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.     

Species diversity causing human cutaneous leishmaniasis in Rio Branco, state of Acre, Brazil

OBJECTIVE: Information on Leishmania species diversity in western Brazilian Amazon and the clinical picture of human cutaneous leishmaniasis it causes is scarce. We describe clinical findings, diagnostic procedures and identification of Leishmania species in patients from that region. METHODS: The sample consisted of 50 patients, prospectively evaluated for epidemiological and clinical characteristics by means of a structured questionnaire. Conventional and molecular tools were applied to confirm the parasitological diagnosis and identify the species responsible for the disease. RESULTS: Patients were predominantly male (76.5%) and living in rural areas. Median average age was 18 years and median average disease evolution was 8 weeks. For the diagnostic procedures of leishmanin skin test, direct visualization of amastigotes in dermal scrapings and parasite culture of aspirates of the ulcer border were positive for 98%, 52% and 34%, respectively. Molecular methods applied to DNA extracted from skin biopsies of the 50 patients yielded 100%, 82% and 44% positivity by PCR minicircle kDNA, PCR-RFLP ITS1rDNA and PCR-glucose-6-phosphate (G6P), respectively. Fourteen samples from 13 patients were successfully isolated and identified. Multilocus enzyme electrophoresis, PCR-RFLP ITS1rDNA and PCR-G6P permitted identification of the Leishmania species responsible for the aetiology of American tegumentary leishmaniasis in 60% of the examined patients: 16 Leishmania (Viannia) braziliensis, 12 Leishmania (Viannia) lainsoni, 1 Leishmania (Viannia) guyanensis and 1 putative hybrid of Leishmania (Viannia) naiffi and L. (V.) lainsoni. CONCLUSION: The clinical and epidemiological behaviour of cutaneous leishmaniasis in Acre, Brazil, is similar to other Amazon scenarios previously described; however Acre's complex parasite diversity may be contributed to the concomitant circulation of at least three distinct Leishmania species. The implementation of control interventions in the studied area must take into consideration the possibility of various expected phlebotomine vectors and reservoirs.     

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.     

Naturally infected Lutzomyia sand flies in a Leishmania-endemic area of Brazil

In Brazil, Leishmania transmission involves several species of phlebotomine sand flies that are closely associated with different parasites and reservoirs, giving rise to different transmission cycles. The present study focused on naturally infected phlebotomines originating from Santa Luzia, a municipality near Belo Horizonte, capital of the Brazilian state of Minas Gerais, in which leishmaniasis are endemic. Systematic and non systematic approaches,involving the use of light traps and direct aspiration from resting sites, respectively, were used to collect females and flies. Identification of the captured insects and determination of natural infection by Leishmania spp. were performed using both conventional dissection methods and polymerase chain reaction (PCR). The dissection of 102 sand flies allowed five species of Lutzomyia to be identified, although no flagellate parasite forms were observed.In addition, 211 sand flies were identified, were separated according to species, and were combined into 11 pools of up to 20 individuals each. PCR analyses showed that two of these pools were infected with Leishmania:one pool of Lu. whitmani was infected with Le. (Viannia) spp. and another of Lu. cortelezzii was infected with Le. chagasi. This suggests that Lu. whitmani may be a possible vector of Leishmania in the study area, and more work needs to be performed to assess the role of Lu. cortelezzii as a vector.     

Molecular mass screening to incriminate sand fly vectors of Andean-type cutaneous leishmaniasis in Ecuador and Peru

Sand flies from the Andean areas of Ecuador and Peru were examined for Leishmania infections by using our recently established molecular mass screening method. Leishmanial minicircle DNA-positive sand flies were detected in 3 of 192 and 1 of 462 samples from Ecuador and Peru, respectively. Sand fly species were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the 18S ribosomal RNA (rRNA) gene, and the positive flies were Lutzomyia (Lu.) ayacuchensis and Lu. peruensis, respectively. Furthermore, cytochrome b and mannose-phosphate isomerase gene sequence analyses identified the parasites from Ecuador and Peru as Leishmania (Leishmania) mexicana and L. (Viannia) peruviana, respectively. Thus, the mass screening method was confirmed to be a powerful tool for sand fly research.     

Multiple hybrid genotypes of Leishmania (viannia) in a focus of mucocutaneous Leishmaniasis

The principal agent of mucocutaneous leishmaniasis (MCL) is the South American protozoan parasite Leishmania (Viannia) braziliensis. This organism is generally considered to be clonal, that is, it does not to undergo genetic exchange. Nevertheless, apparent hybrids between several Leishmania species have been reported in the New World and the Old World. When we characterized isolates of Leishmania (Viannia) from a single focus of cutaneous leishmaniasis (CL) and MCL, we found a remarkable phenotypic and genotypic diversity, with 12 zymodemes and 20 microsatellite genotypes. Furthermore, 26 of the 59 isolates were L. braziliensis/L. peruviana phenotypic hybrids that displayed 7 different microsatellite genotypes. A hybrid genotype was the only organism isolated from 4 patients with MCL. Thus hybrids must be included among the potential agents of MCL. Despite the propensity for clonality, hybrids are also an important feature of Leishmania (Viannia) and may give rise to epidemiologically important emergent genotypes.     

