Relationship of serum complement levels to events of the malarial paroxysm.

Malarial paroxysms due to Plasmodium vivax were studied for alterations in whole serum complement (C') and certain C' components. The objective was to relate C' values with events of the parasite cycle during schizogony and with the febrile pattern. Substantial decreases in C' were found in 9 of 18 paroxysms studied during relapse. In contrast, only one of 22 paroxysms occuring during the primary attack was associated with a striking depression in C', and this case exhibited certain characteristics of a relapse paroxysm. The mean change in C' levels during paroxysms in relapse (-23%) was significantly different from paroxysms of the primary attack (-2%). Depletion of C' was associated directly with degree of parasitemia and presence of complement-fixing (CF) antibody. Lowest levels of C' were found within a few hours after completion of schizont repture and peak fever. C4 levels reflected changes in whole serum C' and appeared to be a more sensitive indicator of C' alterations during malaria. While the alterations in C4 as well as C1 and C2 indicated that the classical C' pathway was involved, some preliminary results showed little or no depletion of late components, C3 and C6. Overall results are compatible with C' activation and depletion during or soon after schizont repture if parasite density is sufficiently high and if CF antibody is present.

American trypanosomiasis (Chagas' disease) in Central American immigrants.

A survey was conducted to determine the prevalence of infection with Trypanosoma cruzi, the protozoan etiologic agent of American trypanosomiasis (Chagas' disease), among Nicaraguan and Salvadoran immigrants living in the Washington, D.C., area. The serum samples of study subjects were tested for reactivity with T. cruzi antigens in an enzyme-linked immunosorbent assay, and also tested for antibody specific for the 72 and 90 kilodalton (kDa) surface glycoproteins of the parasite in an immunoprecipitation and electrophoresis procedure. Xenodiagnosis using reduviid bugs to detect parasites, and clinical evaluations for cardiac and gastrointestinal disease were performed in patients in whom results of both serologic tests were positive. Of 205 subjects studied, 4.9 percent were infected with T. cruzi, and parasites were isolated from 50 percent of those in whom xenodiagnosis was attempted. No significant cardiac or gastrointestinal abnormalities were detected in the six infected patients who were evaluated clinically. These findings suggest that a sizable proportion of persons in this immigrant group are infected with this organism. Thus, routine serologic testing for antibody to T. cruzi may be warranted in immigrants from these countries, especially in view of the potentially serious consequences of infection with this parasite, and also because of the risk of transmission of T. cruzi by blood transfusion.

Antigens from the surface and excretions/secretions of the filariform larva of Strongyloides stercoralis.

The surface and excretory/secretory (ES) antigens of the infective, filariform larva (L3) of Strongyloides stercoralis were identified. These studies provide a basis for the purification of these proteins as diagnostic allergens for human strongyloidiasis. The Mr values of the surface and ES molecules were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, fluorography, or silver staining following the recovery of these molecules after the radiolabelling of living parasites. At least 10 surface proteins were radioiodinated extrinsically using chloroglycoluril as the catalyst for iodination, and then extracted with detergents and/or beta-mercaptoethanol. Several surface molecules of the L3 were immunogenic in humans, as determined by immunoprecipitation with sera (IHS) from infected patients. About 30 proteins were present in the ES preparation. Many ES antigens were labelled biosynthetically during the culture of larvae in media supplemented with either [35S]methionine or [14C]glucose. Furthermore, several of the surface proteins of the L3 were found with the ES antigens recoverable by culturing larvae in vitro. About 10 of the ES proteins were immunogenic as determined by immunoaffinity chromatography using IHS; and two of these antigens with Mr 50,000 and 90,000 incorporated [35S]methionine during culture of larvae. Moreover, some ES proteins were allergenic when tested in an in vitro assay of histamine release from basophils from infected humans or monkeys. The isotype of the homocytophilic antibodies involved in this immediate hypersensitivity assay, which is the basis of a diagnostic skin test for human strongyloidiasis, appears to be IgE.

Conservation of low-copy gene loci in Old World leishmanias identifies mechanisms of parasite evolution and diagnostic markers.

