Publications by authors named "Carlos H N Costa"

23 Publications

  • Page 1 of 1

Assessment of a recombinant protein from Leishmania infantum as a novel tool for Visceral Leishmaniasis (VL) diagnosis in VL/HIV co-infection cases.

PLoS One 2021 17;16(5):e0251861. Epub 2021 May 17.

Instituto Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, Pernambuco, Brazil.

Visceral Leishmaniasis and HIV-AIDS coinfection (VL/HIV) is considered a life-threatening pathology when undiagnosed and untreated, due to the immunosuppression caused by both diseases. Serological tests largely used for the VL diagnosis include the direct agglutination test (DAT), ELISA and immunochromatographic (ICT) assays. For VL diagnosis in HIV infections, different studies have shown that the use of the DAT assay facilitates the VL diagnosis in co-infected patients, since the performance of the most widely used ELISA and ICT tests, based on the recombinant protein rK39, are much less efficient in HIV co-infections. In this scenario, alternative recombinant antigens may help the development of new serological diagnostic methods which may improve the VL diagnosis for the co-infection cases. This work aimed to evaluate the use of the recombinant Lci2 antigen, related to, but antigenically more diverse than rK39, for VL diagnosis in co-infected sera through ELISA assays. A direct comparison between recombinant Lci2 and rK39 was thus carried out. The two proteins were first tested using indirect ELISA with sera from VL afflicted individuals and healthy controls, with similar performances. They were then tested with two different sets of VL/HIV co-infected cases and a significant drop in performance, for one of these groups, was observed for rK39 (32% sensitivity), but not for Lci2 (98% sensitivity). In fact, an almost perfect agreement (Kappa: 0.93) between the Lci2 ELISA and DAT was observed for the coinfected VL/HIV patients. Lci2 then has the potential to be used as a new tool for the VL diagnosis of VL/HIV co-infections.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0251861PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8128258PMC
May 2021

A Flow Cytometry-Based Serological Assay to Detect Visceral Leishmaniasis in HIV-Infected Patients.

Front Med (Lausanne) 2021 30;8:553280. Epub 2021 Apr 30.

Aggeu Magalhães Institute, Oswaldo Cruz Foundation, Recife, Brazil.

Visceral Leishmaniasis (VL) is a severe parasitic disease that has emerged as an important opportunistic condition in HIV-infected patients and whose control is impaired by inaccurate identification. This is mainly due to the serological tests used for VL having a reduced performance in cases of VL-HIV coinfection due to a low humoral response. In this situation, however, a positive test has even greater diagnostic value when combined with the clinical status. This study aimed to evaluate the application and performance of flow cytometry to detect anti- antibodies in HIV-infected patients. Sera from VL/HIV coinfected patients, characterized using "gold standard" techniques, were compared with sera from healthy controls plus sera from HIV-infected individuals. The flow cytometry results were expressed as levels of IgG reactivity, based on the percentage of positive fluorescent parasites (PPFP). A ROC curve analysis of a serum titration indicated a PPFP of 1.26% as being the cutoff point to segregate positive and negative results. At the 1:2,048 dilution, with 89% sensitivity and 83% specificity, flow cytometry showed greater sensitivity in relation to the serological tests evaluated. Futhermore, flow cytometry was the only assay that positively identified all VL-HIV patients with quantified HIV load. Together, these findings suggest that flow cytometry may be used as an alternative serological approach for VL identification and as a tool to characterize the humoral response against in HIV-infected patients.
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http://dx.doi.org/10.3389/fmed.2021.553280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8119745PMC
April 2021

Gene design, optimization of protein expression and preliminary evaluation of a new chimeric protein for the serological diagnosis of both human and canine visceral leishmaniasis.

PLoS Negl Trop Dis 2020 07 27;14(7):e0008488. Epub 2020 Jul 27.

Instituto Aggeu Magalhães (IAM)-Fundação Oswaldo Cruz (Fiocruz), Recife, Pernambuco, Brazil.

Background: Visceral leishmaniasis (VL) is a major neglected disease, potentially fatal, whose control is still impaired by inefficient and/or expensive treatment and diagnostic methods. The most promising approach for VL diagnosis uses serological assays with recombinant proteins, since they are more efficient and easier to perform. Tests developed for the human form of the disease, however, have not been shown to be efficient for its diagnosis in the canine host, the major reservoir for the American VL.

