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.     

A real-time polymerase chain reaction assay for the identification and quantification of American Leishmania species on the basis of glucose-6-phosphate dehydrogenase

A real-time polymerase chain reaction (PCR) test was developed on the basis of the Leishmania glucose-6-phosphate dehydrogenase locus that enables identification and quantification of parasites. Using two independent pairs of primers in SYBR-Green assays, the test identified etiologic agents of cutaneous leishmaniasis belonging to both subgenera, Leishmania (Viannia) and Leishmania (Leishmania) in the Americas. Furthermore, use of TaqMan probes enables distinction between L. (V.) braziliensis or L. (V.) peruviania from the other L. (Viannia) species. All assays were negative with DNA of related trypanosomatids, humans, and mice. The parasite burden was estimated by normalizing the number of organisms per total amount of DNA in the sample or per host glyceraldehyde-3-phosphate dehydrogenase copies. The real-time PCR assay for L. (Leishmania) subgenus showed a good linear correlation with quantification on the basis of a limiting dilution assay in experimentally infected mice. The test successfully identifies and quantifies Leishmania in human biopsy specimens and represents a new tool to study leishmaniasis.     

Natural infection of Lutzomyia tortura with Leishmania (Viannia) naiffi in an Amazonian area of Ecuador

Natural infection of sand flies with Leishmania parasites was surveyed in an Amazonian area in Ecuador where leishmaniasis is endemic. Seventy-one female sand flies were dissected and one was positive for Leishmania protozoa. The species of this sand fly was identified as Lutzomyia (Lu.) tortura on the basis of morphologic characteristics. Analysis of the cytochrome b gene sequence identified the parasite as L. (Viannia) naiffi. We report the distribution of L. (V.) naiffi in Ecuador and detection of a naturally infected sand fly in the Ecuadorian Amazon and natural infection of Lu. tortura with Leishmania parasites in the New World.     

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.     

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.     

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.     

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.     

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.     

Identification, sequencing and expression of peroxidoxin genes from Leishmania aethiopica

Cutaneous leishmaniasis (CL) is a painful, disfiguring and debilitating disease prevalent in Ethiopia and other countries around the world. In Ethiopia, CL is primarily caused by Leishmania aethiopica and less often by L. tropica and L. major. The intracellular survival mechanisms of Leishmania parasites are still not well understood. Recently a new family of antioxidant enzymes called peroxidoxins have been identified that play an important role in parasite survival. In this study, we have identified two distinct peroxidoxin genes (Pxn1 and Pxn2) that are part of a multi-gene family in L. aethiopica. Protein sequence analysis showed that Pxn1 and Pxn2 are highly homologous to peroxidoxins from other Leishmania species. We have found that L. aethiopica Pxn1 is predominantly expressed in amastigotes and stationary phase promastigotes, whereas Pxn2 is constitutively expressed in the different stages of the parasite. This pattern of RNA expression is consistent with patterns seen in some Leishmania species, but not all. Data from this study will be helpful in enhancing vaccine strategies and drug studies targeted towards peroxidoxins.     

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.     

The sensitivity of clinical isolates of Leishmania from Peru and Nepal to miltefosine

Clinical isolates of Leishmania, from visceral leishmaniasis (VL) cases in Nepal and from cutaneous leishmaniasis (CL) cases in Peru, were cultured using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to type species and strain. Promastigotes from 38 isolates, within eight passages from isolation, were used to infect mouse peritoneal macrophage cultures in vitro, and the amastigote sensitivity to miltefosine was determined. The concentration required to kill 50% of intracellular amastigotes from Nepalese VL isolates, all typed as Leishmania (L.) donovani (N = 24) from both Sbv responders and nonresponders, ranged from 8.7 to 0.04 microg/mL. In contrast, the concentration required to kill 50% intracellular amastigotes from isolates from Peru, typed as L.(V.) braziliensis (N = 8), was > 30 to 8.4 microg/mL, L.(V.) guyanensis (N = 2) > 30 to 1.9 microg/mL, L.(L.) mexicana (N = 1) > 30 microg/mL, and L. (V.) lainsoni (N = 4) was 3.4 to 1.9 microg/mL. This demonstrates a notable difference in the intrinsic sensitivity of Leishmania species to miltefosine in vitro. If this model can be correlated to therapeutic outcome, it may have implications for the interpretation of clinical trials.     

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.     

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.     

