The American Journal of Tropical Medicine and Hygiene - Volume 31, Issue 4, July 1982

Mr. Chairman, Dr. Weller, dear members of Dr. Soper's family, ladies and gentlemen, at the outset I should like to say how extremely honored I am to be asked to give this brief talk on a subject with which I have had some practical experience in my country, where my story takes place.

It is my understanding that the invitation to deliver this lecture reflects your interest in hearing the testimony of a Brazilian on some lessons learned as a result of Fred Lowe Soper's long years of work in my country. On that ground, I will leave aside any further comments of his worldwide brilliant career following his leaving Brazil.

It would be perfectly justifiable for some of the young scientists attending this meeting to question whether my timing is opportune in discussing an antique but memorable public health achievement: the consecutive eradication of Aedes aegypti and Anopheles gambiae in Brazil.

Ca++ was shown to be indispensable for the normal growth of cultures of Plasmodium falciparum. Inclusion of ethyleneglycolbis (β-amino-ethylether) N,N′-tetra-acetic acid (EGTA) caused blocking of the asexual cell cycle of the parasite in two sites, the first blockage occurring between 20 and 26 hours after invasion of the erythrocyte. It proved to be irreversible by additions of Mg++ or Ca++, and to lead to morphologically abnormal parasites arrested in the mature trophozoite stage of the cycle. The second site of inhibition was probably one of the steps in the process of invasion of the erythrocyte by the merozoite. When 1 mM EGTA was added 24–30 hours after the culture was synchronized the cell cycle of the parasite continued without any interference in the normal maturation until the development of schizonts and release of merozoites into the medium. However, reinvasion of fresh erythrocytes by these merozoites was impeded. The inhibition of reinvasion caused by EGTA was overcome by the addition of an excess of Ca++ but not by an excess of Mg++. After the addition of Ca++ to cultures blocked just before the invasion phase as schizonts, the merozoites were again rendered fully infective and the rate of invasion was similar to that in an untreated control culture. Implications of the effects of Ca++ depletion on the asexual cell cycle and possible applications of EGTA as a reversible inhibitor of the invasion process are discussed.

The T1-resistant oligonucleotides of the ribosomal RNAs from four isolates of Plasmodium falciparum, the Southeast Asian strain FCR-1/Vietnam, the West African strains FCR-3/Gambia and FCR-8/West Africa, and an isolate from Honduras, were analyzed by two-dimensional polyacrylamide gel electrophoresis. Comparison of the largest 33–35 oligonucleotides indicated that 30 were common to all four of the isolates examined, with each isolate giving a characteristic pattern. Similar analyses were undertaken with cloned preparations of the FCR-3/Gambia isolate to determine if variation within a population of parasites could be detected. The patterns obtained for the five clones examined were identical.

Macrophages from the peritoneal cavities of normal mice or mice previously immunized with epimastigotes of Trypanosoma cruzi were incubated with sera from normal or immunized mice and then infected with T. cruzi epimastigotes or trypomastigotes. After 2 hours of incubation the specimens were prepared for scanning electron microscopy. Macrophages from immunized mice were observed to be qualitatively and quantitatively more effective in binding either parasite form than were macrophages from normal mice. Preincubation with specific antibody appeared to enhance parasite binding in all cases, and extracellular destruction of both epimastigote and trypomastigote forms was noted in these preparations. While extensive destruction of epimastigotes was seen with either normal or immune macrophages pre-incubated with antibody, lysis of trypomastigotes was typically seen only in the presence of both immune macrophages and specific antibody.

Female hamsters, infected intracardially (i.c.) with 1.0–2.0 × 105 amastigotes of Leishmania donovani produced offspring, following mating, which, when immunized subcutaneously with 1.0 × 107 amastigotes at 8 weeks of age, were more resistant to i.c. challenge 6 weeks later than were hamsters born to non-infected mothers. Offspring of mothers infected with as many as 6.0 × 106 amastigotes demonstrated no greater capacity for immunization than did those of mothers infected with 1.0 × 105 amastigotes. Sensitization of offspring of infected mothers apparently is transplacental since the effect could only be seen in offspring of infected mothers and not in those of normal mothers weaned by infected dams. Offspring of female hamsters immunized by footpad inoculation of 1.0 × 107 amastigotes exhibited reduced spleen parasite burdens when challenged at 8.5 weeks of age and reduced spleen and liver parasite burdens when challenged at 16 weeks of age, compared to offspring of non-immunized hamsters; this effect was not noted following challenge of offspring of the two groups within 1 day of weaning. The passage of parasites from mother to young during gestation and/or nursing apparently does not occur since, at 125 days of age, no parasites were observed in spleen or liver impression smears of offspring of hamsters infected i.c. with 6.0 × 106 amastigotes while parasites were seen in such smears of hamsters infected, 120 days previously, with 10 amastigotes i.c. Thus, sensitization to immunization in offspring of infected hamsters and to challenge of offspring of immunized hamsters is apparently transplacental, effected either by soluble leishmanial antigen, soluble lymphocyte (or transfer) factor, or by cells.