Methodology/principal Findings: Here, we describe a systematic approach aimed at the production of a new chimeric protein potentially able to be used for both human and canine VL diagnosis and based both on in silico gene design and experimental data. Starting from the previous identification of Leishmania infantum recombinant antigens efficient for the diagnosis of either human or canine VL, three of the best performing antigens were selected (Lci2, Lci3 and Lci12). After a preliminary evaluation validating the chimeric approach, DNA fragments encoding predicted antigenic regions from each protein, enriched with repeats, were joined in various combinations to generate a total of seventeen chimeric genes optimized for prokaryotic expression. These were assessed for optimal expression and purification yield, with four chimeric proteins being efficiently produced. Their diagnostic potential was then evaluated through ELISA assays with sera from VL afflicted humans and dogs. After two rounds of gene design, the results showed high levels of sensitivity for the best chimeric protein, named Q5, in humans (82%) and dogs (100%) with 100% specificity in comparison with healthy controls. A single non-specific reaction was seen with serum from individuals with tegumentary leishmaniasis.

Conclusion: The newly described chimeric protein is potentially useful for the detection of both humans and dogs afflicted with VL, with its use in rapid tests necessary for validation as a new diagnostic tool.
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http://dx.doi.org/10.1371/journal.pntd.0008488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7410341PMC
July 2020

Performance evaluation of anti-fixed Leishmania infantum promastigotes immunoglobulin G (IgG) detected by flow cytometry as a diagnostic tool for visceral Leishmaniasis.

J Immunol Methods 2019 06 25;469:18-25. Epub 2019 Feb 25.

Fundação Oswaldo Cruz (Fiocruz-Pernambuco), Instituto Aggeu Magalhães, Recife, Pernambuco, Brazil.

Visceral Leishmaniasis (VL) is a severe disease, caused by the protozoans Leishmania infantum and L. donovani that is widely diagnosed using serological tools. These, however, have limitations in performance that limit their use for the correct identification of the cases. This study aimed to evaluate the performance of flow cytometry with fixed parasites for VL diagnosis, comparing it with four other serological tests. Samples from two endemic VL regions in Brazil, diagnosed by direct examination (DG1) and by at least two or one standard serological test (DG2 and DG3, respectively), as well as patients with chronic Chagas' disease (CG1) and healthy controls (CG2) were used in this study. The flow cytometry results were expressed as levels of IgG reactivity, based on the percentage of positive fluorescent parasites (PPFP). Using a 1:4096 serum dilution, a ROC curve analysis of the serum titration on flow cytometry has indicated a PPFP of 2% as the cutoff point to segregate positive and negative results. In the present study, flow cytometry had the best performance for DG1 (sensitivity of 96%) while rK39 (imunocromagraphic rapid test) and DAT (Direct agglutination test) were also associated with high sensitivity and specificity. The substantial agreement and kappa indexes observed suggested similar performances between these two tests and flow cytometry. IFAT (Immunofluorescent antibody test) and ELISA (Enzyme-linked immunosorbent assay) had lower performances and the lower values of agreement with flow cytometry. Together, these findings suggest that although adjustments are needed in order to reduce cross reactivity with other trypanosomatids, flow cytometry has the potential to be a safe serological alternative for the diagnosis of VL.
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http://dx.doi.org/10.1016/j.jim.2019.02.009DOI Listing
June 2019

A Leishmania infantum genetic marker associated with miltefosine treatment failure for visceral leishmaniasis.

EBioMedicine 2018 Oct 27;36:83-91. Epub 2018 Sep 27.

Centre for Immunology and Infection, Department of Biology, University of York, United Kingdom.; Wellcome Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, United Kingdom.. Electronic address:

Background: Miltefosine has been used successfully to treat visceral leishmaniasis (VL) in India, but it was unsuccessful for VL in a clinical trial in Brazil.

Methods: To identify molecular markers that predict VL treatment failure whole genome sequencing of 26 L. infantum isolates, from cured and relapsed patients allowed a GWAS analysis of SNPs, gene and chromosome copy number variations.

