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Background: Although canine visceral leishmaniasis (CVL) transmission primarily occurs through the bite of phlebotomine sand flies infected with Leishmania infantum, alternative routes may exist., Methods: Thirty-four dogs diagnosed with CVL were sampled for parasitological investigation in tissues from the reproductive tract., Results: Amastigotes of Leishmania sp. were present in 79% (27/34) of the reproductive system samples, with distinct infection rates depending on the tissue., Conclusions: Our data confirms that alternative routes, such as horizontal and vertical transmissions, should be considered in the epidemiological chain of CVL.

The present study aimed to verify the impact of etiological treatment on the genotype-specific serological diagnosis of chronic Chagas disease patients (CH), using the Chagas-Flow ATE IgG1 methodology. For this purpose, a total of 92 serum samples from CH, categorized as Not Treated (NT, n = 32) and Benznidazole-Treated (Bz-T, n = 60), were tested at Study Baseline and 5Years Follow-up. At Study Baseline, all patients have the diagnosis of Chagas disease confirmed by Chagas-Flow ATE IgG1, using the set of attributes ("antigen/serum dilution/cut-off"; "EVI/250/30%"). The genotype-specific serodiagnosis at Study Baseline demonstrated that 96% of patients (44/46) presented a serological profile compatible with TcII genotype infection. At 5Years Follow-up monitoring, NT and Bz-T presented no changes in anti-EVI IgG1 reactivity. However, significant differences were detected in the genotype-specific IgG1 reactivity for Bz-T. The most outstanding shift comprised the anti-amastigote TcVI/(AVI), anti-amastigote TcII/(AII) and anti-epimastigote TcVI/(EVI) reactivities. Regardless no changes in the genotype-specific serology of NT (TcI = 6%; TcII = 94%), distinct T. cruzi genotype-specific sero-classification was detected for Bz-T samples at 5Years Follow-up (TcII = 100%) as compared to Baseline (TcII = 97%; TcVI = 3%). The anti-trypomastigote TcI/(TI) was the attribute accountable for the change in genotype-specific sero-classification. In conclusion, our findings of dissimilar T. cruzi genotype-specific serology upon Bz-treatment re-emphasize the relevance of accomplishing the genotype-specific serodiagnosis during clinical pos-therapeutic management of chronic Chagas disease patients., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Alessio et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cutaneous leishmaniasis (CL) is a global public health problem caused by species on the genus Leishmania and is the most prevalent clinical form of leishmaniasis. The aim of this study was to develop a new LAMP assay for Leishmania sp. based on HSP70 gene and evaluate it clinically for molecular diagnosis of CL. The study was carried out in the following stages: i) design of primers based on HSP70 gene of Leishmania sp.; ii) evaluation of detection limit and analytical specificity; iii) estimation of the accuracy of LAMP-Leish/HSP70 assay for diagnosing CL. A total of 100 skin biopsy samples from patients, comprising 60 CL cases and 40 non-cases, were analyzed in this study. One LAMP assay using HSP70 gene as molecular target were standardized, and the observed detection limit was 100fg of L. braziliensis purified DNA. The LAMP-Leish/HSP70 assay was specific for Leishmania spp. The LAMP-Leish/HSP70 assay showed an accuracy of 92%, and positivity rates were not affected by lesion onset time or parasite load. This novel LAMP assay targeting the HSP70 gene of Leishmania sp. has the potential to be a useful tool to integrate into routine diagnosis for suspected cases of CL., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Soares et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The current study aims to contribute to the understanding of leishmaniasis diagnosis by providing an overview of patent filings in this field and analyzing whether the methods revealed are consistent with the needs described by the scientific community, in special the main gaps detected by the World Health Organization's 2021-2030 Roadmap for Neglected Tropical Diseases. To this aim, a patent search was carried out focusing on documents disclosing leishmaniasis diagnostic methods supported by experimental evidence and with earliest priority date from 2010 onwards. Our results show that patenting activity is low and patent families are often formed by individual filings. Most R&D activity occurs in Brazil, which is also the main market of protection. Brazilian academic institutions are the main patent drivers, and collaboration between different institutions is rare. Most patent families describe immunological methods based on ELISA assays, using antibodies directed to K39 and homologues. kDNA is the primary gene for molecular testing. Experimental evidence of test performance in fulfilling critical diagnostic gaps is usually absent. The patent scenario suggests that leishmaniasis diagnostic gaps need to be more closely addressed to drive innovation directed to the control and/or elimination of leishmaniasis. From the public policy point of view, the following strategies are suggested: (i) strengthening collaborative networks, (ii) enhancing the participation of the private sector, and (iii) increasing funding, with special focus on the remaining diagnostic gaps., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 de Avelar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In this serum panel-based study, we evaluated the accuracy of serological tests originally developed for visceral leishmaniasis (VL), for diagnosis of mucosal leishmaniasis (ML). A total of five tests were evaluated, four of which are registered at the National Agency of Sanitary Surveillance (Agência Nacional de Vigilância Sanitária-ANVISA) (RIDASCREEN® Leishmania Ab from R-Biopharm AG., Leishmania ELISA IgG + IgM from Vircell S.L., IFI Leishmaniose Humana-BioManguinhos, and IT-LEISH® from Bio-Rad Laboratories, Inc.), and the other a direct agglutination test (DAT-LPC) prototype kit developed at Fiocruz. The panel was composed of 40 serum samples from patients with confirmed ML and 20 from patients with mucosal involvement and negative parasitological/molecular tests for leishmaniasis and confirmation of another etiology. All cases were treated from 2009 to 2016 in a referral center for leishmaniasis in Belo Horizonte, Minas Gerais, Brazil (Instituto René Rachou, Fiocruz). Diagnostic accuracy, based on the cut-off point for VL diagnosis, was 86.2% with RIDASCREEN® Leishmania Ab, 73.3% with Leishmania ELISA IgG + IgM, and 66.7% with IFI Leishmaniose Humana, while IT-LEISH® and DAT-LPC had the lowest accuracy (38.3%), despite high specificity (100% and 95%, respectively). New cut-off points defined with sera from ML patients improved accuracy from 86.2 to 89% (p = 0.64) and 73.3 to 88% (p = 0.04) for RIDASCREEN® Leishmania Ab and Leishmania ELISA IgG + IgM, respectively. Moreover, these tests presented greater sensitivity and immunoreactivity in patients with moderate/severe clinical ML forms. The data of this study suggest that ELISA assays can contribute to laboratory diagnosis, especially for patients with moderate or severe mucosal involvement., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A rapid and accurate diagnosis is a crucial strategy for containing the coronavirus disease (COVID-19) pandemic. Considering the obstacles to upscaling the use of RT-qPCR, rapid tests based on antigen detection (Ag-RDT) have become an alternative to enhance mass testing, reducing the time for a prompt diagnosis and virus spreading. However, the performances of several commercially available Ag-RDTs have not yet been evaluated in several countries. Here, we evaluate the performance of eight Ag-RDTs available in Brazil to diagnose COVID-19. Patients admitted to tertiary hospitals with moderate or mild COVID-19 symptoms and presenting risk factors for severe disease were included. The tests were performed using a masked protocol, strictly following the manufacturer's recommendations and were compared with RT-qPCR. The overall sensitivity of the tests ranged from 9.8 to 81.1%, and specificity greater than 83% was observed for all the evaluated tests. Overall, slight or fair agreement was observed between Ag-RDTs and RT-PCR, except for the Ag-RDT COVID-19 (Acro Biotech), in which moderate agreement was observed. Lower sensitivity of Ag-RDTs was observed for patients with cycle threshold > 25, indicating that the sensitivity was directly affected by viral load, whereas the effect of the disease duration was unclear. Despite the lower sensitivity of Ag-RDTs compared with RT-qPCR, its easy fulfillment and promptness still justify its use, even at hospital admission. However, the main advantage of Ag-RDTs seems to be the possibility of increasing access to the diagnosis of COVID-19 in patients with a high viral load, allowing immediate clinical management and reduction of infectivity and community transmission., Competing Interests: The authors have declared that no competing interests exist.

