Molecular identification of the parasites causing cutaneous leishmaniasis on the Caribbean coast of Colombia

All clinical manifestations of leishmaniasis exist in Colombia, the cutaneous form being the most frequent in the department of Sucre, where the Leishmania species associated with cutaneous leishmaniasis (CL) is unknown. This study was carried out to determine which Leishmania species was responsible for CL in Sucre, based on amplification and sequencing of the Cyt b gene. Isolates of Leishmania were obtained after CL diagnosis of eight patients who received attention in several health care centers of the study area. The nucleotide sequences obtained from patients were compared to Leishmania reference strains and six of the isolates identified as Leishmania (Viannia) braziliensis, the remaining two being identified as Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis. This represents the first report of the presence of L. (V.) guyanensis on the Caribbean coast of Colombia.     

Comparison of Leishmania OligoC-TesT PCR with Conventional and Real-Time PCR for Diagnosis of Canine Leishmania Infection

There is a need for standardization and simplification of the existing methods for molecular detection of Leishmania infantum in the canine reservoir host. The commercially available OligoC-TesT kit incorporates standardized PCR reagents with rapid oligochromatographic dipstick detection of PCR products and is highly sensitive for use in humans but not yet independently validated for use in dogs. Here we compare the sensitivity of OligoC-TesT with those of nested kinetoplast DNA (kDNA) PCR, nested internal transcribed spacer 1 (ITS-1) PCR, and a PCR-hybridization protocol, using longitudinal naturally infected canine bone marrow samples whose parasite burdens were measured by real-time quantitative PCR (qPCR). The sensitivity of OligoC-TesT for infected dogs was 70% (95% confidence interval [CI], 63 to 78%), similar to that of kDNA PCR (72%; 95% CI, 65 to 80%; P = 0.69) but significantly greater than those of PCR-hybridization (61%; 95% CI, 53 to 69%; P = 0.007) and ITS-1 nested PCR (54%; 95% CI, 45 to 62%; P < 0.001); real-time qPCR had the highest sensitivity (91%; 95% CI, 85 to 95%; P < 0.001). OligoC-TesT sensitivity was greater for polysymptomatic and oligosymptomatic dogs than for asymptomatic dogs (93%, 74%, and 61%, respectively; P = 0.005), a trend also observed for the other qualitative PCR methods tested (P ≤ 0.05). Test positivity increased with increasing parasite burdens, as measured by real-time qPCR: OligoC-TesT and kDNA PCR detected 100% and 99% of positive samples when parasite burdens exceeded 74 and 49 parasites/ml, respectively. OligoC-TesT has high sensitivity for detection of canine Leishmania infections; its ease of operation and ease of interpretation are further advantages for veterinary diagnostic laboratories and for large-scale survey work in developing countries.Zoonotic visceral leishmaniasis (ZVL), a vector-borne disease caused by the protozoan parasite Leishmania infantum [Leishmania (Leishmania) infantum chagasi (11)], results in significant mortality and morbidity in the reservoir host (the domestic dog) and represents a serious public health problem in many regions where it is endemic (the Mediterranean basin, Latin America, and parts of central and eastern Asia). Serological methods are the most technically straightforward of the available tests for diagnosis of canine infection, but these methods lack sensitivity for asymptomatic and early-stage infections (5, 17, 24). Detection of Leishmania parasites in canine clinical samples has traditionally been performed by means of microscopic examination of stained tissue specimens and by parasitological culture, which are known to be insensitive. PCR for amplification of defined parasite DNA sequences is highly sensitive for animals with clinical disease and has higher sensitivity than serology for asymptomatic animals and early-stage infections (8, 9, 19, 23, 29). However, the technical complexity of PCR may reduce its practicality for use in developing countries most affected by ZVL. Furthermore, there is a lack of standardization in the selection of target Leishmania DNA sequences and experimental PCR protocols used in laboratories worldwide, which complicates objective comparisons of test sensitivity and specificity. In order to address some of these issues, a commercially available PCR test kit (Leishmania OligoC-TesT) has been developed and validated for detection of Leishmania parasite DNA in human specimens (3, 6). Sensitivities of the test ranged from 77.8% to 100% for clinical samples from patients with visceral leishmaniasis in Sudan and Kenya, with a limit of detection of 1 parasite in 180 μl blood (3). OligoC-TesT has not yet been validated independently for use in dogs. The aims of this study were therefore (i) to measure the sensitivity of OligoC-TesT compared with those of three conventional PCR procedures (nested PCR for amplification of kinetoplast DNA [kDNA], nested PCR of internal transcribed spacer region 1 [ITS-1] of the rRNA gene, and kDNA/rRNA PCR followed by hybridization with specific oligonucleotide probes) and with that of real-time quantitative PCR (qPCR), (ii) to compare the sensitivities of OligoC-TesT and the PCR methods listed above for samples from infected dogs that were positive or negative for clinical signs of leishmaniasis, and (iii) to determine the analytical sensitivity of OligoC-TesT relative to canine bone marrow parasite burdens measured by real-time qPCR. For this study, we used samples collected in a longitudinal study of a cohort of naturally L. infantum-infected domestic dogs in Brazil.     

