The American Journal of Tropical Medicine and Hygiene - Volume 33, Issue 6, November 1984

Gametocyte production by cloned lines of Plasmodium falciparum and their parental isolates has been studied in culture over periods of several months. Many isolates differed significantly from each other in their capacity for gametocyte production. Clones derived from an individual isolate were also widely different in capacity for gametocyte production. Consistent differences in gametocyte production were observed between clones which had always been grown concurrently and thus had identical culture histories. Levels of gametocytogenesis characteristic of individual clones, although subject to transient fluctuations under environmental influence, were stable over several months.

We characterized a set of eight monoclonal antibodies produced against Plasmodium falciparum. In an indirect fluorescent antibody assay the antibodies produced small dots of fluorescence in schizonts and individual merozoites. This merozoite-associated dot reactivity occurred with 21 different strains of P. falciparum, but there was no reactivity with other human, nonhuman primate, or rodent Plasmodium species. Three of the monoclonal antibodies precipitated proteins of Mr 145,000, 135,000, and 104,000. Five of the monoclonal antibodies precipitated proteins of Mr 78,000, 63,000, 42,000, and 40,000.

Mouse monoclonal antibodies (McAbs) have been used to characterize the proteins of the asexual erythrocytic cycle of Plasmodium falciparum. Three different McAbs react with antigens of the schizont and extracellular merozoite to give a punctate fluorescence pattern. In many cases, such areas of fluorescence were composed of two adjacent, fluorescent bodies; these were distinct from the nuclei. In contrast, McAbs which bound to the ring-stage parasite were not localized, but were diffusely distributed within or around the ring-stage parasite. These McAbs immunoprecipitated five prominent, 35S-methionine-labeled schizont proteins (p) of Mr 82K, 70K, 67K, 39K, and 37K. Only p82, p39, and p37 were immunoprecipitated from schizont-labeled ring-stage parasites; thus, it appears that p70 and p67 are modified, degraded, or secreted some time between intracellular merozoite maturation and erythrocyte invasion.

Isospora belli is an uncommon cause of diarrhea in man. Like the closely related Cryptosporidium, the organism causes disease that may be more severe and chronic in patients with underlying immune deficiency states. We describe three male homosexuals with Isospora enteritis. Each had several months of diarrhea. All three patients had lymphopenia and two had profound T cell helper-suppressor ratio reversal. In addition, two of the patients admitted to oral-anal contact, a practice associated with the acquisition of parasites and other enteric pathogens in the homosexual population. We suggest that Isospora belli may be a sexually transmitted pathogen which should be considered in the diagnosis of homosexual patients with diarrhea.

Ten patients were identified at Jackson Memorial hospital/University of Miami Hospitals and Clinics with enteric coccidial infection due to Cryptosporidium spp. or Isospora belli. All had the acquired immunodeficiency syndrome as manifested by Kaposi's sarcoma or multiple opportunistic infections, or both. They presented with profuse diarrhea associated with weakness, anorexia, and weight loss. Routine examinations of stools for eggs and parasites as performed by the hospital laboratory were negative in all patients. Sugar flotation and modified acid fast techniques were used in the Tropical Disease Laboratory to identify oocysts of Cryptosporidium spp. in stools of seven patients. Malabsorption, characterized by a low 5-hour D-xylose and positive fecal fat, was observed in 6/6 of these patients. In three other patients Isospora belli oocysts were identified in stool specimens or via a duodenal string test. Spiramycin was the only drug found to be effective in treating patients with cryptosporidiosis. Patients with Isospora belli responded to a prolonged course of trimethoprim-sulfamethoxazole.

Using a previously described mouse model of late-stage African trypanosomiasis (i.e., involvement of the central nervous system), we demonstrate that a combination of DL-α-difluoromethylornithine (DFMO) and suramin is curative. In the curative protocol, DFMO is given as a 2% solution in the drinking water for 14 days and suramin is administered as a single dose (20 mg/kg intravenously) on day 1 of DFMO administration. Since: 1) DFMO has very low toxicity, 2) suramin is one of the least toxic of the presently used trypanocides, and 3) suramin and DFMO act synergistically in mouse models of both acute and late stage tryanosomiasis, we conclude that this combination offers special promise in the treatment of African trypanosomiasis in man.

