The American Journal of Tropical Medicine and Hygiene - Volume 32, Issue 4, July 1983

A case of falciparum malaria accidentally acquired in the laboratory is reported here. The infection apparently resulted from sporozoite transmission by mosquitoes which were infected with Plasmodium falciparum gametocytes produced in vitro.

We have previously reported the antimalarial activity of imidazoles and amphotericin B against chloroquine-resistant Plasmodium falciparum. We now report the enhancement of imidazole activity in an atmosphere with 17–18% oxygen (the candle jar) vs. 3% or 0.3% oxygen. Based on both morphologic and radiometric testing, smaller amounts of the imidazoles were required to inhibit parasite growth by 50% in the candle jar vs. 3% or 0.3% oxygen. The use of older (more oxidant-sensitive) red cells also enhanced the antimalarial activity of ketoconazole. Neither increased concentrations of oxygen nor the use of older red cells affected the activity of amphotericin B. These results suggest that the imidazoles may exert their antimalarial effect by increasing the oxidant stress on the red cell-parasite complex.

Three red blood cell membrane fractions (Band 3, macromolecules exhibiting I antigenic determinants, and a delipidated glycoprotein fraction) were separated from red blood cell membranes and tested for their ability to inhibit penetration of red blood cells by Plasmodium falciparum merozoites in an in vitro inhibition assay. The delipidated glycoprotein fraction (containing the major sialoglycoproteins and devoid of Band 3) was the only fraction that inhibited merozoite invasion. This fraction showed 73% and 70% inhibition at 1 mg/ml and 500 µg/ml, respectively, and slight inhibition below these levels.

Rabbits were immunized with exoantigens from the spent medium of Plasmodium falciparum cultures, and the resultant immunologic responses were studied by indirect fluorescent antibody (IFA), hemagglutination (HA), and two-dimensional crossed immunoelectrophoretic (IEP) techniques. By crossed IEP, three parasite antigens, identified and characterized as proteins lacking lipid and carbohydrate moieties, reacted with rabbit antiserum and human immune serum. Pre-immunization sera of the rabbits used in these experiments had 1:80 IFA titers against P. falciparum schizonts which were then boosted eightfold by immunization with parasite exoantigens, in contrast to IFA titers of <1:2 for coccidia- and Pasteurella-free rabbits. Experimental infections of coccidia- and Pasteurella-free rabbits with rabbit Eimeria spp. resulted in 1:80 anti-P. falciparum IFA titers, suggesting cross-reactivity of coccidial and plasmodial antigens. Post-immunization sera demonstrated extremely high HA titers against human erythrocytes, underscoring the potency of human blood components containing parasite antigens prepared from cultures. These results suggest that coccidia- and Pasteurella-free rabbits may be useful in the analysis of antigen of cultured P. falciparum.

Plasmodium falciparum-parasitized erythrocytes obtained from continuous in vitro cultures were fractionated over metrizamide density gradients. Late developmental stage schizonts were isolated from uninfected erythrocytes and other intracellular blood stage forms (rings and trophozoites) by centrifugation through 15% metrizamide. Schizonts comprised, on the average, 85% of the total number of cells recovered from the top fraction of the gradient with the remaining cells being predominantly uninfected erythrocytes. The mean percentage of schizonts recovered was 27% relative to the estimated number of mature forms present before fractionation. The concentrated schizonts were viable based upon their ability to complete schizogony and undergo a new cycle of reinvasion and schizogony in vitro. In addition, in these short term synchronous cultures the newly infected cells were able to incorporate radiolabeled hypoxanthine. This function was inhibited by the addition of chloroquine to these 45- to 46-hour cultures. Metrizamide-purified schizonts should be useful for further study and characterization of the unique metabolic, biochemical, and immunological properties of the malaria parasite.

When inoculated with sporozoites of Plasmodium berghei, a line of hepatoma cells (HepG2-A16) derived from human liver supports the complete asexual developmental cycle of the exoerythrocytic stage. Parasites were shown to resemble parasites in vivo in hepatocytes. Subinoculation of merozoites into mice induced a red blood cell infection.

