Evaluation of blood based quantitative PCR as a molecular diagnostic tool for post kala-azar dermal leishmaniasis (PKDL).

BACKGROUND: Post kala-azar dermal leishmaniasis (PKDL) is a consequential dermal manifestation of visceral leishmaniasis (VL), serving as a parasite reservoir. The traditional diagnostic approach, which requires an invasive skin biopsy is associated with inherent risks and necessitates skilled healthcare practitioners in sterile settings. There is a critical need for a rapid, less invasive method for Leishmania detection. The main objective of this study was to evaluate and compare the diagnostic efficacy of PCR and qPCR in detecting PKDL, utilizing both skin and blood samples and to assess the utility of blood samples for molecular diagnosis. METHODS AND RESULTS: 73 individuals exhibiting clinical symptoms of PKDL and who had tested positive for rK39 rapid diagnostic test (RDT) were enrolled in this study. For the diagnosis of PKDL, both PCR and real-time quantitative PCR (qPCR), employing SYBR Green and TaqMan assays, were performed on blood and skin matched samples. qPCR results using both TaqMan and SYBR Green assay, indicated higher parasite loads in the skin compared to blood, as evident by the Ct values. Importantly, when blood samples were used for PKDL diagnosis by qPCR, an encouraging sensitivity of 69.35% (TaqMan assay) and 79.36% (SYBR Green) were obtained, compared to 8.2% with conventional PCR. CONCLUSION: The findings of the study suggest the potential utility of blood for molecular diagnosis by qPCR, offering a less invasive alternative to skin biopsies in field setting for the early detection of parasitaemia in PKDL patients and effective management and control of the disease.

Prevalence of cutaneous leishmaniasis in the largest populated city Karachi, Pakistan / Prevalência de leishmaniose cutânea na maior cidade populosa de Karachi, Paquistão

The study was undertaken from September 2018 to April 2019 to determine the prevalence of cutaneous leishmaniasis in human beings living in six districts of Karachi. Suspected persons were screened for the disease and positive cases were identified on the basis of skin lesions and blood samples. Samples were observed by mounting their smear. A total of 207 subjects of different ages and sex groups were investigated, however, only 192 (92%) of the suspected cases were found to have the disease 64% of cases were male which were significantly high (p<0.05), than female 36%. The lesion was more frequently detected among the youth ages of 21-30 years (31%) as compared to other groups. In both sexes, legs were found to be more infected (25% male + 20% female) followed by arms (20% male + 0% female) and face (15% male +11% female). The mixed body parts had shown the lowest infections such as (4% in males + 5%) in females. In conclusion, the highest and lowest leishmaniasis infections were observed in District West (23% male + 9% female) followed by District East (15% male + 7% female), District Malir (11% male+ 4% female), District Central (7% male + 5% female), District Korangi (4% male + 7% female) and District South (4% male + 4% female) respectively.

O estudo foi realizado de setembro de 2018 a abril de 2019 para determinar a prevalência de leishmaniose tegumentar em seres humanos que vivem em seis distritos de Karachi. Pessoas suspeitas foram rastreadas para a doença e os casos positivos foram identificados com base em lesões de pele e amostras de sangue. As amostras foram observadas montando seu esfregaço. Um total de 207 indivíduos de diferentes idades e grupos sexuais foi investigado, no entanto apenas 192 (92%) dos casos suspeitos foram encontrados para ter a doença; 64% dos casos eram do sexo masculino, que foram significativamente elevados (p < 0,05), e do sexo feminino 36%. A lesão foi detectada com maior frequência entre os jovens de 21 a 30 anos (31%) em comparação com os outros grupos. Em ambos os sexos, as pernas estavam mais infectadas (25% homens + 20% mulheres), seguidas pelos braços (20% homens + 0% mulheres) e rosto (15% homens + 11% mulheres). As partes mistas do corpo mostraram as infecções mais baixas (4% homens + 5% mulheres). Em conclusão, as infecções de leishmaniose mais altas e mais baixas foram observadas no Distrito Oeste (23% homens + 9% mulheres) seguido pelo Distrito Leste (15% homens + 7% mulheres), Distrito Malir (11% homens + 4% mulheres), Distrito Central (7% homens + 5% mulheres), Distrito Korangi (4% homens + 7% mulheres) e Distrito Sul (4% homens + 4% mulheres), respectivamente.

