. Evaluation of the reactions are particularly difficult because most of the human sera tested are from returned missionaries who have been exposed to a wide spectrum of parasites. The titers obtained are usually low, ranging from 1:5 to 1:1,280 in the BFT but most below 1:40. HA titers range from 1:200 to 1:25,600+, with only rare samples above 1:800. In 1963, we tested 768 sera for filariasis: 37 per cent (281) reacted 1:5 or higher in the BFT, and 28 per cent VOL 55, NO. 11, A.J.P.H.

6 SEROLOGIC TESTING FOR PARASITIC DISEASES (218) reacted above 1:200 by HA. On the basis of previously determined criteria, a titer of 1:5 in the BFT and 1:200 and higher in the HA test, 198 (26 per cent) were reported positive. From the results of a previous smaller evaluation10 we could expect that in 50 per cent of our positive reactors microfilariae would be isolated from the blood. Evaluations have showed that the antigens react with some sera containing Toxocara, Trichinella, A. lumbricoides, and Schistosoma antibodies. The 768 sera tested for filariasis were also tested for schistosomiasis. If both FA and C-L tests for schistosomiasis were positive, the serum was considered positive for this parasitic disease. In this battery 554 were negative with Di antigens, 214 were reactive. A total of 126 was positive for schistosomiasis. In this group of positive sera, only 56 were positive with both antigens. The serology of filariasis has recently been reviewed.1 Cysticercosis Two antigens have been used in the HA test for cysticercosis. One was prepared from whole adult worms of Taenia saginata and the other from cysticerci in pig muscle of T. solium. The test was intially standardized with sera from cows experimentally infected with T. saginata. Serologic results were inconclusive since normal cows showed a relatively high level of reactivity. An evaluation of a serologic test for cysticercosis must await the collection of good positive human control sera. With our evaluation battery of sera (Table 7), the whole worm antigen was much more specific than the cysticercus antigen. As expected, the antigen crossreacted with sera from patients with Echinococcus granulosus (52 per cent). The cross-reactions with sera from Puerto Rican patients with schistosomiasis are difficult to interpret since Chagas' Disease Taenia infections of any kind are rare in that country. A comparison of results obtained in Mexico using three tests, precipitation, HA, and CF, lead Biagi to conclude that the hemagglutination test was the most sensitive serologic procedure for diagnosis of cysticercosis.12 For the diagnosis of Chagas' disease we perform the CF test recommended by the Laboratory Branch of the Communicable Disease Center. We have also evaluated the Freitas' quantitative CF method.13 The antigen employed is a saline extract of defatted culture stages of T. cruzi organisms.14 In a limited study both technics gave similar results with a group of sera from cases of Chagas' disease. The HA test using the same antigen appears to be more sensitive. A latex test is under development. All tests require further evaluation to determine their specificity. Table 6-Evaluation of Sensitivity and Specificity of the Bentonite Flocculation Test with Ascaris and Toxocara Antigen Anti- Anti- Anti- Anti- Anti- Antigen Normal Anti-Tst Anti-Egt Filaria Asc/Tox. Schisto. Protozoa Bact. Ascaris 1/24* 4/33 3/19 2/23 14/21 2/28 3/18 2/39 Toxocara 1/24 7/33 0/19 14/23 8/21 1/28 1/18 2/39 * Number positive/number tested t Trichinella spiralis I Echinococcus granulosus NOVEMBER

7 Table 7-Evaluation of Hemagglutination Test for the Diagnosis of Cysticercosis Anti- Anti- Anti- Anti- Anti- Antigen Normal Anti-Tst Anti-Egt Schisto. Filaria Asc/Tox. Protozoa Bact. T. saginata whole worm 1/24* 0/31 11/21 8/29 0/23 0/23 1/18 0/33 T. solium cysticercus 1/24 1/31 15/21 8/29 4/23 4/23 1/18 0/33 * Number positive/number tested t Trichinella spiralis t Echinococcus granulosus Amebiasis Only recently have we attempted the standardization of serologic tests for amebiasis. We are evaluating the hemagglutination test reported by Lewis and Kessel.15 The antigen is an extract of lyophilized or wet ameba grown in culture. The test is reliable and reproducible, especially in cases of extraintestinal amebiasis and acute amebic dysentery. Titers in such cases are very high and remain so for long periods of time. The test still requires evaluation in patients with chronic amebic colitis or with vague abdominal symptoms. Toxoplasmosis The methylene blue dye test is generally considered the standard procedure for the serologic diagnosis of toxoplasmosis. One can expect as high as 80 per cent reactors in the adult population in the absence of clinical symptoms. For this reason, it is difficult to determine a "significant" titer, but in our laboratory we assume a titer of 1:256 or above to be of clinical significance. Since the introduction of this test by Sabin and Feldman,16 two other major tests have been developed or evaluated in our laboratories. The fluorescence inhibition (FI) test developed by Goldman and Carver17 has proved to be equally as specific but slightly less sensitive than the dye test. In general, titers can be expected to be fourfold lower in the FI than in the dye test. We have recommended this procedure as a satisfactory substitute for the dye test in laboratories where the latter test cannot be used. The indirect hemagglutination test (IHA) has recently been evaluated by Jacobs and Lunde18 and others, and has been suggested as a more practical routine than either of the two previously discussed tests. Using the modification of Lewis and Kessel,15 we evaluated the IHA in reference to the dye test and found that although titers were generally parallel, the IHA was usually fold higher, and in 14 per cent of the sera gave significant titers in the absence of reactivity in the dye test. Further evaluation is necessary before the hemagglutination test can be recommended for routine use or the significance of serologic titers assessed. Malaria The diagnosis of malaria by serologic tests is currently not being performed in our laboratory. There are, however, two procedures which are very promising-the FA test19 and a hemagglutination test20 developed by Stein and Desowitz. These serologic procedures may be very valuable for future epidemiologic and diagnostic studies. Summary and Conclusions Trichina Serology The serologic tests used for the diagnosis of trichinosis are highly specific. VOL. 55, NO. 11. A.J.P.H.

