Identification of antigenically distinct populations of Leishmania (Viannia) guyanensis from Manaus, Brazil, using monoclonal antibodies

Eighty Leishmania isolates from patients who contracted cutaneous leishmaniasis in the Manaus region, Amazonas State, Brazil, were identified as Leishmania (Viannia) guyanensis by the electrophoretic profiles of six enzymes. None reacted with the species-specific monoclonal antibody B19. Two L. (V.) guyanensis subpopulations were detected with the monoclonals B2 and B12. The lack of B19 expression by the L. (V.) guyanensis strains in the present study contrasts with that of the vast majority of the strains of the same parasite from eastern Amazonia and French Guyana that express the epitope.     

Leishmania (Viannia) braziliensis is the prevalent species infecting patients with tegumentary leishmaniasis from Mato Grosso State, Brazil

The frequency of Leishmania (Viannia) braziliensis infection among patients of Mato Grosso, Brazil was estimated by polymerase chain reaction-PCR, DNA hybridization and by isoenzyme electrophoresis. Analysis of DNA polymorphism was carried out using random amplified polymorphic DNA-PCR (RAPDPCR) with five different primers. The patients were attended from May 1997 to February 1998 at the Reference Ambulatory for American Tegumentary Leishmaniasis at Júlio Müller University Hospital of the Federal University of Mato Grosso, Brazil. In a first screening by PCR and DNA hybridization 94.1% of 68 patients, from whom parasites were isolated in culture medium, were found to be infected with species of the Le. braziliensis complex. Only four patients (5.9%) were infected with species of Le. mexicana complex. Thirty-three samples of Le. braziliensis complex and three of Le. mexicana complex were typed by isoenzyme analysis as Le. (V.) braziliensis sensu stricto and Le. (Leishmania) amazonensis, respectively. The predominant species was Le. (V.) braziliensis, although most of the patients of this study came from the northern area of Mato Grosso, which is part of the Amazonian region of Brazil, where other known species of both subgenus Viannia (Le. braziliensis complex) and Leishmania (Le. mexicana complex) are present. The results of RAPD showed higher genetic variability among the Le. (V.) braziliensis samples from Mato Grosso. The importance of these results concerning the taxonomic status of New World Leishmania, and their implications for both clinical and epidemiological data is discussed.     

Influence of Leishmania (Viannia) species on the response to antimonial treatment in patients with American tegumentary leishmaniasis

BACKGROUND: Pentavalent antimonials (SbV) are the first-line chemotherapy for American tegumentary leishmaniasis (ATL). There are, however, reports of the occurrence of treatment failure with these drugs. Few studies in Latin America have compared the response to SbV treatment in ATL caused by different Leishmania species. METHODS: Clinical parameters and response to SbV chemotherapy were studied in 103 patients with cutaneous leishmaniasis (CL) in Peru. Leishmania isolates were collected before treatment and typed by multilocus polymerase-chain-reaction restriction fragment-length polymorphism analysis. RESULTS: The 103 isolates were identified as L. (Viannia) peruviana (47.6%), L. (V.) guyanensis (23.3%), L. (V.) braziliensis (22.3%), L. (V.) lainsoni (4.9%), L. (Leishmania) mexicana (1%), and a putative hybrid, L. (V.) braziliensis/L. (V.) peruviana (1%). L. (V.) guyanensis was most abundant in central Peru. Of patients infected with the 3 former species, 21 (21.9%) did not respond to SbV chemotherapy. The proportions of treatment failure (after 12 months of follow-up) were 30.4%, 24.5%, and 8.3% in patients infected with L. (V.) braziliensis, L. (V.) peruviana, and L. (V.) guyanensis, respectively. Infection with L. (V.) guyanensis was associated with significantly less treatment failure than L. (V.) braziliensis, as determined by multiple logistic regression analysis (odds ratio, 0.07 [95% confidence interval, 0.007-0.8]; P=.03). CONCLUSIONS: Leishmania species can influence SbV treatment outcome in patients with CL. Therefore, parasite identification is of utmost clinical importance, because it should lead to a species-oriented treatment.     

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.     

