Experimental infection of Mystromys albicaudatus with Leishmania braziliensis: pathology.

Thirty-four Mystromys albicaudatus were injected intradermally with Leishmania braziliensis promastigotes and examined and killed during a 12-week period. All animals developed single cutaneous lesions at the sites of inoculation that began as a papular thickening in the dermis and progressed to a 2.0 cm crateriform ulcer. The histopathology of these lesions was characterized by granuloma formation and diffuse infiltrates of lymphocytes and plasma cells. Parasites were most frequently observed in vacuolated macrophages immediately underlying the necrotic debris of the ulcer. Histiocytes in which amastigotes were not identified were noted in aggregates at the margins of the inflammation. Russell's bodies were observed at 6 weeks post-infection, and discrete lymphocytic infiltrates bordered the inflammation at 12 weeks post-infection. It is suggested that M. albicaudatus is an excellent model of American cutaneous leishmaniasis.

Identification of Leishmania spp. by multiple isozyme analysis.

Biochemical data as enzyme profiles which were obtained by cellulose acetate electrophoresis (CAE) are reported on 44 Leishmania isolates. These enzyme profiles contain data from 25 enzyme systems. Calculations from the CAE data on average polymorphism indicated that Leishmania species/types or groups can be expected to be about 25% polymorphic, which suggests that isolate pairs which have profiles about 75% or more identical should be considered samples from the same species/type, and isolates that are significantly less than 75% identical are therefore samples from different species/types. There were five major groupings of isolates according to enzyme profiles, which were for the most part consistent with groupings of the genus based on other criteria: braziliensis, mexicana, donovani, tropica and hertigi profiles. Within these groups there were natural subgroups of isolates among which there was 75% or more allozyme or allomorph (genetic) identity. The braziliensis profile group had two subgroups: panamensis and braziliensis or guayanensis, and the mexicana profile group had three subgroups: mexicana, amazonensis and peruviana. There was an indication that an L. d. infantum isolate might be different from the other L. donovani isolates, and that the L. tropica isolates could be samples from more than one group. The data reported here are consistent with previously reported CAE data, but suggest that isozyme analysis of Leishmania isolates leading to identification should be based on data from many enzyme systems rather than just a few.

Susceptibility of clinically sensitive and resistant Leishmania to pentavalent antimony in vitro.

Standard courses of pentavalent antimonials frequently fail to cure cutaneous, mucocutaneous, and visceral leishmaniasis, and characteristically fail to cure diffuse cutaneous disease. We have determined the in vitro sensitivity of clinical isolates of Leishmania to pentavalent antimony to determine if inherent drug resistance of the parasite is responsible for treatment failures in human beings. Intracellular amastigotes resulting from promastigote-initiated infection of human macrophages were exposed to pentavalent antimony for 6 days at 34.5-35 degrees C. Amastigotes from clinically sensitive simple cutaneous lesions exhibited a range of in vitro sensitivity. Four strains were greater than or equal to 90% eliminated and two strains were 70-75% eliminated in vitro by concentrations of antimony (15-20 micrograms Sb/ml), comparable to peak achievable serum levels in humans. Amastigotes from initially clinically resistant simple cutaneous lesions showed a wider range of sensitivities. Five strains were greater than or equal to 90% eliminated, but one strain was only 40% eliminated and another strain was completely insensitive in vitro. The clinically resistant diffuse cutaneous strain was 61% eliminated. The techniques described herein permit determination of the in vitro antimicrobial susceptibility of Leishmania from all major human forms of leishmaniasis. The data from this series indicate that in a minority of initially resistant cases parasite resistance to the drug may be contributing to clinical resistance, and use of non-antimonial drugs might be recommended for future therapy.

Indigenous human cutaneous leishmaniasis caused by Leishmania tropica in Kenya.