Findings: A strong association was identified (p = 0·0005) between the presence of a genetically stable L. infantumMiltefosine Sensitivity Locus (MSL), and a positive response to miltefosine treatment. The risk of treatment failure increased 9·4-fold (95% CI 2·11-53·54) when an isolate did not have the MSL. The complete absence of the MSL predicted miltefosine failure with 0·92 (95% CI 0·65-0·996) sensitivity and 0·78 (95% CI 0·52-0·92) specificity. A genotyping survey of L. infantum (n = 157) showed that the frequency of MSL varies in a cline from 95% in North East Brazil to <5% in the South East. The MSL was found in the genomes of all L. infantum and L. donovani sequenced isolates from the Old World (n = 671), where miltefosine can have a cure rate higher than 93%.

Interpretation: Knowledge on the presence or absence of the MSL in L. infantum will allow stratification of patients prior to treatment, helping to establish better therapeutic strategies for VL treatment. FUND: CNPq, FAPES, GCRF MRC and Wellcome Trust.
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http://dx.doi.org/10.1016/j.ebiom.2018.09.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197651PMC
October 2018

Evaluation of a new set of recombinant antigens for the serological diagnosis of human and canine visceral leishmaniasis.

PLoS One 2017 28;12(9):e0184867. Epub 2017 Sep 28.

Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz (Fiocruz-Pernambuco), Recife, Pernambuco, Brazil.

Current strategies for the control of zoonotic visceral leishmaniasis (VL) rely on its efficient diagnosis in both human and canine hosts. The most promising and cost effective approach is based on serologic assays with recombinant proteins. However, no single antigen has been found so far which can be effectively used to detect the disease in both dogs and humans. In previous works, we identified Leishmania infantum antigens with potential for the serodiagnosis of VL. Here, we aimed to expand the panel of the available antigens for VL diagnosis through another screening of a genomic expression library. Seven different protein-coding gene fragments were identified, five of which encoding proteins which have not been previously studied in Leishmania and rich in repetitive motifs. Poly-histidine tagged polypeptides were generated from six genes and evaluated for their potential for diagnosis of VL by ELISA (Enzyme Linked ImmunoSorbent Assay) with sera from infected humans and dogs. None of those was valid for the detection of human VL (26-52% sensitivity) although their performance was increased in the canine sera (48-91% sensitivity), with one polypeptide useful for the diagnosis of canine leishmaniasis. Next, we assayed a mixture of three antigens, found to be best for human or canine VL, among 13 identified through different screenings. This "Mix" resulted in similar levels of sensitivity for both human (84%) and canine (88%) sera. With improvements, this validates the use of multiple proteins, including antigens identified here, as components of a single system for the diagnosis of both forms of leishmaniasis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0184867PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5619722PMC
October 2017

Causes and consequences of higher Leishmania infantum burden in patients with kala-azar: a study of 625 patients.

Trop Med Int Health 2017 06 2;22(6):679-687. Epub 2017 May 2.

Laboratório de Leishmanioses, Instituto de Doenças Tropicais Natan Portella, Universidade Federal do Piauí, Teresina, Brazil.

Background: An infected host's Leishmania infantum load in blood is considered to be an estimate of his or her total parasite burden. Therefore, the measurement of blood parasite burden is important in the identification of factors involved in parasite control.

Methods: Quantitative polymerase chain reaction was performed on blood samples from 625 patients with kala-azar consecutively admitted to a reference hospital in Teresina, Brazil. Primers were used to amplify a segment of kDNA using the TaqMan system. Non-parametric statistical tests were applied.

Results: The median blood parasite burden was 499.2 amastigote equivalents (AE)/ml. Children <1 year old (yo) had a high parasite burden, which dropped sharply after the first year of life (192.8, AE/ml at 1 < 2 yo) and remained lower until adolescence. Following adolescence, the parasite burden increased with age, peaking among elderly individuals. Men had a higher parasite burden than women. HIV-infected patients had a much higher parasite burden than non-infected patients. The parasite burden of children under 5 years with acute moderate to severe malnourishment (weight-for-age and body mass index z-scores <-2) was almost three times greater than that of better-nourished children. The parasite burden identified in deceased patients was more than twice that of surviving patients; those with a higher risk of death, sepsis, pneumonia and jaundice also had increased parasite burdens. All of these differences were statistically significant at P-values <0.05.

