Spatio-Temporal Tracking and Phylodynamics of an Urban Dengue 3 Outbreak in Sao Paulo, Brazil

The dengue virus has a single-stranded positive-sense RNA genome of similar to 10.700 nucleotides with a single open reading frame that encodes three structural (C, prM, and E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. It possesses four antigenically distinct serotypes (DENV 1-4). Many phylogenetic studies address particularities of the different serotypes using convenience samples that are not conducive to a spatio-temporal analysis in a single urban setting. We describe the pattern of spread of distinct lineages of DENV-3 circulating in Sao Jose do Rio Preto, Brazil, during 2006. Blood samples from patients presenting dengue-like symptoms were collected for DENV testing. We performed M-N-PCR using primers based on NS5 for virus detection and identification. The fragments were purified from PCR mixtures and sequenced. The positive dengue cases were geo-coded. To type the sequenced samples, 52 reference sequences were aligned. The dataset generated was used for iterative phylogenetic reconstruction with the maximum likelihood criterion. The best demographic model, the rate of growth, rate of evolutionary change, and Time to Most Recent Common Ancestor (TMRCA) were estimated. The basic reproductive rate during the epidemics was estimated. We obtained sequences from 82 patients among 174 blood samples. We were able to geo-code 46 sequences. The alignment generated a 399-nucleotide-long dataset with 134 taxa. The phylogenetic analysis indicated that all samples were of DENV-3 and related to strains circulating on the isle of Martinique in 2000-2001. Sixty DENV-3 from Sao Jose do Rio Preto formed a monophyletic group (lineage 1), closely related to the remaining 22 isolates (lineage 2). We assumed that these lineages appeared before 2006 in different occasions. By transforming the inferred exponential growth rates into the basic reproductive rate, we obtained values for lineage 1 of R(0) = 1.53 and values for lineage 2 of R(0) = 1.13. Under the exponential model, TMRCA of lineage 1 dated 1 year and lineage 2 dated 3.4 years before the last sampling. The possibility of inferring the spatio-temporal dynamics from genetic data has been generally little explored, and it may shed light on DENV circulation. The use of both geographic and temporally structured phylogenetic data provided a detailed view on the spread of at least two dengue viral strains in a populated urban area.

The variability and predictors of quality of AIDS care services in Brazil

Background: Since establishing universal free access to antiretroviral therapy in 1996, the Brazilian Health System has increased the number of centers providing HIV/AIDS outpatient care from 33 to 540. There had been no formal monitoring of the quality of these services until a survey of 336 AIDS health centers across 7 Brazilian states was undertaken in 2002. Managers of the services were asked to assess their clinics according to parameters of service inputs and service delivery processes. This report analyzes the survey results and identifies predictors of the overall quality of service delivery. Methods: The survey involved completion of a multiple-choice questionnaire comprising 107 parameters of service inputs and processes of delivering care, with responses assessed according to their likely impact on service quality using a 3-point scale. K-means clustering was used to group these services according to their scored responses. Logistic regression analysis was performed to identify predictors of high service quality. Results: The questionnaire was completed by 95.8% (322) of the managers of the sites surveyed. Most sites scored about 50% of the benchmark expectation. K-means clustering analysis identified four quality levels within which services could be grouped: 76 services (24%) were classed as level 1 (best), 53 (16%) as level 2 (medium), 113 (35%) as level 3 (poor), and 80 (25%) as level 4 (very poor). Parameters of service delivery processes were more important than those relating to service inputs for determining the quality classification. Predictors of quality services included larger care sites, specialization for HIV/AIDS, and location within large municipalities. Conclusion: The survey demonstrated highly variable levels of HIV/AIDS service quality across the sites. Many sites were found to have deficiencies in the processes of service delivery processes that could benefit from quality improvement initiatives. These findings could have implications for how HIV/AIDS services are planned in Brazil to achieve quality standards, such as for where service sites should be located, their size and staffing requirements. A set of service delivery indicators has been identified that could be used for routine monitoring of HIV/AIDS service delivery for HIV/AIDS in Brazil (and potentially in other similar settings).

