Uso de meio seletivo para identificação de cepas de Burkholderia cepacia em amostras de trato respiratório de pacientes portadores de fibrose cística

Burkholderia cepacia colonizes cystic fibrosis (CF) patients. We evaluated the impact of the use of a selective medium in the rate of B. cepacia recovery from respiratory samples of CF patients. During a 6-month period, respiratory samples were collected from 106 CF patients and cultivated on selective media including a B. cepacia selective medium. Confirmation of the identity of B. cepacia isolates was carried out by species specific PCR and determination of genomovar status performed by a sequential PCR approach. Results of B. cepacia isolation during this period were compared to the preceding two years, when the sample processing was identical except for the lack of the B. cepacia selective medium. B. cepacia was isolated in 11/257 (4.2%) of the samples using the selective medium, in contrast with the preceding two years, when it was isolated in 6/1029 samples (0.58%), p < 0.0001. Identity of all 11 isolates was confirmed by PCR and genomovar determination was accomplished in all but one isolate. These results suggest that the use of a selective medium increases recovery rate of B. cepacia from respiratory samples.A Burkholderia cepacia coloniza os pacientes portadores de fibrose cística (FC). Avaliamos o impacto do uso de um meio seletivo no isolamento de B. cepacia em amostras de secreção respiratória de pacientes portadores de FC. Durante um período de 6 meses, amostras de trato respiratório foram colhidas de 106 pacientes com FC e cultivadas em meios seletivos incluindo um meio para isolamento de B. cepacia. A identidade das cepas de B. cepacia foi confirmada através de PCR espécie específica e a determinação do genomovar ou subespécie realizada através de reações seqüenciais de PCR. Os resultados de isolamento de B. cepacia durante este período foram comparados com os dois anos precedentes, quando o processamento das amostras era idêntico, exceto pela utilização do meio seletivo para B. cepacia. B. cepacia foi isolada em 11/257 (4,2%) amostras usando o meio seletivo, e em apenas 6/1029 (0,58%) nos dois anos precedentes (p < 0,0001). A identidade destas 11 cepas foi confirmada e a determinação do genomovar obtida em 10/11. Estes resultados sugerem que o uso do meio seletivo aumenta a freqüência de isolamento de B. cepacia em amostras de trato respiratório de pacientes portadores de FC

HIV-1 drug resistance genotypic profiles in children with undetectable plasma viremia during antiretroviral therapy

Treatment of HIV-1 infection with highly active antiretroviral therapy has led to sustained viral suppression in the plasma in a large number of children. However, studies have suggested that the integrated provirus in resting CD4+ T lymphocytes could be a source of reactivatable virus and maintain drug-resistant virus. We evaluated the resistance-related mutations in children receiving antiretroviral therapy with prolonged viral suppression. Thirty-two peripheral blood mononuclear cell samples from 16 children with viral loads that had been below detection limits for at least 12 months were obtained at two different time points and the DNAs sequenced. The median CD4 cell count was 1,016 cells/mm³ (347-2,588) and 938 cells/mm³ (440-3,038) at the first and second time points, respectively. The median follow-up time was 15 months (9-27). Six (37.5%) and seven (43.75%) of the 16 patients showed at least one NRTI-associated mutation in the first and second samples, respectively. Two out of 16 (12.5%) had an NNRTI-associated mutation at the first time point and three out of 16 (18.75%) at the second. In addition, 14 out of 16 (87.5%) had at least one PI-associated mutation at both time points. Despite plasma HIV-1 RNA suppression for at least 12 months, resistance-related mutations from previous antiretroviral failures could still be detected in archival virus. Furthermore, viral evolution occurred at the reverse transcriptase region in spite of viral suppression to levels below 400 copies/mL. Persistence of archival resistant virus may be relevant when considering future treatment options

Rhinovirus C and Respiratory Exacerbations in Children with Cystic Fibrosis

To investigate a possible role for human rhinovirus C in respiratory exacerbations of children with cystic fibrosis, we conducted microbiologic testing on respiratory specimens from 103 such patients in São Paulo, Brazil, during 2006–2007. A significant association was found between the presence of human rhinovirus C and respiratory exacerbations

Typical epidemiology of respiratory virus infections in a Brazilian slum

Host population size, density, immune status, age structure, and contact rates are critical elements of virus epidemiology. Slum populations stand out from other settings and may present differences in the epidemiology of acute viral infections. We collected nasopharyngeal specimens from 282 children aged ≤5 years with acute respiratory tract infection (ARI) during 2005 to 2006 in one of the largest Brazilian slums. We conducted real-time reverse transcription-polymerase chain reaction (RT-PCR) for 16 respiratory viruses, nested RT-PCR-based typing of rhinoviruses (HRVs), and collected clinical symptoms. Viruses were common causes of respiratory disease; with ≥1 virus being detected in 65.2% of patients. We detected 15 different viruses during 1 year with a predominance of HRV (33.0%) and human respiratory syncytial virus (hRSV, 12.1%) infections, and a high rate of viral coinfections (28.3%). We observed seasonality of hRSV, HRV and human coronavirus infections, more severe symptoms in hRSV and influenza virus (FLU) infections and prolonged circulation of seven HRV clusters likely representing distinct serotypes according to genomic sequence distances. Potentially unusual findings included the absence of human metapneumovirus detections and lack of typical FLU seasonal patterns, which may be linked to the population size and density of the slum. Nonetheless, most epidemiological patterns were similar to other studies globally, suggesting surprising similarities of virus-associated ARI across highly diverse settings and a complex impact of population characteristics on respiratory virus epidemiology

