Evaluation of systemic immunity in atypical cutaneous leishmaniasis caused by Leishmania (L.) infantum chagasi.

In some central-American countries, Leishmania (L.) infantum chagasi infection can cause non-ulcerated or atypical cutaneous leishmaniasis (NUCL) in addition to the classic clinical form, visceral leishmaniasis (VL). Little is known about the host-parasite relationship that can contribute to the determination of one or another clinical form. The present study had the objective to evaluate the humoral and cellular immunity in the sera of individuals affected by NUCL to improve the comprehension of this atypical host-parasite interaction. Based on clinical and laboratory diagnosis, serum of 80 individuals was collected to evaluate the cytokines and immunoglobulins profile of NUCL (n = 47), VL patients (n = 5), and negative controls (n = 28). Cytokines were detected using Cytokine Bead Array (CBA) Human Th1/Th2/Th17 kit according to the manufacturer's instructions; class (IgG and IgM), and subclass of (IgG1 and IgG2) immunoglobulins was evaluated by ELISA using specific antigens. The concentration of TNF-α, IFN-γ, IL-2 and IL-4 cytokines in NUCL, VL and control was present below the detection threshold of CBA kit. IL-6, IL-10 and IL-17A cytokines was lower in NUCL compared to LV patients. Regarding to immunoglobulins, NUCL patients produced 4.0 times more IgG than the control, while VL patients produced 6.6 times more; and IgM level was 1.6 times higher in NUCL and 2.6 times in VL patients compared to the control. Concerning the immunoglobulins subclass, only VL patients showed positive reaction for IgG1, and IgG2 did not show positive reaction among the groups. The results showed a weak cellular and humoral systemic immune response in NUCL patients.

Increasing putative vector importance of Trichophoromyia phlebotomines (Diptera: Psychodidae).

Despite some phlebotomines being well recognised as vectors of leishmaniasis agents, vector importance of those belonging to the genus Trichophoromyia has not been extensively studied. The present study provides evidence regarding the putative vector role played by some species of Trichophoromyia on leishmanine enzootics, based on literature reports and findings obtained from field experiments conducted in the ecotopes of Pará State, Brazil. The species Th. ubiquitalis, Th. velascoi, Th. auraensis, Th. ininii and Th. brachipyga possess minimal criteria to be included in the list of suspected leishmanine vectors. However, knowledge on man-biting behavior, substantiation of vector competence and determination of epidemiological implications are limited for all of the above mentioned species. Published studies together with present data draw attention to prioritize these phlebotomine species in entomological surveillance programs and studies on experimental susceptibility to Leishmania spp. infection.

Flavopereirine-An Alkaloid Derived from Geissospermum vellosii-Presents Leishmanicidal Activity In Vitro.

Chemotherapy is limited in the treatment of leishmaniasis due to the toxic effects of drugs, low efficacy of alternative treatments, and resistance of the parasite. This work assesses the in vitro activity of flavopereirine on promastigote cultures of Leishmania amazonensis. In addition, an in silico evaluation of the physicochemical characteristics of this alkaloid is performed. The extract and fractions were characterized by thin-layer chromatography and HPLC-DAD, yielding an alkaloid identified by NMR. The antileishmanial activity and cytotoxicity were assayed by cell viability test (MTT). The theoretical molecular properties were calculated on the Molinspiration website. The fractionation made it possible to isolate a beta-carboline alkaloid (flavopereirine) in the alkaloid fraction. Moreover, it led to obtaining a fraction with greater antileishmanial activity, since flavopereirine is very active. Regarding the exposure time, a greater inhibitory effect of flavopereirine was observed at 24 h and 72 h (IC50 of 0.23 and 0.15 µg/mL, respectively). The extract, fractions, and flavopereirine presented low toxicity, with high selectivity for the alkaloid. Furthermore, flavopereirine showed no violation of Lipinski's rule of five, showing even better results than the known inhibitor of oligopeptidase B, antipain, with three violations. Flavopereirine also interacted with residue Tyr-499 of oligopeptidase B during the molecular dynamics simulations, giving a few insights of a possible favorable mechanism of interaction and a possible inhibitory pathway. Flavopereirine proved to be a promising molecule for its antileishmanial activity.

