Falsos positivos na detecção de Legionella spp. seguindo a norma UNE ISO-11731:1998
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2008
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As infecções provocadas por Legionella spp. são consideradas actualmente, um problema emergente de saúde pública e estão associadas a altas taxa de mortalidade e morbilidade, quando não tratadas adequadamente.
Neste estudo foram analisadas 54 amostras de água provenientes de 8 concelhos da região Norte de Portugal, com a finalidade de obter uma colecção de estirpes do género Legionella e caracteriza-las tanto fenotipicamente como geneticamente. Sendo também objectivo deste trabalho, avaliar a eficácia da técnica de cultivo, método padrão de referência de acordo com a norma International Organization for Standardization 11731 (1998), na detecção de espécies de Legionella. O processamento laboratorial das amostras foi realizado segundo a metodologia recomendada pela ISO-11731:1998. Após filtração das amostras (1 litro), o colectado foi ressuspendido em 5 ml de água destilada estéril. Posteriormente, retirou-se 100 μl da suspensão e semeou-se em um meio selectivo, agar GVPC (BD BBL 257007), incubando-se 7 a 10 dias, a 37 ºC em condições aeróbias. Colónias morfologicamente características do género foram subcultivadas em agar BCYE (BD BBL 221808) e agar sangue, para confirmação. Somente as colónias que cresceram em agar BCYE e não em agar sangue foram consideradas presuntivas de Legionella, segundo a ISO 11731. A identificação das mesmas foi realizada por sequenciação do gene 16S rRNA, tendo-se verificado que nenhum dos isolados foi identificado como pertencente ao género Legionella. No entanto, mediante a ISO 11731 estes foram interpretados como positivos, correspondendo portanto, a resultados falsos positivos. Embora a técnica de cultura, seja o método recomendado pela norma ISO 11731 para a detecção de Legionella spp., neste trabalho esta técnica demonstrou-se muito morosa e de baixa sensibilidade, verificando-se inclusivamente a ocorrência de falsos positivos, o que evidência a importância da utilização de métodos genéticos para a correcta identificação dos microrganismos. A metodologia utilizada neste estudo permitiu ainda o isolamento de um conjunto de isolados (n=40), que formam um grupo independente de todos os géneros da família Chitinophagaceae descritos até ao momento e que pela sua distância filogenética poderá corresponder a um género ainda não descrito e portanto a uma nova espécie. Da avaliação do perfil de susceptibilidade, verificou-se que à excepção de três isolados, todas as estirpes testadas apresentaram um perfil de multiresistência aos antibióticos utilizados, entre os quais, os β-lactâmicos, os aminoglicosídeos e as quinolonas, sendo os menos eficazes a amoxicilina (penicilina) e a eritromicina.
Infections caused by Legionella spp. are considered at present, an emerging public health problem and are linked to high rates of mortality and morbidity, if not properly treated. This study analyzed 54 samples of water from 8 counties from Northern Portugal, with the aim of obtaining a collection of strains of the genus Legionella and to characterize them genetically and phenotypically. Another objective of this study was to evaluate the effectiveness of the technique of cultivation, the standard method according to International Organization for Standardization 11731 (1998), for the detection of species of Legionella. The laboratory processing of samples was performed according to procedure recommended by ISO-11731:1998. After filtration of samples (1 L), the filtrate was re-suspended in 5 ml of sterile distilled water. Subsequently, 100 μl of the suspension was spread in GVPC selective agar medium (BD 257007 BBL), and incubated for 7 to 10 days at 37 ºC, under aerobic conditions. Colonies that were morphologically characteristic of the genus were sub-cultured onto BCYE agar (BD 221808 BBL) and blood agar for verification. Only the colonies which grew in BCYE agar and not on blood agar were considered presumptive of Legionella, according to ISO 11731. The identification of these initially selected colonies was performed by sequencing the 16S rRNA gene, which revealed that none of the isolates were identified as belonging to the genus Legionella. However, through the ISO 11731 they were interpreted as positive, corresponding therefore to false-positive results. While the technique of culture is the method recommended by ISO 11731 for the detection of Legionella spp. in this work it was clearly shown that this technique is very slow and has low sensitivity, there is even the occurrence of false-positives, which highlighted the importance of using genetic methods for the proper identification of microorganisms. The methodology used in this study allowed the isolation of a number of isolates (n=40), which form an independent group of all genus of the family Chitinophagaceae written so far and that by their phylogenetic distance may be a genus not yet described and therefore a new species. Assessing the profile of susceptibility, it was found that with the exception of three strains, all strains tested showed a profile of multi-resistance to antibiotics used, including the β-lactams, aminoglycosides and the quinolones, and the least effective were amoxicillin (penicillin) and erythromycin.
Infections caused by Legionella spp. are considered at present, an emerging public health problem and are linked to high rates of mortality and morbidity, if not properly treated. This study analyzed 54 samples of water from 8 counties from Northern Portugal, with the aim of obtaining a collection of strains of the genus Legionella and to characterize them genetically and phenotypically. Another objective of this study was to evaluate the effectiveness of the technique of cultivation, the standard method according to International Organization for Standardization 11731 (1998), for the detection of species of Legionella. The laboratory processing of samples was performed according to procedure recommended by ISO-11731:1998. After filtration of samples (1 L), the filtrate was re-suspended in 5 ml of sterile distilled water. Subsequently, 100 μl of the suspension was spread in GVPC selective agar medium (BD 257007 BBL), and incubated for 7 to 10 days at 37 ºC, under aerobic conditions. Colonies that were morphologically characteristic of the genus were sub-cultured onto BCYE agar (BD 221808 BBL) and blood agar for verification. Only the colonies which grew in BCYE agar and not on blood agar were considered presumptive of Legionella, according to ISO 11731. The identification of these initially selected colonies was performed by sequencing the 16S rRNA gene, which revealed that none of the isolates were identified as belonging to the genus Legionella. However, through the ISO 11731 they were interpreted as positive, corresponding therefore to false-positive results. While the technique of culture is the method recommended by ISO 11731 for the detection of Legionella spp. in this work it was clearly shown that this technique is very slow and has low sensitivity, there is even the occurrence of false-positives, which highlighted the importance of using genetic methods for the proper identification of microorganisms. The methodology used in this study allowed the isolation of a number of isolates (n=40), which form an independent group of all genus of the family Chitinophagaceae written so far and that by their phylogenetic distance may be a genus not yet described and therefore a new species. Assessing the profile of susceptibility, it was found that with the exception of three strains, all strains tested showed a profile of multi-resistance to antibiotics used, including the β-lactams, aminoglycosides and the quinolones, and the least effective were amoxicillin (penicillin) and erythromycin.
Descrição
Dissertação de Mestrado em Biologia Clínica Laboratorial
Palavras-chave
Microbiologia , Bactérias , Legionella