HIV associado a desordens neurocognitivas (HAND) continua a ser um grave problema atualmente devido à alta prevalência de suas formas mais brandas. Indivíduos HIV+ possuem o comprimento dos telômeros significativamente mais curtos nas células mononucleares do sangue periférico e células T CD8+, quando comparados aos indivíduos HIV negativos. Diante do exposto, o objetivo deste estudo foi avaliar a associação do comprimento dos telômeros de leucócitos em indivíduos infectados pelo HIV com deficiências cognitivas, pois ainda é um assunto bastante controverso. Métodos: Um total de 73 pacientes infectados pelo HIV-1 de ambos os sexos, com idades entre 20 a 60 anos, participaram deste estudo. Entre 19 indivíduos HIV(+) sem comprometimento cognitivo e 54 indivíduos HIV(+) com distúrbios neurocognitivos: 29 alteração neurocognitiva assintomático (ANI), 15 comprometimento neurocognitivo leve a moderado (MND) e, 10 demência associada ao HIV (HAD); 118 indivíduos HIV negativos formaram o grupo controle. Todos os participantes foram submetidos a uma série de testes neuropsicológicos previamente validados. Determinou-se a carga viral de HIV-1 nas células do líquido cefalorraquidiano (LCR) e em PBMC. Utilizou-se DNA a partir de leucócitos periféricos para calcular o comprimento de telômeros por PCR em tempo real. Resultados: O comprimento dos telômeros não foi associado com gêneros e diminuiu com a idade, independentemente do status de HIV. Indivíduos infectados pelo HIV-1com formas mais leves de deficiência neurocognitiva apresentaram um comprimento dos telômeros reduzida em comparação com pacientes HIV+ sem comprometimento neurocognitivo. Não houve correlação entre a carga viral plasmática e o tamanho dos telômeros. Conclusões: Nossos resultados sugerem que o comprimento dos telômeros pode ser considerado um marcador de senescência celular em indivíduos com alterações neurocognitivas / HIV associated neurocognitive disorders (HAND) remains a serious problem today because of the high prevalence of its milder forms. HIV + individuals have the length substantially shorter telomeres in peripheral blood mononuclear cells and CD8 + T cells compared to HIV negative individuals. Given the above, the objective of this study was to evaluate the association of telomere length of leukocyte (LTL) in HIV-infected individuals with cognitive disabilities because it is still a very controversial subject. Methods: A total of 73 patients infected with HIV-1 of both sexes, aged 20 to 60 years participated in this study. Among 19 HIV patients (+) without cognitive impairment and 54 HIV patients (+) with neurocognitive disorders: 29 asymptomatic neurocognitive disorder (ANI), 15 mild neurocognitive disorder to moderate (MND) and 10 HIVassociated dementia (HAD); 118 HIV-negative individuals formed the control group. All participants underwent a series of previously validated neuropsychological tests. Determined if the viral load of HIV-1 in cerebrospinal fluid cells (CSF) and in PBMC. We used DNA from peripheral leukocytes to calculate the length of telomeres by real time PCR. Results: The telomere length was not associated with genres and decreased with age, irrespective of HIV status. HIV-1-infected individuals with milder forms of neurocognitive impairment had a significantly length of telomeres reduced compared to HIV + patients without neurocognitive impairment. There was no correlation between plasma viral load and the size of telomeres. Conclusions: Our results suggest that telomere length can be considered a marker of cellular senescence in individuals with neurocognitive abnormalities

A sepse bacteriana constitui a causa mais frequente de óbitos neonatais, e seu diagnóstico é complexo devido à inexistência de um teste laboratorial definitivo. O presente estudo desenvolveu uma técnica de amplificação quantitativa (qPCR) do gene 16S rDNA de bactérias tanto para o diagnóstico de sepse neonatal, quanto para avaliar se a qPCR é capaz de monitorar o tratamento. Para ser recrutado o RN deveria apresentar ao menos dois sinais/sintomas sugestivos de sepse, e dois parâmetros laboratoriais alterados. Amostras de sangue foram colhidas no tempo zero (suspeita de sepse), 48 horas e sete dias após o início da antibioticoterapia. Foram analisados 73 RN (21 RNT e 52 RNPT) com suspeita de sepse neonatal. A hemocultura foi positiva em 32 RN (43,8% - sepse confirmada) e negativa em 41 (56,2% - sepse clínica), enquanto a qPCR foi positiva em 65 RN (89%) e negativa em oito casos (11%). Dentre os 32 RN com sepse confirmada (11 RNT e 21 RNPT), neutrofilia foi encontrada em 22 (68,75%), CRP elevada em 21 (65,62%), plaquetopenia em 15 (46,87%) e leucopenia em 14 (43,75%). Foram analisadas 200 amostras dos 73 casos suspeitos, considerando os três tempos de coleta, resultando em 36 hemoculturas positivas (18,0%) e 135 qPCR positivas (67,5%). Nas 36 hemoculturas positivas houve 38 isolamentos. Bactérias Gram-positivas foram encontradas em 32 amostras (84,21%) e Gram-negativas em seis (15,78%). Staphylococcus coagulase negativa predominou dentre as Gram-positivas (75,0%). No grupo de 32 RN com sepse confirmada a qPCR foi positiva em 30 (30/32 - 93,7%). Em 14 casos (47%) a qPCR antecipou o diagnóstico de sepse quando comparada à hemocultura e foi positiva no tempo zero em 22 casos (68,75%), enquanto a hemocultura foi positiva em 11. Dos 41 casos de sepse clínica, a qPCR foi positiva em 35 (85,4%); em 26 casos (74,3%) já no tempo zero. O teste de McNemar encontrou discordância entre os resultados das hemoculturas e qPCR (p<0,0001, IC de 95%), indicando superioridade da qPCR. Houve nove óbitos na casuística, todos com hemocultura e qPCR positiva. Em seis dos nove óbitos somente a terceira hemocultura foi positiva, enquanto a qPCR foi positiva em cinco casos já no tempo zero e não negativou em seis casos. A