Conclusions: These data indicate that the parasite burden in patients with kala-azar was associated with age- and gender-associated factors and with HIV infection status. Acute malnutrition could be either a cause or a consequence of a higher parasite burden. An individual's parasite burden influences his or her clinical profile, disease severity and mortality risk. The best explanation for the presence of a higher parasite burden in individuals with these immunoregulatory conditions and severe disease is the occurrence of acquired immunosuppression followed by heightened innate immunity.
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http://dx.doi.org/10.1111/tmi.12877DOI Listing
June 2017

DNA barcode for the identification of the sand fly Lutzomyia longipalpis plant feeding preferences in a tropical urban environment.

Sci Rep 2016 07 20;6:29742. Epub 2016 Jul 20.

Federal University of Piauí, Picos, Brazil.

Little is known about the feeding behavior of hematophagous insects that require plant sugar to complete their life cycles. We studied plant feeding of Lutzomyia longipalpis sand flies, known vectors of Leishmania infantum/chagasi parasites, in a Brazilian city endemic with visceral leishmaniasis. The DNA barcode technique was applied to identify plant food source of wild-caught L. longipalpis using specific primers for a locus from the chloroplast genome, ribulose diphosphate carboxylase. DNA from all trees or shrubs within a 100-meter radius from the trap were collected to build a barcode reference library. While plants from the Anacardiaceae and Meliaceae families were the most abundant at the sampling site (25.4% and 12.7% of the local plant population, respectively), DNA from these plant families was found in few flies; in contrast, despite its low abundance (2.9%), DNA from the Fabaceae family was detected in 94.7% of the sand flies. The proportion of sand flies testing positive for DNA from a given plant family was not significantly associated with abundance, distance from the trap, or average crown expansion of plants from that family. The data suggest that there may indeed be a feeding preference of L. longipalpis for plants in the Fabaceae family.
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http://dx.doi.org/10.1038/srep29742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4951712PMC
July 2016

In Vitro and In Vivo Miltefosine Susceptibility of a Leishmania amazonensis Isolate from a Patient with Diffuse Cutaneous Leishmaniasis: Follow-Up.

PLoS Negl Trop Dis 2016 07 14;10(7):e0004720. Epub 2016 Jul 14.

Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, São Paulo, Brazil.

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http://dx.doi.org/10.1371/journal.pntd.0004720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4945063PMC
July 2016

Effectiveness of insecticide spraying and culling of dogs on the incidence of Leishmania infantum infection in humans: a cluster randomized trial in Teresina, Brazil.

PLoS Negl Trop Dis 2014 Oct 30;8(10):e3172. Epub 2014 Oct 30.

Department of Global Health and Population, Harvard School of Public Health, Boston, Massachusetts, United States of America.

Background: To evaluate the effect of insecticide spraying for vector control and elimination of infected dogs on the incidence of human infection with L. infantum, a randomized community intervention trial was carried out in the city of Teresina, Brazil.

Methods/principal Findings: Within each of ten localities in the city, four blocks were selected and randomized to 4 interventions: 1) spraying houses and animal pens with insecticide; 2) eliminating infected dogs; 3) combination of spraying and eliminating dogs, and 4) nothing. The main outcome is the incidence of infection assessed by the conversion of the Montenegro skin test (MST) after 18 months of follow-up in residents aged ≥ 1 year with no previous history of visceral leishmaniasis (VL). Reactions were measured at 48-72 h, induration of ≥ 5 mm considered positive. Interventions were executed after the baseline interview and repeated 6 and 12 months later. The effects of each type of intervention scheme on the incidence of infection were assessed by calculating relative risks and 95% confidence intervals using Poisson population-averaged regression models with robust variance. Among the 1105 participants, 408 (37%) were MST positive at baseline. Of the 697 negatives, only 423 (61%) were reexamined at the end of the follow-up; 151 (36%) of them converted to a positive MST. Only dog culling had some statistically significant effect on reducing the incidence of infection, with estimates of effectiveness varying between 27% and 52%, depending on the type of analysis performed.

Conclusions/significance: In light of the continuous spread of VL in Brazil despite the large scale deployment of insecticide spraying and dog culling, the relatively low to moderate effectiveness of dog culling and the non-significant effect of insecticide spraying on the incidence of human infection, we conclude that there is an urgent need for revision of the Brazilian VL control program.
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http://dx.doi.org/10.1371/journal.pntd.0003172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4214628PMC
October 2014

A case of conventional treatment failure in visceral leishmaniasis: leukocyte distribution and cytokine expression in splenic compartments.

BMC Infect Dis 2014 Sep 9;14:491. Epub 2014 Sep 9.

Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz, Salvador, BA, Brazil.

Background: In this paper we study the distribution of leukocyte populations and of cytokine-producing cells in the spleen of a patient with visceral leishmaniasis resistant to clinical treatment. It is the first attempt to compare the distribution of leukocyte populations and cytokine-producing cells in the splenic compartments of a patient with visceral leishmaniasis with those observed in patients without the disease.

Case Presentation: A 25-year-old male, farmer, was hospitalized on several occasions with diagnosis of visceral leishmaniasis and received all recommended treatments for the disease with only transient improvement followed by relapse. He was eventually subjected to splenectomy in order to control the effects of hypersplenism and to potentially overcome infection. After surgery and combined chemotherapy, the disease evolved to cure. In comparison with the spleens of the other two patients without visceral leishmaniasis, an increase was observed in the CD4/CD8 ratio and in the number of IL-10- and FoxP3-producing cells, while the number of IL-17-producing cells was lower in the spleen of the patient with visceral leishmaniasis.

Conclusion: This report confirms previous data on changes in the CD4/CD8 ratio in the spleens of patients with visceral leishmaniasis. Additionally the data presented herein suggests that splenic FoxP3- and IL-17-producing cells are involved in the chronicity of visceral leishmaniasis.
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http://dx.doi.org/10.1186/1471-2334-14-491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4175220PMC
September 2014

In vitro and in vivo miltefosine susceptibility of a Leishmania amazonensis isolate from a patient with diffuse cutaneous leishmaniasis.

PLoS Negl Trop Dis 2014 Jul 17;8(7):e2999. Epub 2014 Jul 17.

Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, São Paulo, Brazil.

Miltefosine was the first oral compound approved for visceral leishmaniasis chemotherapy, and its efficacy against Leishmania donovani has been well documented. Leishmania amazonensis is the second most prevalent species causing cutaneous leishmaniasis and the main etiological agent of diffuse cutaneous leishmaniasis in Brazil. Driven by the necessity of finding alternative therapeutic strategies for a chronic diffuse cutaneous leishmaniasis patient, we evaluated the susceptibility to miltefosine of the Leishmania amazonensis line isolated from this patient, who had not been previously treated with miltefosine. In vitro tests against promastigotes and intracellular amastigotes showed that this parasite isolate was less susceptible to miltefosine than L. amazonensis type strains. Due to this difference in susceptibility, we evaluated whether genes previously associated with miltefosine resistance were involved. No mutations were found in the miltefosine transporter gene or in the Ros3 or pyridoxal kinase genes. These analyses were conducted in parallel with the characterization of L. amazonensis mutant lines selected for miltefosine resistance using a conventional protocol to select resistance in vitro, i.e., exposure of promastigotes to increasing drug concentrations. In these mutant lines, a single nucleotide mutation G852E was found in the miltefosine transporter gene. In vivo studies were also performed to evaluate the correlation between in vitro susceptibility and in vivo efficacy. Miltefosine was effective in the treatment of BALB/c mice infected with the L. amazonensis type strain and with the diffuse cutaneous leishmaniasis isolate. On the other hand, animals infected with the resistant line bearing the mutated miltefosine transporter gene were completely refractory to miltefosine chemotherapy. These data highlight the difficulties in establishing correlations between in vitro susceptibility determinations and response to chemotherapy in vivo. This study contributed to establish that the miltefosine transporter is essential for drug activity in L. amazonensis and a potential molecular marker of miltefosine unresponsiveness in leishmaniasis patients.
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http://dx.doi.org/10.1371/journal.pntd.0002999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4102453PMC
July 2014

Bone marrow parasite burden among patients with New World kala-azar is associated with disease severity.

Am J Trop Med Hyg 2014 Apr 10;90(4):621-6. Epub 2014 Mar 10.

Laboratory of Leishmaniasis, Institute of Tropical Diseases "Natan Portella", Federal University of Piauí, Teresina, PI, Brazil; Department of Biology, Federal University of Piauí, Floriano at Floriano, PI, Brazil; Maternal and Childhood Department, Federal University of Piauí, Teresina, PI, Brazil; Laboratory of Molecular Biology, Nucleus of Tropical Medicine, Federal University of Pará, Belém, PA, Brazil; Department of Community Medicine, Federal University of Piauí, Teresina, PI, Brazil.