Autologous mixed lymphocyte reactions (AMLR) were studied in 18 individuals chronically infected with Trypanosoma cruzi. These individuals were further classified into three clinical groups: the asymptomatic indeterminate form (n = 5); the mega disease form (n = 5); and the cardiomyopathy form (n = 8). While patients with mega disease showed a normal proliferative response when compared with normal controls, the indeterminate group showed a lowered response in sharp contrast with the heart disease group, which presented a very high proliferative response to autologous non-T cells. These abnormal AMLR represent direct evidence for an immunoregulatory malfunction which may be involved in the inflammatory cardiac damage in chronic Chagas' disease.

Epidemiologic studies to define the domiciliary and extradomiciliary transmission cycles of Trypanosoma cruzi and Trypanosoma rangeli in the Oriental Plains of Colombia were conducted in the gallery forests near Carimagua and El Porvenir. One-hundred and seven palm trees belonging to nine genera were examined; triatomines were found in only three palm species, the leaves of which are locally used for roof thatching: 2/29 Maximiliana elegans, 1/7 Mauritia flexuosa and 7/7 Scheelea sp. Bugs were also found in 5/14 hollow Mauritia inhabited by bats, 4/21 bird nests and 1/4 armadillo burrows. Five species of triatomines were collected: Rhodnius prolixus was the most abundant, 192 of the total 207 (92%) collected; the bugs were found in Maximiliana and Mauritia but especially in Scheelea, and 8% were infected with T. cruzi and T. rangeli; Cavenicola pilosa and Triatoma maculata were found associated with bats; Psammolestes arthuri and Panstrongylus lignarius with bird nests and Panstrongylus geniculatus with armadillos. Although triatomine colonies were not found in human dwellings, flying adults of R. prolixus occasionally reached houses by their own locomotion and fed on man, but did not become established. Only 12 of 199 persons (6%) tested serologically were reactors to T. cruzi antigens and all 12 had lived in areas of domiciliary transmission elsewhere in the country, indicating that domiciliary transmission is not occurring in this region. Whether the presence of domiciliary R. prolixus in houses located in the ecologically altered piedmont of the oriental plains, a known area of domiciliary transmission of T. cruzi, is due to importation of domiciliary bugs from endemic areas or to the domiciliarization of wild R. prolixus remains to be determined.

Monoclonal antibodies have been made against clones of Trypanosoma brucei rhodesiense from the WRATAR 1 serodeme and analyzed by indirect immunofluorescence assay for specificity against homologous and heterologous clones. These antibodies were shown to be variable antigen type (VAT)-specific as they identified the majority of parasites in the homologous clones but few parasites in other clones. The reactivities of these VAT-specific monoclonal antibodies with uncloned human trypanosome isolates from Kenya were compared with the reactivities of polyvalent, VAT-specific rabbit sera on the same isolates. Different reaction patterns were obtained with the two sets of reagents and concordant reactions were less than 20%. Our data indicate that single monoclonal antibodies are not interchangeable for sera in the typing of antigenic variants of African trypanosomes, and multiple monoclonal antibodies for each antigenic variant will probably be needed.

The Dot-ELISA, a rapid, visually read micro enzyme immunoassay for visceral leishmaniasis utilizing minute volumes of antigen “dotted” on nitrocellulose filter discs and precipitable chromogenic substrate, was analyzed under a variety of experimental parameters. Raising assay incubation temperatures from 23°C to 28°C resulted in titer increases in three of five leishmaniasis patient sera; at 37°C, all five patient sera and one of five normal human sera showed titer increases. The amount of antigen used could be reduced 50% by incubating patient serum overnight at 4°C. Antigen discs stored at -20°C were optimally reactive with leishmaniasis sera over a 270-day period. Antigen discs stored at 4°C and 23°C showed reproducible titer decreases at 90 days. Aging either peroxidase-conjugated antibody or substrate for up to 28 days at 4°C did not adversely affect titers of positive and negative control sera and reagent controls. Activated substrate stored at 23°C was optimally reactive in the assay for at least 24 hours. No changes in titers of positive and negative control sera or nonspecific reactions in reagent controls occurred when using different brands of microtiter plates. The long shelf lives and stabilities of Dot-ELISA antigen and reagents indicate this test should prove useful both in the laboratory and in the field.