American visceral leishmaniasis in a state of northeastern Brazil: clinical, epidemiological and laboratory aspects / Leishmaniose visceral americana em estado do nordeste brasileiro: aspectos clínicos, epidemiológicos e laboratoriais

In Brazil, American visceral leishmaniasis (AVL) has become a public health concern due to its high incidence and lethality. This study aimed to analyze the clinical, epidemiological, and laboratory aspects of AVL in a state of Brazil. This descriptive, cross-sectional, retrospective, and quantitative study of notified cases of AVL was carried out in Alagoas between 2008 and 2017 from data obtained from DATASUS/SINAN. Sociodemographic, clinical, and laboratory variables were analyzed. A descriptive analysis was performed using absolute values and valid percentages, using tables and/or graphs. Data processing was performed using Stata 12.0®. Results with P <0.05 were considered statistically significant. During the study period, 352 cases of AVL were reported, of which 6.82% died and 38.92% had met a cure criterion. Male patients were predominant (66.76%). Of the total infected patients, 16.76% had attended only the 1st to the 4th grades, with those most affected aged 1 to 4 years (28.69%). Laboratory diagnostic criteria were most commonly used to confirm the notified cases (76.42%), whereas 51.70% and 8.52% of the cases had positive parasitological and immunofluorescence diagnoses, respectively. Finally, the study showed a higher prevalence of the disease in children, men and in rural residents. Although with low lethality, the expressive frequency of AVL in the State of Alagoas was still verified, since there was an increase in the number of cases during the years of the study.

No Brasil, a leishmaniose visceral americana (LVA) tornou-se uma preocupação de saúde pública devido à sua alta incidência e letalidade. Este estudo teve como objetivo analisar os aspectos clínicos, epidemiológicos e laboratoriais da AVL em um estado brasileiro. Este estudo descritivo, transversal, retrospectivo e quantitativo dos casos notificados de AVL foi realizado em Alagoas entre 2008 e 2017 a partir de dados obtidos do DATASUS/SINAN. Foram analisadas variáveis sociodemográficas, clínicas e laboratoriais. Foi realizada uma análise descritiva utilizando-se valores absolutos e percentuais válidos, utilizando tabelas e/ou gráficos. O processamento dos dados foi realizado por meio do Stata 12.0®. Os resultados com P<0,05 foram considerados estatisticamente significativos. Durante o período de estudo, foram notificados 352 casos de LVA, dos quais 6,82% morreram e 38,92% atenderam a um critério de cura. Os pacientes do sexo masculino foram predominantes (66,76%). Do total de pacientes infectados, 16,76% tinham sido atendidos apenas do 1º ao 4º ano, com os mais afetados entre 1 e 4 anos (28,69%). Os critérios de diagnóstico laboratorial foram mais utilizados para confirmar os casos notificados (76,42%), enquanto 51,70% e 8,52% dos casos apresentaram diagnósticos positivos parasitológicos e imunofluorescência, respectivamente. Por fim, o estudo demonstrou maior prevalência da doença em crianças, homens e nos residentes em zona rural. Embora com letalidade baixa, constatou-se ainda a expressiva frequência da LVA no Estado de Alagoas, uma vez que houve aumento do número de casos durante os anos do estudo.

Immunogenicity and potential protection of DNA vaccine of Leishmania martiniquensis against Leishmania infection in mice.

INTRODUCTION: In Thailand, Leishmania martiniquensis is the predominant species causing cutaneous and visceral leishmaniasis. Its incidence has been increasing among immunocompetent and immunocompromised hosts. We developed a prototype DNA vaccine using a partial consensus sequence of the cysteine protease B (cpb) gene derived from L. martiniquensis from Thai patients. METHODOLOGY: The laboratory inbred strain of albino BALB/c mice were immunized intramuscularly three times at 2-week intervals (weeks 0, 2, and 4) with cpb plasmid DNA (pcDNA_cpb) with or without the adjuvant, monoolein (pcDNA_cpb-MO). Mice were challenged at week 6 with L. martiniquensis promastigotes. Sera were analysed for IgG1, IgG2a, interferon gamma and interleukin 10 (IFN-γ and IL-10, respectively) levels at weeks 0, 4, and 9. Additionally, livers and spleens were also analysed for parasite burden using immunohistochemistry and real-time polymerase chain (qPCR) assays. RESULTS: Three weeks after promastigote challenge, vaccinated mice showed significantly increased levels of IgG2a and IFN-γ while IL-10 level was significantly reduced when compared with those in the control group (p < 0.01). Parasite burden in the livers and spleens of vaccinated mice significantly decreased. In addition, a significant increase in mature granuloma formation in the livers when compared with those of the control group (p < 0.05) was found, indicating increased T-helper cells (Th1)-induced inflammation and destruction of amastigotes. Monoolein produced a booster effect to enhance the mouse Th1 protective immunity. CONCLUSIONS: The prototype DNA vaccine could induce a Th1 immune response that conferred potential protection to the L. martiniquensis promastigote challenge in BALB/c mice.

