The rarity of infection with Leishmania (Viannia) braziliensis among patients from the Manaus region of Amazonas state,Brazil, who have cutaneous leishmaniasis

The frequency of Leishmania ( Viannia) braziliensis infection was assessed in 79 of the 138 patients with cutaneous leishmaniasis who attended a reference outpatient unit in Manaus, Amazonas state, between the August and December of 1997. The disease was characterized by one or more cutaneous ulcers, the skin lesions being frequently associated with satellite lymph-node enlargement. All parasite isolates were identified using monoclonal antibodies and enzyme electrophoresis. Only two (2.8%) of the 71 patients from whom parasites were successfully isolated were found to be infected with L. ( V.) braziliensis, the other 69 isolates being identified, from their isoenzyme profiles, as L. ( V.) guyanensis. In the Manaus region, therefore, almost all human cutaneous leishmaniasis is the result of infection with L. (V.) guyanensis, and L. ( V.) braziliensis is a relatively rare cause of the disease.     

First evidence of transmission of Leishmania (Viannia) lainsoni in a Sub Andean region of Bolivia

Using ubiquitous primers which amplify the variable parts of kDNA minicircle of all Leishmania spp, we obtained for Leishmania (viannia) lainsoni a major band of 605 bp (band 1) shared with L. V. braziliensis and a minor 524 bp band (band 2) specific of L. V. lainsoni. The specificity of the two bands was examined through Southern blot hybridization of kDNA PCR obtained from reference strains belonging to L. braziliensis, L. mexicana, L. donovani complexes with L. V. lainsoni species. Band 1 was not specific of L. V. lainsoni since it hybridized with some isolates belonging to L. braziliensis complex. In contrast, band 2 was L. V. lainsoni specific. PCR-based detection followed by hybridization with the new L. V. lainsoni probe (Band 2) and L. V. braziliensis probe (564 bp), was assayed using sample from a pool of 25 females of Lutzomiya nuneztovari anglesi, blood, skin and liver samples of 18 mammals, spinal cords of four mammals and blood and cutaneous ulcers aspirates from 95 patents from Sub Andean region of La Paz, Bolivia. We observed a ositive hybridization of four patients lesions and the pool of L. nuneztovari anglesi with the L. V. lainsoni probe. It is the first time that L. V. lainsoni is observed in a cycle of transmission in Bolivia. PCR products of three patients lesions and the pool of L. nuneztovari anglesi were also hybridized with the specific probe of L. V. braziliensis suggesting mixed infection in this focus.     

CD4+CD25+ T cells in skin lesions of patients with cutaneous leishmaniasis exhibit phenotypic and functional characteristics of natural regulatory T cells

Endogenous regulatory T (Treg) cells are involved in the control of infections, including Leishmania infection in mice. Leishmania viannia braziliensis is the main etiologic agent of cutaneous leishmaniasis (CL) in Brazil, and it is also responsible for the more severe mucocutaneous form. Here, we investigated the possible involvement of Treg cells in the control of the immune response in human skin lesions caused by L. viannia braziliensis infection. We show that functional Treg cells can be found in skin lesions of patients with CL. These cells express phenotypic markers of Treg cells--such as CD25, cytotoxic T lymphocyte-associated antigen 4, Foxp3, and glucocorticoid-induced tumor necrosis factor receptor--and are able to produce large amounts of interleukin-10 and transforming growth factor- beta . Furthermore, CD4+CD25+ T cells derived from the skin lesions of 4 of 6 patients with CL significantly suppressed in vitro the phytohemagglutinin-induced proliferative T cell responses of allogeneic peripheral-blood mononuclear cells (PBMCs) from healthy control subjects at a ratio of 1 Treg cell to 10 allogeneic PBMCs. These findings suggest that functional Treg cells accumulate at sites of Leishmania infection in humans and possibly contribute to the local control of effector T cell functions.     