Cellular localization and expression of gp63 homologous metalloproteases in Leishmania (Viannia) braziliensis strains

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that encompasses a broad spectrum of clinical manifestations. In a previous study, we showed that Brazilian and Colombian L. braziliensis strains, isolated from patients with distinct clinical manifestations, display different pattern of metalloprotease activities. Following these results, we investigated the cellular localization of these molecules and their relation to the major surface protease (gp63) of Leishmania. Comparative analyses of metalloprotease expression among different clinical isolates as well as an evaluation of the effect of long-term in vitro passage on the expression pattern of these metalloproteases were also performed. Western blot analysis, using an anti-gp63 antibody, revealed polypeptide patterns with a similar profile to that observed in zymographic analysis. Flow cytometry and fluorescence microscopy analyses corroborated the presence of metalloproteases with homologous domains to gp63 in the parasites and revealed differences in the expression level of such molecules among the isolates. The cellular distribution of metalloproteases, assessed by confocal analysis, showed the existence of intracellular metalloproteases with homologous domains to gp63, predominantly located near the flagellar pocket. Finally, it was observed that differential zymographic profiles of metalloproteases exhibited by L. (V.) braziliensis isolates remain unaltered during prolonged in vitro culture, suggesting that the proteolytic activity pattern is a stable phenotypic characteristic of these parasites.     

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.     

PCR-RFLP to identify Leishmania (Viannia) braziliensis and L. (Leishmania) amazonensis causing American cutaneous leishmaniasis

A PCR-RFLP based method was developed to diagnose and identify the Leishmania species causing American cutaneous leishmaniasis (ACL) in a panel of clinical samples obtained from an endemic region of Brazil. The comparison of the results obtained by PCR-RFLP and PCR-hybridization in the identification of Leishmania (Viannia) braziliensis and L. (Leishmania) amazonensis were highly concordant (kappa=91.5%). The PCR-RFLP method was reliable, fast and easy to conduct on biopsies and presents potential value of utmost importance for the diagnosis and identification of Leishmania in clinical specimens, infected reservoirs and vectors.     

Antileishmanial activity of azithromycin against Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, and Leishmania (Leishmania) chagasi

Azithromycin, an azalide antibiotic, is highly concentrated within different phagocytic cells, especially macrophages. The potential antileishmanial activity of azithromycin against three species of Leishmania from the New World was assessed using in vitro models. Azithromycin decreased viability of promastigote cultures of Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, and Leishmania (Leishmania) chagasi as determined by the colorimetric Alamar blue assay. In amastigote intracellular cultures, a significant decrease in infected macrophages counts was observed for all three species with IC(50) of 20.83 (27 micromol/L), 2.18 (2.7 micromol/L), and 6.12 (7.8 micromol/L) microg/mL, respectively. Azithromycin showed in vitro activity against L. (L.) amazonensis, L. (V.) braziliensis, and L. (L.) chagasi and may offer an alternative to current leishmaniasis treatment.     

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.     

Disseminated cutaneous leishmaniasis due to Leishmania(Leishmania) amazonensis in human immunodeficiency virus(HIV)-infected patients: A report of two cases

Rationale: Co-infection of human immunodeficiency virus(HIV) and Leishmania spp. has impact on clinical and therapeutic outcomes of leishmaniases. Most studies do not present the identification of Leishmania species causing American tegumentary leishmaniasis in co-infections. In the Americas, Leishmania(L.) Viannia(V.) braziliensis and L.(V.) guyanensis have been identified. Patient concerns: In this study, two cases of American tegumentary leishmaniasis in patients infected with HIV are described. Patients presented several lesions with rapid dissemination and mucosal involvement. Diagnosis: Disseminated cutaneous leishmaniasis caused by L. amazonensis was identified by molecular test. Interventions: The patients were treated with conventional therapies for HIV infection and American tegumentary leishmaniasis. Outcomes: In co-infection, the clinical manifestations are atypical and the treatment response can be impaired. Lessons: These cases show that HIV infection impacts L. amazonensis infection and point to the relevance of identifying Leishmania species, which can lead to a better patient management.     

Isolation, purification, characterization and antigenic evaluation of GPI-anchored membrane proteins from Leishmania (Viannia) braziliensis

GPI-anchored proteins from the plasma membrane of Leishmania (Viannia) braziliensis promastigotes were isolated, characterized and their migration pattern compared with those from other Leishmania species. In all cases the SDS-PAGE migration patterns were obtained under reducing and non-reducing conditions, using DL-dithiothreitol (DTT) as a reducer agent. Our results reveal that under reducing conditions the SDS-PAGE migration pattern is modified as a consequence of the disruption of disulphur-bonds and protein transformation. This is demonstrated when in non-reducing conditions the L. (V.) braziliensis-GPI-anchored proteins pattern showed a group of bands over the 100kDa, and two more bands of 52kDa and 50kDa in four different isolates, whereas under reducing conditions the major GPI-anchored protein fractions were detected as bands of 63kDa, 50kDa and an increase of peptides between 34kDa and 22kDa. Similar modifications were detected in the SDS-PAGE migration patterns of GPI-anchored protein fractions from L. (Leishmania) donovani, L. (L.) mexicana and L. (L.) amazonensis run under the same reducing conditions. Antigenic evaluation carried out by Western blot revealed the presence of two very specific L. (V.) braziliensis-GPI-anchored protein bands of 50kDa and 28kDa. These bands were specifically recognized by anti-L. (V.) braziliensis-GPI-anchored protein serum from experimentally immunized animals. These two peptides were not detected when GPI-anchored protein fractions from L. (L.) donovani, L. (L.) mexicana and L. (L.) amazonensis, were challenged with the same anti-serum. The present results lead us to suggest the use of these two peptides as biochemical markers to identify and differentiate leishmaniasis caused by L. (V.) braziliensis. The lack of immunogenicity observed here with the peptide gp63, a very common protein detected in Leishmania species, is considered.     