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.     

Preliminary study towards a novel experimental model to study localized cutaneous leishmaniasis caused by Leishmania (Leishmania) mexicana

There is not an experimental model of localized cutaneous leishmaniasis (LCL) caused by Leishmania (Leishmania) mexicana. The aim of the present study was to characterize the clinical and histological features of Peromyscus yucatanicus experimentally infected with L. (L.) mexicana. A total of 54 P. yucatanicus (groups of 18) were inoculated with 1x10(6) promastigotes of L. (L.) mexicana in the base of the tail. They were euthanized at three and six months post experimental infection. The control group was inoculated with RPMI-1640. The predominant clinical sign observed was a single ulcerated lesion in 27.77% (5/18) and in 11.11% (2/18) P. yucatanicus at three and six months respectively. The histological pattern described as chronic granulomatous inflammation with or without necrosis was found in 7/7 (100%) biopsies of euthanized P. yucatanicus at three (n = 5) and six (n = 2) months, respectively. These results resembled clinical and histological features caused by L. (L.) mexicana in humans, and support the possibility to employ P. yucatanicus as a novel experimental model to study LCL caused by this parasite.     

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 .     

A case of multiple lesion mucocutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis infection

A case of leishmaniasis with multiple lesions due to Leishmania (Viannia) braziliensis infections was diagnosed in a male patient from north-east Brazil. The skin lesions were nodular and pustular and progressed to ulcers. Skin and the oral mucosae were also affected. The behaviour in culture and hamsters of the parasite recovered from a skin lesion was similar to other strains of L. (V.) braziliensis and the strain was characterized by monoclonal antibodies as belonging to serodeme I of this species. The histopathology of the skin lesions of the patient and hamster were of an inflammatory nature which extended through the dermis; leishmania in the tissues were more easily detected by the immunoenzymatic PAP method.     

Genetic analysis of leishmania parasites in Ecuador: are Leishmania (Viannia) panamensis and Leishmania (V.) Guyanensis distinct taxa?

In the course of an epidemiologic survey in Ecuador, the following collection of Leishmania stocks was isolated: 28 from patients with clinical signs of leishmaniasis, 2 from sloths, 1 from a dog, and 4 from sand flies. For genetic characterization of these stocks, multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA (RAPD) were used. Twenty six of the 35 stocks were identified as either Leishmania (V.) panamensis or L. (V.) guyanensis, 2 stocks were identified as L. (V.) braziliensis, the 2 stocks from sloths showed specific genotypes, and 5 stocks were characterized as hybrids between L. (V.) braziliensis and L. (V.) guyanensis. These data show that genetic diversity of Leishmania in Ecuador is high and that L. (V.) panamensis/guyanensis is the dominant group in this country. The genetic analysis questioned the distinctness between the two species L.(V.) panamensis and L. (V.) guyanensis, since MLEE and RAPD data did not indicate that L. (V.) panamensis and L. (V.) guyanensis correspond to distinct monophyletic lines. Population genetic analysis performed on the L. (V.) panamensis/guyanensis group favors the hypothesis of a basically clonal population structure.     

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.     

Detection and identification of Leishmania species in field-captured phlebotomine sandflies based on mini-exon gene PCR

Leishmaniasis is one of the most diverse and complex of all vector-borne diseases. Because it involves several overlapping species and sandfly vectors, the disease has a complex ecology and epidemiology. Adequate therapy and follow-up depend on parasitological diagnosis, but classical methods present several constraints when identifying species. We describe a polymerase chain reaction (PCR) which uses primers designed from mini-exon repetitive sequences that are specific for subgenus LeishmaniaViannia (PV), as well as sequences with specificity for genus (PG) that can distinguish between Leishmania species from other insect flagellates with minor differences in PCR products. For standardization, these PCR were tested in experimentally infected sandflies, and Leishmania infection in these insects was successfully confirmed. This methodology identified a 3.9% infection rate in field-captured phlebotomine sandflies from an endemic region in Brazil. Natural infection by Leishmania species was identified in three samples of Lutzomyia longipalpis, of which two were Leishmania (L.) chagasi and one Leishmania (L.) amazonensis. Irrespective of specific epidemiological conclusions, the method used in this study was able to identify Leishmania infections both in experimentally infected and field-captured phlebotomine sandflies, and could be a useful tool in epidemiological studies and strategic planning for the control of human leishmaniasis.