The potential feasibility of using exogenously administered human interferon for the treatment of selected cases of leishmaniasis prompted us to study the effects of murine interferon on the course of Leishmania tropica infection in C57Bl/6 mice. L cell-derived mouse interferon was administered daily by intraperitoneal (1,000 and 10,000 U) or intralesional (100 U) injection in mice inoculated into footpads with L. tropica amastigotes. Footpad swelling and tissue parasite density were assessed over the course of infection. Interferon treatment did not significantly affect these clinical and parasitological parameters. Furthermore, addition of interferon (100–100,000 U) to cultures of amastigote-infected mouse peritoneal macrophages or to axenic cultures of promastigotes did not affect replication. We conclude that interferon lacks intrinsic antileishmanial activity and does not significantly enhance host defense against Leishmania.

A 56-year-old woman with acquired, common variable immunodeficiency was found to have persistent gastrointestinal as well as pulmonary infection with Strongyloides stercoralis. Repeated courses of treatment with thiabendazole led to marked reduction or loss of Strongyloides stercoralis larvae, but cessation of treatment always led to recurrence of Strongyloides infection. Several small bowel biopsies showed normal villous architecture and little inflammatory response to presence of larvae. Interestingly, no definite symptomatology could be attributed to the Strongyloides infection. It was postulated that the lack of signs and symptoms of strongyloidiasis, as well as poor response to treatment, was related to the immunodeficiency state. With low-dose, long-term interrupted courses of thiabendazole treatment, the Strongyloides infection finally seemed to be cured.

Ferrets inoculated subcutaneously with 150–200 infective larvae of Brugia malayi (subperiodic strain) usually developed patent infection during the 3rd month post inoculation. Microfilaremia was transient, and most animals became amicrofilaremic after the 6th month of infection. Ferrets developed a persistent eosinophilia at the time of patency. At necropsy, 5–8 months post infection, adult worms were recovered principally from lymphatic vessels and recovery ranged from 0.5–13% of the inoculated larvae. The inflammatory response of ferrets to microfilariae was characterized by nodules 1–5 mm in diameter in the liver, lungs, spleen, and submucosa of the gastrointestinal tract. The center of these lesions contained a degenerated microfilaria or the cast of a microfilaria embedded in Splendore-Hoeppli substance. The Splendore-Hoeppli substance was surrounded by eosinophils and/or foreign body giant cells. Identical lesions were observed in ferrets experimentally infected with Brugia pahangi. Sera from ferrets infected with B. malayi demonstrated a 3- to 5-fold increase in IgG by the 4th month of infection and these sera produced 2–3 precipitin bands in double gel diffusion assays with an extract of B. malayi microfilarial antigen. Skin tests with B. malayi microfilarial antigen showed that the majority of the infected ferrets had immediate hypersensitivity responses, but none had Arthus or delayed hypersensitivity responses.

A pilot experiment using NaPCP as a molluscicide to control schistosomiasis japonica was initiated in 1950–51 in Nagatoishi-cho, Kurume, Japan, where 1,050 persons lived behind dikes built to prevent flooding from the Chikugo River. They farmed 60 ha of rice, and 72.9% of them were infected with schistosomiasis. At the end of 2 years, 99.5% of the snails (Oncomelania h. nosophora) were controlled and new cases dropped from 30–35 per year to 5 and 0 at the end of the 1st and 2nd year, respectively. Mollusciciding was continued by Japanese, and paddy irrigation ditches were lined with concrete by 1958. Land reclamation, involving conversion of cultivated land in the river bottom to a golf course, resulted in virtual elimination of infected snails from this area and, when found, snails were uninfected. Using immunologic tests (intradermal, complement fixation, and circumoval precipitation) plus multiple stool examinations in 1972 and 1978, it was possible to demonstrate a marked decrease in cases of schistosomiasis in children 15 years of age or younger. These results were significantly different (P < 0.001) from those found in similar tests run in 1940 and 1966. By 1978 all children in the Nagatoishi area of Kurume, Japan, were negative for Schistosoma japonicum infection.

