The American Journal of Tropical Medicine and Hygiene - Volume 35, Issue 2, 1986

I am happy to be on the podium at this time. I have been asked to introduce to you a friend of mine.

He is a man I have known for forty-odd years. He went to the same college that I had attended a few years earlier. I recognize that this is the converse of the statement that we went to different schools together, but it indicates that he was influenced by the same teacher, Dr. George S. Tulloch, who was responsible for pointing a number of his students towards parasitology and vector biology. We met briefly when he took his first job with the USPHS. Later on, we worked in the same Laboratory of Tropical Diseases (later the Laboratory of Parasitic Diseases) of the NIH—and so on, and on, and on ….

This man, who is endowed with the names of two saints, has been professing Biology at the University of Notre Dame for the last sixteen years.

Being President of the Society during the past year has been an enriching experience and I thank you for the privilege and opportunity to serve. My special thanks go to Dr. John Scanlon who shoulders the work of the Society so effectively.

The last time I addressed the entire Society was in this city in 1972 at a joint meeting with the American Society of Parasitologists. I discussed the relationship of government, parasitology and tropical medicine and ended with an assessment of funding for research for these fields. In reviewing the history of extramural funding by the National Institutes of Health for filariasis research it was interesting to note that in 1965 only two grants existed for the total sum of $15,150. Seven years later, largely due to the efforts of the U.S.-Japan Cooperative Medical Science Program, the number of funded grants had increased to eleven for a total sum of $314,975.

A method consisting of filtration of up to 100 macerated mosquitoes in a batch, followed by fixation with glutaraldehyde and concentration of filtrate by centrifugation has been developed to rapidly assess malaria infection in anopheline mosquitoes. Determination of the presence of sporozoites is made by observation of a sample of the final filtrate with a phase microscope. The method is simple and field adaptable, essential factors for the application of any technique to large scale field operations. Application of the technique in El Gancho, Chiapas, Mexico, in February 1984 yielded an infection rate for Anopheles albimanus of 0.9% for intradomicile-collected human bait mosquitoes and 0.1% for peridomicile-collected human bait mosquitoes.

In 1982, 2 of 14 Plasmodium falciparum infections acquired in East Africa and diagnosed in Copenhagen were resistant to treatment with sulfadoxine plus pyrimethamine (Fansidar®), while in 1983, 6 of 18 were so. The in vivo tests were supplemented by determinations of drug concentrations in serum, and 4 isolates from in vivo-sensitive cases and 6 from in vivo-resistant cases were selected for in vivo tests. These were performed in ordinary RPMI 1640 medium and in a medium with physiological p-aminobenzoic acid and folic acid concentrations. Pharmacokinetic aberrations were found to be of possible importance in only 2 of the in vivo-resistant cases. In vitro susceptibility to sulfadoxine was found to be uniformly low in all isolates. Testing with a combination of sulfadoxine and pyrimethamine in the medium with physiological concentrations of cofactors probably reflects the in vivo situation most accurately, but in all but 1 of the isolates studied in vitro the in vivo susceptibility to Fansidar® would be predicted by in vitro susceptibility to pyrimethamine in either medium. The concentration of p-aminobenzoic acid in serum, quantitated by high performance liquid chromatography, was found to be subject to wide variation, and this may have implications for in vitro testing.

Three different regimens were compared for treatment of falciparum malaria in displaced Kampucheans living in encampments on the Thai-Kampuchean border in 1983: single dose 750 mg mefloquine, 1.5 g sulfadoxine, 75 mg pyrimethamine (MSP); 600 mg quinine 8-hourly for 3 days and 500 mg tetracycline 8-hourly for 7 days (Q3T7); or 600 mg quinine 8-hourly for 7 days and 500 mg tetracycline 8-hourly for 7 days (Q7T7).

Radical cure rates were 98% (40/41) for MSP, 76% (32/42) for Q3T7 and 92% (33/36) for Q7T7. The criterion for treatment failure was reappearance of parasites by 35 days after commencement of treatment or no parasite clearance. Treatment failures comprised one case of reduction but no clearance of parasites (RII resistance) for MSP, 10 recrudescences (RI) for Q3T7 and 3 recrudescences (RI) for Q7T7. The radical cure rate for Q3T7 was significantly lower than that for MSP (P < 0.01), whilst Q7T7 did not differ significantly from the other groups.

