J. S. Williams

Pathophysiology of Plasmodium berghei infection in mice

Abstract Significant biochemical changes were detected in the serum of mice infected with P. berghei . Marked increases in SGP-T and SGO-T transaminases were observed as early as 2 days after infection. Lower fasting glucose levels occurred in heavily infected mice 4 days after infection. A moderate reduction in alkaline phosphatase and albumin values was observed. Increases in BSP retention and positive cephalin flocculation reactions were also registered in the infected mice. Minimal or no changes in total protein, non-protein nitrogen, phosphorus, globulins, bilirubin, calcium, creatinine, carbon dioxide, chloride, sodium, and potassium resulted from infection.

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.

Biochemical aspects of Schistosomiasis mansoni in mice in relation to worm burdens and duration of infection

Abstract By means of ultramicrotechniques, quantitative estimations of serum components of mice were made before and at various intervals after exposure to Schistosoma mansoni cercariae. Infected mice were divided into five intensity groups ranging from 1 to 115 worms per animal and the mean biochemical values obtained in the different groups at various times after infection were compared. Significant increases in transaminase values were noticeable as early as 8 weeks after exposure in the lightly infected mice and 2 weeks earlier in animals that had heavier infections. As the infection progressed in all groups there was a relatively greater increase in SGP-T values than SGO-T. Consequently, the SGP-T SGO-T ratios showed significant increases in all groups. Significant increases were also observed in the BSP retention test, even in those mice harboring 5 worms or less. Moderate increases in total protein were observed between 8 and 10 weeks following exposure to infection. A small decrease in albumin and an increase in globulin levels resulted in reduced albumin/globulin ratios that became more marked with time, particularly in the groups of mice with heavy worm burdens. Infections even with relatively heavy worm burdens produced no significant departures from normal values in the serum levels of glucose, alkaline phosphatase, phosphorus, bilirubin, creatinine, calcium, sodium, potassium, chloride, and carbon dioxide.

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.

Serum biochemical changes in mice infected with Trypanosoma rhodesiense and Trypanosoma duttoni.

Abstract Significant biochemical changes were measured in the serum of mice infected with Trypanosoma rhodesiense or T. duttoni. Mice infected with T. rhodesiense were sorted into groups as follows: those with (1) acute infections, (2) drug-suppressed chronic infections, (3) “drug-cured” infections, and (4) drug-treated, no infection. Biochemical tests were performed on the serum of each animal 2 days before inoculation and subsequently on days 1, 3, 5, 8, 15, 22, and 29. Five days after infection glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), creatinine, bromsulphalein retention, total protein, alpha-2 globulin, and gamma-globulin values increased while glucose and beta globulin decreased. These values returned to essentially normal levels when parasites were cleared from the peripheral blood. The following components were not altered significantly: blood-urea nitrogen, sodium, potassium, calcium, chloride, and carbon dioxide. In mice infected with T. duttoni changes in GPT, GOT, creatinine, total protein, and gamma globulin took place later and were not as great as those in animals infected with T. rhodesiense. Some serum biochemical changes appeared to be related directly to the number of parasites in the circulating blood.

A radioactive antigen microprecipitin (RAMP) assay for schistosomiasis.

An assay for measuring antigen binding by antibody in schistosomiasis was developed using a fraction of Schistosoma mansoni antigen labeled with radioactive iodine. This assay which is not de- signed for the routine serological diagnosis of schistosomiasis was designated radioactive antigen micro- precipitin (RAMP). It is highly reproducible, quantitative, and may serve as an indicator of the immune response in schistosomiasis. Serum samples from patients with proven schistosomiasis and other diseases were assayed for their ability to precipitate 125I-labeled S. mansoni antigen. Eighty-one per cent of 104 sera from schistosomiasis patients were reactive in the RAMP assay; no reactions were obtained with 90 sera from healthy persons. A few nonspecific reactions were obtained in other disease states. The RAMP assay and the passive cutaneous anaphylaxis (PCA) tests were compared using 85 serum specimens from infected schistosomiasis patients reactive in the soluble antigen fluorescent antibody (SAFA) technique. The RAMP assay and the PCA test were both reactive with 48 of these specimens. Three specimens in which reaginic antibodies had been detected failed to react in the RAMP assay and 17 sera which reacted in the RAMP assay gave negative results in the PCA test. Fractions of pooled human anti-S. mansoni sera were tested in the RAMP assay and the results were compared with those obtained with the same frac- tions in the SAFA and PCA tests. The first fraction from DEAE-A-25 contained the major amounts of IgG and IgA immunoglobulins and all of the detectable SAFA reaction, while fraction 5 contained RAMP and PCA activity but no detectable SAFA antibodies. Heating or reduction and alkylation of the immune serum destroyed or markedly reduced the antibody reactions in the RAMP assay and PCA test, but did not affect the reactivity of the SAFA technique. When rabbit S. mansoni antiserum was absorbed with goat antirabbit IgE serum, the reactivity of the serum was greatly reduced in the RAMP assay, elimi- nated in the PCA test, and was unchanged in the SAFA technique. The RAMP assay and PCA test cor- related closely in demonstrating the time-course development of antibodies in animals infected with schis- tosomiasis, but the antibodies detected by the SAFA technique followed an entirely different time course. These studies indicate that the RAMP assay can reliably measure antibodies in all immunoglobulin classes and particularly in IgE, and this assay is a means of demonstrating primary binding of antigen by antibody in schistosomiasis.