The aim of this study was to evaluate two methods of nucleic acid extraction (spin-column-based method - commercial kit and direct boil - DB) from swab sampling compared to biopsy sampling for the diagnosis of tegumentary leishmaniasis (TL), (cutaneous - CL and mucocutaneous - MCL forms). The impact of these nucleic acid extraction protocols on different types of PCR and LAMP techniques were compared regarding nucleic acid quality, molecular assays accuracy, indirect quantitation, and costs. The evaluated patients were 57 TL cases (36 CL and 21 MCL) and 34 non-cases. Swab samples extracted by the DB method showed a higher DNA degradation rate and worse DNA quality in comparison to the commercial kit. Molecular tests performed on biopsy samples showed identical or higher performance in all analysis, as compared to their own performance on swab samples for TL (CL and MCL). However, only the SSU rRNA TaqMan™ RT-PCR test showed a significant difference between the performance of biopsy and swab samples extracted by commercial kit. The kDNA-cPCR coupled with swab extracted by commercial kit showed the highest accuracy (95.6%) for TL diagnosis. The sensitivity of the LAMP-RT 18S method in swab samples extracted with a commercial kit (82.5%) was close to that found in biopsy samples (86%) for TL diagnosis. The DB extraction method presented the lowest cost. The use of swab as a minimally-invasive sampling method, associated with an efficient nucleic acid extraction protocol, may represent a low-cost alternative for the diagnosis of CL and MCL., (Copyright © 2021. Published by Elsevier B.V.)

Cutaneous leishmaniasis (CL) remains a globally spreading public health problem. Among Latin America countries, Brazil has the greatest number of recorded CL cases with several Leishmania species being associated with human cases. Laboratory diagnosis is one of the major challenges to disease control due to the low accuracy of parasitological techniques, the restricted use of molecular techniques, and the importance of differential diagnosis with regard to several dermatological and systemic diseases. In response, we have developed and validated an immunohistochemistry (IHC) technique for CL diagnosis using anti-mTXNPx monoclonal antibody (mAb). Recombinant Leishmania -mTXNPx was produced and used as an immunogen for mAb production through the somatic hybridization technique. The viability of mAb labeling of Leishmania amastigotes was tested by IHC performed with skin biopsies from hamsters experimentally infected with Leishmania amazonensis , Leishmania braziliensis , and Leishmania guyanensis . The enzymes horseradish peroxidase (IHC-HRP) and alkaline phosphatase (IHC-AP), both biotin-free polymer detection systems, were used in the standardization step. The IHC was further validated with skin biopsies from 49 CL patients diagnosed by clinical examination and quantitative real-time polymerase chain reaction and from 37 patients presenting other dermatological infectious diseases. Other parasitological techniques, such as direct examination and culture, were also performed for confirmed CL patients. Histopathology and IHC were performed for all included patients. Overall, the highest sensitivity was observed for IHC-AP (85.7%), followed by IHC-HRP (79.6%), direct examination (77.6%), histopathological examination (HE; 65.3%), and in vitro culture (49%). Only IHC and HE presented specificity over 90% and were able to detect CL patients regardless of parasite burden (odds ratio > 1.94; 95%CI: 0.34-11.23). A significant increase in positivity rates was observed when IHC-AP was combined with direct examination (95.9%) and HE (93.9%). The IHC techniques evaluated in here detected the main Leishmania species causing CL in Brazil and can support diagnostic strategies for controlling this neglected disease, especially if used in combination with other approaches for an integrative laboratorial diagnosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Freire, Rego, Lopes, Coutinho, Grenfell, Avelar, Cota, Pascoal-Xavier and Oliveira.)

Sensitive, reliable and fast diagnostic tools that are applicable in low-resource settings, at the point of care (PoC), are seen as crucial in the fight against visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Addressing the need for a PoC test, several diagnostic tests, including serological and molecular methods, have been developed and evaluated in the past. One promising molecular method, already implemented for diagnosis of a range of diseases, is the loop-mediated isothermal amplification (LAMP) protocol. In this systematic review and meta-analysis, using a comprehensive search strategy, we focus on studies evaluating the performance of LAMP for the diagnosis of leishmaniasis in humans and other mammals such as dogs, compared with microscopy and/or any other molecular diagnostic method. A meta-analysis, pooling sensitivity and specificity rates and calculating areas under the curve (AUCs) in summary receiver operating characteristic (SROC) plots, was conducted on datasets extracted from studies, grouped by clinical condition and sample type. We found high sensitivity and specificity for LAMP when compared with microscopy and PCR using blood samples, with pooled estimate values of > 90% for all subgroups, corresponding to calculated AUC values > 0.96, except for LAMP compared to microscopy for diagnosis of CL. However, only a limited number of studies were truly comparable. Most of the observed heterogeneity is likely based on true differences between the studies rather than sampling error only. Due to simple readout methods and low laboratory equipment requirements for sample preparation compared to other molecular methods, LAMP is a promising candidate for a molecular (near-)PoC diagnostic method for VL and CL., (© 2022. The Author(s).)

We performed a detailed analysis of immunophenotypic features of circulating leukocytes and spleen cells from cynomolgus macaques that had been naturally infected with Trypanosoma cruzi , identifying their unique and shared characteristics in relation to cardiac histopathological lesion status. T. cruzi- infected macaques were categorized into three groups: asymptomatic [CCC(-)], with mild chronic chagasic cardiopathy [CCC(+)], or with moderate chronic chagasic cardiopathy [CCC(++)]. Our findings demonstrated significant differences in innate and adaptive immunity cells of the peripheral blood and spleen compartments, by comparison with non-infected controls. CCC(+) and CCC(++) hosts exhibited decreased frequencies of monocytes, NK and NKT-cell subsets in both compartments, and increased frequencies of activated CD8 + T-cells and GranA + /GranB + cells. While a balanced cytokine profile (TNF/IL-10) was observed in peripheral blood of CCC(-) macaques, a predominant pro-inflammatory profile (increased levels of TNF and IFN/IL-10) was observed in both CCC(+) and CCC(++) subgroups. Our data demonstrated that cardiac histopathological features of T. cruzi -infected cynomolgus macaques are associated with perturbations of the immune system similarly to those observed in chagasic humans. These results provide further support for the validity of the cynomolgus macaque model for pre-clinical research on Chagas disease, and provide insights pertaining to the underlying immunological mechanisms involved in the progression of cardiac Chagas disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sathler-Avelar, Vitelli-Avelar, Mattoso-Barbosa, Pascoal-Xavier, Elói-Santos, Costa-Rocha, Teixeira-Carvalho, Dick, VandeBerg, VandeBerg and Martins-Filho.)