Use of Recombinant Antigens for Sensitive Serodiagnosis of American Tegumentary Leishmaniasis Caused by Different Leishmania Species.

American tegumentary leishmaniasis (ATL) (also known as cutaneous leishmaniasis [CL]) is caused by various species of protozoa of the genus Leishmania The diagnosis is achieved on a clinical, epidemiological, and pathological basis, supported by positive parasitological exams and demonstration of leishmanin delayed-type hypersensitivity. Serological assays are not routinely used in the diagnosis because many are considered to have low sensitivity and the particular Leishmania species causing the disease can lead to variable performance. In the present study, we generated recombinant versions of two highly conserved Leishmania proteins, Leishmania (Viannia) braziliensis-derived Lb8E and Lb6H, and evaluated both in enzyme-linked immunosorbent assays (ELISA). Recombinant Lb6H (rLb6H) had better performance and reacted with 100.0% of the ATL and 89.4% of the VL samples. These reactions with rLb6H were highly specific (98.5%) when compared against those for samples from healthy control individuals. We then assessed rLb6H against sera from ATL patients infected with different species of Leishmania prevalent in Brazil [Leishmania (Leishmania) amazonensis, L (Viannia) braziliensis, and L (V) guyanensis] and samples from patients with other infectious diseases. In analyses of 500 sera, ELISA using rLb6H detected all 219 ATL samples (sensitivity of 100.0%) with an overall specificity of 93.9% (considering healthy individuals and other infectious diseases patients). Only a minority of samples from Chagas disease patients possessed antibodies against rLb6H, and all of these responses were low (with a highest reactivity index of 2.2). Taken together, our data support further evaluation of rLb6H and the potential for its routine use in the serological diagnosis of ATL.

Immunopathological characterization of human cutaneous leishmaniasis lesions caused by Leishmania (Viannia) spp. in Amazonian Brazil.

American cutaneous leishmaniasis (ACL) is a chronic infectious disease caused by different protozoan species of Leishmania, and it is endemic in both tropical and subtropical countries. Using immunohistochemistry, we investigate the density of CD68+, lysozyme+, CD1a+, factor XIIIa+, CD4+, CD8+, CD56+, interferon (IFN)-γ+, and inducible NO synthase (iNOS+) cells. These cells were analyzed from 22 biopsy samples obtained from the lesions of ACL patients, whose infection was caused by Leishmania (Viannia) spp. Histopathological analysis showed dense mononuclear inflammatory infiltration in the dermis, which was composed of lymphocytes, macrophages, plasma cells, and discrete tissue parasitism. Granulomatous reactions were also present in the majority of cases. The density of the activated macrophages was higher than that of inactivated macrophages in the lesions. The density of Langerhans cells (CD1a+) was lower than that of dermal dendrocytes (factor XIIIa+). The density of CD8+ T lymphocytes was higher than that of CD4+ T lymphocytes. The cellular density of these immunological markers in relation to the species of Leishmania demonstrated that L. (Viannia) sp. lesions had higher IFN-γ expression than that Leishmania (Viania) braziliensis lesions. The evaluation of these markers, according to disease progression, did not reveal any significant differences. L. (Viannia) sp. infection leads to a favorable immune response in the host, as predominantly represented by lysozyme+, factor XIIIa+, CD8+ T cells, and the expression of (IFN)-γ+ at the lesion site.

Comparative analysis of carbohydrate residues in the midgut of phlebotomines (Diptera: Psychodidae) from colony and field populations from Amazon, Brazil.

