The phlebotomine fauna (Diptera: Psychodidae) of Guaraí, state of Tocantins, with an emphasis on the putative vectors of American cutaneous leishmaniasis in rural settlement and periurban areas

Phlebotomine sandflies were captured in rural settlement and periurban areas of the municipality of Guaraí in the state of Tocantins (TO), an endemic area of American cutaneous leishmaniasis (ACL). Forty-three phlebotomine species were identified, nine of which have already been recognised as ACL vectors. Eleven species were recorded for the first time in TO. Nyssomyia whitmani was the most abundant species, followed by Evandromyia bourrouli, Nyssomyia antunesi and Psychodopygus complexus. The Shannon-Wiener diversity index and the evenness index were higher in the rural settlement area than in the periurban area. The evaluation of different ecotopes within the rural area showed the highest frequencies of Ev. bourrouli and Ny. antunesi in chicken coops, whereas Ny. whitmani predominated in this ecotope in the periurban area. In the rural settlement area, Ev. bourrouli was the most frequently captured species in automatic light traps and Ps. complexus was the most prevalent in Shannon trap captures. The rural settlement environment exhibited greater phlebotomine biodiversity than the periurban area. Ps. complexus and Psychodopygus ayrozai naturally infected with Leishmania (Viannia) braziliensis were identified. The data identified Ny. whitmani as a potential ACL vector in the periurban area, whereas Ps. complexus was more prevalent in the rural environment associated with settlements.

Host genetic factors in American cutaneous leishmaniasis: a critical appraisal of studies conducted in an endemic area of Brazil

American cutaneous leishmaniasis (ACL) is a vector-transmitted infectious disease with an estimated 1.5 million new cases per year. In Brazil, ACL represents a significant public health problem, with approximately 30,000 new reported cases annually, representing an incidence of 18.5 cases per 100,000 inhabitants. Corte de Pedra is in a region endemic for ACL in the state of Bahia (BA), northeastern Brazil, with 500-1,300 patients treated annually. Over the last decade, population and family-based candidate gene studies were conducted in Corte de Pedra, founded on previous knowledge from studies on mice and humans. Notwithstanding limitations related to sample size and power, these studies contribute important genetic biomarkers that identify novel pathways of disease pathogenesis and possible new therapeutic targets. The present paper is a narrative review about ACL immunogenetics in BA, highlighting in particular the interacting roles of the wound healing gene FLI1 with interleukin-6 and genes SMAD2 and SMAD3 of the transforming growth factor beta signalling pathway. This research highlights the need for well-powered genetic and functional studies on Leishmania braziliensis infection as essential to define and validate the role of host genes in determining resistance/susceptibility regarding this disease.

Ecology of cutaneous leishmaniasis in Sinai: linking parasites, vectors and hosts

Cutaneous leishmaniasis (CL) is a neglected clinical form of public health importance that is quite prevalent in the northern and eastern parts of Egypt. A comprehensive study over seven years (January 2005-December 2011) was conducted to track CL transmission with respect to both sandfly vectors and animal reservoirs. The study identified six sandfly species collected from different districts in North Sinai: Phlebotomus papatasi, Phlebotomus kazeruni, Phlebotomus sergenti, Phlebotomus alexandri, Sergentomyia antennata and Sergentomyia clydei. Leishmania (-)-like flagellates were identified in 15 P. papatasi individuals (0.5% of 3,008 dissected females). Rodent populations were sampled in the same districts where sandflies were collected and eight species were identified: Rattus norvegicus (n = 39), Rattus rattus frugivorous (n = 13), Rattus rattus alexandrinus (n = 4), Gerbillus pyramidum floweri (n = 38), Gerbillus andersoni (n = 28), Mus musculus (n = 5), Meriones sacramenti (n = 22) and Meriones crassus (n = 10). Thirty-two rodents were found to be positive for Leishmania infection (20.12% of 159 examined rodents). Only Leishmania major was isolated and identified in 100% of the parasite samples. The diversity of both the vector and rodent populations was examined using diversity indices and clustering approaches.

Polymerase chain reaction detection of LeishmaniaDNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detectLeishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmaniagenus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.

The microbiological signature of human cutaneous leishmaniasis lesions exhibits restricted bacterial diversity compared to healthy skin

Localised cutaneous leishmaniasis (LCL) is the most common form of cutaneous leishmaniasis characterised by single or multiple painless chronic ulcers, which commonly presents with secondary bacterial infection. Previous culture-based studies have found staphylococci, streptococci, and opportunistic pathogenic bacteria in LCL lesions, but there have been no comparisons to normal skin. In addition, this approach has strong bias for determining bacterial composition. The present study tested the hypothesis that bacterial communities in LCL lesions differ from those found on healthy skin (HS). Using a high throughput amplicon sequencing approach, which allows for better populational evaluation due to greater depth coverage and the Quantitative Insights Into Microbial Ecology pipeline, we compared the microbiological signature of LCL lesions with that of contralateral HS from the same individuals.Streptococcus, Staphylococcus,Fusobacterium and other strict or facultative anaerobic bacteria composed the LCL microbiome. Aerobic and facultative anaerobic bacteria found in HS, including environmental bacteria, were significantly decreased in LCL lesions (p < 0.01). This paper presents the first comprehensive microbiome identification from LCL lesions with next generation sequence methodology and shows a marked reduction of bacterial diversity in the lesions.

