The American Journal of Tropical Medicine and Hygiene - Volume 15, Issue 6, 1966

Total lipid extracts of Plasmodium lophurae and P. berghei were separated into lipid classes by thin-layer chromatography (TLC). Phospholipids, free fatty acids, triglycerides, and sterol esters were scraped from TLC plates, transmethylated, and the component fatty acids of each fraction were separated by gas-liquid chromatography.

P. berghei generally has ratios of saturated to unsaturated acids similar to or higher than P. lophurae. This fact and the results obtained from analyses of the sterol ester fractions may be due to uptake of host lipids.

A total of 27 fatty acids were observed in the various fractions assayed from the two organisms. Both organisms have large percentages of the fatty acids accounted for by the groups with carbon chain lengths of 16 and 18 in all fractions except the sterol esters. Fatty acids with longer carbon chain lengths predominate this fraction.

Levels of reduced and oxidized pyridine nucleotides have been determined for the avian malaria parasite, Plasmodium lophurae, and its host cell the duck erythrocyte. During infection the levels of DPN, TPN and DPNH increase 1.5- to 2-fold, whereas the TPNH remains at a level similar to that of the uninfected red blood cell. The TPNH, DPNH and TPN content of erythrocyte-free P. lophurae is much like that of the normal duck erythrocyte, but the parasite DPN level is 1.5 times greater. These changes in the levels of pyridine nucleotides suggest that they may function in the regulation of metabolic pathways in malaria parasites.

Turkey erythrocytes infected with Plasmodium fallax were incubated with C14-phosphatides, -acetate, and -fatty acids in the presence and absence of chloroquine. The C14-labeled lipids were fractionated on silicic acid columns and by paper chromatography, and the radioactivity for each fraction was assayed. The drug depressed uptake of acetate into fatty acids and phospholipids in both malaria-infected and normal erythrocytes. C14-stearic and palmitic acid incorporation into phospholipids of infected erythrocytes was increased by chloroquine while uptake of C14-oleic acid was depressed by the drug. Incorporation of C14-sodium acetate into fatty acids and phospholipids of turkey heart muscle homogenates was inhibited by the drug, and uptake of C14-stearic acid into phospholipids was stimulated by chloroquine.

The evaluation of sulfadiazine, sulfamethoxypyridazine (Midicel®) and sulphorthomidine (Fanzil®) alone or combined with pyrimethamine against drug-resistant strains of Plasmodium falciparum is reported. The results indicate that (1) combination of a sulfonamide with pyrimethamine enhanced the curative effect of either one alone, (2) when combined with pyrimethamine, Midicel and Fanzil were superior to sulfadiazine, and (3) of the regimens tested, 1.0 g Fanzil as a single oral dose given together with 50 mg pyrimethamine showed the most promise for the successful treatment of the multi-drug-resistant falciparum malarias. Factors limiting the usefulness of sulfonamides as antimalarials are discussed.

The evaluation of quinine sulfate and diaminodiphenyl sulfone (DDS) as suppressants against sporozoite-induced Chesson strain Plasmodium vivax is reported. The results indicate that quinine sulfate, 7.5 grams daily, is inadequate to offer complete protection in that 3 of 4 volunteers developed patent infections. DDS, when given in daily doses of 25 mg, failed to protect 5 of 5 volunteers.

The repository antimalarial cycloguanil pamoate, known as CI-501 (Camolar®) was administered to 173 subjects living on a malarious island near Rabaul, New Guinea. It was given as a deep intragluteal injection at a dosage of 9.3 to 11.0 mg per kg body weight to children and at a total dose of 350 mg to subjects weighing over 32 kg-Concurrently, a single oral dose of a 4-aminoquinoline (amodiaquine) was given on the basis of 600 mg base for adults and proportionately less for children.

The drug was well accepted by the community and there were no observed systemic side-effects. Many subjects showed some local injection site reactions but these were mostly mild in nature and subsided quickly.

Asexual forms of both Plasmodium falciparum and P. vivax disappeared within 72 hours of drug administration. High attendance was maintained at the monthly follow-up blood surveys. Patent infections of P. vivax first reappeared on day 90, but P. falciparum did not reappear until 150 days after treatment.

The entomological observations, as well as the blood surveys conducted on the untreated observation group, showed that transmission of both species occurred throughout the study period.