Localized skin inflammation during cutaneous leishmaniasis drives a chronic, systemic IFN-γ signature.

Cutaneous leishmaniasis is a localized infection controlled by CD4+ T cells that produce IFN-γ within lesions. Phagocytic cells recruited to lesions, such as monocytes, are then exposed to IFN-γ which triggers their ability to kill the intracellular parasites. Consistent with this, transcriptional analysis of patient lesions identified an interferon stimulated gene (ISG) signature. To determine whether localized L. braziliensis infection triggers a systemic immune response that may influence the disease, we performed RNA sequencing (RNA-seq) on the blood of L. braziliensis-infected patients and healthy controls. Functional enrichment analysis identified an ISG signature as the dominant transcriptional response in the blood of patients. This ISG signature was associated with an increase in monocyte- and macrophage-specific marker genes in the blood and elevated serum levels IFN-γ. A cytotoxicity signature, which is a dominant feature in the lesions, was also observed in the blood and correlated with an increased abundance of cytolytic cells. Thus, two transcriptional signatures present in lesions were found systemically, although with a substantially reduced number of differentially expressed genes (DEGs). Finally, we found that the number of DEGs and ISGs in leishmaniasis was similar to tuberculosis-another localized infection-but significantly less than observed in malaria. In contrast, the cytolytic signature and increased cytolytic cell abundance was not found in tuberculosis or malaria. Our results indicate that systemic signatures can reflect what is occurring in leishmanial lesions. Furthermore, the presence of an ISG signature in blood monocytes and macrophages suggests a mechanism to limit systemic spread of the parasite, as well as enhance parasite control by pre-activating cells prior to lesion entry.

Low Serum Levels of Selenium, Zinc, Iron, and Zinc/Copper Ratio in an Endemic Region of Cutaneous Leishmaniasis in Southwest Iran.

Leishmaniasis is a widespread tropical infection; cutaneous leishmaniasis (CL) is the most common form of this disease known to cause significant morbidity. Trace metals, including selenium, zinc, iron, and copper, are required for the activity of several enzymes involved in immune system responses. The aim of this research was to measure the serum levels of selenium (Se), zinc (Zn), copper (Cu), iron (Fe), and Zn/Cu ratio in patients with CL. In this case-control study, 80 patients with CL and 80 healthy volunteers (not exposed to CL) from a CL endemic region in southwest Iran agreed to participate. Both clinical and parasitological verifications were made to include each subject as a CL-positive case. A questionnaire was completed for each participant which included the following criteria: age (year), height (cm), weight (kg), body mass index (kg/m2), and duration of disease (day). The biochemical assays were performed according to the standard protocols, and the values of Zn, Cu, Se, and Fe were expressed in micrograms per deciliter (µg/dl). All results were expressed as mean ± standard deviation (SD), and the statistical significance level was defined to be less than 0.05 (P < 0.05). There were no statistically significant differences in terms of mean values of age, body weight, height, and body mass index between CL patients and the control group (P > 0.05). The mean ± SD concentrations of Zn, Fe, and Se in the control group were found to be 118.87 ± 6.35 µg/dl, 123.00 ± 8.40 µg/dl, and 11.26 ± 1.88 µg/dl, respectively. These trace elements (TEs) were statistically lower (P < 0.001) in patients with CL (case group) with values of 83.05 ± 7.32 µg/dl for Zn, 86.51 ± 10.09 µg/dl for Fe, and 3.83 ± 1.20 µg/dl for Se. We have also observed that serum levels of Cu in CL-positive group were significantly higher than in the controls (P < 0.001). Furthermore, CL patients had significantly lower Zn/Cu ratio than controls (0.63 ± 0.05 µg/dl vs. 1.11 ± 0.10; P < 0.001). The alternation in serum levels of TEs may be a part of the defense strategy of the organism. Based on these results, it can be suggested that serum levels of these TEs can be a useful marker to estimate the prognosis of CL infection.

