Biomarkers of disease severity in patients with visceral leishmaniasis co-infected with HIV.

Visceral leishmaniasis (VL) is caused by the protozoan Leishmania spp, transmitted by sand fly bites. VL is one of the deadliest tropical infection diseases, yet the coinfection with HIV virus drastically increases relapses, treatment failure and mortality. The concomitant action of these two pathogens leads to high cellular activation independently of the progression to AIDS. In addition, microbial translocation and bacterial infections are thought to contribute worsening the clinical picture. Identifying biomarkers associated with disease severity is of interest for clinical management of patients with VL-HIV/AIDS. Thus, we analyzed in the sera several markers including interleukins (IL-1ß, IL-6, IL-8, and IL-17), interferon-γ (IFN- γ), tumor necrosis factor (TNF), lipopolysaccharide (LPS), soluble CD14 (sCD14), macrophage migration inhibitory factor (MIF) and intestinal fatty acid-binding protein (IFABP). These markers were compared with disease severity in 24 patients with VL/HIV presenting different clinical outcomes. Disease severity was defined by the probability of death calculated using a score set system derived by the Kala-Cal® software. Probability of death ranged from 3.7% to 97.9%, with median of 28.8%. Five patients died (20%). At the univariate analysis, disease severity was correlated with TNF, IFN-γ and sCD14. LPS was positively correlated with sCD14 specifically in patients with low CD4+ count (CD4+ T-cell <200 cells/mL). Most importantly, the multivariate analysis including LPS, CD4+count and sCD14 showed that sCD14 was the only independent predictor for disease severity and death. Altogether, our results indicated that sCD14 is a powerful marker of pathogenicity and death for patients with VL-HIV/AIDS.

Evidence map of diagnosis, treatment, prognosis, prevention, and control in visceral leishmaniasis.

Objective: To develop an evidence map on visceral leishmaniasis prevention, control, diagnosis, treatment, and prognosis. Methods: Systematic reviews on visceral leishmaniasis were searched using MEDLINE/PubMed and Virtual Health Library. After selection, each included systematic review was assessed, characterized, and categorized by intervention type and by outcomes, according to the methodology offered by the PAHO/WHO Latin American and Caribbean Center on Health Sciences Information (BIREME). The methodological quality was assessed using the AMSTAR2 tool to determine the confidence level of the evidence obtained. Results: Among the prevention and control interventions, insecticide spraying, bednets, dog collars, and dog culling were the most assessed, emphasizing that insecticidal dog collars can reduce visceral leishmaniasis incidence in dogs. Regarding diagnosis, polymerase chain reaction (PCR), rK39 immunochromatographic test (rK39 ICT), and direct agglutination test (DAT) presented high sensitivity and specificity. As for treatment, pentavalent antimonials and amphotericin B were the most analyzed drugs and showed therapeutic success; however, serious adverse events can occur due to their use. The prognostic factors identified were anemia, edema, bleeding, jaundice, age, and HIV coinfection. Conclusions: The evidence map developed shows rK39 ICT and DAT as promising diagnostic alternatives and reinforces the efficacy of liposomal amphotericin B and pentavalent antimonials. Insecticide-impregnated dog collars appear as a promising measure for the control of visceral leishmaniasis, but there is also a need for future studies and reviews with higher methodological quality, especially on prevention and control interventions.

A Novel Antigen, Otubain Cysteine Peptidase of Leishmania donovani, for the Serodiagnosis of Visceral Leishmaniasis and for Monitoring Treatment Response.

Tests for visceral leishmaniasis (VL) are not uniformly effective for all endemic regions. In a serological assay, a novel antigen, otubain cysteine peptidase, compared with rK39, showed comparable sensitivity with Indian VL serum samples and prominently increased sensitivity with Brazilian samples, as well as improved monitoring of the treatment response.

Influence of sex hormones on the immune response to leishmaniasis.