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.     

Epidemiology of cutaneous leishmaniasis in Suriname: a study performed in 2006

Cutaneous leishmaniasis (CL) is a widespread disease in Suriname caused by Leishmania Viannia guyanensis. It is argued that other Leishmania species are also responsible for CL and that the incidence is increasing. This study aimed to identify the species causing the disease and to estimate the annual detection rate of CL in Suriname in 2006. In Paramaribo, 152 patients were registered, of whom 33 were tested in two polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) methods. Twenty-seven patients were infected with L. (V.) guyanensis (complex), one with L. (V.) lainsoni, and one with L. (Leishmania) amazonensis. In the hinterland, 162 CL suspected patients were registered by questionnaires; of these, 24 of 27 tested positive by PCR-RFLP (88.9%; 95% CI, 77.1-100%). With extrapolation of collected data, a detection rate was calculated of 5.32 to 6.13 CL patients per 1,000 inhabitants for the hinterland and 0.64 to 0.74 patients per 1,000 inhabitants for the whole country.     

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.     

Leishmania (Viannia) braziliensis is the predominant species infecting patients with American cutaneous leishmaniasis in the State of Minas Gerais, Southeast Brazil

Skin biopsies from 53 patients with American cutaneous leishmaniasis (ACL) from the State of Minas Gerais, Brazil, were used for a characterization of the Leishmania parasites. A pair of primers flanking the conserved region of the Leishmania minicircle kDNA was used to obtain amplified DNA via the polymerase chain reaction. The amplified products were subsequently hybridized with Leishmania subgenus-specific radiolabeled probes. Parasites from 49 out of 53 samples (92.5%) were characterized as belonging to the subgenus Viannia and four (7.5%) as belonging to the subgenus Leishmania. Clinical, epidemiological and molecular evidence allow us to conclude that Leishmania (V.) braziliensis and Leishmania (L.) amazonensis are the species present in the patients studied and that L. (V.) braziliensis is the predominant species in the State of Minas Gerais, Brazil.     

Immunopathogenic competences of Leishmania (V.) braziliensis and L. (L.) amazonensis in American cutaneous leishmaniasis

The immunopathogenic competences of Leishmania (V.) braziliensis and L. (L.) amazonensis were reviewed in the light of more recent features found in the clinical and immunopathological spectrum of American cutaneous leishmaniasis. It was shown a dichotomy in the interaction between these Leishmania species and human T-cell immune response; while L. (V.) braziliensis shows a clear tendency to lead infection from the localized cutaneous leishmaniasis (LCL), a moderate T-cell hypersensitivity form at the centre of the spectrum, toward to the mucocutaneous leishmaniasis (MCL) at the T-cell hypersensitivity pole and with a prominent Th1-type immune response, L. (L.) amazonensis shows an opposite tendency, leading infection to the anergic diffuse cutaneous leishmaniasis (ADCL) at the T-cell hyposensitivity pole and with a marked Th2-type immune response. Between the central LCL and the two polar MCL and ADCL, the infection can present an intermediary form known as borderline disseminated cutaneous leishmaniasis, characterized by an incomplete inhibition of T-cell hypersensitivity but with a evident supremacy of Th1 over Th2 immune response (Th1 ≥ Th2). These are probably the main immunopathogenic competences of L. (V.) braziliensis and L. (L.) amazonensis regarding the immune response dichotomy that modulates human infection outcome by these Leishmania parasites.     