qPCR is being used for the quantification of parasite load in different tissues of dogs infected by Leishmania infantum with or without clinical manifestations. It may be employed in the diagnosis, monitoring of the infection during treatment, and clinical studies for validation of vaccines. Aimed at enhancing the molecular diagnosis and the subsequent monitoring of the infection, this study evaluated the parasite load in several tissues from dogs infected by Leishmania infantum, showing different clinical status. Thus, the qPCR was performed on skin, conjunctival swab, popliteal lymph node, and bone marrow puncture samples taken from 65 dogs naturally infected by L. infantum. Dogs were divided into three groups per clinical score: group 1 (n = 12), included animals with zero points and no clinical manifestations of the disease; group 2 (n = 35), included animals with a score ranging from 1 to 5 points and moderate clinical manifestations; and group 3 (n = 18), included dogs with a score ranging from 6 to 11 points and intensive clinical manifestations. Another analysis was performed classifying the animals into two groups, considering the presence of, or lack of clinical signs of the disease. Analyses of these results showed that the skin was the tissue with a higher parasite load, followed by popliteal lymph node and bone marrow punctures, and conjunctival swab samples having the lowest loads. Furthermore, the skin was also the tissue with the highest parasite load when evaluating the groups individually. Animals in group 3, with intensive clinical manifestations, showed a higher parasite load in different tissues when compared to animals from groups 1 and 2. Finally, animals with clinical manifestations of the disease showed a higher parasite load when compared to dogs with no manifestations. The importance of the dog as a reservoir of L. infantum in nature is reinforced by the demonstration of skin having the highest amount of parasites/μL in this study's analysis, as well as the fact that skin is the main point of access to the parasite vector. Also, a strong and positive correlation between the intensity of clinical manifestations and the increase of parasite load in the skin was observed. In conclusion, skin was the tissue that was demonstrated to be the best option for the molecular diagnosis of L. infantum infection in dogs with varying clinical statuses used in this study., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Timely and accurate laboratory testing is essential for managing the global COVID-19 pandemic. Reverse transcription polymerase chain reaction remains the gold-standard for SARS-CoV-2 diagnosis, but several practical issues limit the test's use. Immunoassays have been indicated as an alternative for individual and mass testing., Objectives: To access the performance of 12 serological tests for COVID-19 diagnosis., Methods: We conducted a blind evaluation of six lateral-flow immunoassays (LFIAs) and six enzyme-linked immunosorbent assays (ELISAs) commercially available in Brazil for detecting anti-SARS-CoV-2 antibodies., Results: Considering patients with seven or more days of symptoms, the sensitivity ranged from 59.5% to 83.1% for LFIAs and from 50.7% to 92.6% for ELISAs. For both methods, the sensitivity increased with clinical severity and days of symptoms. The agreement among LFIAs performed with digital blood and serum was moderate. Specificity was, in general, higher for LFIAs than for ELISAs. Infectious diseases prevalent in the tropics, such as HIV, leishmaniasis, arboviruses, and malaria, represent conditions with the potential to cause false-positive results with these tests, which significantly compromises their specificity., Conclusion: The performance of immunoassays was only moderate, affected by the duration and clinical severity of the disease. Absence of discriminatory power between IgM/IgA and IgG has also been demonstrated, which prevents the use of acute-phase antibodies for decisions on social isolation., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Introduction: Canine visceral leishmaniasis (CVL) is an endemic disease in Brazil, and integrated control actions have been adopted by the Brazilian Ministry of Health to control its spread. However, the transmission profile is unknown in areas with recent CVL cases, including Itaúna, located in the Brazilian state of Minas Gerais, where the present study was carried out., Methods: A total of 2,302 dogs from 12 neighborhoods were serologically tested for canine VL using the current diagnostic protocol adopted by the Brazilian Ministry of Health. Test positivity rate (TPR) and CVL prevalence were determined for each neighborhood. The presence of Leishmania was assessed in 60 seropositive dogs which had been recommended for euthanasia. Twenty-two of them (37%) were asymptomatic, and 38 (63%) were symptomatic for CVL. Parasitological (myeloculture and smear/imprint) and molecular (PCR) methods were employed for Leishmania detection in bone marrow, spleen, mesenteric lymph nodes, and ear skin. The infecting Leishmania species was identified by DNA sequencing., Results: CVL prevalence (per 1,000 dogs) varied from 0.0-166.67, depending on the neighborhood, with a mean of 68.96 (SD 51.38). Leishmania DNA was detected in at least one tissue from all seropositive dogs, with comparable TPR among tissues. Leishmania parasites were identified in most (54/60) seropositive dogs, and the infecting parasite was identified as Leishmania infantum in all of these., Conclusions: Prevalence of CVL is a contributor to the spread of visceral leishmaniasis in Itaúna.

Visceral leishmaniasis (VL) is considered a major public health concern in Brazil and several regions of the world. A recent advance in the diagnosis of infectious diseases was the development of loop-mediated isothermal amplification (LAMP). The aim of this study was to develop and evaluate a new LAMP assay for detection of K26 antigen-coding gene of L. donovani complex. A total of 219 blood samples of immunocompetent patients, including 114 VL cases and 105 non-VL cases, were analyzed for the diagnosis of VL in the present study. Diagnostic accuracy was calculated against a combination of parasitological and/or serological tests as a reference standard. The results were compared to those of kDNA Leishmania- PCR. The detection limit for the K26-Lamp assay was 1fg L. infantum purified DNA and 100 parasites/mL within 60 min of amplification time with visual detection for turbidity. The assay was specific for L. donovani complex. Sensitivity, specificity, and accuracy were 98.2%, 98.1%, and 98.2%, respectively, for K26-LAMP and 100%, 100%, and 100%, respectively, for kDNA Leishmania -PCR. Excellent agreement was observed between K26-LAMP and kDNA Leishmania- PCR assays (K = 0.96). A highly sensitive and specific LAMP assay targeting K26 antigen-coding gene of L. donovani complex was developed for diagnosis in peripheral blood samples of VL patients., Competing Interests: The authors declare no conflicts of interest.

Background: Visceral Leishmaniasis (VL) is an infectious disease that is a significant cause of death among infants aged under 1 year and the elderly in Brazil. Serodiagnosis is a mainstay of VL elimination programs; however, it has significant limitations due to low accuracy., Objective: This study aimed to evaluate three recombinant Leishmania infantum proteins (rFc, rC9, and rA2) selected from previous proteomics and genomics analyses to develop enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests (ICT) for the serodiagnosis of human VL (HVL) and canine VL (CVL)., Methods: A total of 186 human (70 L. infantum-infected symptomatic, 20 other disease-infected, and 96 healthy) and 185 canine (82 L. infantum-infected symptomatic, 27 L. infantum-infected asymptomatic, and 76 healthy) sera samples were used for antibody detection., Findings: Of the three proteins, rA2 (91.5% sensitivity and 87% specificity) and rC9 (95.7% sensitivity and 87.5% specificity) displayed the best performance in ELISA-HVL and ELISA-CVL, respectively. ICT-rA2 also displayed the best performance for HVL diagnosis (92.3% sensitivity and 88.0% specificity) and had high concordance with immunofluorescence antibody tests (IFAT), ELISA-rK39, IT-LEISH®, and ELISAEXT. ICT-rFc, ICT-rC9, and ICT-rA2 had sensitivities of 88.6%, 86.5%, and 87.0%, respectively, with specificity values of 84.0%, 92.0%, and 100%, respectively for CVL diagnosis., Main Conclusions: The three antigens selected by us are promising candidates for VL diagnosis regardless of the test format, although the antigen combinations and test parameters may warrant further optimisation.

The systems biology approach has become an innovative tool when it comes to shedding light on the complex immune response underlying the development/maintenance of distinct clinical forms of Chagas disease. The goal of this study was to describe an integrative overview of Fc-γR expression, cytokine microenvironment and anti- Trypanosoma cruzi IgG interface in indeterminate-(IND) and cardiac-(CARD) patients. Data demonstrated that IND displayed an overall higher Fcγ-R expression (CD16; CD32; CD64) on neutrophils-(NEU), along with (CD16; CD64) on monocytes-(MON) as compared to CARD. Additionally, CARD presented an increased expression of CD32 in B-cells. While preserved frequency of IL-10-producing cells was observed in IND, decreased levels of IL-10 + phagocytes and enhanced TNF + MON and NK-cells were observed in CARD. T. cruzi -antigen recall in vitro induces a general decrease of Fc-γR expression in Chagas disease patients, especially in CARD. Moreover, T. cruzi -antigen stimuli triggered a concomitant increase of IFN-γ + NEU/TNF + NK-cells and IL-10 + MON/IL-10 + B-cells in IND. Biomarker signatures further emphasized the contrasting Fc-γR expression and cytokine microenvironment observed in Chagas disease patients with distinct clinical forms. Up-regulation of Fc-γR expression (CD16 on NEU;MON;NK) was observed in IND, whereas a general decrease was reported for CARD. Moreover, while a mixed cytokine microenvironment (TNF; IL-10) was observed in IND, CARD presented a contrasting profile with up-regulation of TNF + NEU and IL-12 + NEU. Integrative network analysis revealed a distinct assemblage of biomarkers, with CARD presenting a large number of negative internode connectivity in comparison with IND. The relevant gaps in Fc-γR expression and impaired regulatory cytokine microenvironment interfaced with the anti- T. cruzi IgG reactivity throughout an exacerbated negative connectivity may account for the development/maintenance of the clinical status of cardiac Chagas disease.