The differences in morbidity and mortality patterns and life expectancy between the sexes are well established in different infectious and parasitic conditions, such as in leishmaniases, in which biological, genetic, sexual and hormonal variations can modulate the immune response indicating greater infectivity, prevalence and clinical severity in men. In this regard, in seeking the understanding of factors related to protection and susceptibility to infection, this review aimed to discuss the influence of sex hormones on the immune response to leishmaniases. In the literature, sex hormone variations promote differences in the innate, humoral and cell-mediated immune response, leading to greater susceptibility, mortality and complications in males. Epidemiological estimates confirm these results, showing a predominance of the disease, in its different clinical forms, in men and suggesting that sexual variations influence immunomodulatory mechanisms since the prevalence of cases comprises the post-puberty and adulthood period. In this perspective, the action of sex hormones has been investigated in different clinical models, highlighting the potential of testosterone in immunosuppression, given its association with greater susceptibility and poor control of parasite load and the induction of cell apoptosis and attenuation of pro-inflammatory signalling pathways. Therefore, hormonal variations influence the immune response among males and females against leishmaniases, in which androgens may present immunosuppressive potential, while steroids present immunomodulatory characteristics.

Biomarkers of the early response to treatment of visceral leishmaniasis: A prospective cohort study.

BACKGROUND: Early biomarkers of the response to treatment are lacking and may help to reduce mortality by the vector-borne disease visceral leishmaniasis (VL). METHODS: A prospective cohort study was conducted to investigate plasma cytokines and clinical laboratory data as biomarkers of the early response to specific treatment for VL in 36 patients. RESULTS: The mean interleukin 6 (IL-6) concentration on the 7th day was 2.3% of the pre-treatment concentration, interleukin 10 (IL-10) was 8.0%, and interleukin 8 (IL-8) was 8.2%. On the 7th day, IL-10 was below half of the pre-treatment concentration in 100.0%, IL-8 in 95.5% and IL-6 in 90.9%. The spleen and liver sizes, haemoglobin, interleukin 1 beta (IL-1ß) and tumour necrosis factor alpha (TNF-α) showed a slower recovery. Fever disappeared in 91% on the 7th day, 69.4% had a normal white cell count, and 77.8% had a normal platelet value by this time. CONCLUSIONS: The plasma cytokines IL-6, IL-10 and IL-8 were demonstrated to be excellent markers of the early response to VL treatment and if tested before the 7th day, will likely prove to be better than fever measurement.

Potential role of dengue virus, chikungunya virus and Zika virus in neurological diseases.

This study showed that laboratory markers of recent infection by dengue, Zika or chikungunya arboviruses were detected in the biological samples of approximately one-third of patients with encephalitis, myelitis, encephalomyelitis or Guillain-Barré syndrome, in a surveillance programme in Piauí state, Brazil, between 2015-2016. Fever and myalgia had been associated with these cases. Since in non-tropical countries most infections or parainfectious diseases associated with the nervous system are attributed to herpesviruses, enteroviruses, and Campylobacter jejuni, the present findings indicate that in tropical countries, arboviruses may now play a more important role and reinforce the need for their surveillance and systematic investigation in the tropics.

Performance of two immunochromatographic tests for diagnosis of visceral leishmaniasis in patients coinfected with HIV.

Because of visceral leishmaniasis (VL) urbanization and spreading of the human immunodeficiency virus (HIV) infection to rural areas, coinfection has become more common. Here, we compared the accuracy of Kalazar Detect® (KD), an rK39-based immunochromatographic (IC) test, and OrangeLife® (OL), an rK39 + rK28 IC test, for diagnosing VL in patients coinfected with HIV in an endemic area in Brazil. Seventy-six VL patients and 40 patients with other diseases, of which 31 and 21 patients, respectively, were infected with HIV, were examined. The sensitivity of OL and KD tests was 88.89 and 95.45%, respectively, in patients without HIV. The sensitivity dropped to 67.74 and 61.29%, respectively, in coinfected patients. The decrease in sensitivity was not related to a decrease in the production of Leishmania-specific IgG. Because of the low sensitivity of rk39 test in HIV-infected patients, we suggest that patients with negative rK39 results should undergo further investigation with additional serological tests that are not based only on the rK39 antigen and examination of bone marrow aspirates.