Fauna of phlebotomine sand flies (Diptera, Psychodidae) in areas with endemic American cutaneous leishmaniasis in the State of Mato Grosso do Sul, Brazil

Fauna of phlebotomine sand flies (Diptera, Psychodidae) in areas with endemic American cutaneous leishmaniasis in the State of Mato Grosso do Sul, Brazil. The aim of this study was to investigate the ecological aspects of the main vectors of American cutaneous leishmaniasis (ACL) in four monitoring stations situated in the municipalities of Naviraí, Nova Andradina, Novo Horizonte do Sul and Rio Verde de Mato Grosso. For each monitoring station, the captures of sand flies were undertaken each month from July 2008 to June 2010 using CDC and Shannon traps. The CDC traps were installed simultaneously for three consecutive nights in three collection sites: intradomicile, peridomicile and edge of the forest. A Shannon trap was installed from dusk to 10 pm, inside the forest, one night per month. A total of 7,651 sand flies belonging to nine genera and twenty-nine species were captured. Nyssomyia neivai (52.95%), Psathyromyia hermanlenti (10.91%), Psathyromyia runoides (9.16%), Nyssomyia whitmani (7.95%), Psathyromyia aragaoi (4. 89%), Nyssomyia antunesi (3.14%) and Evandromyia bourrouli (2.20%) were the most frequent species. Approximately 65% of the sand flies were collected in the forest environment. The municipalities presented significantly different indexes of species diversity. Naviraí presented the lowest species diversity index, however, it showed the highest abundance. Novo Horizonte do Sul had the highest species diversity index, but the lowest abundance (< 5%). It is noteworthy the occurrence of vector species of Leishmania in the areas studied, especially in Naviraí, where Ny. neivai presented high frequencies which may explain the increased number of ACL cases in this municipality.

Immunologic patterns associated with cure in human American cutaneous leishmaniasis

Patients with American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-g) and interleukin 4 (IL-4) produced were also determined in the culture supernatants. Two different patterns of Lb-induced T cell responses were observed: a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-g and IL-4) during the active disease, and b) similar proportions of responding CD4+ and CD8+ cells, and type 1 cytokine production (presence of IFN-g and very low IL-4) at the end of therapy (healed lesions). This last pattern is probably associated with a beneficial T cell response

Electrocardiographic changes during low-dose, short-term therapy of cutaneous leishmaniasis with the pentavalent antimonial meglumine

The pentavalent antimonial (Sb5+) meglumine is the drug of choice for the treatment of cutaneous leishmaniasis (CL) in Brazil. Although the cardiotoxicity of high-dose, long-term Sb5+ therapy is well known, the use of low-dose, short-term meglumine has been considered to be safe and relatively free from significant cardiac effects. In order to investigate the cardiotoxicity of low-dose, short-term therapy with meglumine in cutaneous leishmaniasis, 62 CL patients treated with meglumine were studied. A standard ECG was obtained before and immediately after the first cycle of treatment (15 mg Sb5+ kg-1 day-1). The electrocardiographic interpretation was carried out blindly by two investigators using the Minnesota Code. There were no significant differences in qualitative ECG variables before and after meglumine treatment. However, the corrected QT interval was clearly prolonged after antimonial therapy (420.0 vs 429.3 ms, P<10-6). QTc augmentation exceeded 40 ms in 12 patients, 7 of whom developed marked QTc interval enlargement (500 ms) after meglumine therapy. This previously unrecognized cardiac toxicity induced by short-term, low-dose antimonial therapy has potentially important clinical implications. Since sudden death has been related to QTc prolongation over 500 ms induced by high-dose antimonial therapy, routine electrocardiographic monitoring is probably indicated even in CL patients treated with short-term, low-dose meglumine schedules. Until further studies are conducted to establish the interactions between pentavalent antimonials and other drugs, special care is recommended when using meglumine in combination with other medications, in particular with drugs that also increase the QTc interval.

The role of natural killer cells in the early period of infection in murine cutaneous leishmaniasis

In order to study the role of natural killer (NK) cells during the early period of Leishmania infection, BALB/c mice were selectively and permanently depleted of NK cells by injection with 90Sr and subsequently infected with Leishmania (Leishmania) amazonensis (HSJD-1 strain). 90Sr is known to selectively deplete NK cells, leaving an intact T- and B-cell compartment and preserving the ability to produce both interferon alpha and IL-2. This method of depletion has advantages when compared with depletion using anti-NK cell monoclonal antibodies because the effect is permanent and neither activates complement nor provokes massive cell death. In the present study, after one month of treatment with 90Sr, the depletion of NK cells was shown by a more than ten-fold reduction in the cytotoxic activity of these cells: 2 x 106 spleen cells from NK-depleted animals were required to reach the same specific lysis of target cells effected by 0.15 x 106 spleen cells from normal control animals. The histopathology of the skin lesion at 7 days after Leishmania infection showed more parasites in the NK cell-depleted group. This observation further strengthens a direct role of NK cells during the early period of Leishmania infection.

