Clinic-epidemiologic study of human infection by Granada virus, a new phlebovirus within the sandfly fever Naples serocomplex.

Granada virus (GRV), a new phlebovirus within the Naples serocomplex, has been recently described in phlebotomine sandflies from Spain. The presence of anti-GRV immunoglobulin G (IgG) antibodies was investigated by indirect fluorescence assay (IFA) and neutralization test (NT) in 920 serum samples from the Granada population. By IFA, an overall GRV seroprevalence of 15.8% (N = 145) was observed, significantly increasing up to 65 years. NT was positive in 18% of anti-GRV IFA-positive samples. IgG antibodies against Toscana virus (TOSV), a hyperendemic phlebovirus within Granada province, were detected in 40% of anti-GRV-positive cases. Anti-GRV IgM antibodies were detected in 36 (6.6%) of 547 acute-phase serum samples from individuals with febrile illness, exanthema, and/or acute respiratory infection. All positives were anti-TOSV IgM-negative. GRV may infect humans, with most cases being asymptomatic. The codetection of anti-GRV and anti-TOSV IgG antibodies could be attributable to cross-reactivity or exposure to the same transmission vector.

Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting

Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis. We detected the C526T and C531T mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene with qPCR-HRM using plasmid-based controls. A segment of the RRDR region from M. tuberculosis H37Rv and from strains carrying C531T or C526T mutations in the rpoB were cloned into pGEM-T vector and these vectors were used as controls in the qPCR-HRM analysis of 54 M. tuberculosis strains. The results were confirmed by DNA sequencing and showed that recombinant plasmids can replace genomic DNA as controls in the qPCR-HRM assay. Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts.

Apoptotic markers in protozoan parasites.

ABSTRACT: The execution of the apoptotic death program in metazoans is characterized by a sequence of morphological and biochemical changes that include cell shrinkage, presentation of phosphatidylserine at the cell surface, mitochondrial alterations, chromatin condensation, nuclear fragmentation, membrane blebbing and the formation of apoptotic bodies. Methodologies for measuring apoptosis are based on these markers. Except for membrane blebbing and formation of apoptotic bodies, all other events have been observed in most protozoan parasites undergoing cell death. However, while techniques exist to detect these markers, they are often optimised for metazoan cells and therefore may not pick up subtle differences between the events occurring in unicellular organisms and multi-cellular organisms.In this review we discuss the markers most frequently used to analyze cell death in protozoan parasites, paying special attention to changes in cell morphology, mitochondrial activity, chromatin structure and plasma membrane structure/permeability. Regarding classical regulators/executors of apoptosis, we have reviewed the present knowledge of caspase-like and nuclease activities.

Impact of a community-based bug-hunting campaign on Chagas disease control: a case study in the department of Jalapa, Guatemala

Chagas disease control requires an innovative approach to strengthen community participation in vector surveillance. This paper presents a case study of a community-based bug-hunting campaign in Guatemala. The campaign was implemented in 2007 in the following three stages: (i) a four week preparation stage to promote bug-hunting, (ii) a one week bug-hunting stage to capture and collect bugs and (iii) a 10 week follow-up stage to analyse the bugs and spray insecticide. A total of 2,845 bugs were reported, of which 7% were Triatominae vectors, such as Rhodnius prolixus and Triatoma dimidiata. The bug-hunting campaign detected a five-six-fold higher amount of vectors in one week than traditional community-based surveillance detects in one year. The bug-hunting campaign effectively detected vectors during a short period, provided information to update the vector infestation map and increased community and political awareness regarding Chagas disease. This approach could be recommended as an effective and feasible strategy to strengthen vector surveillance on a larger scale.

Phlebotomine sandflies (Diptera: Psychodidae) from Lábrea, state of Amazonas, Brazil, with a description of Evandromyia (Aldamyia) apurinan Shimabukuro, Figueira & Silva, sp. nov.

An entomological survey was conducted from July-December 2009 and September-December 2010, as part of the epidemiological monitoring of American cutaneous leishmaniasis (ACL) in the municipality of Lábrea, state of Amazonas (AM), Brazil. Sandflies were collected using CDC light traps installed in intra and peridomiciliary locations, as well as the border of forested areas around houses where autochthonous cases of ACL were recorded. A total of 510 sandflies belonging to 26 species were collected. The most abundant species was Nyssomyia antunesi (44.5%) followed by Evandromyia walkeri (10.6%) and Micropygomyia rorotaensis (9.8%). Here we also describe Evandromyia (Aldamyia) apurinan sp. nov. and report new records for Trichophoromyia flochi and Evandromyia sipani in AM and Brazil, respectively. Our results describe the composition of the sandfly fauna in the south of AM and suggest Ny. antunesi as the putative vector in the transmission of Leishmania in this area of the Amazon Region.

