The American Journal of Tropical Medicine and Hygiene - Volume 33, Issue 3, 1984

Members of the Society, Distinguished Guests, Ladies and Gentlemen—

First, I would like to thank the Society for the privilege of being President for the past year and give special thanks to our Secretary-Treasurer, Dr. John Scanlon, for doing so much of the work necessary to the successful functioning of the Society.

I found that one of the most difficult tasks as President is choosing a topic for today's speech. I had an initial impulse to discuss field research and use my experiences in Pakistan, Southeast Asia, the Caribbean, and the eastern shore as a basis for the talk. That approach would have allowed me to give proper credit and recognition to several colleagues who have been very important in my research career. However, that approach would have been, of necessity, anecdotal and on some reflection I discarded the idea.

I also considered an in-depth analysis of a scientific issue, but I decided that no matter which subject I chose I would run an almost certain risk of boring a large segment of the audience who do not share my personal scientific interests.

Two strains of Plasmodium falciparum were tested in vitro for sensitivity to the dihydrofolate reductase inhibitor pyrimethamine, the p-aminobenzoic acid (PABA) analogue sulfadoxine, and combinations of both drugs. One strain was sensitive and one resistant to pyrimethamine in vitro. Parasites cultured in medium containing neither folic acid nor PABA were inhibited by 10-6 M sulfadoxine, a concentration well below that achievable after therapeutic dosage. Folic acid added to this medium at a physiological concentration of 0.01 mg/liter caused a 1,000-fold reduction in sulfadoxine activity; a 100-fold higher concentration of folic acid caused a 10-fold reduction in pyrimethamine activity. Sulfadoxine in a concentration of 10-7 M was able to potentiate pyrimethymine activity in PABA-free medium with no added folic acid or with 0.01 mg folic acid/liter. These data indicate that P. falciparum can utilize exogenous folic acid, and suggest that sulfadoxine may potentiate pyrimethamine activity by simultaneous inhibition of dihydrofolate reductase.

The erythrocytes of the Colombian owl monkey Aotus trivirgatus griseimembra can be used for the long-term in vitro cultivation of Plasmodium falciparum employing a modified Trager-Jensen method. Cultures are grown in HEPES-buffered RPMI-1640 using a 4% suspension of monkey erythrocytes and 10% pooled heat-inactivated human AB serum, with initial parasitemias in a range between 0.2 and 0.5%. Adaptation of new strains from human erythrocyte cultures can be performed by simply subculturing from human to owl monkey erythrocytes in a stepwise manner. When 5% human AB serum is included in cultures to support growth, as much as 5% monkey serum can be added in order to investigate serum effects, such as antibody activity against P. falciparum. The Aotus trivirgatus continuous culture system has provided a stable, consistent source of infected erythrocytes for in vitro experimentation, and the techniques developed have been used to further refine and support the animal experiments in progress.

Numerous exoerythrocytic forms of Plasmodium falciparum (PFEEF) were obtained from the liver of the South American monkey, Cebus apella, for analysis of the antigens on this stage. As antigen for the fluorescent assay, 5-µm sections of liver fragments collected on day 5 following sporozoite inoculation and fixed in Carnoy's solution or kept in liquid nitrogen were used. Two types of fluorescent labeling of the PFEEF were identified: diffuse and peripheral. Each of 23 sera from individuals with P. falciparum infection acquired naturally by mosquito bite showed the diffuse and peripheral patterns of fluorescence at low serum dilutions (i.e., 1:10–1:100), but only peripheral staining at higher serum dilutions (i.e., 1:200–1:1,600). All other polyclonal sera tested showed only the diffuse pattern of fluorescence whatever the serum dilution used; this was true for P. falciparum infections acquired accidentally by blood transfusion, heterologous human infections with P. vivax, P. ovale, P. malariae or P. cynomolgi, and experimental animal infections with P. berghei, P. gallinaceum, or P. cynomolgi. Fluorescent antibody titers on PFEEF were generally 1–4 dilutions lower than on blood stages. No age-dependent pattern of fluorescence titers was found in 30 sera from individuals ranging in age from 2–78 years living in a malaria-endemic area. Twenty-six monoclonal antibodies directed to P. falciparum blood stages which reacted at high titers with rings, schizonts, merozoites, and gametocytes did not react with PFEEF antigen even when using the undiluted ascitic fluid. Results were also negative when using a monoclonal antibody directed to P. falciparum sporozoite surfaces and one to P. flaciparum gametes. The results indicate that, in addition to antigens of broad specificity which are shared with blood stages and other malaria species, the hepatic stage of P. falciparum exhibits stage- and species-specific antigens which are located at the periphery of the schizont.