Detection of early apoptosis and cell death in T CD4+ and CD8+ cells from lesions of patients with localized cutaneous leishmaniasis

Human localized cutaneous leishmaniasis (LCL), induced by Leishmania braziliensis, ranges from a clinically mild, self-healing disease with localized cutaneous lesions to severe forms which can present secondary metastatic lesions. The T cell-mediated immune response is extremely important to define the outcome of the disease; however, the underlying mechanisms involved are not fully understood. A flow cytometric analysis of incorporation of 7-amino actinomycin D and CD4+ or CD8+ T cell surface phenotyping was used to determine whether different frequencies of early apoptosis or accidental cell death occur at different stages of LCL lesions. When all cells obtained from a biopsy sample were analyzed, larger numbers of early apoptotic and dead cells were observed in lesions from patients with active disease (mean = 39.5 ± 2.7%) as compared with lesions undergoing spontaneous healing (mean = 17.8 ± 2.2%). Cells displaying normal viability patterns obtained from active LCL lesions showed higher numbers of early apoptotic events among CD8+ than among CD4+ T cells (mean = 28.5 ± 3.8 and 15.3 ± 3.0%, respectively). The higher frequency of cell death events in CD8+ T cells from patients with LCL may be associated with an active form of the disease. In addition, low frequencies of early apoptotic events among the CD8+ T cells were observed in two patients with self-healing lesions. Although the number of patients in the latter group was small, it is possible to speculate that, during the immune response, differences in apoptotic events in CD4+ and CD8+ T cell subsets could be responsible for controlling the CD4/CD8 ratio, thus leading to healing or maintenance of disease.

Comparison of the specificity of PCR and the histopathological detection of leishmania for the diagnosis of American cutaneous leishmaniasis

More precise and rapid diagnostic methods for American cutaneous leishmaniasis (ACL) are necessary because of the growing number of cases observed in Brazil, including the northeastern region of the State of São Paulo. We applied PCR to 54 skin or mucosal biopsies from patients with a clinical and/or laboratory diagnosis of ACL using primers 13A and 13B, with positive results being obtained for 82% of the samples. When the PCR results were compared to those of histopathological leishmania detection, PCR showed superior results with 81.5% sensitivity and 95% CI of 68.0-95.1%. The Montenegro skin test (MST) was positive in 88.7% of patients. Since MST cannot be used as a diagnostic tool in endemic areas, the present results strongly suggest the use of PCR for the etiological confirmation of ACL, with emphasis on the mucosal form.

What about Th1/Th2 in cutaneous leishmaniasis vaccine discovery?

The T helper cell type 1 (Th1) response is essential to resist leishmaniasis, whereas the Th2 response favors the disease. However, many leishmanial antigens, which stimulate a Th1 immune response during the disease or even after the disease is cured, have been shown to have no protective action. Paradoxically, antigens associated with an early Th2 response have been found to be highly protective if the Th1 response to them is generated before infection. Therefore, finding disease-associated Th2 antigens and inducing a Th1 immune response to them using defined vaccination protocols is an interesting unorthodox alternative approach to the discovery of a leishmania vaccine.

Further support for a palaearctic origin of Leishmania

The fossil record and systematics of murid rodents, reservoirs of zoonotic cutaneous leishmaniasis in the Palaearctic, Oriental, African, Nearctic and Neotropical, strongly support a Palaearctic origin of Leishmania. The fossil record and systematics of phlebotomine sand flies reinforce this idea. Interpretations of molecular data that place the origin of Leishmania in the Neotropical are inconsistent with the natural histories of reservoirs and vectors. The evolutionary pattern of New World rats (Sigmodontinae) indicates that they may be the most important reservoirs of zoonotic cutaneous leishmaniasis throughout their range.

Retention of Leishmania (Leishmania) Mexicana in naturally infected rodents from the State of Campeche, Mexico

In the State of Campeche, Mexico, zoonotic cutaneous leishmaniasis is mainly due to Leishmania (L.) mexicana. The parasite population is maintained in a mammalian species, a reservoir in which the ideal course of infection should be long and relatively nonpathogenic. The objective of the present study was to document the retention of L. (L.) mexicana in 29 naturally infected rodents. These cricetids lived in captivity for up to two years and were tested monthly for the presence of the parasite, by cultures of needle aspirates from the base of the tail. Peromyscus yucatanicus and Ototylomys phyllotis were incriminated as the primary reservoir hosts. The finding that the multiplication of parasites in P. yucatanicus might be triggered by temperature, suggests that this animal would be a good choice for further research on L. (L.) mexicana.