8 SEROLOGIC TESTING FOR PARASITIC DISEASES They may detect residual antibodies from earlier infections, but usually a changing titer obtained over several weeks time is suggestive of acute and active disease. The tests vary in sensitivity, but even the most sensitive may be negative during the first two weeks of the disease. Low titered reactions should be confirmed by other tests since some sera are more reactive with different antigens. Echinococcus Serology Because of differences in antigens, it is desirable to test sera by more than one test or more than one antigen. Those sera giving high titers with evaluated tests (almost always) indicate hydatid disease. Very low titers must be questioned, since very low level reactions can be obtained in collagen diseases or liver cirrhosis. Cysts of the lung seldom produce a measurable antibody level with hemagglutination or flocculation tests. Positive Casoni skin tests, especially in conjunction with positive serology, are highly diagnostic. A positive skin test and negative serology may indicate sensitization or the presence of a calcified cyst. Visceral Larva Migrans Serology A. lumbricoides and Toxocara sp. antigens cross-react with sera from patients with VLM. There is, however, a strong tendency for a serum to have a much higher titer with one antigen than with the other, depending on the species of larvae invading the body. Since ascariasis is endemic in almost all parts of the world and an infected individual may produce measurable amounts of antibodies to crude parasitic antigens, it is impossible to state that a positive reaction is indicative of current disease. Suspected cases of VLM-eye infections with positive serology should be studied carefully for the presence of intestinal infection. A high serologic titer should be considered suggestive, but not pathognomic. Schistosomiasis In schistosomiasis, depending in part upon the age of the individual and status of the disease, serologic tests give variable results. There are a variety of tests and antigens used in diagnosis, but Ino single test detects all infections. Skin tests are sensitive21 and all positive reactors should be examined for infection. A combination of serologic tests is recommended. The possibility of trichinosis must be ruled out with positive sera, since cross-reactions occur in these two diseases. The length of time after treatment that the patient retains antibodies is not known precisely, but titers may be expected to drop slowly only after one or two years. Cysticercosis Because of the limited number of cases in the United States, the tests for cysticercosis have not been evaluated thoroughly and need more study, especially in regard to their sensitivity in cerebral infection (spinal fluid). Antigens prepared from cysticerci and adult Taenia worms cross-react with Echinococcus and Schistosoma sera. Titers in proved cases of cysticercosis generally have been high. Judging from the results obtained with experimentally infected cows, the tests are quite sensitive and a negative reaction may have some significance. Filariasis The diagnosis of filariasis by hemagglutination and flocculation tests is a good procedure when working with sera of missionaries or other Americans returning from an endemic area after a period of several months to several years. In hyperendemic areas these technics may not be specific enough to separate active infection from sensitization. Chagas' Disease In acute infections serologic tests are sensitive. The chronic disease found in NOVEMBER,

9 Figure 1-Immunodiagnostic Tests for Parasitic Infections TESTS Particle Agglutination tests.0~~~~~~~~~~~~~~~~~6 - a c 0 c. a - C 6)~~~~0 00 PARASITIC - - m u J u ~- DISEASES \ U' -C0 o c ~~E U 0 < ~~~~~< LL U~U-U Trichinosis * 0 Echinococcosis o 0 A r X Toxocariasis (III) (:3 ( ( )4 Filariasis 0o #1 1 [ 3 Cysticercosis * * Clonorchiasis l) (3 ) CI) Paragonimiasis 1 _ _ _ Chagas' disease Q * Q 0*0 Q 0 Leishmaniasis *T 0 Toxoplasmosis 0 Amebi asi s 0 [A_Ae_A Tests availa6le in the U.S. Q Under experimental investigation Used for diagnosis but requires further evaluation for routine use * Generally accepted useful routine diagnostic test L c South America is also detected immunologically with considerable accuracy. There is some evidence of cross-reactions with leishmaniasis. Whether or not the tests are sensitive enough to reveal very low-grade infections, suspected to exist in the United States, is still not known. Studies to date do indicate that anti- VOL. 55, NO. 11, A.J.P.H.