The production of two hybridoma cell lines secreting monoclonal antibodies (MAb), both of which react specifically with erythrocytic merozoites of Plasmodium yoelii in the indirect immunofluorescence assay, has been reported earlier. MAb 25.77 was reactive with a localized region within each merozoite, while MAb 25.1 appeared to be specific for the plasma membrane of schizonts and merozoites. The parasite antigens recognized by antibodies 25.77 and 25.1 are proteins of 235,000 and 230,000 molecular weight, respectively, both of which induce protective immunity against P. yoelii in mice. In order to establish the precise localization of these protective antigens within erythrocyte merozoites, ultrathin cryomicrotomy was used in conjunction with the MAb and protein A-gold. This technique showed that gold particles were exclusively concentrated over the rhoptries when erythrocytic merozoites were incubated with MAb 25.77. On the other hand, gold particles were distributed uniformly over the merozoite surface when parasites were incubated with MAb 25.1. These results demonstrate, for the first time, that a protective antigen of the erythrocytic stage of P. yoelii is localized within the rhoptries as well as on the merozoite surface.

Humoral immunity to Plasmodium berghei infection of F1 hybrid B6D2 (C57Bl/6 × DBA/2) mice was investigated using an immune serum prepared from mice which survived a lethal challenge of erythrocytic stage P. berghei because of previous vaccination with formalin-killed P. berghei. Immune serum, but not normal serum, if injected intraperitoneally or intravenously soon after infection, during rapidly increasing parasitemia, transiently inhibited the progress of infection in a pattern which was directly related to the dose and timing of serum injection. However, the level of restriction of parasitemia caused by the intravenous injection of 1 ml of immune serum could not be improved upon by subsequent additional 1-ml injections of the same serum, and parasitemia in all mice, whether delayed by immune serum or not, ultimately progressed to death. The protective capacity of the immune serum was markedly specific for plasmodial species but absorption with formalin-killed homologous parasites failed to remove the protective component. No variants of P. berghei insensitive to inhibition by immune serum were detected. The results support the view that humoral immunity to P. berghei may be mediated by more than one soluble factor and effected through at least two different pathways.

Of 16 patients with kala-azar treated with sodium stibogluconate (0.1 ml/kg body weight a day), one died on the 12th day of treatment and nine were cured by a 30-day course, although two subsequently relapsed. Extending the course cured a further five patients, and in one patient allopurinol was used in addition before a cure was achieved. Clinical and hematological recovery began within a few days of the start of treatment, but parasites continued to be seen in splenic aspirates for 3 weeks or more.

Antigens derived from the amastigote or the epimastigote stages of Trypanosoma cruzi and prepared by sonication or formalinization were examined and compared in the immunofluorescent antibody (IFA) test and enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to the parasite in sera of infected humans. The results revealed that antigens derived of amastigotes from cell cultures can be used for the detection of antibodies to T. cruzi in both tests. In the IFA test, 46.3% of the sera had higher titers with amastigote antigens, 41.5% had equal titers with antigens from both stages, and 12.2% had higher titers with epimastigote antigens. In the ELISA, 43.9% had higher titers with amastigote antigens, 43.9% had equal titers with antigens of both stages and 12.2% had higher titers with the epimastigote antigens. The differences in titers, however, were not of a magnitude sufficient to indicate a higher sensitivity for the amastigote antigens. The ELISA was performed successfully with formalinized or sonicated organisms of both stages. The use of formalinized parasites introduces a new advantage over previously reported ELISA methods. Formalinized antigens are easier to prepare and can be stored for prolonged periods of time at 4°C. The reason for the higher titers with amastigote antigens was examined by using 125I-labeled antigens to determine the binding of antigens to the plastic solid-phase used in the ELISA and to compare the antigens of each stage that could be immunoprecipitated by antibodies to T. cruzi. The ability of amastigote antigens to bind to the plastic solid phase appeared to be slightly higher than that of epimastigote antigens although the differences were not statistically significant. On the other hand, the amount of antigens in the amastigote preparation immunoprecipitated by antibodies in 6 of 10 sera examined was higher than the amount of antigens in the epimastigote preparation immunoprecipitated by antibodies in the same sera. In two sera the amount was similar, and in the remainder more antigens in the epimastigote antigen preparation were immunoprecipitated. These results are of interest and may suggest clinical implications.

The congenitally athymic nude (nu/nu) mouse was studied as a model for amebic liver disease, using Entamoeba histolytica. Despite intrahepatic inoculation of massive numbers of amebae, we could not produce sustained infections in nu/nu mice. We also failed to induce hepatic amebiasis in thymus-intact nu/+ and +/+ littermates, or in nu/+ mice pretreated with rabbit antimouse thymocyte globulin. Humoral response was measured in both the nu/nu and nu/+ mice. Nu/+ but not nu/nu animals developed a specific IgG response after challenge with E. histolytica. There were no significant IgM responses. Pretreatment with silica increased susceptibility of both nu/nu and nu/+ mice to development of liver abscesses. These studies suggest that host resistance in murine amebiasis is critically dependent upon the macrophage, but not upon T cell-mediated defenses.