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.     

Epidemiology of Andean cutaneous leishmaniasis: incrimination of Lutzomyia ayacuchensis (Diptera: psychodidae) as a vector of Leishmania in geographically isolated, upland valleys of Peru

The southernmost limit of the distribution of endemic Andean cutaneous leishmaniasis (CL), commonly known as Uta, is localized in the western Andean valleys of Ayacucho, Peru. This area is completely isolated from other regions endemic for this disease. Identification of the insect vector for Andean CL was carried out by combining entomologic and parasitologic approaches. Two Lutzomyia species were captured: Lutzomyia ayacuchensis and Lu. noguchii. The former species was considered responsible for transmission of Leishmania because 1) there was a coincidence in space and time between the presence of this insect and the distribution of Andean CL, 2) it was shown to be highly anthropophilic, 3) Leishmania parasites of the subgenus Viannia were detected by a specific polymerase chain reaction assay, 4) promastigotes isolated from this insect were shown by multilocus enzyme electrophoresis and molecular karyotyping to belong to the same deme of Leishmania (Viannia) peruviana as the one circulating in humans living in the study area, and 5) the complete cycle of L. (V.) peruviana was observed in experimental infections of Lu. ayacuchensis. Parasite and vector homogeneity found in Ayacucho contrasted with the heterogeneity reported for other areas endemic for Andean CL. The potential influence of ecologic determinants on this geographically isolated area is discussed.     

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 neivai with Leishmania spp. in northwestern Argentina

The natural infection of Lutzomyia neivai with Leishmania in the endemic area of American cutaneous leishmaniasis (ACL) in northwestern Argentina was analyzed by the polymerase chain reaction (PCR)-hybridization technique. Phlebotominae sand flies were captured in the provinces of Tucumán and Salta between 1999 and 2003. From a sample of 440 Lu. neivai females analysed for the detection of the Leishmania (Viannia) and Leishmania (Leishmania) subgenera, 9.1% of the samples resulted infected with a parasite of the subgenus Viannia and none with the Leishmania. This is the first report of naturally infected sand flies in Argentina besides the first report of infected Lu. neivai sensu strictu. Our results contributed to further incrimination of this specie as vector of leishmaniasis in the area and the identification of the main circulating parasite as belonging to the Leishmania (Viannia) subgenera.     

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.     

Leishmania (Viannia) infection in the domestic dog in Chaparral, Colombia

Peridomestic transmission of American cutaneous leishmaniasis is increasingly reported and dogs may be a reservoir of Leishmania (Viannia) in this setting. We investigated the prevalence of infection in dogs in Chaparral County, Colombia, the focus of an epidemic of human cutaneous leishmaniasis caused by Leishmania (Viannia) guyanensis. Two (0.72%) of 279 dogs had lesions typical of cutaneous leishmaniasis that were biopsy positive by kinetoplast DNA polymerase chain reaction-Southern blotting. Seroprevalence was 2.2% (6 of 279) by enzyme-linked immunosorbent assay. Buffy coat and ear skin biopsy specimens were positive by polymerase chain reaction-Southern blotting in 7.3% (10 of 137) and 11.4% (12 of 105) of dogs, respectively. Overall 20% of dogs (21 of 105) showed positive results for one or more tests. Amplification and sequencing of the Leishmania 7SL RNA gene identified L. guyanensis in one dog and L. braziliensis in two dogs. No association was identified between the risk factors evaluated and canine infection. Dogs may contribute to transmission but their role in this focus appears to be limited.     

Clinical features and diagnosis of 42 travellers with cutaneous leishmaniasis

BACKGROUND: Leishmania species that occur within different geographical areas may cause different clinical manifestations, virulence and drug sensitivity. Patients/Methods. All patients with a clinical diagnosis of cutaneous leishmaniasis seen at the Hospital for Tropical Diseases from 1997 to 2000 were identified and clinical details recorded onto a database, with emphasis on clinical presentation, risk factors, travel history and laboratory diagnosis. RESULTS: Forty-two patients were identified, 23 of whom had travelled to New World and 19 to Old World countries. Clinical presentation typically consisted of a single nodule with ulceration. In 50% infection was caused by L. (Viannia) braziliensis. PCR was performed in specimens from 34 patients and species identification was possible in 32 cases (sensitivity 94%), the two PCR negative patients had amastigotes demonstrated by histology and culture. Patients were treated with established therapies. Seventy one percent were cured by treatment, 12% had a spontaneous cure, 7% were lost to follow-up and the remaining 10% required a second-line therapy. No relapses were reported during a mean follow-up period of 27 months. CONCLUSIONS: Our study highlights the need for comprehensive investigations and the advantages of PCR in the diagnosis of patients with suspected leishmaniasis in non-endemic regions of the world.     

