The American Journal of Tropical Medicine and Hygiene - Volume 35, Issue 5, September 1986

This Editor's Page had been reserved for a report on Plasmodium falciparum resistance to chloroquine, mefloquine, and quinine in scattered foci in several countries in West Africa.

The manuscript was received late and exceeded the specified length. It had to be shortened. On 16 September the authors expressed dissatisfaction with the results of the Editor's red pen and in consequence I have withdrawn the report.

During the period from May 1983 to July 1985 we conducted an epidemiological study to determine potential vectors of malaria in 6 districts in the state of Pará in northern Brazil. The examination of random human blood smears, prepared at the time of mosquito capture, indicated overall human infection rates of 16.7% and 10.9% for Plasmodium falciparum and P. vivax, respectively. Two immunoassays, the immunoradiometric assay (IRMA) and the enzyme-linked immunosorbent assay (ELISA), based on the use of species-specific antisporozoite monoclonal antibodies, were used to analyze a total of 9,040 field-collected Anopheles mosquitoes for plasmodial infection. P. falciparum sporozoite antigen was detected in A. darlingi at rates varying from 2.7% to 4.2%, and in small numbers of A. oswaldoi collected in 1 of the districts. In contrast, sporozoite antigen of P. vivax was found in A. darlingi, A. triannulatus, A. nuneztovari, and A. albitarsis at rates ranging from 0.9% to 12.0%. By dissection, sporozoites were found in the salivary glands of these same 4 species at rates ranging from 0.8% to 2.2%. The latter 3 species had not previously been implicated as malaria vectors of any significance in northern Brazil.

We tested the hypothesis that cerebral malaria is caused by blood-brain barrier inflammation and cerebral edema. In a group of 157 Thai patients with strictly defined cerebral malaria, cerebrospinal fluid (CSF) opening pressures were normal in 79% and were lower in fatal cases than in survivors (means ± 1 SD, 144 ± 58 and 167 ± 51 mm CSF, respectively, P = 0.051). CSF: serum albumin ratios (× 103) in 39 of them were significantly higher than in 61 British controls (medians 8.5 and 5.5, respectively, P = 0.04), but were no higher in 7 fatal cases. In a group of 12 patients this ratio was not significantly higher during coma than after full recovery (means ± 1 SD, 9.0 ± 6.2 and 6.7 ± 4.2, respectively, P > 0.1). CSF α2-macroglobulin concentrations were always normal. CSF: serum 77Br- ratios were elevated in 11/19 comatose cases but fell to normal 4 to 9 days later in 11/11 cases. Dexamethasone treatment had no significant effect on bromide partition. The percentage of an intravenously administered dose of 125I-human serum albumin detectable per ml of CSF 6 hr after intravenous injection was 2.4 ± 1.3 × 10-5 in 14 comatose patients and 4.4 ± 4.0 × 10-5 in 9 of them during convalescence (P > 0.1). These results demonstrate that the blood-CSF barrier is essentially intact in patients with cerebral malaria and give no support to the idea that cerebral edema is the cause of coma.

Human retroplacental serum (RPS) containing polyamine oxidase inhibited the growth of the Camp strain of Plasmodium falciparum in vitro as assayed by the parasite's decreased incorporation of 3H-hypoxanthine. Inhibition was dose-dependent on the concentrations of serum polyamine oxidase and added polyamines. Almost complete inhibition was seen in 96-hr asynchronous cultures containing 10% RPS and in those containing 1.2% RPS plus 50 µM polyamine. Subtle morphologic changes in mature stages and decreased numbers of new rings were associated with inhibition seen in 19-hr synchronous cultures initiated at the trophozoite stage. These incubation times were longer than in previous reports showing inhibition of malaria parasites by bovine polyamine oxidase but not by human polyamine oxidase. Macrophages contain polyamine oxidase, the reaction products of which are known to be similar to those of RPS polyamine oxidase but different from those of bovine polyamine oxidase. It remains to be determined whether human polyamine oxidase, acting upon ubiquitous polyamines, contributes to host defenses against malaria.