Six Leishmania isolates from 3 indigenous Kenyans (2 isolates from one patient) and 2 Canadian visitors in Kenya were characterized by cellulose acetate electrophoresis. The isolates were compared among themselves and with reference strains of Leishmania donovani, L. aethiopica, L. major, L. tropica, and L. arabica using 9 enzymes: malate dehydrogenase (MDH), malic enzyme (ME), phosphogluconate dehydrogenase (6PGD), glucose-6-phosphate dehydrogenase (G6PD), aspartate aminotransferase (ASAT), adenylate kinase (AK), mannose phosphate isomerase (MPI), glucose phosphate isomerase (GPI), and phosphoglucomutase (PGM). Enzyme migration patterns of isolates from the 3 indigenous Kenyans were indistinguishable from those of 2 L. tropica reference strains. The isolates from the 2 Canadians yielded migration patterns of 7 enzymes that were indistinguishable from those of 2 L. tropica reference strains. However, migration patterns of 2 enzymes, PGM and ME, differed from all migration patterns of the 10 reference strains. Balb/c mice were inoculated with stationary phase promastigotes cultured from 3 stabilates from the lesions of 2 of the Kenyan patients. The mice developed no gross pathological lesions in 6 months time. All of the study patients developed cutaneous leishmaniasis while living in or visiting districts in Central and Rift Valley Provinces, Kenya. This is the first report of human cutaneous leishmaniasis caused by L. tropica indigenous to Africa south of the Sahara.

Biochemical characterization and zymodeme classification of Leishmania isolates from patients, vectors, and reservoir hosts in Kenya.

A total of 407 Leishmania and other Leishmania-like isolates obtained from patients, other vertebrates, sand fly vectors, and other arthropods from Kenya and other countries were characterized and compared with several World Health Organization and other well-characterized reference strains of Leishmania, Trypanosoma, Crithidia, Herpetomonas, and Leptomonas by cellulose acetate electrophoresis (CAE), using 20 enzyme systems. Analysis of the isoenzyme banding patterns (IBP) of the isolates generated isoenzyme profiles that were resolved as zymodemes and tabulated. Isolates that produced similar isoenzyme profiles in all 20 enzyme systems were placed into a particular Leishmania isoenzyme taxon, with the zymodeme designated numerically as Zn. A total of 66 zymodemes were recorded for the 407 isolates studied. To obviate the need to draw all 66 representative IBP for each of the 20 enzyme systems, the 66 zymodemes (Z1-Z66) were again placed into similarity groups represented by pattern number or Pn. This resulted in 23-50 IBP (Pn) per enzyme system. The highest number of IBP scored was for malate dehydrogenase (MDH) (P1-50) and the lowest score was for glucose-6-phosphate isomerase (GPI) (P1-23). From these different isoenzyme profiles or zymodemes, IBP of 14 (MDH, GPI, nucleoside hydrolase, phosphoglucomutase, malic enzyme, isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, mannose-6-phosphate isomerase, 6-phosphogluconate dehydrogenase, glutamate oxaloacetate transferase/aspartate aminotransferase, glutathione reductase, superoxide dismutase, fumarase, and glyceraldehyde-3-phosphate dehydrogenase) of the 20 enzyme systems were selected for computer-calculated numerical taxonomy. Consistent individual isoenzyme bands with similar relative mobilities of the 14 enzyme systems were scored into groups (allelomorphs, allozymes, or electromorphs) and used in cluster analysis. For each pattern in every profile, the presence of a consistent band was entered as 1 and its absence as 0. A total of 419 allozyme characters (variables) were scored for the 14 enzyme systems. Lastly, all different zymodemes sharing a particular IBP (Pn) within an enzyme system were counted and the total number was shown as a zymodeme frequency (Zf). Final analysis of the CAE isoenzyme profiles and cluster-dendrograms resulted in the identification of several potentially new species and subspecies of Leishmania and other Leishmania-like isolates from patients, sand flies, and animal reservoir hosts collected from Kenya and other locations in Africa. Zymodeme analysis of the Kenyan visceral and cutaneous leishmaniasis isolates resulted in the identification of 11 subpopulations of the L. donovani species complex and six subpopulations of the L. tropica species complex endemic to different geographic areas of Kenya.

Cutaneous leishmaniasis associated with jungle training.

In November 1977, 627 soldiers belonging primarily to the First Battalion, 82nd Airborne Division, stationed at Fort Bragg, were sent to the Canal Zone, Panama, for jungle warfare training. A medical surveillance program incorporating pre- and post-evaluations over a 6-month period with dermatologic examinations, questionnaires, and serologic tests was established. Ten cases of cutaneous leishmaniasis (1.6/100 men) were diagnosed by positive Leishmania culture. The demonstrated lack of sensitivity and specificity of the indirect fluorescent antibody test and the direct agglutination test render these serological methods useless as diagnostic screening methods in the early stages of this disease.

