Robert I. Anderson

A technique for the use of minute amounts of dried blood in the fluorescent antibody test for schistosomiasis.

Abstract A simple method for the collection, preservation, shipment, and testing of minute amounts of dried blood for the diagnosis of Schistosomiasis is described. A drop of blood obtained from a finger puncture and collected on filter paper was extracted in saline by use of flexible tube and carpenters vise. The extracted blood was tested by the indirect fluorescent antibody technique employing Schistosoma mansoni cercariae as antigen. The dried filter paper blood specimens were preserved at room temperature for as long as 90 days without detectable changes in antibody response. This technique is now being evaluated for field use in epidemiological surveys.

Fluorescent Antibody Test for the Serodiagnosis of African and American Trypanosomiasis in Man.

A fluorescent antibody technique for the serodiagnosis of African and American trypanosomiasis in man is described. The test can be performed simply and rapidly through the use of trypanosomes in blood smears as antigen. There were extensive cross-reactions with sera from patients with other species of trypanosomes. Tests of sera from healthy controls and from patients with nontrypanosomal diseases and disorders revealed relatively few cross-reactions, indicating a high degree of specificity. The finding that dried blood on absorbent paper could be tested successfully with the FA technique may indicate its usefulness for studies in endemic areas. Human infections with African and American trypanosomiasis constitute important clinical and public health problems in large regions of the world. An unequivocal diagnosis of trypanosomiasis is often difficult to obtain since the clinical picture is not always well defined and the organisms frequently cannot be recovered by blood examinations. Consequently, there is a need for reliable, rapid, and inexpensive procedures which could provide the basis for an adequate diagnosis of these infections, especially during the latent and chronic phases of the disease. The fluorescent antibody technique (Coons et al., 1941) is rapidly developing into a practical, sensitive, and specific diagnostic method for several parasitic infections. Fife and Muschel (1959) described a fluorescent antibody technique (FA) for the serodiagnosis of Trypanosoma cruzi infections. T. cruzi cultured on a diphasic blood agar medium was utilized as a source of antigen. This technique, which required all the reactions to be carried out in test tubes to prevent drying of organisms, appeared to be less specific than the complementfixation test using purified T. cruzi antigen. Studies with T. rhodesiense and T. gambiense in experimental animals (Williams et al., 1963) have resulted in a rapid and practical FA technique which can be run on slides using as Received for publication 21 December 1962. antigen thin blood smears from infected rats. A technique for the use of minute amounts of dried blood in the fluorescent antibody test was developed recently (Anderson et al., 1961). This technique, which permitted mailing of specimens under adverse conditions, was found to be particularly useful in epidemiological surveys of schistosomiasis and trichinosis (Sadun et al., 1961, 1962). The current report summarizes results of studies in which the FA technique was used to stain fixed blood forms of T. rhodesiense, T. gambiense, and T. cruzi toward the development of a reliable and practical test for the laboratory diagnosis of African and American trypanosomiasis. The procedures were evaluated with sera obtained from humans in endemic areas. Furthermore, in the present work attempts were made to determine the degree of cross-reactivity of different trypanosome species and to determine whether blood smears dried on absorbent paper could be used in the serological diagnosis of trypanosomiasis. MATERIALS AND METHODS Sera: Human sera were obtained from individuals with well-documented trypanosomiasis infections. The diagnosis of trypanosomiasis in the patients was established by the recovery and identification of organisms from the blood or spinal fluid. To determine the specificity of the FA test, control sera from individuals with viral, bacterial, and parasitic infections other than trypanosomiasis were used. To test whether the presence of auto-

Fluorescent antibody test for the serological diagnosis of trichinosis.

Abstract A fluorescent antibody test for trichinosis, employing T. spiralis larvae as antigen and possessing a high degree of sensitivity and specificity, is described. Reliable qualitative and quantitative results were obtained either with fresh sera or dried blood specimens on absorbent paper.

Fluorescent Antibody Technic for Sero-diagnosis of Schistosomiasis in Humans.∗

Summary Use of fluorescein-labelled antiglobulin as an indicator of cercarial antibody is described. Sensitivity of the procedure appears to be great for all human schistosome infections. While non-specific staining occurs regularly with sera from trichinosis patients, the specificity with other sera suggests that this procedure may be useful in serological diagnosis of human schistosomiasis.