Two human cases of infection with Lagochilascaris are described from Colombia. One patient was a 21 year-old woman who suffered from repeated attacks of tonsillitis and passage of worms from the nose. When a tonsillectomy was performed, numerous worms were found in the tissues. Treatment with thiabendazole and mebendazole was ineffective. She was cured after treatment with levamisole. The second patient was a 7 year-old girl who had a painful abscess on the neck that contained adult worms. She was also cured of this infection after treatment with levamisole. These are the first cases described from Colombia, and bring the total number of human cases recorded to twenty-three.

This study evaluated the efficacy and tolerance of a single oral 400-mg dose of albendazole on Necator americanus larvae, and compared its efficacy when administered between meals or with a meal. Twenty-nine healthy and hookworm-free male volunteers were exposed on the forearm to approximately 45 8-day-old N. americanus larvae. All subjects developed discrete maculopapular eruptions at the site of larval application. Following a random double-blind study design, each subject received at the end of the 6th post-infection day either the investigational drug or a placebo as follows: Group I (n = 8)-placebo; Group II (n = 11)-400 mg albendazole with a meal; Group III (n = 10)-400 mg albendazole 3 or more hours after or before a meal. On day 56 post-infection, the stools of all subjects who received placebo were positive for N. americanus eggs (by zinc sulfate flotation technique), compared with 48% positivity (10/21) in those who received albendazole (P = 0.01). By day 63 post-infection, an additional three subjects in the treatment group became positive, for an overall 62% rate of positivity (13/21), i.e., albendazole prevented patent infection in 38%. Administration of albendazole with a meal did not alter drug efficacy. In those subjects in whom patent infections were not prevented, egg output was one-fourth that of the placebo group. There was no difference in viability of eggs appearing in feces of treated and untreated subjects as judged by larval development in Harada-Mori cultures. Our data indicate that albendazole is active against pre-intestinal stages of N. americanus in human infections.

The World Health Organization's Collaborating Centre for the Histopathology of Filarial Diseases of Man contains specimens of lymph node from 13 patients infected by the filarial nematode Loa loa. Ten of these nodes have distinctive microscopic features characterized by distended sinuses that contain histiocytes and eosinophils and by atrophy of lymphoid follicles. Less striking features include fibrosis of capsule and trabeculae, dilated lymphatic vessels of capsule and medulla, and inflammatory cell infiltrates. We believe that these changes, although in themselves nonspecific, are characteristic of lymphadenitis caused by Loa loa. These 10 lymph nodes were removed from the inguinal region — one from each of 10 native Zairians. At the time of herniorrhaphy the nodes in eight patients were found to be enlarged and were removed for diagnosis. Seven of the 10 patients were infected with Dipetalonerna perstans as well as Loa loa, and one of these seven had three filarial infections — L. loa, D. perstans and D. streptocerca. Lymph nodes from other patients infected by other filariae that were available for study at the AFIP did not have these histopathologic features.

A survey for the prevalence of onchocerciasis was conducted on selected divisions of the Firestone Rubber Plantation, Harbel, Liberia, in November 1980. Divisions 19, 22, 23, and 36 are located in the north-central portion of the plantation, and were selected to complement transmission studies which were performed there in 1979 and 1980. Biopsy of the calf, iliac crest, and shoulder was done on a total of 583 individuals. No lateral difference in microfilarial density was observed. A prevalence of 80.8% was found. Prevalence appeared to be equal between the sexes, and to plateau above 35 years of age. Of those presenting positive skin biopsies, 20.2% had palpable onchocercomata. The geometric mean wet weight microfilarial density/mg of skin for the population sampled was 19. Onchocerciasis is considered hyperendemic in the Harbel area. Further investigations are warranted to define its dimensions in relation to clinical sequelae on the plantation.

Simulium quadrivittatum Loew (Diptera: Simuliidae), a man-biting black fly, was shown, for the first time, to be capable of supporting development of Onchocerca volvulus Leuckart (Nematoda: Filarioidea) from microfilariae to third-stage (infective) larvae. The black flies were collected in Chiriqui Province, Panama and transported alive to Guatemala, where they were allowed to feed on a human subject infected with O. volvulus. Samples of these flies were dissected over an 11-day period to assess morphogenesis of the parasite. Vigorously motile microfilariae were recovered from the mid-gut during the first 24 hours postfeeding; second-stage larvae were found in the thoracic musculature on day 4; and fully developed third-stage larvae were obtained from the cephalic capsule by day 10. This rate of larval development is similar to that observed in Guatemalan S. ochraceum. Onchocerciasis is not known to occur in Panama. The results of the present study direct attention to a potential public health hazard there and possibly elsewhere in Central America.

