The American Journal of Tropical Medicine and Hygiene - Volume 32, Issue 5, 1983

Retinal hemorrhages were seen in 21 patients among a group of 144 with strictly-defined cerebral malaria. Hemorrhages were multiple in 17 cases and bilateral in 14. There was subhyaloid extension in two. Soft exudates were seen in two, the retinae were considered edematous in four and in one there was bilateral papilledema. Retinal hemorrhages were significantly associated with several indices of severity of Plasmodium falciparum infection: high parasitemia with schizontemia, anemia, elevated serum creatinine and reduced plasma antithrombin III. Only two patients with hemorrhages were both severely anemic and thrombocytopenic. It is suggested that retinal hemorrhages, a frequent finding in cerebral malaria, may be visible evidence of vascular lesions involved in the pathogenesis of this condition.

A 48-hour in vitro test for determining the chloroquine sensitivity of Plasmodium falciparum isolates was evaluated in Kisumu and Malindi, Kenya. P. falciparum isolates from 14 children, aged 5 to 13 years, were studied. In vivo and 48-hour in vitro tests were done on all 14. Successful Rieckmann macro and micro in vitro tests for chloroquine sensitivity were completed in nine isolates each. All 14 infections cleared within 3 days of beginning chloroquine treatment, and none recrudesced during a 7-day (8 patients) or 28-day (6 patients) follow-up period. The three in vitro tests gave comparable results. Although all isolates tested were chloroquine sensitive in vitro, different response patterns were observed. In the 48-hour test, 10 isolates were inhibited at chloroquine concentration ≤0.03 nmol/ml medium. These isolates were inhibited by ≤0.5 nmol of chloroquine per ml blood in the Rieckmann macro test and by 2–6 pmol/well in the micro test. The other four isolates had response patterns intermediate between those of previously reported sensitive and resistant strains. Complete inhibition did not occur until chloroquine concentrations of ⩾0.03 nmol/ml medium in the 48-hour test, ⩾0.5 nmol/ml blood in the macro test, and 6 pmol/well in the micro test. The results demonstrate that the 48-hour test is a useful addition to existing in vivo and in vitro methods for determining the chloroquine sensitivity of P. falciparum in the field.

Forty-two infants, aged 6 to 24 months, infected with Plasmodium falciparum were identified in Kisumu, Kenya. Because of their age, all were presumably not semi-immune to malaria. Each infant was treated with 25 mg/kg chloroquine base and followed for 7 days. Forty-one infections were sensitive to chloroquine in vivo; asexual parasites disappeared in all by day 4 and were not present on days 5, 6, or 7. One infection was resistant in vivo; parasites disappeared by day 3 but recrudesced on day 4. Rieckmann micro in vitro tests for chloroquine were done on the 42 isolates. Interpretable results were found in 25. In vitro resistance was demonstrated in 18 (72%) isolates, including the patient with in vivo resistance; ⩾99% inhibition of schizont development only occurred in wells containing ⩾8 pmol chloroquine base (compared with ≤5.7 pmol/well for known sensitive isolates). This is the first demonstration of in vivo and in vitro chloroquine-resistant P. falciparum in a Kenyan. Comparison of these results with results from other studies carried out in the same area on older individuals suggests that the immune response may be playing a role in modifying the expression of resistance.

An indirect immunofluorescence test with fresh non-fixed infected blood as antigen was used to show that antibody in human sera from the Gambia recognized antigens on the surface of Plasmodium falciparum-infected human erythrocytes. Surface immunofluorescence was detected on 90% of erythrocytes infected with trophozoites and schizonts produced in continuous culture of isolates from the Gambia (FCR 3/K+), Brazil and Thailand. Fluorescence was equally strong with a Gambian parasite clone (FCR 3/K-) that lacked knobs, an ultrastructural modification of the erythrocyte membrane associated with parasite sequestration. Immunofluorescence could not be detected with an isolate from Uganda. The surface antigenicity of parasitized erythrocytes was eliminated by chymotrypsin and trypsin treatment. Fluorescence was specific for the surface of trophozoite- and schizont-infected cells on the condition that fresh erythrocytes were added to cultures every 4–5 days (subculture); if fresh erythrocytes were not added for over 2 weeks, a large percentage of non-infected erythrocytes also bound antibody. Normal erythrocytes incubated with media from these cultures also gave positive surface immunofluorescence. Thus, there are two types of antigenicity on erythrocytes: one expressed on infected erythrocytes and another passively absorbed from media to normal erythrocytes when parasites are not subcultured for long periods.