qPCR empregou a técnica de touchdown, com temperaturas de annealing decaindo de 66 a 62oC, limiar de detecção entre 1-10 UFC/mL. As cargas bacterianas foram em geral baixas (< 50 UFC/mL) mesmo nos casos com sepse confirmada e óbitos, porém quando as medianas das cargas bacterianas no tempo zero dos grupos com sepse confirmada (37,10 UFC/mL) e sepse clínica (24,49 UFC/mL) foram comparadas, foi encontrada uma diferença estatisticamente significante (p=0,0402). O estudo concluiu que a qPCR é capaz de detectar mais casos de sepse neonatal que a hemocultura, antecipando o diagnóstico na maior parte deles. Em relação à monitorização do tratamento, a qPCR apresentou associação com o sucesso ou falha terapêutica, negativou em casos que tiveram evolução favorável, não negativou na maior parte dos óbitos, porém há necessidade de confirmação destes dados / Bacterial sepsis constitutes one of the most frequent causes of neonatal deaths and its diagnosis is difficult due to the lack of a definitive laboratorial approach. The present study developed a bacterial 16S rDNA-based quantitative real time polymerase chain reaction (qPCR) both to the diagnosis of neonatal sepsis and to evaluate if qPCR is capable of monitoring antimicrobial treatment. For enrollment, the newborn (NB) should present, at least, two signs/symptoms suggestive of sepsis, and two abnormal laboratory parameters. Blood samples were collected on day zero (suspected sepsis), 48 hours and 7 days after the initiation of antibiotic therapy. Seventy-three newborns with suspected sepsis were recruited (21 term NB and 52 preterm NB), blood culture was positive in 32 (43.8% - confirmed sepsis) and negative in 41 (56.2% - clinical sepsis), while qPCR was positive in 65 (89.0%) and negative in 8 cases (11.0%). Considering the group of 32 NB with confirmed sepsis (11 TNB and 21PTNB), qPCR was positive in 30 (30/32 - 93.7%). Neutrophilia was found in 22 NB (68.75%), elevated CRP in 21 (65.62%), thrombocytopenia in 15 (46.87%) and leukopenia in 14 (43.75%). Of the 73 cases, taking into account the three collected samples (day zero, 48h and 7 days), 200 samples were analyzed, with 36 positive blood culture (18.0%) and 135 positive qPCR (67.5%). Of the 36 positive blood cultures, there were 38 bacterial isolations. Gram-positive bacteria were found in 32 samples (84.21%) and Gram-negative in 6 (15.78%). Coagulase-negative Staphylococcus was predominant in the Grampositive group (75.0%). In 14 cases, qPCR anticipated the diagnosis when compared with blood culture, and was positive in 22 cases on day zero (68.75%), whereas blood culture was positive in 11. Among the 41 cases of clinical sepsis, qPCR was positive in 35 (85.4%); of these 26 (74.3%) on day zero. McNemar test found discordance between the results of blood cultures and qPCR (p < 0.0001, CI of 95%), indicating superiority of qPCR. There were nine deaths in the casuistic, all with positive blood culture and qPCR. In six of the nine deaths only the third blood culture was positive, while qPCR was positive in five cases already on day zero, and was still positive in the third sample in 6 cases. The qPCR employed the touchdown technique, with annealing temperatures decreasing from 66 to 62oC, detection threshold between 1-10 CFU/ml. Bacterial loads were generally low ( < 50 CFU/ml), even in those cases with confirmed sepsis and deaths, however when bacterial load medians on day zero were compared between confirmed (37.1 CFU/ml) and clinical (24.49 CFU/ml) sepsis groups, a statistically significant difference was found (p = 0.0402). The study concluded that qPCR can detect more cases of neonatal sepsis than blood culture, anticipating the diagnosis in most of them. Regarding the monitoring of treatment, qPCR was associated with success or treatment failure, became negative in cases that progressed favorably, remained positive in the majority of the deaths, however these data need to be confirmed

HIV associado a desordens neurocognitivas (HAND) continua a ser um grave problema atualmente devido à alta prevalência de suas formas mais brandas. Indivíduos HIV+ possuem o comprimento dos telômeros significativamente mais curtos nas células mononucleares do sangue periférico e células T CD8+, quando comparados aos indivíduos HIV negativos. Diante do exposto, o objetivo deste estudo foi avaliar a associação do comprimento dos telômeros de leucócitos em indivíduos infectados pelo HIV com deficiências cognitivas, pois ainda é um assunto bastante controverso. Métodos: Um total de 73 pacientes infectados pelo HIV-1 de ambos os sexos, com idades entre 20 a 60 anos, participaram deste estudo. Entre 19 indivíduos HIV(+) sem comprometimento cognitivo e 54 indivíduos HIV(+) com distúrbios neurocognitivos: 29 alteração neurocognitiva assintomático (ANI), 15 comprometimento neurocognitivo leve a moderado (MND) e, 10 demência associada ao HIV (HAD); 118 indivíduos HIV negativos formaram o grupo controle. Todos os participantes foram submetidos a uma série de testes neuropsicológicos previamente validados. Determinou-se a carga viral de HIV-1 nas células do líquido cefalorraquidiano (LCR) e em PBMC. Utilizou-se DNA a partir de leucócitos periféricos para calcular o comprimento de telômeros por PCR em tempo real. Resultados: O comprimento dos telômeros não foi associado com gêneros e diminuiu com a idade, independentemente do status de HIV. Indivíduos infectados pelo HIV-1com formas mais leves de deficiência neurocognitiva apresentaram um comprimento dos telômeros reduzida em comparação com pacientes HIV+ sem comprometimento neurocognitivo. Não houve correlação entre a carga viral plasmática e o tamanho dos telômeros. Conclusões: Nossos resultados sugerem que o comprimento dos telômeros pode ser considerado um marcador de senescência celular em indivíduos com alterações neurocognitivas / HIV associated neurocognitive disorders (HAND) remains a serious problem today because of the high prevalence of its milder forms. HIV + individuals have the length substantially shorter telomeres in peripheral blood mononuclear cells and CD8 + T cells compared to HIV negative individuals. Given the above, the objective of this study was to evaluate the association of telomere length of leukocyte (LTL) in HIV-infected individuals with cognitive disabilities because it is still a very controversial subject. Methods: A total of 73 patients infected with HIV-1 of both sexes, aged 20 to 60 years participated in this study. Among 19 HIV patients (+) without cognitive impairment and 54 HIV patients (+) with neurocognitive disorders: 29 asymptomatic neurocognitive disorder (ANI), 15 mild neurocognitive disorder to moderate (MND) and 10 HIVassociated dementia (HAD); 118 HIV-negative individuals formed the control group. All participants underwent a series of previously validated neuropsychological tests. Determined if the viral load of HIV-1 in cerebrospinal fluid cells (CSF) and in PBMC. We used DNA from peripheral leukocytes to calculate the length of telomeres by real time PCR. Results: The telomere length was not associated with genres and decreased with age, irrespective of HIV status. HIV-1-infected individuals with milder forms of neurocognitive impairment had a significantly length of telomeres reduced compared to HIV + patients without neurocognitive impairment. There was no correlation between plasma viral load and the size of telomeres. Conclusions: Our results suggest that telomere length can be considered a marker of cellular senescence in individuals with neurocognitive abnormalities

A sepse bacteriana constitui a causa mais frequente de óbitos neonatais, e seu diagnóstico é complexo devido à inexistência de um teste laboratorial definitivo. O presente estudo desenvolveu uma técnica de amplificação quantitativa (qPCR) do gene 16S rDNA de bactérias tanto para o diagnóstico de sepse neonatal, quanto para avaliar se a qPCR é capaz de monitorar o tratamento. Para ser recrutado o RN deveria apresentar ao menos dois sinais/sintomas sugestivos de sepse, e dois parâmetros laboratoriais alterados. Amostras de sangue foram colhidas no tempo zero (suspeita de sepse), 48 horas e sete dias após o início da antibioticoterapia. Foram analisados 73 RN (21 RNT e 52 RNPT) com suspeita de sepse neonatal. A hemocultura foi positiva em 32 RN (43,8% - sepse confirmada) e negativa em 41 (56,2% - sepse clínica), enquanto a qPCR foi positiva em 65 RN (89%) e negativa em oito casos (11%). Dentre os 32 RN com sepse confirmada (11 RNT e 21 RNPT), neutrofilia foi encontrada em 22 (68,75%), CRP elevada em 21 (65,62%), plaquetopenia em 15 (46,87%) e leucopenia em 14 (43,75%). Foram analisadas 200 amostras dos 73 casos suspeitos, considerando os três tempos de coleta, resultando em 36 hemoculturas positivas (18,0%) e 135 qPCR positivas (67,5%). Nas 36 hemoculturas positivas houve 38 isolamentos. Bactérias Gram-positivas foram encontradas em 32 amostras (84,21%) e Gram-negativas em seis (15,78%). Staphylococcus coagulase negativa predominou dentre as Gram-positivas (75,0%). No grupo de 32 RN com sepse confirmada a qPCR foi positiva em 30 (30/32 - 93,7%). Em 14 casos (47%) a qPCR antecipou o diagnóstico de sepse quando comparada à hemocultura e foi positiva no tempo zero em 22 casos (68,75%), enquanto a hemocultura foi positiva em 11. Dos 41 casos de sepse clínica, a qPCR foi positiva em 35 (85,4%); em 26 casos (74,3%) já no tempo zero. O teste de McNemar encontrou discordância entre os resultados das hemoculturas e qPCR (p<0,0001, IC de 95%), indicando superioridade da qPCR. Houve nove óbitos na casuística, todos com hemocultura e qPCR positiva. Em seis dos nove óbitos somente a terceira hemocultura foi positiva, enquanto a qPCR foi positiva em cinco casos já no tempo zero e não negativou em seis casos. A qPCR empregou a técnica de touchdown, com temperaturas de annealing decaindo de 66 a 62oC, limiar de detecção entre 1-10 UFC/mL. As cargas bacterianas foram em geral baixas (< 50 UFC/mL) mesmo nos casos com sepse confirmada e óbitos, porém quando as medianas das cargas bacterianas no tempo zero dos grupos com sepse confirmada (37,10 UFC/mL) e sepse clínica (24,49 UFC/mL) foram comparadas, foi encontrada uma diferença estatisticamente significante (p=0,0402). O estudo concluiu que a qPCR é capaz de detectar mais casos de sepse neonatal que a hemocultura, antecipando o diagnóstico na maior parte deles. Em relação à monitorização do tratamento, a qPCR apresentou associação com o sucesso ou falha terapêutica, negativou em casos que tiveram evolução favorável, não negativou na maior parte dos óbitos, porém há necessidade de confirmação destes dados / Bacterial sepsis constitutes one of the most frequent causes of neonatal deaths and its diagnosis is difficult due to the lack of a definitive laboratorial approach. The present study developed a bacterial 16S rDNA-based quantitative real time polymerase chain reaction (qPCR) both to the diagnosis of neonatal sepsis and to evaluate if qPCR is capable of monitoring antimicrobial treatment. For enrollment, the newborn (NB) should present, at least, two signs/symptoms suggestive of sepsis, and two abnormal laboratory parameters. Blood samples were collected on day zero (suspected sepsis), 48 hours and 7 days after the initiation of antibiotic therapy. Seventy-three newborns with suspected sepsis were recruited (21 term NB and 52 preterm NB), blood culture was positive in 32 (43.8% - confirmed sepsis) and negative in 41 (56.2% - clinical sepsis), while qPCR was positive in 65 (89.0%) and negative in 8 cases (11.0%). Considering the group of 32 NB with confirmed sepsis (11 TNB and 21PTNB), qPCR was positive in 30 (30/32 - 93.7%). Neutrophilia