The results from this area indicate that the combined administration of cycloguanil pamoate and a 4-aminoquinoline provided better protection against P. falciparum than against P. vivax. The duration of protection afforded by CI-501 against P. falciparum makes it conceivable that this species could be eliminated from certain isolated communities provided all the inhabitants receive the drug.

Three groups of 60 schoolchildren living in a hyperendemic zone in East Africa, where pyrimethamine-resistant Plasmodium falciparum is prevalent, were injected with a single dose of the following repository antimalarial agents:

cycloguanil pamoate, at a mean dosage of 10.8 mg (base)/kg body weight;

4,4′ - diacetyldiaminodiphenylsulfone (DADDS), at a mean dosage of 6.9 mg (base)/kg body weight;

a combination of equal parts of these drugs, at a mean dosage of 12.9 mg (base)/kg body weight.

All three preparations were effective in clearing patent infections, but DADDS acted more slowly than the other preparations. Each preparation had repository action but DADDS acted for the shortest period of time. There were indications that a 1:1 combination of cycloguanil pamoate and DADDS acted longer than cycloguanil pamoate and was well tolerated. Most of the patients given the combination remained relatively parasite free for four months.

Immunoelectrophoresis was carried out with eleven amebic antigens and three antiamebic sera in order to determine the antigenic moiety of these strains and to describe the antigenic relationships between the various strains. Antigens were prepared from amebae grown in MS-F medium, washed in saline, concentrated and lyophilized. Nitrogen determinations were carried out and each antigen was titered to determine the optimum concentration for immunoelectrophoretic (IEP) analysis. Antisera were prepared in rabbits. Results indicate that strains JS and Laredo have similar IEP patterns, as do strains NRS and 200, Huff and Entamoeba invadens, K9 and JH, and F22 and BH.

Considering the mobilities of the proteins and the similarities of the antigen-antibody arcs, the strains of amebae with similar IEP patterns have several factors in common. The pathogenic strains showed similarities not shared by the non-pathogens and E. invadens. E. invadens was more closely related to Huff which also shares a cultural characteristic, namely growth at room temperature. The strains isolated from extraintestinal infection were similar and those isolated from cases of amebic colitis show similar patterns. Immunoelectrophoretic analysis of these amebic strains demonstrate that immunologic differences do occur.

Drug sensitivities of several strains of Entamoeba histolytica grown at 37°C and strain Laredo and E. invadens grown at room temperature in the CLG medium were compared. Strain Laredo and E. invadens were similar in response in that they were more resistant to 3 or 4 drugs tested than were strains which were propagated at 37°C. In this respect and the fact that it no longer propagates at 37°C in our laboratory the Laredo strain is more like E. invadens than like E. histolytica. We have previously shown that the same is true for its free amino acid content. One recently isolated strain of E. histolytica (D1) grown at 37°C was, in general, more resistant than were several strains which have been grown in synthetic medium for years in this laboratory. This supports the idea that the testing of recent isolates is important in studies of this kind, since such isolates have not been subjected to any selective pressures that culture factors may exert.

One drug, paromomycin was equally effective at low concentrations against all strains of Entamoeba tested. It is suggested that testing of drugs at both 37°C and room temperature may be useful as a preliminary screening device for drugs to be used in the treatment of amebiasis, since effectiveness at both temperature extremes might indicate that a drug has a “broad spectrum” of activity against Entamoeba.

Bacteroides sp. grown for 24 hours in the CLG medium releases a considerable quantity of ammonia into the medium. Such cultures containing bacterial cells which are rendered non-multiplying by penicillin are used in our laboratory for the routine propagation of Entamoeba histolytica. Further experiments revealed that the quantity of ammonia in such cultures does not show detectable increases or decreases during the period of propagation of amebae. It is suggested that these levels of ammonia produced during the period of bacterial growth might be one of the factors responsible for the prevention of luxurious growth in cultures of amebae in which Bacteroides is the associate organism.