American tegumentary leishmaniasis and HIV-AIDS association in a tertiary care center in the Brazilian Amazon

American tegumentary leishmaniasis (ATL) and human immunodeficiency virus (HIV) are both common infectious diseases in the Brazilian Amazon with overlapping expansion areas, which leads to the occurrence of Leishmania/HIV coinfection. Most ATL/HIV-acquired immunodeficiency syndrome (AIDS) association cases have been reported from areas where Leishmania (Viannia) braziliensis is the main pathogen; this finding is in contrast with the Amazon region, where L. (V.) guyanensis is the most implicated agent, implying distinct clinical and therapeutic aspects. We describe 15 cases of ATL/HIV coinfection treated in a tertiary care center in the Brazilian Amazon between 1999 and 2008. Thirteen patients presented with diverse clinical manifestations of cutaneous leishmaniasis, and four of them had disseminated forms; two patients presented with mucosal leishmaniasis (ML). Seven patients required more than one course of treatment. The particularities of ATL/HIV-AIDS association in L. (V.) guyanensis-endemic areas require efforts for an increased understanding of its burden and subsequent improvements in case management.     

Natural infection of Nyssomyia neivai by Leishmania (Viannia) spp. in the state of Paraná, southern Brazil, detected by multiplex polymerase chain reaction

Natural sandfly infection by Leishmania spp. in an area endemic for American cutaneous leishmaniasis was analyzed using multiplex polymerase chain reaction (PCR). The sandflies were captured using Falc?o light traps in an endemic area of the municipality of Doutor Camargo during March, April, and June 2008. In total, 1803 females were analyzed; 1755 were Nyssomyia neivai (Pinto) and 48 were Nyssomyia whitmani (Antunes and Coutinho). Multiplex PCR analyses using MP3H-MP1L and 5Llcac-3Llcac primers showed the presence of Leishmania (Viannia) spp. in 4/181 pools of sandflies, all Ny. neivai, that is, a minimal infection rate of 0.22%. This study showed, for the first time, the presence of DNA of Leishmania (Viannia) spp. in Ny. neivai. This suggests the existence of natural infection by Leishmania (Viannia) spp. in Ny. neivai in the state of Paraná. Multiplex PCR is an important tool in the detection of Leishmania infection in sandflies.     

Epidemiology of imported cutaneous leishmaniasis at the Hospital for Tropical Diseases, London, United Kingdom: use of polymerase chain reaction to identify the species

This study reviewed all patients diagnosed with imported cutaneous leishmaniasis (CL) at the Hospital for Tropical Diseases in London, United Kingdom, over an 11-year period. Diagnostic and epidemiologic information was collected prospectively for all patients with imported CL to this hospital during 1998-2009. A total of 223 patients were given a diagnosis of CL. Ninety patients were diagnosed with Old World CL, which was caused most commonly by Leishmania donovani complex (n = 20). A total of 71% were tourists to the Mediterranean region, 36% were migrants or visiting friends and relatives, and 17% were soldiers. One hundred thirty-three patients were given a diagnosis of New World CL. The Leishmania subgenus Viannia caused 97 of these cases; 44% of these were in backpackers and 29% were in soldiers. Polymerase chain reaction was more sensitive and faster for detecting Leishmania DNA (86% for Old World CL and 96% for New World CL) than culture. This is the largest study of imported leishmaniasis, and demonstrates that tourists to the Mediterranean and backpackers in Central and South America are at risk for this disease.     

The transmission dynamics of canine American cutaneous leishmaniasis in Huánuco, Peru

The epidemiology of canine American cutaneous leishmaniasis (ACL) due to Leishmania (Viannia) spp. was investigated in Huánuco, Peru to 1) describe the natural course of canine L. (Viannia) infections and 2) assess the role of domestic dogs as ACL reservoir hosts. Over a three-year period 1,022 dogs were surveyed, with cumulative village L. (Viannia) prevalence being 26% (range = 0-100%). The incidence of L. (Viannia) was estimated to be 0.285 dogs/year (95% confidence interval [CI] = 0.160-0.410) using cross-sectional data and 0.291 dogs/year (95% CI = 0.195-0.387) using data from 108 dogs that were surveyed prospectively. The recovery rate was estimated to be 0.456 dogs/year (95% CI = 0.050-0.862) and 0.520 dogs/year (95% CI = 0.302-0.738), respectively. Using those findings, the basic reproduction number was estimated to be R0 approximately to 1.9; if dogs were the principal ACL reservoirs, the mean yearly effort (i.e., coverage or elimination) of a dog control intervention (e.g., collaring, culling, or vaccination) to ensure the elimination of L. (Viannia) spp. transmission would be as low as 47%.     