Preserved cercariae in the fluorescent antibody (FA) test for schistosomiasis

Abstract 1. 1. Cercariae stained with rhodamine bovine albumin and preserved with 10% formalin for several weeks gave excellent results in the fluorescent antibody test for schistosomiasis, thus obviating the necessity of using fresh cercariae. 2. 2. Cercariae so preserved were found to be suitable regardless of whether they were maintained in wet storage at 3 to 6 ° C, frozen and stored at −20 ° C, or lyophilized. 3. 3. Shipment of preserved wet cercariae through the regular mails without refrigeration was also possible. 4. 4. The use of preserved cercariae in conjunction with filter paper—dried blood methods makes the fluorescent antibody test one of the simplest and most convenient sero-diagnostic techniques for Schistosomiasis.

Fluorescent Antibody Test for the Laboratory Diagnosis of Schistosomiasis in Humans by using Dried Blood Smears on Filter Paper.

Abstract Use of the fluorescent antibody test for the laboratory diagnosis of human schistosomiasis on minute amounts of dried blood specimens is described. Sensitivity and specificity of the test appear to be comparable to the best described serological tests. Mailing of specimens under adverse conditions did not seem to influence the results. Determinations of antibody titers were possible. These findings and the ease with which specimens can be obtained, delivered to the laboratory and tested suggest that the procedure may be useful in epidemiologic surveys offering some advantages over the known methods of stool examinations, serology, and intradermal testing.

Cross absorption studies performed with Schistosoma mansoni and Trichinella spiralis antigens in sera from patients with trichinosis.

Abstract The apparent one-way cross reaction between Schistosoma mansoni antigens and sera from patients with trichinosis was studied by absorption technics. When the Trichinella antiserum was absorbed with Trichinella slide flocculation test antigen, the homologous antibody was removed but the antibody reactive against cercarial slide flocculation test antigen remained. The converse was also true in that absorption of the Trichinella antiserum with schistosome antigen removed antibody serologically reactive with the schistosome antigen but failed to remove antibody reactive with Trichinella antigen.

SEROLOGICAL CHARACTERISTICS AND GENERAL CHEMICAL NATURE OF THE IN VITRO EXOANTIGENS OF T. CRUZI.

Serologic, immunologic, and chemical properties of substances produced during in vitro cultivation of Trypanosoma cruzi were investigated. After cultivation of the organisms, the medium contained an antigen which fixed complement in the presence of homologous antisera and produced detectable circulating antibody when injected into experimental animals. Different serologic patterns were observed in an evaluation of the antigen in parallel complement fixation tests using standard somatic antigens as reference. Chemical analyses of the product indicate that it is a complex substance, possibly a glycoprotein. The findings suggest that exoantigen produced during culture of the organism differs from previously described somatic antigens. Complement fixation tests are the methods of choice for serodiagnosis of Trypanosoma cruzi infection, particularly in cases of chronic infection (Pifano, 1960; Maekelt, 1960). However, the crude somatic antigens of T. cruzi used in the CF tests have been shown to lack specificity (Kelser, 1936; Liem and van Thiel, 1941; Romana and Gil, 1946; Chaffee et al., 1956). Treatment of somatic antigen with benzene and chloroform (de Freitas and Almeida, 1949) increased specificity but resulted in a loss of sensitivity. Recently, Fife and Kent (1960) prepared a purified protein antigen which gave excellent results in diagnostic tests, virtually eliminating cross reactivity except with sera from patients with mucocutaneous leishmaniasis. Development of feasible methods for mass cultivation of T. cruzi within cellulose sacs (Fife and Kent, 1960) made possible studies of an alternative, and previously unexplored T. cruzi antigen source, namely the exoantigens which might be produced during the in vitro cultivation of the parasite. Antigens liberated by parasites maintained in media have been Received for publication 7 August 1964. * From work done by the senior author in partial fulfillment of the M.S. degree at Howard University, Washington, D. C. t This work was presented, in part, at the 1st International Congress of Parasitology, Rome, 21-26 September 1964. previously studied (Sadun and Norman, 1957; Minning et al., 1958; Kagan and OliverGonzalez, 1958; Anderson et al., 1962; Sleeman et al., 1963). In addition, limited chemical analyses of similar products have been performed (Stirewalt, 1959; Stirewalt, 1963; Sleeman et al., 1963). In general, the investigations indicate that the antigenic materials liberated by organisms maintained in vitro are less complex than the somatic antigens, and that similar products liberated in vivo may play an important role in the immunogenic response of the host. The present study was conducted to dedermine whether detectable antigenic substances were produced by T. cruzi during in vitro cultivation and, if so, to study their serologic characteristics and general chemical

