Complete sequencing of the Xylella fastidiosa genome revealed characteristics that have not been described previously for a phytopathogen. One characteristic of this genome was the abundance of genes encoding proteins with adhesion functions related to biofilm formation, an essential step for colonization of a plant host or an insect vector. We examined four of the proteins belonging to this class encoded by genes in the genome of X. fastidiosa: the PilA2 and PilC fimbrial proteins, which are components of the type IV pili, and XadA1 and XadA2, which are afimbrial adhesins. Polyclonal antibodies were raised against these four proteins, and their behavior during biofilm development was assessed by Western blotting and immunofluorescence assays. In addition, immunogold electron microscopy was used to detect these proteins in bacteria present in xylem vessels of three different hosts (citrus, periwinkle, and hibiscus). We verified that these proteins are present in X. fastidiosa biofilms but have differential regulation since the amounts varied temporally during biofilm formation, as well as spatially within the biofilms. The proteins were also detected in bacteria colonizing the xylem vessels of infected plants.

The efficacy of conjunctival swab (CS) as a sampling method for visceral leishmaniasis (VL) diagnosis by PCR of asymptomatic dogs was evaluated. The CS was compared to blood samples (B) and skin biopsies (SB), two less invasive samples potentially useful for massive screening of dogs. Thirty asymptomatic dogs, with serological and parasitological positive tests, were used. The samples were analyzed by two PCR methods: kDNA PCR-hybridization and ITS-1 nPCR. The DNA sample volume used was of 1.0 microL and 10.0 microL respectively. Using CS samples the kDNA PCR-hybridization was able to detected parasite DNA in 24/30 dogs (80%) using the right conjunctiva (RC) and 23/30 dogs (76.6%) with the left conjunctiva (LC), 17/30 dogs (56.7%) by means of SB and 4/30 dogs (13.3%) with B. The CS positivity obtained combining RC and LC results was of 90% (27/30 dogs). The assay of CS samples by ITS-1 nPCR revealed that 25/30 dogs (83.3%) were positive when using RC and 20/30 dogs (66.6%) were positive when using LC. Via the same method 15/30 dogs (50.0%) were positive by SB and 17/30 dogs (56.7%) with B. The CS positivity obtained by ITS-1 nPCR combining RC and LC was of 83.3%. The CS positivities for RC and LC were significantly higher (p<0.05) than SB and B for kDNA PCR-hybridization method. Statistical difference in relation to SB and B was verified by ITS-1 nPCR only for RC. The kDNA PCR-hybridization and ITS-1 nPCR methods showed similar sensitivities for CS and SB samples. On the other hand, for blood samples, the positivity of ITS-1 nPCR was significantly higher than the one obtained by the kDNA PCR-hybridization, indicating that sensitivity of PCR methods can vary according to the biological sample examined. Our results showed that CS was suitable to detect Leishmania DNA in asymptomatic animals when comparing to other low-invasive samples. The CS sensitivities obtained in this study were similar to the ones observed in other studies for VL diagnosis in symptomatic dogs. We concluded that the use of CS for regular screenings of dogs by PCR should be considered.

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The study aims to build a general overview of the Nursing Graduation Programs in Brazil, in the decade of the National Curriculum Guidelines for Nursing Graduation, period 2001-2011. This is an exploratory, descriptive study, based on data from the e-MEC, Higher Education Census, ENADE. The results evidenced: the privatization of the Nursing Graduation Programs, the oversupply of courses and places day and night; sharp expansion of the number of vacancies for distance learning, not meeting the minimum quality criteria evaluated by ENADE, among other respects. It is recommended that strategies for the regulation of the expansion already installed must be defined, in addition to the revision of indicators drawn from the INEP Single Form, to meet the necessities of nursing, as an specific area, particularly with regard to: the number of vacancies, integration with the local and regional health system and the SUS, education in health care, teaching practical activities, working arrangements and experience of the faculty of the course. It is recommended, though, the immediate intervention of the MEC in the poles of Distance Education, suspending the training of nurses in that modality of education.