was found in 22 NB (68.75%), elevated CRP in 21 (65.62%), thrombocytopenia in 15 (46.87%) and leukopenia in 14 (43.75%). Of the 73 cases, taking into account the three collected samples (day zero, 48h and 7 days), 200 samples were analyzed, with 36 positive blood culture (18.0%) and 135 positive qPCR (67.5%). Of the 36 positive blood cultures, there were 38 bacterial isolations. Gram-positive bacteria were found in 32 samples (84.21%) and Gram-negative in 6 (15.78%). Coagulase-negative Staphylococcus was predominant in the Grampositive group (75.0%). In 14 cases, qPCR anticipated the diagnosis when compared with blood culture, and was positive in 22 cases on day zero (68.75%), whereas blood culture was positive in 11. Among the 41 cases of clinical sepsis, qPCR was positive in 35 (85.4%); of these 26 (74.3%) on day zero. McNemar test found discordance between the results of blood cultures and qPCR (p < 0.0001, CI of 95%), indicating superiority of qPCR. There were nine deaths in the casuistic, all with positive blood culture and qPCR. In six of the nine deaths only the third blood culture was positive, while qPCR was positive in five cases already on day zero, and was still positive in the third sample in 6 cases. The qPCR employed the touchdown technique, with annealing temperatures decreasing from 66 to 62oC, detection threshold between 1-10 CFU/ml. Bacterial loads were generally low ( < 50 CFU/ml), even in those cases with confirmed sepsis and deaths, however when bacterial load medians on day zero were compared between confirmed (37.1 CFU/ml) and clinical (24.49 CFU/ml) sepsis groups, a statistically significant difference was found (p = 0.0402). The study concluded that qPCR can detect more cases of neonatal sepsis than blood culture, anticipating the diagnosis in most of them. Regarding the monitoring of treatment, qPCR was associated with success or treatment failure, became negative in cases that progressed favorably, remained positive in the majority of the deaths, however these data need to be confirmed

A triagem clínica para malária em candidatos à doação de sangue em uma região não endêmica é um recurso empregado na seleção do doador que visa minimizar o risco transfusional uma vez que não há triagem laboratorial para Plasmodium spp. Este trabalho analisou questionário empregado na triagem clínica para malária em um hemocentro público no período de 2008 até 2010. Além dos critérios preconizados pela legislação nacional, os candidatos eram submetidos a perguntas específicas sobre residência e/ou visitação de região de Mata Atlântica preservada com transmissão de malária ou ainda se participaram de algum estudo de malária. A resposta afirmativa a uma destas questões selecionou um grupo com 500 candidatos (risco para malária), possíveis portadores de malária subclínica e a resposta negativa a todas estas perguntas, outro grupo com 606 doadores, denominado grupo controle. Verificou-se que a correlação é significativa entre essas respostas e os resultados laboratoriais obtidos na pesquisa do DNA do Plasmodium falciparum (Pf), do Plasmodium malariae (Pm) e o do Plasmodium vivax (Pv). O DNA foi detectado por reação de cadeia de polimerase (PCR) em tempo real, conforme protocolo de Gama et al. No grupo de risco para malária com n=500, observou-se uma taxa de positividade de 61 (12,2%) no PCR para Plasmodium ssp e de 23 (3,79 %) no grupo controle com n=606. Dos 61 PCR positivos do grupo de risco, 53 (86,9%) foram identificados como P. falciparum, 7(11,5%) P. vivax, 1 (1,6%) misto para P. falciparum e P. vivax e 0 (0%) P. malariae. No grupo controle com n=606 e taxa positiva de PCR de 23 (3,79%), foi isolado P. vivax em 18 (78,3%) dos casos, P. falciparum em 4 (17,4%), infecção mista pelo P. vivax e P. falciparum em 1 (4,3%) e em nenhum caso o P. malariae. Verificou-se que o emprego deste questionário foi capaz de selecionar um número maior de candidatos infectados por Plasmodium spp (p < 0,001) / Clinical screening for malaria on blood donation candidates of a non-endemic region is a resource used in donors selection, which aims to minimize transfusion risks, since there is no laboratory screening for Plasmodium spp. The project analyzed a questionnaire used in the clinical screening for malaria for the selection of blood donation candidates at a public blood center in São Paulo, 2008 to 2010. In addition to the criteria recommended by national legislation, candidates were subjected to specific questions about where they reside and / or visitation of non-endemic regions with malaria transmission, or whether they were aware of any study of malaria there. A positive answer to at least one of those questions selected a group of 500 candidates who could be carriers of malaria, and negative answers defined a control group of 606 donors without risk of malaria. It was found that there is a significant correlation between these responses and the results obtained in laboratory in DNA research to the P falciparum, P malariae and P. vivax. DNA was detected by polymerase chain reaction (PCR) in real time according to the protocol of Gamma et al. In the risk group for malaria (500) was we observed a positive rate of 61 (12.2%) polymerase chain reaction for Plasmodium spp and a rate of 23 (3.79%) in the control group. Of the 61 (12.2%) found positive in the risk group, 53 (86.9%) were identified as P falciparum, 7(11,5%) P vivax, 1(1.6%) mixed for both P falciparum and P vivax and 0 (0%) P malariae. In the control group (606) with positive PCR rate of 23 (3.79%), the P vivax was isolated in 18 (78.3%), P falciparum in 4 (17.4%), mixed infection 1 (4.3%) for P vivax and P falciparum and none P malariae. It was found that the use of this questionnaire in screening for blood donors was able to select more candidates infected by Plasmodium spp, (p < 0.001).

