The American Journal of Tropical Medicine and Hygiene - Volume 37, Issue 2, September 1987

The results of this study in Thailand indicate that the early response of falciparum infections to a single dose of pyrimethamine-sulfadoxine is influenced by the developmental stages of the parasite present at the time of treatment. Parasite clearance is slower when young rings predominate at the time of treatment. This should be taken into account when considering the clinical management of patients and the comparative efficacy of antimalarials in clearing parasites from the peripheral blood. The 36–48 hr delay in schizonticidal action observed after treatment of febrile infections and the associated decline in blood concentrations of pyrimethamine suggest that a single dose may not be the ideal way of administering this drug combination and may encourage the emergence of drug-resistant parasites.

In June 1986, Plasmodium falciparum parasites were collected from 33 children presenting at the Mama Yemo Hospital in Kinshasa (Zaire) and were successfully tested in vitro by a 48-hr reinvasion test for their susceptibility to various antimalarial drugs. In vitro resistance to chloroquine was found in 82% of the isolates, a marked increase over findings obtained by the same technique 3 years ago in Kinshasa. In vitro chloroquine resistance was not associated with a history of previous drug intake. The inhibitory endpoints for quinine varied from 0.03 to 1 µM, and correlated with the chloroquine endpoints in the corresponding isolates (r = 0.64). Pyrimethamine resistance in vitro was demonstrated in 52% of the isolates tested.

The presence of antibody to the repeating epitope of the circumsporozoite protein of Plasmodium falciparum was determined in children 1 month to 10 years old from three villages in western Kenya using the synthetic peptide (PNAN)5 in an enzyme-linked immunosorbent assay. The percentage of antibody-positive children increased with age and differed in the three villages. The village with the lowest percentage of antibody-positive children had the lowest percentage of infections as determined by detection of blood stage parasites. The villages also differed in the age at which antibody first appeared. In one village, only 12% of the children had antibody by the age of 5; while in the other two villages, 60% and 73% had antibody by 4 years of age.

A newly developed radioimmunoassay for the diagnosis of malaria has been tested in South Africa. The radioimmunoassay is an antibody binding-inhibition assay, based on a monoclonal antibody (D5) cross-reacting with Plasmodium berghei and P. falciparum. Washed solubilized red blood cells were incubated with the antibody and the residual binding activity was tested on antigen-coated microtiter plates. A sample was considered positive if it inhibited binding of the antibodies to an extent exceeding that of the microscopically negative blood samples. Blood was collected on 3 separate occasions from a total of 530 individuals living in a malaria-endemic area and was examined by radioimmunoassay and microscopy. Group I, consisting of 194 samples, yielded 12 samples positive by microscopy and 10 of these (83%) were also positive by radioimmunoassay. One sample in this group was “positive” in the radioimmunoassay but negative on microscopy (false positive). In the 320 samples of group 2, 13 were positive by microscopy and 6 (46%) by radioimmunoassay. Group 3, which included 16 samples preselected as positive by microscopic examination and 16 controls, was examined after 4 weeks storage at −20°C. Twelve samples (75%) were positive by radioimmunoassay. Tests carried out to determine the effect of blood storage on the activity of the antigen indicated that activity was preserved with little loss over a 3-month period.

A DNA probe consisting of 21 base pair repeats obtained from a Tanzanian isolate of Plasmodium falciparum, cloned in pBR322 and labeled with 32P by nick translation was used to detect malaria parasitemia in samples obtained during a malaria survey undertaken in The Gambia. In an initial trial the hybridization assay had a specificity for P. falciparum of 100% and a sensitivity of 68%. False negative results were obtained only on samples with low parasitemia. Assay of red cells collected during an earlier malaria survey which had been stored for 1 year at ™20°C gave a higher level of sensitivity (85%), suggesting a beneficial effect from freezing and thawing. This was confirmed by examining in the same assay red cells processed immediately after collection and after 2 weeks of storage at ™20°C. Freezing and thawing gave a 21% increase in positivity, and a sensitivity of 100% was achieved with the frozen samples. Quantitation of autoradiographs by visual inspection and by scintillation counting gave a reasonable correlation with parasite counts. The DNA hybridization assay has considerable promise as an epidemiological tool.