Case Report: Cutaneous Leishmaniasis Misdiagnosed as Pyoderma Gangrenosum.

With an estimated prevalence of 0.7%, pyoderma gangrenosum (PG) is the most frequent type of reactive skin lesions seen in the setting of inflammatory bowel disease, together with metastatic Crohn's disease. However, in the case of persistent cutaneous ulcerations in patients with Crohn's disease under biologic and/or immunosuppressive therapy, infectious or malignant etiologies should be excluded. We report a case of multiple difficult-to-treat skin ulcerations due to Leishmania in a patient with Crohn's disease treated with antitumor necrosis factor-alpha drugs, misinterpreted for a long time as PG.

Evaluation of Leishmanization Using Iranian Lizard Leishmania Mixed With CpG-ODN as a Candidate Vaccine Against Experimental Murine Leishmaniasis.

Background and Objectives: The live non-pathogenic Leishmania tarantolae has recently provided a promising approach as an effective vaccine candidate against experimental leishmaniasis (ILL). Here, we evaluated the immunoprotective potential of the live Iranian Lizard Leishmania mixed with CpG adjuvant against L. major infection in BALB/c mice. Methods: Four groups of female BALB/c mice were included in the study. The first and second groups received PBS and CpG, respectively. The immunized groups received 2 × 105 ILL promastigotes and the CpG-mixed ILL (ILL+CpG). Injections were performed subcutaneously in the right footpad. Three weeks later, all mice were challenged with 2 × 105 metacyclic promastigotes of Leishmania majorEGFP ; inoculation was done in the left footpad. The measurement of footpad swelling and in vivo fluorescent imaging were used to evaluate disease progress during infection course. Eight weeks after challenge, all mice were sacrificed and the cytokines levels (IFN-γ, IL-4, and IL-10) and sera antibodies concentrations (IgG2a and IgG1) using ELISA assay, nitric oxide production using Griess assay, and arginase activity in cultured splenocytes, were measured. In addition, direct fluorescent microscopy analysis and qPCR assay were used to quantify the splenic parasite burden. Result: The results showed that mice immunized with ILL+CpG were protected against the development of the dermal lesion. Moreover, they showed a significant reduction in the parasite load, in comparison to the control groups. The observed protection was associated with higher production of IFN-γ, as well as a reduction in IL-4 level. Additionally, the results demonstrated that arginase activity was decreased in ILL+CpG group compared to other groups. Conclusion: Immunization using ILL+CpG induces a protective immunity; indicating that ILL with an appropriate adjuvant would be a suitable choice for vaccination against leishmaniasis.

Programmed Cell Death Ligand (PD-L)-1 Contributes to the Regulation of CD4+ T Effector and Regulatory T Cells in Cutaneous Leishmaniasis.

Cutaneous Leishmaniasis (CL) affects up to one million people every year and treatments are costly and toxic. The regulation of the host immune response is complex and the knowledge of how CD4+ T cells are activated and maintained during Leishmania infection is still limited. Current therapies aim to target programmed cell death (PD)-1 and programmed cell death ligand (PD-L)-1 in order to boost T cell activity. However, the role of the PD-1/PD-L1 axis during Leishmania infection is still unclear. In this study, we found that patients with active and post-treatment CL displayed different subsets of CD4+PD-1+ T cells. Accordingly, L. major-infected mice upregulated PD-1 on activated CD4+ T effector cells and PD-L1 on resident macrophages and infiltrating monocytes at the site of infection. L. major-infected Pdl1-/- mice expressed lower levels of MHCII and higher levels of CD206 on macrophages and monocytes and, more importantly, the lack of PD-L1 contributed to a reduced frequency of CD4+Ly6Chi T effector cells and an increase of CD4+Foxp3+ regulatory T cells at the site of infection and in draining lymph nodes. Additionally, the lack of PD-L1 was associated with lower production of IL-27 by infiltrating monocytes and lower levels of the Th1 cytokines IFN-γ and TNF-α produced by CD4+ T effector cells. Pdl1-/- mice initially exhibited larger lesions despite having a similar parasite load. Our results describe for the first time how the interruption of the PD-1/PD-L1 axis influences the immune response against CL and suggests that this axis regulates the balance between CD4+Ly6Chi T effector cells and CD4+Foxp3+ regulatory T cells.

Cutaneous Leishmaniasis of Lip and Role of Polymerase Chain Reaction: A Case Report.