Epidemiologic, clinical, diagnostic and therapeutic aspects of visceral leishmaniasis in renal transplant recipients: experience from thirty cases.

BACKGROUND: Visceral leishmaniasis is a disease caused by the protozoan Leishmania sp. and is transmitted by Lutzomyia longipalpis (sand fly). In renal transplant recipients, visceral leishmaniasis causes severe damage to the liver, spleen, and hematopoietic system, as well as poor outcomes for patients with transplanted kidneys. This study describes the largest series of cases of visceral leishmaniasis in renal transplant recipients, providing important information about the diagnostic routines and therapeutic strategies in this patient population. METHODS: A retrospective, descriptive study was performed to analyze the distribution and evaluate the extent of the epidemiologic, clinical, diagnostic and therapeutic aspects of 30 renal transplant recipients from endemic regions who presented with visceral leishmaniasis in the post-transplantation period. RESULTS: In this study, visceral leishmaniasis was more frequent in men (80%). The mean age of presentation was 40 ± 10.5 years. The majority of patients worked in urban areas (66.7%), cohabitated with domestic animals (90%), and were from low-income households. In 73.3% of cases, diagnosis was made by direct isolation of Leishmania forms. Patients were treated with liposomal amphotericin, resulting in a high degree of disease remission (80%). CONCLUSIONS: This study describes the largest series of visceral leishmaniasis in renal transplant recipients and expands clinical-epidemiological knowledge for transplantation teams to perform adequate disease management for this specific patient population.

Lipoprotein lipase and PPAR alpha gene polymorphisms, increased very-low-density lipoprotein levels, and decreased high-density lipoprotein levels as risk markers for the development of visceral leishmaniasis by Leishmania infantum.

In visceral leishmaniasis (VL) endemic areas, a minority of infected individuals progress to disease since most of them develop protective immunity. Therefore, we investigated the risk markers of VL within nonimmune sector. Analyzing infected symptomatic and, asymptomatic, and noninfected individuals, VL patients presented with reduced high-density lipoprotein cholesterol (HDL-C), elevated triacylglycerol (TAG), and elevated very-low-density lipoprotein cholesterol (VLDL-C) levels. A polymorphism analysis of the lipoprotein lipase (LPL) gene using HindIII restriction digestion (N = 156 samples) (H+ = the presence and H- = the absence of mutation) revealed an increased adjusted odds ratio (OR) of VL versus noninfected individuals when the H+/H+ was compared with the H-/H- genotype (OR = 21.3; 95% CI = 2.32-3335.3; P = 0.003). The H+/H+ genotype and the H+ allele were associated with elevated VLDL-C and TAG levels (P < 0.05) and reduced HDL-C levels (P < 0.05). An analysis of the L162V polymorphism in the peroxisome proliferator-activated receptor alpha (PPARα) gene (n = 248) revealed an increased adjusted OR when the Leu/Val was compared with the Leu/Leu genotype (OR = 8.77; 95% CI = 1.41-78.70; P = 0.014). High TAG (P = 0.021) and VLDL-C (P = 0.023) levels were associated with susceptibility to VL, whereas low HDL (P = 0.006) levels with resistance to infection. The mutated LPL and the PPARα Leu/Val genotypes may be considered risk markers for the development of VL.

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene.

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

Association between clinical outcomes, peripheral blood and cytomorphologic features of bone marrow in visceral leishmaniasis.