Leishmaniasis are worldwide diseases that occur in 98 countries including Brazil, transmitted by the bite of female phlebotomines during blood feeding. In Brazil it is known that some species of sand flies as Lutzomyia longipalpis sensun latum (vector of Leishmania infantum chagasi), Lutzomyia flaviscutellata (vector of Leishmania (Leishmania) amazonensis) and Lutzomyia antunesi [suspected vector of Leishmania (Viannia) lindenbergi] are incriminated of transmitting the parasite Leishmania for the vertebrate host. The phlebotomine-parasite is mediated by the attachment of the promastigote lipophosphoglycan (LPG) to the midgut epithelium. However, another mechanism that is LPG-independent and mediated by N-acetyl-galactosamine (GalNAc) seems to occur in some species of phlebotomines that are classified as permissive. The aim of this study was to characterize the carbohydrate residues that, probably, play a role in parasite attachment to the midgut of phlebotomine from colony and field populations from the Brazilian Amazonian region. We observed the presence of GalNAc, mannose, galactose and GlcNAc in all phlebotomine species. A binding assay between L. (L.) amazonensis and L. i.chagasi to the midguts of different species of phlebotomines was performed. The attachment of both Leishmania and vector species suggests the presence of GalNAc on the midgut surfaces. Thus, these results suggested that GalNAc is a possible binding sites of Leishmania in sand flies from the Brazilian Amazonian region.

In vitro cytokines profile and ultrastructural changes of microglia and macrophages following interaction with Leishmania.

In the present study, we assessed morphological changes and cytokine production after in vitro interaction with causative agents of American cutaneous leishmaniasis and compared the microglia and macrophage immune responses. Cultures of microglia and macrophages infected with stationary-phase promastigotes of Leishmania (Viannia) shawi, Leishmania (Viannia) braziliensis or Leishmania (Leishmania) amazonensis were evaluated 24, 48 and 72 h after interaction. Macrophages only presented the classical phagocytic process while microglia also displayed large cytoplasmic projections similar to the ruffles described in macropinocytosis. In the macrophage cultures, the percentage of infected cells increased over time, in a fashion that was dependent on the parasite species. In contrast, in microglial cells as the culture time progressed, there was a significant reduction in the percentage of infected cells independent of parasite species. Measurements of cytokines in macrophage cultures 48 h after interactions revealed distinct expression patterns for different parasites, whereas in microglial cultures they were similar for all Leishmania tested species. Taken together, our results suggest that microglia may have a higher phagocytic ability and cytotoxic potential than macrophages for all investigated species. The robust response of microglia against all parasite species may suggest microglia have an important role in the defence against cerebral leishmaniasis.

Assessment of light-emitting diodes for sampling phlebotomines (Diptera: Psychodidae) from an urban park of the Brazilian Amazon.

The present study aimed to assess different light sources for sampling phlebotomines (Diptera: Psychodidae) from Bosque Rodrigues Alves, a forested park surrounded by the urban area of Belém in the Brazilian Amazon. Centers for Disease Control traps, baited with blue, green, and warm white light-emitting diodes (LEDs), as test group, and incandescent light, as control group, were used. The electromagnetic spectra and luminous intensities of the light sources were characterized. Fractional vegetation cover at each sampling site was also estimated. Abundance, richness, rarefaction curves, Shannon and Simpson diversity indices, phlebotomines/trap/hour, and phlebotomines/trap/night were estimated and compared. The light sources of the test group presented greater luminous intensity than the control, but were similar to each other. There were no differences in vegetation cover at each site. A total of 1,346 phlebotomines comprising 11 species were sampled. The most abundant species were as follows: Nyssomyia antunesi (Coutinho, 1939), Trichophoromyia ubiquitalis (Mangabeira, 1942), Bichromomyia flaviscutellata (Mangabeira, 1942), and Th. brachipyga (Mangabeira, 1942). Light traps with LEDs had richness, abundance, and Shannon diversity indices similar to those obtained with incandescent light. The warm white LED had a higher Simpson's index than the other light sources. Phlebotomine responses to incandescent light were similar to those to LEDs in most analyses, confirming the applicability of these light sources as alternative devices for entomological surveillance. Low consumption ensures greater autonomy of the traps, providing better operability during fieldwork.