Objective: The aim of this study was to develop an immunomagnetic/real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay and assess its clinical value for the molecular detection of circulating tumor cells (CTCs) in peripheral blood of pancreatic cancer patients. Methods: The presence of CTCs was evaluated in 34 pancreatic cancer patients before systemic therapy and in 40 healthy controls, through immunomagnetic enrichment, using the antibodies BM7 and VU1D9 [targeting mucin 1 and epithelial cell adhesion molecule (EpCAM), respectively], followed by real-time RT-PCR analysis of the genes KRT19, MUC1, EPCAM, CEACAM5 and BIRC5. Results: The developed assay showed high specificity, as none of the healthy controls were found to be positive for the multimarker gene panel. CTCs were detected in 47.1% of the pancreatic cancer patients before the beginning of systemic treatment. Shorter median progression-free survival (PFS) was observed for patients who had at least one detectable tumor-associated transcript, compared with patients who were CTC negative. Median PFS time was 66.0 days [95% confidence interval (CI) 44.8–87.2] for patients with baseline CTC positivity and 138.0 days (95% CI 124.1–151.9) for CTC-negative patients (p = 0.01, log-rank test). Conclusion: Our results suggest that in addition to the current prognostic methods, CTC analysis represents a potential complementary tool for prediction of outcome in pancreatic cancer patients. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

In previous studies silicon has been associated with reduced disease severity and incidence, the enhanced accumulation of phenolic compounds and lignin, and with changes in the defence-related enzyme activity and transcript abundance of defence and stress related genes. All of these aspects of plant defence were considered in this study on cotton infected with Fusarium oxysporum f. sp. vasinfectum (Fov), and the results obtained have greatly enhanced our understanding of the effects of silicon on this interaction. In all experiments conducted, defence responses were only significantly enhanced by silicon treatment following inoculation with Fov, strongly suggesting that silicon can prime defence responses in cotton infected with Fov. Sicot F-1 was the cultivar most resistant to Fov infection at the commencement of this research, whilst Sicot 189 was considered to have moderate resistance to the pathogen. Vascular discolouration was significantly reduced in the more resistant cultivar, Sicot F-1 following treatment with potassium silicate, compared to mock inoculated plants and inoculated plants treated with potassium sulphate or calcium sulphate. No significant differences between treatments were observed in the moderately resistant cultivar, Sicot 189, though further trials may need to be conducted to confirm this result. In both cultivars, silicon content was significantly greater in plants which had been treated regularly with liquid potassium silicate, rather than with calcium silicate powder. Histological investigation of cotton infected with Fov, with and without silicon treatment, was conducted to ascertain the effects of this element on the accumulation of fungitoxic phenolic compounds, cell ultrastructural changes and fungal infection structures. Fov proliferated through the cortex and stele of plants from both the resistant (Sicot F-1), and moderately resistant (Sicot 189) cultivars, regardless of silicon treatment. However, defences were more rapidly and intensely induced in endodermal and vascular regions of inoculated, potassium silicate treated Sicot F-1 plants. Significantly more phenolic compounds were present at seven days post infection (dpi) in root extracts of inoculated, potassium silicate treated Sicot F-1 plants. Phenolic compounds were not significantly increased in inoculated, potassium silicate treated root extracts of Sicot 189 plants at three or seven dpi. Lignin assays demonstrated that the dry weight percentage of lignin in root material from inoculated, potassium silicate treated Sicot F-1 plants was significantly higher than that of extracts from inoculated plants not receiving silicon treatment at three dpi. This trend was also observed at seven dpi; however lignin content was not significantly different in this case. Percentage lignin content in the roots of Sicot 189 plants was not significantly different between inoculated potassium silicate treated plants and those not treated with silicon. Histological alterations were not observed in mock inoculated water or potassium silicate treated plants, nor were any significant increases in phenolic compounds or lignin accumulation detected in control treatments not inoculated with the pathogen. The expression of several defence related genes was assessed with quantitative reverse transcriptase real-time polymerase chain reaction. The results obtained verify that potassium silicate can enhance defence responses in Sicot 189 and Sicot F-1 plants inoculated with Fov, with silicon having a more pronounced effect on the more resistant cultivar, Sicot F-1. Genes upregulated at three and four dpi in potassium silicate treated, Fov inoculated Sicot F-1 plants included peroxidase, cadinene synthase and polygalacturonase inhibiting protein (PGIP), with peroxidase associated with phenol oxidation and lignification and cadinene synthase with phytoalexin biosynthesis. Osmotin-like protein and chitinase class I were consistently upregulated in potassium silicate treated, inoculated Sicot 189 plants; both genes coding for pathogenesis related (PR) proteins, with chitinase also classified as an antifungal protein. In both cultivars, silicon treatment without Fov inoculation did not result in the significant up-regulation of any of the defence genes assessed, providing further evidence for the role of silicon in priming in this interaction. The activities of three defence related enzymes, peroxidase, chitinase and β-1, 3- glucanase was assessed in root and shoot material by colourimetric assays. Regular application of potassium silicate significantly increased the activity of peroxidase in root extracts from the highly resistant cultivar Sicot F-1, at three, four and seven dpi with Fov, and in root extracts from the moderately resistant Sicot 189 at three and four dpi. Significant increases in chitinase activity in inoculated, silicon treated Sicot 189 plants were observed in root extracts at three dpi, and in shoot extracts at four dpi. Soluble potassium silicate treatment resulted in significant increases in β-1, 3- glucanase activity in Sicot 189 root extracts at four dpi. Few significant differences between treatments in terms of chitinase and β-1, 3- glucanase activity were detected in Sicot F-1 plants, though higher levels of each of these enzymes were present in root and shoot extracts from this cultivar. In this study the effects of acibenzolar-S-methyl, applied in the form of Bion®, on defence gene expression and enzyme activity in cotton infected with Fov were more pronounced in plants cultivated from treated seed, rather than in plants treated via foliar spray; a finding which is particularly relevant to the industry presently. Significant up-regulation of chitinase class I, peroxidase, and β-1, 3-glucanase transcripts and enzyme activities occurred in the Bion® seed soak treatment with Fov inoculation compared to all other treatments. It was possible to compare the actions of silicon with those of Bion® in this study. Bion® primed defence responses in cotton infected with Fov, in a manner similar to that observed in silicon treated cotton. The use of silicon and Bion® treatments, both alone and in combination as part of integrated disease management programmes, may potentially contribute to increased protection against this pathogen in Australian cotton fields in the future.

