The American Journal of Tropical Medicine and Hygiene - Volume 34, Issue 3, 1985

Five artemisinine-resistant mutants of Plasmodium falciparum have been isolated from a cloned parent following mutagenic treatment with N-methyl-N′-nitronitrosoguanidine. This observation may influence the strategy of the use of artemisinine or its derivatives. Mutant isolation should prove useful in the study of drug resistance to other antimalarials.

Today, research into tropical medicine is in a ferment of activity with new, almost magical technologies being applied to the study of age-old problems. Immunology has come of age, and has sired new disciplines of great promise. The immortalization of the antibody-producing cell has greatly facilitated the antigenic analysis of complex parasitic pathogens and currently nourishes prospects of targeted chemotherapy of zymotic diseases. Recombinant DNA procedures have made possible the production of complex antigen molecules uncontaminated by unwanted by-products and have facilitated the creation of vaccines of exceptional purity. It is, therefore, not surprising that universities, both ancient and modern, have stretched their resources to welcome and accommodate this new learning, or that young scientists of ability are increasingly seeking careers in such glittering disciplines. Insofar as these developments have rekindled interest in diseases which are of great prevalence and importance in the tropical world they are to be applauded.

The ultrastructural changes induced by the administration of the antimalarial drug, qinghaosu, were studied in monkeys (Macaca assamensis) infected with Plasmodium inui. Significant changes, notably mitochondrial swelling within the parasites but not within host cells, were first observed 2.5 hr after exposure to qinghaosu. This suggests that the target of qinghaosu may be the parasite's mitochondria, as occurs with primaquine. This is in contrast to the most widely used antimalarial drug, chloroquine.

A test system that uses infective gametocytes from in vitro cultures was developed for evaluating the sporontocidal activity of antimalarial compounds. In evaluating the system, pyrimethamine and cycloguanil (dihydrofolate reductase inhibitors) and primaquine (8-aminoquinoline) were tested against pyrimethamine-sensitive and pyrimethamine-resistant strains of Plasmodium falciparum. The drugs were administered to Anopheles either in a blood meal containing infective gametocytes or in a noninfective meal 2–4 days later. The mosquitoes were dissected 9–10 days after they received the infective blood meal, and the sporontocidal effect of the drugs was evaluated by the number of oocysts found in the gut. Both cycloguanil and pyrimethamine had marked sporontocidal activity. The susceptibility pattern of the strains to the sporontocidal effect of pyrimethamine and cycloguanil was similar to the susceptibility of their asexual blood stages in vitro to the schizontocidal effect of the compounds. The sporontocidal effect was observed only when the compounds were administered at the same time as the infective blood meal, but not when they were given 2–4 days later. No sporontocidal activity was observed with primaquine. This system permits more reliable quantitative observations than have been possible with previous methods.

A clinical field trial was conducted to determine if mefloquine is effective in the treatment of malaria due to Plasmodium vivax. Forty patients with P. vivax malaria were treated with either mefloquine, chloroquine or chloroquine plus primaquine and followed for 28 days. All patients responded rapidly and were cured. There were no significant side effects.

Promastigotes from four cutaneous leishmaniasis cases from Colombia were tested by cellulose acetate electrophoresis using nine enzyme systems. The isoenzyme profiles of the Colombian isolates were indistinguishable from each other and from Panamanian Leishmania braziliensis panamensis controls, but were distinct from an isolate of Leishmania braziliensis guyanensis from Brazil and three isolates from the Leishmania mexicana complex for the enzyme phosphogluconate dehydrogenase.

In order to determine the vectors of leishmaniasis in Ecuador, 1,054 manbiting sand flies from the Department of Cañar were dissected and examined for promastigotes. There were 2 man-biting species, Lu. trapidoi and Lu. hartmanni in this endemic area of the disease. The infection rates were 7.7% in the former and 3.9% in the latter species, demonstrating the different rates in various localities and altitudes of the study areas. There was an association between infection rates and the time of day, suggesting some connection with biting activity of sand fly species. In collections using human bait at 7 study areas in 5 Departments, 6 man-biting species were recognized, indicating different dominant species in each area. It was assumed that the dominant species would play an important role as the principal vector of leishmaniasis in each endemic area. As to species determination of the present Leishmania promastigotes, suffice it to say that the parasites are Leishmania sp., presumably L. braziliensis s.l., until the isolates have been typed.