Transmission potential, skin inflammatory response, and parasitism of symptomatic and asymptomatic dogs with visceral leishmaniasis

Background: Visceral leishmaniasis in Brazil is caused by the protozoan Leishmania (Leishmania) chagasi and it is transmitted by sandfly of the genus Lutzomyia. Dogs are an important domestic reservoir, and control of the transmission of visceral leishmaniasis (VL) to humans includes the elimination of infected dogs. However, though dogs are considered to be an important element in the transmission cycle of Leishmania, the identification of infected dogs representing an immediate risk for transmission has not been properly evaluated. Since it is not possible to treat infected dogs, they are sacrificed when a diagnosis of VL is established, a measure that is difficult to accomplish in highly endemic areas. In such areas, parameters that allow for easy identification of reservoirs that represents an immediate risk for transmission is of great importance for the control of VL transmission. In this study we aimed to identify clinical parameters, reinforced by pathological parameters that characterize dogs with potential to transmit the parasite to the vector. Results: The major clinical manifestations of visceral leishmaniasis in dogs from an endemic area were onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. The transmission potential of these dogs was assessed by xenodiagnosis using Lutzomyia longipalpis. Six of nine symptomatic dogs were infective to Lutzomyia longipalpis while none of the five asymptomatic dogs were infective to the sandfly. Leishmania amastigotes were present in the skin of all clinically symptomatic dogs, but absent in asymptomatic dogs. Higher parasite loads were observed in the ear and ungueal region, and lower in abdomen. The inflammatory infiltrate was more intense in the ears and ungueal regions of both symptomatic and asymptomatic dogs. In clinically affected dogs in which few or none Leishmania amastigotes were observed, the inflammatory infiltrate was constituted mainly of lymphocytes and macrophages. When many parasites were present, the infiltrate was also comprised of lymphocytes and macrophages, as well as a larger quantity of polymorphonuclear neutrophils (PMNs). Conclusion: Dogs that represent an immediate risk for transmission of Leishmania in endemic areas present clinical manifestations that include onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. Lymphadenopathy in particular was a positive clinical hallmark since it was closely related to the positive xenodiagnosis.

Incidence and predictors of severe liver fibrosis in HIV-infected patients with chronic hepatitis C in Brazil

The aim of this study was to examine the incidence and factors associated with the severity of liver fibrosis in 234 coinfected patients in Brazil. Patients were cared for in our clinic, from 1996 to 2004. Eligible patients were defined as patients with documented HIV and hepatitis C virus (HCV) infections and had previously undergone a liver biopsy. Patients with persistently normal alanine aminotransferase (ALT) were also included. The variables selected for study were age, gender, risk category, history of high alcohol consumption, CD4(+) T cell count, antiretroviral therapy usage, HCV genotype and duration of HCV infection. Stage of fibrosis was scored as follows: F0, no fibrosis; F1, portal fibrosis with no septa; F2, portal fibrosis with few septa; F3, bridging fibrosis with many septa; and F4, cirrhosis. The liver fibrosis stage was F3 in 39 (16.6%) and F4 in 20(8.5%) patients. Among patients with normal ALT, the liver fibrosis stage was F3-F4 in three patients (5.6%). Predictors of severe liver fibrosis (17344) by multivariate analysis were age (older patients) and genotype 3 (genotype I odds ratio [OR], 0.28; 95% confidence interval [0], 0.12 0.65). In summary, in the present study severe liver fibrosis was found in 25% of our patients and was associated with an age of more than 38 years at the time of liver biopsy as well as, HCV genotype 3. No differences were found with respect to CD4(+) T cell counts although patients with a CD4(+) T cell count greater than 50 were excluded.