The Panama II strain of Plasmodium falciparum, acquired at the second passage level in splenectomized Colombian owl monkeys, was adapted to owl monkeys of Panamanian origin. Patent infections were induced in 22 of 27 unaltered and 20 of 21 splenectomized recipients during 19 serial passages. The infections were significantly more virulent in splenectomized than normal Panamanian owl monkeys, however recrudescences in seven normal monkeys achieved peak parasitemias 48 times greater than in the primary attack. These results describe the first reproducible infections of indigenous falciparum malaria in Panamanian owl monkeys.

The susceptibility of Anopheles superpictus for Plasmodium vivax was described quantitatively under laboratory conditions. Of the 697 laboratory females studied in 16 groups, 513 (73.5%) females took a bloodmeal, of which 88.4% developed ookinetes on day 1, 56.1% oocysts between days 3 and 11, and 52.5% sporozoites in the salivary gland on days 15 to 63 post-infection. Sporogony was completed in 10–11 days post-infection. There was no difference in the longevity of uninfected and infected females. Infected females survived an average of 30 days (maximum 63 days). Sporozoites survived up to 50 days in the salivary glands without any observable changes in structure and motility. These data indicate that An. superpictus is an efficient laboratory vector of P. vivax and should not be ignored in future entomological field studies.

Procoagulant activity of erythrocytes infected with Plasmodium falciparum was determined by measuring the effect of infected erythrocytes on the clotting time of platelet-poor human plasma in the presence of Russell's viper venom. We found that erythrocytes infected with P. falciparum enhanced clotting. In the presence of the infected erythrocytes, clotting times were significantly shortened compared to clotting times in the presence of uninfected erythrocytes. Procoagulant activity of infected erythrocytes, especially at high parasitemia, may be a factor in the pathogenesis of disease in falciparum malaria.

Sera collected from 176 individuals residing in three malaria-endemic regions of Cameroon, West Africa, were tested against Plasmodium falciparum isolates from different areas of the world by enzyme-linked immunosorbent assay (ELISA), growth inhibition studies, and immunoprecipitation assays. A wide range of parasite proteins with apparent molecular weights of 14 to 250 kilodaltons (Kd) were immunoprecipitated by the sera. Of these, two polypeptides of 41 and 96 Kd could be associated with parasite growth inhibition in vitro.

The use of recombinant peptides based upon the repeated amino acid sequences of Plasmodium has been proposed for malaria vaccines. By reducing homologies of such peptide vaccines to host proteins, the possibility of autoimmune complications may be reduced, and the effective immune response may be enhanced. The Wilbur and Lipman Wordsearch algorithm was used to identify homologous amino acid sequences between tandemly repeated Plasmodium amino acid sequences and the human and human viral sequences compiled in the National Biomedical Research Foundation database. Six published repetitive immunogenic amino acid sequences from the circumsporozoite (CS) antigen, ring-infected erythrocyte surface antigen (RESA), soluble (S) antigen, and falciparum interspersed repetitive antigen (FIRA) of P. falciparum, and the CS protein of P. vivax, were analyzed by computer. Matches of at least 4 amino acids were found for all sequences. In the database, 29 matches were found for human proteins and 26 matches were found for human viruses with the 6 antigen sequences. Most of the matched proteins, and many of the matched human viruses, are found in blood. The biological significance of these matches remains to be clarified.

Soluble antigens from Leishmania donovani chagasi were studied in terms of their ability to react with sera from human visceral leishmaniasis. Thirty-six polypeptides, with molecular weights ranging from 14,400 to 123,000 were demonstrated by Western blot analysis. An extensive cross-reactivity with sera from patients with cutaneous leishmaniasis and Chagas' disease also was observed. Two polypeptides (Mr 119,000 and 123,000) reacted with all the sera from visceral leishmaniasis patients. When they were electroeluted from gels and evaluated with respect to specificity to the L. donovani chagasi subspecies, these components were expressed in all strains of Leishmania tested, but not in those of Trypanosoma cruzi. These results indicated that these components are shared by Trypanosomatidae of genus Leishmania. The eluted polypeptides did not react with sera from patients with Chagas' disease, indicating the feasibility of using purified antigens to discriminate between the humoral immune responses in T. cruzi and Leishmania infections.