Made available in DSpace on 2015-07-22T20:50:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-01-28. Added 1 bitstream(s) on 2015-08-11T03:26:00Z : No. of bitstreams: 1 Publico-119.pdf: 1451822 bytes, checksum: feaf2803cde522b94132a62f8fde6def (MD5) / Introdução: A identificação de 42 formas recombinantes circulantes (CRF) de HIV-1, juntamente com suas inúmeras formas recombinantes únicas, evidencia ainda mais o papel da recombinação gênica para esta epidemia. No Brasil, os subtipos B, F e C co-circulam, sendo que cinco CRF entre eles foram recém descobertas. A PCR em tempo real é uma ferramenta rápida, confiável e capaz de detectar diferentes subtipos de HIV-1 e suas formas recombinantes. Objetivo: O objetivo deste trabalho foi desenvolver sistemas de PCR em tempo real capazes de detectar vírus dos subtipos B e F, bem como recombinantes B/F gerados por ensaios de competição in vitro, e, assim, obter uma ferramenta para triagem das CRF brasileiras, 28 e 29, também recombinantes B/F. No futuro, estes sistemas serão testados para discriminação de subtipos de amostras clínicas. Metodologia: Células MT-4 foram infectadas separadamente pelos isolados virais BZ167 (subtipo B) e BR020 (subtipo F), e o sobrenadante foi coletado para otimização dos sistemas de PCR em tempo real (TaqMan®) desenvolvidos para detectar o subtipo de diferentes regiões genômicas, incluindo o gene pol (protease, transcriptase reversa, integrase) e o gene env (gp120 e gp41). Os primers desenhados deveriam amplificar igualmente o subtipo B e F; por outro lado, foram necessárias sondas subtipo-específicas capazes de detectar o subtipo presente no sobrenadante da cultura. As células MT-4 também foram co-infectadas por ambos os isolados, B e F, em iguais condições, para averiguação da possível geração de recombinantes. Para futura validação do uso destes sistemas em amostras clínicas, 157 amostras provenientes do Município de Santos, submetidas à genotipagem, foram seqüenciadas e subtipadas para as cinco regiões genômicas estudadas, utilizando o pacote Philip 3.5, sendo Neighbor-Joining o algoritmo de escolha. Resultados: Os sistemas desenvolvidos apresentaram-se capazes de detectar especificamente os isolados virais. A eficiência estimada para cada sistema, sendo um cálculo para a sonda do subtipo B e outro para F foram de, respectivamente: 80,97 e 85,16% para a região da protease; 89,80 e 75,09% para a região da transcriptase reversa; 80,90 e 83,83% para a região da integrase; 93,49 e 98,93% para a região da gp120; e 88,45 e 80,19% para a região da gp41. Nos ensaios de co-cultivo, a detecção de cada subtipo na primeira e na quinta passagem aconteceu de forma diferente, com variações na média dos valores de Cycle threshold (Ct) de intensidades diferentes. De uma forma geral, a concentração inicial do subtipo B pareceu diminuir, chegando a ficar indetectável em algumas regiões, enquanto do subtipo F pareceu aumentar ao longo das passagens para todas as regiões amplificadas (protease, transcriptase reversa, gp120 e gp41). A exceção foi a região da integrase, para qual foi detectado sinal somente para o subtipo B, sendo este sinal crescente ao longo do tempo. Do seqüenciamento e subtipagem das 157 amostras provenientes da cidade de Santos, foram obtidas seqüências de todas as regiões estudadas para 71 amostras. Destas, 43 foram subtipadas como B em todas as regiões, e somente três como F. De acordo com o padrão de distribuição de subtipos para as regiões, foram encontradas 12 seqüências com o padrão da CRF_28 e 9 no padrão da CRF_29. Nenhum subtipo C foi encontrado em nenhuma das regiões em estudo. Conclusão: O uso da técnica de PCR em tempo real para identificação de subtipos de fragmentos em cultura celular e para avaliação da dinâmica replicativa da recombinação em culturas co-infectadas reforça seu potencial uso em futuros testes in vivo para identificação de estruturas recombinantes. Esta metodologia mostrou ser eficiente, de mais fácil manipulação e mais econômica que o seqüenciamento de DNA. Os ensaios de co-cultivo amplificados pelos sistemas desenvolvidos sugeriram uma distribuição divergente nos subtipos resultantes para as diferentes regiões, sendo um grande indicativo de recombinação. O seqüenciamento das amostras clínicas evidenciou a importância das CRF em estudo, devido sua notável presença na amostragem utilizada, sendo um reflexo da epidemia de um local de grande circulação de mais de um subtipo. / Background: The discovery of 42 HIV-1 circulating recombinant forms (CRF) together with the innumerous unique HIV recombinants forms, makes clear the role of genetic recombination for the epidemic. In Brazil, clades B, F, and C co-circulate, with 5 recently described CRFs. Real Time PCR is a rapid and reliable tool capable of detecting different HIV-1 subtypes and recombinant profiles. Objective: The aim of this study was to develop real time PCR systems in order to detect the Brazilian CRF_28 and CRF_29, which are B/F recombinants, as well as detect B/F recombinants generated by in vitro competition assays. In future, these systems should be able to discriminate subtypes in clinical surveys. Methodology: MT-4 cells were separately infected with the viral strains BZ167 (subtype B) and BR020 (subtype F), and supernatant was collected in order to optimizing the real time PCR systems (TaqMan®) developed to detect the subtype profile of different genomic regions, including pol gene (protease, reverse transcriptase, integrase) and env gene (gp120 and gp41). The designed primers should be able to equally amplify the subtype B and F, which should be discriminated by subtype-specific probes. For future validation of these