Parasite clearance time was shorter (2.4 days) with MSP than with Q3T7 (3.5 days) and Q7T7 (3.3 days). There was little difference in side effects between the regimens, and tolerance was good.

The MSP and Q7T7 regimens are both effective for treatment, but the single dose of MSP is much easier to manage than 7 days of quinine and tetracycline.

Plasmodium vivax and Plasmodium ovale schizont-infected erythrocytes were separated from peripheral blood by centrifugation using discontinuous Percoll (colloidal silica) gradients. Infected Aotus monkey or chimpanzee blood was diluted and placed on a discontinuous gradient containing 30%, 40%, 45%, and 50% Percoll (v/v in media) layers before centrifugation at 1,450 × g. Parasitized erythrocytes were concentrated to greater than 95% schizont-infected cells in two bands that contained an average of one leukocyte per 500 infected cells. Mononuclear cells and trophozoites were isolated in another band and noninfected red cells, ring-infected cells, and granulocytes were pelleted to the bottom. The yield of parasitized erythrocytes ranged from 50% to close to 100% of the estimated number of infected cells in the original whole blood. Use of this Percoll procedure results in a high yield of concentrated parasitized erythrocytes and separation of these cells from host white blood cells.

The results of a double-blind serological study of 15 sera sampled in a residual focus of vivax malaria transmission in Algeria, and of 7 sera from patients with slideproven P. vivax infections acquired in India, are analyzed. The reactivity of each of these serum samples was tested by indirect immunofluorescence using 6 different batches of antigen, including 3 batches of P. vivax antigen prepared with isolates from Zaire (Africa), India and the Solomon Islands, respectively.

The geometric mean of reciprocal titers (GMRT) calculated on the 7 sera from proven vivax infections fell from 289.8 using the homologous antigen from the same geographic origin (India) to 48.7 using a homologous (vivax) antigen originating from a different continent (Africa). Among the 15 samples from Algeria, the percentage of seropositives decreased from 100% using the homologous P. vivax antigen originating from the same continent (Africa) to 53.3% using a homologous antigen from India. Two aspects are included in the discussion: in seroepidemiological studies, sensitivity could be improved by the use of a homologous antigen from the same geographic origin; in detection of clinical cases of malaria and species identification based on serology, our results stress the need for caution in interpreting serological titers and for taking into account the geographic origin of the isolates used as antigen.

The effects of chloroquine, amodiaquine and pyrimethamine-sulfadoxine (SP) (Fansidar) on the infection rate and density of Plasmodium flaciparum gametocytes were studied in 198 patients with falciparum malaria from an area in the Punjab where malaria is endemic but seasonally transmitted. One month following treatment of 100 patients, SP had reduced the gametocyte carrier rate from 37% to 6% and the mean gametocyte density from 80 to 1.4 per mm3 of blood. Chloroquine and amodiaquine were much less effective. Since SP has no gametocytocidal properties and the reduction in gametocytes coincided with clearance of asexual parasitemias, gametocytes were probably reduced subsequent to the cure of the asexual malaria infections. If used during the nontransmission season, SP might be an effective component of an integrated program for reducing malaria transmission in the Punjab and other areas where 4-aminoquinoline-resistant and SP-sensitive falciparum malaria exists.

In a continuing reexamination of plasmodial tissue stages within the context of the hypnozoite theory of malarial relapse, 2 strains of Plasmodium vivax with distinct and disparate relapse characteristics in humans were studied in chimpanzees. Following intravenous inoculation of massive numbers of salivary gland sporozoites, both the frequently relapsing Chesson strain and a North Korean strain characterized by predominantly delayed relapses exhibited relapse patterns and antimalarial sensitivity in the splenectomized chimpanzee essentially indistinguishable from those seen in humans. Examination of hepatic biopsies obtained at 7 and 10 days after infection revealed both pre-erythrocytic (pre-e) schizonts and hypnozoites in tissue obtained from the animal infected with the Chesson strain, but only rare hypnozoites (no pre-e schizonts) at 7 days in the animal infected with the North Korean strain. These findings, combined with the comparability of relapse behavior—which indicates the suitability of the chimpanzee as a model for the natural (human) host-parasite relationship—are essentially as predicted by the hypnozoite theory, despite the small numbers of tissue forms seen. Pre-erythrocytic schizogony of the Chesson strain in the liver was essentially indistinguishable from that of other strains studied, also underlining the suitability of this model system for tissue stage studies of P. vivax.

