The American Journal of Tropical Medicine and Hygiene - Volume 32, Issue 6, November 1983

The indirect immunofluorescence (IIF) test was used to detect persons with malaria antibody, with or without patent parasitemia, in a campaign to detect imported malaria and to prevent its introduction into the vector in the Sutter-Yuba area of California over the period 1975–1979. In addition to 176 slide-proven imported cases of Plasmodium vivax, 94 other persons reactive serologically but no patent parasitemia was found. Slide and serologic results indicated that the dominant malaria species involved was P. vivax. Seven patients who were both serologically and parasite negative when first tested subsequently reported with patent parasitemia and high antibody titer. One of these quiescent cases had resided in the United States at least 7 months prior to the first negative serology, and for some period thereafter before development of parasitemia and antibody response. Serologic results indicated that some persons had had contact with P. falciparum. When parasitemia is rapid in onset and serum is taken immediately, serologic response may be lacking. For detection of presumptive malaria infections, serology may have certain advantages not available with slide examinations. Blood slide examination and malaria serology are used to best advantage together, but either may be valuable in itself, alone, in efforts toward case detection and transmission control in hypoendemic malaria.

Serum samples from 120 individuals living in a malaria-endemic area, 31 patients with Plasmodium falciparum infection, and 58 healthy blood donors were tested for antibodies against P. falciparum and P. vivax sporozoites. Specific antibodies were determined by the circumsporozoite precipitation (CSP) reaction and indirect immunofluorescent (IFA) tests for IgG and IgM antibodies. It was found that a high proportion of adults living in the endemic area had IFA anti-sporozoite antibodies, usually IgG. Children and healthy donors were either negative or had low antibody titers. A positive correlation was found between IgG antibody titers against P. falciparum sporozoites and those against P. vivax sporozoites. CSP reactivity was demonstrated in 5 of 31 sera from patients with falciparum malaria, and was always associated with a high level of IFA antibodies. The anti-sporozoite antibodies were found to be stage- and species-specific.

Serologic assessment was carried out in a sample of the population living in an area endemic for malaria in which control measures were being applied. The study area was in El Salvador, Central America, and antimalarial activities consisted of the residual application of the pesticide, propoxur, and drug distribution through a combination of active and passive case detection. Indirect fluorescent antibody (IFA) responses to Plasmodium vivax and P. falciparum antigens were determined on four occasions, at approximately 6-month intervals. The study population consisted of one resident from each of 268 widely distributed houses. Each person was treated with a curative course of chloroquine and primaquine at the beginning of the investigation. An increase in the proportion of positive IFA responses (⩾ 1:20) and an increasing number of serologic conversions (i.e., from negative to positive or a 2-fold increase in titer) during the course of the 18-month study confirmed continued transmission of malaria. The presence of parasites in some individuals without associated serologic changes is discussed.

Amodiaquine, a 4-aminoquinoline which has been shown to be effective in treating infections with chloroquine-resistant strains of Plasmodium falciparum, was evaluated against chloroquine-resistant infections in children in Zanzibar, Tanzania, during July 1982. A 25-mg base/kg dosage of amodiaquine produced parasite clearance in 34 of 38 (89%) children in a mean of 2.8 days. When followed for 28 days, 15 of 38 (39%) children were completely cured of their infection as judged by the absence of renewed parasitemia. The parasite clearance rates produced by amodiaquine were significantly higher than those observed in a comparison group of children treated with 25 mg base/kg chloroquine. There was, however, no difference in the cure rates in the chloroquine and amodiaquine groups. Despite the enhanced parasite clearance rate, amodiaquine is not sufficiently more effective against Zanzibari strains of P. falciparum to replace chloroquine. Other alternative drugs must be evaluated to define the optimal malaria therapy regimen on Zanzibar.

In the search for antibodies correlating with host-protective immunity to Plasmodium falciparum in man, sera from individuals in Papua New Guinea were analyzed at the time of infection and in the convalescent period following infection. Titers of antibody were determined by enzyme linked immunoassay (ELISA), and the specificities of antibodies was examined by gel electrophoresis of immunoprecipitates. In the majority of cases, convalescence was associated with an increase in antibody titer and one-dimensional gel analysis of immunoprecipitated biosynthetically labeled parasite antigens demonstrated the variability in specificity of the antibody response in the two types of serum samples from different individuals. A protein of Mr 96,000 which has previously been identified as a candidate host-protective antigen was not clearly seen in immunoprecipitates generated with acute serum, even in samples with high titers of antibody assessed by ELISA. Antibodies to a protein Mr 96,000 were present in some, but not all convalescent sera. Two-dimensional gel analysis was more sensitive in detecting a boost in antibody response to minor antigens (e.g., an acidic protein Mr ≅ 230,000). This approach has not led to identification of antibody specificities to major antigens which are invariably boosted by infection and drug cure, but has identified antibody specificities in acute sera which are inadequate in quantity or quality to inhibit parasite growth.