The intake and development of Onchocerca volvulus in Simulium pintoi from the Parima mountain region of the Federal Territory of Amazonas in Venezuela, were studied experimentally. When wild females fed on the lower half of the legs and lower third of the back of an onchocerciasis patient harboring 23 and 264 microfilariae per skin snip, respectively, at each site, an average (median) of 14 (range, 1–77) and 245 (range, 58–495) microfilariae was ingested. However, within 24 hours of microfilarial ingestion a mortality of 47% (16/34 flies) was observed in the group of flies which fed on the back, as compared with 2% (2/101 flies) in the other group which fed on the legs. At a temperature varying between 16°C and 24°C, the development of O. volvulus larvae in S. pintoi was synchronous and orderly; no abnormal nor deformed larvae were observed. Third-stage larvae were first seen in the head of flies dying between 8 and 9 days after microfilarial ingestion, and 98 of 100 larvae recovered from days 10–16 were in the third stage. The proportions of females harboring third-stage larvae among flies which lived through day 8 in the two groups which fed on the legs and back, respectively, were 55% (21/38 flies) and 63% (5/8 flies). Although only two of five positive flies in the latter group contained third-stage larvae in the head (1 and 12, respectively), 71% (15/21 positive flies) of the former group had an average of 2.7 third-stage larvae in the head (range, 1–10). In conclusion, it is suggested that S. pintoi is an efficient vector of O. volvulus due to its high susceptibility, in spite of the high mortality caused by an excessive intake of microfilariae.

A monoclonal antibody to surface antigens of Brugia malayi microfilariae promotes the adherence of peripheral blood cells to microfilariae in vitro and reduces microfilaremia in vivo. The antibody reacts with epitopes present on two stage-specific antigens with estimated molecular weights of 70,000 and 75,000 daltons.

Circulating Dirofilaria immitis antigen was detected in sera from 24 of 24 infected dogs by counterimmunoelectrophoresis (CIE). Parasite antigen was not detected in sera from uninfected dogs or dogs with Dipetalonema reconditum infection. In experimentally infected dogs, the antigen was first detectable 6.5–8.5 months after infection. Preliminary evidence suggests that the antigen is present in male and female adult worms but not in microfilariae. Sera from dogs with microfilaremic and amicrofilaremic infections contained statistically equivalent amounts of D. immitis antigen. However, a significant correlation was observed between serum parasite antigen content and the number of adult worms present in individual dogs at necropsy. Previous studies from several laboratories have shown that microfilarial counts and serum antibody titers are not related to adult worm counts in canine dirofilariasis or other filarial infections. Thus, CIE detection of D. immitis antigenemia represents a significant improvement over previously available diagnostic techniques because it is more sensitive than microfilarial tests, more specific than antibody tests, and the only test that has been related to infection intensity.

Fatal disseminated disease was induced in ten patas monkeys infected with two Southeast Asian strains of Strongyloides stercoralis. While some animals died within 6 weeks after infection, others controlled their infections until placed on high doses of corticosteroids. Larvae were first noted in the stools 11–20 days after transcutaneous exposure to filariform larvae. Daily larval counts tended to increase as the infections progressed, but the number of larvae in the stool was not predictive of whether a monkey would control his infection or succumb to fatal disease. Hyperinfection was confirmed in the six monkeys in which counts were made of the adult female parasites in the duodenum at postmortem, as well as by pathologic findings in all animals. Clinical signs of disease were vague until dyspnea induced by terminal pulmonary hemorrhage occurred. Eosinophilia and/or basophilia were noted intermittently in some infections. Severe necrotizing duodenitis, colitis, and pulmonary hemorrhage were the most conspicuous postmortem findings. Hyperinfection has been predictably induced in a cercopithecoid monkey for the first time; a species which may lend itself to further investigations into the pathogenesis of disseminated strongyloidiasis.

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.

Natural killer (NK) activity was assayed in peripheral blood mononuclear cells of patients with schistosomiasis, of patients following treatment, and of uninfected control subjects. The patient populations were from villages in the Qalyub Province, Egypt and around Belo Horizonte, Brazil. NK activity was assayed by the cytotoxicity of 51Crlabelled K562 target tumor cells. In neither infected population were significant alterations from normal levels found in the percent cytotoxicity per 106 cells, or in the lytic units that expressed 25% cytotoxicity. Likewise, prior treatment (2 and 6 months previously) did not alter the group NK activity detected. Similarly, in the Egyptian study there was no difference in the percentage of large granular lymphocytes between the infected and uninfected groups. In parallel studies in Egyptian and Brazilian schistosomiasis patients we did not find any evidence that this chronic infection consistently altered circulating NK activity.