The attachment and entry of Plasmodium berghei sporozoites to cultured human lung fibroblast (WI38) or hepatoma (HepG2-A16) cells in vitro has been visualized using an immunoperoxidase technique coupled with light microscopy. Attachment and entry was substantially more frequent with HepG2-A16 cells, and appeared to be mediated by the Pb44 sporozoite surface protective antigen. When sporozoites were incubated with intact monoclonal antibodies to Pb44 or their monovalent Fab fragments, attachment was inhibited, suggesting that this technique may be an in vitro assay of protective immunity. Sporozoites appeared to enter cells actively, rather than by cell phagocytosis. Once within a membrane-bound vacuole, the sporozoite transformed into a trophozoite. It was suggested that Pb44 recognized a specific cell receptor, and that this technique may permit receptor characterization.

The course of Plasmodium inui and Babesia microti infections was studied in seven splenectomized squirrel monkeys (Saimiri sciureus) of Guyanan or Bolivian origin. Three of the monkeys were infected with P. inui either by the inoculations of parasitized blood or by the bite of infected mosquitoes. The remaining four monkeys were infected by the inoculation of parasitized blood, containing P. inui and B. microti in three and with B. microti only in one. The infection in all seven animals was severe, terminating fatally.

Five isolates of Leishmania tropica from southwest Iran were studied to identify correlates among human disease, animal infectivity, and surface biochemistry. Clinical patterns of the disease in humans differed. One striking strain, LT-249, produced a small dry lesion which did not heal during four years of observation. Infectivity of these L. tropica for mice was correlated with lectin agglutination patterns and interaction with macrophages. There was also a significant difference among the five isolates regarding infectivity for BALB/c mice; isolate LT-249 was not infective whereas all the others were. All isolates agglutinated with Concanavalin A (Con A), Ricinus communis and soybean agglutinin but not with four other lectins listed. However, Leishmania isolate LT-249 showed much poorer agglutination with all lectins than did the other four isolates. Two isolates were selected for detailed study of attachment to macrophages, one, LT-249, which was not infective and one, LT-252, which was infective for BALB/c mice. The number of promastigotes which attached to macrophages in vitro was the same, but the mechanism of attachment differed since only the LT-252 bound predominately by Con A-mannose receptor interaction. These results indicate a correlation among animal infectivity, lectin agglutination, and promastigote-macrophage attachment. In particular one isolate of L. tropica which caused prolonged infection in humans was not infective in BALB/c mice, showed poor agglutination with lectins, and bound to macrophages by a different mechanism than did other isolates from the same region.

Biochemical data as enzyme profiles which were obtained by cellulose acetate electrophoresis (CAE) are reported on 44 Leishmania isolates. These enzyme profiles contain data from 25 enzyme systems. Calculations from the CAE data on average polymorphism indicated that Leishmania species/types or groups can be expected to be about 25% polymorphic, which suggests that isolate pairs which have profiles about 75% or more identical should be considered samples from the same species/type, and isolates that are significantly less than 75% identical are therefore samples from different species/types. There were five major groupings of isolates according to enzyme profiles, which were for the most part consistent with groupings of the genus based on other criteria: braziliensis, mexicana, donovani, tropica and hertigi profiles. Within these groups there were natural subgroups of isolates among which there was 75% or more allozyme or allomorph (genetic) identity. The braziliensis profile group had two subgroups: panamensis and braziliensis or guayanensis, and the mexicana profile group had three subgroups: mexicana, amazonensis and peruviana. There was an indication that an L. d. infantum isolate might be different from the other L. donovani isolates, and that the L. tropica isolates could be samples from more than one group. The data reported here are consistent with previously reported CAE data, but suggest that isozyme analysis of Leishmania isolates leading to identification should be based on data from many enzyme systems rather than just a few.

The antigenic constitution of two Trypanosoma cruzi strains and six cloned stocks was determined immunoelectrophoretically. Five to seven antigens common to all of the T. cruzi stocks were found. However, each stock also contained “strain-specific antigens,” and five of the six cloned stocks presented “clone-specific antigens.” These data demonstrate that a T. cruzi strain is composed of an antigenically heterogeneous population of organisms.