Experimental Susceptibility of Nyssomyia antunesi and Lutzomyia longipalpis (Psychodidae: Phlebotominae) to Leishmania (Viannia) lainsoni and L. (V.) lindenbergi (Trypanosomatidae: Leishmaniinae).

The present work assessed the experimental susceptibility of Nyssomyia antunesi and Lutzomyia longipalpis to Leishmania (Viannia) lainsoni and L. (V.) lindenbergi. A L. (Leishmania) chagasi-Lu. longipalpis combination was used as a susceptible control. Wild-caught Ny. antunesi and laboratory-bred Lu. longipalpis were membrane-fed on blood with a 5 × 106/mL log-phase promastigote culture suspension and dissected on days 2 and 8 post-blood meal (pbm) for analysis focused on the assessment of parasitoses, as well as placement and promastigote morphotyping. Survival curves were constructed. In all combinations, promastigotes were observed on day 8 pbm. For both Leishmania species, in Lu. longipalpis, the presence of parasites was observed up to the stomodeal valve, while in Ny. antunesi, the presence of parasites was observed up to the cardia. There were no significant differences in parasitosis between L. (V.) lainsoni and L. (V.) lindenbergi in either Ny. antunesi or Lu. longipalpis. Six morphological promastigote forms were distinguished in Giemsa-stained gut smears. The survival curves of all combinations decreased and were affected differently by several Lu. longipalpis-parasite combinations, as well with Lu. longipalpis-uninfected blood. These findings stress Lu. longipalpis as experimentally susceptible to Leishmania spp. and suggest the putative susceptibility of Ny. antunesi to L. (V.) lainsoni and L. (V.) lindenbergi.

Anti-leishmanial activity of Eleutherine plicata Herb. and predictions of isoeleutherin and its analogues.

Introduction: Leishmaniasis is caused by protozoa of the genus Leishmania, classified as tegumentary and visceral. The disease treatment is still a serious problem, due to the toxic effects of available drugs, the costly treatment and reports of parasitic resistance, making the search for therapeutic alternatives urgent. This study assessed the in vitro anti-leishmanial potential of the extract, fractions, and isoeleutherin from Eleutherine plicata, as well as the in silico interactions of isoeleutherin and its analogs with Trypanothione Reductase (TR), in addition to predicting pharmacokinetic parameters. Methods: From the ethanolic extract of E. plicata (EEEp) the dichloromethane fraction (FDEp) was obtained, and isoeleutherin isolated. All samples were tested against promastigotes, and parasite viability was evaluated. Isoeleutherin analogues were selected based on similarity in databases (ZINK and eMolecules) to verify the impact on structural change. Results and Discussion: The extract and its fractions were not active against the promastigote form (IC50 > 200 µg/mL), while isoeleutherin was active (IC50 = 25 µg/mL). All analogues have high intestinal absorption (HIA), cell permeability was moderate in Caco2 and low to moderate in MDCK. Structural changes interfered with plasma protein binding and blood-brain barrier permeability. Regarding metabolism, all molecules appear to be CYP3A4 metabolized and inhibited 2-3 CYPs. Molecular docking and molecular dynamics assessed the interactions between the most stable configurations of isoeleutherin, analogue compound 17, and quinacrine (control drug). Molecular dynamics simulations demonstrated stability and favorable interactions with TR. In summary, fractionation contributed to antileishmanial activity and isoleutherin seems to be promising. Structural alterations did not contribute to improve pharmacokinetic aspects and analogue 17 proved to be more promising than isoeleutherin, presenting better stabilization in TR.

ELISA with recombinant antigen Lb6H validated for the diagnosis of American tegumentary leishmaniasis.