Genome plasticity has been hypothesized to be a driving force behind parasite speciation. We have evaluated divergence in single and low-copy genes in terms of locus organization, chromosomal localization and gene expression in Leishmania infantum, L. major, L. tropica and three widely divergent geographic isolates of L. donovani. Seventeen genes of low to moderate copy number (1-4 copies/haploid genome) were analyzed to identify restriction fragment length polymorphisms (RFLPs) providing heritable markers distinguishing Old World (OW) leishmanias. These RFLP markers were conserved in parasite isolates from primary infections demonstrating their utility as diagnostic tools. The species designations established by RFLP analysis of field isolates was confirmed by use of monoclonal antibodies. All 17 genes were present in each OW leishmania analyzed except LSIP (A45), which was absent from L. infantum. The 17 genes were found to be distributed among 9 distinct chromosomes. However, in spite of variations in chromosome karyotypes among the various OW leishmanias, individual gene probes localized to a similar sized chromosome from each isolate. These observations coupled with a molecular tree derived from RFLP data suggest that the OW leishmanias comprise a monophyletic lineage, with species associated with cutaneous disease exhibiting the greatest level of divergence. Data from this study supports previous observations that species causing cutaneous and visceral disease have diverged primarily by nucleotide substitutions. Such nucleotide divergence may not only lead to changes in protein function and antigenicity, but may also alter gene regulation programs as exemplified by the finding that the LdI-9-5 and LdE-6-1 genes were expressed only in visceralizing leishmanias.

A complement-fixing antigen from Trypanosoma cruzi grown in cell cultures.

A complement-fixing (CF) antigen was prepared from amastigotes and trypomastigotes of Trypanosoma cruzi (Ernestina strain) grown in beef embryo cell cultures. Multiple lots of the antigen, which consisted of a supernate of washed and disrupted organisms, required material from 10(6) to 10(7) total organism per ml for optimum CF activity. Antibody at dilutions up to 1:256 was demonstrable in various sera from infected animals or patients. Contaminating beef cells from infected cultures were shown to be partly responsible for crossreactions of the antigen by CF with sera from cases of cutaneous leishmaniasis in whom concomitant infection with T. cruzi could be excluded. There were no cross-reactions with syphilitic sera and the frequency of positive reactions with normal sera was very low. Some characterisitics of the antigen included stability to storage at -20 degrees C and -70 degrees C for months, inactivation at 60 degrees C and by lyophilization, and an estimated molecular size of between 50,000 and 100,000 on the basis of membrane filtration.

Comparison of cell culture with epimastigote antigens of Trypanosoma cruzi.

Various epimastigote antigens of Trypanosoma cruzi were compared with an amastigote-trypomastigote antigen from infected cell cultures by complement fixation (CF) and gel diffusion (Ouchterlony). CF results with human Chagasic sera showed that the amastigote-trypomastigote antigen was usually more sensitive than epimastigote antigens tested. In addition, cross-reactions with normal and other sera were no greater and perhaps less frequent than with crude epimastigote antigens. Specificity of the amastigote-trypomastigote antigen, however, was less than with a protein extract of epimastigotes. Gel diffusion results with human Chagasic and hyper-immunized rabbit sera indicated differences between epimastigote and amastigote-trypomastigote antigens whereas differences by CF with the same sera were equivocal.

Molecular differences between several species of Strongyloides and comparison of selected isolates of S. stercoralis using a polymerase chain reaction-linked restriction fragment length polymorphism approach.

The relationships between the parasitic nematodes of medical importance belonging to the genus Strongyloides was studied using a polymerase chain reaction (PCR)-linked restriction fragment length polymorphism approach. We used several human and dog isolates of S. stercoralis, a monkey isolate of S. fulleborni, and S. ratti, a rodent parasite. The molecular analysis was based on amplification of the internal transcribed spacer and the 5' portion of the 23S-like rRNA gene followed by restriction enzyme digests. The length of the PCR product was specific to each species and varied around 1.5 kilobase pairs. Using nine restriction enzymes, we were able to analyze both interspecific and intraspecific variations. With four restriction enzymes (Taq I, Dde I, Dra I, and Mwo I), human isolates of S. stercoralis from different parts of the world showed identical patterns and could be differentiated from the dog isolate of S. stercoralis. Interspecific differences were readily observed with these and other enzymes. In addition to providing new information on the genomic characteristics of Strongyloides parasites, the results suggest that this technique could be useful for diagnostic and epidemiologic investigations.