Nineteen cases of “localized” leishmania lymphadenitis without any evidence of visceral leishmaniasis are reported. Fifteen males and 4 females aged 5 to 30 years have presented with localized lymphadenopathy of up to 3 months duration. The disease has a seasonal incidence of late summer to mid-winter. Cutaneous leishmaniasis even when present, usually was overlooked. Lesions of cutaneous leishmaniasis were absent in 3; healed in 1; small, similar to insect bites in 5; classic in 3; lupoid in 1 and unknown in 6 patients. Serum leishmanial antibody determination by IFAT performed on 12 cases were positive. Toxoplasma serology was negative. Histological picture of one lymph node biopsy showed an (anergic) intact histiocytic response characterized by thousands of intracellular amastigotes, no necrosis and inconspicuous plasma cells. In 17 biopsies the picture was that of histiocytic granulomata with varying degrees of necrosis, moderate numbers of amastigotes in several foci, fibrosis and varying numbers of plasma cells. One biopsy from a lupoid case shows numerous epitheloid granulomata, no organisms, no necrosis and inconspicuous plasma cells. Electron microscopy has been performed on 8 biopsies to confirm leishmania amastigotes. Differential diagnosis from toxoplasmosis and cat-scratch disease is discussed. Histological types of responses in the lymph nodes are comparable to those described in cutaneous leishmaniasis: 1) an anergic response with intact macrophage granuloma, 2) a histiocytic response with necrosis, and 3) a lupoid type of response with epithelioid granulomas.

Midgut promastigotes were obtained from Phlebotomus papatasi and Lutzomyia longipalpis on days 3–7 after infection with cloned isolates of Leishmania major and Leishmania mexicana amazonensis, respectively, and examined as to their ability to initiate cutaneous infections in BALB/c mice. Sequential development of midgut promastigotes from a noninfective to an infective stage was confirmed for both the New World and Old World species. The generation of infective promastigotes from rapidly dividing avirulent populations occurred as early as day 3 and was well under way by day 4 after infective feed. Optimally infective promastigotes were recovered from midguts shortly after bloodmeal passage, coinciding with the time at which another bloodmeal is sought by the fly.

Acetylcholinesterase (AChE) activity was measured in skeletal muscle from susceptible (A/J) and resistant (C57BL/6) mice infected with the Brazil strain (myotropic) of Trypanosoma cruzi. There was a 60% decrease in activity in skeletal muscle obtained from A/J mice 20 days post-infection as compared to controls. There was no decrease in AChE activity in skeletal muscle obtained from infected C57BL/6 mice 20 and 150 days post-infection. Histologic examination of skeletal muscle from infected A/J mice revealed marked necrosis, pseudocysts, and minimal inflammation. Similar examinations in C57BL/6 mice revealed marked inflammation in the absence of necrosis and parasites. These data provide additional biochemical support that denervation hypersensitivity is an important concomitant of Chagas' disease and that it is already present during the acute stage. Additionally, it may support the notion that the presence of the parasite mediates these abnormalities.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Entamoeba histolytica in human feces, using both a monoclonal antibody and rabbit antisera. It detected from less than 1 to 57 trophozoites of 6 E. histolytica strains. Stool specimens were positive by ELISA in 18 of 22 (82%) patients with E. histolytica and in 3 of 186 (2%) of patients without demonstrable E. histolytica in their stools. The latter included one from a child living near an asymptomatic cyst carrier and another from a traveler with giardiasis who had recently taken antibiotics. One hundred eight of 183 microscopy- and ELISA-negative specimens contained other parasites including Giardia (49 specimens), Endolimax nana (24), Entamoeba coli (21), Iodamoeba butschlii (2), and Entamoeba hartmanni (1). This ELISA for E. histolytica is a simple, sensitive and specific diagnostic tool.