Germfree guinea pigs monocontaminated with each of seven species of bacteria were inoculated intracecally with the strain 200 Entamoeba histolytica from ameba-trypanosome cultures. Amebic lesions occurred with each bacterial species tested but lesions developed more rapidly and extensively with some bacterial symbionts than with others. All animals with Bacillus subtilis succumbed to amebic enteritis as did varying percentages of animals with Clostridium perfringens, Staphylococcus aureus, Streptococcus faecalis, and Lactobacillus acidophilus. None of the amebic infections proved fatal in animals with Escherichia coli and a species of Micrococcus during the period of observation. The inflammatory response varied remarkably from an acute reaction with B. subtilis and S. aureus to a typically chronic reaction in germfree animals. The ameba and other bacterial symbionts elicited a basically chronic type of reaction with various degrees of superimposed bacterial inflammation.

The course of an infection with the BEV strain of Toxoplasma gondii was observed in splenectomized mice, and in mice treated with cortisone and 6-MP—procedures known to modify or suppress the normal immune response. It was observed that the pathogenesis of the disease was greatly altered in the experimental animals, being distinguished by signs of severe neurological involvement, and by the eventual death of most of the mice from a disseminated toxoplasmic meningoencephalitis. Signs of the disease were ruffled fur, shuddering, weakness of limbs, diarrhea, head-tilting, choreiform movements, arched spine and sinuous rolling motions, posterior paraplegia, emaciation, coma, and death. Also observed were persistence of trophozoites in peritoneal exudate, earlier appearance and greater numbers of cysts in the brain, development of many cysts in large clusters, and widespread inflammation of the brain and meninges.

Challenge of the survivors of the four groups of experimental mice with the virulent RH strain after 1 month of primary infection indicated that immunity had developed to a lesser degree in the treated mice. It is hypothesized that as a direct consequence of the immuno-suppression, there was continuous parasite proliferation in tissues and hematogenous transport of trophozoites to the brain.

A portable clinical centrifuge has been modified so that it can be used as a continuous-flow centrifuge. An adapter fitted to the head of the instrument delivers the test liquid through four flexible tubes inserted into centrifuge shields used as collecting vessels. The waste liquid overflows the vessels and is discharged through a drain in the centrifuge bowl.

Cercariae of Schistosoma mansoni were successfully recovered alive from 1 to 20 liters of water. The centrifuge is suitable for field studies and, since a large proportion of the cercariae was recovered from test runs, the instrument may be useful for study of natural cercaria populations.

The centrifuge may also be useful for concentration of other microorganisms from water and so may be of value to oceanographers and limnologists.

The decline in infectivity of Schistosoma mansoni cercariae was studied under controlled laboratory conditions considered favorable for cercarial survival. Cercariae of known age were tested by mouse exposure challenge at intervals up to 30 hours from the time they were shed from snails. Infectivity decreased with time and the decrease was rapid. About eight hours after the cercariae were shed, one-half the infectivity had been lost. Since there was no detected loss of infectivity in the first three hours, the time for infectivity to fall to one-half of the maximum level was less than five hours. Infectivity was about one-tenth the initial level after 14–15 hours.

Since conditions for cercarial survival in the experiments were relatively favorable, it can be expected that under field conditions, which are almost always more rigorous, the cercarial half-life is probably only a few hours even in the absence of predators.

Review of the comparative pathology of the Hoeppli phenomenon shows that it is correlated with substantial egg loads, appears during the later part of the acute primary state of infection, arises around mature eggs, occurs selectively in organs with large egg loads and affects a fraction of eggs not exceeding 10 percent at any time.

Immunofluorescent studies in Mastomys coucha show that the Hoeppli corona contains both egg antigen and fixed host globulin, and is therefore an antigen-antibody complex. Within the complex, there is a gradient of antigen-antibody concentration, with antibody excess in the periphery. The precipitate pursues a sequence of maturation and degradation concomitant with the evolution of the pseudotubercle, during which additional host and parasite components may be incorporated into it. The characteristics of the Hoeppli phenomenon in three schistosome species, its relationship to the in vitro circumoval precipitate, and to stellate precipitates in other granulomatous diseases are discussed, and it is postulated that the Hoeppli phenomenon is an auxiliary mechanism of antigen sequestration invoked by the heavily infected host when antigen production against certain egg fractions attains a critical concentration and ratio resulting in precipitation of an antigen-antibody complex in vivo.