When schizont-infected erythrocytes were incubated with N-acetyl glucosamine coupled to bovine serum albumin (GluNAc-BSA), the number of new ring forms which appeared several hours later was reduced and the number of abnormal and unruptured schizont-infected erythrocytes was increased compared with controls, indicating that GluNAc-BSA prevents invasion by a toxic effect on schizonts rather than by receptor blockade. Invasion of erythrocytes by Plasmodium falciparum was inhibited by a monoclonal antibody against glycophorin A, but inhibition also occurred with P. knowlesi, a parasite that is known to invade independently of glycophorin A. Inhibition of invasion with anti-glycophorin A is unlikely to be related to receptor blockade and is probably related to decreased deformability of the erythrocyte membrane caused by the binding of this antibody. Previous studies suggesting that GluNAc-BSA and anti-glycophorin A antibodies inhibit invasion by receptor blockade should be reevaluated. Erythrocytes deficient in glycophorin C and band 4.1 were also resistant to invasion by both P. falciparum and P. knowlesi.

Measurements of mean corpuscular hemoglobin (MCH) in Plasmodium falciparum-infected red cells cultured in vitro revealed that malarial pigment (hemozoin) interferes with a true estimate of the actual hemoglobin content in Drabkin's reagent. When the hemozoin pigment was removed by passage of the lysate over a Biorex 70 column, a lower MCH value was obtained which allowed one to estimate that, under these conditions, the parasite consumes about 25% of the red cell's initial hemoglobin. Because spectrophotometric examinations of infected red cell lysates in Drabkin's reagent detect the unchanging heme content of infected red cells (hemoglobin + hemozoin), it can be used for expressing enzymatic activity or metabolite content. Results agree with simultaneous measurements on a per cell basis. However, it is suggested that instead of per gram hemoglobin, the activity should be stated as per mmole (or µmole) heme pigment. The ability to estimate accurately the consumption of intracellular hemoglobin will be useful in metabolic and pharmacologic studies of the parasite/red cell interaction.

A radiolabeled synthetic oligonucleotide was evaluated as a diagnostic probe specific for Plasmodium falciparum using blood samples lysed directly on nitrocellulose filters. The probe technique successfully diagnosed malaria in experimentally infected chimpanzees that had 0.001% parasitemias (50 parasites/µl) as determined by blood smears, and in 1 chimpanzee whose blood smear was negative, but whose blood was culture-positive for P. falciparum. In a double blind study of 50 patient samples from the Philippines, the probe results correlated well with blood smear results when the autoradiographs were read after 4–8 hr exposure. The results indicate that the oligonucleotide probe may be useful in the rapid and specific diagnosis of P. falciparum infection.

An indirect immunofluorescence (IIF) test was performed with human sera to detect cross-reactivity of Babesia microti antibodies with other species of Babesia parasites, with other blood and tissue parasites, and with various tick-borne organisms. Antisera to B. microti cross reacted with other Babesia species, but at lower dilutions than with the homologous antigens, and occurred most often during the acute phase of the disease. Cross-reactions with antibodies to malaria, Colorado tick fever, and a variety of other parasitic diseases were uncommon. However, acute and convalescent phase sera from 4 patients with suspected or confirmed Rocky Mountain spotted fever showed a rise in titer to B. microti antigen. In addition, 6 of 185 serum samples from children on an Indian reservation in North Carolina had IIF titers of ≥1:256, suggesting a possible focus of B. microti infections in humans.

To test the effects of post-bloodmeal nutrition of sand flies on the transmission of Leishmania major, groups of infected P. papatasi females maintained on diets of sucrose, trehalose, albumin or a mixture of sucrose and albumin, were subjected to forced feeding with capillaries. Transmission was evaluated by counting the parasites egested; numbers ranged from 0 to over 1,000 promastigotes. Infections of the anterior midgut were seen in the majority of flies from all the experimental groups but the percentage of transmitting females was significantly higher in the group maintained on a mixture of sucrose and albumin. There were no attached parasites in the pharynx and cibarium of the flies and the presence of free promastigotes in these parts was not itself indicative of infectivity. However, transmission was positively correlated with apparent inability to engorge. The parasites egested were typical infective form promastigotes and identical to those observed in the esophagus and the anterior thoracic midgut.