Kala-azar or visceral leishmaniasis, found mostly throughout the Indian Subcontinent, East Africa, and Brazil, kills 20,000-40,000 persons annually. The agents, Leishmania donovani and Leishmania infantum, are obligatory intracellular protozoa of mononuclear phagocytes found principally in the spleen and bone marrow. Protracted fever, anemia, wasting, hepatosplenomegaly, hemorrhages, and bacterial co-infections are typical features. One hundred and twenty-two (122) in-hospital patients were studied to verify if higher bone marrow parasite load estimated by quantitative polymerase chain reaction is associated with severe disease. The estimated median parasite load was 5.0 parasites/10(6) human nucleated cells. It is much higher in deceased than among survivors (median 75.0 versus 4.2). Patients who lost more weight had a higher parasite burden, as well as patients with epistaxis, abdominal pain, edema, and jaundice. This study suggests that higher parasite load is influenced by wasting, which may lead to more severe disease.
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http://dx.doi.org/10.4269/ajtmh.13-0376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3973504PMC
April 2014

Anti-leishmanial activity of the antimicrobial peptide DRS 01 observed in Leishmania infantum (syn. Leishmania chagasi) cells.

Nanomedicine 2014 Feb 2;10(2):483-90. Epub 2013 Oct 2.

Núcleo de Pesquisa em Biodiversidade e Biotecnologia, BIOTEC, Campus de Parnaíba, Universidade Federal do Piauí, UFPI, Parnaíba, PI, Brazil.

Unlabelled: Leishmaniasis is one of the most serious diseases in the world and can be lethal if untreated. This is especially the case for visceral leishmaniasis, which is commonly caused by Leishmania (L.) infantum and for which available medication is still inadequate. A recently described antimicrobial peptide DRS 01 has been reported to kill L. infantum promastigotes, but nothing is known about its mode of action or effect on the cell. In this paper we report the visualization of the interaction between DRS 01 and L. infantum promastigotes using two high resolution microscopic techniques: atomic force microscopy and scanning electron microscopy. The results show considerable morphological changes at and above the IC50 in the treated cells. Both membrane damage and flagella alterations were observed. The results strongly suggest a membrane-directed action for DRS 01 on the Leishmania species studied.

From The Clinical Editor: In this paper, the effects of DRS 01, an antimicrobial peptide, is studied in Leishmania infantum using atomic force microscopy as well as standard scanning electron microscopy techniques, with the conclusion of a membrane-based effect by DRS 01 on the parasites.
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http://dx.doi.org/10.1016/j.nano.2013.09.003DOI Listing
February 2014

Identification and diagnostic utility of Leishmania infantum proteins found in urine samples from patients with visceral leishmaniasis.

Clin Vaccine Immunol 2012 Jun 18;19(6):935-43. Epub 2012 Apr 18.

DetectoGen Inc., Grafton, Massachusetts, USA.

Despite the clear need to control visceral leishmaniasis (VL), the existing diagnostic tests have serious shortcomings. Here, we introduce an innovative approach to directly identify Leishmania infantum antigens produced in vivo in humans with VL. We combined reverse-phase high-performance liquid chromatography (RP-HPLC) with mass spectrometry and categorized three distinct L. infantum proteins presumably produced in bone marrow/spleen/liver and excreted in the urine of patients with VL. The genes coding for these proteins (L. infantum iron superoxide dismutase, NCBI accession number XP_001467866.1; L. infantum tryparedoxin, NCBI accession number XP_001466642.1; and L. infantum nuclear transport factor 2, NCBI accession number XP_001463738.1) were cloned, and the recombinant molecules were produced in Escherichia coli. Antibodies to these proteins were produced in rabbits and chickens and were used to develop a capture enzyme-linked immunosorbent assay (ELISA) designed to detect these L. infantum antigens in the urine of VL patients. Specificity of the antibodies was confirmed by a Western blot analysis using both recombinant proteins and whole parasite extract. Importantly, a urinary antigen detection assay assembled with pairs of antibodies specific for each of these antigens identified 17 of 19 patients with VL. These results indicate that an improved antigen detection assay based on L. infantum proteins present in the urine of patients with VL may represent an important new strategy for the development of a specific and accurate diagnostic test that has the potential to both distinguish active VL from asymptomatic infection and serve as an important tool to monitor therapy efficacy.
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http://dx.doi.org/10.1128/CVI.00125-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3370439PMC
June 2012

Infectious disease control in Brazil.