Among 884 hospital patients with gastroenteritis, 36 (4.1%) were excreting Cryptosporidium oocysts in their stools; only 5 of the 36 patients were also excreting other enteropathogens, while none of 320 hospital patients without gastroenteritis were excreting Cryptosporidium oocysts. Children were more commonly infected with Cryptosporidium (4.8%) than were adults (1.6%). The prevalence of infection was higher (7%) during the summer period of February–May 1981 than in the remainder of the observation period to the beginning of June 1982 (1.9%). The most common clinical manifestation of gastroenteritis in Cryptosporidium-infected patients was diarrhea, lasting from 3 to over 14 days, accompanied by vomiting, anorexia, and abdominal pain. The results show that a small proportion of patients with gastroenteritis are infected with Cryptosporidium, and the importance of the infection needs to be examined.

A random survey of 1% of the total population of La Guadeloupe, French West Indies, for toxoplasmosis was carried out. Blood specimens from 3,238 individuals were collected on filter paper strips and tested for Toxoplasma antibodies by the fluorescent antibody technique. Sixty percent were positive. A gradual increase in positivity with increasing age, and high prevalence in children (50% in the 6- to 10-year age group) were observed. No differences were found among the different ethnic groups, nor among persons in different occupations. Economic status showed a highly negative correlation with antibody prevalence rates. The infection rate was not higher in individuals who consumed raw or undercooked meat, and no differences in antibody prevalence related to the sources of meat were observed. The seropositivity rates varied over a wide range (40–76%) according to the locality; higher Toxoplasma antibody rates were found in areas with higher rainfall, and toxoplasmosis prevalence rates correlated positively with mean annual rainfall but not with altitude or with rural or urban residence. A significant difference was observed between seropositivity rates when people living in houses with or without cats were compared. A higher prevalence of hookworm and Strongyloides infections—considered as evidence of closer contact with moist soil—were found in children with Toxoplasma antibodies than in age-adjusted control children without such antibodies. These results provide evidence that meat plays a negligible role in the transmission of Toxoplasma to man, and support the hypothesis that oocysts shed in cat feces are the almost exclusive mode of human infection in Guadeloupe.

A simplified enzyme-linked immunosorbent assay (ELISA) was evaluated as a diagnostic test for visceral leishmaniasis in the field on 222 individuals with splenomegaly and 110 controls. The test was shown to have a sensitivity of 98.4% and specificity of 100% when compared with parasite identification by splenic aspiration. The data indicate that the ELISA is an accurate, safe, and economical alternative to splenic aspiration for the diagnosis of visceral leishmaniasis.

Because of the need for orally active antileishmanial agents, orally administrable drugs have sometimes been used to treat human leishmaniases without prior demonstration of efficacy in experimental models. The antileishmanial activity of such agents was tested against Leishmania tropica (a cause of cutaneous leishmaniasis) within human macrophages in vitro. Although trimethoprim + sulfamethoxazole and isoniazid + rifampin have been reported as efficacious orally in certain human studies of cutaneous disease, these drugs were ineffective in vitro (≤40% parasite elimination) at peak achievable serum levels. The combination of allopurinol and Pentostam® is being tested in humans. In vitro, allopurinol (5 µg/ml) augmented the antileishmanial effect of a low concentration of Pentostam (5 µg/ml) but not of a higher concentration of Pentostam (20 µg/ml). Nifurtimox is a nitrofuran which has questionable activity against human cutaneous disease. Nifurtimox was similarly only 50% effective in vitro at peak achievable serum levels (1.0–3.0 µg/ml). However, furazolidone, another orally administered nitrofuran, eliminated 92% of parasites at 1.0 µg/ml. Chlorpromazine and quinacrine are concentrated in tissues that are susceptible to infection by Leishmania. Chlorpromazine and quinacrine eliminated only 15% and 35% of organisms in vitro at achievable serum levels (≤0.3 µg/ml), but eliminated virtually all organisms in vitro at possible achievable tissue levels. Both the negative and the positive data of this report may aid in selection of effective orally active agents for in vivo trials.