Sandflies (Diptera: Psychodidae) in rural and urban environments in an endemic area of cutaneous leishmaniasis in southern Brazil

The high proportion of cases of cutaneous leishmaniasis reported amongst residents in the city of Bandeirantes, in the state of Paraná, Brazil, led the authors to investigate the phlebotomine fauna in both urban and rural environments. The sandflies were captured with automatic light traps from 07:00 pm-07:00 am fortnightly in 11 urban peridomiciles from April 2008-March 2009 and monthly in three ecotopes within four rural localities from April 2009-March 2010. In one of these latter localities, sandfly capture was conducted with white/black Shannon traps during each of three seasons: spring, summer and fall. A total of 5,729 sandflies of 17 species were captured. Nyssomyia neivai (46.7%) and Nyssomyia whitmani (35.3%) were the predominant species. In this study, 3,865 specimens were captured with automatic light traps: 22 (0.083 sandflies/trap) in the urban areas and 3,843 (26.69 sandflies/trap) in the rural areas. Ny. neivai was predominant in urban (68.2%) and rural (42.8%) areas. A total of 1,864 specimens were captured with the white/black Shannon traps and Ny. neivai (54.5%) and Ny. whitmani (31.4%) were the predominant species captured. The small numbers of sandflies captured in the urban areas suggest that the transmission of Leishmania has occurred in the rural area due to Ny. neivai and Ny. whitmani as the probable vectors.

Chromosomal divergence and evolutionary inferences in Rhodniini based on the chromosomal location of ribosomal genes

In this study, we used fluorescence in situ hybridisation to determine the chromosomal location of 45S rDNA clusters in 10 species of the tribe Rhodniini (Hemiptera: Reduviidae: Triatominae). The results showed striking inter and intraspecific variability, with the location of the rDNA clusters restricted to sex chromosomes with two patterns: either on one (X chromosome) or both sex chromosomes (X and Y chromosomes). This variation occurs within a genus that has an unchanging diploid chromosome number (2n = 22, including 20 autosomes and 2 sex chromosomes) and a similar chromosome size and genomic DNA content, reflecting a genome dynamic not revealed by these chromosome traits. The rDNA variation in closely related species and the intraspecific polymorphism in Rhodnius ecuadoriensis suggested that the chromosomal position of rDNA clusters might be a useful marker to identify recently diverged species or populations. We discuss the ancestral position of ribosomal genes in the tribe Rhodniini and the possible mechanisms involved in the variation of the rDNA clusters, including the loss of rDNA loci on the Y chromosome, transposition and ectopic pairing. The last two processes involve chromosomal exchanges between both sex chromosomes, in contrast to the widely accepted idea that the achiasmatic sex chromosomes of Heteroptera do not interchange sequences.

Leishmania infection and host-blood feeding preferences of phlebotomine sandflies and canine leishmaniasis in an endemic European area, the Algarve Region in Portugal

The Algarve Region (AR) in southern Portugal, which is an international tourist destination, has been considered an endemic region of zoonotic leishmaniasis caused by Leishmania infantum since the 1980s. In the present study, phlebotomine and canine surveys were conducted to identify sandfly blood meal sources and to update the occurrence of Leishmania infection in vectors and dogs. Four sandfly species were captured: Phlebotomus perniciosus, Phlebotomus ariasi, Phlebotomus sergenti and Sergentomyia minuta. In one P. perniciosus female, L. infantum DNA was detected. Blood meal tests showed that this species had no host preferences and was an opportunistic feeder. An overall canine leishmaniasis (CanL) seroprevalence of 16.06% was found; the seroprevalence was 3.88% in dogs housed in kennels and 40.63% in dogs that attended veterinary clinics. The simultaneous occurrence of dogs and P. perniciosus infected with L. infantum in the AR indicates that the region continues to be an endemic area for CanL. Our results reinforce the need for the systematic spatial distribution of phlebotomine populations and their Leishmania infection rates and the need to simultaneously perform pathogen monitoring in both invertebrate and vertebrate hosts to investigate the transmission, distribution and spreading of Leishmania infection.