Usefulness of sampling with cotton swab for PCR-diagnosis of cutaneous leishmaniasis in the New World

In this study, we tested the polymerase chain reaction (PCR)-method to diagnose cutaneous leishmaniasis (CL) by taking exudate materials from lesions with cotton swabs, using our previously tested (PCR) panel comprised of Leishmania (Viannia) panamensis, L. (V.) braziliensis, L. (V.) guyanensis, L. (Leishmania) mexicana and L. (L.) amazonensis. The objectives of the present study were to improve the sampling method convenient for the patients and to test the usefulness of samples taken with cotton swabs. Sixteen patients were clinically diagnosed to have CL including one case of diffuse cutaneous leishmaniasis (DCL) in Ecuador and the causative Leishmania parasites were identified by PCR. All the 12 samples from CL patients of La Mana, positive for Leishmania DNA, were identified as L. (V.) panamensis, while two from CL of Huigra and one from DCL of San Ignacio were L. (L.) mexicana. In the field condition, taking biopsy material is not only painful but sometimes causes iatrogenic bacterial infections. Considering the sensitivity of the test, and convenient sampling procedure, it may be suggested that collection of exudates using cotton swabs may be a better alternative to biopsy sample for PCR-diagnosis of CL.     

Species assignation of Leishmania from human and canine American tegumentary leishmaniasis cases by multilocus enzyme electrophoresis in North Argentina

Sixteen Leishmania stocks, 15 isolated from patients with cutaneous (CL), mucocutaneous (MCL), or recurrent cutaneous leishmaniasis, plus one from a dog with CL in Salta and Corrientes Provinces, Argentina, were studied by multilocus enzyme electrophoresis. Thirteen of the stocks from humans were grouped in two zymodemes; nine termed as KMS 1, four as KMS 2, and assigned to Leishmania (Viannia) braziliensis. Two additional stocks from CL cases expressed a KMS 4 enzyme profile, corresponding to L. (V.) guyanensis. Although the parasites from the dog were also assigned to L. (V.) braziliensis, its zymodeme, KMS 3, was not expressed in any of the current human isolates. The characterization of Leishmania from a dog was done for the first time in Argentina. The importance of the intraspecific polymorphism in the induction of clinical forms and in the host-reservoir concept is briefly discussed, based on the zymodeme data of isolates from humans and dogs. The presence of L. (V.) guyanensis was confirmed in the country.     

In vitro sensitivity of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis Brazilian isolates to meglumine antimoniate and amphotericin B

Resistance of Leishmania parasites to specific chemotherapy has become a well-documented problem in the Indian subcontinent in recent years but only a few studies have focused on the susceptibility of American Leishmania isolates. Our susceptibility assays to meglumine antimoniate were performed against intracellular amastigotes after standardizing an in vitro model of macrophage infection appropriate for Leishmania (Viannia) braziliensis isolates. For the determination of promastigote susceptibility to amphotericin B, we developed a simplified MTT-test. The sensitivity in vitro to meglumine antimoniate and amphotericin B of 13 isolates obtained from Brazilian patients was determined. L. (V.) braziliensis isolates were more susceptible to meglumine antimoniate than Leishmania (Leishmania) amazonensis . EC₅₀, EC₉₀ and activity indexes (calculated over the sensitivity of reference strains), suggested that all isolates tested were susceptible in vitro to meglumine antimoniate, and did not show association with the clinical outcomes. Isolates were also uniformly susceptible in vitro to amphotericin B.     

Species diversity of Leishmania (Viannia) parasites circulating in an endemic area for cutaneous leishmaniasis located in the Atlantic rainforest region of northeastern Brazil

Objectives  To identify the aetiological agents of cutaneous leishmaniasis and to investigate the genetic polymorphism of Leishmania (Viannia) parasites circulating in an area with endemic cutaneous leishmaniasis (CL) in the Atlantic rainforest region of northeastern Brazil.
Methods  Leishmania spp. isolates came from three sources: (i) patients diagnosed clinically and parasitologically with CL based on primary lesions, secondary lesions, clinical recidiva, mucocutaneous leishmaniasis and scars; (ii) sentinel hamsters, sylvatic or synanthropic small rodents; and (iii) the sand fly species Lutzomyia whitmani . Isolates were characterised using monoclonal antibodies, multilocus enzyme electrophoresis (MLEE) and polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region rDNA locus.
Results  Seventy-seven isolates were obtained and characterised. All isolates were identified as Leishmania (Viannia) braziliensis serodeme 1 based on reactivity to monoclonal antibodies. MLEE identified 10 zymodemes circulating in the study region. Most isolates were classified as zymodemes closely related to L. (V.) braziliensis, but five isolates were classified as Leishmania (Viannia) shawi . All but three of the identified zymodemes have so far been observed only in the study region. Enzootic transmission and multiclonal infection were observed.
Conclusions  Our results confirm that transmission cycle complexity and the co-existence of two or more species in the same area can affect the level of genetic polymorphism in a natural Leishmania population. Although it is not possible to make inferences as to the modes of genetic exchange, one can speculate that some of the zymodemes specific to the region are hybrids of L. (V.) braziliensis and L. (V.) shawi .