Concurrent infection with Leishmania donovani and Leishmania major in a Kenyan patient: clinical description and parasite characterization.

Leishmania isolates aspirated a few months apart from the spleen of an indigenous adult male kala-azar patient from Baringo District, Kenya, were biochemically characterized and compared. The patient lived within a dual focus of L. donovani kalazar and L. major cutaneous leishmaniasis. A primary Leishmania isolate from splenic aspirates was cryopreserved (NLB-294). The patient was treated with sodium stibogluconate for kala-azar and discharged. Three months later, he had clinical relapse and returned for retreatment. During his second visit, the patient participated in a diagnostic study in which urine and nasopharyngeal samples were cultured for leishmaniasis. Urine, nasopharyngeal, and splenic samples were positive for Leishmania. Secondary isolates from splenic (NLB-294-I) and urine (NLB-318) cultures were cryopreserved and characterized by cellulose acetate electrophoresis (CAE) using 20 enzymes. Whereas the urine isolate was typed as L. donovani, the splenic aspirate culture revealed a mixed infection with L. donovani and L. major. The primary isolate (NLB-294) was then characterized and also showed a mixed infection. To exclude the possibility of protein post-translational modifications in electrophoretic assays, the primary and secondary isolates were grown and processed under identical cultural and lysis conditions, and compared using CAE. The results were identical to the first electrophoretic assays showing mixed promastigote banding patterns. Stationary-phase promastigotes of the secondary splenic isolate (NLB-294-I) inoculated subcutaneously, intraperitoneally, and intracardially into Syrian hamsters and BALB/c mice produced both kala-azar and cutaneous leishmaniasis within 6.5 months.(ABSTRACT TRUNCATED AT 250 WORDS)

Leishmania donovani parasites in the nasal secretions, tonsillopharyngeal mucosa, and urine centrifugates of visceral leishmaniasis patients in Kenya.

In the early 1930s, investigators of visceral leishmaniasis stated that Leishman-Donovan bodies are found in body fluids of kala-azar patients, for example, in urine, feces, semen, and nasal and pharyngeal secretions. Based on this finding, we investigated the diagnostic potential of nasal secretions, tonsillopharyngeal mucosal swabs, and urine centrifugates inoculated into Schneider's Drosophila Medium (containing antibiotics and antifungal agents) as well as with Giemsa-stained smears. Consequently, 64 randomly selected patients with visceral leishmaniasis from Kenya (59 who were splenic culture or Giemsa stain positive and five who were culture negative but Giemsa stain positive) were tested by three noninvasive methods. These tests were all performed before the patients were treated with Pentostam. Cultures of nasal and tonsillopharyngeal swabs and urine centrifugates produced 28 positive samples representing 24 patients (37.5%). Moreover, a set of 25 Giemsa-stained slide smears made from the nasal and tonsillopharyngeal mucosa of 25 patients with visceral leishmaniasis who had not tested positive in cultures produced nine positives. Therefore, the overall total of patients who tested positive by all of the above methods was 33 or 51.6%. The cryopreserved Leishmania isolates were characterized by cellulose acetate electrophoresis using 20 enzyme systems. The isoenzyme profiles produced by the parasites were represented in five different L. donovani s.l. zymodemes. Representatives of these isolates were also characterized by DNA Southern blotting analysis, which corroborated the isoenzyme results.

Evaluation of promastigote and amastigote antigens in the indirect fluorescent antibody test for American cutaneous leishmaniasis.

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 less than or equal to 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, syphilis, 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.

American cutaneous leishmaniasis: a comparison of three sodium stibogluconate treatment schedules.

Thirty-six patients with American cutaneous leishmaniasis were randomized to receive intravenous sodium stibogluconate for 10 days at a dose of 600 mg antimony (Sb) per day by one of three schedules: once daily by rapid infusion (A), by continuous 24 hr infusion (B), or in divided doses every eight hours by rapid infusion (C). Patients not cured after initial treatment were rerandomized to one of the other treatment schedules. An additional 19 patients who were not part of the randomized study received standard (STD) sodium stibogluconate treatment (600 mg Sb once daily by rapid infusion for 10 days, identical with schedule A). In the randomized study, the overall cure rate after the first course of treatment was 64%, but was higher for schedule A (100%) than for B (50%) or C (42%) (P less than 0.01). Considering all courses of treatment, schedule A was more effective (94%) than B (53%) or C (43%) (P less than 0.01). Paradoxically, patients in group A had a higher cure rate than patients in group STD (42% after the first course of treatment and 51% when all courses of treatment were considered). Side effects were mild and well tolerated. Total side effects were more frequent in groups B + C (52%) than A + STD (23%) due to an increased incidence of subjective complaints (26% vs. 10%, P less than 0.05) in patients receiving other than once daily rapid infusion. We conclude that giving the same total amount of sodium stibogluconate in three divided doses or by continuous infusion offers no advantage over standard, once daily treatment of American cutaneous leishmaniasis.