Integration and visualization of systems biology data in context of the genome

Background: High-density tiling arrays and new sequencing technologies are generating rapidly increasing volumes of transcriptome and protein-DNA interaction data. Visualization and exploration of this data is critical to understanding the regulatory logic encoded in the genome by which the cell dynamically affects its physiology and interacts with its environment. Results: The Gaggle Genome Browser is a cross-platform desktop program for interactively visualizing high-throughput data in the context of the genome. Important features include dynamic panning and zooming, keyword search and open interoperability through the Gaggle framework. Users may bookmark locations on the genome with descriptive annotations and share these bookmarks with other users. The program handles large sets of user-generated data using an in-process database and leverages the facilities of SQL and the R environment for importing and manipulating data. A key aspect of the Gaggle Genome Browser is interoperability. By connecting to the Gaggle framework, the genome browser joins a suite of interconnected bioinformatics tools for analysis and visualization with connectivity to major public repositories of sequences, interactions and pathways. To this flexible environment for exploring and combining data, the Gaggle Genome Browser adds the ability to visualize diverse types of data in relation to its coordinates on the genome. Conclusions: Genomic coordinates function as a common key by which disparate biological data types can be related to one another. In the Gaggle Genome Browser, heterogeneous data are joined by their location on the genome to create information-rich visualizations yielding insight into genome organization, transcription and its regulation and, ultimately, a better understanding of the mechanisms that enable the cell to dynamically respond to its environment.

Ovariole structure and oogenesis in queens and workers of the stingless bee Melipona quadrifasciata (Hymenoptera: Apidae, Meliponini) kept under different social conditions

The high variability in the reproductive biology of stingless bees makes them very amenable for comparative studies with other eusocial bee taxa. We investigated the structural organization of the ovaries of Melipona quadrifasciata queens and workers kept under different social conditions by analyzing their general histology, mitotic activity, and microfilament organization. The overall dynamics of ovarian activity were similar in the two castes, and at emergence their ovarioles contained a previtellogenic follicle. Stingless bees and honey bees differ in the structural organization in the lower germarium, but they have in common synchronized mitotic activity and putative germ line stem cells in the terminal filament. Unlike honey bees, stingless bee workers lay trophic eggs in addition to reproductive eggs. The overall similarities in oogenesis between the two taxa suggest that the decision to form trophic eggs should only occur in the late stages of oogenesis.

Genome Survey Sequence Analysis and Identification of Homologs of Major Surface Protease (gp63) Genes in Trypanosoma rangeli

In this study, 222 genome survey sequences were generated for Trypanosoma rangeli strain P07 isolated from an opossum (Didelphis albiventris) in Minas Gerais State, Brazil. T. rangeli sequences were compared by BLASTX (Basic Local Alignment Search Tool X) analysis with the assembled contigs of Leishmania braziliensis, Leishmania infantum, Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi. Results revealed that 82% (182/222) of the sequences were associated with predicted proteins described, whereas 18% (40/222) of the sequences did not show significant identity with sequences deposited in databases, suggesting that they may represent T. rangeli-specific sequences. Among the 182 predicted sequences, 179 (80.6%) had the highest similarity with T. cruzi, 2 (0.9%) with T. brucei, and 1 (0.5%) with L. braziliensis. Computer analysis permitted the identification of members of various gene families described for trypanosomatids in the genome of T. rangeli, such as trans-sialidases, mucin-associated surface proteins, and major surface proteases (MSP or gp63). This is the first report identifying sequences of the MSP family in T. rangeli. Multiple sequence alignments showed that the predicted MSP of T. rangeli presented the typical characteristics of metalloproteases, such as the presence of the HEXXH motif, which corresponds to a region previously associated with the catalytic site of the enzyme, and various cysteine and proline residues, which are conserved among MSPs of different trypanosomatid species. Reverse transcriptase-polymerase chain reaction analysis revealed the presence of MSP transcripts in epimastigote forms of T. rangeli.

A scientific note on the isolation and characterization of microsatellite loci of Frieseomelitta varia (Hymenoptera, Apidae, Meliponini)

FAPESP[BIOTA 2004/15801-0]

Tumor slices as a model to evaluate doxorubicin in vitro treatment and expression of trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 in canine mammary gland cancer