American tegumentary leishmaniasis (ATL) diagnosis is an open question, and the search for a solution is urgent. The available tests that detect the etiological agent of the infection are specific for ATL diagnosis. However, they present disadvantages, such as low sensitivity and the need for invasive procedures to obtain the samples. Immunological methods (leishmanin skin test and search for anti-Leishmania antibodies) are good alternatives to the etiological diagnosis of ATL. Presently, we face problems with disease confirmation due to the discontinuity in the production of leishmanin skin test antigen, particularly in resource-poor settings. Aiming to diagnose ATL, we validated rLb6H-ELISA for IgG antibodies using 1,091 samples from leishmaniasis patients and healthy controls, divided into four panels, living in 19 Brazilian endemic and non-endemic states. The rLb6H-ELISA showed a sensitivity of 98.6% and a specificity of 100.0%, with the reference panel comprising 70 ATL patient samples and 70 healthy controls. The reproducibility evaluation showed a coefficient of variation of positive samples ≤ 8.20% for repeatability, ≤ 17,97% for reproducibility, and ≤ 8.12% for homogeneity. The plates sensitized with rLb6H were stable at 4°C and -20°C for 180 days and 37°C for seven days, indicating 12 months of validity. In samples of ATL patients from five research and healthcare centers in endemic and non-endemic areas, rLb6H-ELISA showed a sensitivity of 84.0%; no significant statistical difference was observed among the five centers (chi-square test, p = 0.13). In samples of healthy controls from four areas with different endemicity, a specificity of 92.4% was obtained; lower specificity was obtained in a visceral leishmaniasis high endemicity locality (chi-square test, p<0.001). Cross-reactivity was assessed in 166 other disease samples with a positivity of 13.9%. Based on the good diagnostic performance and the reproducibility and stability of the antigen, we suggest using ELISA-rLb6H to diagnose ATL.

Can domestic dogs be considered a good reservoir of Leishmania (L.) infantum chagasi in an endemic area of nonulcerated cutaneous leishmaniasis in Southern Honduras?

Dogs are considered to be the main domestic reservoir associated with the transmission of Leishmania (L.) infantum chagasi to humans in endemic areas of visceral leishmaniasis in America. However, little is known about the role of canines as a source of infection in endemic areas of nonulcerated cutaneous leishmaniasis (NUCL). Therefore, the objective of the present study was to investigate the role of dogs as a possible reservoir of the parasite in Southern Honduras. Dogs (n = 107) living with individuals affected by NUCL were clinically examined and biological material was collected for parasitological and immunological diagnosis. Most animals showed a healthy appearance and a few presented slight weight loss (64%), alopecia (7%), onychogryphosis (5%) and skin lesions (1%). The overall seroprevalence of Leishmania infection based on the DDP ® quick test and/or in-house ELISA serological test was 41%. The presence of the parasite's DNA was confirmed in 94% of the dogs; however, the average parasite load in the buffy coat was low at 6.09 parasites/µL, ranging between 0.221 and 50.2. The skin of seropositive dogs examined by histopathology using paraffin sections stained by hematoxylin and immunohistochemistry did not show cutaneous lesions or parasite amastigotes. Based on the absence of parasites in the skin and the low parasite load detected in the buffy coat, it seems that the dog does not represent a good source of infection for the vector in the endemic area of NUCL transmission in Southern Honduras. Other domestic and/or wild animals should be investigated.

Human cutaneous leishmaniasis: interferon-dependent expression of double-stranded RNA-dependent protein kinase (PKR) via TLR2.