This double-blind trial was initiated to test, in a controlled study, the value of single-day drug treatment of amebic liver abscess. Patients were treated with 2 g either of ornidazole or tinidazole, two newly developed 5-nitroimidazole derivatives, given in two equal doses during the course of 1 day. A success rate of over 94% in the 72 patients from both treatment groups who were followed up for 6 months demonstrates the efficacy of the single-day drug treatment.

The antibody patterns—obtained by enzyme immunoassay (EIA), complement fixation (CF), indirect hemagglutination (IHA), and latex agglutination (LA)—of 216 patients with amebic liver abscess (ALA) were related to the time of onset of illness and to epidemiological data. The usual period between the stay in an Entamoeba histolytica-endemic area and clinical manifestation of the disease was 8–20 weeks. Increasing antibody concentration affected the sensitivity rates for CF and LA in the first 3 weeks of clinical ALA. In comparison with CF, IHA, and LA, EIA was the most sensitive test, showed the most substantial changes in antibody concentration according to the stage of disease, and measured persisting antibodies for the longest period. Neither the duration of previous stay in tropical countries nor the time when specific chemotherapy was started influenced development or persistence of antibodies in ALA patients. ALA occurred with similar frequency regardless of whether the patient was a long-term resident of the tropics or a short-term visitor to an E. histolytica-endemic area.

A procedure for visualizing and quantifying motility of Entamoeba histolytica by migration under agarose is described. Agarose suspended in tissue culture medium 199 supplemented with bovine albumin was poured into plastic dishes and allowed to harden. Six pairs of wells were cut out in a circular configuration. To the inner wells a suspension of E. histolytica in Eagle's Medium (24 × 106 cells/ml) was added, and to the outer wells a chemoattractant or the control medium. After overnight incubation at 37°C, the amebae were fixed and stained. The chemotactic and spontaneous migrations were measured in an enlarging projector. Escherichia coli filtrates, suspensions of intact and lysed erythrocytes, and the complement factor C5a acted as good chemoattractants. Both the random and chemotactic motility were correlated to the time of the incubation. Cytochalasin B effected a dose-related inhibition of both chemotactic and random migration, while colchicine caused a decrease of the chemotaxis only. The reproducibility of the method, measured by 10 intra-assay tests, was good. Thus, the described method can be useful for comparative determinations of the motility of different ameba populations. Furthermore, different factors affecting the motility of amebae can be studied.

In March 1981, a 32-year-old male naturalist involved with collecting mammals for study was found to have an unexpected eosinophilia. Serum parasitic screening done at the Centers for Disease Control showed filarial titer by indirect hemagglutination of 1:1,024 and of 1:40 by bentonite flocculation. His travel history disclosed 3 months spent in Cameroon in 1978. He also gave a history of intermittent arm swelling for at least a year. Peripheral blood, collected repeatedly at mid-day and midnight and tested for microfilariae by the Knott technique, was negative. He was begun empirically on diethylcarbamazine, and on day 11 of treatment he removed a worm, identified as a male Loa loa, from his leg. He completed treatment without difficulty and has done well.

To determine the rate of appearance of new nodules during a specified period, a follow-up study on onchocercomas that had been removed was carried out in San Vicente Pacaya, Guatemala, between June 1976 and April 1977. Incidence rate, or rate of nodules appearing in initially negative subjects, was studied in relation to the degree of endemicity. Rates in a period of 7–8 months in high, medium, and low endemic areas were 0.231, 0.083, and 0.022, respectively. The rate of nodule appearance in persons nodulectomized, or rate of nodules appearing in subjects who were surgically rendered negative during the first survey, was 45.3%, 36.4%, and 4.5% in those areas. The highest rate of nodule appearance was observed in patients with microfilariae in both the skin and nodules, followed by patients with either microfilariae or nodules. In highly endemic areas, new nodules appeared in 13.2% of persons without microfilariae or nodules, while in low endemic areas even patients with nodules earlier were less likely to have developed new ones. The performance of the nodulectomy teams (brigadas) was evaluated by confirming the worms in nodules. By gross examination alone, about 6% of nodules removed by brigadas did not contain worms. Nodules containing worms were examined for microfilariae to clarify their role as a source of microfilariae. Microfilariae were not detected in some of these nodules, especially in small ones. In contrast, microfilariae emerged from 80% of those larger than 10 mm. Number of worms per nodule, their sex, and the fecundity of female worms were examined by a collagenase technique. The average number of worms per nodule was 0.6 males and 1.2 females. The highest burden in any one nodule was seven worms, four females and three males. Most nodules had one female and either one male worm or none. Intrauterine microfilariae were found in most females from nodules shared with a male, while solitary females were not gravid. Solitary females were likely to be found in small nodules and large nodules were more likely to include both sexes.