INTRODUCTION: An intracellular parasite of mononuclear phagocytes, mainly distributed in the bone marrow and the spleen, causes visceral leishmaniasis. Complete blood count (CBC) reveals the poorly understood pathogenesis of anemia, leukopenia and thrombocytopenia. Our study aimed to compare the CBC with bone marrow cytomorphological features and their association with clinical outcomes to clarify this relevant issue. METHODS: The CBC and bone marrow of 118 patients were described by two hematologists and compared to check their association with each other and mortality. RESULTS: Peripheral cytopenias were common findings, particularly anemia, as seen in almost all patients. No relationship was found between values of hemoglobin, neutrophils and platelet count with fatal outcomes. The bone marrow was normocellular in 61.9% of the cases. Dysplasia figures were frequent and 49.1% of the samples had dysgranulopoiesis. Additionally, erythroid hyperplasia was found in 72% of the patients with severe anemia. Patients with reduced bone marrow cellularity, erythroid hypercellularity and dyserythropoiesis seem to have a riskier disease. CONCLUSION: The study results suggest that the bone marrow of patients with visceral leishmaniasis manifests a reactional pattern to the inflammatory event, thereby modulating cytokines and other colony growth factors. This compensatory response may be dysplastic and ineffective and generate peripheral cytopenias of varying intensity. Further studies are needed to clarify the signaling pathways involved, which may be used as therapeutic tools in the future.

The spleen is the graveyard of CD4+ cells in patients with immunological failure of visceral leishmaniasis and AIDS.

BACKGROUND: Visceral leishmaniasis (VL), or kala-azar, is a common comorbidity in patients with AIDS in endemic areas. Many patients continue to experiences relapses of VL despite virological control, but with immunological failure. These patients remain chronically symptomatic with hypersplenism, for example with anemia, leukopenia, and thrombocytopenia, and are at risk of severe co-infection due to low CD4+ count. Therefore, in this study, splenectomized patients with VL and HIV infection were investigated to understand why the CD4+ count fails to recover in these patients, evaluating the importance of spleen mass for hypersplenism and immunological failure. METHODS: From a retrospective open cohort of 13 patients who had previously undergone splenectomy as salvage therapy for relapsing VL, 11 patients with HIV infection were investigated. This study compared the patients' complete blood cell count (CBC) and CD4+ and CD8+ cell counts before and after splenectomy with respect to spleen weight. RESULTS: CBC was substantially improved after splenectomy, indicating hypersplenism. However, to the best of our knowledge, this is the first study to show that spleen mass is strongly and negatively correlated with CD4+ cell count (ρ = -0.71, P = 0.015). CONCLUSIONS: This finding was unexpected, as the spleen is the most extensive lymphoid tissue and T-lymphocyte source. After reviewing the literature and reasoning, we hypothesized that the immunological failure was secondary to CD4+ loss initially by apoptosis in the spleen induced by productive HIV infection and, subsequently, by pyroptosis sustained by parasitic infection in spleen macrophages.

Scientometric review of research on Neglected Tropical Diseases: a 31-year perspective from the Journal of the Brazilian Society of Tropical Medicine.

BACKGROUND: To analyze the temporal evolution of research on Neglected Tropical Diseases (NTDs) published by the Journal of the Brazilian Society of Tropical Medicine (JBSTM). METHODS: We performed an analysis of the scientific production in JBSTM on NTDs using an advanced search, which included authors' descriptors, title, and abstract, and by combining specific terms for each NTDs from 1991 to 2021. Data related to authors, countries of origin, institutions, and descriptors, were evaluated and analyzed over time. Bibliographic networks were constructed using VOSviewer 1.6.16. RESULTS: The JBSTM published 4,268 scientific papers during this period. Of these 1,849 (43.3%) were related to NTDs. The number of publications on NTDs increased by approximately 2.4-fold, from 352 (total 724) during 1991-2000 to 841 (total 2,128) during 2011-2021, despite the proportional reduction (48.6% versus 39.5%). The most common singular NTDs subject of publications included Chagas disease (31.4%; 581/1,849), leishmaniasis (25.5%, 411/1,849), dengue (9.4%, 174/1,849), schistosomiasis (9.0%; 166/1,849), and leprosy (6.5%, 120/1,849), with authorship mostly from Brazil's South and Southeast regions. CONCLUSIONS: Despite the proportional reduction in publications, JBSTM remains an important vehicle for disseminating research on NTDs during this period. There is a need to strengthen the research and subsequent publications on specific NTDs. Institutions working and publishing on NTDs in the country were concentrated in the South and Southeast regions, requiring additional investments in institutions in other regions of the country.