The “aerobic” and “facultative” sewage stabilization ponds, according to the laboratory findings, are not likely to suppress the hatching of the eggs or significantly affect the survival of miracidia of Schistosoma mansoni or the vector snails. The results indicate, however, that the deep, heavily loaded, anaerobic pond will curtail the hatching of the eggs, significantly affect the survival of the miracidia, and discourage the survival of vector snails. Under tropical conditions the inclusion of a preliminary anaerobic chamber in the stabilization pond design should effectively remove the eggs and miracidia of S. mansoni.

In support of the above conclusion, the following experimental results are summarized:

The viability of S. mansoni eggs harvested from livers of infected mice does not extend beyond the second day in sewage or sewage stabilization ponds of any classification.

All waters devoid or nearly devoid of dissolved oxygen were found to inhibit hatching and reduce the viability of the eggs.

Environmental factors in raw sewage other than the dissolved oxygen content inhibited the hatching of eggs.

Eggs remaining in the bottom sludge of sewage stabilization ponds for more than nine hours cannot be induced to hatch.

The maximum and mean survival times of miracidia in the “anaerobic pond” water were 6 and 2 hours, while in the “aerobic pond” water they were 10 and 4 hours.

A number of monophasic and diphasic media, with different gas phases and pH ranges, were used in the cultivation of Mesocestoides tetrathyridia in vitro. Growth and multiplication were evaluated by determining increases in the number of organisms and increases in protein after different periods of culture. In diphasic media containing whole human blood, asexual multiplication in vitro occurred consistently at a rate comparable to that in the mouse host. Good growth and some multiplication was obtained in medium containing human plasma.

Six cases of accidentally diagnosed cystic calcification of the liver were studied. Four of the patients were Colombian and the other two, an Argentinian and a German, had lived in Colombia for years. The calcifications were all very similar and in endemic areas of hydatid disease would have been considered to be calcified hydatids. Five of the patients were alive and in good health but one of the Colombian patients died of a carcinoma of the prostate. A non-specific calcified cyst containing no hydatid elements was found at autopsy.

It is believed that the non-Colombian patients may have calcified hydatid cysts (dead in one case and alive in the other) because of the endemicity of this disease in their countries of origin, their history, and the results of the intradermal and serological tests for hydatidosis. On the other hand, it is thought that the Colombian patients may have calcified abscesses, possibly amebic, because of the pathological findings in one of them, a history compatible with poorly treated amebic liver abscess in two of them (one still having a positive hemagglutination test for amebiasis), and the negative results of the serological study for hydatid disease. Furthermore, in Colombia Echinococcus granulosus hydatid disease has not been recognized but amebic liver abscess is fairly common.

Clinical and biochemical findings on 921 persons harboring Opisthorchis viverrini were compared with similar data from 191 persons free of infection, all examinees being from northeastern Thailand. Edema, anemia, diarrhea, dysentery, nausea, flatulence, malaise, anorexia and hepatomegaly were found no more frequently in the infected than in the non-infected group. No significant differences were noted in WBC (neutrophil or lymphocyte) counts, or in hematocrit or hemoglobin. There were no significant differences in any of the following liver function tests: total protein, albumin, globulin, zinc turbidity, bilirubin, cholesterol and esters, thymol turbidity, alkaline phosphatase or the transaminases (SGOT and SGPT). Statistically significant higher abnormal results were correlated with infection by O. viverrini only in the cases of dizziness, slight jaundice and eosinophilia in the central-count range. These data indicate that infection with O. viverrini in northeastern Thailand does not elicit specific symptoms or conditions by which infected individuals may be differentiated from non-infected controls living under identical conditions.

Sensitivity to injected (exogenous) antigens was determined at various intervals after primary and challenging infections with Trichinella spiralis. Infected mice showed marked reactions (including congestion of the small intestine) after intravenous injections of homologous but not heterologous antigens. Sensitivity to specific antigen was demonstrated before the immune elimination of intestinal worms (from both primary and challenging infections) began and reached its peak after most of these adult worms had been eliminated. Injections of cortisone inhibited both immunity to infection and reactivity to exogenous antigen.

Host sensitivity to exogenous antigens has been associated with the interval of immune elimination of intestinal worms. It is suggested that some of the intestinal pathology associated with worm elimination may represent host allergic responses to endogenous worm antigens, antigens naturally released during the course of infection.