The American Journal of Tropical Medicine and Hygiene - Volume 38, Issue 2, March 1988

Anticipation of the shift to publication of the Journal twelve times each year has provoked considerable thought on ways to improve the efficiency of procedures used in this office. Four years of experience provide a background that can be gained in no other manner.

In any publication devoted to science the primary consideration must be accuracy, with promptness of publication running a close second. While initially the two may seem to be opposites, it nevertheless is true, that accuracy and timeliness in publishing are served by the same practices. A manuscript submitted in the exact format specified by the Journal, supported by appropriately designed figures, tables, and references, will move rapidly through this office and will be easily processed by those turning it into print. It will become a printed product requiring a minimum of proofreading. A desideratum is the manuscript that requires no intervention by this office beyond the insertion of the printer's codes.

In the greater part of Brazil's extensive territory climatic conditions are propitious for the establishment of endemic malaria, which has been causing pain and death among the country's inhabitants for generations. In Brazil the epidemiology of malaria has presented some peculiar aspects: besides being transmitted in most areas by anophelines breeding in groundwater collections, its dissemination in a restricted but economically important zone was entirely due to mosquitoes breeding in water accumulated in plants of the family Bromeliaceae. It is the so called “bromeliad malaria.” To the native vector species, the alien Anopheles gambiae, introduced to the country, added a new problem.

Considerable efforts have been made by Brazilians and foreigners to understand malaria epidemiology and control its transmission. Among the foreigners one immediately thinks of Fred Lowe Soper. Following his contribution to the elimination of Aedes aegypti and urban yellow fever in Brazil, Soper was one of those most directly responsible for the eradication of An. gambiae from this part of the world.

We studied the effects of daily proguanil compared to weekly chloroquine as malaria prophylaxis in 170 children living in a malaria-endemic area along the Thai-Burmese border. Children aged 5–10 years were matched for age, weight, and presence of splenomegaly then randomly assigned to receive either proguanil (equivalent of 200 mg daily adult dose) or chloroquine (equivalent of 300 mg base weekly). All medications were administered by the investigators and malaria smears were performed on a weekly basis. Among 85 children taking proguanil for 524 human-weeks, there were 17 cases of falciparum malaria and 11 cases of vivax. Of 85 children on chloroquine for 537 human-weeks, there were 24 cases of falciparum and 1 case of vivax. There were no statistically significant differences between the two regimens when analyzed either as suppressive or as causal Plasmodium falciparum prophylactics. The data were suggestive that proguanil may have some causal prophylactic effect against falciparum malaria. There were significantly more vivax prophylactic failures (P < 0.01) in the proguanil group. Side effects were infrequent, mild, and comparable in both groups.

The standard chloroquine treatment for Plasmodium falciparum malaria is 25 mg (base)/kg (C25) given over 3 days. In Rwanda, 50 mg/kg (C50) administered over 6 days has been recommended by the Faculty of Medicine, Ministry of Health. The present study compared clinical and parasitological efficacy and side effects of C25 and C50 in children ≤5 years of age. In vitro studies with chloroquine, mefloquine, pyrimethamine, and quinine were also performed. Ninety children were given a 3-day treatment of C25 and 48 a 5-day treatment of C50. Cases were followed for a total of 15 days (D0 to D14). At day 14, 73% of the C25 and 67% of the C50 children were still parasitemic, but the mean geometric parasite density had decreased by at least 96% in both groups. Clinically, 44 C25 and 12 C50 children had fever on day 0; by day 14 only 4 (9%) C25 and 4 (33%) C50 children still had fever. Side effects were found to be minimal. The chloroquine in vitro tests corroborated the in vivo findings. P. falciparum was found to be quite sensitive to mefloquine and quinine, but showed a high (59%) resistance to pyrimethamine.

The efficacy of amodiaquine and sulfadoxine-pyrimethamine combination as a second-line therapy for chloroquine-resistant Plasmodium falciparum infections was investigated in Rwanda in September 1986. Children ≤5 years old presenting with a P. falciparum parasitemia 14 days after treatment with chloroquine were administered either amodiaquine (25 mg/kg over 3 days, 64 patients) or sulfadoxine-pyrimethamine (as a single dose with tablets containing 500 mg of sulfadoxine and 25 mg of pyrimethamine: ¼ tablet for children under 1 year, ½ for those 1–3 years old, and 1 tablet for those 4–5 years old; 34 patients) and followed for 7 days. Seven days after starting treatment with amodiaquine, 50 (76%) children were aparasitemic. All the children who had received sulfadoxine-pyrimethamine were aparasitemic 7 days after initiation of therapy.