The diagnosis of cutaneous leishmaniasis is mostly confirmed by the identification of parasite in a skin smear or biopsy. However, this method may not always be sensitive enough to detect the disease when parasitic load is low. Molecular test such as polymerase chain reactions can be useful in such circumstances. Here, we report a case of cutaneous leishmaniasis diagnosed by a polymerase chain reaction test when both smear and biopsy failed to confirm the diagnosis. A 17-years-old female from mountainous district of Nepal, presented with a crusted plaque over the upper lip for a duration of 6 months. Both skin smear and biopsy from the lesion failed to demonstrate Leishmania parasite but a polymerase chain reaction test was positive for Leishmania donovani. This case emphasizes on the importance of molecular testing such as polymerase chain reaction when commonly performed diagnostics test fails to support confirmation of clinical diagnosis.

Characterization of serum cytokines and circulating microRNAs that are predicted to regulate inflammasome genes in cutaneous leishmaniasis patients.

Leishmaniasis is a neglected disease caused by an intracellular protozoan parasite of the genus Leishmania. Infection starts when this protozoan replicates in a phagolysosomal compartment in macrophages, after evading host immune responses. The balance of Th1 and Th2 immune responses is crucial in leishmaniasis because it will determine whether the infection will be under control or if clinical complications will occur. The inflammasome, which is activated during Leishmania infection, involves the action of caspase-1 and release of the proinflammatory cytokines interleukin-1ß and interleukin-18. Together, they contribute to the maintenance of an inflammatory response and pyroptosis. Here, we evaluated the serum levels of cytokines and the expression of circulating microRNAs related to inflammasome regulation in twenty-seven patients with cutaneous leishmaniasis in comparison to nine healthy individuals, in the context of the inflammasome activation. Evaluation of serum cytokines activation (IL-1ß, IL-2, IL-4, IL-6, IL-10, and IL-17) was performed by flow cytometry using CBA kits (cytometric beads array) while the expression of circulating microRNAs (miR-7, miR-133a, miR-146b, miR-155, miR-223, miR-328, and miR-342) in plasma was measured by quantitative polymerase chain reaction. Our results showed an increase of the expression of miR-7-5p (p < 10-5), miR-133a (p = 0.034), miR-146b (p = 0.003), miR-223-3p (p = 10-5), and miR-328-3p (p = 0.002), and cytokine levels for IL-1ß (p = 0.0005), IL-6 (p = 0.001), and IL-17 (p = 0.001) in patients with cutaneous leishmaniasis compared to the controls. These results suggest that microRNAs and cytokines can play an important role in regulating the human immune responses to Leishmania infection. Our findings may contribute to the understanding of the mechanisms of the gene regulation during the cutaneous leishmaniasis and to the identification of possible biomarkers of the infection.

Repeated intravenous injection of adipose tissue derived mesenchymal stem cells enhances Th1 immune responses in Leishmania major-infected BALB/c mice.

Mesenchymal stem cell (MSCs) therapy are among new strategies that are used to combat infections through immunomodulation. Cell number, route and frequency of injection and the duration of exposure to the infectious agent are of the main factors to determine the effectiveness of cell therapy. The current study was aimed to assess the effect of multiple intravenous (i.v.) injection of adipose tissue derived (AD)-MSCs on immune response of Leishmania (L.) major-infected BALB/c mice. Therefore, infected mice received AD-MSCs four times during the early phase of infection through i.v. route. They were then monitored weekly for footpad swelling and lesion development. Parasite burden, nitric oxide (NO) and cytokine production were measured in the spleen and lymph node 90 days post-infection. Delayed lesion development, significant reduction in footpad swelling and lower parasite burden in the spleen of AD-MSCs-treated mice showed the relative effect of AD-MSCs therapy in the control of L. major dissemination. In addition, MSCs were able to manage direct cytokine responses toward T-helper 1 (Th1). Although the level of interleukin (IL)-10 was still higher than the associated level of tumor necrosis factor (TNF)-α, a shift towards higher level of TNF-α was also observed.

Recombinant Cysteine Proteinase B from Leishmania braziliensis and Its Domains: Promising Antigens for Serodiagnosis of Cutaneous and Visceral Leishmaniasis in Dogs.