C3HeB/FeJ peritoneal exudate macrophages elicited with a variety of sterile inflammatory agents or with Mycobacterium bovis strain BCG were exposed to Leishmania tropica amastigotes in vitro. The percentages of L. tropica-infected macrophages were similar in resident and inflammatory macrophage populations over 72 hours in culture. Inflammatory macrophages supported intracellular replication of L. tropica in excess of that by resident macrophages. These macrophages also failed to demonstrate cytotoxicity to tumor cells in vitro, a defined nonspecific effector function of activated macrophages. However, macrophages from BCG-treated mice were significantly more resistant to initial infection with L. tropica, killed intracellular amastigotes by 72 hours in culture and were cytotoxic to tumor cells, indicating full immunologic activation. The inability of inflammatory macrophages to kill L. tropica parasites suggests that inflammation may actually contribute to the pathogenesis of leishmaniasis.

The oral administration of ketoconazole to mice protected them against death caused by infection with Trypanosoma cruzi. The addition of ketoconazole to cultures of macrophages infected with the organism markedly inhibited the intracellular multiplication of amastigotes. These observations suggest that ketoconazole may be a potent agent against T. cruzi and should be evaluated more extensively as a chemotherapeutic agent for Chagas' disease.

Isolation of Trypanosoma cruzi in hemoculture was done in media with different glucose concentrations. The three strains studied developed into epimastigotes 2 weeks after seeding in media with less than 4.5 mM hexose. Inhibition of growth of trypanosomes in media containing a greater amount of glucose was not caused by the higher osmotic pressures. Cultures containing more than 4.5 mM glucose showed a greater depletion of pH and higher lactic acid production than did those with lower concentrations, resulting in damage to amastigote clusters or to initial epimastigote development. The maximum recommended level of glucose in the culture medium is determined by the detrimental acidification resulting from excessive quantities.

Adult Microtus montanus were inoculated with a recently isolated strain of Trypanosoma brucei rhodesiense of human origin. The animals developed subacute to chronic infection and low-grade parasitemia. Histopathological examination of the heart revealed a severe pancarditis resulting in pronounced weight loss, and survival times of 5–8 weeks, preventing development of meningoencephalitis. In the brain a moderate meningitis was found, usually associated with moderate numbers of parasites in the choroid plexus and leptomeninges; however, trypanosomes were also found without tissue inflammation. Meningoencephalitis was found after 7 weeks, with parasites in the cerebral parenchyma. Chronic inflammation was present in lungs and kidney, often associated with trypanosomes; in one animal glomerulonephritis was found. Spleen and lymph glands showed a variable degree of lymphoid hyperplasia but no extravascular parasites. In the liver of all animals plasmolymphoid infiltrates were observed in the periportal connective tissue; no extravascular parasites were observed. A variable degree of lymphoplasmohistiocytic infiltrate in the connective tissue and occasional rare trypanosomes were seen in mesenterium, pancreas, epididymis, striated muscle, and skin. Experimental infection in M. montanus appears to be a suitable model for study of the acute trypanosomiasis of T. b. rhodesiense, but not for chronic African sleeping sickness with cerebral involvement.

Axenically grown Entamoeba histolytica was found to contain adenosine kinase. This organism lacks de novo purine biosynthetic pathways. Adenosine kinase provides the amoeba with a method for salvaging adenosine from ingested nucleosides or from degraded nucleotides. Adenosine kinase was purified 64-fold, by chromatography on Sephacryl S-200, DEAE-cellulose, and (C-8)-adenosine-agarose. The latter separated it from amebal adenylate kinase. Adenosine kinase has a molecular weight of 38,000 and requires glycerol for stability. It utilizes adenosine triphosphate to phosphorylate adenosine, and 7-deazaadenosine (tubercidin), but adenine 9-β-D-arabinofuranoside (ara-A) is not detectably phosphorylated. It requires Mg++ as a cofactor.

Prolonged oral high-dose mebendazole therapy provided an effective anthelmintic regimen for trichinosis unresponsive to steroid therapy in one patient. Side effects were limited to a Herxheimer-like reaction. Serum mebendazole levels documented gastrointestinal absorption. Repeat muscle biopsies and fluorescein angiography substantiated objective improvement.