PCR systems, 157 clinical samples from the city of Santos were sequenced and phylogeneticaly analyzed in order to perform the clade assignment with Neighbor-Joining algorithm (Phylip software package v3.5). Results: The designed systems were able to differentiate the utilized viral strains. The estimated efficiencies for each system, for each probe, subtypes B and F separately, were respectively: 80,97 and 85,16% for protease region; 89,80 and 75,09% for reverse transcriptase region; 80,90% and 83,83% for integrase region; 93,49 and 98,93% for gp120 region; and 88,45 and 80,19% for gp41 region. For the co-infected cell culture, the detection of each subtype was performed in the first and fifth passages. Generally, the initial concentration of subtype B appeared to have decreased, some of them becoming undetectable, whereas subtype F seemed to increase with the passages, for protease, reverse transcriptase, gp120 and gp41 regions. The integrase region was an exception, since only the subtype B was detected, with increasing Cycle threshold (Ct) values over time. Sequencing results revealed that 65 out of 157 samples had the subtype profile defined for all regions. 43 out of 71 were defined as B, whereas 3 were F in all regions. 12 samples presented the CRF_28 profile, and 9 samples presented the CRF_29 profile. There were no subtype C samples in any genomic regions analyzed. Conclusion: The use of real time PCR technique for identification of fragments’ subtypes in cell culture and for evaluation of replicative dynamics of recombination in co-infected cultures warrants its potential use in future in vivo surveys. This methodology proved to be efficient, fast, less cumbersome and less expensive than DNA sequencing. The newly designed systems performed for supernatant of competition assays had suggested a divergent distribution of subtypes for the different regions, which reflects the possibility of genetic recombination. Results from clinical samples revealed a high prevalence of CRF_28/29 in this geographic region, thus reflecting the resulting consequence of different co-circulating strains and pointing to the need for a careful surveillance. / TEDE / BV UNIFESP: Teses e dissertações

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Over the past few years, there has been increasing evidence of the effect of the oral health over the general health of individuals, supported by a series of biological and epidemiological studies that show a relation between the mouth and many diseases, including cardiovascular diseases. Structural deficiencies and functional abnormalities of heart valves represent an important cause of cardiovascular morbidity and mortality in Brazil, and a few defects have been recently associated with infectious agents. The aim of this study was to identify cariogenic and periodontopathogenic bacteria in dental plaque, saliva and heart valves, without clinical endocarditis, of patients with heart valve diseases, and correlate these findings with the oral health status of the patients. Oral exams using the DMTF (decayed, missing and filled teeth) and PSR (Periodontal Screening and Recording) indexes to evaluate caries and periodontal disease, respectively, were performed. Samples of supragingival and subgingival dental plaque, saliva and cardiac valves were evaluated, through Real Time Polymerase Chain Reaction, for the presence of DNA of Streptococcus mutans (S. mutans), Prevotella intermedia (P. intermedia), Porphyromonas gingivalis (P. gingivalis) and Treponema denticola (T. denticola). A total of 114 samples were collected from 42 patients with a mean age of 55.6  13.8 years. The average number of missing teeth due to caries was 23.52  9.41 teeth per patient, and according to the highest score of periodontal disease observed for each patient, excluding edentulous patients (44.0%), periodontal pockets over 4mm (43.4%) and dental calculus (34.7%) were detected in a higher number of patients. The molecular analysis of the oral samples revealed high frequency of S. mutans and P. intermedia in supragingival dental plaques, subgingival dental plaques and saliva of dentate and edentulous patients (variation 60.0% - 100.0%), while P. gingivalis and T. denticola were detected in a smaller number of oral samples (variation 17.6% - 64.0%). The microorganism most frequently detected in heart valve samples was the S. mutans (89.3%), followed by P. intermedia (19.1%), P. gingivalis (4.2%) e T. denticola (2.1%). Significant difference was observed between the frequency of P. intermedia, P. gingivalis and T. denticola in the heart valve and dental plaque, as oposed to S. mutans. The identification of oral bacteria, especially S. mutans, in heart valves of patients with a previous history of dental caries and gingivitis/periodontitis suggests the possible involvement of these pathogens in the etiopathogenesis of heart valve diseases. / Atualmente, cada vez mais se tem evidÃncias do efeito da condiÃÃo oral na saÃde geral dos indivÃduos, atravÃs de uma sÃrie de estudos epidemiolÃgicos e biolÃgicos que mostram uma relaÃÃo entre a boca e diversas doenÃas, incluindo as doenÃas cardiovasculares. Desordens estruturais e nas funÃÃes das vÃlvulas cardÃacas representam uma importante causa de morbidade e mortalidade cardiovascular no Brasil, sendo alguns processos, como a estenose aÃrtica degenerativa, mais recentemente associados a agentes infecciosos. O objetivo desta pesquisa foi identificar bactÃrias cariogÃnicas e periodontopatogÃnicas na placa dental, saliva e vÃlvulas cardÃacas, sem endocardite clÃnica, de pacientes com doenÃa valvar, correlacionando esses achados à condiÃÃo bucal dos