We investigated the type I interferon (IFN-1)/PKR axis in the outcome of the Leishmania (Leishmania) amazonensis infection, along with the underlying mechanisms that trigger and sustain this signaling pathway. Reporter assays of cell extracts from RAW-264.7 macrophages infected with L. (L.) amazonensis or HEK-293T cells cotransfected with TLR2 and PKR promoter constructions were employed. Primary macrophages of TLR2-knockout (KO) or IFNR-KO mice were infected, and the levels of PKR, IFN-1, and superoxide dismutase 1 (SOD1) transcript levels were investigated and compared. Immunohistochemical analysis of human biopsy lesions was evaluated for IFN-1 and PKR-positive cells. Leishmania infection increased the expression of PKR and IFN-ß on induction of PKR-promoter activity. The observed effects required the engagement of TLR2. TLR2-KO macrophages expressed low IFN-ß and PKR levels postinfection with a reduced parasite load. We also revealed the requirement of PKR signaling for Leishmania-induced IFN-1 expression, responsible for sustaining PKR expression and enhancing infection. Moreover, during infection, SOD1 transcripts increased and were also enhanced when IFN-1 was added to the cultures. Remarkably, SOD1 expression was abrogated in infected, dominant-negative PKR-expressing cells. Finally, lesions of patients with anergic diffuse cutaneous leishmaniasis exhibited higher levels of PKR/IFN-1-expressing cells compared to those with single cutaneous leishmaniasis. In summary, we demonstrated the mechanisms and relevance of the IFN-1/PKR axis in the Leishmania infection.

Artificial blood-feeding of phlebotomines (Diptera: Psychodidae: Phlebotominae): is it time to repurpose biological membranes in light of ethical concerns?

BACKGROUND: The aims of the present study were to evaluate and compare the efficacy of blood-feeding in phlebotomines through industrially processed membranes from the small intestine of pigs (used for the production of commercial sausages) and the skin of euthanized chicks. METHODS: Laboratory-bred Lutzomyia longipalpis and different field-caught phlebotomine species were subjected to the artificial feeding systems under similar conditions. Paired tests were performed using the control (skin from euthanized chicks) and test membranes (pig small intestine). The feeding rates were compared by paired t-test, and Pearson correlation was used to examine the relationship between the thickness of the membranes and feeding rate. RESULTS: The feeding rate was greater with the test membrane than with the control membrane for L. longipalpis (t-test, t = -3.3860, P = 0.0054) but not for the most frequent field-caught species, Nyssomyia antunesi (t-test, t = 0.7746, P = 0.4535). The average thicknesses of the control and test membranes were 184 ± 83 µm and 34 ± 12 µm, respectively (Mann-Whitney U-test, U = 0.00, Z = 2.8823, P = 0.0039); however, there was no correlation between feeding rate and membrane thickness. A moderate positive correlation was observed between the number of phlebotomines that fed and the total number of phlebotomines in the cage for each type of membrane and for each species. CONCLUSIONS: The test membrane is a viable alternative for the artificial blood-feeding of phlebotomines, and is thus a potential substitute for the skin of animals that are euthanized for this purpose. Feeding rate was independent of membrane thickness.

Blood Feeding Sources of Nyssomyia antunesi (Diptera: Psychodidae): A Suspected Vector of Leishmania (Kinetoplastida: Trypanosomatidae) in the Brazilian Amazon.

Present work aimed to identify blood feeding sources and attempt to detect Leishmania DNA in Nyssomyia antunesi, suspected vector of Leishmania sp., from a park in the urban center of Belém, the capital of Pará State, in the Brazilian Amazon. Entire bodies and gut contents of Ny. antunesi engorged females, previously captured in the urban park with Centers for Disease Control (CDC) light traps and aspiration on tree bases, were subjected to Leishmania and vertebrate DNA detection through amplification of the Leishmania mini-exon and vertebrate cytochrome b (cyt b) gene regions, respectively. The quality of DNA extraction from entire bodies was ensured through amplification of the dipteran cyt b region. The vertebrate cyt b amplicons were sequenced and compared with those available on GenBank. A maximum likelihood phylogenetic tree was constructed to assess the clustering patterns of these sequences. Leishmania DNA was not detected. The sequences of 13 vertebrate cyt b amplicons were considered informative, exhibiting similarity and clustering with the following six vertebrate species: Dasyprocta leporina (1), Cuniculus paca (1), Tamandua tetradactyla (4), Choloepus didactylus (4), Pteroglossus aracari aracari (2), Homo sapiens (1). The samples of D. leporina and C. paca were obtained from the CDC canopy, whereas the others were by aspiration from tree bases. The present results revealed the eclectic and opportunist blood-feeding behavior of Ny. antunesi, with birds and mammals, these last ones acting as potential reservoirs for Leishmania species, distributed throughout the vertical forest strata.