The cellular and humoral interactions that contribute to protective immunity in Plasmodium yoelii malaria were studied by adoptive transfer of selective cell populations or hyperimmune serum into sublethally irradiated syngeneic C57BL/6 mice. For some experiments pools of mononuclear spleen cells were depleted of T or B lymphocytes and cells that take up silica were inactivated by standard procedures. Unfractionated immune spleen cells, but not nonimmune spleen cells, protected recipients from lethal P. yoelii challenge. Analysis of the protective capacity of subpopulations of immune spleen cells showed that levels of immunity similar to those seen after transfer of unfractionated immune cells were present only in those instances where immune macrophages, i.e., cells not previously inactivated with silica, were transferred concomitantly with either immune T (supplemented with nonimmune B) or immune B (supplemented with nonimmune T) cells. The requirement for immune macrophages could not be met by transferring mononuclear cells from a nonimmune donor. The results support the hypothesis that an immune 5,000 R-radioresistant, silica-inactivated, non-T, non-B cell, probably a macrophage, must act in concert with immune T and B lymphocytes in the optimal expression of transferred immunity to P. yoelii.

The effect of passive antibody transfer against infection with vector-borne, metacyclic forms of Trypanosoma cruzi, and possible mechanisms of immunologic lysis of these organisms, were examined in this work. Anti-T. cruzi antibodies from mice surviving an infection conferred marked protection against challenge with a lethal dose (2,000 organisms) of metacyclic flagellates isolated from the reduviid insect Rhodnius prolixus. In vitro lysis of these parasites by immune sera from mice or chronic chagasic patients was found to require complement (C) activity since the phenomenon was abrogated by heating the sera at 56°C, adding the C inactivator cobra venom factor or in the absence of divalent cations. The lytic activity was provided via the alternative pathway of C activation since it was readily demonstrable in the absence of calcium ions. The metacyclic trypanosomes were also killed by human lymphocytes, neutrophils and eosinophils in the presence of anti-T. cruzi antibodies. Minimal or insignificant cytotoxicity was afforded by the cells or the antibodies when tested separately. These results emphasize the beneficial role of the humoral immune response in host defense against challenge with the form of T. cruzi responsible for natural infections.

Experimental studies were undertaken in tissue culture and mice infected with a cloned derivative of Trypanosoma cruzi, Y strain to determine the efficacy of two 2-substituted 5-nitroimidazole compounds, MK-436 and L634,549. The use of an X-irradiated myoblast culture system proved better than a conventional fibroblast culture for assaying the activity of compounds against intracellular parasite stages. MK-436 showed activity against amastigotes at a level of 25 µg/ml and L634,549 a dihydroxy metabolite of MK-436 showed activity 2 µg/ml. Neither compound caused morphological damage to the host cells at levels tested (250 µg/ml). By contrast, nifurtimox, which was active at 2 µg/ml, caused significant host cell damage at 100 µg/ml. In mice, studies in the chronic infection showed that MK-436 was curative at a level of 30 mg/kg if given daily for 20 days. Neither nifurtimox nor benznidazole were fully curative when given at a level of 100 mg/kg daily for 20 days. These studies showed that administration of MK-436 with a suitable solvent, PEG 400, enhanced its efficacy fourfold, and that efficacy was also enhanced by increasing the treatment interval. Since MK-436 showed better efficacy in chronic rodent infections than either nifurtimox or benznidazole, such compounds should be evaluated for efficacy in human Chagas' disease.

A total of 46 Trypanosoma rangeli stocks were isolated from naturally infected mammals and triatomine vectors. Twenty-two stocks were from the common opossum (Didelphis marsupialis), one from the brown “4-eyed” opossum (Metachirus nudicaudatus), one from the anteater (Tamandua tetradactyla), one from the coati (Nasua nasua), seven from Rhodnius pictipes and 14 from Rhodnius robustus. Two stocks were also isolated from recently fed sandflies (Lutzomyia sp., Shannoni group). The stocks were identified as T. rangeli on the basis of natural or experimental salivary gland infections in Rhodnius, inoculative (anterior station) transmission to mice, morphological parameters in parasitemic mice and comparisons of isozyme profiles with a known stock of T. rangeli isolated from man. Three other trypanosome stocks from D. marsupialis, T. tetradactyla and the three-toed sloth (Bradypus tridactylus) were morphologically similar to T. rangeli in culture but had quite different isozyme profiles and were not identified. It is concluded that T. rangeli is widely distributed in Amazonas, Pará and Rondonia States of Brazil, and probably extends into other regions where R. pictipes and R. robustus are known to occur. R. pictipes is light-attracted into houses and occasionally transmits Chagas' disease to man. It is likely that T. rangeli is also occasionally transmitted to man in the Amazon basin.