A North Korean strain of Plasmodium vivax invaded HepG2-A16 cells, and differentiated mostly into persisting nondividing parasites.

Because salivary function and blood location are impaired in sporozoite-infected mosquitoes, we determined whether such pathology also could lead to an increased biting rate. For 5 days, we compared relative daily biting rates of Plasmodium gallinaceum sporozoite-infected mosquitoes (Aedes aegypti) and noninfected mosquitoes with an olfactometer. Mosquitoes then were exposed for 5 min to an anesthetized guinea pig. Infected mosquitoes exhibited a significant increase in olfactometer response which was also reflected in a decreased egg output. We conclude that if duration of contact with a host is limited, then infected mosquitoes may make more attempts at probing before being successful, and thus enhance transmission.

The synthetic imidazole, itraconazole, was examined for in vitro and in vivo activity against Trypanosoma cruzi. Mice treated with concentrations as low as 15 mg itraconazole/kg/day were completely protected against death due to infection with any of three different and highly virulent strains of T. cruzi. Treatment of infected mice with 120 mg itraconazole/kg/day for seven to nine weeks apparently resulted in the parasitologic cure as determined by negative hemocultures and subinoculations, negative serology for T. cruzi, and absence of parasites in histologic sections following completion of therapy. Peak serum levels of itraconazole after treatment of mice with the dose of the drug that provided protection against death were less than 1 µg/ml. Experiments in vitro revealed that concentrations of itraconazole as little as 0.001 µg/ml inhibited replication of intracellular amastigotes in macrophages. These results indicate that itraconazole has a remarkable activity against T. cruzi. Further investigation of intraconazole as a therapeutic agent for Chagas' disease may be warranted.

Examination of blood films for trypanosomes in primates housed at the Tulane University Delta Regional Primate Research Center showed that 47% (32/68) of the Bolivian Saimiri sciureus and 51% (68/135) of Brazilian Saguinus mistax harbored one or more types: Trypanosoma (Schizotrypanum) cruzi in 6% to 7% and Trypanosoma (Herpetosoma) spp. or Trypanosoma (Megatrypanum) spp. in 39% and 45%, respectively. Trypanosomes were isolated from a sample of the infected monkeys and morphobiological studies were carried out. Both T. cruzi and Trypanosoma rangeli were demonstrated. The transmission of T. rangeli by Rhodnius prolixus, Rhodnius neglectus, and Triatoma infestans was assessed: only R. prolixus transmitted the infection by bite. T. rangeli has not been found previously in Bolivia, nor has any trypanosome been reported previously in Saguinus mistax.

Sera from 32 patients who became ill after jungle combat training were tested for antibodies to Toxoplasma gondii using the indirect immunofluorescence test. Swift rises of both IgG and IgM antibodies occurred within 2 weeks of infection. Reduction in IgM titers, due to competitive suppression by IgG antibody, occurred in most but not all cases. Suppression of IgM reaction by IgG antibody could be prevented by adsorption of serum with Staphylococcus aureus containing protein A. Antibody of the IgM class could be detected at ≥ 1:256 level in many sera at 6-month and 1-year intervals after exposure. In groups with exposures such as were experienced in this study, the presence of IgM antibody titers in single serum specimens cannot be used to indicate recent exposure. Both IgG and IgM antibody may rise together to high levels very rapidly after infection; IgM did not precede IgG antibody in our 32 subjects.

A recent approach to the chemotherapy of visceral leishmaniasis has been the encapsulation of clinical agents within macrophage directed carriers such as liposomes. Because in mammals Leishmania are obligate intramacrophage microorganisms, injection of an encapsulated drug should deliver large quantities of drug to the organisms, thus decreasing both the number of drug administrations needed for cure and drug toxicity. Drugs contained within red cell ghosts have been used clinically to treat other macrophage disorders. We encapsulated the clinical antileishmanial agent pentamidine within human red cell ghosts and administered it to hamsters infected with Leishmania donovani. The ED50 and ED90 of single injections of this preparation were 231–240 times lower than that of the positive control drug, sodium stibogluconate (Pentostam), and essentially all parasites could be eliminated by 2.5 mg encapsulated drugs/kg. Against splenic parasites, the ED50 was 195 times lower than that of antimony, although only 80% of parasites were eliminated by the highest doses of encapsulated drug (2.5–6.4 mg/kg). The difference in liver vs. splenic parasite killing is probably related to the greater uptake of encapsulated drug by the liver (11–14 µg/g tissue) vs. the spleen (2–3 µg/g). If activity in this model is comparable to activity in humans, these results suggest that a single injection of a preparation consisting of ghosts of a patient's own red cells and the amount of pentamidine in one standard dosage (4 mg/kg) would eliminate 80%-100% of L. donovani from the spleen and liver.