Activity of alkaloids from Aspidosperma nitidum against Leishmania (Leishmania) amazonensis.

This study evaluated the morphological changes caused by fractions and subfractions, obtained from barks of Aspidosperna nitidum, against L. (L.) amazonensis promastigotes. The ethanolic extract (EE) obtained through the maceration of trunk barks was subjected to an acid-base partition, resulting the neutral (FN) and the alkaloid (FA) fractions, and fractionation under reflux, yielded hexane (FrHEX), dichloromethane (FrDCL), ethyl acetate (FrACoET), and methanol (FrMEOH) fractions. The FA was fractionated and three subfractions (SF5-6, SF8, and SF9) were obtained and analyzed by HPLC-DAD and 1H NMR. The antipromastigote activity of all samples was evaluated by MTT, after that, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for the active fractions were performed. Chromatographic analyzes suggest the presence of alkaloids in EE, FN, FA, and FrDCL. The fractionation of FA led to the isolation of the indole alkaloid dihydrocorynantheol (SF8 fractions). The SF5-6, dihydrocorynantheol and SF-9 samples were active against promastigotes, while FrDCL was moderately active. The SEM analysis revealed cell rounding and changes in the flagellum of the parasites. In the TEM analysis, the treated promastigotes showed changes in flagellar pocket and kinetoplast, and presence of lipid inclusions. These results suggest that alkaloids isolated from A. nitidum are promising as leishmanicidal.

Comparative Genomic Analyses of New and Old World Viscerotropic Leishmanine Parasites: Further Insights into the Origins of Visceral Leishmaniasis Agents.

Visceral leishmaniasis (VL), also known as kala-azar, is an anthropozoonotic disease affecting human populations on five continents. Aetiologic agents belong to the Leishmania (L.) donovani complex. Until the 1990s, three leishmanine parasites comprised this complex: L. (L.) donovani Laveran & Mesnil 1903, L. (L.) infantum Nicolle 1908, and L. (L.) chagasi Lainson & Shaw 1987 (=L. chagasi Cunha & Chagas 1937). The VL causal agent in the New World (NW) was previously identified as L. (L.) chagasi. After the development of molecular characterization, however, comparisons between L. (L.) chagasi and L. (L.) infantum showed high similarity, and L. (L.) chagasi was then regarded as synonymous with L. (L.) infantum. It was, therefore, suggested that L. (L.) chagasi was not native to the NW but had been introduced from the Old World by Iberian colonizers. However, in light of ecological evidence from the NW parasite's enzootic cycle involving a wild phlebotomine vector (Lutzomyia longipalpis) and a wild mammal reservoir (the fox, Cerdocyon thous), we have recently analyzed by molecular clock comparisons of the DNA polymerase alpha subunit gene the whole-genome sequence of L. (L.) infantum chagasi of the most prevalent clinical form, atypical dermal leishmaniasis (ADL), from Honduras (Central America) with that of the same parasite from Brazil (South America), as well as those of L. (L.) donovani (India) and L. (L.) infantum (Europe), which revealed that the Honduran parasite is older ancestry (382,800 ya) than the parasite from Brazil (143,300 ya), L. (L.) donovani (33,776 ya), or L. (L.) infantum (13,000 ya). In the present work, we have now amplified the genomic comparisons among these leishmanine parasites, exploring mainly the variations in the genome for each chromosome, and the number of genomic SNPs for each chromosome. Although the results of this new analysis have confirmed a high genomic similarity (~99%) among these parasites [except L. (L.) donovani], the Honduran parasite revealed a single structural variation on chromosome 17, and the highest frequency of genomic SNPs (more than twice the number seen in the Brazilian one), which together to its extraordinary ancestry (382,800 ya) represent strong evidence that L. (L.) chagasi/L. (L.) infantum chagasi is, in fact, native to the NW, and therefore with valid taxonomic status. Furthermore, the Honduran parasite, the most ancestral viscerotropic leishmanine parasite, showed genomic and clinical taxonomic characteristics compatible with a new Leishmania species causing ADL in Central America.