Relationship Between Markers of Chronic Inflammation and Copper Nutritional Status in Obese Women.

Adipose tissue dysfunction causes the development of metabolic complications, such as low-grade chronic inflammation, which may to alter copper homeostasis in obese individuals. Thus, the objective of this study is to analyze the relationship between markers of chronic inflammation and copper nutritional status in obese women. Cross-sectional study involved women aged 20-50 years, divided into two groups: case (BMI > 35 kg/m2) and control (18.5 > BMI > 24.9 kg/m2). Plasma and erythrocyte copper concentrations were determined by inductively coupled plasma optical emission spectrometry (ICP-OES) method. Activity of superoxide dismutase (SOD) enzyme in the erythrocytes was determined with an automatic biochemical analyzer. Serum concentrations of interleukin (IL)-6, IL-8, IL-12, IL-10, and IL-1ß and tumor necrosis factor-alpha (TNF-α) were determined by using flow cytometer. Serum IL-6 concentrations were 105% higher in the case group compared to eutrophic women. Plasma copper concentrations were 20.5% higher, and erythrocyte copper concentrations were 23.5% lower in patients with obesity. In addition, erythrocyte SOD activity was 20% lower in obese participants when compared to eutrophic women. Our study identified significant negative correlation between the cytokines TNF-α and IL-10 and the SOD activity in the case group, suggesting a possible influence of chronic inflammation on copper distribution in obese individuals.

Recombinant protein KR95 as an alternative for serological diagnosis of human visceral leishmaniasis in the Americas.

In the Americas, visceral leishmaniasis (VL) is caused by the protozoan Leishmania infantum, leading to death if not promptly diagnosed and treated. In Brazil, the disease reaches all regions, and in 2020, 1,933 VL cases were reported with 9.5% lethality. Thus, an accurate diagnosis is essential to provide the appropriate treatment. Serological VL diagnosis is based mainly on immunochromatographic tests, but their performance may vary by location, and evaluation of diagnostic alternatives is necessary. In this study, we aimed to evaluate the performance of ELISA with the scantily studied recombinant antigens, K18 and KR95, comparing their performance with the already known rK28 and rK39. Sera from parasitologically confirmed symptomatic VL patients (n = 90) and healthy endemic controls (n = 90) were submitted to ELISA with rK18 and rKR95. Sensitivity (95% CI) was, respectively, 83.3% (74.2-89.7) and 95.6% (88.8-98.6), and specificity (95% CI) was 93.3% (85.9-97.2) and 97.8% (91.8-99.9). For validation of ELISA with the recombinant antigens, we included samples from 122 VL patients and 83 healthy controls collected in three regions in Brazil (Northeast, Southeast, and Midwest). When comparing the results obtained with the VL patients' samples, significantly lower sensitivity was obtained by rK18-ELISA (88.5%, 95% CI: 81.5-93.2) compared with rK28-ELISA (95.9%, 95% CI: 90.5-98.5), but the sensitivity was similar comparing rKR95-ELISA (95.1%, 95% CI: 89.5-98.0), rK28-ELISA (95.9%, 95% CI: 90.5-98.5), and rK39-ELISA (94.3%, 95% CI: 88.4-97.4). Analyzing the specificity, it was lowest with rK18-ELISA (62.7%, 95% CI: 51.9-72.3) with 83 healthy control samples. Conversely, higher and similar specificity was obtained by rKR95-ELISA (96.4%, 95% CI: 89.5-99.2), rK28-ELISA (95.2%, 95% CI: 87.9-98.5), and rK39-ELISA (95.2%, 95% CI: 87.9-98.5). There was no difference in sensitivity and specificity across localities. Cross-reactivity assessment, performed with sera of patients diagnosed with inflammatory disorders and other infectious diseases, was 34.2% with rK18-ELISA and 3.1% with rKR95-ELISA. Based on these data, we suggest using recombinant antigen KR95 in serological assays for VL diagnosis.