When using a genus-specific monoclonal antibody (83-J3D2) as the primary reagent in an indirect immunofluorescent antibody assay (IFA), intracellular amastigotes of Leishmania were easily identified in 9 of 9 biopsies and in 11 of 12 needle aspirates taken from human lesions. In contrast, only 5 of the biopsies and 4 ofthe aspirates yielded promastigotes upon culture in vitro. Similarly, all but 2 of the aspirates and one-half of the biopsies were reported as negative for parasites when stained with Wright's and hematoxylin-eosin, respectively. Serum antibody titers, ranging from 1:8 to 1:128, corroborated the results of the amastigote detection assays when histopathology and isolation were negative.

These findings support the practicality of using the genus-specific monoclonal IFA in those field situations where it becomes necessary to differentiate leishmaniasis from other skin infections.

Stool specimens from 3,038 children in rural and urban areas near Harare were examined for Giardia lamblia cysts. Preliminary studies, using specimens collected on three consecutive days from 157 known cyst passers, showed that over 89% of infections could be diagnosed on single stool specimens by examination of Gomori-stained smears. The overall prevalence of giardial infection was 19.4% with significantly more urban children (21.1%) passing cysts than rural children (16.7%). In urban areas the highest prevalence was in young (5-6 year) children, while in rural areas, the highest prevalence was in older (9-10 year) children. Of 132 children treated with Entamizole® a metronidazole-diloxanide combination, 127 (96.2%) had ceased excreting cysts by the fifth treatment day. Followup examination of these children showed a high rate of reinfection, with 29.6% excreting cysts during the year following treatment. During the same period 13.3% of previously uninfected children had started passing cysts, while over half of infected, but untreated, children had undergone apparent “self-cure.” Younger children were more likely to be reinfected than older children, and continued excreting cysts for a longer period of time.

Rabbit antisera to 11 Giardia lamblia isolates were reacted with 8 G. lamblia isolates using single antibody ELISA and indirect immunofluorescent antibody techniques (IFA). Using living trophozoite organisms, IFA showed marked surface fluorescence with homologous antisera-organism pairs while heterologous pairs showed reduced or no reactivity. Using formalin -fixed trophozoites, the pattern of fluorescence changed to include diffuse internal fluorescence with both homologous and heterologous pairs. In contrast to the variability in surface fluorescence, similar reactivity was noted for homologous and heterologous antisera-isolate pairs with the ELISA. In addition, a double antibody enzyme immunoassay using rabbit antisera prepared to 2 G. lamblia isolates was performed with 8 different isolates as test antigen. All 8 were equally well detected. These data confirm previous findings that G. lamblia isolates have both different and common antigens. The different antigens appear to be on the surface of the organism while the common antigens appear to be internal or somatic.

Fifty sera from 17 cases of invasive amebiasis (15 hepatic localizations, ameboma, 1 diffused colic localization) were studied by enzyme-linked immunofiltration assay (ELIFA). IgM and IgE anti-Entamoeba histolytica were detected in 10 and 15, respectively, of the 17 patients studied. IgM and IgE antibodies were found simultaneously in 10 cases; these 2 isotypes were only recognized twice in the same serum by the same antigenic components. During post-therapy monitoring, in less than 90 days IgE or IgM-Ab regressed completely in half the cases. If they appeared or reappeared after the third month, prognosis was bad. In addition to its value for diagnosis and prognosis, the ELIFA allowed us to detect the functional antigenic components revealed more particularly by some IgG, IgM, IgE, or IgA antibodies.

The isoenzyme patterns of 92 isolates of Entamoeba histolytica from British Columbia and 28 from Ontario were determined. Seropositivity for E. histolytica was assessed by indirect hemagglutination and enzyme-linked immunosorbent assay in the one center and by ELISA and amebic gel diffusion in the other.

In both British Columbia and Ontario nonpathogenic zymodemes I and III were most common. A newly described isoenzyme pattern was identified in Ontario. Only 9 of 120 zymodeme patterns identified were found to be pathogenic strains of E. histolytica. Pathogenic isolates were strongly correlated with clinical symptoms and seropositivity.

