The American Journal of Tropical Medicine and Hygiene - Volume 34, Issue 4, 1985

A Pan American Health Organization (PAHO) workshop, held in 1962 and just recently reported was concerned with malaria in the Americas. The principal reason for holding the workshop was the recognition of the failure of present programs to control the malarias in the Americas, particularly falciparum malaria. The prime causes claimed were insecticide failure and parasite drug resistance, a combination that the participants seem to infer led to a reorientation of antimalarial measures by the World Health Organization in 1969 and by PAHO in 1982.

There was considerable discussion of Plasmodium falciparum resistance to the 4-aminoquinolines and to mefloquine. The participants also took note of the increasing number of cases that were failing to respond to a sulfadoxine-pyrimethamine combination.

Much attention was paid to the apparent absence of 4-aminoquinoline resistance in much of Central America (less Panama) and in Hispaniola.

Members of the American Society of Tropical Medicine and Hygiene, the Royal Society of Tropical Medicine, and guests, it is my privilege to have been asked to introduce Dr. Karl M. Johnson, President of the American Society. This is the fourth time I have been asked to introduce a president of this Society and it was with some relief that I noted Paul Weinstein, a parasitologist, is the President-Elect. I doubt that he will ask me to introduce him next year, and especially after I finish with Karl today.

My first contact with Karl was in the 1960s when the National Institute of Allergy and Infectious Diseases sent him to Berkeley from a meeting in Bethesda — the mission being to convince me that I should chair a Contract Review Committee on Arbovirus Reagents. There was some doubt that I would accept this assignment.

Dr. William Reeves' introduction, a searing yet loving statement, moves me to propose him as this Society's official roastmaster! It also serves as counterpoint to my remarks today which represent a theme chosen with difficulty, a melody not yet perfected. In fact, I seem to recall while attempting to discuss hemorrhagic fevers with one of his graduate classes that Dr. Reeves advised, “Don't just talk — say something.” I intend to try.

Faced with the need to say something, I decided that I must choose among three principal interests: zoonotic virology, the ethos of tropical medicine, or this Society itself. Like all but one of my predecessors, Dr. Martin Young, who was numero uno and therefore immune to historical influence, I examined the record. A frequency chart by theme is shown in Table 1. Note that science has been the first choice, and that virologists have been slightly, but not significantly, more likely than parasitologists to discuss philosophy.

A rapid and simple assay for detecting Plasmodium falciparum in human blood was developed. The assay is based on DNA-DNA spot hybridization, using radiolabeled P. falciparum DNA as a probe and finger prick blood as the assay sample. It is very sensitive, able to detect parasitemia levels of 0.0001% in 10 µl of blood. The assay can be quantified and used to estimate parasitemia levels. Several hundred blood samples can be processed simultaneously, and the entire procedure is completed within 24 hr. This assay can be useful for epidemiological surveys, for screening of blood by blood banks and for health authorities examining immigrants and tourists coming from malaria infested areas.

A variety of known or suspected inducers of crisis form parasites in cultivated Plasmodium falciparum were examined. Sera from Sudanese residents of malaria-endemic areas, sera from American tuberculosis patients, and rabbit sera containing tumor necrosis factor were assayed in vitro for cytotoxic activities against P. falciparum and mouse L-M cell cultures. Inhibition was determined by measurement of incorporation of radiolabeled nucleic acid precursors. When compared to normal serum, parasites grown in the presence of a 1:4 dilution of rabbit sera containing tumor necrosis factor, TB patient sera, or Sudanese sera were metabolically inhibited 73%, 75%, and 95%, respectively. However, only the rabbit sera containing tumor necrosis factor were cytotoxic to L-M cells, inhibiting radiolabel incorporation by 80% at a 1:1,000 serum dilution. These findings suggest that tumor necrosis factor is apparently not responsible for the induction of parasite crisis forms by the inhibitory human sera tested. In addition, human gamma-interferon had no effect on parasite growth.

Sensitivity of Plasmodium falciparum to several antimalarial drugs was determined by in vitro and in vivo tests. Chloroquine resistance in vitro was detected in 97 of 101 patients from different geographic areas of Colombia. Sensitivity to amodiaquine in vitro was observed in 29 of 30 P. falciparum isolates. In vitro sensitivity to amodiaquine was observed in 16 patients infected with chloroquine-resistant P. falciparum. In vitro sensitivity to quinine was demonstrated in 57 P. falciparum isolates. Two infections from the Amazon base (2/24) were resistant to mefloquine in vitro at concentrations of 5.7 and 16 pmol/well.