Leishmania braziliensis panamensis promastigotes, temperature-induced in vitro-cultivated amastigotes, Vero cell-derived amastigotes, and rodent lesion-derived amastigotes were evaluated as antigens in the indirect immunofluorescent antibody (IFA) test for American cutaneous leishmaniasis. Test sensitivity was determined using sera from 34 U.S. soldiers with leishmaniasis diagnosed by demonstrating parasites in their skin lesions. Sera were collected from 3 to 24 months after exposure to Leishmania. Positive IFA reactions among patient sera were 82% with promastigotes or lesion amastigotes, 79% with in vitro amastigotes, and 76% with Vero cell amastigotes (P = N.S.). Positive titers ranged from 1: 8 to 1:128 using all antigens. Test specificity was determined with 30 sera from healthy individuals. False positive reactions ranged from 0–5% depending on the antigen and all titers were ⩽1:8. Test cross-reactivity was assessed with 47 sera from patients with other diseases. Depending on the antigen, cross-reactions occurred with sera from patients with Chagas' disease, lupus erythematosus, malaria, toxoplasmosis and amebiasis. None of the antigens cross-reacted with sera from patients with viral hepatitis, coccidioidomycosis, syphillis, schistosomiasis, and trichinosis. In replicate experiments, 99–100% of the sera varied no more than ±1 titer dilution. As sensitivity, specificity, cross-reactivity, and reproducibility of the four antigens were statistically similar, promastigotes, which can be easily and economically cultured in large numbers in vitro are recommended for use in the IFA test for American cutaneous leishmaniasis.

Previously described monoclonal antibodies IX-IF9-D8, IX-2H7-E10 and IX-5H9-C1 recognize promastigote stage-specific determinants present on externally exposed membrane proteins of axenically cultured Leishmania mexicana amazonensis. In the present study, these antigens were demonstrated by indirect immunofluorescence to be present on promastigotes found in the gut lumen of infected Lutzoymia longipalpis. The presence of these antigens on promastigotes found in infected sandflies suggests that the same antigens should be relevant for protective immunity studies and for species identification of Leishmania encountered in epidemiological studies.

A man with massive Capillaria hepatica infection survived after treatment with prednisone, disophenol, and pyrantel tartrate. He remained well during an 8-year follow-up.

Various preparations of crude and a purified preparation of Trichinella spiralis antigens were compared in a rapid, micro-enzyme immunoassay (EIA) for detecting trichinellosis in swine. The crude antigen preparations (XM-300 or S3 fraction) were lipid-free, cell-free fractions of muscle larvae, and the purified antigen was prepared by immunoaffinity chromatography of the soluble fraction of stichocyte secretory granules from rat muscle larvae. The antigens were tested against normal and immune swine sera for sensitivity and specificity, and for their ability to detect seroconversions early in the immune response. At optimum concentrations, absorbance values from immune and nonimmune sera produced sample to noise (S/N) ratios three-fold higher for the column antigen than for XM-300. Tests of sequential sera from experimentally-infected pigs showed that the column antigen produced lower absorbances with pre-infection sera and, from 18 days post-infection, higher absorbances with positive sera. From 21–28 days post-infection, absorbances and S/N ratios with column antigen were nearly twice those with XM-300. Column antigen detected antibodies more often than XM-300 antigen in sera collected prior to the appearance of larvae. Crude antigen did not distinguish all true negatives from weakly positives in a study involving 100 sera from muscle digestion-negative pigs and 75 sera from experimentally infected pigs, whereas the column antigen distinguished all negatives from positives. In a larger scale test of the column antigen, 1,130 pigs from Puerto Rico were tested in the micro-EIA test. Puerto Rico has no endogenous trichinellosis, and all 1,130 pigs were shown to be muscle digestion negative. These same pigs were all negative using the column antigen. These results show that the column antigen out-performs the crude antigens in sensitivity, specificity, and early detection. The column antigen is therefore a major improvement in the EIA for swine trichinellosis.

This is a report of a case of massive cutaneous larva migrans in a 20-year-old man who also had pulmonary symptoms and larval invasion of the skeletal muscles. In sections of a muscle biopsy specimen taken 3 months after the initial cutaneous lesions, a third-stage Ancylostoma larva, probably A. caninum, was found within a muscle fiber.

The small intestine is the usual site of chronic infection with Strongyloides stercoralis. Colonic involvement has generally been seen in the hyperinfection syndrome in immunosuppressed individuals. We describe an immunologically competent female who underwent multiple abdominal operations over a 5-year period for hematochezia and diffuse abdominal pain of obscure etiology. Eosinophilia had been present but never investigated until 1981, at which time stool examination revealed S. stercoralis larvae. Pathological specimens from operations in 1977 and 1981 demonstrated extensive colonic wall invasion with filariform larvae consistent with S. stercoralis. Involvement of other organs was never documented. We believe this case is consistent with chronic colitis due to strongyloidiasis. This entity has not been previously described, and expands the spectrum of this disease.