Background: Cutaneous leishmaniasis (CL) is a world-wide health problem which currently lacks effective, affordable and easy to use therapy. Recently, the meglumine antimoniate (MA) intralesional infiltration was included among the acceptable therapies for New World leishmaniasis. While this approach is attractive, there is currently little evidence to support its use in Americas., Objectives: The aim of this study was to provide information about effectiveness and safety of a standardised MA intralesional infiltration technique for the treatment of CL., Methods: It is a single-arm phase II clinical trial conducted at a Brazilian referral centre. CL cases with parasitological confirmation presenting a maximum of three CL-compatible skin lesions were treated with weekly MA intralesional infiltration by using a validated technique, up to a maximum of eight infiltrations., Results: A total of 53 patients (62 lesions) were included. Overall, patients received a median of seven infiltrations (IQR25-75% 5-8) over a median treatment period of 43 days (IQR25-75% 28-52 days). The definitive cure rate at D180 was 87% (95% CI:77-96%). The majority of adverse events were local, with mild or moderate intensity. Bacterial secondary infection of the lesion site was observed in 13% of the treated patients, beside two intensity-three adverse events (hypersensitivity reactions).

Immunochromatographic tests based on the recombinant antigen K39 represent a major advance in diagnosing visceral leishmaniasis (VL) in recent years. Some performance variations are expected and have occurred in the use of several commercial rapid tests, especially in different geographical settings. This is the first evaluation in the Americas of the test recently provided by the public health system in Brazil for the diagnostic of VL, the OnSite™ Leishmania IgG/IgM Combo. In this first clinical test evaluation, 113 VL-positive patient samples and 73 negative controls were tested and a sensitivity of 91.2% and specificity of 94.5% were observed. These results indicate the need for further analysis and comparisons with the performance of other available commercial tests in order to define the impact of this new test on the quality of VL diagnosis in Brazil.

Visceral leishmaniasis (VL) can cause large-scale and tenacious epidemics with high fatality rates. Current seroprevalence and circulating Leishmania species were evaluated in dogs domiciled in the municipality of Sabará, a small historic and touristic city in the Brazilian state of Minas Gerais. A total of 3926 dogs domiciled in seven different districts of Sabará were serologically tested for canine visceral leishmaniasis (CVL) by indirect enzyme-linked immunosorbent (ELISA) and immunofluorescence (IFA) assays, in a two-years census survey (2011-2012). The average positivity rate of canine infection was 3.4%. Three additional diagnostic tests - imprint/smear direct parasitological, molecular (LnPCR) and myeloculture - were performed in a random sample of fifty seropositive dogs composed of symptomatic (39) and asymptomatic (eleven) animals. LnPCR showed 100% of positivity for Leishmania DNA in, at least, one among four tissue samples tested (mesenteric lymph node, skin, spleen and bone marrow), independently of the clinical canine group. Higher and statistically equivalent positivity rates (98% and 96%) for Leishmania DNA were found in canine lymph node and spleen. Asymptomatic dogs showed expressive positivity rates in all three additional diagnostic techniques. Leishmania infantum was confirmed as the etiological agent of CVL in Sabará.

Background: Non-human primates have been shown to be useful models for Chagas disease. We previously reported that natural T. cruzi infection of cynomolgus macaques triggers clinical features and immunophenotypic changes of peripheral blood leukocytes resembling those observed in human Chagas disease. In the present study, we further characterize the cytokine-mediated microenvironment to provide supportive evidence of the utility of cynomolgus macaques as a model for drug development for human Chagas disease., Methods and Findings: In this cross-sectional study design, flow cytometry and systems biology approaches were used to characterize the ex vivo and in vitro T. cruzi-specific functional cytokine signature of circulating leukocytes from TcI-T. cruzi naturally infected cynomolgus macaques (CH). Results showed that CH presented an overall CD4+-derived IFN-γ pattern regulated by IL-10-derived from CD4+ T-cells and B-cells, contrasting with the baseline profile observed in non-infected hosts (NI). Homologous TcI-T. cruzi-antigen recall in vitro induced a broad pro-inflammatory cytokine response in CH, mediated by TNF from innate/adaptive cells, counterbalanced by monocyte/B-cell-derived IL-10. TcIV-antigen triggered a more selective cytokine signature mediated by NK and T-cell-derived IFN-γ with modest regulation by IL-10 from T-cells. While NI presented a cytokine network comprised of small number of neighborhood connections, CH displayed a complex cross-talk amongst network elements. Noteworthy, was the ability of TcI-antigen to drive a complex global pro-inflammatory network mediated by TNF and IFN-γ from NK-cells, CD4+ and CD8+ T-cells, regulated by IL-10+CD8+ T-cells, in contrast to the TcIV-antigens that trigger a modest network, with moderate connecting edges., Conclusions: Altogether, our findings demonstrated that CH present a pro-inflammatory/regulatory cytokine signature similar to that observed in human Chagas disease. These data bring additional insights that further validate these non-human primates as experimental models for Chagas disease.

Background: Immunosenescence is associated with several changes in adaptive and innate immune cells. Altered cytokine production is among the most prominent of these changes. The impact of age-related alterations on cytokine global profiles produced by distinct populations of leukocytes from healthy Brazilian individuals was studied. We analysed frequencies of cytokine-producing lymphocytes and innate immune cells from individuals at several ages spanning a lifetime period (0-85 years)., Results: Healthy adult individuals presented a balanced profile suggestive of a mature immune system with equal contributions of both innate and adaptive immunity and of both categories of cytokines (inflammatory and regulatory). In healthy newborns and elderly, innate immune cells, especially neutrophils and NK-cells, contributed the most to a balanced profile of cytokines., Conclusions: Our results support the hypothesis that ageing is not associated with a progressive pro-inflammatory cytokine production by all leukocytes but rather with distinct fluctuations in the frequency of cytokine-producing cells throughout life.

Background: Cynomolgus macaques (Macaca fascicularis) represent a feasible model for research on Chagas disease since natural T. cruzi infection in these primates leads to clinical outcomes similar to those observed in humans. However, it is still unknown whether these clinical similarities are accompanied by equivalent immunological characteristics in the two species. We have performed a detailed immunophenotypic analysis of circulating leukocytes together with systems biology approaches from 15 cynomolgus macaques naturally infected with T. cruzi (CH) presenting the chronic phase of Chagas disease to identify biomarkers that might be useful for clinical investigations., Methods and Findings: Our data established that CH displayed increased expression of CD32+ and CD56+ in monocytes and enhanced frequency of NK Granzyme A+ cells as compared to non-infected controls (NI). Moreover, higher expression of CD54 and HLA-DR by T-cells, especially within the CD8+ subset, was the hallmark of CH. A high level of expression of Granzyme A and Perforin underscored the enhanced cytotoxicity-linked pattern of CD8+ T-lymphocytes from CH. Increased frequency of B-cells with up-regulated expression of Fc-γRII was also observed in CH. Complex and imbricate biomarker networks demonstrated that CH showed a shift towards cross-talk among cells of the adaptive immune system. Systems biology analysis further established monocytes and NK-cell phenotypes and the T-cell activation status, along with the Granzyme A expression by CD8+ T-cells, as the most reliable biomarkers of potential use for clinical applications., Conclusions: Altogether, these findings demonstrated that the similarities in phenotypic features of circulating leukocytes observed in cynomolgus macaques and humans infected with T. cruzi further supports the use of these monkeys in preclinical toxicology and pharmacology studies applied to development and testing of new drugs for Chagas disease.

