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.     

Development of a Multilocus Microsatellite Typing Approach for Discriminating Strains of Leishmania (Viannia) Species

A multilocus microsatellite typing (MLMT) approach based on the analysis of 15 independent loci has been developed for the discrimination of strains belonging to different Viannia species. Thirteen microsatellite loci were isolated de novo from microsatellite-enriched libraries for both Leishmania braziliensis and L. guyanensis. Two previously identified markers, AC01 and AC16, were modified and added to our marker set. Markers were designed to contain simple dinucleotide repeats flanked by the minimal possible number of nucleotides in order to allow variations in repeat numbers to be scored as size variations of the PCR products. The 15 markers in total were amplified for almost all of the strains of Viannia tested; one marker did not amplify from the two L. peruviana strains included in the study. When 30 strains of L. braziliensis, 21 strains of L. guyanensis, and 2 strains of L. peruviana were tested for polymorphisms, all strains except two strains of L. guyanensis had individual MLMT types. Distance-based analysis identified three main clusters. All strains except one strain of L. guyanensis grouped together. Two clusters consisted of strains of L. braziliensis according to their geographical origins. The two strains of L. peruviana grouped together with strains of L. braziliensis from Peru and the adjacent Brazilian state of Acre. MLMT has proven capable of individualizing strains even from the same areas of endemicity and of detecting genetic structures at different levels. MLMT is thus applicable for epidemiological and population genetic studies of strains within the subgenus Viannia.Cutaneous leishmaniasis (CL) is a serious but neglected public health problem that is endemic in 22 countries of Latin America, with Brazil and Colombia being among the six countries reporting 90% of the worldwide cases. Species of the subgenus Viannia cause the majority of cases of CL and mucocutaneous leishmaniasis (MCL) in South America and often overlap in their distribution (22). The subgenus is subdivided into species complexes representing Leishmania braziliensis, L. guyanensis, and L. naiffi (8). The L. guyanensis complex includes some named species, such as L. panamensis and L. shawi, which were found to be very similar to each other (7). On the other hand, L. peruviana was always considered to be closely related to L. braziliensis (14, 30). L. lainsoni was shown to be very distinct from the other species (8). In addition, numerous interspecies hybrids have been reported, such as L. braziliensis/L. peruviana (26), L. braziliensis/L. guyanensis (10), L. braziliensis/L. panamensis (4), and L. lainsoni/L. naiffi (35).Many of the Leishmania (Viannia) species are capable of producing a wide spectrum of diseases, with the severity varying from self-limiting CL to severe MCL (9, 15). MCL is principally caused by L. braziliensis, but less than 5% of patients with a primary cutaneous lesion will develop metastatic mucosal involvement during their life course (17, 23, 24). Other species of the subgenus Leishmania (Viannia), such as L. guyanensis, may be associated with MCL (34), but the exact risk and frequency are unknown. Interestingly, L. peruviana, the species that is the closest to L. braziliensis, has not yet been reported to cause MCL.The transmission of leishmaniasis in South America occurs through sand flies of the genus Lutzomyia, with each Leishmania species usually being transmitted by more than one sand fly species, and the preferred mammalian hosts also vary according to the Leishmania species (15). For some of the Leishmania (Viannia) species, such as L. guyanensis and L. panamensis, the patterns of transmission are well understood, but for other species they are not. Knowledge of the patterns of transmission are necessary, however, to develop disease control measures and surveillance activities.Markers that are able to discriminate Leishmania (Viannia) organisms to the species and strain levels are needed to resolve the diversity and structure of Leishmania (Viannia) populations. Multilocus microsatellite typing (MLMT) has proven highly discriminatory in the typing of strains (5) and has been successfully used in population genetic studies of different Old World species of Leishmania (2, 19, 20, 32). This approach could be performed directly with clinical materials without prior cultivation of the parasites. Its results are reproducible, can be stored in databases, and can be exchanged between different laboratories. The first three microsatellite markers for the subgenus Leishmania (Viannia) were published in 1999 (30). Two of them, AC01 and AC52, were polymorphic in L. braziliensis, L. peruviana, L. guyanensis, and L. panamensis; however, marker AC16 was monomorphic for the seven strains of L. guyanensis analyzed. Marker AC52 had a composite repeat and needed to be sequenced because electrophoretic alleles of the same size could consist of different repeat compositions. This marker was also not very reliable when it was used to type strains of L. braziliensis and L. peruviana from Peru (26). Recently, an additional eight microsatellites designed by using genomic libraries of L. braziliensis genome project loci were found to be polymorphic in L. braziliensis (28). However, the variation of these markers in other Leishmania (Viannia) species was not tested.The study described here aimed to develop an MLMT approach that could be used for epidemiological and population studies of different Leishmania (Viannia) species. For this purpose, 13 microsatellite loci were identified by searching microsatellite-enriched libraries for L. braziliensis and L. guyanensis. Two previously identified markers, AC01 and AC16, were modified and added to our marker set. Variations in repeat numbers should be scorable as size variations of the PCR products. The 15 markers in total were amplified for all Leishmania (Viannia) species tested and tested for polymorphisms in 31 strains of L. braziliensis, 20 strains of L. guyanensis, and 2 strains of L. peruviana.     