A mechanism by which infective stage promastigotes from the esophagus and the stomodeal valve may be transmitted by bite is proposed.

An outbreak of 20 cases of acute Chagas' disease followed the movement of Triatoma infestans into the county of Riacho de Santana, Bahia, Brazil. The outbreak was unusual in that the majority of cases occurred in adults. Vector control measures were implemented. Three years after the outbreak, a rural community was examined to determine the extent of human infection and disease due to Trypanosoma cruzi. Ninety of 440 residents (20.5%) had serologic evidence of infection, but rates of electrocardiographic (EKG) abnormalities were low. Comparison of age-specific rates of seropositivity and EKG abnormalities with rates from areas with endemic Chagas' disease supported the hypothesis of a recent epidemic. Control measures appear to have interrupted transmission in the region.

Past attempts to immunize mice against a fatal Trypanosoma cruzi infection utilizing related hemoflagellates have been unsuccessful. In the present study, C57BL/6 mice received a footpad inoculation of 107 promastigotes of Leishmania braziliensis panamensis. Six and 9 weeks subsequent to this inoculation mice were infected intraperitoneally with the Tulahuen strain of T. cruzi. All immunized mice survived infection over the 6-month period of observation whereas control mice regularly died. There was an early transient T. cruzi parasitemia in the immunized mice. Culture of blood and organs as well as histopathological examination of various organs 6 months post-challenge failed to yield any evidence of T. cruzi. Heat-killed and freeze-thawed extracts of promastigotes did not confer any protection. These observations raise the possibility that certain leishmanial species might confer natural protection against a T. cruzi infection and that this information could be useful in the development of a vaccine.

A liquid medium without blood or serum was developed for cultivation of hemoflagellates. To a basic LIT medium containing liver infusion broth and tryptose, a mixture of RPMI 1640 and Medium 199 was added. This combination permitted high parasite yields useful for biochemical and immunological studies.

Single oral doses of amoscanate (4-isothiocyanato-4′-nitrodiphenylamine), an experimental antiparasitic agent, are highly effective in animals against the four major species of schistosomes which infect humans. Two prospective, randomized, double blinded, placebo controlled Phase I studies were designed to evaluate the tolerance and safety of the 5% aqueous suspension of 2-µ particles of amoscanate administered to healthy male volunteers. In addition to routine safety monitoring, particular attention was directed toward detecting hepatic, neurological, cardiovascular or ocular toxicity. Three of four men who received 3.5 mg/kg of amoscanate developed mild, reversible hepatotoxicity, which could not be unequivocably attributed to the drug. In the second study, of 1 mg/kg amoscanate, there was no statistically significant evidence of hepatotoxicity, although 1 of 12 drug recipients developed transient liver chemistry changes. Despite intensive monitoring, there was no evidence in either study of significant symptomatic complaints, or of neurological, cardiovascular or ocular toxicity. No mutagenic activity attributable to amoscanate was detectable in the urine. These results suggest that this formulation of amoscanate, at 1 mg/kg, is sufficiently well tolerated and safe to justify evaluation for efficacy in patients with schistosomiasis.

Proteinuria was studied in 128 children aged 6 to 18 years with Schistosoma haematobium infection in the People's Republic of Congo. Urinary protein concentration in spontaneously voided midday urine of patients with > 100 ova/10 ml was significantly higher than in 24-hr urine specimens. Median daily urinary protein loss in patients with moderate intensity of infection (100–350 ova/10 ml) was 300 mg and 584 mg/1.73 m2 body surface in heavily infected patients (> 350 ova/10 ml). A significant correlation existed between egg excretion at noon and protein concentration in spontaneous urine samples as well as daily urinary protein loss (r = 0.76 and r = 0.68, respectively). Heavily infected patients had a daily protein loss of up to 3.3 g/1.73 m2, total serum protein and albumin concentration, however, were within normal limits. This may indicate adaptive mechanisms in patients with urinary schistosomiasis and high proteinuria which maintain a balanced serum protein concentration.