Pentamidine is an antileishmanial agent that is often toxic at therapeutic dosages. The obligate intramacrophage localization of Leishmania indicates that encapsulation of pentamidine within a carrier phagocytized by macrophages (IgG-coated sheep red cell ghosts) might improve activity. In in vitro experiments, treatment of infected mouse macrophages for 1 hour with a mean of 1.4 µg of encapsulated drug resulted in a calculated drug concentration of 180 µg/ml macrophage, and in 73% suppression of organism multiplication within the macrophages after 4–5 days of further cultivation. In comparison, 27 µg unencapsulated drug/ml was needed for similar suppression. Electron microscopic examination 5 hours after phagocystosis of IgG-ghosts revealed that 95% of organisms were adjacent to ghosts in phagolysosomes. Fusion of drug carrier with phagolysosome containing drug target is therefore an important step in carrier-mediated parasite suppression in this model. These results suggest that IgG-coated erythrocyte ghosts containing pentamidine have potential as an antileishmanial formation.

Bancroftian filariasis has been reported in several areas of Papua New Guinea. The epidemiologic features and natural history of Wuchereria bancrofti infection in this geographic region, however, have not been well-defined. The objective of this study was to assess the parasitological and clinical features of bancroftian filariasis in a community in East Sepik Province, Papua New Guinea. In a village of 99 individuals, the overall prevalence of microfilaremia was 68%. The microfilarial carrier rate was high in those ≤ 10 years (62%), remained elevated in the 11–20, 21–30, and 31–40 age groups (42–55%), and peaked in subjects ≥41 years old (90%). The geometric mean level of parasitemia in all subjects with patent infection was 3,198 microfilariae/ml blood. This value was 78 parasites/ml in the ≤10-year-old age group, increased to 1,753 in 21 to 30-year-olds and was markedly elevated in subjects ≥41 years old (6, 792 microfilariae/ml). Acute symptoms of filariasis (lymphadenitis and lymphangitis) were initially noted in individuals between the ages of 11 and 20 years (30%). Obstructive disease, manifested as elephantiasis and hydroceles, was present in 64 and 79% of 31–40 and ≥41-year-olds, respectively. These data suggest that intense transmission of W. bancrofti infection occurs at an early age in this area of East Sepik Province; patent infection remains high in older age groups. Irreversible lymphatic obstruction develops 20–30 years after initial infection and may be associated with either amicrofilaremia or microfilaremia.

A survey for Wuchereria bancrofti in Limbe, Haiti (est. pop. = 10,500) revealed that 17% (231/1,450) had a patent infection. Nearly half of those surveyed harbored fewer than 10 microfilariae (mf) per 20 mm3 of finger-prick blood; the median mf density for females and males was 12.4 and 9.5, respectively. Parasitemias occurred as early as age 4. Antibody titers ≥1:20 against adult D. viteae antigen were observed in 38% of microfilaremic individuals and in 29% of amicrofilaremic individuals. Peak antibody responsiveness (40%) was observed between 5 and 9 years of age. In all age groups there was no correlation between mf density and antibody titer. Among the mf carriers, 5.6% had no clinical symptoms. Lymphangitis was a common feature with 14.3% having lymphedema, 8.2% with edema of the lower extremities, and 1.3% reporting episodes of chyluria. Genital involvement among women was rare, but in males 5.4% had genital swelling and 4.5% had hydroceles. Culex pipiens quinquefasciatus (Say) was observed to support the complete development of W. bancrofti in Limbe.

A monoclonal antibody designated Gib 13-5-2 (Gib 13) and directed against the cattle parasite Onchocerca gibsoni was used in a two-site immunoradiometric assay (IRMA) for detection of circulating antigen in the sera of Wuchereria bancrofti-infected individuals from Sri Lanka and Papua New Guinea. The microfilaremic patients were, in general, serum antigen positive by the Gib 13 IRMA. Among the amicrofilaremic patients, 47% of those with lymphedema, lymphangitis, hydrocele, etc., and 25% of those with elephantiasis had circulating antigen. Correlation of the presence of serum antigen with clinical status indicated that the Gib 13 target antigen in serum is probably an indicator of either active or early infection, or of both. The antigen was also detected in the urine of some patients. By sodium dodecyl sulphate polyacrylamide gel electrophoresis immunoblotting, Gib 13 target antigens of molecular weights 67,000 and 52,000 were identified.