Background: In women with breast cancer submitted to neoadjuvant chemotherapy based in doxorubicin, tumor expression of groups of three genes (PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2) have classified them as responsive or resistant. We have investigated whether expression of these trios of genes could predict mammary carcinoma response in dogs and whether tumor slices, which maintain epithelial-mesenchymal interactions, could be used to evaluate drug response in vitro. Methods: Tumors from 38 dogs were sliced and cultured with or without doxorubicin 1 mu M for 24 h. Tumor cells were counted by two observers to establish a percentage variation in cell number, between slices. Based on these results, a reduction in cell number between treated and control samples >= 21.7%, arbitrarily classified samples, as drug responsive. Tumor expression of PRSS11, MTSS1, CLPTM1 and SMYD2, was evaluated by real time PCR. Relative expression results were then transformed to their natural logarithm values, which were spatially disposed according to the expression of trios of genes, comprising PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. Fisher linear discrimination test was used to generate a separation plane between responsive and non-responsive tumors. Results: Culture of tumor slices for 24 h was feasible. Nine samples were considered responsive and 29 non-responsive to doxorubicin, considering the pre-established cut-off value of cell number reduction = 21.7%, between doxorubicin treated and control samples. Relative gene expression was evaluated and tumor samples were then spatially distributed according to the expression of the trios of genes: PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. A separation plane was generated. However, no clear separation between responsive and non-responsive samples could be observed. Conclusion: Three-dimensional distribution of samples according to the expression of the trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 could not predict doxorubicin in vitro responsiveness. Short term culture of mammary gland cancer slices may be an interesting model to evaluate chemotherapy activity.

Vectorial Competence of Larvae and Adults of Alphitobius diaperinus in the Transmission of Salmonella Enteritidis in Poultry

Introduction: The ingestion of food products originating from poultry infected with Salmonella spp. is one of the major causes of food poisoning in humans. The control of poultry salmonellosis is particularly difficult since birds are asymptomatic and numerous factors may expedite the maintenance of bacteria in poultry production facilities. Objective: The aim of the study was to determine the vectorial capacity of adults and larvae of Alphitobius diaperinus (Coleoptera: Tenebrionidae) in the experimental transmission of Salmonella Enteritidis phage type 4 to 1-day-old specific pathogen-free White Leghorn chicks. Methods: Adult insects and larvae were starved for 1 day, fed for 24 h or 7 days on sterile ration that had been treated with Salmonella Enteritidis phage type 4, and the levels of bacterial infection were determined. Infected adult insects and larvae were fed to groups of day-old chicks, after which bacteria were recovered from cecum, liver, and spleen samples over a 7-day period. Results: Infected larvae were more efficient than adult insects in transmitting Salmonella Enteritidis to chicks. Higher concentrations of bacteria could be reisolated from the cecum, liver, and spleen of chicks that had ingested infected larvae compared with those that had ingested infected adults. Conclusions: The control of A. diaperinus, and particularly of the larvae, represents a critical factor in the reduction of Salmonella spp. in poultry farms.

SEROPREVALENCE AND ISOLATION OF TOXOPLASMA GONDII FROM SHEEP FROM SAO PAULO STATE, BRAZIL

Sheep are important in the epidemiology of Toxoplasma gondii infection, but little is known of ovine toxoplasmosis in Brazil. Antibodies to T. gondii were assayed in sera of 495 sheep from 36 countries of Sao Paulo state. Brazil, using the modified agglutination test (MAT titer >= 1:25); 120 (24.2%) sheep tested positive. Samples of brain, heart, and diaphragm of 85 seropositive sheep were pooled, digested in pepsin, and bioassayed in mice. Toxoplasma gondii was isolated from tissue homogenated of 16 sheep, and the isolated were designated TgShBr 1-16. Six of the 16 T. gondii isolated killed 100% of infected mice. Results indicate that asymptomatic sheep can harbour mouse-virulent T. gondii; hence, they can serve as a source of infection for humans.

DESCRIPTION OF ADULTS AND NYMPH, AND REDESCRIPTION OF THE LARVA, OF ORNITHODOROS MARINKELLEI (ACARI: ARGASIDAE), WITH DATA ON ITS PHYLOGENETIC POSITION