In order to understand the determinants of human infection by Leishmania chagasi in an urban area, a cross-sectional population based study was conducted using molecular and serologic methods to identify infection. Participants were interviewed using a pre-coded questionnaire. Two criteria were tested to identify risk factors: Model 1-including all participants positive in hybridization by Leishmania donovani complex probe; Model 2-including all participants positive for hybridization and at least one serologic test. In Model 1, the variables associated with infection were: ownership of birds, time spent outside house between 6:00-10:00 PM and garbage not collected. In Model 2, the variables associated with infection were: family with knowledge of the vector, garbage not collected, garbage not removed or buried, ownership of birds and eroded areas in the neighborhood. The risk factors identified were associated with household conditions, presence of animals and the likelihood of contact with phlebotomine sandflies. Key-words: Visceral leishmaniasis. Kala-Azar. Leishmania chagasi. Asymptomatic infection. Risk factors. RESUMOCom o objetivo de identificar os determinantes da infecção humana por Leishmania chagasi em uma área urbana, foi realizado um estudo seccional de base populacional utilizando-se métodos moleculares e sorológicos para identificar a infecção. Os participantes foram entrevistados utilizando-se questionário pré-codificado. Dois critérios foram testados para identificar os fatores de risco: Modelo 1-incluindo todos os participantes positivos na hibridização com sonda para o complexo Leishmania donovani; Modelo 2-incluindo todos os participantes positivos na hibridização e em pelo menos um teste sorológico. No Modelo 1, as variáveis associadas à infecção foram: criar pássaros, encontrar-se fora de casa entre 18:00-22:00h e não ter o lixo coletado. No Modelo 2, as variáveis associadas à infecção foram: família conhecer o vetor, não ter o lixo coletado, lixo não removido ou queimado, criar pássaros, proximidade de áreas erodidas. Os fatores de risco identificados foram associados às condições das moradias, presença de animais e probabilidade de contato com flebotomíneos. Palavras-chaves: Leishmaniose visceral. Calazar. Leishmania chagasi. Infecção assintomática. Fatores de risco.

BackgroundOne of the most important drawbacks in visceral leishmaniasis (VL) population studies is the difficulty of diagnosing asymptomatic carriers. The aim of this study, conducted in an urban area in the Southeast of Brazil, was to evaluate the performance of serology to identify asymptomatic VL infection in participants selected from a cohort with a two-year follow-up period.MethodologyBlood samples were collected in 2001 from 136 cohort participants (97 positive and 39 negatives, PCR/hybridization carried out in 1999). They were clinically evaluated and none had progressed to disease from their asymptomatic state. As controls, blood samples from 22 control individuals and 8 patients with kala-azar were collected. Two molecular biology techniques (reference tests) were performed: PCR with Leishmania-generic primer followed by hybridization using L. infantum probe, and PCR with specific primer to L. donovani complex. Plasma samples were tested by ELISA using three different antigens: L. infantum and L. amazonensis crude antigens, and rK39 recombinant protein. Accuracy of the serological tests was evaluated using sensitivity, specificity, likelihood ratio and ROC curve.FindingsThe presence of Leishmania was confirmed, by molecular techniques, in all kala-azar patients and in 117 (86%) of the 136 cohort participants. Kala-azar patients showed high reactivity in ELISAs, whereas asymptomatic individuals presented low reactivity against the antigens tested. When compared to molecular techniques, the L. amazonensis and L. infantum antigens showed higher sensitivity (49.6% and 41.0%, respectively) than rK39 (26.5%); however, the specificity of rK39 was higher (73.7%) than L. amazonensis (52.6%) and L. infantum antigens (36.8%). Moreover, there was low agreement among the different antigens used (kappa<0.10).ConclusionsSerological tests were inaccurate for diagnosing asymptomatic infections compared to molecular methods; this could lead to misclassification bias in population studies. Therefore, studies which have used serological assays to estimate prevalence, to evaluate intervention programs or to identify risk factors for Leishmania infection, may have had their results compromised.