Dexamethasone has recently been shown to block the production of cachectin (implicated in the pathogenesis of cerebral malaria) if administered prior to endotoxin induction of mouse macrophages. Using the hamster cheek pouch-cerebral malaria model, we tested the hypothesis that dexamethasone is effective as a therapeutic agent in severe malaria if given before some yet undefined trigger point in the disease. Infected hamsters were treated with dexamethasone (0.7 mg/kg) daily on days 7–12, 4–12, or 1–12 post-challenge. When treatment was started on day 1, whole body oxygen consumption (used as a measure of erythrocyte transport to sites of diffusion) on day 12 was greater than (P < 0.05) that of infected control animals, though the degree of anemia was no different in treated and untreated groups. Furthermore, treatment produed a reduction in monocyte accumulation, capillary malfunction, and monocyte/red blood cell aggregate formation observable in the cheek pouch in vivo and a similar reduction in monocyte presence, capillary pathologic change, and multifocal hemorrhage in the brain on postmortem. These data suggest that mediator(s), whose production can be blocked by pretreatment with dexamethasone, are involved in the pathogenesis of disease leading to death of the Plasmodium berghei infected hamster.

A new and rapid malaria immunoperoxidase assay using the enzyme horseradish peroxidase in place of fluorescein isothiocyanate was developed to allow the serological measurement of antimalarial antibody by light microscopy. Acetone-fixed thin blood films prepared from cultured Plasmodium falciparum were used as the source of antigen. This malaria immunoperoxidase assay is as sensitive as, and occasionally more sensitive than, the indirect fluorescent antibody assay. It is easy to perform and the antigen used does not show cross-reactivity with sera from nonmalarial diseases.

The stage specificity and localization of 12 Plasmodium falciparum antigens were determined by immunofluorescence using acetone-fixed parasites reacted with monospecific antibodies against cloned antigens. Antibodies were prepared by immunization of rabbits with recombinant proteins or by affinity purification of human plasma against cloned antigen adsorbents. Most of the antigens occurred predominantly in mature asexual parasites, two were abundant in ring stages and three were absent in rings. Four of the 12 antigens were detected in asexual stages but not in gametocytes. Grouping of antigens by localization within blood stages was difficult because of the complexity of fluorescence patterns observed. With some antibodies, fluorescence was apparently distributed evenly over the parasites, but in other cases label was concentrated within discrete compartments or organelles. Extraparasitic intraerythrocytic fluorescence was also observed.

A protocol was developed for the testing of blood stage vaccines against Plasmodium falciparum using Peruvian Aotus vociferans and the Indochina I/CDC strain of the parasite. Three different fused polypeptide vaccines containing elements of the ring-infected erythrocyte surface antigen molecule combined with Freund's complete and Freund's incomplete adjuvants were tested to determine their ability to protect against overwhelming infection following challenge with this highly virulent strain of P. falciparum, and to invoke antibody responses as measured by a standard indirect immunofluorescence technique. Nine of 14 immunized animals exhibited some protection. Presented are the test procedures developed for the conduct of such trials with New World monkeys and the analysis of results that led to the identification of variables selected for study in future trials.

A monoclonal antibody specific for a repeated epitope of the circumsporozoite protein of Plasmodium malariae sporozoites has been used to develop a two-site, single antibody-based enzyme-linked immunosorbent assay that can detect P. malariae sporozoites in mosquitoes. The assay uses a purified monoclonal antibody produced against sporozoites of the Uganda I/CDC strain of P. malariae to capture the antigen and the same monoclonal antibody labeled with horseradish peroxidase as the detector. Sporozoites have been detected in laboratory-infected mosquitoes stored at room temperature in the presence of a desiccant for as long as 18 months. The detection limit of the assay is approximately 50 P. malariae sporozoites per test well. Cross-reaction has not been observed with mosquitoes infected with P. falciparum, P. vivax, or P. ovale sporozoites.