Amplification of Emerging Viruses in a Bat Colony

Concentration and prevalence of coronaviruses and astroviruses increase when bats form maternity roosts and bear young

Depression and suicidality among psychiatric residents - results from a multi-country study

Background Previous studies have highlighted risks for depression and suicide in medical cohorts, but evidence regarding psychiatric residents is missing. This study aimed to determine rates of depression, suicide ideation and suicide attempt among psychiatric residents and to identify associated individual, educational and work-related risk factors. Methods A total of 1980 residents from 22 countries completed the online survey which collected data on depression (PHQ-9), suicidality (SIBQ), socio-demographic profiles, training, and education. Generalized linear modeling and logistic regression analysis were used to predict depression and suicide ideation, respectively. Results The vast majority of residents did not report depression, suicide ideation or attempting suicide during psychiatric training. Approximately 15% (n=280) of residents met criteria for depression, 12.3% (n=225) reported active suicide ideation, and 0.7% (n=12) attempted suicide during the training. Long working hours and no clinical supervision were associated with depression, while more completed years of training and lack of other postgraduate education (e.g. PhD or psychotherapy training) were associated with increased risk for suicide ideation during psychiatric training. Being single and female was associated with worse mental health during training. Limitations Due to the cross-sectional nature of the study, results should be confirmed by longitudinal studies. Response rate was variable but the outcome variables did not statistically significantly differ between countries with response rates of more or less than 50%. Conclusion Depression rates among psychiatric residents in this study were lower than previously reported data, while suicide ideation rates were similar to previous reports. Poor working and training conditions were associated with worse outcomes. Training programmes should include effective help for residents experiencing mental health problems so that they could progress through their career to the benefit of their patients and wider society

Otimização da PCR em tempo real - Sybr Green para detecção do Herpes Vírus Humano tipo 6 (HHV-6)

HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/µL (equivalent to 2465.8 molecules/µL) to 10-9 (equivalent to 2.46 molecules/µL). Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3)'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3') and by conventional nested PCR using primers HHV1 (outer): 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner) and HHV4 (inner) 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/µL and for the nested PCR was 2.46 molecules/µL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination.HHV-6 é o agente etiológico do Exantema Súbito e considerado a sexta doença mais comum na infância. Em indivíduos imunocomprometidos, a reativação da infecção latente pode causar doença aguda ou morte. Padronizamos PCR em Tempo Real utilizando a química Sybr Green na detecção do HHV-6 e comparamos os resultados com a PCR convencional. Um fragmento de 214 pb foi clonado através do kit pGEM-T do sistema Promega. Com este clone, foram feitas diluições seriadas em um pool de leucócitos negativos a partir de 10-6 ng/µL (equivalente a 2465,8 moleculas/µL) até 10-9 (equivalente a 2,46 moleculas/µL). As diluições foram amplificadas por PCR em Tempo Real utilizando Sybr Green, com primers HHV3 5' TTG TGC GGG TCC GTT CCC ATC ATA 3' e HHV4 5' TCG GGA TAG AAA AAC CTA ATC CCT 3' e pelo método convencional, PCR nested usando primers HHV1 (externo): 5' CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (externo): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3', HHV3 (interno) e HHV4 (interno): 5' TCG GGA TAG AAA AAC CTA ATC CCT 3'. O limite de detecção foi determinado pelas diluições seriadas do plasmídio contendo um fragmento de HHV6: para o ensaio com Sybr Green, foi de 24,6 moleculas/µL e para a PCR nested, 2,46 moleculas/µL. Elegemos o PCR em Tempo Real - Sybr Green como método diagnóstico e quantitativo do HHV-6 devido a sua boa sensibilidade e menor risco de contaminação

Molecular Characterization of Strains of Respiratory Syncytial Virus Identified in a Hematopoietic Stem Cell Transplant Outpatient Unit Over 2 Years: Community or Nosocomial Infection?

Respiratory syncytial virus (RSV) is recognized as the leading cause of nosocomial respiratory infection among hematopoietic stem cell transplant (HSCT) recipients, causing considerable morbidity and mortality. RSV is easily transmitted by contact with contaminated surfaces, and in HSCT units, more than 50% of RSV infections have been characterized as of nosocomial origin. From April 2001 to October 2002, RSV was identified by direct immunofluorescent assay in 42 symptomatic HSCT recipients. Seven RSV strains from 2001 and 12 RSV strains from 2002 were sequenced. RNA extraction, cDNA synthesis, and seminested polymerase chain reaction (PCR) with primers complementary to RSV genes G and F were pet-formed. PCR products were analyzed by nucleotide sequencing of the C-terminal region of gene G for typing (in group A or B). Of the 7 strains analyzed in 2001, only 2 belonged to group B; the other 5 belonged to group A. Of these 7 strains, 3 were identical and were from recipients receiving outpatient care. In 2002, of the 12 strains analyzed, 3 belonged to group A and the other 9 belonged to group B. Of these 9 strains, 7 were genetically identical and were also from recipients receiving outpatient care. Therefore, multiple strains of RSV cocirculated in the hematopoietic stem cell transplant units (ward and outpatient units) between 2001 and 2002. Nosocomial transmission was more likely to occur at the HSCT outpatient unit than in the HSCT ward. Infection control practices should also be implemented in the outpatient setting.Fapesp[2001/11087-2]Fapesp[2002 08465-8]CAPESBrazilian Ministry of Educatio