Leishmaniasis represents a group of parasitic diseases caused by a protozoan of the genus Leishmania and is widely distributed in tropical and subtropical regions. Leishmaniasis is one of the major tropical neglected diseases, with 1.5 to 2 million new cases occurring annually. Diagnosis remains a challenge despite advances in parasitological, serological, and molecular methods. Dogs are an important host for the parasite and develop both visceral and cutaneous lesions. Our goal was to contribute to the diagnosis of canine cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) using the recombinant cysteine proteinase B (F-CPB) from Leishmania braziliensis and its N- and C-terminal domains (N-CPB and C-CPB) as antigens in an enzyme-linked immunosorbent assay (ELISA). Sera from dogs from Northwest Argentina diagnosed with CL were tested by ELISA against a supernatant of L. braziliensis lysate, the F-CPB protein, and its domains. We found values of sensitivity (Se) of 90.7%, 94.4%, and 94.3% and specificity (Sp) of 95.5%, 90.9%, and 91.3% for F-CPB and its N- and C-terminal domains, respectively. In sera from dogs diagnosed with VL from Northeast Argentina, we found Se of 93.3%, 73.3%, and 66.7% and Sp of 92.3%, 76.9%, and 88.5% for F-CPB and its N- and C-terminal domains, respectively. These results support CPB as a relevant antigen for canine leishmaniasis diagnosis in its different clinical presentations. More interestingly, the amino acid sequence of CPB showed high percentages of identity in several Leishmania species, suggesting that the CPB from L. braziliensis qualifies as a good antigen for the diagnosis of leishmaniasis caused by different species.

Immunodetection and molecular determination of visceral and cutaneous Leishmania infection using patients' urine.

The diagnosis of leishmaniasis relies mainly on the use of invasive processes, to collect the biological material for detecting Leishmania parasites. Body fluids, which can be collected by non-invasive process, would greatly facilitate the leishmaniasis diagnosis. In the present study, we investigated the potency of urine immunoblotting to diagnose cutaneous and visceral leishmaniasis and we compared with routine molecular methods. A total of 80 samples, including 40 sera and their 40 corresponding urine samples were collected from 37 suspected patients with cutaneous and visceral leishmaniasis, and 3 healthy individuals (as control), in Ilam and Ardabil provinces of Iran. All sera and urine samples were analyzed, using immunoblotting. The confirmation of leishmaniasis infection was performed, using conventional and quantitative PCRs as well as by sequencing the amplicons. Among 37 suspected patients, 23 patients presented cutaneous lesions (CL) and 14 exhibited clinical symptoms reminiscent of visceral leishmaniasis (L. infantum). Among cutaneous patients, 15 were positive for zoonotic cutaneous leishmaniasis (L. major), and eight for anthroponotic cutaneous leishmaniasis (L. tropica). Molecular quantification of Leishmania parasites was performed on sera, urines and cutaneous biopsies of CL and VL patients, demonstrating that parasite load is lower in urines, compared to sera or biopsy. DNA can be detected in 20 out of 23 (86.9%) CL urine samples and in 13 out of 14 (92.8%) VL urine samples. Immunodetection analysis demonstrates that 22 out of 23 (95.6%) sera from CL patients and all patients suspected with VL are positive. For urine samples, 18 out of 23 (78.2%) urine of CL patients and 13 out of 14 (92.8%) urine of VL patients were positive, using Western blot. Therefore, immunodetection and molecular analysis using urine samples can be used as a diagnostic tool for surveying cutaneous and visceral leishmaniasis.

A secondary wave of neutrophil infiltration causes necrosis and ulceration in lesions of experimental American cutaneous leishmaniasis.

We evaluated the importance of neutrophils in the development of chronic lesions caused by L. Viannia spp. using the hamster as experimental model of American Cutaneous Leishmaniasis (ACL). Neutrophils infiltrated the lesion within the first six hours post-infection. Inhibition of this early infiltration using a polyclonal antibody or cyclophosphamide was associated with transient parasite control but the protective effect vanished when lesions became clinically apparent. At lesion onset (approximately 10 days p.i.), there was an increased proportion of both uninfected and infected macrophages, and subsequently a second wave of neutrophils infiltrated the lesion (after 19 days p.i.) This second neutrophil infiltration was associated with lesion necrosis and ulceration (R2 = 0.75) and maximum parasite burden. Intradermal delivery of N-formylmethionyl-leucyl-phenylalanine (fMLP), aimed to increase neutrophil infiltration, resulted in larger lesions with marked necrosis and higher parasite burden than in mock treated groups (p<0.001 each). In contrast, reduced neutrophil infiltration via cyclophosphamide-mediated depletion led to more benign lesions and lower parasite loads compared to controls (p<0.001 each). Neutrophils of the second wave expressed significantly lower GM-CSF, reactive oxygen species and nitric oxide than those of the first wave, suggesting that they had less efficient anti-leishmania activity. However, there was increased inflammatory cytokines and expression of neutrophil proteases (myeloperoxidase, cathepsin G and elastase) in lesions during the second wave of neutrophil infiltration compared with the levels reached during the first wave (6h p.i.). This suggests that augmented neutrophil proteases and inflammatory cytokines during the secondary wave of neutrophils could contribute to skin inflammation, ulceration and necrosis in ACL. The overall results indicate that neutrophils were unable to clear the infection in this model, and that the second wave of neutrophils played an important role in the severity of ACL.