Miracidial nuclei within calcified schistosome eggs are frequently mummified. Calcified eggs are often decalcified prior to or during the routine processing and staining of tissues, and mummified miracidial nuclei then stain clearly and are often arranged in the patterns seen in live eggs. These miracidia may be incorrectly assumed to be alive or to have died recently and thus be taken as evidence of an active infection requiring chemotherapy. The present observations demonstrate the decalcification of mineralized eggs by hematoxylin preparations used for routine staining of tissue sections.

School attendance records of all primary schools in a guinea worm-endemic village in southwestern Nigeria were examined to determine the cause of missed school days and school drop-outs. At the time of the survey, 1,495 pupils (768 boys and 727 girls) were registered in the 4 primary schools in the village, of which 21% of the pupils were infected with guinea worm disease (GWD). Female pupils had a higher infection rate than their male counterparts. Guinea worm-infected pupils missed up to 25% of school year days compared to a non-guinea worm-infected absence of 2.5%. At the height of guinea worm season in the study area, guinea worm-related absences contributed virtually all of the absenteeism recorded in the schools. Implications of the findings within the context of educational attainment of the pupils are discussed.

New levels of reproducibility and sensitivity have been achieved in the detection of anticysticercus antibodies in human sera by using cysticercus vesicular fluid as the source of antigens for both ELISA and hemagglutination assays. Reproducibility both between tests on a serum and between similar sera was significantly improved over typical results using antigens from whole parasite extracts. Sera collected from uninfected individuals in endemic areas gave somewhat elevated values over those collected in nonendemic areas. This necessitated the use of a higher threshold in endemic areas to avoid false positives. With the threshold appropriate for a nonendemic area, both ELISA and hemagglutination were sensitive enough to detect infection in 95% of cases. With the threshold value for sera from an endemic area, these sensitivities were reduced to 80%–90%. A prominent 103-Kd protein of vesicular fluid, not related to antigen B, elicited the strongest antibody response in neurocystercotic patients.

The distribution of excretory-secretory antigens of larval Toxocara canis (TEX) in tissue was studied in cases of both acute and chronic experimental murine toxocariasis. Liver samples were collected from BALB/c BYJ mice killed either 2 days (acute) or 8 months (chronic) after intragastric inoculation with infective T. canis eggs. Cryostat sections of liver were stained using biotin-conjugated, rabbit anti-TEX antibody with an avidinfluorescein isothiocyanate indicator. In both acute and chronic infections larval sections were brightly fluorescent. The patterns of antigen deposition in acute infections were indicative of larval migration. In tissue sections from chronic infections, antigens were observed mainly within the core of granulomas centered around larvae and within the inner rim of the collagen capsule of granulomas in which a larva or larval remnant was not detected. When present within the collagenous capsule, TEX appeared to be extracellular. These findings suggest that an accurate histopathological diagnosis of larval toxocariasis may be possible in biopsy or autopsy material lacking an obvious etiological agent.

Vaccination of inbred jirds (Meriones unguiculatus) with 60cobalt radiation-attenuated Brugia malayi infective stage larvae (L3) protected against homologous challenge given either subcutaneously (sc) or by the intraperitoneal route (ip). At necropsy numerous nodules were recovered from the peritoneal cavities of jirds which had been vaccinated sc and challenged ip. Histopathologic analysis showed these to be granulomas containing dead and dying larvae and transmission electron microscopy showed that eosinophils were present in high numbers around and within the larvae. Structural damage to the L3 cuticle was apparent in discrete areas and eosinophils actively entering the breached cuticle at the time of fixation were observed. Coalescence of eosinophil secretion granules and the formation of degranulation vacuoles were seen in eosinophils throughout the granulomas. Degranulation resulted in the deposition of electron-dense material on the surface of the larval cuticle. The jird vaccine model for B. malayi thus appears to be a potentially useful tool for investigation of immune mechanisms in filariasis.

A 4.5-cm male Dirofilaria indistinguishable from D. immitis was removed from the tip of the index finger of an 18-year-old farmer in Costa Rica. The case is reminiscent of a report of a larger (9-cm) but otherwise similar Dirofilaria, identified as D. spectans, a heartworm of otters, causing Raynaud's syndrome by occluding an artery of the fifth finger of a farm woman in Brazil. As in the present case, the worm was removed from a painful papule on the volar surface at the extreme tip of the affected finger.