A 28-year-old Indian female presented with shortness of breath, white cell count of 27,400 with 39% eosinophils, and increased interstitial markings and bilateral pleural effusions on chest x-ray. Although the clinical presentation was consistent with tropical pulmonary eosinophilia (TPE), to our knowledge there has been only one previous report in the world's literature of pleural effusion associated with TPE. We suggest that pleural effusions are not incompatible with the diagnosis of TPE and that TPE be added to the list of conditions causing eosinophilic pleural effusions.

Congenitally athymic nude C3H/HeN mice, microfilaremic with Brugia pahangi, were treated with diethylcarbamazine citrate (DEC). A single oral dose (100 mg/Kg body weight) of DEC resulted in the rapid reduction of numbers of circulating microfilariae in nude, thymus-grafted nude and complement-depleted nude mice. Antibodies of the IgM and IgG isotypes were not detected in the serum of microfilaremic nudes or on the microfilarial surface. These results suggest that DEC-mediated clearance of microfilariae from the circulation of nude mice is probably independent of thymus-dependent immunological mechanisms.

A standardized microtest plate enzyme-linked immunosorbent assay was developed using the microsomal fraction of adult worms of Schistosoma mansoni (MAMA) as antigen. The standard reference serum pool was prepared from acutely and chronically infected rhesus monkeys and was shown to be appropriate as a standard for measuring the levels of reactivity of the unknowns. The standard serum pool was arbitrarily designated as having 100 activity units per µl. The levels of reactivity of the unknowns were expressed as activity units per µl. Serum specimens were obtained from 190 patients infected with S. mansoni in the Caribbean, South America, and Africa. Serum was obtained from small numbers of patients infected with S. haematobium, S. japonicum, or S. mekongi. Controls were 136 patients with other helminthic infections, 142 patients with protozoal or other diseases with liver involvement, and 81 healthy serum donors. The J index (or predictability) of the assay was calculated to determine the significant level of reactivity. The assay has a predictability of 95% for both patients with S. mansoni infections and those with other infections. The sensitivity of the assay for S. mansoni infections was 96%, and the specificity (in terms of cross-reactions with infections with other parasite genera or with other liver diseases) was 99%. The heterologous Schistosoma species showed a markedly lower level of reactivity, with an overall sensitivity of 55%. This is in accord with the species-specificity previously recognized in MAMA, and emphasizes the need for standard reference pools of human sera prepared from patients infected with single species of each of the Schistosoma. Use of these pools in assays with antigens of the respective schistosome species would allow optimum serologic evaluation.

The progression of humoral immune responses exhibited by mice during the year following exposure to Schistosoma mansoni cercariae was established by studying radioimmunoprecipitations of adult male and cercarial glycoproteins. 35S-methionine metabolically-labeled adult S. mansoni male worm glycoproteins precipitated by sera of 14 mice were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The initial specific antibody response (week 5) was directed against 3 worm glycoproteins of 55,000, 52,000 and 35,000 molecular weight (Mr). As infection progressed, all major worm glycoproteins (ranging from 400,000 to 12,000 Mr) were precipitated by sera from each mouse and only minor individual variations in titer were noted in the antibody responses of the mice against these glycoproteins. Maximal immunoreactivity toward the radiolabeled glycoproteins occurred at week 20 and remained at this level through week 50. Analogous experiments with sera from acutely- and chronically-infected humans resulted in immunoprecipitation patterns almost identical to those obtained with sera from the corresponding experimentally-infected mice. The kinetics of the antibody response against 125Iodine-labeled cercarial glycoproteins was the same as that observed with worm glycoproteins.

A glycoprotein antigen of 80,000 apparent molecular weight expressed by S. mansoni eggs, cercariae, and male and female worms has been characterized by use of a monoclonal antibody prepared against cercarial glycoproteins. The antigen was expressed on the surface of both male and female worms. Its expression on the male surface, however, was restricted to the gynecophoral canal of male worms derived from fully patent infections. This pattern of surface expression suggests a role in schistosome sexual reproduction.