Lancet 2011 Sep;378(9797):1135-6; author reply 1136

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http://dx.doi.org/10.1016/S0140-6736(11)61499-2DOI Listing
September 2011

Multi-centric prospective evaluation of rk39 rapid test and direct agglutination test for the diagnosis of visceral leishmaniasis in Brazil.

Trans R Soc Trop Med Hyg 2011 Feb 20;105(2):81-5. Epub 2010 Oct 20.

Laboratório de Pesquisas Clínicas, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, Minas Gerais, Brazil.

The diagnosis of visceral leishmaniasis (VL) is still a major problem in Brazil and several other countries where the disease is endemic. The use of an easy-to-use and interpret, sensitive, and specific method that requires no complex infrastructure or specialized professionals, such as direct agglutination test (DAT) and the rK39-based rapid immunochromatographic test may enhance the diagnosis of disease. This study evaluated the performance of a rapid test (DiaMed- IT-LEISH®) and the DAT for the diagnosis of VL in 213 parasitologically confirmed cases and 119 controls with clinical suspicion of VL and confirmation of another etiology. The sensitivities and specificities of the rapid test were 93% and 97%, respectively and those of the DAT were 90% and 96%, respectively. The positive predictive values of the rapid test and the DAT were 98% and 97%, respectively and the negative predictive values were 89% and 84%, respectively. The Kappa index showed agreement between both methods classified as substantial (0.77). This study showed that the DAT and the rapid test can be used to diagnose VL in Brazil, following a pilot study for implementation of the rapid test in the health services.
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http://dx.doi.org/10.1016/j.trstmh.2010.09.004DOI Listing
February 2011

Discovery of markers of exposure specific to bites of Lutzomyia longipalpis, the vector of Leishmania infantum chagasi in Latin America.

PLoS Negl Trop Dis 2010 Mar 23;4(3):e638. Epub 2010 Mar 23.

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, United States of America.

Background: Sand flies deliver Leishmania parasites to a host alongside salivary molecules that affect infection outcomes. Though some proteins are immunogenic and have potential as markers of vector exposure, their identity and vector specificity remain elusive.

Methodology/principal Findings: We screened human, dog, and fox sera from endemic areas of visceral leishmaniasis to identify potential markers of specific exposure to saliva of Lutzomyia longipalpis. Human and dog sera were further tested against additional sand fly species. Recombinant proteins of nine transcripts encoding secreted salivary molecules of Lu. longipalpis were produced, purified, and tested for antigenicity and specificity. Use of recombinant proteins corresponding to immunogenic molecules in Lu. longipalpis saliva identified LJM17 and LJM11 as potential markers of exposure. LJM17 was recognized by human, dog, and fox sera; LJM11 by humans and dogs. Notably, LJM17 and LJM11 were specifically recognized by humans exposed to Lu. longipalpis but not by individuals exposed to Lu. intermedia.

Conclusions/significance: Salivary recombinant proteins are of value as markers of vector exposure. In humans, LJM17 and LJM11 emerged as potential markers of specific exposure to Lu. longipalpis, the vector of Leishmania infantum chagasi in Latin America. In dogs, LJM17, LJM11, LJL13, LJL23, and LJL143 emerged as potential markers of sand fly exposure. Testing these recombinant proteins in large scale studies will validate their usefulness as specific markers of Lu. longipalpis exposure in humans and of sand fly exposure in dogs.
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http://dx.doi.org/10.1371/journal.pntd.0000638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2843637PMC
March 2010

Antibodies against Lutzomyia longipalpis saliva in the fox Cerdocyon thous and the sylvatic cycle of Leishmania chagasi.

Trans R Soc Trop Med Hyg 2007 Feb 2;101(2):127-33. Epub 2006 Aug 2.

Faculdade de Medicina, Universidade Federal da Bahia, Salvador, Bahia, Brazil.