To further investigate factors responsible for the recently documented changes in schistosomiasis patterns in the Nile Delta, questionnaire-derived information on antischistosomal drug usage was obtained from a 25% systematic sample of 609 residents of a stable village in the south-central Delta. Ten percent of the population had received antischistosomal drugs during the previous 4 years. Most of the drugs administered were injectable compounds, and 92% of individuals receiving them failed to complete the treatment regimen. Additional sources of information from a village physician, a pharmacist and a major pharmaceutical corporation confirmed and expanded the survey findings, indicating that antischistosomal drug usage cannot explain the recently observed changing patterns of human schistosome infections in the Nile Delta region.

Oltipraz was administered orally to 62 hospitalized male Sudanese infected with Schistosoma mansoni. The patients were split into two equal groups; one group received a total dose of 25 mg/kg body weight, the other group received 35 mg/kg. Half of the total dose was given with breakfast, the second half with supper. In general, the drug was well tolerated although some vomiting was observed 3–5 hours after the second half-dose. Blood chemistry and hematology remained normal 24 hours after administration of oltipraz. The cure rate was above 95% for both groups at 1, 3, and 6 months after treatment. Stratification of patients by eggs/g feces clearly indicated that the drug was equally efficacious for patients excreting high, medium or low numbers of eggs. Our results indicate that further trials will be necessary with lower doses of oltipraz in order to determine its antischistosomal potency.

Necropsy was performed in 32 cases of schistosomal cor pulmonale, and the lungs were studied by means of conventional histologic techniques, by serial sectioning, and by the method of plastic vascular casting. Fibrin deposition and marked endothelial cell hyperplasia were the main early changes evoked by schistosome eggs within small arteries and arterioles. A complex fibrin thrombus organization and revascularization, followed by congestion and dilatation of focal blood vessels, resulted in plexiform lesions. Vascular casts showed them to be purely arterial, to have a glomeruloid appearance, and to originate laterally or terminally in small arteries.

A serum was prepared by immunizing rabbits with circumoval precipitin (COP) immune complexes isolated by Protein A Sepharose affinity chromatography. This serum, positive in the COP test, was used to isolate by antibody affinity chromatography the COP antigens contained in crude schistosome egg antigens (SEA). The COP antigens were then applied to the enzyme-linked immunosorbent assay (ELISA) and compared to SEA. In a rabbit immunized repeatedly with the COP immune complexes the reactivity in the ELISA to both SEA and the COP antigens increased with time. In a primary infection of S. mansoni the reactivity to both antigen preparations rose in parallel; however, SEA showed higher reactivity. Interestingly, with the COP antigens a dramatic increase in antibody response was obtained in the serum of the 4th week in two separate groups of infected mice. This is probably a reflection of the common antigens between worms and eggs since two of the three major antibodies to the COP antigens identified previously are absorbable with lyophilized S. mansoni worms.

Peripheral blood mononuclear cells (PBMC) from five of six baboons (Papio cynocephalus) with chronic schistosomiasis mansoni showed a marked reduction in the ability to generate anti-sheep erythrocyte (SRBC) plaque-forming cells (PFC) after primary in vitro immunization as compared to the PFC responses of PBMC from normal (non-infected) baboons. Removal of the plastic-adherent (PLAD) cells from the PBMC of these animals results in a population of cells capable of responding to in vitro immunization with SRBC at a level equal to or higher than their normal counterparts. Reconstitution of plastic-non-adherent cells with PLAD cells re-establishes suppression. In contrast, the single apparently non-suppressed infected baboon showed reduced responses after PLAD cell removal, but, upon reconstitution with PLAD cells, responses were higher than those obtained before cell separation. No evidence of PLAD suppressor cells in normal animals was found, and indeed the data suggest that PBMC from normal animals may require a PLAD accessory cell for full responsiveness.