Trypanosoma cruzi was isolated from 98 patients, 59 Triatoma infestans, 51 Triatoma spinolai, and 1 Octodon degus from northern Chile. With few exceptions, stocks originating from domestic hosts were classified, based on their isozyme profile, as principal zymodeme (Z)2, while sylvatic stocks from T. spinolai and the rodent O. degus showed Z1 profiles. These results indicate the existence of separate domestic and sylvatic transmission cycles.

Clinical data and T. cruzi isozyme profiles from 107 chronic Chagas' disease patients showed no association between infecting T. cruzi zymodeme and the prevalence of chagasic cardiopathy. However, the age distributions of two groups of patients carrying different zymodemes were significantly different.

In vitro studies against epimastigotes and intracellular amastigotes and in vivo studies in inbred C3H/He mice infected with Trypanosoma cruzi Brazil strain were performed to determine the activities of two N-substituted imidazole compounds, RS- 49676 and ketoconazole. Both compounds are extremely active in vitro against the amastigotes (ED50 <0.0001 μg/ml) yet inactive against the epimastigotes. In vivo, RS-49676 increased the mean survival time over 11 weeks beyond the untreated control when given subcutaneously twice daily at 100 mg/kg/day. Ketoconazole increased the mean survival time 11-18 days beyond the untreated control (mean survival time 22 days) when given subcutaneously twice daily at 100 mg/kg or orally once daily at 100 mg/kg. Approximately 20%-25% of the RS-49676 treated mice were cured as determined by culturing the blood of infected mice with fibroblast lung cells. None of the ketoconazole mice were cured.

Tyrosine aminotransferase (TAT), glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), and alkaline phosphatase (ALP) were measured in the serum and livers of Microtus montanus infected with Trypanosoma brucei gambiense. Only liver TAT and serum ALP showed significant changes. In addition, blood glucose, pyruvate and lactate, and liver glycogen levels were assayed. All four compounds showed significant changes, strongly suggesting increased glycogen mobilization and increased catabolic activity. Interestingly, the serum ketone levels were very low and no significant changes were observed. These chronically infected animals had an organic aciduria in which pyruvate, lactate, β-hydroxybutyrate, α-ketoglutarate, phenylpyruvate, and p-hydroxyphenylpyruvate were significantly increased. The possible significance of these observations is discussed.

Antibodies to a cysteinyl proteinase of the trematode Schistosoma mansoni have been detected in serum from infected mice and humans. We have evaluated antiproteinase responses in infected baboons and in baboons vaccinated with irradiated, cryopreserved schistosomuJes prior to challenge. Prechallenge sera and normal, uninfected control sera were nonreactive by ELISA and immunoblots. Serum antibodies were first detectable by ELISA at two months post-challenge in both challenged (C) and vaccinated-challenged (V-C) baboons (serum dilution 1:200). By four months post-challenge, ELISA absorbance values for subgroup C baboons were significantly higher than for V-C counterparts.

The immunoblot technique provided a more sensitive means of detecting antibody early in the infection. One month post-challenge, 7 of 12 C and V-C sera (diluted 1:100) contained measurable anti-proteinase antibody. By month two, 12 of 12 were immunoblot-positive. Baboons vaccinated but not challenged (subgroup V) remained negative. The presence of the anti-proteinase antibody appears to be a sensitive and early marker for infection by S. mansoni.

The effect of schistosome infection on in vivo gluconeogenesis was studied in mice. Mice infected for 6 weeks displayed no nutritional pathology, regardless of nutritional treatment. All mice with patent infections (9 weeks) displayed decreased rates of growth and food consumption as well as significantly reduced gross conversion efficiencies.

The glucogenic capacity, % pyruvate incorporated/g liver, was markedly reduced in infected fasted mice when compared with fasted controls. Total glucogenesis, % pyruvate incorporated/100 g body water, was the same in infected and control groups and the compensatory response in infected individuals was correlated with increased liver mass. The level of hepatic glycogen was significantly greater in infected fasted mice than in their unparasitized counterparts.