Real-Time PCR Assay for Detection and Quantification of Leishmania (Viannia) Organisms in Skin and Mucosal Lesions: Exploratory Study of Parasite Load and Clinical Parameters

Earlier histopathology studies suggest that parasite loads may differ between cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) lesions and between acute and chronic CL. Formal demonstration requires highly sensitive detection and accurate quantification of Leishmania in human lesional tissue. In this study, we developed a quantitative real-time PCR (qPCR) assay targeting minicircle kinetoplast DNA (kDNA) to detect and quantify Leishmania (Viannia) parasites. We evaluated a total of 156 lesion biopsy specimens from CL or ML suspected cases and compared the quantitative performance of our kDNA qPCR assay with that of a previously validated qPCR assay based on the glucose-6-phosphate dehydrogenase (G6PD) gene. We also examined the relationship between parasite load and clinical parameters. The kDNA qPCR sensitivity for Leishmania detection was 97.9%, and its specificity was 87.5%. The parasite loads quantified by kDNA qPCR and G6PD qPCR assays were highly correlated (r = 0.87; P < 0.0001), but the former showed higher sensitivity (P = 0.000). CL lesions had 10-fold-higher parasite loads than ML lesions (P = 0.009). Among CL patients, the parasite load was inversely correlated with disease duration (P = 0.004), but there was no difference in parasite load according to the parasite species, the patient''s age, and number or area of lesions. Our findings confirm that CL and recent onset of disease (<3 months) are associated with a high parasite load. Our kDNA qPCR assay proved highly sensitive and accurate for the detection and quantification of Leishmania (Viannia) spp. in lesion biopsy specimens. It has potential application as a diagnostic and follow-up tool in American tegumentary leishmaniasis.     

Use of FTA Cards for Direct Sampling of Patients' Lesions in the Ecological Study of Cutaneous Leishmaniasis