indivÃduos. AvaliaÃÃo, quanto Ãs doenÃas cÃrie e periodontal, foi realizada, atravÃs dos Ãndices CPO-D (Dentes Permanentes Cariados, Perdidos e Obturados) e PSR (Registro Periodontal Simplificado), respectivamente. Amostras de placa dental supragengival, subgengival, saliva e vÃlvula cardÃaca foram coletadas para investigaÃÃo da presenÃa de DNA, atravÃs de PCR (ReaÃÃo em Cadeia de Polimerase) em tempo real, de Streptococcus mutans (S. mutans), Prevotella intermedia (P. intermedia), Porphyromonas gingivalis (P. gingivalis) e Treponema denticola (T. denticola). Um total de 114 amostras foi coletado de 42 pacientes com mÃdia de idade de 55.6  13.8 anos. A mÃdia de dentes perdidos devido à cÃrie, por paciente, foi em torno de 23.52  9.41 e, segundo o maior grau de doenÃa periodontal observado no indivÃduo, excluindo-se os pacientes desdentados totais (44.0%), bolsa superior a 4 mm (43.4%) e o cÃlculo dental (34.7%) esteve presente em um maior nÃmero de pacientes. A anÃlise molecular das amostras bucais revelou alta frequÃncia de S. mutans e P. intermedia nas placas supragengival, subgengival e saliva de pacientes dentados e desdentados (variando entre 60.0% e 100.0%), enquanto que P. gingivalis e T. denticola estiveram presentes em menor nÃmero de amostras bucais (variando entre 17.6% e 64.0%). O micro-organismo mais frequentemente encontrado nas amostras valvares foi o S. mutans (89.3%), seguido da P. intermedia (19.1%), P. gingivalis (4.2%) e T. denticola (2.1%). DiferenÃa significativa foi encontrada entre a presenÃa de P. intermedia, P. gingivalis e T. denticola na vÃlvula e na placa dental, diferentemente do S. mutans. A identificaÃÃo de bactÃrias orais, principalmente S. mutans, em vÃlvulas cardÃacas de pacientes com elevada experiÃncia prÃvia de cÃrie e ocorrÃncia de gengivite/periodontite, sugere o possÃvel envolvimento desses patÃgenos nas doenÃas valvares.

A triagem clínica para malária em candidatos à doação de sangue em uma região não endêmica é um recurso empregado na seleção do doador que visa minimizar o risco transfusional uma vez que não há triagem laboratorial para Plasmodium spp. Este trabalho analisou questionário empregado na triagem clínica para malária em um hemocentro público no período de 2008 até 2010. Além dos critérios preconizados pela legislação nacional, os candidatos eram submetidos a perguntas específicas sobre residência e/ou visitação de região de Mata Atlântica preservada com transmissão de malária ou ainda se participaram de algum estudo de malária. A resposta afirmativa a uma destas questões selecionou um grupo com 500 candidatos (risco para malária), possíveis portadores de malária subclínica e a resposta negativa a todas estas perguntas, outro grupo com 606 doadores, denominado grupo controle. Verificou-se que a correlação é significativa entre essas respostas e os resultados laboratoriais obtidos na pesquisa do DNA do Plasmodium falciparum (Pf), do Plasmodium malariae (Pm) e o do Plasmodium vivax (Pv). O DNA foi detectado por reação de cadeia de polimerase (PCR) em tempo real, conforme protocolo de Gama et al. No grupo de risco para malária com n=500, observou-se uma taxa de positividade de 61 (12,2%) no PCR para Plasmodium ssp e de 23 (3,79 %) no grupo controle com n=606. Dos 61 PCR positivos do grupo de risco, 53 (86,9%) foram identificados como P. falciparum, 7(11,5%) P. vivax, 1 (1,6%) misto para P. falciparum e P. vivax e 0 (0%) P. malariae. No grupo controle com n=606 e taxa positiva de PCR de 23 (3,79%), foi isolado P. vivax em 18 (78,3%) dos casos, P. falciparum em 4 (17,4%), infecção mista pelo P. vivax e P. falciparum em 1 (4,3%) e em nenhum caso o P. malariae. Verificou-se que o emprego deste questionário foi capaz de selecionar um número maior de candidatos infectados por Plasmodium spp (p < 0,001) / Clinical screening for malaria on blood donation candidates of a non-endemic region is a resource used in donors selection, which aims to minimize transfusion risks, since there is no laboratory screening for Plasmodium spp. The project analyzed a questionnaire used in the clinical screening for malaria for the selection of blood donation candidates at a public blood center in São Paulo, 2008 to 2010. In addition to the criteria recommended by national legislation, candidates were subjected to specific questions about where they reside and / or visitation of non-endemic regions with malaria transmission, or whether they were aware of any study of malaria there. A positive answer to at least one of those questions selected a group of 500 candidates who could be carriers of malaria, and negative answers defined a control group of 606 donors without risk of malaria. It was found that there is a significant correlation between these responses and the results obtained in laboratory in DNA research to the P falciparum, P malariae and P. vivax. DNA was detected by polymerase chain reaction (PCR) in real time according to the protocol of Gamma et al. In the risk group for malaria (500) was we observed a positive rate of 61 (12.2%) polymerase chain reaction for Plasmodium spp and a rate of 23 (3.79%) in the control group. Of the 61 (12.2%) found positive in the risk group, 53 (86.9%) were identified as P falciparum, 7(11,5%) P vivax, 1(1.6%) mixed for both P falciparum and P vivax and 0 (0%) P malariae. In the control group (606) with positive PCR rate of 23 (3.79%), the P vivax was isolated in 18 (78.3%), P falciparum in 4 (17.4%), mixed infection 1 (4.3%) for P vivax and P falciparum and none P malariae. It was found that the use of this questionnaire in screening for blood donors was able to select more candidates infected by Plasmodium spp, (p < 0.001).