The argasid tick Ornithodoros marinkellei Kohls, Clifford, and Jones, 1969 was described 4 decades ago based on larval specimens collected from bats (Pteronotus spp.) in Colombia and Panama. Thereafter, larval O. marinkellei parasitizing bats were reported from Venezuela, Guyana, and Brazil. Herein, we describe the adults and nymph, and redescribe the larva of O. marinkellei based on specimens recently collected in the western Brazilian Amazon region. In contrast to all other known adult argasids, the idiosoma of both males and females of O. marinkellei is covered with sclerotized plaques. The idiosoma of the nymph of O. marinkellei is entirely micromamillated, and differs from the adults by the absence of plaques. The larva of O. marinkellei is morphologically similar to the larvae of the 2 other species belonging to the subgenus Subparmatus, i.e., Ornithodoros viguerasi Cooley and Kohls, 1941 and Ornithodoros mormoops Kohls, Clifford, and Jones, 1969. Because of the long and narrow dorsal plate, the larva of O. marinkellei is readily distinguished from O. viguerasi and O. mormoops. Comparison of our larvae from Brazil with O. marinkellei paratype specimens from Colombia confirmed their taxonomic identification. However, a few morphological differences, particularly in the size of the gnathosoma, were observed. Further studies are necessary to clarify whether O. marinkellei is a complex of different species, or a single species represented by morphologically polymorphic, and geographically distinct populations. Partial mitochondrial 16S rDNA gene sequences were generated for O. marinkellei specimens from Brazil, and compared with available homologous sequences in GenBank. Phylogenetic analyses revealed O. marinkellei to be distinct from the remaining argasid species available in GenBank, including other bat-associated tick species that are found in sympatry with O. marinkellei in the Neotropical region.

Experimental Infection of the Opossum Didelphis aurita by Rickettsia felis, Rickettsia bellii, and Rickettsia parkeri and Evaluation of the Transmission of the Infection to Ticks Amblyomma cajennense and Amblyomma dubitatum

This work evaluated the infection of opossums (Didelphis aurita) by Rickettsia felis, Rickettsia bellii, and Rickettsia parkeri and their role as amplifier hosts for horizontal transmission to Amblyomma cajennense and/or Amblyomma dubitatum ticks. Infection in D. aurita was induced by intraperitoneal inoculation with R. felis (n = 4 opossums), R. bellii (n = 4), and R. parkeri (n = 2). Another group of six opossums were inoculated intraperitoneally with Leibovitz-15 sterile culture medium, representing the uninfected groups (n = 2 opossums simultaneously to each infected group). Opossum blood samples collected during the study were used for DNA extraction, followed by real-time polymerase chain reaction targeting the rickettsial gene gltA, hematology, and detection of Rickettsia spp.-reactive antibodies by indirect immunofluorescence assay. Opossums were infested with uninfected A. cajennense and/or A. dubitatum for 30 days postinoculation (DPI). Flat ticks molted from ticks fed on opossums were allowed to feed on uninfected rabbits, which were tested for seroconversion by immunofluorescence assay. Samples of flat ticks were also tested by real-time polymerase chain reaction. Inoculated opossums showed no clinical abnormalities. Antibodies to Rickettsia spp. were first detected at the second to fourth DPI, with detectable titers until the 150th DPI. Rickettsemia was detected only in one opossum inoculated with R. parkeri, at the eighth DPI. Only one A. cajennense tick (2.0%) previously fed on a R. parkeri-inoculated opossum became infected. None of the rabbits infested with opossum-derived ticks seroconverted. The study demonstrated that R. felis, R. bellii, and R. parkeri were capable to produce antibody response in opossums, however, with undetectable rickettsemia for R. felis and R. bellii, and very low rickettsemia for R. parkeri. Further studies must be done with different strains of these rickettsiae, most importantly the strains that have never gone through in vitro passages.

T cells, adhesion molecules and modulation of apoptosis in visceral leishmaniasis glomerulonephritis