IN VIVO AND IN VITRO ANTILEISHMANIAL EFFECTS OF METHANOLIC EXTRACT FROM BARK OF BURSERA APTERA.

BACKGROUND: Cutaneous leishmaniasis lacks effective and well-tolerated treatments. The current therapies mainly rely on antimonial drugs that are inadequate because of their poor efficacy. Traditional medicine offers a complementary alternative for the treatment of various diseases. Additionally, several plants have shown success as anti-leishmanial agents. Therefore, we sought to evaluate the in vitro and in vivo activity of MEBA against Leishmania mexicana. MATERIALS AND METHODS: Methanolic extract of B. aptera was obtained by macetration, after we determined in vitro anti-leishmanial activity of MEBA by MTT assay and the induced apoptosis in promastigotes by flow cytometry. To analyze the in vivo anti-leishmanial activity, we used infected mice that were treated and not treated with MEBA and we determined the levels of cytokines using ELISA. The phytochemical properties were determined by CG-MS and DPPH assay. RESULTS: We determined of LC50 of 0.408 mg/mL of MEBA for in vitro anti-leishmanial activity. MEBA induced apoptosis in promastigotes (15.3% ± 0.86). Treated mice exhibited smaller lesions and contained significantly fewer parasites than did untreated mice; in addition, we found that IFN-γ and TNF-α increased in the sera of MEBA-treated mice. GC-MS analysis showed that podophyllotoxin was the most abundant compound. Evaluation of the activity by DPPH assay demonstrated an SC50 of 11.72 µg/mL. CONCLUSION: Based on the above data, it was concluded that MEBA is a good candidate in the search for new anti-leishmanial agents.

Maxadilan-simile expression in Nyssomyia neivai, a sandfly vector in an endemic region of Brazil, and its immunogenicity in patients with American tegumentary leishmaniasis

BACKGROUND Maxadilan (Max) is a salivary component in the sandfly Lutzomyia longipalpis (Lutz & Neiva 1912), a vector of visceral leishmaniasis. Max has a powerful vasodilatory effect and is a candidate vaccine that has been tested in experimental leishmaniasis. Nyssomyia neivai (Pinto 1926) is a vector of the pathogen responsible for American tegumentary leishmaniasis (ATL) in Brazil. OBJECTIVE We searched for Max expression in Ny. neivai and for antibodies against Max in ATL patients. METHODS cDNA and protein were extracted from the cephalic segment, including salivary glands, of Ny. neivai and analysed by polymerase chain reaction, DNA sequencing, and blotting assays. The results were compared with data obtained from Lu. longipalpis samples. We quantified antibodies against Max in serum samples from 41 patients with ATL (31 and 10 with the cutaneous and mucocutaneous forms, respectively) and 63 controls from the endemic northeastern region of São Paulo state, using enzyme-linked immunosorbent assay. FINDINGS Recognition of a Max-simile peptide by specific antibodies confirmed expression of a Max sequence in Ny. neivai (GenBank EF601123.1). Compared to controls, patients with ATL presented higher levels of antibodies against Max (p = 0.004); 24.4% of the patients with ATL and 3.2% of the controls presented anti-Max levels above the cutoff index (p = 0.014). The anti-Max levels were not associated with the specific clinical form of ATL, leishmanin skin test response, absence or presence of amastigotes in histopathologic exam, results of indirect immunofluorescence testing for leishmaniasis, or duration of cutaneous form disease. MAIN CONCLUSION High serum anti-Max levels did not protect patients against ATL, but confirmed previous natural exposure to Ny. neivai bites in this ATL endemic region.