Comparative sensitivity and specificity of ELISA and IHA for serodiagnosis of strongyloidiasis with larval antigens.

Sera from 68 patients with parasitologically proven strongyloidiasis were tested by the ELISA and IHA tests using larval antigens prepared from Strongyloides stercoralis and Strongyloides ratti. The ELISA using the S. stercoralis antigen detected the greatest number of sero-reactors (83.8%), whereas the IHA using the S. ratti antigen detected the fewest (55.9%). In addition, the S. stercoralis antigen had higher geometric mean titers than the S. ratti antigen in both the ELISA and the IHA tests. Sera from 37 presumed normal individuals also were tested by IHA and ELISA and nonspecific reactions were seen only with the IHA test. When sera from patients with parasitic infections other than strongyloidiasis were tested, the only consistent cross-reactions were with those sera from patients who had occult filariasis and acute schistosomiasis.

Skin test antigens for immediate hypersensitivity prepared from infective larvae of Strongyloides stercoralis.

More rapid and simplified diagnostic procedures are needed for the diagnosis of strongyloidiasis. One approach is the use of an immediate hypersensitivity skin test that would reliably identify infected people. Accordingly, somatic and excretion/secretion (E/S) antigens were prepared from filariform larvae of Strongyloides stercoralis and were treated to remove possible adventitious agents. By use of a quantitative method for measurement of skin reactions, several preparations of the 2 antigens were tested in uninfected controls and in various groups of patients. Doses of 0.35 microg of E/S and 4 microg of somatic antigens elicited positive skin tests in 82-100% of infected people, depending on clinical status. A lower frequency of positive skin tests was found in strongyloidiasis patients also infected with human T-cell lymphotropic virus type 1. Cross-reactions, especially to somatic antigens, were frequently found in patients with filarial infections. Despite these limitations and the need for further study of specificity, these results provide a basis for future development of a diagnostic skin test antigen for strongyloidiasis.

Experimental disseminated strongyloidiasis in Erythrocebus patas. II. Immunology.

Parasite-specific humoral and cellular immune responses were evaluated in seven Erythrocebus patas monkeys experimentally infected with a Southeast Asian strain of Strongyloides stercoralis. Most animals developed high titers of anti-larval surface IgG antibody (as evaluated by the indirect immunofluorescence test), and all animals tested developed specific IgE antibody (as shown by the in vitro histamine release test). Modest lymphoproliferative responses to S. stercoralis antigens were demonstrated in most animals during the early phase of the infection (days 20-40), but disappeared later. Steroid treatment (prednisone, 12.5 mg/kg on alternate days) was given to three animals, but did not appear to significantly affect the immune parameters tested. The degree of the immune responses to S. stercoralis larval antigens did not correlate well with the course of the infection, and several animals died of disseminated disease in spite of demonstrable humoral and cellular responses to these antigens. We suggest therefore that other factors, such as local intestinal immune and nonimmune mechanisms may be of importance in protection against disseminated strongyloidiasis.

Diffuse cutaneous leishmaniasis in Mexico.

In Mexico, 6 cases of diffuse cutaneous leishmaniasis (DCL) were found in widely separated geographic regions. Information was also available on 2 other cases. In addition to the typical clinical features, half of the patients had evidence of nasopharyngeal mucosal involvement. All isolates from the DCL patients were identified as Leishmania mexicana mexicana by isoenzyme analysis and monoclonal antibody typing. In 1 region of Tabasco state where DCL was found, uncomplicated cutaneous leishmaniasis appeared to be highly endemic, and isolates from a few such patients were identified as L. mexicana mexicana. An incidental finding was the recovery of an isolate of L. braziliensis braziliensis from a patient with chiclero ulcer in Oaxaca state. The clinical and epidemiological significance of the reported cases are discussed.

Antibody levels to Trypanosoma cruzi in infected patients with and without evidence of chronic Chagas' disease.