10 SEROLOGIC TESTING FOR PARASITIC DISEASES bodies against T. cruzi antigens may be detected in the southern part of the United States.22 Amebiasis The HA test for amebiasis is presently undergoing a thorough evaluation to determine its sensitivity and specificity in diagnosing intestinal infections. Its reliability with sera from patients with extraintestinal infection is high. Toxoplasmosis Although the methylene blue dye test is still the reference test for toxoplasmosis, FI and IHA tests have been shown to be of value. The FI test correlates directly with the dye test, but the IHA is much more sensitive. The latter test therefore is recommended only as a screening procedure. In Figure 1 the status of immunodiagnostic tests for parasitic infections is summarized. REFERENCES 1. Donaldson, A. W. Serological Diagnosi of Paraidc and Mycotic Infections. Pub. Health Lab. 9 : Zimmermann, W. J. Personal communication. 3. Bozicevich, J. Immunological Diagnosis of Parasitic Diseases in Parasitic Infections in Man. New York, N. Y.: Columbia University Prea, Norman, L., and Kagan, I. G. Bentonite, Latex and Cholesterol Flocculation Tests for the Diagnosi of Trichinosis. Pub. Health Rep. 78: , Norman, L.; Sadun, E. H.; Redding, R. W.; and Cooperrider, D. E. Flocculation Test in Sera from Hogs Experimentally and Naturally Infected with Trichinella spiralis. J. Parasitol. 41: , Anderson, R. I.; Sadun, E. H.; and Schoenbechler, M. J. Cholesterol-lecithin Slide (TsSF) and Charcoal Card (TsCC) Flocculation Teats Using an Add Soluble Fraction of Trichinella spirali larvae. Ibid. 49:6-647, Szyfres, B., and Kagn, I. G. A Modified Slido Latex Screening Teat for Hydatid Diaeae. Ibid. 49: 69-72, Anderson, R. I. Serologic Diagnods of Schistosom mansoni Infectious. I. Development of a Cercarial Antigen Slide Flocculation Test. Am. J. Trop. Mod. 9: , Kagan, I. G. Hemagglutination Teats with Aacaris Antigens. J. ImmunoL 80:t96-399, Adolph, P. E.; Kagan, L. G.; and McQuay, R. M. Diagnosis and Treatment of Acanthocheilonema perstans Filariais. Am. J. Trop. Med. 11:76-88, Kagan, I. G. A Review of Immunological Methods for the Diagnosis of Filariadsc. J. Parasitol. 49: , Biagi, F.; Navarrete, F.; Pisia, A.; Santiago, A. M.; and Tapia, L. Estudio de Tres Reacciones Serologicas en el Diagnostico do la Ciaticercosis. Rev. Mid. Hoop. Gon. (Mexico, D. F.) 25: , Freitas, J. L. P. de Reacio de Fixagio do Com. plemento para Diagn6stico de Molestia de Chagas pla Tecnica Quantitativo. Arq. Hig. e Safide Pub. (Sbo Paulo, Brazil) 48:55-94, Maekelt, G. A. Die Komplementbindungareaktion der Chagaskrankheit. Stuttgart, Germany: Ztschr. Tropenmed. u Parasitol. 11: , Lewi, W. P., and Kassel, J. F. Hemagglutination in the Diagnosis of Toxoplasmosis and Amebiasis. Arch. Ophth. 66: , Sabin, A. B., and Feldman, H. A. Dyes as Microchemical Indicators of a New Immunity Phenomenon Affecting a Protozoan Parasite (Toxoplasmosis). Science , Goldman, M.; Gordon, M. A.; and Carver, R. K. Comparison of Tites by Dye and Fluorescence Inhibition Tests in the Serological Diagnosis of Toxoplasmosis. Am. J. Clin. Path. 37: , Jacobs, L.. and Lunde, M. N. A Hemagglutination Test for Toxoplasmosis. J. Parasitol. 43: , Tobie, J. E. Detection of Malaria Antibodies-Im. munodiagnosis. Supplement to Am. J. Trop. Med. 13(Part 2) : , Stein, B., and Desowitz, R. S. The Measurement of Antibody in Human Malaria Infections by a Formalized Tanned Sheep Cell Hemagglutination Test. Bull. World Health Organ. 30:45-51, Kagan, I. G., and Pellegrino, J. A Critical Review of Immunological Methods for the Diagnosis of Bil. harziasis. Ibid. 25: , Farrar, W. E.; Kagan, I. G.; Everton, F. D.; and Sellers, T. F. Serologic Evidence of Human Infec. tion with Trypanosoma cruzi in Georgia. Am. J. Hyg. 78: Dr. Kagan is chief, Parasitology Unit, Mycology and Parasitology Section, Communicable Disease Center, Public Health Service, U. S. Department of Health, Education, and Welfare, Atlanta, Ga. This paper was presented before the Laboratory Section of the American Public Health Association at the Ninety-Second Annual Meeting in New York, N. Y., October 7, NOVEMBER l1829