Use of DNA-based diagnostic methods for human leishmaniasis in Minas Gerais, Brazil

DNA hybridisation was used to type 26 samples from lesions of human patients from the Rio Doce Valley (Minas Gerais, Brazil) clinically diagnosed as having cutaneous leishmaniasis, using kinetoplast DNA (kDNA) cloned mini-circle probes specific for the Leishmania mexicana and Leishmania braziliensis complexes. All samples were found to belong to the L. braziliensis complex. When biopsies were pressed directly onto touch blot membranes 38.5% of the samples were positive. The positivity and specificity obtained were both 100% when cultured blotted parasites were used. The results were confirmed by polymerase chain reaction (PCR) analysis using primers specific for the L. mexicana and L. braziliensis complexes.     

The aetiological agents of American cutaneous leishmaniasis in the municipality of Monte Negro, Rondônia state, western Amazonia, Brazil

Although American cutaneous leishmaniasis (ACL) is one of the most important endemic diseases in the Brazilian state of Rond?nia, there is very little information on the species of parasite involved. The objective of the present study was to identify the Leishmania species causing ACL in the Monte Negro municipality of the state. Over a 6-year period (1997-2002), the skin lesions of 233 patients were examined while the patients were attending an outpatients' clinic at the University of S?o Paulo's Advanced Research Unit in Monte Negro. ACL was diagnosed in 137 (58.8%) of the patients and leishmanial parasites were successfully isolated from 14 of the ACL cases. Using a panel of 24 monoclonal antibodies, 12 of the 14 isolates were identified, as L. (Viannia) braziliensis (seven), L. (V.) lainsoni (one), a L. (V.) lainsoni-like species (two), a L. (V.) guyanensis-like species (one), or a L. (Viannia) species that was different from all named species (one). These are the first records of human infection with L. (V.) braziliensis and L. (V.) lainsoni in Rond?nia.     

Cutaneous leishmaniasis in Guatemala: isoenzyme characterization of isolates from humans

Leishmania organisms cultivated from cutaneous lesions of humans in Guatemala were characterized by cellulose acetate electrophoresis. Six isolates had electrophoretic enzyme patterns identical to World Health Organization reference strains of Leishmania braziliensis braziliensis, and 5 had patterns identical to reference strains of Leishmania mexicana mexicana.     

Antibody response in patients with cutaneous leishmaniasis infected by Leishmania (Viannia) braziliensis or Leishmania (Viannia) guyanensis in Brazil

The antibody response against Leishmania (Leishmania) amazonensis crude antigen was measured through the indirect immunofluorescent assay (IFA) and the immunoenzymatic assay (ELISA) in 114 patients with cutaneous leishmaniasis (CL) in Brazil. Fifty-four patients were infected by Leishmania (Viannia) braziliensis, and 60 patients had L. (V.) guyanensis infection. Patients were comparable by age, sex, disease duration and the Montenegro skin test diameter. L. (V.) braziliensis-infected patients showed significant lower number of ulcerated lesions, greater ulcerated area and higher proportion of lymph node enlargement. Sensitivity of IFA was 79.6% (95% CI 66.1-88.9) and 71.7% (95% CI 58.4-82.2) for L. (V.) braziliensis and L. (V.) guyanensis-infected patients, respectively (P=0.324). Sensitivity of ELISA was 98.2% (95% CI 88.8-99.9) and 85.0% (95% CI 72.9-92.5) for L. (V.) braziliensis and L. (V.) guyanensis-infected patients, respectively (P=0.018). Significant differences were observed in the magnitude of the antibody response before treatment with higher levels detected in L. (V.) braziliensis-infected patients by both serologic techniques. Eighty-four patients had serologic evaluations before and 12 weeks after treatment with meglumine antimoniate, 20 mg/kg/day for 20 days. Significant lower optic density values were observed after treatment with both species independent of cure or failure. Our data showed that L. (V.) braziliensis induces a higher antibody response against L. (L.) amazonensis antigens than L. (V.) guyanensis and that down-modulation of the antibody response occurs shortly during disease evolution after treatment. Moreover the data support the use of ELISA as a better tool for detection of antibodies in CL.     