The FTA card (Whatman) was assessed for its utility as a molecular epidemiological tool in collecting samples from patients with leishmaniasis in Peru because the card has a variety of merits; it is less invasive for patients and easy to handle for both physicians and other medical personnel for sample collection or diagnosis, in addition to its simplicity and easy countrywide and/or intercountry transportation for analysis. Samples were collected from 132 patients suspected of having leishmaniasis, and Leishmania species were successfully identified in samples from 81 patients in 15 departments of Peru by cytochrome b and mannose phosphate isomerase gene analyses. Of these, 61.7% were identified as Leishmania (Viannia) peruviana, 22.2% as L. (V.) braziliensis, 12.3% as L. (V.) guyanensis, 2.5% as L. (V.) shawi, and 1.2% as L. (V.) lainsoni. The three predominant species, L. (V.) peruviana, L. (V.) braziliensis, and L. (V.) guyanensis, were mainly found in the Andean highlands, in the tropical rainforest, and in northern and central rainforest regions, respectively. This is the first time L. (V.) shawi has been identified outside Brazil. The present study showed that the FTA card will be a useful tool for the ecological study of different forms of leishmaniasis. Furthermore, collecting samples directly from patients'' lesions by using the FTA card eliminates (i) the possibility of contamination of Leishmania isolates during short- and/or long-term passages of culture in vitro in each laboratory and (ii) pain and suffering of patients from taking samples by skin biopsy.Leishmaniasis is caused by protozoan parasites of the genus Leishmania, which is further divided into two subgenera, Leishmania (Leishmania) and Leishmania (Viannia) (10). The disease is widely distributed around the world, especially in tropical and subtropical areas, affecting at least 12 million people in 88 countries (6). Approximately 20 Leishmania species are known to be pathogenic to humans, and the species is the major determinant of clinical outcome (cutaneous, mucocutaneous, and visceral forms) (6). Therefore, identification of the parasite species in areas of endemicity is important for both appropriate treatment and prognosis.The standard method for the classification of Leishmania species is multilocus enzyme electrophoresis (MLEE), which requires the isolation and mass culture of the parasites (4, 17). This process has several disadvantages: (i) risk of contamination with bacteria and/or fungus and even other Leishmania isolates in the laboratory; (ii) maladaptation of the parasites to the artificial medium; (iii) difficulty in cultivation due to low numbers of parasites in cutaneous lesions, especially for the subgenus Leishmania (Viannia); and (iv) the time, several weeks or months, required to obtain a result after sample collection. All of these factors affect epidemiological studies in spite of considerable efforts to collect patient specimens from different areas of endemicity, especially for specimens from remote locations. To overcome these problems, molecular biological methods have been developed for the detection and identification of Leishmania species using DNA extracted from clinical samples (7, 16, 21). However, sampling procedures, such as skin biopsy, are sometimes painful for patients and become a burden to both patients and physicians. Therefore, alternative sampling procedures with less invasiveness, simple and easy handling, and greater convenience are required for the detection and identification of Leishmania species and continuous monitoring of endemic species of causative organisms.FTA technology (Whatman) is a rapid and safe method for extracting nucleic acids from blood, cell, and pathogen samples without using any organic solvent or specialized equipment. When the samples are spotted onto an FTA card, the cells are readily lysed and the nucleic acids are fixed on the card, resulting in protection from nuclease, oxidative, and UV damage and prevention of the growth of bacteria and other microorganisms. The card is also suitable for long-term storage and the transportation of materials at room temperature, eliminating the possibility of contamination from isolates in vitro during the laboratory phase. In the present study, the utility of FTA cards was assessed for sample collection for the countrywide molecular epidemiological study of leishmaniasis in Peru.     

Leishmania sp. identification by PCR associated with sequencing of target SSU rDNA in paraffin-embedded skin samples stored for more than 30 years

Paraffin-embedded samples commonly stored at educational and research institutions constitute tissues banks for follow-up or epidemiological studies; however, the paraffin inclusion process involves the use of substances that can cause DNA degradation. In this study, a PCR protocol was applied to identify Leishmania strains in 33 paraffin-embedded skin samples of patients with American cutaneous leishmaniasis. DNA was obtained by the phenol-chloroform protocol following paraffin removal and then used in PCR or nested PCR based on the nucleotide sequence of the small subunit ribosomal RNA (SSU rDNA). The amplicons obtained were cloned and sequenced to determine the single nucleotide polymorphism that distinguishes between different Leishmania species or groups. This assay allowed to distinguish organisms belonging to the subgenus Viannia and identify L. (Leishmania) amazonensis and L. (L.) chagasi of the Leishmania subgenus. Of the 33 samples, PCR and nested PCR identified 91% of samples. After sequencing the PCR product of 26 samples, 16 were identified as L. (L.) amazonensis, the other 10 contain organisms belonging to the L. (Viannia) sub-genus. These results open a huge opportunity to study stored samples and promote relevant contributions to epidemiological studies.     

Comparison of Tetrazolium Salt Assays for Evaluation of Drug Activity against Leishmania spp.

In French Guiana, leishmaniasis is an essentially cutaneous infection. It constitutes a major public health problem, with a real incidence of 0.2 to 0.3%. Leishmania guyanensis is the causal species most frequently encountered in French Guiana. The treatment of leishmaniasis is essentially drug based, but the therapeutic compounds available have major side effects (e.g., liver damage and diabetes) and must be administered parenterally or are costly. The efficacy of some of these agents has declined due to the emergence of resistance in certain strains of Leishmania. There is currently no vaccine against leishmaniasis, and it is therefore both necessary and urgent to identify new compounds effective against Leishmania. The search for new drugs requires effective tests for evaluations of the leishmanicidal activity of a particular molecule or extract. Microculture tetrazolium assays (MTAs) are colorimetric tests based on the use of tetrazolium salts. We compared the efficacies of three tetrazolium salts—3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8)—for quantification of the promastigotes of various species of Leishmania. We found that the capacity of Leishmania to metabolize a tetrazolium salt depended on the salt used and the species of Leishmania. WST-8 was the tetrazolium salt best metabolized by L. guyanensis and gave the best sensitivity.     