Characterization of Leishmania spp. causing cutaneous leishmaniasis in Manaus, Amazonas, Brazil

In the State of Amazonas, American tegumentary leishmaniasis is endemic and presents a wide spectrum of clinical variability due to the large diversity of circulating species in the region. Isolates from patients in Manaus and its metropolitan region were characterized using monoclonal antibodies and isoenzymes belonging to four species of the parasite: Leishmania (Viannia) guyanensis, 73% (153/209); Leishmania (Viannia) braziliensis, 14% (30/209); Leishmania (Leishmania) amazonensis, 8% (17/209); and Leishmania (Viannia) naiffii, 4% (9/209). The most prevalent species was L. (V.) guyanensis. The principal finding of this study was the important quantity of infections involving more than one parasite species, representing 14% (29/209) of the total. The findings obtained in this work regarding the parasite are further highlighted by the fact that these isolates were obtained from clinical samples collected from single lesions.     

The therapeutic potential of immune cross-talk in leishmaniasis

Veterans of infection, Leishmania parasites have been plaguing mammals for centuries, causing a morbidity toll second only to that of malaria as the most devastating protozoan parasitic disease in the world. Cutaneous leishmaniasis (CL) is, by far, the most prevalent form of the disease, with symptoms ranging from a single self-healing lesion to chronic metastatic leishmaniasis (ML). In an increasingly immunocompromised population, complicated CL is becoming a more likely outcome, characterized by severely inflamed, destructive lesions that are often refractory to current treatment. This is perhaps because our ageing arsenal of variably effective antileishmanial drugs may be directly or indirectly immunomodulatory and may thus have variable effects in each type and stage of CL. Indeed, widely differing immune biases are created by the various species of Leishmania, and these immunological watersheds are further shifted by extrinsic disturbances in immune homeostasis. For example, we recently showed that a naturally occurring RNA virus (Leishmania RNA virus (LRV)) within some Leishmania parasites creates hyperinflammatory cross-talk, which can predispose to ML: a case of immunological misfire that may require a different approach to immunotherapy, whereby treatments are tailored to underlying immune biases. Understanding the intersecting immune pathways of leishmaniasis and its co-infections will enable us to identify new drug targets, and thereby design therapeutic strategies that work by untangling the immunological cross-wires of pathogenic cross-talk.     

Accuracy of qPCR for quantifying Leishmania kDNA in different skin layers of patients with American tegumentary leishmaniasis

Objectives

Superficial swab sampling of American tegumentary leishmaniasis (ATL) lesions shows higher amounts of Leishmania than those from biopsy. Subcutaneous involvement is also important in ATL, but parasite quantification according to lesion depth has not been evaluated. We aim to present the best depth at which sampling should be performed for molecular exams of ATL.

