Identification of Pseudomonas aeruginosa, Burkholderia cepacia complex, and Stenotrophomonas maltophilia in respiratory samples from cystic fibrosis patients using multiplex PCR.

A multiplex PCR method was developed to identify P. aeruginosa, B. cepacia complex, and S. maltophilia directly in sputum and oropharyngeal samples from CF patients. One hundred and six patients (53 male, and 53 female) attending our pulmonology clinic were studied from September 2000-April 2001. Two hundred and fifty-seven samples were cultured in selective media and submitted to multiplex PCR reactions, using three primer pairs targeting specific genomic sequences of each species, with an additional primer pair targeting a stretch of ribosomal 16S DNA, universal for bacteria, to act as a control. P. aeruginosa was isolated by culture in 56% of samples, B. cepacia complex in 4.3%, and S. maltophilia in 2.7%, while multiplex PCR identified P. aeruginosa in 78.7%, B. cepacia complex in 3.9%, and S. maltophilia in 3.1% of samples. Multiplex PCR results were verified by PCR reactions using different species-specific primers described in the literature and DNA sequencing of amplicons from a few samples. Comparing to culture results, the sensitivity and specificity values of multiplex PCR for bacterial identification were, respectively, 97.2% and 45.5% for P. aeruginosa, 45.5% and 97.9% for B. cepacia complex, and 40% and 97.6% for S. maltophilia. All 10 multiplex PCR-positive results for B. cepacia complex were confirmed using other species-specific primers described in the literature, while this approach confirmed results for S. maltophilia identification in 7/8 samples (87.5%). Sequencing of amplicons from samples culture-negative but multiplex PCR-positive for P. aeruginosa and B. cepacia complex confirmed their identity, while minor nucleotide differences among amplicons ruled out the hypothesis of PCR contamination.

Use of selective medium for Burkholderia cepacia isolation in respiratory samples from cystic fibrosis patients.

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.

Phenotypic and genotypic characteristics of shiga toxin-producing Escherichia coli strains isolated from children in São Paulo, Brazil.

The biochemical and serological characteristics, virulence properties, and genetic relatedness of Shiga toxin-producing Escherichia coli (STEC) strains isolated in S o Paulo, from April 1989 through March 1990, were determined. This is also the first report on clinic findings of human STEC infections in Brazil. The only three STEC strains identified in that period were lysine decarboxylase negative, belonged to serotype O111ac: non-motile, were Stx1 producers, carried the eae and astA genes, and 2 of them also presented the EHEC-hly sequence. The children carrying STEC were all boys, with less than two years old, and had no previous history of hospitalization. None of them presented blood in stools. Vomiting, cough and coryza were the most common clinical manifestations observed. Although the STEC strains were isolated during summer months, and presented similar phenotypic and genotypic characteristics, carbohydrate fermentation patterns and PFGE analysis suggested that these diarrheal episodes were not caused by a single clone.

Phenotypic and genotypic characteristics of shiga toxin-producing Escherichia coli strains isolated from children in São Paulo, Brazil

The biochemical and serological characteristics, virulence properties, and genetic relatedness of Shiga toxin-producing Escherichia coli (STEC) strains isolated in São Paulo, from April 1989 through March 1990, were determined. This is also the first report on clinic findings of human STEC infections in Brazil. The only three STEC strains identified in that period were lysine decarboxylase negative, belonged to serotype O111ac: non-motile, were Stx1 producers, carried the eae and astA genes, and 2 of them also presented the EHEC-hly sequence. The children carrying STEC were all boys, with less than two years old, and had no previous history of hospitalization. None of them presented blood in stools. Vomiting, cough and coryza were the most common clinical manifestations observed. Although the STEC strains were isolated during summer months, and presented similar phenotypic and genotypic characteristics, carbohydrate fermentation patterns and PFGE analysis suggested that these diarrheal episodes were not caused by a single clone

Meningite bacteriana no período neonatal: evoluçäo clínica e complicaçöes em 109 casos / Bacterial meningitis in the neonatal period: clinical evaluation and complications in 109 cases

As meningites bacterianas apresentam caracteristicas peculiares durante o período neonatal. A infecçäo bacteriana que se assesta no SNC, em fase rápida de crescimento, ocasiona complicaçöes e sequelas graves, na maioria das crianças que contraem a doença, prejudicando seu desenvolvimento neuropsicomotor. A análise de 109 crianças com meningite bacteriana neonatal no período janeiro/1977-abril/1987 demonstrou mortalidade de 34,8//. Na maioria dos casos näo foram observados fatores de risco relacionados a antecedentes perinatais e 80,5// dessas crianças foram caracterizadas como recém-nascido de termo. Os sinais mais encontrados à internaçäo foram convulsöes (53,2//), fontanela abaulada (37,6//) e apnéia (20//); os sintomas mais frequentes foram depressäo sensorial (64,2//), recusa alimentar (64,2//), febre (50,5//) e irritabilidade (35,8//). As complicaçöes verificadas durante a internaçäo foram, por ordem de frequência, ventriculite (34,9//), SSHAD (27,5//), coleçäo subdural (8,3//), abscesso cerebral (4,6//) e enfarte cerebral (2,8//). A presença de SSHAD e de ventriculite foram associadas a maior mortalidade. Das 71 crianças que sobreviveram à doença 44 (62//) apresentaram-se com exame neurológico alterado e 29 (40,8//) con hidrocefalia que exigiu a instalaçäo de sistema de derivaçäo ventriculo-peritoneal em 18 (62//) dos casos. O seguimento neurológico dessas crianças é imprescindível, pois poderá haver modificaçäo no prognóstico a longo prazo

Use of selective medium for Burkholderia cepacia isolation in respiratory samples from cystic fibrosis patients

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 percent) of the samples using the selective medium, in contrast with the preceding two years, when it was isolated in 6/1029 samples (0.58 percent), 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