Immunocytochemical and immunohistochemical methods as auxiliary techniques for histopathological diagnosis of cutaneous leishmaniasis

A significant increase in cutaneous leishmaniasis (CL) and its geographic expansion has motivated the development of techniques to help with diagnosis of the disease. Here we describe immunocytochemical (ICC) and immunohistochemical (IHC) techniques for the diagnosis of CL in the laboratory. Polyclonal antibodies and a modified avidin-biotin complex (Ultra Streptavidin^®) for Leishmania (V.) braziliensis or Leishmania (L.) amazonensis were developed for the present study. In vitro culture and histological sections from experimentally infected tissues were submitted to ICC/IHC techniques. The polyclonal antibody specificity, stability and immunostaining were evaluated. The polyclonal antibodies purified by chromatography (Sephadex^®) and obtained from L. (V.) braziliensis and L. (L.) amazonensis insoluble antigens presented 83.3% sensitivity, when the presence of antigens was evaluated, i.e., higher than histopathology or any equivalent method (in vitro culture). The polyclonal antibody presented 100% specificity when used against species frequently found in CL lesions. The ICC/IHC techniques developed in the current study were able to recognize amastigotes and antigens from in vivo and in vitro cultures and from biopsies, offering additional help in the diagnosis of CL. This methodology could be beneficially adopted in public health laboratories.     

<Emphasis Type="Italic">Leishmania</Emphasis> (<Emphasis Type="Italic">Viannia</Emphasis>) <Emphasis Type="Italic">shawi</Emphasis> purified antigens confer protection against murine cutaneous leishmaniasis

Objective  

Leishmania (Viannia) shawi was characterized only recently, and few studies concerning the immunogenic and protective properties of its antigens have been performed. The present study aimed to evaluate the protective potential of the five antigenic fractions isolated from L. (V.) shawi promastigotes in experimental cutaneous leishmaniasis.     

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.     

Improving Leishmania Species Identification in Different Types of Samples from Cutaneous Lesions

The discrimination of Leishmania species from patient samples has epidemiological and clinical relevance. In this study, different gene target PCR-restriction fragment length polymorphism (RFLP) protocols were evaluated for their robustness as Leishmania species discriminators in 61 patients with cutaneous leishmaniasis. We modified the hsp70-PCR-RFLP protocol and found it to be the most reliable protocol for species identification.     

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.     

Proteins Selected in Leishmania (Viannia) braziliensis by an Immunoproteomic Approach with Potential Serodiagnosis Applications for Tegumentary Leishmaniasis

The serodiagnosis of human tegumentary leishmaniasis (TL) presents some problems, such as the low level of antileishmanial antibodies found in most of the patients, as well as the cross-reactivity in subjects infected by other trypanosomatids. In the present study, an immunoproteomic approach was performed aimed at identification of antigens in total extracts of stationary-phase promastigote and amastigote-like forms of Leishmania (Viannia) braziliensis using sera from TL patients. With the purpose of reducing the cross-reactivity of the identified proteins, spots recognized by sera from TL patients, as well as those recognized by antibodies present in sera from noninfected patients living in areas where TL is endemic and sera from Chagas disease patients, were discarded. Two Leishmania hypothetical proteins and 18 proteins with known functions were identified as antigenic. The study was extended with some of them to validate the results of the immunoscreening. The coding regions of five of the characterized antigens (enolase, tryparedoxin peroxidase, eukaryotic initiation factor 5a, β-tubulin, and one of the hypothetical proteins) were cloned in a prokaryotic expression vector, and the corresponding recombinant proteins were purified and evaluated for the serodiagnosis of TL. The antigens presented sensitivity and specificity values ranging from 95.4 to 100% and 82.5 to 100%, respectively. As a comparative antigen, a preparation of Leishmania extract showed sensitivity and specificity values of 65.1 and 57.5%, respectively. The present study has enabled the identification of proteins able to be employed for the serodiagnosis of TL.     

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.     

Place de l’identification des leishmanies par la polymerase chain reaction – restriction fragment length polymerase dans l’étude de l’épidémiologie des leishmanioses cutanées en Tunisie

Aim

Three forms of cutaneous leishmaniasis (CL) are endemic in Tunisia. The identification of the causative species is useful to complete epidemiological data and to manage the cases. The aim of this study is to assess PCR-RFLP technique in the identification of Leishmania species responsible of CL in Tunisia and to compare the results of this technique to those of isoenzyme analysis.