Relationship Between Zinc, Selenium, and Magnesium Status and Markers of Metabolically Healthy and Unhealthy Obesity Phenotypes.

Our objective was to investigate the relationship between zinc, selenium, and magnesium status and markers of metabolically healthy and unhealthy obesity phenotypes. This was a cross-sectional study with 140 women: metabolically healthy obese women (n = 35), metabolically unhealthy obese women (n = 28), and normal-weight women (n = 77). We have calculated the body mass index, waist-hip ratio, waist-height ratio and some adiposity indices. Additionally, we evaluated endocrine-metabolic parameters and estimated the dietary intake of energy, macronutrients, zinc, selenium, and magnesium. The mineral concentrations in plasma, erythrocytes, and urine were assessed. In obese patients, there was a significant decrease in dietary zinc, selenium, and magnesium intake per kilogram of body weight, as well as lower mineral concentrations in both plasma and erythrocytes. Additionally, these patients exhibited higher urinary mineral levels compared to the control group, regardless of whether they had healthy or unhealthy phenotypes. We observed a significant correlation between deficiencies in zinc, selenium, and magnesium and obesity-associated metabolic disorders, including dyslipidemias and redox status disturbances. This study highlights a connection between deficiencies in zinc, selenium, and magnesium and metabolic disorders linked to obesity, including dyslipidemias, alterations in redox status, and thyroid hormonal dysfunction.

High seroprevalence of Leishmania infantum is linked to immune activation in people with HIV: a two-stage cross-sectional study in Bahia, Brazil.

Visceral leishmaniasis is an opportunistic disease in HIV-1 infected individuals, unrecognized as a determining factor for AIDS diagnosis. The growing geographical overlap of HIV-1 and Leishmania infections is an emerging challenge worldwide, as co-infection increases morbidity and mortality for both infections. Here, we determined the prevalence of people living with HIV (PWH) with a previous or ongoing infection by Leishmania infantum and investigated the virological and immunological factors associated with co-infection. We adopted a two-stage cross-sectional cohort (CSC) design (CSC-I, n = 5,346 and CSC-II, n = 317) of treatment-naïve HIV-1-infected individuals in Bahia, Brazil. In CSC-I, samples collected between 1998 and 2013 were used for serological screening for leishmaniasis by an in-house Enzyme-Linked Immunosorbent Assay (ELISA) with SLA (Soluble Leishmania infantum Antigen), resulting in a prevalence of previous or ongoing infection of 16.27%. Next, 317 PWH were prospectively recruited from July 2014 to December 2015 with the collection of sociodemographic and clinical data. Serological validation by two different immunoassays confirmed a prevalence of 15.46 and 8.20% by anti-SLA, and anti-HSP70 serology, respectively, whereas 4.73% were double-positive (DP). Stratification of these 317 individuals in DP and double-negative (DN) revealed a significant reduction of CD4+ counts and CD4+/CD8+ ratios and a tendency of increased viral load in the DP group, as compared to DN. No statistical differences in HIV-1 subtype distribution were observed between the two groups. However, we found a significant increase of CXCL10 (p = 0.0076) and a tendency of increased CXCL9 (p = 0.061) in individuals with DP serology, demonstrating intensified immune activation in this group. These findings were corroborated at the transcriptome level in independent Leishmania- and HIV-1-infected cohorts (Swiss HIV Cohort and Piaui Northeast Brazil Cohort), indicating that CXCL10 transcripts are shared by the IFN-dominated immune activation gene signatures of both pathogens and positively correlated to viral load in untreated PWH. This study demonstrated a high prevalence of PWH with L. infantum seropositivity in Bahia, Brazil, linked to IFN-mediated immune activation and a significant decrease in CD4+ levels. Our results highlight the urgent need to increase awareness and define public health strategies for the management and prevention of HIV-1 and L. infantum co-infection.