Cutaneous leishmaniasis associated with extensive lymphadenopathy during an epidemic in Ceará State, northeast Brazil

During an epidemic of cutaneous leishmaniais in northeast Brazil a prospective study was conducted in order to assess clinical, parasitological and immunological features of cutaneous leishmaniasis in a representative sample of patients. A general examination was done and demographic and anthropometric data as well as numbers, duration, location, size, consistency and tenderness of enlarged lymph nodes and lesions recorded. Hematologic and biochemical parameters as well as Leishmania-specific antibodies were measured in the blood. Lymph node aspirations were performed to detect Leishmania-specific DNA by PCR. Cellular immunity was assessed by a leishmanin skin test. Ninety-seven patients with cutaneous leishmaniasis presented with a total of 181 lesions and 127 enlarged regional lymph nodes. In 36% the lymph node enlargement had been observed 2 days to 2 months before the development of a lesion. In 59% the lesions were followed by lymphadenopathy by 3 days to 3 months. The skin test was positive in all patients tested. PCR of the lymph node aspirates was positive in 63.2%. No significant correlations were found between positive PCR results, antibody levels or number of lesions. In northeast Brazil cutaneous leishmaniasis due to Leishmania braziliensis is constantly accompanied by an extensive lymphadenopathy. This may help to identify patients when lesions are still small or have not even developed and lymphadenopathy is the only clinical sign.     

Transforming growth factor beta as a virulence mechanism for Leishmania braziliensis.

Transforming growth factor beta (TGF-beta) has potent down-regulating effects on macrophages and is thus capable of influencing the fate of intramacrophage parasites, including leishmanias. We report the development of a mouse model for the study of the human pathogen Leishmania braziliensis and demonstrate, both in vitro and in vivo, a key regulatory role for TGF-beta in the pathogenesis of infection with this parasite. Recombinant TGF-beta added to cultures of murine peritoneal macrophages led to increased intracellular L. braziliensis replication, whereas addition of neutralizing anti-TGF-beta monoclonal antibody decreased levels of infection. Macrophages infected with L. braziliensis produced biologically active TGF-beta, with a direct correlation between amounts of TGF-beta induced by two parasite isolates and their relative virulence. In vivo, treatment with recombinant TGF-beta rendered avirulent parasites virulent and activated latent L. braziliensis infection. Activation of parasite replication was observed in mice which had been infected with L. braziliensis 15 weeks previously but had not developed lesions or had healed lesions, depending on the parasite isolate used to infect the mice. The exacerbation of L. braziliensis infection in vivo was associated with an increase of interleukin 10 mRNA in the draining lymph node. These results demonstrate that TGF-beta is able to alter the course of in vitro and in vivo infections with L. braziliensis, the latter being characterized by an increase in interleukin 10, an important Th2 helper-T-cell cytokine.     

Infectivity of the subspecies of the Leishmania braziliensis complex in vivo and in vitro

The infectivity of Leishmania braziliensis ssp. in relation to their growth kinetics in Senekjie's medium was determined using the human macrophage cell line U937 and inbred hamsters. In both systems, infectivity was shown to be distinctive for each subspecies. While L. b. panamensis promastigotes from 6-day-old cultures (early stationary phase) were more infective than parasites from any other culture day, L. b. guyanensis and L. b. braziliensis reached maximum infectivity on days 8-10 and day 10 (late stationary phase of growth), respectively. Although maximum infectivity occurred during stationary growth, strict growth phase dependency was not observed. The populations of parasites on these culture days were composed mostly of small, highly motile promastigotes with flagella 2-3 times the length of their cell bodies. These promastigotes resembled the infective forms transmitted by the sand fly vector. A distinct pathological picture characterized the disease caused by the different WHO reference strains for these subspecies in hamsters: L. b. guyanensis developed the most severe lesions, while moderate and inconspicuous lesions were observed when L. b. panamensis and L. b. braziliensis, respectively, constituted the inocula.     