Resistance to Fansidar®, a sulfadoxine-pyrimethamine combination, was described in 9 patients from the Amazon region. One patient showed recrudescence of the infection 41 days after treatment.

The current distribution and degree of resistance of P. falciparum to widely used antimalarial drugs requires the evaluation of therapeutic schemes based on combinations of fast blood schizontocides with slow acting drugs. These associations may reduce the development of multidrug-resistant isolates and retard the spread of resistant populations of P. falciparum parasites.

Plasma protein binding of quinine was measured in 12 patients with cerebral malaria on the first and seventh day of treatment, and in 7 patients with uncomplicated falciparum malaria on admission and also one month later. Binding was significantly higher and therefore the proportion of free drug was lower in cerebral malaria patients (free:total quinine concentration; 7.2 ± 3.5%, mean ± SD, on admission; 7.4 ± 5.3% on day 7) compared with uncomplicated malaria patients on admission (10.2 ± 5.8%) or following recovery (11.0 ± 5.5%, n = 6) P = 0.011. Binding was significantly correlated with the red cell/total concentration ratio r = 0.56, P < 0.0001. The ratio of cerebrospinal fluid to free (unbound) plasma quinine was 0.55 ± 0.33 which suggests that quinine does not freely cross the blood brain barrier. These findings are relevant to the interpretation of total plasma or serum concentration, and may explain the rarity of serious quinine toxicity in severe falciparum malaria.

Oocysts of Plasmodium knowlesi developed normally on the gut of the mosquito Anopheles freeborni, rupturing and releasing sporozoites at 10–14 days post-infection. Subsequently, however, sporozoites were never found in this mosquito's salivary glands. Heterologous transplants of whole salivary glands from uninfected An. freeborni and An. dirus, a completely susceptible mosquito, into the abdomens of insects heavily infected with mature oocysts were done. Sporozoites failed to infect An. freeborni glands implanted in An. dirus but did enter An. dirus glands implanted in An. freeborni. These experiments suggest that P. knowlesi sporozoites are unable to recognize An. freeborni glands.

In a volunteer with infection induced by injection of the mefloquine-sensitive, multidrug-resistant Vietnam Smith isolate of P. falciparum, parasitemia recurred following treatment with the candidate antimalarial drug enpiroline. Parasitemia also recurred after subsequent treatment with mefloquine and again after retreatment with the same drug. All recurrences were at the RI level. Parasite drug sensitivities determined by a semi-automated isotope microdilution method after the second and third recurrences revealed a progressive decrease in sensitivity to all arylaminoalcohols tested (halofantrine, enpiroline, and mefloquine). Decreased sensitivity persisted after 30 days of isolate culture. The parallel changes in parasite sensitivity to the synthetic arylaminoalcohols argue for development of drugs which are chemically dissimilar.

Hybridoma cell lines secreting monoclonal antibodies against the Tulahuén strain of Trypanosoma cruzi were produced by the fusion of SP2/O-Ag14 myeloma cells with spleen cells from mice immunized with irradiated metacyclic trypomastigotes. Twenty of the monoclonals synthesized by the hybridomas were identified as IgM, 2 as IgG1, 10 as IgG2a, 3 as IgG2b, 4 as IgG3, 1 as IgE, and 1 as IgA. Twenty-three of these antibodies had κ light chains and 18 showed λ chains. Twelve of the monoclonals agglutinated metacyclic trypomastigotes without additional concentration and 4 of these precipitated antigens in extracts of T. cruzi metacyclic or epimastigote stages. One monoclonal precipitated an epimastigote antigen, while another reacted with a metacyclic antigen, and 2 antibodies formed precipitin lines with antigens of both stages. Agglutinin assays performed to characterize surface antigenic specificities of the 12 monoclonal antibodies showed that 2 reacted only with the metacyclic stage of the Tulahuén strain. Two monoclonals agglutinated both metacyclic trypomastigotes and epimastigotes of the Tulahuén strains. Three antibodies caused clumping of metacyclics and epimastigotes of the Tulahuén, Raccoon V, and Corpus Christi strains of T. cruzi, while a fourth also reacted with bloodstream trypomastigotes. One monoclonal detected identical epitopes on metacyclics and epimastigotes of T. cruzi and epimastigotes of Trypanosoma musculi, while 2 antibodies reacted with metacyclics, epimastigotes, and bloodstream trypomastigotes of the Tulahuén and Raccoon V strains and the bloodstream stage of T. musculi. One antibody agglutinated all stages and strains of T. cruzi, T. musculi, and Trypanosoma lewisi which were tested. None of the 12 monoclonals reacted with amastigotes of T. cruzi. The precipitin reactions between the monoclonals and parasite extracts indicate that more than 1 identical epitope is present on each precipitating antigen molecule, while agglutinin analyses demonstrated antigenic specificities and relationships among stages, strains, and species of these trypanosomes.