High neutrophil and eosinophil chemotactic activities of soluble extract of Schistosoma japonicum cercariae (SjCe-ext) were detected in vitro by using blind-well chemotaxis chambers and Millipore filters in a dose-dependent manner. Low doses (total of 0.25 and 2.5 µg) of SjCe-ext induced significant neutrophilic infiltrations in normal and S. japonicum-infected (8 week) guinea pig tissues beginning at 2 hr after the intradermal injection. Eosinophilic responses were observed in the infected animals especially in early phase (2–8 hr post-injection of SjCe-ext). SjCe-ext elicited strong immediate skin reactions (at 30 min) in the infected guinea pigs. Possible roles of these chemotactic activities and hypersensitivity reaction in S. japonicum infection are discussed.

Relationships between hemoglobin level and S. hematobium, hookworm, and malarial infection before and six months after metrifonate treatment were studied in Kenyan primary school children in an area where anemia, S. hematobium and hookworm are common (prevalences 61%, 46%, and 95%, respectively) and malaria is holoendemic. The mean hemoglobin level in children from one school, both with and without S. hematobium infection (n = 250), was significantly lower in children with higher S. hematobium egg counts, heavier hookworm infections, positive Plasmodium slides, and larger spleens. All children with light-moderate S. hematobium infection (1–500 eggs/10 ml adj) in four schools were examined (Exam 1), allocated at random to either placebo (MIP, n = 198) or metrifonate treatment (MIT, n = 202) groups, treated, and examined again six months later (Exam 2). Hemoglobin levels rose significantly in both groups between exams, but the rise in the MIT group was 30% higher than in the MIP group (1.3 vs. 1.0 g/dl, P < 0.014). The increase in hemoglobin level in the MIT group was significantly and positively correlated with decreases between exams in S. hematobium and hookworm egg counts and with higher malarial parasite counts at Exam 1 (Pearson r's 0.21, 0.20, 0.20, respectively, P < 0.01). A stepwise multiple regression equation using hemoglobin rise between exams as the dependent variable showed that decreases in S. hematobium and hookworm egg counts were equally important determinants of hemoglobin rise and that malarial parasite count was almost as important as the changes in intensities of the helminth infections. These results show that treatment for S. hematobium with metrifonate can increase hemoglobin levels in children in an area where S. hematobium and anemia are common. They also emphasize the importance of measuring multiple parasitic infections and using multivariate statistical techniques such as multiple regression analysis in order to define the relationships between parasitic infections and morbidity.

To determine definitively whether or not the severity of the Mazzotti reaction was correlated with infection intensity, as determined by skin snip quantification, 21 infected Ghanian patients were evaluated during 7 days of treatment with 200 mg/day of diethylcarbamazine. Serial blood, urine and skin biopsy samples were collected during the progression of the Mazzotti reaction. Hypotension, fever, adenitis and pruritus were all correlated with infection intensity in these patients while arthralgia and tachycardia were not. Peripheral blood eosinopenia and neutrophilia also correlated with intensity of infection and appeared to reflect the accumulation of degranulating eosinophils around “mobilized” microfilariae that migrated from the dermis to the epidermis after diethylcarbamazine (DEC). Other mobilized microfilariae apparently were cleared by the liver and resulted in abnormal liver enzyme levels in the serum which, again, were directly correlated with the patients' microfilarial density. Though the severity of the Mazzotti reaction clearly correlated with intensity of infection, the different times of onset of symptoms, and cellular and serum chemistry changes indicate that there are probably multiple infection intensity-dependent mechanisms responsible for mediating this complex reaction.

The authors report clinical and histopathologic changes in six patients with symptomatic loiasis. One patient had cutaneous swellings, three patients presented with hydrocele, one patient developed bowel obstruction, and one had generalized fatal loiasis. The first five patients had localized lesions provoked by adult worms; all were surgically removed. The sixth patient died of disseminated loiasis that included a severe loal encephalitis. The authors discuss the mechanism of “Calabar” swellings, the reaction to adult Loa loa worms and loal encephalitis.