Patients and methods

Sixty-one CL lesions were sampled. Dermal samples were tested by culture on NNN medium and analyzed by PCR-RFLP assay targeting the ITS1 region of ribosomal DNA. Species identification was performed by both iso-enzymatic typing for positive cultures and analysis of restriction profiles after enzymatic digestion by HaeIII of the obtained amplicons.

Results

Thirty-eight (62%) samples were positive by culture. The iso-enzymatic typing of 32 isolates identified 3 L. infantum, 23 L. major MON-25 and 6 L. tropica MON-8. Sixty samples were positive by PCR. The PCR-RFLP digestion profiles of the 56 PCR products identified 12 L. infantum, 38 L. major and 6 L. tropica. The results of both techniques were concordant in the 32 strains identified by both techniques. Species identification correlated with the geographical distribution of CL forms endemic in Tunisia.

Conclusion

Results of PCR-RFLP revealed highly concordant with those of isoenzyme electrophoresis. Thanks to its simplicity, rapidity and ability to be performed directly on biological samples, this technique appears as an interesting alternative for the identification of Leishmania strains responsible of CL in Tunisia.     

Usefulness of a PCR-based method in the detection and species identification of Leishmania from clinical samples

The aim of this study is to assess the usefulness of a simple, low-cost method for the detection and species identification of Leishmania isolated by in vitro culture or detected directly from clinical samples. A total of 110 samples were used in this study. Among these, 21 were human and canine peripheral bloods, 63 skin lesion material samples, eight reference strains and 18 Leishmania culture. Detection of Leishmania DNA with PCR using primers designed to amplify the internal transcribed spacer 1 (ITS1) region of the rRNA gene proved sufficiently sensitive at the level of 0.1 parasites per PCR reaction. Furthermore, followed by single-strand conformational polymorphism (SSCP), the PCR-ITS1 allowed the species identification of Leishmania. The inter-specific polymorphism of Leishmania was first validated on reference strains, and then this method was applied on clinical samples and culture. Typing identified all human and canine visceral leishmaniasis samples (21 samples) as L. infantum, 95.23% of the cutaneous leishmaniasis samples as L. major and 3.17% as L. killicki and 1.58% as L. infantum. A scheme of the PCR diagnosis procedure for the detection and identification of Leishmania parasites is proposed in this study.     

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.     

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.     

PCR for identification of species causing American cutaneous leishmaniasis

Parasites of the complexes Leishmania (Leishmania) mexicana, Leishmania (Viannia) braziliensis, and Leishmania (Leishmania) chagasi coexist within the same endemic areas of the American Continent. They produce similar clinical manifestations, yet not all respond well to treatment with anti-leishmania drugs. Thus, high specificity and sensitivity are needed to improve diagnosis and treatment. We developed a highly specific and sensitive polymerase chain reaction based diagnostic method that permits the identification of parasites belonging to the genus Leishmania and the differentiation between parasites belonging to the L. (L.) mexicana and L. (V.) braziliensis complexes and the identification of species of the L. (L.) mexicana complex, such as L. (L.) mexicana, Leishmania (L.) amazonensis, and Leishmania (L.) venezuelensis. This PCR permits the specific identification of Leishmania species in tissues of patients with different clinical forms of leishmaniasis. Its high sensitivity and specificity allow a precise diagnosis in lesions of patients that harbor few parasites, where the microscopic evaluation is unreliable. Additionally, this PCR could be a valuable tool for the identification of Leishmania species in mammalian reservoirs and sand fly vectors present in the American Continent. This work was supported by the CONACyT 47256-M and DGAPA IN221806-3     

Contribution of molecular diagnosis to the management of cutaneous leishmaniasis in travellers

Cutaneous leishmaniasis is one of the most frequent skin diseases occurring after travelling in endemic areas. Optimal management requires identification of the species of Leishmania involved. In this study we aimed to evaluate the use of molecular diagnosis as routine, in comparison with direct examination and culture. Thirty positive diagnoses were carried out between 2007 and 2013. Classical PCR enabled 11 positive cases to be identified that were found to be negative by conventional methods. Sequencing led to the identification of eight different species. Routine use of PCR and sequencing appears very efficient in the management of cutaneous leishmaniasis.