Disseminated leishmaniasis: a new and emerging form of leishmaniasis observed in northeastern Brazil

During the past decade, there has been an increase in the number of patients with disseminated leishmaniasis (DL), which is characterized by a large number of acneiform and papular skin lesions, with very few or no parasites in the skin tissue. The present report describes 42 cases of DL identified between 1992 and 1998 in an area where Leishmania braziliensis transmission is endemic; 8 of the patients were prospectively diagnosed. In a contrast to localized cutaneous leishmaniasis (LCL), acquisition of DL was associated with age >19 years (P<.05), male sex (P<.05), and agricultural occupation (P<.001). Patients with DL presented with 10-300 lesions that were a mixture of acneiform, papular, nodular, and ulcerated types. Twelve (29%) of 42 patients had mucosal involvement. Patients with DL had lower levels of interferon-gamma (P<.05) and tumor necrosis factor-alpha (P<.05) production, compared with patients with LCL. DL is an emerging clinical distinct form of leishmaniasis associated with agricultural activities and host immunological response.     

Placebo-controlled clinical trial of meglumine antimonate (glucantime) vs. localized controlled heat in the treatment of cutaneous leishmaniasis in Guatemala

Sixty-six Guatemalans with parasitologically proven cutaneous leishmaniasis were randomly and equally divided into 3 treatment groups: those receiving meglumine antimonate (Glucantime), 850 mg antimony/day im for 15 days; those receiving localized controlled heat from a radio-frequency generator, 50 degrees C for 30 sec, 3 treatments at 7 day intervals; and those receiving treatment with a placebo. Of 53 isolates identified, 40 were Leishmania braziliensis braziliensis and 13 were L. mexicana mexicana. Thirteen weeks after beginning treatment, the number of patients from each group with completely healed and parasitologically negative lesions were as follows: meglumine antimonate, 16 (73%); localized heat, 16 (73%); and placebo, 6 (27%). The cure rate for those with infections due to L. b. braziliensis in each group was as follows: meglumine antimonate, 11 out of 14 (79%); controlled heat, 9 out of 14 (64%); and placebo, 0 out of 11.     

Polymerase chain reaction detection of Leishmania kDNA from the urine of Peruvian patients with cutaneous and mucocutaneous leishmaniasis

We hypothesized that Leishmania kDNA may be present in urine of patients with cutaneous leishmaniasis (CL). Urine samples and standard diagnostic specimens were collected from patients with skin lesions. kDNA polymerase chain reaction (PCR) was performed on samples from patients and 10 healthy volunteers from non-endemic areas. Eighty-six of 108 patients were diagnosed with CL and 18 (21%) had detectable Leishmania Viannia kDNA in the urine. Sensitivity and specificity were 20.9% (95% confidence interval [CI] 12.3-29.5%) and 100%. Six of 8 patients with mucocutaneous involvement had detectable kDNA in urine versus 12 of 78 patients with isolated cutaneous disease (P < 0.001). L. (V.) braziliensis (N = 3), L. (V.) guyanensis (N = 6), and L. (V.) peruviana (N = 3) were identified from urine. No healthy volunteer or patient with an alternate diagnosis had detectable kDNA in urine. Sensitivity of urine PCR is sub-optimal for diagnosis. On the basis of these preliminary data in a small number of patients, detectable kDNA in urine may identify less localized forms of infection and inform treatment decisions.     

Comparison of cutaneous leishmaniasis due to Leishmania (Viannia) braziliensis and L. (V.) guyanensis in Brazil: clinical findings and diagnostic approach.

We compared the clinical findings and diagnostic methods for 66 patients with cutaneous leishmaniasis in the state of Bahia, Brazil, who were infected by Leishmania (Viannia) braziliensis (group A), with those for 68 patients in the state of Amazonas, Brazil, who were mainly infected by Leishmania (Viannia) guyanensis (group B). Differences were observed with regard to number, size, and location of skin lesions and to the pattern of lymphatic involvement. Patients in group B had smaller and more numerous lesions, which were frequently located above the waist, versus the larger but less numerous lesions among patients in group A, which were usually located on the lower limbs. Lymphatic involvement was present in 55 (83.3%) of the 66 patients in group A and in 42 (61.8%) of the 68 patients in group B (P=0.005). The positivity rates of imprints and skin culture procedures were higher in group B. Sensitivity of in vitro culture of skin aspirates was 47.0% and 91.2% for groups A and B, respectively (P<.001). Although hamster inoculation showed similar results in both groups, the interval before development of disease was shorter in group B. Our data provide substantial evidence that indicate that the disease caused by these species differs with regard to clinical presentation and diagnostic approach.     