Sera from individuals in an area of Haiti endemic for Mansonella ozzardi were analyzed for reactivity to antigens of Brugia pahangi, Dirofilaria immitis, Mansonella llewellyni or Ascaris lumbricoides using either an enzyme-linked immunosorbent assay or an indirect immunofluorescent antibody test. IgM and IgG reactivity to all antigens was observed with sera from both microfilaremic and amicrofilaremic individuals when compared to reactivity of sera from individuals from nonendemic areas. Antibody reactivity to B. pahangi was greater than that to other antigens. IgG reactivity of sera from endemic patients to filarial antigens was consistently greater than that of IgM. Antibody reactivity was not correlated with age or microfilarial density.

Analysis of human serum reactivities to antigenic components of soluble Taenia solium metacestode proteins showed the predominant presence of determinants shared by T. solium, Echinococcus multilocularis and E. granulosus. Two polypeptides were demonstrated by SDS-PAGE and Western blot or enzyme-linked immunoelectrotransfer blot (EITB) assay to bind serum and CSF antibodies only from T. solium cysticercosis patients. These species-specific antigenic polypeptides focused between pH 4.6 and 3.9 after resolution by isoelectric focusing followed by EITB. The high species-specificity demonstrated by the present techniques offers the opportunity to confirm serologically an infection by T. solium metacestode.

More than 796 Ascaris lumbricoides worms weighing 550 g were recovered at autopsy from a 2-year-old black South African girl. Most of the worms were taken from necrotic small intestine, but worms were also in the stomach, esophagus, intrahepatic and extrahepatic bile ducts, and gallbladder. The worms had caused torsion and gangrene of the ileum, which was interpreted as the cause of death. Worms were formalin-fixed and individually weighed. There were 796 intact worms and 112 appreciably large (>0.2 g) fragments of worms. Statistical analysis of the weights revealed 2 distinct populations of worms: 16 large worms (0.5–2.3 g) and 778 small worms (0.03–0.95 g). The difference in weight between these 2 groups of worms was significant (male and female worms treated separately; P < 0.05 to P < 0.001). These observations reveal that the patient acquired a massive and fatal infection with A. lumbricoides while hosting a relatively light worm burden.

From January to April 1984, 63 Rattus rattus and 40 R. norvegicus were trapped in northeastern Puerto Rico and examined for Angiostrongylus cantonensis adults. Nineteen (47.5%) of the R. norvegicus and 10 (15.9%) of the R. rattus were infected, giving an overall infection rate of 28.2%. Four species of terrestrial snails and one species of brown slug were examined for A. cantonensis larvae. Two snail species, Subulina octona and Aquebana belutina, were found infected with third stage larvae of A. cantonensis. These larvae were harvested and inoculated per os into adult white mice. Immature adult worms were found in the brain tissue of all mice inoculated. This is the first report of A. cantonensis in the rat and snail populations of Puerto Rico.

In order to examine the effect of metrifonate, 156 patients with mixed Schistosoma haematobium and mansoni infection were randomly divided into three groups and treated with metrifonate (twice 10 mg/kg body weight), oxamniquine (60 mg/kg) and praziquantel (40 mg/kg), respectively. The output of S. haematobium and S. mansoni ova were quantitatively assessed in urine and stool. Application of metrifonate resulted in a similar reduction of S. haematobium and S. mansoni eggs in the urine, whereas no effect on egg excretion was observed in the stool irrespective of the parasite species. In contrast, oxamniquine influenced the output of S. mansoni ova in stool and urine, but showed no effect on S. haematobium egg excretion. Praziquantel was equally effective against both parasite species. The chemotherapeutic effects were not of transient nature since the number of ova of both parasite species remained unchanged five months after treatment. The results clearly indicate that metrifonate acted exclusively on adult worms located in the perivesical plexus irrespective of the parasite species.