The cat flea, Ctenocephalides felis felis (Bouché, 1835), is an important ectoparasite of dogs and cats throughout the world, causing annoyance to the animals and acting as a vector of infections and a cause of allergic dermatitis in dogs and cats. Although climatic variability and seasonality are known to influence the diversity and abundance of fleas, few investigations of seasonal prevalence of cat flea infestation have involved the same group of dogs being examined regularly over an extended period. The aim of this study was to determine the effects of temperature, rainfall, and relative humidity on the infestation by C. felis felis on 88 outdoor dogs in southeastern Brazil. The dogs, which were of mixed breed, sex, and age, were examined for ectoparasites every month during the period August 2011 to July 2012, and samples of fleas were randomly collected and identified. Meteorological data, comprising mean temperature, total rainfall, and mean relative humidity, were recorded for the calendar month prior to that in which the examinations were performed. Dogs were found to be infested only with C. felis felis, with a higher prevalence in the months with lowest rainfall (July, August, and September). The data obtained in this investigation can be used in control programs in order to establish an efficient strategy for environmental management and the application of insecticides, particularly during the driest months of the year based on the seasonal pattern of infestation of dogs by C. felis felis., (© The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

About 97% of the human cases of the American visceral leishmaniasis (VL) occur in Brazil. In the last few years, the disease expanded to medium- and large-sized cities, in which surveillance and control actions have been intensified, in an effort to control VL spreading. Our two-year study was conducted in Belo Horizonte, the sixth most populous city in Brazil, which is endemic for VL. We focused in two particular districts of recent transmission of the disease, with no reported human cases and submitted to minor surveillance and control actions. Our aim was to draw an epidemiological profile of the local situation concerning Lutzomyia vector, Leishmania parasites, and the main domestic reservoirs (dogs). Lutzomyia longipalpis comprised 96.5% of the total phlebotomine sand flies captured and displayed an expressive minimal infection rate by Leishmania infantum (16.7%). Positive correlations were found between the population densities of L. longipalpis, rainfall and temperature. L. infantum was also detected in the cortelezzii complex and, for the first time, in Lutzomyia lloydi. Leishmania braziliensis, an etiological agent of the American cutaneous leishmaniasis, was also identified in L. longipalpis. Among the 1408 dogs serologically tested by standard enzyme-linked and fluorescence immune assays (ELISA/IFA) 3.6% were positive for VL. L. infantum DNA and Leishmania parasites were identified in 100% and 72.5% of the seropositive dogs, respectively. The co-positivity of other diagnostic tests for VL-Leishmania-nested PCR, imprint and myeloculture-was compared to the standard serology. Both symptomatic or asymptomatic dogs displayed an equal average number of positive diagnostic tests for VL. The districts studied display favorable conditions for the rapid spreading of human infection, in terms of L. longipalpis population density, and presence of L. infantum in both vector and main reservoir., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A new species of Tunga (Siphonaptera: Tungidae) collected from armadillos in Argentina is described. The new species is characterized by large and pigmented eyes, the presence of two bristles on antennal segment II, two bristles at the base of the maxilla, and a discoid neosome compressed anteroposteriorly. The gravid female is located in the carapace of the host, perforating the osteoderms. The new species resembles Tunga penetrans and Tunga terasma in general appearance. However, it differs by the greater anteroposterior compression of the neosome, a more angular head, and a manubrium with a pointed proximal end and convex ventral margin (the proximal end of the manubrium is rounded or slightly pointed in T. terasma, and the ventral margin is straight in both T. penetrans and T. terasma). In addition, specimens of T. penetrans have more bristles in antennal segments II and III, and lack bristles in the posterior tibia. This is the first report of a species of Tunga perforating the osteoderms of its host and thereby showing a high degree of specialization. Tunga terasma is recorded for the first time in Argentina; the male is described again and the characteristics of the species amended. This information may be useful in epidemiological studies of diseases caused by species of Tunga., (© 2015 The Royal Entomological Society.)

In the present study, we characterized the phagocytic capacity, cytokine profile along with the FCγ-R and TLR expression in leukocytes from Chagas disease patients (indeterminate-IND and cardiac-CARD) before and one-year after Bz-treatment (INDT and CARDT). A down-regulation of IL-17, IFN-γ and IL-10 synthesis by neutrophils was observed in CARDT. The Bz-treatment did not impact on the expression of phagocytosis-related surface molecules or monocyte-derived cytokine profile in INDT. Although CARDT showed unaltered monocyte-phagocytic capacity, up-regulated expression of Fcγ-RI/III and TLR-4 may be related to their ability to produce IL-10 and TGF-β. Down-regulation of lymphocyte-derived cytokine was observed in INDT whereas up-regulated cytokine profile was observed for lymphocytes in CARDT. Analysis of cytokine network revealed that IND displayed a multifaceted cytokine response characterized by strong connecting axes involving pro-inflammatory/regulatory phagocytes and lymphocytes. On the other hand, CARD presented a modest cytokine network. The Bz-treatment leads to distinct cytokine network: decreasing the links in INDT, with a pivotal role of IL-10(+) monocytes and expanding the connections in CARDT. Our findings highlighted that the Bz-treatment contributes to an overall immunomodulation in INDT and induces a broad change of immunological response in CARDT, eliciting an intricate phenotypic/functional network compatible with beneficial and protective immunological events., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Differential serological diagnosis of Chagas disease and leishmaniasis is difficult owing to cross-reactivity resulting from the fact that the parasites that cause these pathologies share antigenic epitopes. Even with optimized serological assays that use parasite-specific recombinant antigens, inconclusive test results continue to be a problem. Therefore, new serological tests with high sensitivity and specificity are needed. In the present work, we developed and evaluated the performance of a new flow cytometric serological method, referred to as FC-TRIPLEX Chagas/Leish IgG1, for the all-in-one classification of inconclusive tests. The method uses antigens for the detection of visceral leishmaniasis, localized cutaneous leishmaniasis, and Chagas disease and is based on an inverted detuned algorithm for analysis of anti-Trypanosomatidae IgG1 reactivity. First, parasites were label with fluorescein isothiocyanate or Alexa Fluor 647 at various concentrations. Then serum samples were serially diluted, the dilutions were incubated with suspensions of mixed labeled parasites, and flow cytometric measurements were performed to determine percentages of positive fluorescent parasites. Using the new method, we obtained correct results for 76 of 80 analyzed serum samples (95% overall performance), underscoring the outstanding performance of the method. Moreover, we found that the fluorescently labeled parasite suspensions were stable during storage at room temperature, 4 °C, and -20 °C for 1 year. In addition, two different lots of parasite suspensions showed equivalent antigen recognition; that is, the two lots showed equivalent categorical segregation of anti-Trypanosomatidae IgG1 reactivity at selected serum dilutions. In conclusion, we have developed a sensitive and selective method for differential diagnosis of Chagas disease, visceral leishmaniasis, and localized cutaneous leishmaniasis.

Introduction: The present study was designed to assess the occurrence of co-infection or cross-reaction in the serological techniques used for detecting the anti-Leishmania spp., -Babesia canis vogeli and -Ehrlichia canis antibodies in urban dogs from an area endemic to these parasites., Methods: The serum samples from dogs were tested for the Babesia canis vogeli strain Belo Horizonte antigen and Ehrlichia canis strain São Paulo by immunofluorescence antibody test (IFAT) and by anti-Leishmania immunoglobulin G (IgG) antibody detection to assess Leishmania infection. We used the following four commercial kits for canine visceral leishmaniasis: ELISA, IFAT, Dual Path Platform (DPP) (Bio Manguinhos(r)/FIOCRUZ/MS) and a rK39 RDT (Kalazar Detect Canine Rapid Test; Inbios)., Results: Of 96 serum samples submitted to serological assays, 4 (4.2%) were positive for Leishmania as determined by ELISA; 12 (12.5%), by IFAT; 14 (14.6%) by rK39 RDT; and 20 (20.8%), by DPP. Antibodies against Ehrlichia and Babesia were detected in 23/96 (23.9%) and 30/96 (31.2%) samples, respectively. No significant association was identified between the results of tests for detecting Babesia or Ehrlichia and those for detecting Leishmania (p-value>0.05)., Conclusions: In the present study, we demonstrated co-infection with Ehrlichia or Babesia and Leishmania in dogs from Minas Gerais (Brazil); we also found that the serological tests that were used did not cross-react.