Application of serial analysis of gene expression to the study of the gene expression profile of Leishmania infantum chagasi promastigote.

This study describes the application of the LongSAGE methodology to study the gene expression profile in promastigotes of Leishmania infantum chagasi. A tag library was created using the LongSAGE method and consisted of 14,208 tags of 17 bases. Of these, 8,427 (59.3%) were distinct. BLAST research of the 1,645 most abundant tags showed that 12.8% of them identified the coding sequences of genes, while 82% (1,349/1,645) identified one or more genomic sequences that did not correspond with open reading frames. Only 5.2% (84/1,645) of the tags were not aligned to any position in the L. infantum genome. The UTR size of Leishmania and the lack of CATG sites in some transcripts were decisive for the generation of tags in these regions. Additional analysis will allow a better understanding of the expression profile and discovering the key genes in this life cycle.

Genetic diversity of Leishmania infantum field populations from Brazil.

Leishmania infantum (syn. Leishmania chagasi) is the etiological agent of visceral leishmaniasis (VL) in Brazil. The epidemiology of VL is poorly understood. Therefore, a more detailed molecular characterization at an intraspecific level is certainly needed. Herein, three independent molecular methods, multilocus microsatellite typing (MLMT), random amplification of polymorphic DNA (RAPD) and simple sequence repeats-polymerase chain reaction (SSR-PCR), were used to evaluate the genetic diversity of 53 L. infantum isolates from five different endemic areas in Brazil. Population structures were inferred by distance-based and Bayesian-based approaches. Eighteen very similar genotypes were detected by MLMT, most of them differed in only one locus and no correlation was found between MLMT profiles, geographical origin or the estimated population structure. However, complex profiles composed of 182 bands obtained by both RAPD and SSR-PCR assays gave different results. Unweighted pair group method with arithmetic mean trees built from these data revealed a high degree of homogeneity within isolates of L. infantum. Interestingly, despite this genetic homogeneity, most of the isolates clustered according to their geographical origin.

Development of Immunological Assays Based on Leishmania donovani Antigen for Diagnosis of Canine Visceral Leishmaniasis and Their Multicenter Evaluation in Brazil and Italy.

Canine visceral leishmaniasis (CVL) due to Leishmania infantum infection is a zoonotic disease prevalent in the areas of South America and the Mediterranean. Infected dogs as reservoirs can contribute to disease transmission and can be a scourge to public health. Therefore, early diagnosis of infected dogs may play a pivotal role in circumscribing disease progression. Invasive tissue aspiration and insufficient serological methods impair a single assay for prompt CVL diagnosis. In the present study, we aimed to evaluate the potential of Leishmania donovani isolated membrane protein, LAg, for the diagnosis of CVL through immunological assays. Initially, enzyme-linked immunosorbent assay was done with Brazilian dog sera to evaluate the performance of LAg in diagnosing CVL and found sensitivity and specificity of 92.50% and 95%, respectively. The study further confirmed the diagnostic efficacy of LAg in a dipstick format. The dipstick test of canine sera from three centers in Brazil and one center in Italy collectively showed sensitivity values in the range of 53.33% to 100% in recognizing symptomatic dogs and specificity values between 75% and 100% to rule out healthy dogs. Moreover, a rapid immunochromatographic test was developed and optimized using LAg. This test was able to identify 94.73% of CVL of Brazilian origin with specificity of 97.29%. The current results highlight the reactive potential of the L. donovani antigen, LAg, for L. infantum CVL diagnosis and support our previous findings, which suggest the utility of LAg for the diagnosis of both L. donovani and L. infantum human VL in a variety of endemic regions. LAg as a diagnostic candidate may be employed to identify comprehensive CVL cases in epidemiological areas.