Immunopathogenesis of non-healing American cutaneous leishmaniasis and progressive visceral leishmaniasis

The outcomes of Leishmania infection are determined by host immune and nutrition status, parasite species, and co-infection with other pathogens. While subclinical infection and self-healing cutaneous leishmaniasis (CL) are common, uncontrolled parasite replication can lead to non-healing local lesions or visceral leishmaniasis (VL). It is known that infection control requires Th1-differentiation cytokines (IL-12, IL-18, and IL-27) and Th1 cell and macrophage activation. However, there is no generalized consensus for the mechanisms of host susceptibility. The recent studies on regulatory T cells and IL-17-producing cells help explain the effector T cell responses that occur independently of the known Th1/Th2 cell signaling pathways. This review focuses on the immunopathogenesis of non-healing American CL and progressive VL. We summarize recent evidence from human and animal studies that reveals the mechanisms of dysregulated, hyper-responses to Leishmania braziliensis, as well as the presence of disease-promoting or the absence of protective responses to Leishmania amazonensis and Leishmania donovani. We highlight immune-mediated parasite growth and immunopathogenesis, with an emphasis on the putative roles of IL-17 and its related cytokines as well as arginase. A better understanding of the quality and regulation of innate immunity and T cell responses triggered by Leishmania will aid in the rational control of pathology and the infection.     

Molecular diagnosis of Old World cutaneous leishmaniasis and species identification by use of a reverse line blot hybridization assay

Reverse line blot hybridization assays (RLB) have been used for the rapid diagnosis and genotyping of many pathogens. The leishmaniases are caused by a large number of species, and rapid, accurate parasite characterization is important in deciding on appropriate therapy. Fourteen oligonucleotide probes, 2 genus specific and 12 species specific (2 specific for Leishmania major, 3 for L. tropica, 1 for L. infantum, 3 for L. donovani, and 3 for L. aethiopica), were prepared by using DNA sequences in the internal transcribed spacer 1 (ITS1) region of the rRNA genes. Probe specificity was evaluated by amplifying DNA from 21 reference strains using biotinylated ITS1 PCR primers and the RLB. The genus-specific probes, PP and PP3′, recognized all Leishmania species examined, while the species-specific probes were able to distinguish between all the Old World Leishmania species. Titrations using purified parasite DNA showed that the RLB is 10- to 100-fold more sensitive than ITS1 PCR and can detect <0.1 pg DNA. The RLB was compared to kinetoplast DNA (kDNA) and ITS1 PCR by using 67 samples from suspected cutaneous leishmaniasis (CL) patients in Israel and the West Bank. The RLB accurately identified 58/59 confirmed positive samples as CL, a result similar to that found by kDNA PCR (59/59) and better than that by ITS1 PCR (50/59). The positive predictive value and negative predictive value of the RLB were 95.1% and 83.3%, respectively. L. major or L. tropica was identified by the RLB in 55 of the confirmed positive cases, a level of accuracy better than that of ITS1 PCR with restriction fragment length polymorphism (42/59). Thus, RLB can be used to diagnose and characterize Old World CL.     

Presence of parasite DNA in clinically unaffected nasal mucosa during cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis

Objectives

We aimed to detect Leishmania DNA carriage in nasal mucosa of individuals with cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) braziliensis.

Synergy between activated Leishmania major-specific CD4+ T lymphocytes and bone-marrow-derived cells in the exacerbation of murine cutaneous leishmaniasis

Mechanisms of exaberbation of murine cutaneous leishmaniasis mediated by Leishmania major-specific CD4⁺ T lymphocytes were studied. Using a limiting dilution assay for the quantification of Leishmania parasites, the infected tissues (footpad) of lethally irradiated mice were found to contain tenfold less parasites at four days of infection than the footpads of infected unirradiated animals. Injection of bone marrow cells depleted of T cells into irradiated mice at the site of infection led to an increase in parasite numbers to levels equivalent to those seen in unirradiated mice. After injection of either L. major-specific CD4⁺ T cells, previously shown to exacerbate cutaneous leishmaniasis, into the infected footpad or the intravenous (i.v.) injection of bone marrow cells depleted of T cells, the numbers of parasites in lesions of irradiated mice never reached the values found in unirradiated control mice. In contrast, the concomitant transfer of CD4⁺ T-cell populations in situ and bone marrow cells depleted of T cells intravenously led to an increase in parasite loads in irradiated mice up to levels comparable to those of the unirradiated mice. This suggested that recruitment of myelomonocytic cells at the site of the lesions plays a role in the exacerbation of murine cutaneous leishmaniasis mediated by these CD4⁺ T lymphocytes. Finally, a similar effect was observed with T cells specific for an antigen unrelated to Leishmania, provided that this antigen was added to the L. major infecting inoculum.     