Albendazole, a new anthelmintic drug was evaluated in Malaysia in 91 patients, with single or mixed infections of Ascaris, Trichuris, and hookworm. Albendazole was administered as a single dose of 400 mg, 600 mg, or 800 mg. The cure rate for Ascaris at all three doses was 100% at days 14 and 21 post-treatment; for hookworm it was 98.8%, 100% and 98%, respectively, at day 14 and 68.8%, 100% and 84%, respectively, at day 21; for Trichuris it was 31.2%, 57.1% and 42.3%, respectively, at day 14 and 27.3%, 60.9% and 48.0%, respectively, at day 21. The egg reduction rate at day 21 was 100% at all three doses for Ascaris, 94.5%, 100% and 96.1%, respectively, for hookworm; and 39.2%, 85.1% and 72.8%, respectively, for Trichuris. There were no side effects, and biochemical examination of blood and urine did not indicate any unfavourable changes. Based on this trial, the recommended dosage for Ascaris and hookworm is a 400 mg single dose, and for Trichuris is a 600 mg single dose. Albendazole appears to be more effective than other available anthelmintic drugs.

Peripheral lymphocytes from 16 patients with chronic uncomplicated strongyloidiasis and 15 non-infected controls were stimulated in vitro with Strongyloides stercoralis larval antigens, other non-parasite antigens and the T cell mitogen phytohemagglutinin (PHA). In the presence of autologous plasma the patients' responses to Strongyloides antigens were similar to those of controls. When lymphocytes from nine patients were cultured in the presence of normal human serum, responses to parasite antigens were enhanced, while responses to other antigens and to PHA were unaffected. Lymphoproliferative responses to PHA were significantly lower in the patients' group than in the controls. These findings suggest that in chronic strongyloidiasis, in addition to a depression of T cell activity, factors are present in the patients' serum that inhibit parasite-specific cellular responses in vitro.

Infection of humans by Mammomonogamus is rare, with approximately 80 cases reported in the literature. When present in man, the worms cause violent coughing until their removal. The diagnosis of Mammomonogamus infection in the past has been based on morphologic characteristics of intact adults. We report a case diagnosed by features of the worms in histologic section.

The pinworm prevalence among 302 children tested in 1980 in five Southern California elementary schools was determined to be 11.6%. The range was 7.3–15.4%. In a 1982 study involving 158 children in six schools, the prevalence was 21.6% (range 11.1–38.9%). The results of these studies are compared with those of a similar prevalence study done in the same area and in some of the same schools during the 5-year period 1960–1964. The prevalence for the earlier period, involving 700 children in six schools, was 34.6%, and ranged from 29.2–43.0%. A new, flexible, plastic pinworm slide was used in the 1980 and the 1982 studies. In the 1982 study, this diagnostic method was evaluated and compared for efficiency and use against the standard cellulose tape/glass slide. The two are equally effective in picking up eggs, and in reading quality. The plastic slide is easier to use and does not break.

Adherence assays were used to demonstrate the in vitro effect of serum-dependent cellular adherence of human buffy coat cells to infective larvae of Brugia malayi in filariasis patients treated with antifilarial drugs. In this study, microfilaraemic patients were treated with either diethylcarbamazine citrate (DEC), mebendazole or levamisole hydrochloride. It was found that DEC and mebendazole decreased the motility of infective larvae due to a direct action of the drugs. Sera of levamisole-treated patients caused increased adherence of human buffy coat cells to infective larvae, leading to a decrease in motility and cuticular damage as confirmed by scanning electron microscopic studies. However, serum of levamisole-treated patients alone could cause a similar lethal effect on infective larvae. Studies with the indirect fluorescent antibody test suggested that IgM was involved in this phenomenon. Complement did not appear to be important.

Experimental studies in Bayeux, Haiti showed that the biting midge, Leptoconops bequaerti, is capable of supporting the complete development of Mansonella ozzardi but only on a very limited scale. This suggests that the species may not be involved in the natural transmission cycle despite its abundance and pestiferous nature in certain areas of Haiti. A midge-holding container is described which markedly enhanced the survival of engorged L. bequaerti in the laboratory.