Background: Immune complex deposition is the accepted mechanism of pathogenesis of VL glomerulopathy however other immune elements may participate. Further in the present study, no difference was seen between immunoglobulin and C3b deposit intensity in glomeruli between infected and non-infected dogs thus T cells, adhesion molecules and parameters of proliferation and apoptosis were analysed in dogs with naturally acquired VL from an endemic area. The dog is the most important domestic reservoir of the protozoa Leishmania (L.) chagasi that causes visceral leishmaniasis (VL). The similarity of VL manifestation in humans and dogs renders the study of canine VL nephropathy of interest with regard to human pathology. Methods: From 55 dogs with VL and 8 control non-infected dogs from an endemic area, kidney samples were analyzed by immunohistochemistry for immunoglobulin and C3b deposits, staining for CD4+ and CD8+ T cells, ICAM-1, P-selectin and quantified using morphometry. Besides proliferation marker Ki-67, apoptosis markers M30 and TUNEL staining, and related cytokines TNF-alpha, IL-1 alpha were searched and quantified. Results: We observed similar IgG, IgM and IgA and C3b deposit intensity in dogs with VL and non-infected control dogs. However we detected the Leishmania antigen in cells in glomeruli in 54, CD4+ T cells in the glomeruli of 44, and CD8+ T cells in 17 of a total of 55 dogs with VL. Leishmania antigen was absent and T cells were absent/scarse in eight non-infected control dogs. CD 4+ T cells predominate in proliferative patterns of glomerulonephritis, however the presence of CD4+ and CD8+ T cells were not different in intensity in different patterns of glomerulonephritis. The expression of ICAM-1 and P-selectin was significantly greater in the glomeruli of infected dogs than in control dogs. In all patterns of glomerulonephritis the expression of ICAM-1 ranged from minimum to moderately severe and P-selectin from absent to severe. In the control animals the expression of these molecules ranged from absent to medium intensity. It was not observed any correlation between severity of the disease and these markers. There was a correlation between the number of Leishmania antigen positive cells and CD4+ T cells, and between the number of CD4+ T cells and CD8+ T cells. In dogs presenting different histopathological patterns of glomerulonephritis, parameters of proliferation and apoptosis were studied. Ki-67, a proliferative marker, was not detected locally, but fewer apoptotic cells and lower TNF-alpha expression were seen in infected animals than in non-infected controls. Conclusion: Immunopathogenic mechanisms of VL glomerulonephritis are complex and data in the present study suggest no clear participation of immunoglobulin and C3b deposits in these dogs but the possible migration of CD4+ T cells into the glomeruli, participation of adhesion molecules, and diminished apoptosis of cells contributing to determine the proliferative pattern of glomerulonephritis in VL.

Experimental Infection of Opossums Didelphis aurita by Rickettsia rickettsii and Evaluation of the Transmission of the Infection to Ticks Amblyomma cajennense

The present study evaluated the infection of opossums (Didelphis aurita) by Rickettsia rickettsii and their role as amplifier hosts for horizontal transmission of R. rickettsii to Amblyomma cajennense ticks. Three groups of opossums were evaluated: on day 0, group 1 (G1) was inoculated intraperitoneally with R. rickettsii; group 2 (G2) was infested by R. rickettsii-infected ticks; and group 3 (G3) was the uninfected control group. Opossum rectal temperature was measured daily. Blood samples were collected every 2 to 4 days during 30 days, and used to (1) inoculate guinea pigs intraperitoneally; (2) extract DNA followed by real-time polymerase chain reaction (PCR) targeting the rickettsial gene gltA; (3) study hematology; (4) detect R. rickettsii-reactive antibodies by indirect direct immunofluorescence assay (IFA). Blood was also collected every 10 days from days 30 to 180, to be tested by serology. Opossums were infested by uninfected A. cajennense larvae and nymphs from days 3 to 15. Engorged ticks were collected and allowed to molt in an incubator. Thereafter, the subsequent flat ticks were allowed to feed on uninfected rabbits, which were tested for seroconversion by IFA. Samples of flat ticks were also tested by real-time PCR. All G1 and G2 opossums became infected by R. rickettsii, as demonstrated by real-time PCR or/and guinea pig inoculation, but they showed no clinical abnormality. Rickettsemia was first detected at days 2 to 8, lasting intermittently till days 1 to 30. Approximately 18% and 5% of the flat ticks previously fed on G1 and G2 opossums, respectively, became infected by R. rickettsii, but only the rabbits infested with G1-derived ticks seroconverted. The study demonstrated that R. rickettsii was capable of infecting opossums without causing illness and developing rickettsemia capable of causing infection in guinea pigs and ticks, although the infection rate in ticks was low.