Evaluation of a hypothetical protein for serodiagnosis and as a potential marker for post-treatment serological evaluation of tegumentary leishmaniasis patients

The serodiagnosis for tegumentary leishmaniasis (TL) presents problems related to the sensitivity and/or specificity of the tests. In the present study, an enzyme-linked immunosorbent assay (ELISA) technique was used to evaluate the performance from a Leishmania braziliensis hypothetical protein, LbHyM, in an attempt to compare its serological reactivity with a soluble Leishmania antigenic preparation (SLA) for the serodiagnosis of cutaneous (CL) and mucosal (ML) leishmaniasis. LbHyM was predicted to be a kinesin-like protein by bioinformatics tools. Serum samples were collected from both CL and ML patients, as well as from those with Chagas disease and from healthy subjects living in endemic or non-endemic areas of TL. Also, sera were collected from patients before and after the treatments, seeking to evaluate their serological follow-up in relation to the anti-protein and anti-parasite antibody levels. When an ELISA-rLbHyM assay was performed, it proved to be significantly more sensitive than ELISA-L. braziliensis SLA in detecting both CL and ML patients. Also, when using sera from Chagas disease patients, the ELISA-rLbHyM proved to be more specific than ELISA-SLA. The anti-protein and anti-parasite antibody levels were also evaluated 6 months after the treatments, and treated patients showed significantly lower levels of specific-rLbHyM antibodies, when compared to the anti-parasite antibody levels. In conclusion, the ELISA-rLbHyM assay can be considered a confirmatory serological technique for the serodiagnosis of L. braziliensis infection and can also be used in the serological follow-up of treated patients, aiming to correlate the low anti-protein antibody levels with the improvement of the healthy state of the patients.     

Comparative Evaluation of Lesion Development,Tissue Damage,and Cytokine Expression in Golden Hamsters (Mesocricetus auratus) Infected by Inocula with Different Leishmania (Viannia) braziliensis Concentrations

The golden hamster (Mesocricetus auratus) is a susceptible model to Leishmania (Viannia) spp.; however, available studies employ different infection protocols, which account for clinical and pathological presentation differences. Herein, L. (V.) braziliensis preparations were standardized to contain 10⁴, 10⁵, or 10⁶ parasites to determine an optimal inoculum that ensured cutaneous lesions without causing a disseminated infection in hamsters. Lesion development was followed for 105 days by size measurements, and skin, draining lymph node, spleen, and sera were investigated to check parasite load, spleen visceralization, cytokine expression, histopathological changes, and anti-Leishmania IgG levels. The lesion emergence time was inversely proportional to the parasite concentration in the inocula. Animals infected by 10⁴ parasites presented nodular lesions, while those infected with 10⁶ parasites often exhibited ulcerated lesions. The differences in the final lesion sizes were observed between 10⁴ and 10⁵ inocula or 10⁴ and 10⁶ inocula. High IFNG expression, anti-Leishmania IgG levels, and parasite load occurred independently of the inoculum used. A mild inflammatory skin involvement was observed in animals infected with 10⁴ parasites, while extensive tissue damage and parasite spleen visceralization occurred with 10⁵ and 10⁶ parasites. These results indicate that inocula with different concentrations of parasites generate differences in the time of lesion emergence, clinical presentation, and systemic commitment, despite high and similar IFNG expression and parasite load. This suggests that a modulation in the immune response to different parasite numbers occurs in an early phase of the infection, which could dictate the establishment and magnitude of the chronic phase of the disease.     