An Indirect Fluorescent Antibody Technique Using Soluble Antigens for Serodiagnosis of Trypanosoma cruzi Infection

Summary A soluble antigen fluorescent antibody (SAFA) technic for the serorecognition of American trypanosomiasis is described. The technic has the following advantages over the conventional whole organism indirect fluorescent antibody tests: 1) permits the investigator to objectively select the antigen; 2) provides for mechanical reading of test results; 3) eliminates the problem of fading; and 4) accounts for nonspecific fluorescence contributed by the patients serum (e.g., drugs) and/or by free fluorescein in the conjugated antiserum. Preliminary investigations revealed that exo, somatic carbohydrate and somatic protein antigens from T. cruzi would adhere to the artificial matrix. The sensitivity and specificity of the somatic protein and exoantigens were evaluated, The findings indicate that the SAFA technic with either of these 2 antigens should yield excellent results.

CHOLESTEROL-LECITHIN SLIDE (TSSF) AND CHARCOAL CARD (TSCC) FLOCCULATION TESTS USING AN ACID SOLUBLE FRACTION OF TRICHINELLA SPIRALIS LARVAE.

Two simple sensitive flocculation tests were developed employing acid soluble T. spiralis larvae antigen absorbed onto cholesterol-lecithin crystals. Washing of the emulsion greatly increased the sensitivity of the antigen in the slide flocculation test. The addition of charcoal to the antigen emulsion permitted its use in a simple card test using serum or minute amounts of plasma collected from finger puncture. Absorption of reactive sera with a single dose of washed, packed, and essentially dry antigen-cholesterol-lecithin complex resulted in a complete removal of serologically detectable antibody indicating that the tests were based upon a true antigen-antibody system. Sensitivity of both tests was excellent. In the slide flocculation test, 64 sera from 68 patients were reactive, 2 were weakly reactive and 2 were nonreactive. All 21 sera from trichinosis patients were reactive in the charcoal card test. The tests also appear to be relatively specific. Only 3 sera from a total of 96 normal persons and patients with other diseases were reactive in the slide flocculation test; 13 sera showed weak reactions. With the charcoal card test 3 reactions were obtained with sera from a total of 64 normal individuals and patients with other diseases. The tests are especially well suited to small laboratory and field conditions because they are simple to perform and utilize antigenic emulsions which may be kept for prolonged periods without apparent loss of reactivity. Slide flocculation tests are among the simplest procedures for the serodiagnosis of infections. These techniques have not yet found wide application in the field of parasitology primarily because of the difficulty in many instances of combining the parasitic antigens with the carrier (cholesterol, latex, or bentonite). Suessenguth and Kline (1944) found that an aqueous extract of Trichinella larvae coated onto cholesterol crystals could be used as antigen in a simple and rapid flocculation slide test for trichinosis. They reported that this test was more sensitive than the complement fixation test (Suessenguth et al., 1957). However, other investigators have presented evidence indicating that this test lacks the specificity of other serological tests for trichinosis (Bozicevich et al., 1951; Sadun and Norman, 1955a; Greene and Brazeale, 1951). Recently Anderson (1960) developed a slide Received for publication 26 April 1963. flocculation test for schistosomiasis using antigen prepared by coating a buffered saline extract of cercariae onto cholesterol-lecithin particles and subsequently washing this complex to remove the excess antigen. This method has been shown to be one of the most sensitive and specific serodiagnostic procedures for schistosomiasis (Anderson, 1960; Anderson and Naimark, 1960; Jachowski and Anderson, 1961). Following the development of the rapid plasma reagin card test (Portnoy et al., 1962) for syphilis, the schistosome slide flocculation te t antigen was adapted to the card procedure and evaluated under laboratory and field conditions (Sadun et al., 1963). The card test in schistosomiasis gave results comparable to those obtained with the standard slide flocculation procedure and permitted the test to be completed within a few minutes on plasma obtained by finger puncture. The purpose of the current study was to determine whether similar procedures would provide a slide flocculation test for trichinosis