Urine qPCR diagnoses over the entire clinical-immunological spectrum of human Leishmania (L.) infantum chagasi-infections in the Brazilian Amazon.

The clinical-immunological spectrum of human Leishmania (L.) infantum chagasi-infections in the Brazilian Amazon has been defined using DTH/IFAT-IgG immune assays and the clinical statuses of infected individuals, revealing five profiles: three asymptomatic [Asymptomatic Infection (AI), Subclinical Resistant Infection (SRI), and Indeterminate Initial Infection (III)], and two symptomatic profiles [Subclinical Oligosymptomatic Infection (SOI) and Symptomatic Infection (SI = American visceral leishmaniasis/AVL)]. We evaluated the diagnostic potential of urine qPCR over the entire spectrum of infection. Resine Instagene Matrix® was used for DNA extraction from urinary sediment, with amplification carried out using SYBR® Green Taq with the RV1 and RV2 primers. We examined urine samples from 151 individuals from an endemic area of AVL in Pará State in the Brazilian Amazon, including: 91 (60.3%) with diagnoses of previous infections [13 (14.3%) sharing the AI profile, 13 (14.3%) with the SRI profile, 43 (47.2%) with III, 12 (13.2%) with SI (treated AVL), and 10 (11%) with SI (untreated AVL)]; sixty (39.7%) were DTH(-)/IFAT-IgG(-) (the uninfected group). The urine qPCR was positive in 61.5% of both the AI and SRI profiles, 65% of the III profile, 50% of treated AVL, 100% of untreated AVL, and 6.7% of the uninfected group. Those results confirmed the urine qPCR diagnosis in 100% of untreated AVL cases as well as in more than 60% of the cases with asymptomatic AI, SRI, and III profiles - indicating it as a promising tool for monitoring the evolution of human L. (L.) infantum chagasi-infections in endemic areas.

Whole-Genome Sequencing of Leishmania infantum chagasi Isolates from Honduras and Brazil.

This work reports on the whole-genome sequencing of Leishmania infantum chagasi from Honduras (Central America) and Brazil (South America).

Further evidences on a new diagnostic approach for monitoring human Leishmania (L.) infantum chagasi infection in Amazonian Brazil.

This was a prospective study carried out during a period over 2 years (May/2006-September/2008) with a cohort of 1,099 individuals of both genders, aged 1 year old and older, from an endemic area of American visceral leishmaniasis (AVL) in Pará state, Brazil. The object was to analyze the prevalence and incidence of human Leishmania (L.) infantum chagasi infection as well as the dynamics evolution of its clinical-immunological profiles prior identified: (1) asymptomatic infection (AI); (2) symptomatic infection (SI = AVL); (3) sub-clinical oligosymptomatic infection (SOI); (4) sub-clinical resistant infection (SRI) and; (5) indeterminate initial infection (III). The infection diagnosis was performed by using both the indirect fluorescent antibody test and leishmanin skin test with amastigotes and promastigotes antigens of L. (L.) i. chagasi, respectively. A total of 187 cases of infection were recorded in the prevalence (17%), 117 in the final incidence (6.9%), and 304 in the accumulated prevalence (26.7%), which provided the following distribution into the clinical-immunological profiles: AI, 51.6%; III, 22.4%; SRI, 20.1%; SOI, 4.3%; and SI (=AVL), 1.6%. The major finding regarding the dynamics evolution of infection was concerned to III profile, from which the cases of infection evolved to either the resistant profiles, SRI (21 cases, 30.8%) and AI (30 cases, 44.1%), or the susceptible SI (=AVL; 1 case, 1.5%); the latter 16 cases remained as III till the end of the study. These results provided the conclusion that this diagnostic approach may be useful for monitoring human L. (L.) i. chagasi infection in endemic area and preventing the high morbidity of severe AVL cases.