Leishmania OligoC-TesT as a Simple,Rapid, and Standardized Tool for Molecular Diagnosis of Cutaneous Leishmaniasis in Peru

Molecular methods such as PCR have become attractive tools for diagnosis of cutaneous leishmaniasis (CL), both for their high sensitivity and for their specificity. However, their practical use in routine diagnosis is limited due to the infrastructural requirements and the lack of any standardization. Recently, a simplified and standardized PCR format for molecular detection of Leishmania was developed. The Leishmania OligoC-TesT is based on simple and rapid detection using a dipstick with PCR-amplified Leishmania DNA. In this study, we estimated the diagnostic accuracy of the Leishmania OligoC-TesT for 61 specimens from 44 CL-suspected patients presenting at the leishmaniasis clinic of the Instituto de Medicina Tropical Alexander von Humboldt, Peru. On the basis of parasitological detection and the leishmanin skin test (LST), patients were classified as (i) confirmed CL cases, (ii) LST-positive cases, and (iii) LST-negative cases. The sensitivities of the Leishmania OligoC-TesT was 74% (95% confidence interval (CI), 60.5% to 84.1%) for lesion aspirates and 92% (95% CI, 81.2% to 96.9%) for scrapings. A significantly higher sensitivity was observed with a conventional PCR targeting the kinetoplast DNA on the aspirates (94%) (P = 0.001), while there was no significant difference in sensitivity for the lesion scrapings (88%) (P = 0.317). In addition, the Leishmania OligoC-TesT was evaluated for 13 CL-suspected patients in two different peripheral health centers in the central jungle of Peru. Our findings clearly indicate the high accuracy of the Leishmania OligoC-TesT for lesion scrapings for simple and rapid molecular diagnosis of CL in Peru.Leishmaniasis is a vector-borne disease caused by obligatory intracellular parasites of the genus Leishmania. Several clinical manifestations are classified under the term leishmaniasis, but three are the most prominent: visceral leishmaniasis (VL), cutaneous leishmaniasis (CL), and mucocutaneous leishmaniasis (MCL), which result from replication of parasites in macrophages in the internal organs, dermis, and naso-oropharyngeal mucosa, respectively (15).In Latin America, CL and MCL are important health problems, and Brazil and Peru are the two most affected countries (9). An increase of cases has been reported for Colombia, Ecuador, and Argentina (4, 25, 26). Human-made risk factors, such as migration, urbanization, and deforestation, likely contribute to the spread of the disease (10).Over 10,000 CL cases per year are reported to occur in Peru, and more than a million people are at risk for infection (11, 22). Furthermore, this disease is endemic in 70% of Peruvian territory, causing high morbidity, lifelong scars, and major health problems for many communities (19).Diagnosing CL is challenging because of its wide spectrum of clinical presentations. Lesions may vary in severity, clinical appearance, and duration (23). Moreover, differential diagnosis with other cutaneous diseases is often difficult (14, 15). In addition, CL can be caused by different Leishmania species. In Peru, the disease is mainly caused by Leishmania (Viannia) braziliensis, Leishmania (Viannia) peruviana, and Leishmania (Viannia) guyanensis, but Leishmania (Viannia) lainsoni and Leishmania (Leishmania) amazonensis infections have also been reported (1, 18).Routine diagnosis of CL is still based on demonstration of amastigotes in skin lesion scrapings through microscopic analysis of direct smears or prior in vitro culture of the parasite (15, 23). Both methods require skilled personnel, and their sensitivities tend to be low and variable (12, 21, 27). Furthermore, in vitro culture is cumbersome and time-consuming. The leishmanin skin test (LST) detects cell-mediated immunity and is frequently used in Peru to support clinical diagnosis of CL. However, it cannot distinguish between past and present infections (12).The PCR is a useful tool for detection of Leishmania parasites in clinical specimens, since high sensitivity and specificity have been reported. Attractive PCR targets are high-copy-number sequences, such as kinetoplast DNA (kDNA) (2, 8, 30) and the ribosomal small subunit (20, 28). Several PCR formats have been designed, but there is actually a demand for simplified and standardized approaches (24). Access to sophisticated equipment such as real-time PCR machines is often limited in Peru. Recently, a simple and rapid dipstick format for detection of amplified Leishmania DNA was developed (Leishmania OligoC-TesT) (7). The test is based on PCR amplification of a small sequence of the 18S rRNA gene followed by visualization of the PCR products on a dipstick by hybridization with a gold-conjugated probe. PCR product detection can be performed in 10 min, and no equipment other than a heating block and a pipette is needed. The test is a promising “low-tech” standardized PCR application for diagnosis of leishmaniasis and can be applied in a mid- to low-level-equipped laboratory (6).In this report, we estimated the sensitivity of the Leishmania OligoC-TesT for 61 skin lesion scrapings from 44 Peruvian patients suspected of having CL. To assess the performance and to demonstrate the applicability of the test in low-level-equipped laboratories, two trials in rural hospitals in the Peruvian jungle were conducted.     