An enzyme-linked immunosorbent assay (ELISA) using a crude soluble antigen from adult Onchocerca volvulus was developed and used as a serodiagnostic test for Guatemalan onchocerciasis. A total of 456 sera from residents in an area with endemic onchocerciasis were tested, and results were compared with previous findings by the indirect fluorescent antibody (IFA) test on the same population. Control “normal” sera from 50 U.S. residents were also tested, as were 144 sera from persons who lived in an area not endemic for onchocerciasis but who had a variety of other helminthiases. None of the “normal” sera were ELISA-positive at a serum dilution of 1:160; however, sera from persons with other intestinal helminths showed cross-reactivity in 17.4% of the individuals tested. A total of 84.4% of the individuals from the endemic area had ELISA endpoints of 1:160 or greater. Only 2.6% of the individuals who were shown to be infected by the presence of microfilariae in skin snips had ELISA titers of less than 1:160. Upon partial separation of proteins of O. volvulus by isoelectric focusing, four of the six major protein peaks were found in the acidic range (pH 3.1 to 6.8) while two were in the alkaline range (pH 9.55 to 10.9). Four major antibody reactive peaks were observed, all of which were in the acidic range. In these findings the ELISA test proved to be more sensitive for the detection of antibodies than the IFA test and could be employed in the future as a valuable tool for seroepidemiologic studies. In addition, the use of partially separated antigenic components may improve the specificity of the test.

Antileishmanial activity of 8-aminoquinolines with substitutions on the quinoline ring or on the 8-amino side-chain was assessed in the Leishmania tropica-infected human macrophage in vitro model of leishmaniasis. The 4-methyl-5,6-dimethoxy compounds were more active than 6-methoxy compounds, which were approximately as active as 4-methyl-6-methoxy compounds. Certain 6-hydroxy compounds were the most active drugs tested. The precise composition of substituents on the 8-amino side-chain had little effect on activity. These investigations identify WR 226292 (a 4-methyl-5,6-dimethoxy compound) and WR 6881 and WR 49577 (6-hydroxy compounds) as the most active 8-aminoquinolines in this in vitro model. The 6-hydroxy compounds also can be considered to be the 6-demethyl derivatives of 6-methoxy-8-aminoquinolines. This study therefore indicates that 6-demethyl-8-aminoquinolines may be generally more active in vitro than the parent 6-methoxy structures against macrophage-contained Leishmania.

Nodule examination, the Mazzotti test (a provocative challenge with diethylcarbamazine), and the enzyme-linked immunosorbent assay (ELISA) were compared for diagnostic efficacy in children who were born in an area endemic for onchocerciasis in Guatemala and were skin-biopsy negative for microfilaria of Onchocerca volvulus. Four groups of children under 16 years of age were formed based upon the possible combinations of nodule positivity or negativity and ELISA positivity or negativity. Only those children with a positive (⩾1:160) ELISA titer had a positive Mazzotti test (9 of 20 tested), regardless of the presence or absence of nodules. These results suggest that the current ELISA serology is highly sensitive for low density infections, and should be considered in surveys for the incidence of new Onchocerca infections.

A survey of 1,551 inhabitants in six villages of West Irian was undertaken to determine the presence and distribution of Wuchereria bancrofti and other filariae. Results confirmed the presence of bancroftian filariasis only in low-lying inland villages and its absence from coastal villages. Microfilariae were detected in thick blood films taken during evening hours in 48 of 168 (28.5%) persons in Senggo, and 65 of 190 (34.2%) in Tamnem, a neighboring village situated to the south of the central mountain range. Males were more frequently infected than females, and infection rates tended to be highest in those of both sexes 20 years or older. Filariasis was not found in the coastal villages of Amamapare, Kokonao, Timika or the mountain village of Waa.