Characterization of Simulium (Edwardsellum) damnosum s.l. populations from six river systems in Kenya by cellulose acetate electrophoresis.

Isoenzyme characterization of the Simulium damnosum Theobald sibling species complex from two widely separated geographical areas in Kenya is presented based on 10 enzymatic loci. Four river systems in Western and Nyanza Provinces, namely, the Yala, Lusumu, Isiukhu and the Nzoia harbouring S. damnosum s.l. were compared among themselves and with S. damnosum s.l. collected from the Thiba and the Nyamindi river systems in the Mt. Kenya area. The two populations were easily separable using PGM, HK and, more than 73% of the time, with PGI. Using the first two enzymatic loci, PGM and HK, all the western Kenya S. damnosum s.l. belong to the same population while those from Mt. Kenya areas belong to a different population. In both geographical zones there was less than 20% qualitative and quantitative polymorphism within infraspecific forms in any given breeding area of S. damnosum s.l. Three enzymes, ME, XDH, and G-6-PDH had isomorphic mobilities for both the Mt. Kenya and western Kenya populations. Four other enzyme/substrate systems tested had no satisfactory resolution as a diagnostic value.

Mouse foot-pad inoculation as an aid to the isolation of Leishmania spp. from patients.

Portions of splenic or subcutaneous saline aspirates from suspected visceral or cutaneous leishmaniasis patients were inoculated into NNN media with an overlay of Schneider's medium or Schneider's medium alone for routine parasitological diagnosis. The remaining portions of the aspirates were used for preparing Giemsa-stained smears and for subcutaneous inoculation into hind foot-pads of Balb/c mice. Saline aspirates obtained from the foot-pads 2-14 d after inoculation were inoculated into Schneider's medium and examined for promastigotes. Parasite isolation was achieved from 90% of confirmed leishmaniasis patients by either culture method alone. Mouse foot-pad aspiration demonstrated parasites in 95% of all patients, and in over 80% of the confirmed cases of leishmaniasis. Combined culturing and aspirate smear examination was more efficient than foot-pad inoculation alone for the demonstration of leishmanial infection. Foot-pad aspiration does not entail killing animals and was sensitive for parasite isolation; it may be a useful short-term adjunct to existing parasite isolation methods, especially under field conditions where the risks of culture contamination may be high.

Cutaneous leishmaniasis caused by Leishmania tropica in Kenya.

9 leishmanial strains, isolated from cutaneous papulonodular lesions on 3 patients, were characterized by cellulose acetate electrophoresis using 7 enzymes. The patterns obtained were indistinguishable from those of a Leishmania tropica reference strain and these 9 strains were similar to L. tropica in failing to infect mice. Although these 3 patients were Americans, their only potential exposure to sandflies was in Kenya, and thus they are believed to be the first cases of cutaneous leishmaniasis due to L. tropica in Kenya.

Human cutaneous leishmaniasis caused by Leishmania donovani s.l. in Kenya.

Our laboratory is characterizing Leishmania stabilates and isolates from active leishmaniasis cases. Smears and cultures from aspirates made on different dates from a single lesion on the bridge of the nose of an 18 years old Kenyan male from Nyandarua District contained Leishmania. The isolates, NLB-271 and NLB-271-IA, were characterized by cellulose acetate electrophoresis (CAE) using 20 enzyme systems and by Southern analysis using 2 deoxyribonucleic acid (DNA) probes (pDK10 and pDK20) from a Dakar strain of L. major (MHOM/SN/00/DK1) and a third probe, p7-059 from L. infantum strain ITMAP-263. Digestion of the two Leishmania DNAs with endonucleases HindIII and PstI, followed by hybridization with the 3 probes, revealed DNA fragment banding patterns indistinguishable from those of the L. donovani species complex. The CAE isoenzyme profiles of these 2 Kenyan isolates were indistinguishable from those of Kenyan L. donovani strains we designated as zymodeme Z6. Excluding post-kala-azar dermal leishmaniasis, this constitutes the first human case of cutaneous leishmaniasis caused by L. donovani s.l. in Kenya. Previously, cutaneous leishmaniasis cases in Kenya have been due to L. aethiopica, L. major and L. tropica only.