Plasmodium fragile infection of the toque monkey is a natural host-parasite association in which parasite sequestration occurs as during P. falciparum infection of humans. We have studied parasite sequestration of P. fragile and demonstrated the existence of a new property of cytoadherence of infected erythrocytes, “rosetting,” which is defined as the agglutination of uninfected erythrocytes around parasitized erythrocytes. Rosetting in vitro and sequestration in vivo appear simultaneously as the parasite matures. The spleen plays a role in modulating cytoadherence; both sequestration and rosetting, which occur with cloned parasites from spleen-intact animals, are markedly reduced in splenectomized animals infected with parasites derived from the same clone. Sequestration and rosetting can be reversed by immune serum. Protease treatment of infected blood abolishes rosetting; however, if treatment is performed at an early stage of schizogony, rosetting reappears if parasites are allowed to further develop in the absence of protease. These results indicate that with P. fragile in its natural primate host, rosetting and sequestration are related to the presence on the infected erythrocyte surface of a parasite-derived antigenic component, the expression of which is modulated by the spleen.

Protein kinase was isolated from both amastigotes and promastigotes of Leishmania mexicana amazonensis. Unlike the previously described enzyme from L. donovani promastigotes, activity of the L. mexicana kinases was 2–3 times higher at low ionic strength than at high ionic strength, and was 3–10-fold augmented by removal of endogenous low molecular weight inhibitors. The Km of the L. mexicana kinases was 123–223 µM, compared to the value of 70 µM for the beef heart kinase. Purine nucleoside analogs are potent antileishmanial agents that are phosphorylated to their respective triphosphates. The mechanism of the analogs is thought to involve competition of the triphosphates with ATP. Cordycepin triphosphate inhibited the amastigote, promastigote, and beef heart protein kinases approximately equally. However, the Kis of formycin A triphosphate for the leishmanial kinases (Ki 40–120 µM) were far less than that of the beef heart kinase (Ki 1,380 µM). The mechanisms of certain chemotherapeutic purine nucleosides may involve specific inhibition of leishmanial protein kinase by the nucleoside triphosphate.

We have developed a simple in vitro method of infecting a continuous human macrophage cell line (U937) with promastigotes of several species of Leishmania. These include L. braziliensis braziliensis, L. b. panamensis, L. donovani, L. mexicana mexicana, L. m. pifanoi, L. tropica, and L. major. The growth kinetics of these species are presented as well as drug sensitivity data. The U937 cell system can be used to determine drug efficacy and eliminates the need to use amastigotes from animal tissues to infect the tissue culture.

We describe conditions for dot blot DNA hybridization studies using biotinylated kDNA probes from Leishmania. The sensitivity and specificity attained with biotinylated or 32P-labeled probes were equivalent. The lower level of detection obtained was 100 parasites that were blotted on nitrocellulose paper and then treated with Proteinase K. Studies were performed with 112 Leishmania isolates from Andean (uta) and sylvatic mucocutaneous (espundia) patients and all were determined to belong to the Leishmania braziliensis complex.

We studied the prevalence of Toxoplasma antibody over a 10-year period in a rural population of 326 people in Chorrera Province of Panama using the dye test. Fifty-five seroconversions were found in 108 people at risk, and 48 (87%) in children between 2 and 13 years with a mean incidence rate of 8.6% per year. Antibody prevalence rose from 25% at 5 years to 50% at 10 years of age, and increased gradually, reaching 90% by 60 years. Mean antibody levels after seroconversion were 1:6,000 in the dye test; they fell to 1:1,000 after 1 year, 1:800 after 2 years, 1:200 after 3 years, and 1:333 after 7–9 years. About 10% of antibody titers ranged between 1:4 and 1:32. Toxoplasma antibody prevalence was also studied in the metropolitan Panama City population using 590 sera collected in the fall of 1981. Age-specific incidence rates were similar in the urban and rural setting (correlation coefficient 0.71). The number of cats observed in the rural area and in the city and the degree of soil contact appeared compatible with a hypothesis of transmission by oocysts.

A study was made of the prevalence of Trypanosoma cruzi infection in armadillos at a site near New Orleans, Louisiana, where the flagellate was known to occur. Blood cultures, microscopic examination of blood, and direct agglutination tests on sera were employed in 80 armadillos. T. cruzi was isolated in culture from 23 of 80 animals; identity of the parasite was confirmed in mice inoculated with each of the isolates. Only 2 animals were positive by direct examination of blood. Serologic evidence of infection was obtained for the 23 animals that were positive by culture, and for at least 7 of those animals with negative or contaminated cultures. These results suggest that the armadillo is a reservoir of this zoonosis.