Tunga is the most specialized genus among the Siphonaptera because adult females penetrate into the skin of their hosts and, after mating and fertilization, undergo hypertrophy, forming an enlarged structure known as the neosome. In humans and other warm-blooded animals, neosomes cause tungiasis, which arises due to the action of opportunistic agents. Although its effects on humans and domestic animals are well described in the literature, little is known about the impact of tungiasis on wild animals. This review focuses on the morphology, taxonomy, geographical distribution, hosts, prevalence, sites of attachment, and impact of tungid neosomes on wild and domestic animals. Because neosomes are the most characteristic form of the genus Tunga and also the form most frequently found in hosts, they are here differentiated and illustrated to aid in the identification of the 13 currently known species. Perspectives for future studies regarding the possibility of discovering other sand flea species, adaptation to new hosts, and the transfer of tungids between hosts in natural and modified habitats are also presented.

Background: Preeclampsia (PE) is associated with platelet activation, which may be involved in its pathogenesis promoting coagulation and mediating inflammation. We investigated whether the platelet activation status together with the frequency of platelet-leukocyte aggregates/PLA and monocyte tissue factor/TF expression could be used as laboratorial biomarkers for PE diagnosis and prognosis., Methods: Ninety-seven women were evaluated including severe PE/sPE (N=15), mild PE/mPE (N=20), normotensive pregnant/NP (N=31) and non-pregnant women/nonP (N=31). Platelet markers were analyzed by flow cytometry., Results: Platelet counts and CD41a expression by platelets were lower in NP and sPE vs nonP. The expression of CD61 was lower during pregnancy. Altered balance of platelet marker expression was also observed in NP and sPE vs nonP. No significant differences in the PLA and TF expression by monocytes were observed among the groups. There are several correlations between platelet activation markers, especially in sPE, which suggest a relevant role of the hemostatic/immunological cross-talk in this disease., Conclusions: PE is not associated with increased platelet activation markers. It cannot rule out a role of platelet activation in the PE pathophysiology. Despite those correlations, we did not find a putative laboratorial biomarker that could be useful by itself for PE diagnosis and prognosis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This review focuses on the neosomes, morphology, and taxonomy of adult species of the genus Tunga, complementing the previously published data on the phylogeny, ecology, and pathogenic role. Neosomes are structures formed after penetration of adult females into the skin of hosts resulting in significant enlargement, being the most characteristic and most frequently observed form in hosts. Neosomes can be differentiated by shape, measurements, and sites of attachment to principal hosts. The taxonomic value and morphometric data of the most widely used characteristics to separate species - such as frontal curvature, head chaetotaxy, preoral internal sclerotization, ventral and dorsal genal lobes, eyes, maxillary palps, fusion of pronotum and mesonotum, metacoxae, metatarsi chaetotaxy, spermatheca (females), manubrium, basimere, telomere, and phallosome (males) - are comparatively analyzed. The sexes, individual variations, undescribed species, higher taxa, as well as a proposal for division of the genus into two subgenera (Tunga and Brevidigita) are presented (as previously given by Wang). A key for females, males, and gravid females (neosomes) also is included for identifying the 13 known species. Data on host specificity and geographical distribution may also support the identification of Tunga species because some sand fleas and their hosts may have co-evolved., (© P.M. Linardi et al., published by EDP Sciences, 2014.)

It has been proposed that the transmission of canine visceral leishmaniasis might involve the participation of mechanical vectors, including ticks of the family Ixodidae, in particular the brown dog tick Rhipicephalus sanguineus, and the cat flea Ctenocephalides felis felis. Here, the association between the infestation by R. sanguineus and C. felis felis and the occurrence of anti-Leishmania antibodies was evaluated in an observational case-control study of dogs living in a Brazilian endemic area for canine visceral leishmaniasis. Blood samples were taken once every three months for one year from 96 initially seronegative domestic dogs, and submitted to indirect immunofluorescence antibody assay. All dogs were evaluated for the presence of ticks and fleas, and the results were expressed qualitatively as infested or non-infested, irrespective of the intensity of infestation. At the end of follow-up, twenty dogs had turned seropositive, while 68 remained seronegative and 8 were excluded because of incomplete data. All the dogs were asymptomatic. The odds of infection was significantly greater (OR=3.54, CI95%=1.10-12.53) for dogs infested by C. felis felis compared to their non-infested counterparts. In contrast, the odds of infection showed no significance difference between non-infested and R. sanguineus-infested groups of dogs (OR=0.31, CI95%=0.03-1.52). This study provides further evidence for the potential role of C. felis felis in mechanically transmitting Leishmania among the canine population., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Tunga trimamillata is a species of sand flea occurring in Ecuador and Peru parasitizing cattle, goat, sheep, swine, and man. This is the first report of this species in Brazil, having been found on the hooves of cows in Barretos, São Paulo State, and Felixlândia, Minas Gerais State, and previously misidentified as Tunga penetrans. A previous report concerning Hydrochoerus hydrochaeris from Rio Novo, Minas Gerais State, may also be attributed to that species of sand flea, a possible the primary host. Given the large geographical distribution of T. trimamillata, the vast cattle population in Brazil, and the high number of individuals subject to the risk of tungiasis, the number of cases attributed to this sand flea will most likely increase over time.

Tunga hexalobulata (Siphonaptera: Tungidae), new species oftungid sand flea belonging to the penetrans group, is described with illustrations of adult female parasitizing Bos indicus (L., 1758) from Brazil. It differs from the 12 other known species of Tunginae by the presence of six anterior humps in the neosome. It also can be differentiated from other species of the penetrans group by lesser size of the neosome, presence of three posterodorsally bristles in antennal segment II, and the extension of the posterior arm of the preoral internal sclerotization.

Background: Benznidazole (Bz)-chemotherapy is recommended to prevent Chagas disease progression, despite its limited efficacy during chronic disease. However, the host mechanisms underlying these benefits still remain to be elucidated., Methods: In this study, we have used short-term whole blood cultures to describe the cytokine profile of Bz-treated Indeterminate Chagas disease patients-(INDt) as compared to untreated patients-(IND)., Results: Our findings showed that IND presented increased levels of IL-10+neutrophils, IL-12⁺ and IL-10⁺ monocytes and IFN-γ⁺NK-cells. Moreover, IND showed slight increase of IL-4⁺CD4⁺T-cells and enhanced levels of IL-10⁺CD8⁺T-cells and B-cells. Additional analysis of cytokine Low and High producers also highlighted the presence of High cytokine producers within IND, including IL-10 from CD4⁺ T-cells and IFN-γ from CD8⁺ T-cells, as compared to NI. The Bz-treatment lead to an overall cytokine down-regulation in the innate and adaptive compartments, including low levels of IL-12⁺ and IL-10⁺ neutrophils and monocytes, IFN-γ⁺NK-cells, IL-12⁺, TNF-α⁺, IFN-γ⁺ and IL-5⁺CD4⁺T-cells and IL-10⁺B-cells, along with basal levels of cytokine-expressing CD8⁺T-cells in INDt as compared to IND. The in vitro antigen stimulation shifted the cytokine profile toward a type 1-modulated profile, with increased levels of IL-12⁺ and IL-10⁺ monocytes, IFN-γ⁺ and IL-4⁺NK-cells along with TNF-α⁺ and IFN-γ⁺CD8⁺T-cells. Analysis of Low and High cytokine producers, upon in vitro antigen stimulation, further confirm these data., Conclusion: Together, our findings showed that the Bz treatment of Indeterminate Chagas' disease patients shifts the cytokine patterns of peripheral blood monocytes, NK-cells and CD8⁺ T-cells towards a long-lasting Type-1-modulated profile that could be important to the maintenance of a non-deleterious immunological microenvironment.