Examination of blood from randomly selected residents in four subdivisions of Puerto Limón, Costa Rica, demonstrated Wuchereria bancrofti infection in 1.8% of 2,879 persons. Previous studies in four other subdivisions of the city revealed infection in 2.7% of 2,338 persons. In both studies the microfilaremia rate in males was approximately twice that in females. Distribution by race was similar in the two surveys, with microfilaremia in 1% of 1,052 white and 4.1% of 1,286 black residents in the first survey and 0.8% of 2,216 white and 5.4% of 663 black residents in the second. The highest rate of infection was in the 10- to 19-year-old and over 40-year-old age groups. In 13 rural communities near Puerto Limón, infection was found in only three (0.1%) of 3,448 individuals (27% of the population). Dissection revealed filarial larvae in 164 (6%) of 2,714 female Culex pipiens fatigans captured in 137 of 140 urban houses and in none of 936 from rural houses. Precipitin tests on stomach contents of 643 C. p. fatigans showed that 85% had taken human blood. The results indicate that endemic bancroftian filariasis in the Puerto Limón area is restricted to the urban population.

Of three species of Nearctic black flies tested, Simulium pictipes Hagen proved the most efficient laboratory vector of the bovine parasite Onchocerca lienalis. Among flies inoculated intrathoracically with 40 microfilariae, numbers of 3rd-stage larvae per fly were 7.63 for S. pictipes, 7.54 for S. vittatum Zetterstedt, and 0.83 for S. decorum Walker. S. pictipes survived the longest under laboratory conditions, with 83.3% of the females remaining alive 10 days after inoculation with 40 microfilariae of O. lienalis. Using an artificial membrane feeding system, S. pictipes could be routinely infected with O. lienalis by mouth. This black fly was also susceptible to infection with the Guatemalan strain of O. volvulus. Among flies injected with 10 microfilariae the rate of infection with 3rd-stage larvae was 93%, with a mean of three 3rd-stage larvae per fly. Successful techniques for the large-scale recovery and cryopreservation of 3rd-stage larvae of O. lienalis were also developed. A motility rate of 92.7% was observed in larvae cryopreserved within vector black flies.

Patency of Brugia malayi infection in gerbils born of infected parents was compared to that in gerbils from noninfected parents. Following infection with third stage larvae, female progeny of infected parents generally became microfilaremic whereas those of noninfected parents rarely did so. Male progeny developed microfilaremia regardless of parentage, although descendants of infected parents tended to have higher parasitemia. We conclude that B. malayi infection in the mother predisposes the offspring to patent infection subsequent to inoculation with infective larvae.

A patient from Southeast Asia with cough and hemoptysis was documented to have pulmonary paragonimiasis. Initial treatment with bithionol failed. The patient was then treated with praziquantel 75 mg/kg body weight a day for 2 days under an investigational protocol. Subsequent follow-up studies showed clinical improvement and indicated parasitologic cure. A concurrent asymptomatic Clonorchis infection was also cured following praziquantel treatment. Side effects were minor.

To determine whether prevalence and intensity of infection are factors in morbidity in schistosomiasis japonica, a cross-sectional study was undertaken in three villages in Leyte, Philippines, namely, Santol (A), Santa Rosa (B), and Macanip (C). Kato thick-smear fecal examination and egg counts were made on 289 of 341 residents in Village A (85%), 824 of 1,008 in Village B (82%), and 1,113 of 1,241 in Village C (90%). Prevalences of 26%, 39%, and 44%, respectively, were found in the three villages, the majority of their populations (56–74%) remaining uninfected. Most of the infected persons (17–30% of the total population) had light infections (10–100 eggs/g feces). Moderately infected persons (101–400 eggs/g) comprised a smaller segment (7–14%), while a very small proportion (2–7%) had heavy infections (⩾401 eggs/g). Age prevalence and egg excretion peaked earlier in the areas with higher prevalence (B and C) than in the area with the lowest prevalence (A). There was no relationship between area prevalence and mean egg count. Symptoms of inability to work, weakness, abdominal pain, and diarrhea correlated with the presence of infection in the area with the highest prevalence (C), but not in the area with the lowest prevalence (A). Except for diarrhea, there was no relationship between symptoms and intensity of infection. Very few persons presented with hepatomegaly and/or splenomegaly (1–5%). The frequency of liver enlargement on the midsternal (measuring 3–6 cm and 6 cm or more) and midclavicular line (2–4 cm), as well as spleen enlargement (Hackett 2 or greater), correlated with the presence but not with the intensity of infection. Hepatomegaly was sex- and age-related, being most common among males and among adolescents aged 10–14 years.