Molecular, cytological, and immunocytochemical study and kDNA sequencing of laryngeal Leishmania infantum infection

Mucosal leishmaniasis is a well-known clinical manifestation of infections mainly caused by New World Leishmania species, especially Leishmania braziliensis (Viannia) in Central and South America. It is extremely uncommon in the world, even in the endemic areas such as Fars Province, Southern Iran. Two male immunocompetent subjects who developed Leishmania mucosal lesion mimicking a laryngeal tumor presented with a several-months history of dysphonia, dyspnea, hoarseness, and odynophagia. Multiple smears from the lesions showed structures resembling the amastigote form of Leishmania. Nested PCR analysis to amplifying a fragment of Leishmania infantum kinetoplastid DNA from the Giemsa-stained smear resulted in a fragment of 680 bp. Sequence analysis of one of the strains showed 98 % similarity to L. infantum strain IranJWinf (GenBank accession no. AB678348.1) and 96 % similarity to L. infantum isolate MCAN/ES/98/10445 (GenBank accession no. EU437407.1), while another strain showed 97 % similarity with two L. infantum strains from kala-azar patient (GenBank accession nos. AJ223725.1 and AF027577.1). Immunocytochemical staining with anti-L. infantum mAb (D2) was positive. Primary mucosal leishmaniasis (ML) may occur in the immunocompetent patients who reside in or travel to endemic areas of leishmaniasis. Mucosal leishmaniasis contracted in endemic areas, such as Iran, has to be considered in the differential diagnosis of lesions in the other mucosa and may occur in previously healthy persons. Therefore, cytology, PCR, and immunocytochemistry-based methods with anti-Leishmania mAb are helpful in the diagnosis of ML.     

T-Cell-Mediated Immune Responses in Patients with Cutaneous or Mucosal Leishmaniasis: Long-Term Evaluation after Therapy

T-cell immune responses in patients with cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) were studied during the active disease, at the end of therapy, and 1 to 17 years posttherapy (long-term follow-up). Lymphocyte proliferative responses, phenotypic characterization of CD4⁺ and CD8⁺ Leishmania-reactive T cells, and cytokine production were assayed. Patients with active ML and CL showed higher proportions of CD4⁺ than CD8⁺ T cells. In CL, the healing process was associated with a decrease of CD4⁺ and an increase of CD8⁺, leading to similar CD4⁺ and CD8⁺ proportions. This pattern was only seen in ML after long-term therapy. Long-term follow-up of patients with CL showed a positive CD4⁺/CD8⁺ ratio as observed during the active disease, although the percentages of these T cell subsets were significantly lower. Patients with CL did not show significant differences between gamma interferon (IFN-γ) and interleukin-5 (IL-5) production during the period of study. Patients with active ML presented higher IFN-γ and IL-5 levels compared to patients with active CL. IL-4 was only detected during active disease. Patients long term after cure from ML showed increasing production of IFN-γ, significant decrease of IL-5, and no IL-4 production. Two apparently beneficial immunological parameters were detected in tegumentary leishmaniasis: (i) decreasing proportions of CD4⁺ Leishmania-reactive T cells in the absence of IL-4 production associated with cure of CL and ML and (ii) decreasing levels of IL-5 long after cure, better detected in patients with ML. The observed T-cell responses maintained for a long period in healed patients could be relevant for immunoprotection against reinfection and used as a parameter for determining the prognosis of patients and selecting future vaccine preparations.     

Chronic infection by Leishmania amazonensis mediated through MAPK ERK mechanisms

Leishmania amazonensis is an intracellular protozoan parasite responsible for chronic cutaneous leishmaniasis (CL). CL is a neglected tropical disease responsible for infecting millions of people worldwide. L. amazonensis promotes alteration of various signaling pathways that are essential for host cell survival. Specifically, through parasite-mediated phosphorylation of extracellular signal regulated kinase (ERK), L. amazonensis inhibits cell-mediated parasite killing and promotes its own survival by co-opting multiple host cell functions. In this review, we highlight Leishmania-host cell signaling alterations focusing on those specific to (1) motor proteins, (2) prevention of NADPH subunit phosphorylation impairing reactive oxygen species production, and (3) localized endosomal signaling to up-regulate ERK phosphorylation. This review will focus upon mechanisms and possible explanations as to how Leishmania spp. evades the various layers of defense employed by the host immune response.     