T-cells play an important role controlling immunity against pathogens and therefore influence the outcome of human diseases. Although most T-lymphocytes co-express either CD4 or CD8, a smaller T-cell subset found the in the human peripheral blood that expresses the αβ or γδ T-cell-receptor (TCR) lacks the CD4 and CD8 co-receptors. These double negative (DN) T-cells have been shown to display important immunological functions in human diseases. To better understand the role of DN T-cells in human Mycobacterium tuberculosis, we have characterized their frequency, activation and cytokine profile in a well-defined group of tuberculosis patients, categorized as severe and non-severe based on their clinical status. Our data showed that whereas high frequency of αβ DN T-cells observed in M. tuberculosis-infected patients are associated with disease severity, decreased proportion of γδ DN T-cells are found in patients with severe tuberculosis. Together with activation of CD4(+) and CD8(+) T-cells, higher frequencies of both αβ and γδ DN T-cells from tuberculosis patients also express the chronic activation marker HLA-DR. However, the expression of CD69, an early activation marker, is selectively observed in DN T-cells. Interestingly, while αβ and γδ DN T-cells from patients with non-severe tuberculosis display a pro-inflammatory cytokine profile, characterized by enhanced IFN-γ, the γδ DN T-cells from patients with severe disease express a modulatory profile exemplified by enhanced interleukin-10 production. Overall, our findings suggest that αβ and γδ DN T-cell present disparate immunoregulatory potentials and seems to contribute to the development/maintenance of distinct clinical aspects of TB, as part of the complex immunological network triggered by the TB infection.

Tunga bossii new species of tungid sand flea belonging to the caecata group is described with illustrations of the adult female, parasitizing the wild rodent Delomys dorsalis (Hensel) from Brazilian Atlantic Forest. Tunga bossii differs from the ten other known species of Tunginae by the size of the first segment of the maxillary palp and the presence of two bristles at the base of the maxilla. Tunga bossii also can be differentiated from other species of the T. caecata group by the eye morphology. A key to the adult species and neosomes of the genus Tunga also is included.

To confirm the taxonomic identification of a trypanosomatid found in the hindgut, rectum and Malpighian tubules of dog fleas captured in Belo Horizonte, Minas Gerais, Brazil, between April and November of 2005, 910 specimens of Ctenocephalides felis felis were removed from street dogs and dissected, and isolates from their digestive tracts were cultivated in NNN-alpha-MEM medium. Four different morphological forms were observed in culture: long, slender, twisted promastigotes with a long flagellum; short, stubby, non-twisted promastigotes; rounded amastigotes; and cyst-like bodies. Twisted and non-twisted promastigotes were frequently seen forming rosettes, and these two forms presented significant differences (P<0.01) in terms of their morphological characteristics. Unlike the promastigote forms observed throughout the culture period, rounded amastigotes were seen only in the lag phase, and the cyst-like bodies were only seen in the decline phase. The trypanosomatid DNA obtained from the culture was analyzed by the polymerase chain reaction (PCR) method and found to be negative for Leishmania infantum chagasi. Based on the growth pattern, morphological parameters and molecular analysis, the flagellates were confirmed to be Leptomonas ctenocephali. The significance of this infection for animals is also commented., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Treatment success of chronically infected Chagas disease patients is laborious and a positive prognosis often is made only after repetitive serological and/or parasitological examinations with continuous negative results. Recently, we have developed a non-conventional flow-cytometric method in order to detect immunoglobulin G antibodies against live trypomastigote forms of Trypanosoma cruzi and showed its usefulness in the prognosis of treatment success. In the present study, we investigated the performance of flow-cytometric anti-live trypomastigote IgG antibodies (FC-ALTA) and flow-cytometric anti-fixed epimastigote IgG antibodies (FC-AFEA), as well as conventional serological methods, for early monitoring of benznidazole treated Chagas disease patients, e.g. 5years after treatment. The analysis of individual FC-ALTA reactivity along the titration curve before and after treatment, we were able to show, that between 4% and 13% of treated patients under evaluation presented with reduced serological reactivity and segregated from the other patient groups. Similar results were obtained with semi-quantitative, conventional indirect hemagglutination or indirect immunofluorescence. Our data therefore suggest that the combined use of conventional and non-conventional serological methods could provide more suitable cure criteria in early post-therapeutic prognosis of Chagas disease., (Copyright © 2011 Elsevier B.V. All rights reserved.)

One of the challenges on immunodiagnostic of Chagas disease in endemic areas has been the search for more practical and safe antigenic preparation that provides tests with higher sensitivity and specificity, with low cross-reactivity. A new approach using fixed Trypanosoma cruzi epimastigotes to detect IgG reactivity was investigated previously. In order to continue this investigation, this study aimed at optimizing the flow cytometry-based method to the diagnosis of Chagas disease patients after specific chemotherapy. To achieve our goal, serum samples from 93 subjects - 52 adults chronically infected by T. cruzi, and 41 uninfected controls were tested by flow cytometry. Secondly, serum samples from patients Treated Cured and Treated Uncured from Chagas disease were also tested to evaluate the potential of the method on assessing cure. After establishing the ideal serum dilution and cut off, 121 serum samples from patients with other endemic infections were tested to check cross-reactivity. The results showed that parasite staining with Evan's blue dye eliminated debris, allowing trustworthy analysis of anti-fixed epimastigote IgG reactivity. The applicability of the method to diagnose Chagas disease was confirmed by the high sensitivity (98.1%) and specificity (100%) found. This method also contributed for post-therapeutic assessment of cure, identifying 94.1% of Treated Uncured and 83.3% of Treated Cured patients. Cross-reactivity was observed in a very low number (6.7%). On the whole, these data highly recommend the use of anti-fixed T. cruzi epimastigote IgG reactivity by flow cytometry to the diagnosis and cure monitoring of Chagas disease in endemic areas., (Copyright © 2011 Elsevier B.V. All rights reserved.)

CD25(High) CD4+ regulatory T cells (Treg cells) have been described as key players in immune regulation, preventing infection-induced immune pathology and limiting collateral tissue damage caused by vigorous anti-parasite immune response. In this review, we summarize data obtained by the investigation of Treg cells in different clinical forms of Chagas' disease. Ex vivo immunophenotyping of whole blood, as well as after stimulation with Trypanosoma cruzi antigens, demonstrated that individuals in the indeterminate (IND) clinical form of the disease have a higher frequency of Treg cells, suggesting that an expansion of those cells could be beneficial, possibly by limiting strong cytotoxic activity and tissue damage. Additional analysis demonstrated an activated status of Treg cells based on low expression of CD62L and high expression of CD40L, CD69, and CD54 by cells from all chagasic patients after T. cruzi antigenic stimulation. Moreover, there was an increase in the frequency of the population of Foxp3+ CD25(High)CD4+ cells that was also IL-10+ in the IND group, whereas in the cardiac (CARD) group, there was an increase in the percentage of Foxp3+ CD25(High) CD4+ cells that expressed CTLA-4. These data suggest that IL-10 produced by Treg cells is effective in controlling disease development in IND patients. However, in CARD patients, the same regulatory mechanism, mediated by IL-10 and CTLA-4 expression is unlikely to be sufficient to control the progression of the disease. These data suggest that Treg cells may play an important role in controlling the immune response in Chagas' disease and the balance between regulatory and effector T cells may be important for the progression and development of the disease. Additional detailed analysis of the mechanisms on how these cells are activated and exert their function will certainly give insights for the rational design of procedure to achieve the appropriate balance between protection and pathology during parasite infections.