Molecular detection of Leishmania species in human and animals from cutaneous leishmaniasis endemic areas of Waziristan,Khyber Pakhtunkhwa,Pakistan

Objectives: To detect Leishmania species in human patients, animal reservoirs and Phlebotomus sandflies in Waziristan, Pakistan. Methods: Tissue smears and aspirates from 448 cutaneous leishmaniasis(CL) suspected patients were analyzed. To sort out role of the reservoir hosts, skin scrapings, spleen and liver samples from 104 rodents were collected. Furthermore, buffy coat samples were obtained from 60 domestic animals. Sandflies were also trapped. All human, animals and sandfly samples were tested by microscopy, kinetoplastic PCR and internal transcribed spacer 1(ITS1) PCR followed by restriction fragment length polymorphism for detection of Leishmania species. Results: An overall prevalence of 3.83% and 5.21% through microscopy and ITS1 PCR respectively was found. However, the statistically non-significant correlation was found between area, gender, and number of lesions. The presence of rodents, sandflies, domestic animals and internally displaced people increased the risk of CL. Using ITS1-PCR-RFLP, Leishmania tropica(L. tropica) was confirmed in 106 samples while 25 of the isolates were diagnosed as Leishmania major(L. major). Similarly, 3/104 rodents were positive for L. major and 14 pools of DNA samples containing Phlebotomus sergenti sandflies were positive for L. tropica. None of samples from domestic animals were positive for leishmaniasis. Conclusions: In the present study, L. tropica and L. major are found to be the main causative agents of CL in study area. Movement of internally displaced people from CL endemic areas presents a risk for nearby CL free areas. To the best of our knowledge, we report for the first time L. major infection in rodents(Rattus rattus) and L. tropica in Phlebotomus sergenti sandflies trapped in Waziristan, Pakistan.     

Detection of natural infection in Lutzomyia cruzi and Lutzomyia forattinii (Diptera: Psychodidae: Phlebotominae) by Leishmania infantum chagasi in an endemic area of visceral leishmaniasis in Brazil using a PCR multiplex assay

In order to identify Lutzomyia spp. naturally infected by Leishmania parasites a PCR multiplex assay coupled to non-isotopic hybridization was used for the analysis of insect samples collected by CDC light traps in an endemic area of visceral leishmaniasis (VL) in the municipality of Corumbá, Mato Grosso do Sul State, Brazil in May/June 2006. Wild sand flies were identified and grouped into pools of 10 female specimens and 27 groups in total were collected. Positive results were obtained from Lutzomyia cruzi (2 out of 13 pools) and Lutzomyia forattinii (1 out of 14 pools). The positive pools were confirmed as being infected by Leishmania infantum chagasi after hybridizing the PCR products with a species-specific biotinylated probe derived from the kinetoplast minicircle conserved sequence. Given that we detected infection in 3 out of 27 groups and that there was at least 1 infected insect in each, it was possible to infer an infection rate of 1.5% for Lu. cruzi and 0.7% for Lu. forattinii in the analyzed samples. These results confirm the vectorial role of Lu. cruzi in transmitting L. infantum chagasi and suggest Lu. forattinii as a potential VL vector in the municipality of Corumbá, where notifications of the disease in humans and dogs have increased over the last two decades.     

Activity of a paromomycin hydrophilic formulation for topical treatment of infections by Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis

Studies on in vitro skin permeation and in vivo anti-leishmanial activity in mice experimentally infected with Leishmania (Leishmania) major pointed out to the potential of a new paromomycin (PA) formulation (hydrophilic gel) for treatment of cutaneous leishmaniasis (CL). In this study, the activity of this formulation was evaluated in animals experimentally infected by Leishmania species that prevail in the New World. PA gel activity was compared to antimony treatment, since it is still the first choice treatment to the different clinical forms of leishmaniasis. The topical treatment activity with 10% PA gel in BALB/c mice infected by Leishmania (Leishmania) amazonensis was higher than that observed for parenteral antimony treatment, while the efficacy of these two regimes in hamsters infected by Leishmania (Viannia) braziliensis was similar. These results suggest that this formulation could be suitable for clinical studies and may represent an alternative novel formulation for topical treatment of CL.