Serial electrocardiograms (ECGs) were obtained during 65 courses of sodium stibogluconate treatment in 59 Kenyan patients with leishmaniasis (56 visceral and 3 cutaneous). ECG abnormalities developed during 54% of the treatment courses. The frequency with which abnormalities occurred was related to the total daily dose of antimony (Sb), increasing from 2/9 patients treated with 10 mg Sb/kg/d to 25/48 treated with 20–30 mg Sb/kg/d and 8/8 treated with 40–60 mg Sb/kg/d. The frequency with which ECG abnormalities developed was also related to the duration of treatment, increasing from 11/65 patients after 7 days to 18/44 after 15 days, 26/39 after 30 days and 11/12 after 60 days. ECG abnormalities were similar to those previously described during treatment with trivalent antimonial drugs, the most common being flattening and/or inversion of T waves. Prolongation of the corrected QT interval occurred in 13 patients, all of whom were treated for more than 30 days or with more than 20 mg Sb/kg/d. One patient died suddenly during the fourth week of treatment with 60 mg Sb/kg/d, and 2 patients died of measles after 9 or 10 days of treatment with 30 mg Sb/kg/d. QT prolongation and a concave ST segment developed in all 3 patients who died. We conclude that minor ECG abnormalities are common when sodium stibogluconate is used at doses above 20 mg Sb/kg/d for more than 15 days, and that life-threatening arrhythmias may occur if very high doses are used.

The treatment of two patients with severe mucosal leishmaniasis due to Leishmania braziliensis braziliensis is described. Both patients had received much prior antimonial therapy and one had relapsed after a total dose of 2.5 g of Amphotericin B. Both patients responded to prolonged continuous Pentostam therapy at a daily dose of 20 mg Sbv/kg/day for 62 days in one case and for 85 days in the other. Pentavalent antimonials can be curative in such protracted courses in selected patients unresponsive to standard chemotherapy.

It is generally held that with rare exception Leishmania braziliensis braziliensis is the parasite responsible for the metastatic development of mucocutaneous leishmaniasis in the New World. Yet the infrequency of mucocutaneous disease compared with cutaneous manifestations, and the difficulty of isolating parasites from mucocutaneous lesions have restricted the study of the organisms involved. We here report the biologic, isoenzymatic, and monoclonal antibody specificity characteristic of eight Leishmania isolates obtained from the mucosal lesions of the same number of patients. Individually and collectively, the identifying criteria implicate at least two L. braziliensis subspecies as etiologic agents of mucocutaneous leishmaniasis in Colombia and suggest that a spectrum of intrinsically distinguishable organisms may be involved in this disease form.

Thirteen of 120 Venezuelan children with acute diarrhea were found to be excreting Cryptosporidium oocysts in their stools. This confirms that Cryptosporidium can infect immunocompetent children, and the relatively high frequency found suggests that this protozoan may be an important cause of diarrhea in Venezuela.

A new experimental model for the production of early stages of invasive intestinal amebiasis in hamsters or guinea pigs with axenic or monoxenic cultures of Entamoeba histolytica of the HMI:IMSS strain is reported. The model is called the washed-closed cecal loop, because it involves the washing out of the cecum contents of conventionally raised animals and the formation of a closed loop, into which amebas are inoculated. Colonization occurred in all inoculated animals. After 48 hr, macroscopic ulcerations of the cecal mucosa were found in approximately one-half (5/12) of the animals inoculated with axenic amebas and in more than two-thirds (10/12) of those inoculated with monoxenic parasites. The amebic nature of the mucosal ulcerations was confirmed by light and transmission electron microscopy, as well as by scanning electron microscopy (SEM) of fractured samples of cecal mucosa. SEM provided a rapid and reliable technique to assess both the extent and the nature of the lesions. This model is particularly useful for the study of the pathogenesis of early intestinal lesions produced by virulent amebas, and may also be applied to experimental studies on the immunology of invasive intestinal amebiasis.

Natural vectors of onchocerciasis (S. yahense) were collected. Ninety-eight were intrathoracically inoculated with 30 freshly obtained skin-dwelling microfilariae. Of the 82 flies dissected, 56 were infected and 28 harbored infective larvae. A total of 193 infective O. volvulus larvae were obtained. The mean infective worm burden was 6.9.