The recently developed Multiple Displacement Amplification technique (MDA) allows for the production of a large quantity of high quality genomic DNA from low amounts of the original DNA. The goal of this study was to evaluate the performance of the MDA technique to amplify genomic DNA of siphonapterids that have been stored for long periods in 70% ethanol at room temperature. We subjected each DNA sample to two different methodologies: (1) amplification of mitochondrial 16S sequences without MDA; (2) amplification of 16S after MDA. All the samples obtained from these procedures were then sequenced. Only 4 samples (15.4%) subjected to method 1 showed amplification. In contrast, the application of MDA (method 2) improved the performance substantially, with 24 samples (92.3%) showing amplification, with significant difference. Interestingly, one of the samples successfully amplified with this method was originally collected in 1909. All of the sequenced samples displayed satisfactory results in quality evaluations (Phred ≥ 20) and good similarities, as identified with the BLASTn tool. Our results demonstrate that the use of MDA may be an effective tool in molecular studies involving specimens of fleas that have traditionally been considered inadequately preserved for such purposes.

There is a general consensus that during chronic Trypanosoma cruzi infection, the host immune system induces complex processes to ensure the control of parasite growth while preserving the potential to mount and maintain a life-long controlled humoral and cellular immune response against the invading pathogen. This review summarises evidence in an attempt to elucidate 'what must be understood' to further clarify the role of innate immunity in the development/maintenance of clinical Chagas disease and the impact of etiological treatment on host immunity, highlighting the contributions of the innate immunity and regulatory T (Treg) cells. Recently, increasing focus on innate immunity suggest that chronic T. cruzi infection may cause morbidity when innate effector functions, or the down-regulation of adaptive regulatory mechanisms are lacking. In this context, stable asymptomatic host-parasite interactions seem to be influenced by the effector/regulatory balance with the participation of macrophages, natural killer (NK) and CD8+ T cells in parallel with the establishment of regulatory mechanisms mediated by NKT and Treg cells. Moreover, a balanced innate immune activation state, apart from Treg cells, may play a role in controlling the adverse events triggered by the massive antigen release induced by trypanosomicidal agents during Chagas disease etiological treatment.

In total, 1500 specimens (448 males and 1052 females) of the flea Ctenocephalides felis felis (Bouché (Siphonaptera: Pulicidae) were collected over a period of a year from 150 dogs captured by the Centro de Controle de Zoonoses de Belo Horizonte, Minas Gerais, Brazil. Microscopic examination of the dissected fleas revealed that 180 fleas were infected with a species of gregarine that was subsequently identified as a member of the genus Steinina. The relative abundances, prevalence rates, and seasonal variation of the different developmental stages of this endoparasite in C. felis felis were determined. Both gamonts and gametocysts presented significant seasonal variation.

Herein we have employed an alternative strategy to assess the cytokine patterns of circulating leukocytes and correlate dominant cytokine profiles with indeterminate-IND and cardiac-CARD clinical forms of Chagas disease. We have first calculated median percentages of cytokine-positive leukocytes of our study sample to establish, for each cytokine-positive cell population, the cut-off edge that would segregate 'low' and 'high' cytokine producers to build colour diagrams and draw a panoramic cytokine chart. Using this approach we demonstrated that most IND individuals presented a dominant regulatory cytokine profile, whereas CARD individuals displayed a dominant inflammatory cytokine pattern. In addition, radar chart analysis confirmed the dichotomic cytokine balance between IND and CARD groups and further allowed the identification of the relative contribution of each cell population for the global cytokine pattern. Data analysis demonstrated that CD4+ T cells were the major cell population defining the regulatory profile in IND, whereas monocytes and CD4+ T cells determined the inflammatory cytokine pattern in CARD individuals. Interestingly, in vitro stimulation with trypomastigote Trypanosoma cruzi antigen was able to invert the cytokine balances in IND and CARD groups. Upon antigenic stimulation, changes in the frequencies of IL-10-producing CD4+ T cells and monocytes drove IND individuals towards an inflammatory pattern and CARD towards a regulatory cytokine profile. A similar inversion could be found after in vivo treatment of IND and CARD individuals with benzonidazole. Altogether, these findings shed some light into the complex cytokine network underlying the immunopathogenesis of Chagas disease and provide putative immunological biomarkers of disease severity and therapeutic response.

Objectives: To assess different methodologies to better define an early post-therapeutic cure criterion after benznidazole treatment in BALB/c mice following mixed infection with dual Trypanosoma cruzi genotypes., Methods: According to the classical cure criteria, animals were classified as treated not cured (TNC = 76.4%), treated cured (TC = 12.5%) and dissociated (DIS = 11.1%) using parasitological [fresh blood examination (FBE), blood culture (BC) and blood PCR] and serological methods [conventional serology (CS-ELISA) and non-conventional serology (NCS-FC-ALTA)]. Tissues were also evaluated by PCR., Results: FBE was able to detect patent parasitaemia in only 18.1% of TNC and therapeutic failure was detected in 79.1% and 97.2% of TNC by BC and blood PCR, respectively. CS-ELISA should not be used before 3 months after treatment since it may lead to false-negative results. At 3 months after treatment with benznidazole, NCS-FC-ALTA was more efficient for categorizing the groups of treated mice. In the TNC group, although a decreased frequency of PCR-positive tissue was observed in several host tissues, increased positivity was also observed, despite the T. cruzi genotype combination. All TC animals presented at least two positive tissue-PCR results., Conclusions: Our results confirm that NSC-FC-ALTA and blood PCR are the most suitable methods to early detect therapeutic failure in acute murine T. cruzi infection. Additionally, our data show that BC positivity is highly dependent upon the T. cruzi genotype combination. Moreover, our findings demonstrated that PCR tests performed on tissues from animals considered cured after benznidazole treatment still detected T. cruzi DNA, most probably indicating residual infection.

Pro-inflammatory immune response is usually associated with Chagas disease pathogenesis, but is also relevant to treatment effectiveness. Cross-sectional studies have suggested that this activated state may persist for years after therapeutic intervention. However, short-term longitudinal investigation has suggested that the Benznidazole treatment (Bz-treatment) leads to decreased immunological activation. In order to elucidate this issue, we performed a longitudinal study to evaluate the immunological status following Bz-treatment during early indeterminate Chagas disease. Our results demonstrated that Bz-treatment led to higher activation status of circulating monocytes but was negatively associated with the number of IL-12(+)CD14(+) cells. Moreover, Bz-treatment triggered a high frequency of circulating CD3(-)CD16(+)CD56(-) NK cells, in addition to elevated activation status associated with a type 1-modulated cytokine pattern. Bz-treatment induced substantial T and B-cell activation status associated with an overall IL-10 modulated type 1 cytokine profile. In summary, these findings provide new information regarding immune activation status following the etiological treatment of Chagas disease. These results suggest that in addition to the increased number of activated leukocytes in the peripheral blood, Bz-treatment may also involve a qualitative change in their functional capacity that drives their activation state toward a modulated cytokine profile. These changes may account for the benefits of etiological treatment of Chagas disease.

Immunological alterations associated with aging (immunosenescence) do not represent a simple unidirectional decline in all functions but develop as a complex remodeling of the immune system, involving multiple reorganization and developmentally regulated changes. In general, most data available about aging were obtained at particular age intervals and most of them come from Caucasian individuals from either Europe or the United States. Here, we report the frequencies of major lymphocyte subsets in healthy Brazilian individuals from 2 distinct geographic regions (Southeast and South) at several age intervals spanning a lifetime period (0-86 years). Overall, we demonstrated that changes in the frequencies of cells related to both innate and adaptive immunity clearly occur with aging in these individuals. These changes were not progressive and equally steady for all cell populations tested but instead showed an oscillatory or rhythmic behavior that was distinctive of each population at different age intervals. We also observed that abrupt changes in the frequencies of immune cells may occur in healthy individuals over 75 years old, suggesting there is an impaired flexibility of the immune system at late stages of life to sustain homeostasis via immune mechanisms. We presented reference ranges for healthy Brazilian individuals at all ages. The knowledge of these parameters in further detail will allow interventions to optimize immune function in advanced age and to improve the quality of life in the elderly., (Copyright 2008 S. Karger AG, Basel.)