Comparative infectivity of a Kenyan strain of Leishmania donovani amastigotes for Rattus rattus and the laboratory white rat.

Amastigotes of a Kenyan strain of Leishmania donovani from a previously infected hamster were used to inoculate Rattus rattus and the laboratory white rat intracardially. The animals were sampled at 2, 4, 6, and 12 weeks post-inoculation to determine infectivity and total parasite burdens in the liver and spleen. Higher parasite burdens were observed in the livers and spleens of R. rattus. Parasite culture indicated more generalized parasite dissemination compared to the white rat. Demonstration of the parasite in dermal tissue of R. rattus at 2 and 4 weeks suggests that the parasite may be accessible to sandfly vectors. Transient susceptibility to systemic infection and parasite survival in dermal tissue suggests a potential role of R. rattus in the transmission cycle of Kenyan visceral leishmaniasis.

Host range characteristics of the primate coccidian, Isospora arctopitheci Rodhain 1933 (Protozoa: Eimeriidae).

Studies were conducted on 35 primates, 12 carnivores, and 2 marsupials to determine their susceptibility to the primate coccidian, Isospora arctopitheci. Patent oocyst infections resulted in 12 of the 14 species of animals investigated. These included 6 genera of New World primates native to Panama: Saguinus geoffroyi, Aotus trivirgatus, Ateles fusciceps, Cebus capucinus, Alouatta villosa, and Saimiri sciureus. In addition 4 families of carnivores (2 domestic and 2 sylvatic) and 1 species of marsupial became infected following experimental exposure. These animals are represented respectively by the following 6 genera and species: Canis familiaris; Felis catus; Nasua nasua, and Potos flavus; Eiria barbara; and Didelphis marsupialis. Four Old World rhesus monkeys, Macaca mulatta, and 1 carnivore, Bassaricyon gabbii, did not become oocyst positive. This unusually large host range makes this isosporan unique among the coccidia that have been investigated to date.

Antileishmanial activity of liposome-encapsulated meglumine antimonate in the dog.

Experimental infections of Leishmania donovani in mixed-breed dogs were induced to determine the antileishmanial efficacy of liposome-encapsulated meglumine antimoniate ( LEMA ). Each dog was inoculated IV with 1.0 +/- 0.2 X 10(8) amastigotes of a Khartoum strain of L donovani/kg of body weight. The antileishmanial agents ( LEMA or unencapsulated meglumine antimoniate ) were given once daily, IV, for 1, 4, or 10 consecutive days beginning the 12th day after inoculation. The dogs were killed 3 or 4 days after completion of therapy, and parasites in the spleens were quantified. A single injection of LEMA (0.61 mg of Sb/kg of body weight) resulted in 89% suppression and 4 consecutive daily injections of LEMA (1.94 mg of Sb/kg/day) resulted in 95.8% suppression of splenic parasites. The dose of LEMA that would give 50% suppression ( SD50 ) was estimated as approximately 0.029 mg of Sb/kg. The SD50 for unencapsulated drug was estimated as approximately 24 mg of Sb/kg. The liposome-encapsulated drug was estimated to be more than 700 times more efficacious than the unencapsulated drug. Seemingly, liposomes can markedly reduce the drug dosage required for equivalent treatment of visceral leishmaniasis in dogs.

Isozyme variation in Simulium (Edwardsellum) damnosum s.l. (Diptera: Simuliidae) from Kenya.

Isozyme variation of the Simulium damnosum sibling species complex was studied by cellulose acetate electrophoresis (CAE) from four Kenyan river systems. Two enzymes, PGM and HK, were diagnostic and differentiated the larvae collected in Western and Nyanza provinces from the larvae collected at Mt. Kenya. Allele frequency differences of the enzyme PGI allowed about 75% separation of the geographically distinct populations.