The processing limits the presentation of the melanoma-associated antigen Melan-A in vitro and in vivo

SUMMARY Both proteasomes and additional proteases play an essential role in the generation of most antigenic peptides presented by MHC class I molecules. Therefore, it is of major importance to characterize the mechanisms leading to the production of correct antigenic peptides to improve the design of vaccines. As a model determinant we used the melanoma-associated protein Melan-A, which contains the immunodominant CTL-epitope Melan-A26/27-35/HLA-A*0201 and against which a high frequency of T lymphocytes has been detected in many melanoma patients. In a first part, we have studied the effects of antigen processing on the induction of a specific T cell response in vivo. Our results have shown that the immunoproteasome, expressed in most cells after exposure to Interferon-γ (IFN-γ) and constitutively in some specialized cells such as dendritic cells, does not efficiently process the HLA¬A2-restricted peptide Melan-A26-35. We have produced recombinant lentiviral vectors (rec. 1v) and vaccinia virus (rec. vv) encoding either preprocessed Melan-A26-35(A27L) peptide or full-length Melan-A(A27L). The immunization of HLA-A2/Kb mice with thoses viruses indicates that immunoproteasomes negatively affect the induction of anti-Melan-A T cell responses in animals immunized with vectors coding for the full- length protein. This negative effect was abrogated in HLA-A2/Kb LMP2-/- mice, lacking the immunoproteasomes. Therefore, we can conclude that the expression of immunoproteasomes limits the induction of the anti-Melan-A T cell response. In a second part, we show that the in vitro degradation of a Melan-A26/27-35 precursor by the proteasomes produces both the final antigenic peptide and N-terminally extended intermediates. When human melanoma cells expressing the corresponding fragments were exposed to specific CTL, those expressing the minimal antigenic sequence were recognized more efficiently than those expressing the N-terminally extended intermediates. We demonstrated that the N-terminally extended intermediates were inefficiently trimmed by cytosolic proteases. These results imply that both proteasomes and post-proteasomal peptidases influence the availability of antigenic peptides and that the efficiency of presentation may be affected by conditions that alter the ratio between fully and partially processed proteasomal products. RESUME Le protéasome ainsi que d'autres protéases jouent un rôle essentiel dans l'apprêtement de la plupart des peptides antigéniques présentés par les molécules de MHC classe I. Il est donc particulièrement important de connaître les mécanismes menant à la production du peptide antigénique correct afin de pouvoir mieux définir de futurs vaccins. Nous avons utilisé la protéine associée au mélanome, Melan-A, contenant un épitope immunodominant Melan-A26/27-35/HLA-A*0201 contre lequel une fréquence élevée de lymphocytes T a été detectée dans plusieurs patients atteints de mélanome. Dans une première partie, nous avons étudié les effets de l'apprêtement du peptide antigéniques Melan-A26-35 sur l'induction de cellules T spécifiques dans la souris. Nos résultats ont démontré que l'immunoprotéasome, exprimé dans la plupart des cellules après exposition à de l'IFN-γ et exprimé constitutivement dans certaines cellules spécialisées, telles les cellules dendritiques, n'apprête pas efficacement le peptide antigénique Melan-A26-35 restreint par HLA-A2 in vitro. Nous avons produit des vecteurs lentiviraux recombinants ainsi que des virus vaccinia codant pour le peptide antigénique Melan-A26-35(A27L) et pour la protéine entière Melan-A(A27L). L'immunisation de souris HLA-A2/Kb avec ces virus démontre que l'immunoprotéasome affecte négativement l'induction d'une réponse T contre Melan¬-A dans les souris immunisées avec des virus contenant la séquence de la protéine entière. Cet effet négatif est complètement aboli dans les souris HLA-A2/Kb LMP2-/- qui n'expriment pas l'immunoprotéasome. Deuxièmement, nous avons demontré que la dégradation d'un peptide précurseur contenant Melan-A26/27-35 par le protéasome produit à la fois le peptide antigénique ainsi que des peptides rallongés à leurs extrémités N-terminales. Lorsque ces fragments sont exprimés dans des cellules humaines et exposés à des cellules T cytotoxiques (CTL), celles qui expriment le peptide antigénique final sont reconnus plus efficacement que celles exprimant les peptides rallongés en N-terminus. Nous avons démontré que les peptides rallongés en N-terminus ne sont pas apprêtés efficacement par les peptidases du cytosol. L'inefficacité de l'apprêtement des peptides rallongés dans le cytosol offre un certain avantage pour les peptides directement produits par le protéasome. Ces résultats impliquent donc que le protéasome ainsi que les peptidases post-proteasomales influencent l'accessibilité des peptides antigéniques.

The first detection of Leishmania major in naturally infected Sergentomyia minuta in Portugal

Phlebotomine sandflies of the genus Sergentomyia are widely distributed throughout the Old World. It has been suggested that Sergentomyia spp are involved in the transmission of Leishmania in India and Africa, whereas Phlebotomus spp are thought to be the sole vectors of Leishmania in the Old World. In this study, Leishmania major DNA was detected in one Sergentomyia minuta specimen that was collected in the southern region of Portugal. This study challenges the dogma that Leishmania is exclusively transmitted by species of the genus Phlebotomus in the Old World.