Mixed Mucosal Leishmaniasis Infection Caused by Leishmania tropica and Leishmania major

Mixed infections with different Leishmania species could explain differences in the clinical courses of these infections. On identification of Leishmania parasites from Iranian patients with mucosal leishmaniasis (ML), a patient with both oral and nasal lesions was found to be concomitantly infected with Leishmania tropica and L. major. Mixed infection was identified by PCR amplification of Leishmania kinetoplast DNA on scraping of cytological smears and histopathological sections. L. major and L. tropica were isolated from the nasal and oral lesions, respectively. These species were also confirmed by immunohistochemistry. This seems to be the first reported case of concurrent ML infection with two Leishmania species. It indicates that, at least in this patient, previous infection with one of these Leishmania species did not protect against infection with the other. This result has important implications for the development of vaccines against leishmaniases and implies careful attention in the treatment of this infectious disease.     

Characterization of regulatory T cell (Treg) function in patients infected with Leishmania braziliensis

Th1 immune responses are crucial for eliminating Leishmania parasites. However, despite strong Th1 responses, cutaneous leishmaniasis (CL) patients infected with Leishmania braziliensis develop the disease, while milder Th1 responses are found in sub-clinical (SC) infections. Therefore, CL patients may experience impaired regulatory T cell (Treg) function, causing excessive Th1 responses and tissue damage. To address this hypothesis, we characterized the function of circulating Tregs in L. braziliensis infected CL patients and compared them to Tregs from uninfected controls (UC) and SC subjects. The frequency of circulating Tregs was similar in CL patients, UC and SC subjects. Moreover, CL patients Tregs suppressed lymphocyte proliferation and PBMC pro-inflammatory cytokine production more efficiently than UC Tregs, and also produced higher levels of IL-10 than UC and SC Tregs. Furthermore, PBMC and mononuclear cells from lesions of CL patients responded normally to Treg-induced suppression. Therefore, the lesion development in CL patients infected with L. braziliensis is not associated with impairment in Treg function or failure of cells to respond to immunomodulation. Rather, the increased Treg activation in CL patients may impair parasite elimination, resulting in establishment of chronic infection. Thus, immunological strategies that interfere with this response may improve leishmaniasis treatment.     

Detection of Leishmania infantum in Rhipicephalus sanguineus ticks from Brazil and Italy

Canine leishmaniosis is a widespread disease caused by Leishmania parasites, which are transmitted by phlebotomine sand flies. However, in some areas where canine leishmaniosis is endemic, but the primary vectors have not been found, ticks have been suspected to play a role in transmitting the infection. Herewith, we report the detection of Leishmania infantum kinetoplast minicircle DNA (kDNA) in ticks collected from naturally infected dogs living in rural areas of Southern Italy (site A) and Northeastern Brazil (site B). Between March and October 2007, ticks were collected from 26 dogs positive to anti-Leishmania antibodies (one from site A and 25 from site B) and either placed directly into vials containing 70% ethanol or maintained alive for identification and subsequent dissection. All the 95 ticks collected were morphologically identified as Rhipicephalus sanguineus. After identification, their genomic DNA was extracted (either individually or in pools) and processed by polymerase chain reaction (PCR) for the detection of L. infantum kDNA. Two pools of salivary glands from ticks (one from five females and other from five males) found on a dog from site A and tested by a conventional PCR were positive. Amplicon sequencing confirmed the identity of the parasite. In addition, nine (12.3%) out of the 73 ticks found on dogs from site B and tested by a real-time PCR were positive, with a low parasite load (less than 1 parasite/ml). The retrieval of L. infantum kDNA in salivary glands of R. sanguineus ticks has been here reported for the first time. Therefore, further studies are needed to assess the competence of ticks as vectors of Leishmania parasites from dog to dog.