The eosinophil granule major basic protein, the eosinophil cationic protein, and the eosinophil-derived neurotoxin were found to be lytic for Trypanosoma cruzi trypomastigotes from blood, cell cultures, or insect vectors and for cultured amastigotes. The toxic effects of the major basic and cationic proteins were inhibited by the polyanions heparin and dextran sulfate, in keeping with the cationic nature of these proteins, or by heat denaturation, suggesting that molecular conformation was also relevant. The lytic activity of the neurotoxin was not inhibited by heating at 56°C for 4 hr, establishing an additional difference with the eosinophil cationic protein. Heparin had only a slight inhibitory effect on the toxicity of the neurotoxin, and dextran sulfate was inactive even at 25 mg/ml. Although both the eosinophil cationic protein and the neurotoxin possess ribonuclease activity, only the toxicity of the latter was abolished by the ribonuclease inhibitor RNasin® (Promega, Madison, Wisconsin) or by a competitive substrate, yeast ribonucleic acid. Eosinophil peroxidase significantly increased the extent of trypomastigote or amastigote killing by hydrogen peroxide in the presence of iodide. This effect was abrogated by sodium azide, bovine serum albumin, or gelatin, known inhibitors of the eosinophil peroxidase + halide + hydrogen peroxide system. These results suggest that the destruction of T. cruzi trypomastigotes and amastigotes by eosinophils may result from toxic mechanisms involving several granule proteins.

To describe the epidemiologic and clinical features associated with invasive amebiasis in Bangladesh, 85 hospitalized diarrheal patients with hematophagous trophozoites of Entamoeba histolytica in their stools were compared to a control group of 84 hospitalized diarrheal patients without amebiasis. Postmortem examinations were carried out in 22 deaths due to amebiasis. For the patients with amebiasis, there was a bimodal age distribution with peaks at 2–3 years and >40 years, whereas the control patients had a unimodal distribution with the peak at 0–1 year. The sex distribution was equal in childhood but young adults were predominantly female and older adults predominantly male. The clinical features significantly associated with amebiasis were prolonged dysentery, prior measles rash, malnutrition, hyponatremia, hypokalemia, and hypoproteinemia (all P < 0.05). The case fatality rate in amebiasis was 29%, which was significantly higher than 11% for the controls (P < 0.05). Postmortem findings included extensive colitis with deep ulcers and complications, including colonic perforation in 2 cases, peritonitis in 4 cases, pneumonia in 9 cases, and septicemia in 5 cases. These results indicate that invasive amebiasis in this population differs from other diarrheal diseases, affecting mainly children > 2 years and adults and causing severe and fatal illness characterized by extensive colitis with diverse systemic consequences.

The rate of phosphorylation of 2-deoxyglucose (2DG) was determined by sequential pulsing of schistosomes (male and female Schistosoma mansoni) with [3H- and 14C]-2-deoxy-D-glucose. The relative phosphorylation rate of 2-[3H]-2DG to 1-[14C]-D-glucose (i.e., the phosphorylation coefficient) was also measured in male and female schistosomes. Even though 2DG is taken up more rapidly than glucose, it is phosphorylated at a much slower rate in S. mansoni. Mated schistosomes phosphorylate 2DG and glucose at a greater rate than do unmated worms. In contrast, the phosphorylation coefficient is greater in separated than mated schistosomes. In schistosomes exposed to oltipraz for short time periods (6 min, at a concentration of 10 µg/ml) glucose utilization rates were significantly reduced in (both mated and separated) female S. mansoni and by a similar magnitude (not significant) in males.

Sera of patients with either hookworm infections or ascariasis, who are from areas nonendemic for Schistosoma mansoni, contain antibody reactivity against extracts of S. mansoni schistosomula. Such patients with hookworm, but not those with Ascaris lumbricoides, also express considerable antibody activity against intact schistosomula and schistosomular membrane preparations. Sera from patients with schistosomiasis mansoni also contain these activities. Because these three helminthic infections often occur in the same areas, and concomitantly in patients, anti-schistosomular reactivity should not be ascribed only to exposure to schistosomes. The demonstration and definition of these surface-specific cross-reactivities point out the problems of serodiagnosis, and the potential of interactive influences in the common occurrence of multiple helminthic infections in humans.