BackgroundThe aim of this study was to evaluate the potential use of nasal, oral, and ear swabs for molecular diagnosis of canine visceral leishmaniasis (CVL) in an endemic urban area in Brazil.Methodology/Principal FindingsSixty-two naturally infected and ten healthy dogs were enrolled in this study. Bone marrow aspirates, peripheral blood, skin biopsy, and conjunctival, nasal, oral, and ear swabs were collected. All samples, except blood, were submitted to conventional PCR (cPCR) and quantitative real time PCR (qPCR) to detect and quantify Leishmania infantum DNA, respectively. All dogs were submitted to thorough clinical analysis and were included based on a combination of serological (ELISA immunoassay and immunofluorescent antibody test) and parasitological methods. The cPCR positivity obtained from nasal swab samples was 87% (54/62), equivalent to those from other samples (P>0.05). Positive results were obtained for 79% (22/28) in oral swabs and 43% (12/28) in ear swab samples. A significant difference was observed between these data (P = 0.013), and the frequency of positive results from oral swab was equivalent to those from other samples (P>0.05). The use of ear swab samples for cPCR assays is promising because its result was equivalent to skin biopsy data (P>0.05). The qPCR data revealed that parasite loads in mucosal tissues were similar (P>0.05), but significantly lower than the parasite burden observed in bone marrow and skin samples (P<0.05).ConclusionsNasal and oral swab samples showed a high potential for the qualitative molecular diagnosis of CVL because their results were equivalent to those observed in samples collected invasively. Considering that mucosae swab collections are painless, noninvasive, fast and practical, the combination of these samples would be useful in massive screening of dogs. This work highlights the potential of practical approaches for molecular diagnosis of CVL and human leishmaniasis infections.

A population-based cross-sectional study was set up in Sabará country, Southeastern Brazil, to identify asymptomatic human visceral leishmaniasis in an urban area of low disease prevalence. Blood was collected on filter paper (n=1,604 inhabitants) and examined by indirect immunofluorescent test, enzyme-linked immunosorbent assay and immunochromatographic strip test. The prevalence rates of infection ranged from 2.4 to 5.6% depending on the test used. One year later, venous blood was collected in a subset of 226 participants (102 seropositive and 124 seronegative). The tests performed were IFAT, ELISA, rk39-ELISA, polymerase chain reaction and hybridization with Leishmania donovani complex probe. No clinical signs or symptoms of leishmaniasis were observed. Using hybridization as a reference test, the sensitivity and specificity of serology were respectively: 24.8 and 71% (ELISA); 26.3 and 76.3% (rk-39); 30.1 and 63.4% (IFAT). Due to disagreements, different criteria were tested to define the infection and hybridization should be considered in epidemiological studies. Key-words: Asymptomatic visceral leishmaniasis. Leishmania chagasi. Prevalence rate. Diagnostic. Hybridization. RESUMOUm estudo seccional de base populacional foi desenvolvido no município de Sabará, região sudeste do Brasil, para identificar a leishmaniose visceral humana assintomática em uma área urbana de baixa prevalência da doença. Foi coletado sangue em papel filtro (n=1.604 moradores), sendo examinados pela reação de imunofluoresência indireta, ensaio imunoenzimático e teste imunocromatográfico (strip test). As taxas de prevalência da infecção variaram de 2,4 a 5,6%, dependendo do teste utilizado. Um ano depois foi coletado sangue venoso de um subgrupo de 226 participantes (102 soropositivos e 124 soronegativos). Os testes realizados foram IFAT, ELISA, rk39-ELISA, reação em cadeia da polimerase e hibridização com sonda específica para o complexo Leishmania donovani. Não foi observado nenhum sinal clínico ou sintoma de leishmaniose. Usando a hibridização como teste de referência, a sensibilidade e especificidade dos testes sorológicos foram, respectivamente: 24.8 e 71% (ELISA); 26,3 e 76,3% (rk39-ELISA); 30,1 e 63,4% (IFAT). Devido a discordâncias, diferentes critérios foram testados para definir a presença da infecção e a hibridização deveria ser considerada em estudos epidemiológicos. Palavras-chaves: Leishmaniose visceral assintomática. Leishmania chagasi. Taxa de prevalência. Diagnóstico. Hibridização.