Antibody levels to Trypanosoma cruzi were compared in asymptomatic individuals infected with the parasite as well as those with different forms of chronic Chagas' disease of varying severity. The following three serologic tests were used: complement fixation, direct agglutination with previous treatment of the serum with 2-mercaptoethanol, and the enzyme-linked immunosorbent assay. The clinical groups tested included individuals with (a) a positive serology but no symptoms and without evidence of chronic disease (indeterminate form); (b) mega disease (groups I, II, III, and IV); (c) cardiomyopathy (mild, moderate, and severe); and (d) those with both mega disease and cardiomyopathy (combined form). The mean enzyme-linked immunosorbent assay and complement fixation antibody levels among the various clinical groups showed no statistical differences. With the direct agglutination test patients with mega disease and those with severe cardiomyopathy had slightly higher mean titers than patients in the indeterminate group and those with mild or moderate cardiomyopathy. While there may be possible reasons for these differences, the biological relevance of the findings was concluded to be of dubious significance.

The immune response to nematode parasites: modulation of mast cell numbers and function during Strongyloides stercoralis infections in nonhuman primates.

Mucosal mast cell numbers are modulated in the intestines of rodents during parasitic infections. These mast cells can degranulate in response to worm antigens, and this event has been suggested to play a protective role for the host. To examine whether mast cells in higher animals play a role in protecting from disseminated parasitic disease, mast cell numbers and responsiveness to parasite antigens were evaluated in 5 Erythrocebus patas infected with the human intestinal nematode Strongyloides stercoralis. Initial infection and subsequent challenge infections were associated with increase in jejunal histamine and mast cell numbers, and these mast cells could release histamine in response to parasite antigens. Jejunal mast cell numbers returned to normal during a chronic phase of infection. The cells lost their ability to respond to antigenic stimulation following limited steroid treatment. Subsequent activation of chronic infections to fatal disseminated disease by more prolonged steroid treatment was associated with a marked decrease in jejunal mast cell numbers and histamine. In one animal which succumbed to severe disease without steroid treatment, jejunal mast cells were refractory to worm antigens.

Resistance to treatment in Kala-azar: speciation of isolates from northeast India.

Kala-azar in India is becoming increasingly difficult to treat, which may be due to the presence of species other than Leishmania donovani; Leishmania tropica was reported to cause the same clinical syndrome in the area. Over the past 3 years, we have collected samples from 241 patients with visceral leishmaniasis from across the region. Of the 189 isolates that grew on diphasic medium, 159 were successfully transferred to liquid medium for typing. Clinically, 80% of these were resistant to antimony. Lipophosphoglycan-specific monoclonal antibodies were used to distinguish the 2 species by agglutination of promastigotes; all 159 were shown to be L. donovani. Eighty-three isolates were confirmed to be L. donovani by isoenzyme analysis, by amplification of kinetoplast DNA, or both, in comparison with multiple reference strains; none were L. tropica. Thus, the rising incidence of clinical resistance to treatment is unlikely to be due to a different species causing kala-azar in north Bihar.

Parasite-specific humoral responses in different clinical forms of strongyloidiasis.

Total IgE, specific IgE and IgG antibodies directed against Strongyloides stercoralis antigens were measured in the serum of 8 asymptomatic individuals with parasitologically proven strongyloidiasis, 10 patients with symptomatic strongyloidiasis and 8 with a severe form of the disease. No correlation was found between any of the immunological quantities evaluated and the clinical form of strongyloidiasis. We suggest that other responses may be involved in determining clinical manifestations of this parasitosis.

A randomized trial of single- and two-dose ivermectin versus thiabendazole for treatment of strongyloidiasis.

A randomized trial is described comparing ivermectin and thiabendazole for treatment of chronic infection with Strongyloides stercoralis. Subjects received ivermectin (200 micrograms/kg) in a single dose, ivermectin (200 micrograms/kg) on 2 consecutive days, or thiabendazole (50 mg/kg/day) twice daily for 3 consecutive days. Most subjects (94%) had intermittent symptoms, including urticaria, epigastric pain, and diarrhea. Stools were examined 7 days and 1, 3, 6, 10, and 22 months after treatment. Fifty-three subjects completed at least 3 months of follow-up. Only 1 of 34 and 2 of 19 ivermectin and thiabendazole subjects, respectively, had a stool positive for larvae after treatment. Symptoms were relieved in all 3 groups and eosinophil levels returned to normal in 90% of all subjects by 12 months. Nearly 95% of thiabendazole subjects had short-term adverse effects during therapy versus only 18% of those treated with ivermectin. One dose of ivermectin provides safety and efficacy equivalent to thiabendazole with a much lower prevalence of side effects and, consequently, better compliance.