Sera of 11 wild Cerdocyon thous foxes from an endemic area for American visceral leishmaniasis were tested for the presence of antibodies against salivary gland homogenates (SGH) of Lutzomyia longipalpis. All foxes had higher levels of anti-Lu. longipalpis SGH antibodies than foxes from non-endemic areas, suggesting contact between foxes and the vector of visceral leishmaniasis. Sera of humans and dogs living in the same area were also tested for reactivity against Lu. longipalpis SGHs and had a lower proportion of reactivity than foxes. Antibodies against Leishmania chagasi were not detected in any of the foxes, but three foxes showed the presence of parasites in the bone marrow by direct examination, PCR or by infecting the vector. Both humans and dogs had higher levels of anti-Le. chagasi IgG antibodies than C. thous. The finding of an antibody response against saliva of Lu. longipalpis among C. thous together with the broad distribution of the vector in resting areas of infected foxes suggests that the natural foci of transmission of Le. chagasi exists independently of the transmission among dogs and humans.
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http://dx.doi.org/10.1016/j.trstmh.2006.06.002DOI Listing
February 2007

Predictors of an unsatisfactory response to pentavalent antimony in the treatment of American visceral leishmaniasis.

Rev Soc Bras Med Trop 2002 Nov-Dec;35(6):629-33. Epub 2003 Feb 26.

Departamento de Medicina Comunitária, Hospital de Doenças Infecto-Contagiosas, Universidade Federal do Piauí, Teresina, PI, Brasil.

Although treatment of visceral leishmaniasis with pentavalent antimony is usually successful, some patients require second-line drug therapy, most commonly with amphotericin B. To identify the clinical characteristics that predict an inadequate response to pentavalent antimony, a case-control study was undertaken in Teresina, Piaui, Brazil. Over a two-year period, there were 19 cases of VL in which the staff physicians of a hospital prescribed second-line therapy with amphotericin B after determining that treatment with pentavalent antimony had failed. The control group consisted of 97 patients that were successfully treated with pentavalent antimony. A chart review using univariate and multivariate analysis was performed. The cure rate was 90% with amphotericin B. The odds ratio for the prescription of amphotericin B was 10.2 for children less than one year old, compared with individuals aged over 10 years. Patients who presented coinfection had an OR of 7.1 while those on antibiotics had an OR of 2.8. These data support either undertaking a longer course of therapy with pentavalent antimony for children or using amphotericin B as a first-line agent for children and individuals with coinfections. It also suggests that chemoprophylaxis directed toward bacterial coinfection in small children with VL may be indicated.
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http://dx.doi.org/10.1590/s0037-86822002000600014DOI Listing
May 2003

Asymptomatic human carriers of Leishmania chagasi.

Am J Trop Med Hyg 2002 Apr;66(4):334-7

Department of Immunology and Infectious Disease, Harvard School of Public Health, Boston, Massachusetts, USA.

In Brazil, programs based on elimination of infected dogs have not curtailed the spread of visceral leishmaniasis (VL), suggesting that other reservoirs of infection exist. Persons with active VL can infect the sand fly vector, but in endemic areas, persons with asymptomatic infections, whose infectivity to sand flies is unknown, are far more numerous. In this study, a polymerase chain reaction-based assay detected kinetoplast DNA of Leishmania chagasi in the blood of eight of 108 asymptomatic persons living with patients with recently diagnosed VL. These eight persons had low or unmeasurable levels of IgG antibodies to Leishmania, demonstrating the insensitivity of serology for subclinical infection. All eight persons had positive leishmanin skin test results, as did 70% of persons living in households of persons with active VL. Even if a small proportion of such asymptomatic persons are infective to sand flies, they represent a formidable reservoir of infection in endemic areas.
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http://dx.doi.org/10.4269/ajtmh.2002.66.334DOI Listing
April 2002

The urban spread of visceral leishmaniasis: clues from spatial analysis.

Epidemiology 2002 May;13(3):364-7

Department of Immunology and Infectious Disease, Harvard School of Public Health, Boston, MA, USA.

Background: The pattern of spread of visceral leishmaniasis in Brazilian cities is poorly understood.

Methods: We used geographic information systems and spatial statistics to evaluate the distribution of 1061 cases of visceral leishmaniasis in Teresina, Brazil, in 1993 through 1996.

Results: A locally weighted (LOESS) regression model, which was fit as a smoothed function of spatial coordinates, demonstrated large-scale variation, with high incidence rates in peripheral neighborhoods that bordered forest land and pastures. Moran's I indicated small-scale variation and clustering up to 300 m, roughly the flight range of the sand fly vector.

Conclusions: Spatial analytical techniques can identify high-risk areas for targeting control interventions.
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http://dx.doi.org/10.1097/00001648-200205000-00020DOI Listing
May 2002