Patterns of fibrosis within hepatic and intestinal granulomas of Schistosoma mansoni-infected mice were analyzed by indirect immunofluorescence. Deposition of collagen isotypes, laminin, and fibronectin was evaluated semiquantitatively between 8 and 20 weeks of the infection. Liver granulomas were the largest at 8 weeks and contained low amounts of type I and higher amounts of type III collagen and fibronectin. Collagen deposition became pronounced as infection progressed. The relative amounts of type I collagen deposits rose and equalled that of type III. In the smaller immunomodulated granulomas at 20 weeks both types I and III were high, and type IV collagen deposition was observed. Fibronectin and laminin deposits were also detected. The small ileal granulomas did not change their size during the course of the infection. At 8 weeks, connective tissue matrix deposition was barely detectable within these lesions. Gradually, small deposits of types I and III appeared in equal amounts and attained highest levels by 20 weeks of the infection. Fibronectin deposits at that time were very prominent but laminin and type IV collagen were absent. Colon granulomas at 8 weeks of the infection were only somewhat smaller than those of the liver, yet contained very sparse deposits of types I and III collagen. During the ensuing weeks collagen deposits rose only slightly. By 20 weeks the granulomas diminished in size and within those lesions type III collagen was predominant. Whereas the presence of fibronectin was pronounced, type IV collagen and laminin were detectable only in trace amounts. These observations indicate the existence of important organ-related differences in the intragranulomatous deposition of connective tissue matrix.

This study describes three monoclonal antibodies against the excretory system of Schistosoma mansoni. Immunofluorescence revealed antigens forming part of the excretory system of cercariae, adult worms, and miracidia, which were located on the luminal membranes of flame and first tubule cells by immunoelectron microscopy. These antigens are either structural components of the membranes or they derive from excretory fluid and are absorbed during transport and ultrafiltration. Binding specificity of the monoclonal antibodies was tested by immunoelectrophoresis and competitive immunofluorescence; one or two antigens were recognized by each. Reactivity of the antigens after treatment with 7.5% trichloroacetic acid or Rossman's fixative demonstrates at least partial polysaccharide content.

A nodule removed from the cecum of a 25-year-old Zairian man contained a degenerated adult nematode. The surrounding tissue contained larvae and eggs in various stages of cleavage. Eggs and larvae were indistinguishable from those of Angiostrongylus costaricensis. These morphological features are described. The diameter and cuticle, and the anatomic location of the adult worm is consistent with A. costaricensis. The tissue reaction was chronic with granulomatous inflammation and numerous eosinophils. This is the fust report of abdominal angiostrongylosis of a human in Africa.

Two cases of subcutaneous dirofilariasis acquired in Ontario and Vermont are reported. The parasites in these and eight cases previously reported from the northern United States and Canada are classified as resembling Dirofilaria ursi, a primarily subcutaneous parasite of bears, or D. subdermata of porcupines, in the same region. A distinguishing morphological feature of the D. ursi-like group is the presence of distinct longitudinal cuticular ridges regularly and widely spaced on the outer surface, and usually evident even when the worms are necrotic. In the 10 known cases, all patients were women, and the usual location of nodules containing the worms was the scalp or a covered part of the upper body where blackfl.ies, the intermediate hosts of D. ursi, normally feed.

Calves were immunized twice in 4 weeks with a Fasciola/Schistosoma cross-reactive, cross-protective defined immunity antigen (denoted FhSmIII(M) isolated from F. hepatica adult worm extracts by antibody affinity chromatography and challenged 7 weeks later with F. hepatica metacercariae. F1ukes were recovered at 16 weeks of infection at which time the immunized calves had 55% less F. hepatica than the controls. All of the immunized calves developed high antibody levels of FhSmIII(M), detectable in the ELISA, by 4 weeks after a single immunization. By 9 weeks of infection with F. hepatica the immunized calves had lower sorbitol dehydrogenase levels than the unimmunized, F. hepatica-infected control calves, indicating less liver damage in the vaccinated group. These studies demonstrate that subcellular F. hepatica macromolecules cross-reactive and cross-protective against S. mansoni also have the potential to serve as vaccines in cattle exposed to this parasitic disease.

Third stage larvae of Wuchereria bancrofti obtained from laboratory-infected mosquitoes grew and molted to the fourth stage in vitro. The culture medium which supported the best growth and development consisted of a 1:1 mixture (v/v) of two commercially available cell culture media, NCTC 135 and Iscove's modified Dulbecco's medium supplemented with 10% human serum or plasma and an antibacterial/antimycotic mixture. Cultures were incubated at 37°C in an atmosphere of either 5% or 8% CO2 in air. After 35 days of culture, 65% to 100% of the larvae were fourth stage. They were motile and in excellent morphological condition with development of the reproductive system in males and females. This culture system will provide an important tool for biochemical and immunological studies.