A study was made of the Meyers-Kouwenaar (MK) body in the livers of experimentally infected ferrets. Meyers-Kouwenaar bodies, the carcasses of microfilariae (mff) covered by deposits of Splendore-Hoeppli (SH) material, were found in small abscesses of eosinophils and in granulomas. The SH deposits varied from an eosinophilic, hyaline fringe around intact mff to multilayered deposits surrounding an unrecognizable granular remnant. In abscesses, peroxidase activity was intense in SH deposits and the surrounding eosinophils. The presence and localization of IgG were variable in MK bodies, as detected by an enzyme-linked immunohistologic assay; and antigens of mff were not detected in the SH deposits. Electron microscopy of the MK body demonstrated a layered, radial deposition of amorphous and granular material on the mff and a structural heterogeneity which apparently included leukocyte granules and other cell organelles. Leukocytes surrounding MK bodies in abscesses were often degranulated and degenerate; incorporation of lysosomes of eosinophils and cellular debris into the SH deposits at the periphery of the MK bodies was indicated.

This study, made between November 1982 and January 1983 to ascertain the prevalence of onchocerciasis in the Babana District of Borgu Local Government Area, Kwara State, Nigeria, showed that 483 (48.6%) of the 993 subjects skin-snipped in 12 communities were infected. The infection rate was significantly higher (P < 0.001) for males (57.7%) than for females (37.0%), for subjects above 10 years of age (54.0%) than for those who were younger (10.7%), and for the Fulanis (63.8%) than for the Bokos (48.3%). Although various indications suggest that this district has a history of long-standing onchocerciasis, the knowledge of river blindness and its association with blackflies was poor throughout the district and, except for two teachers, no other subjects had ever received onchocercal chemotherapy. These factors, apparently, account for the frequent occurrence and severity of various clinical manifestations and complications of onchocerciasis identified in the district.

A 28-year-old Marine Corps officer developed Katayama fever with central nervous system (CNS) manifestations 6 weeks after swimming on Leyte Island in the Philippines. Symptoms consisted of fever, nausea and vomiting, focal visual field deficits and mild confusion. CAT-scan of the patient's head initially revealed multiple lucencies and severe edema in the left frontal, parietal and occipital lobes. No schistosome eggs were found in the patient's stool, and therefore he was treated with a 10-week course of dexamethasone with resolution of all symptoms over 3 months. Repeat CAT-scan after symptoms cleared showed complete resolution of the focal abnormalities seen earlier. The diagnosis was subsequently established by positive serology and by finding eggs in the patient's stool and in tissue obtained by liver biopsy. This is the first report of CAT-scan-detected focal CNS lesions in a patient with acute schistosomiasis japonica, and resolution of the CNS abnormalities, temporally related to non-specific steroid treatment, is documented as well.

Three cases of intestinal schistosomiasis acquired in northern Laos are reported. All three patients swam regularly for many years in the Mekong River near the cities of Vientiane and Luang Prabang. Morphology of the eggs found on rectal biopsy indicated infection with Schistosoma mekongi. One patient had severe portal hypertension with bleeding esophageal varices requiring a splenorenal shunt; studies of her liver biopsy showed S. mekongi eggs and periportal fibrosis. A second patient had abnormal liver function tests, and a third patient was asymptomatic. In two of the three cases, no schistosome eggs were found on stool examination, and the diagnosis was made by rectal biopsy. The implications of this new focus of infection are discussed.

Twenty-five African children from Libreville had concomitant typhoid or paratyphoid fever (Salmonella typhi, 4 children; Salmonella paratyphi A, 1; S. paratyphi B, 5; s. paratyphi C, 15) and Schistosoma intercalatum infection. In 19 children treated for both infections, no relapse occurred. In the six others, antibiotics alone were given and the Salmonella infection relapsed after 1 month. No relapse occurred after a second course of antibiotics together with treatment for the S. intercalatum infection. This observation suggests that S. intercalatum prolongs Salmonella infection, as do other species of schistosomes. The concomitant treatment of both infections is recommended.

Egyptian school children infected with Schistosoma haematobium and treated with a single dose of metrifonate, 10 mg/kg body weight, had a marked reduction in urine egg counts reaching 90% during a 30-week follow-up. While cure rate was higher among lightly infected persons, percentage reduction in egg counts was greater among the heavily infected. After a summer period of probable high risk exposure reinfection rate in those children who were treated and cured was 4.7% as compared to 6% in previously uninfected children.