Cross-immunity experiments between different species or strains of Leishmania in rhesus macaques (Macaca mulatta)

This study evaluates cross-immunity in rhesus monkeys (Macaca mulatta) previously infected with one species of Leishmania and have had self-cured disease or were cured by antimony-based therapy upon development of full-blown disease. We found that a self-healing cutaneous leishmaniasis (CL) following experimental infection with Leishmania (Leishmania) major induces significant protection for L. (L.) amazonensis and L. (Viannia) guyanensis, and was dependent on time of re-challenge by L (L.) amazonensis after animals had recovered from primary lesions, but lacked protection against L. (V.) braziliensis. In contrast, monkeys that recovered from L. (V.) braziliensis CL or L. (L.) chagasi visceral leishmaniasis following chemotherapeutic intervention were protected by challenge with L. (V.) braziliensis and L (L.) amazonensis. These findings indicate the relative variability in protection after self-cure or drug-cured experimental leishmaniasis to challenge by heterologous leishmanial parasites. Further studying the immune response may provide information regarding relevant factors influencing cross-protective immunity.     

First case of cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Suriname

The main causative agent of cutaneous leishmaniasis (CL) in Suriname is Leishmania (Viannia) guyanensis. This case report presents a patient infected with Leishmania (Viannia) braziliensis, a species never reported before in Suriname. This finding has clinical implications, because L. braziliensis has a distinct clinical phenotype characterized by mucocutaneous leishmaniasis, a more extensive and destructive form of CL that requires different treatment. Clinicians should be aware that chronic cutaneous ulcers in patients from the Guyana region could be caused by L. braziliensis.     

Diffuse cutaneous leishmaniasis in Mexico

In Mexico, 6 cases of diffuse cutaneous leishmaniasis (DCL) were found in widely separated geographic regions. Information was also available on 2 other cases. In addition to the typical clinical features, half of the patients had evidence of nasopharyngeal mucosal involvement. All isolates from the DCL patients were identified as Leishmania mexicana mexicana by isoenzyme analysis and monoclonal antibody typing. In 1 region of Tabasco state where DCL was found, uncomplicated cutaneous leishmaniasis appeared to be highly endemic, and isolates from a few such patients were identified as L. mexicana mexicana. An incidental finding was the recovery of an isolate of L. braziliensis braziliensis from a patient with chiclero ulcer in Oaxaca state. The clinical and epidemiological significance of the reported cases are discussed.     

Leishmania braziliensis from the Pacific coast region of Colombia: foci of transmission, clinical spectrum and isoenzyme phenotypes

Tegumentary leishmaniasis is highly prevalent in the Pacific coast region of Colombia. We have identified 90 foci of transmission in this region based on 179 parasitologically diagnosed patients. Human transmission occurred in mangrove forests, secondary growth and intervened tropical rain forest. A parasitological diagnosis, that is, either isolation or visualization of Leishmania was made in 68.6% of suspected cases. Three phenotypically distinguishable groups of L. braziliensis were encountered based on isoenzymes: L. b. panamensis variants (82%), variants of L. b. braziliensis (14.5%), and stocks intermediate between L. b. panamensis and L. b. guyanensis reference strains (3.5%). The L. b. braziliensis variants produced cutaneous disease alone relatively infrequently (12% of classified cutaneous stocks) but were more frequently (38% of all mucosal stocks) isolated from mucosal lesions. Leishmania infection of the mucous membranes caused a wide spectrum of disease, severity being closely related to time of evolution. Both contiguous and metastatic spread to the mucous membranes was supported by the clinical course of 19 mucosal cases.     

Mucocutaneous leishmaniasis in Colombia: Leishmania braziliensis subspecies diversity

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