The human eosinophil granule contains several distinctive cationic proteins that have been purified to homogeneity, including major basic protein (MBP), eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN). Two earlier studies have shown that MBP and ECP both damage schistosomula of Schistosoma mansoni in vitro in a dose-dependent fashion. The present study expands upon these observations by comparing the toxicity of MBP, ECP, as well as EDN when tested at equimolar concentrations (0.03–2 × 10-5 M). On a molar basis, ECP was 8 to 10 times more potent than MBP, and the ECP-mediated killing of schistosomula was qualitatively different than that of MBP. Purified ECP produced complete fragmentation and disruption of schistosomula, whereas MBP produced a distinctive ballooning and detachment of the tegumental membrane. In contrast, EDN was only marginally toxic at high concentrations and caused crinkling of the tegumental membrane. Heating MBP and ECP for four hr at 56°C caused precipitation and loss of toxicity for MBP, but not for ECP. Native MBP (with reactive sulfhydryl groups intact) and stabilized, reduced and alkylated MBP had comparable toxicity. To determine the relative contribution of MBP, ECP and other potentially helminthotoxic eosinophil granule constituents to schistosomulum damage, fractions of acid soluble granule extracts prepared by chromatography on Sephadex G-50 columns were analyzed for toxicity to schistosomula and for MBP and ECP levels by radioimmunoassay. Schistosomula were killed by fractions containing MBP, and to a much lesser and more variable extent by fractions containing EDN and a 21,000 dalton protein, but not by fractions coincident with the elution of ECP, which contained concentrations of ECP below that required to produce significant killing of schistosomula by the purified protein. Therefore, although ECP is a more potent helminthotoxin for schistosomula than MBP on a molar basis, MBP, by virtue of its abundance in the granule, accounts for the bulk of the toxicity in fractions of acid solubilized granules obtained from eosinophils of patients with marked eosinophilia.

Egg excretion of school children with urinary schistosomiasis treated with a single 10 mg/kg dose of metrifonate was monitored over a period of 18 months. At 18 months 68.8% of infected children showed a >90% reduction in egg excretion and 23.7% had ceased excreting eggs. During the study period 25% of children with no evidence of infection at the start of the trial had become infected, while 45.5% of children apparently cured by the metrifonate treatment had recommenced egg excretion. The evidence suggested that reinfection rather than recovery of adult worms was responsible. Children with scanty or light infections, in general, showed increasing egg excretion rates during the following period, while those with heavy or severe infections showed a sustained reduction. In view of this, single dose metrifonate may be a useful approach to mass treatment in a schistosomiasis control program, resulting in significant reduction in egg excretion in those most likely to be important sources of transmission.

Coupled adult pairs of Schistosoma mansoni were incubated in medium containing either peripheral or portal serum from rat, rabbit, hamster or humans. Daily egg production was measured. In all cases egg production was significantly increased for pairs in the presence of portal sera compared with that in the presence of peripheral sera. Fractionation of rabbit portal serum according to molecular weight demonstrated that the most active component(s) were in the range of 2,000 to 50,000. These fractions were as effective in stimulating oviposition as whole portal serum. Conclusions: 1) portal serum factor(s) that stimulate S. mansoni oviposition are present in susceptible and non-susceptible hosts; 2) the molecular weight range for the active components is larger than would be expected for simple carbohydrates, amino acids or free fatty acids absorbed from the gastrointestinal tract.

High oral doses of mebendazole were given for a mean period of 23 months to 22 patients with inoperable alveolar or cystic echinococcosis (Echinococcus multilocularis n = 18, E. granulosus n = 4). Clinical, morphological, biochemical and serological findings and plasma mebendazole levels were monitored. Clinical and biochemical improvement or stabilization was observed in 17 patients but the parasitic lesions did not decrease in size in most instances. One patient died shortly after onset of therapy with hemorrhage of esophageal varices. Three patients with alveolar and one with cystic echinococcosis had evidence of progressive disease such as increase of cholestasis, destruction of lumbar vertebrae and growth of an intraperitoneal cyst. The plasma mebendazole levels (4 hr after the morning dose) of the latter 4 patients were 0.09 ± SD 0.02 µmol/l, while in those with clinical stabilization or improvement it was 0.30 ± SD 0.14 µmol/l (P < 0.001). These preliminary data indicate 1) a good clinical response to chemotherapy in most patients despite unchanged size of the parasitic lesions, and 2) a direct correlation of clinical response with plasma mebendazole levels.