Stool surveys were conducted on children 2–10 years of age in 27 villages within Dacca District and around this index area (1,668 children were sampled), revealing an endemic focus of Fasciolopsis buski infection to the south and the east of Dacca District. In order to determine the seasonal variation in the total snail populations and the natural rate of F. buski infection in the snails, two species of planorbid snails, Segmentina (Trochorbis) trochoideus and Hippeutis (Helicorbis) umbilicalis, were periodically sampled for 12 months from a village endemic for F. buski infection. Gymnocephalous cercariae were found in S. (T.) trochoideus snails during August, September and October. The size of the snail population (n = 1,275) was significantly correlated with inches of rainfall (r = +0.62; P < 0.05) and water temperature (r = +0.59; P < 0.05). The natural infection rate of F. buski in the snails ranged from 0.5–2%. Snails from non-endemic areas were exposed to 3–10 miracidia. A total of 13 of 49 (27%) of H. (H.) umbilicalis and 6 of 14 (43%) of S. (T.) trochoideus had gymnocephalous cercariae present 4 to 6 weeks after exposure to miracidia. Thus, snail strain variation is unlikely to be a barrier to F. buski transmission.

14C-praziquantel penetrates the cyst wall of Cysticercus fasciolaris and kills the cysticercus within the cyst, although the uptake of praziquantel by the encysted larva was slower than by an isolated one. This fact is in good agreement with earlier in vitro chemotherapeutic studies. Scanning and transmission electron microscopic studies have shown that praziquantel causes a marked destruction of the tegument along the whole pseudostrobila and the scolex of C. fasciolaris. The type of tegumental damage is identical to that produced in adult tapeworms and trematodes.

Thirty-five cases of cysticercosis, including 31 of neurocysticercosis, three subcutaneous and one ocular case, were studied and treated with praziquantel (PZQ). The three subcutaneous cases were cured within 2–3 months and the ocular case did not improve. In 31 patients with neurocysticercosis the diagnosis was made by specialized technics, including computerized tomography (CT) and hemagglutination test (HA). Four main clinical syndromes were found: epilepsy in 14, intracraneal hypertension (ICH) in seven, a combination of the two syndromes in seven and headache in three. Fourteen of these patients underwent surgery and in 11 of them cysts were removed. PZQ treatment was given to these patients to treat remaining cysts. Seventeen patients without prior surgery were treated with the drug. Among 29 cases with follow-up the following clinical results were observed: ICH and headache were cured in 13 of 15 patients, and in two cases there was improvement, epilepsy was under control in 14 of 19 cases receiving antiepileptic drugs; in four patients the seizures were still present, although less frequent and in one patient there was no improvement of the epileptic syndrome. Only one patient died. CT revealed cysticerci in different stages of development in each of 15 patients, as shown by the presence of two or more different patterns in all of them. All calcified lesions remained unchanged. In 11 cases that presented with non-calcified lesions and had CT before and after therapy, the lesions disappeared in seven, cysts were reduced in number or in size in two, and in the remaining two cases no changes were observed. HA was positive at significant titers of 1:128 or higher in 21 cases of the neurological disease (67%). The patients presenting with ICH showed in general higher titers. In 16 cases serum HA was done before and after treatment with the following results: in six the titers decreased, in three they remained unchanged, in four there were variations up and down, and in three cases the titers were raised. Blood and urine examinations before and after treatment did not reveal significant changes. Side effects were observed in 10 of 35 patients treated; symptoms were transient and of low intensity in five of them, consisting of headache, nausea and vomiting that did not require treatment. In the other five patients symptoms were more intense due to increase of the ICH, and parenteral steroids, and in one case mannitol, were required. It is concluded that praziquantel is effective in the treatment of cysticercosis, a disease that previously did not have a specific medical treatment. The mortality rate in this series of 31 cases of neurocysticercosis treated with PZQ was only 3.2%, which contrasts with previous 50–80% case fatality rate in the same Colombian institution.

Severe staphylococcal pneumonia developed secondary to pyomyositis of the buttock in an adult male Melanesian in East New Britain, Papua New Guinea. Penicillin-resistant Staphylococcus aureus was cultured from the abscess, and S. aureus, together with Gram-negative organisms, were cultured from sputum. Despite treatment with high dose cloxacillin together with supportive chemotherapy, progressive deterioration of lung function occurred and acute respiratory failure developed. As suitable ventilators were unavailable, tracheal intubation was performed, and manual assisted ventilation was carried out for 4 days. Progressive improvement in ventilation and general status resulted, and the patient recovered without sequelae.

In a retrospective study undertaken to document the frequency of thrombocytopenia in cases of leptospirosis, 18 of 32 patients (56.3%) had a platelet count of 100 × 103/mm3 or less. Renal failure occurred in 72.2% of thrombocytopenic patients and in 21.4% of patients with normal platelet counts. The association of thrombocytopenia and renal failure was significant (P < 0.02). The common occurrence of thrombocytopenia and its association with acute renal failure in patients with leptospirosis has not previously been reported.