Elevated Serum ADA Activity as a Marker for Diagnosis and Prognosis of Visceral Leishmaniasis and Post Kala-Azar Dermal Leishmaniasis in Indian Patients.

Serum adenosine deaminase (ADA) activity increases in diseases where cellular immunity is involved. Since cell-mediated immune responses play a paramount role in the pathogenesis and healing of the visceral leishmaniasis, therefore, the present study was undertaken to evaluate the serum ADA activity in different pathological conditions. Adenosine deaminase was determined in sera of active visceral leishmaniasis (VL) patients (n = 39), active postkala-azar dermal leishmaniasis (PKDL) cases (n = 34) at the point of diagnosis and after treatment stages along with healthy controls (n = 30), endemic healthy subjects (n = 34) and endemic asymptomatic subjects (n = 34).Our in-vitro result revealed that monocytes secrete significant ADA level in response to Leishmania donovani (L.donovani) stimulation. The serum ADA activity in active VL and PKDL subjects were found to be significantly higher than that of respective treated cases and healthy controls. We also observed a marginal number (17.6%) of endemic asymptomatic subjects showed elevated serum ADA activity. Further, the ADA activity in PKDL was found to be decreased gradually during the different phases of treatment. Interestingly, 2 out of 32 treated VL cases found to have high serum ADA activity during follow up period were relapsed within few days. These results suggest the possibility of ADA as a marker of clinical pathogenesis and can be used as a surrogate marker in the diagnosis and prognosis of VL and PKDL.

Tumour Necrosis Factor-alpha (TNF-α) and its soluble receptor type 1 (sTNFR I) in human active and healed leishmaniases.

The role of tumour necrosis factor-alpha (TNF-α) is not fully understood in human leishmaniasis. We analysed the alterations in the levels of TNF-α, soluble TNF receptor type 1 (sTNFR I), IL-17 and IL-22 productions in active and healed leishmaniases. Blood samples were collected from volunteers with active cutaneous leishmaniasis (ACL), the same subjects after lesion healing (healed CL = HCL), volunteers with active visceral leishmaniasis (AVL), healed VL (HVL) and healthy controls. Levels of cytokines were titrated on Leishmania Ag-stimulated PBMC culture. The mean level of TNF-α production from stimulated cells was significantly higher in ACL than controls (P < 0·001) and significantly reduced after treatment in HCL volunteers (P < 0·05). The mean level of sTNFR I production was significantly higher in ACL than controls (P < 0·001) and significantly reduced after treatment in HCL volunteers (P < 0·05). The mean level of IL-22 production in AVL was significantly higher than controls (P < 0·05) and was significantly lower in HVL compared with AVL (P < 0·001) and controls (P < 0·05). The levels of TNF-α (P = 0·0025) and sTNFR I (P < 0·01) productions from PBMCs showed significant decreasing trend after treatment in each CL volunteer. Reduction in TNF-α is associated with clinical response to treatment and healing of CL lesions due to L. major.

Improving serodiagnosis of human and canine leishmaniasis with recombinant Leishmania braziliensis cathepsin l-like protein and a synthetic peptide containing its linear B-cell epitope.

BACKGROUND: The early and correct diagnosis of human leishmaniasis is essential for disease treatment. Another important step in the control of visceral leishmaniasis is the identification of infected dogs, which are the main domestic reservoir of L. infantum. Recombinant proteins and synthetic peptides based on Leishmania genes have emerged as valuable targets for serodiagnosis due to their increased sensitivity, specificity and potential for standardization. Cathepsin L-like genes are surface antigens that are secreted by amastigotes and have little similarity to host proteins, factors that enable this protein as a good target for serodiagnosis of the leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: We mapped a linear B-cell epitope within the Cathepsin L-like protein from L. braziliensis. A synthetic peptide containing the epitope and the recombinant protein was evaluated for serodiagnosis of human tegumentary and visceral leishmaniasis, as well as canine visceral leishmaniasis. CONCLUSIONS/SIGNIFICANCE: The recombinant protein performed best for human tegumentary and canine visceral leishmaniasis, with 96.30% and 89.33% accuracy, respectively. The synthetic peptide was the best to discriminate human visceral leishmaniasis, with 97.14% specificity, 94.55% sensitivity and 96.00% accuracy. Comparison with T. cruzi-infected humans and dogs suggests that the identified epitope is specific to Leishmania parasites, which minimizes the likelihood of cross-reactions.