A genomic library of a savanna isolate of Onchocerca volvulus was screened to detect recombinant plasm ids containing highly repeated DNA sequences of this parasite. Four recombinant plasmids were identified which hybridized specifically to Onchocerca DNA, but not to DNA from humans, black flies, Brugia malayi, B. pahangi, or Wuchereria bancrofti. The recombinant plasm ids had a low level of homology to Dirofilaria immitis. All recombinant plasmids contain related DNA sequences based on Southern hybridization analysis. Sequences related to these recombinant plasmids are present in different geographic isolates of O. volvulus and O. ochengi, an animal parasite. Two of the recombinant plasmids contain sequences also found in O. lienalis. One recombinant plasmid, puOvs3, has been characterized in detail, including DNA sequence determination. Radiolabeled puOvs3 is able to detect 100 pg of genomic DNA isolated from O. volvulus worms from both savanna and forest regions. It can differentiate O. volvulus from O. ochengi by Southern blot analysis.

Three African green monkeys (Cercopithecus aethiops) were inoculated intravenously and intracutaneously with Mycobacterium leprae derived from a naturally infected mangabey monkey. All developed cutaneous lesions at inoculation sites. One developed disseminated cutaneous lesions, while the cutaneous lesions in the other two regressed and eventually disappeared. The animals were examined at necropsy five years after inoculation. All three had active leprosy infection in peripheral nerves with extensive inflammation and fibrosis. The disease histologically resembled borderline-lepromatous leprosy. These findings add a new dimension to animal models of leprosy.

Seven monoclonal antibodies were prepared against a South African strain of Crimean-Congo hemorrhagic fever (CCHF) virus and were found to be directed against viral nucleocapsid protein. Five of the monoclonal antibodies reacted to high titer in indirect immunofluorescence (IF) tests and enzyme-linked immunosorbent assays (ELISA) with 22 strains of CCHF virus and failed to cross-react with the closest antigenic relative of CCHF, Hazara virus, or with 4 other nairoviruses which need to be distinguished from CCHF virus in Africa. These antibodies, used in the IF technique, readily detected antigens induced by all strains of CCHF virus included in the study in cell culture monolayers and mouse brain tissue, which represent the systems commonly used for isolation of CCHF virus. The IF technique with monoclonal antibodies constitutes a rapid and specific means of identifying newly isolated strains of CCHF virus.

Male Aedes albopictus experimentally infected with dengue virus types 1, 2, 3, or 4 transmitted their infection sexually to female Ae. albopictus. Such transmission was enhanced if the females had taken a bloodmeal 2 to 7 days prior to mating. Male Ae. albopictus also transmitted dengue virus vertically to their F, progeny. Infected progeny were found among those derived from eggs laid ≥73 hr after mating but not among those derived from eggs laid prior to that time. This suggests that virus probably was not transmitted directly to ova but, rather, underwent prior replication in the female genital tract. Female Ae. albopictus experimentally infected with dengue type 1 virus did not transmit their infection sexually to males. This finding supports the hypothesis that male mosquitoes naturally infected with dengue virus acquired their infection vertically.

The effect of Rift Valley fever (RVF) viral infection on the survival of female Culex pipiens was examined. In 3 experiments in which mosquitoes ingested RVF virus, there was a 44% decrease in survival to days 14–16 for transmitting vs. nontransmitting mosquitoes, and a 48% decrease in survival for individuals with disseminated vs. nondisseminated infections. These results were corroborated by other experiments in which survival of mosquitoes intrathoracically inoculated with RVF virus was compared with that of those inoculated with diluent. In both the per os and inoculation tests, uninfected mosquitoes survived significantly longer than infected mosquitoes. Even though mosquitoes with disseminated infections had a lower survival rate than did uninfected mosquitoes, dissemination and transmission rates were similar at days 7 and 14–18 after the infectious bloodmeal. This suggests that nondisseminated individuals were developing disseminated infections and becoming capable of transmitting virus between days 7 and 14–18 at approximately the same rate older transmitters were dying. The decreased survival associated with RVF viral infection should be considered in predictive models of this disease.