| Área: Microbiologia Clinica ( Divisão A ) COMPARISON BETWEEN CONVENTIONAL, IMMUNOLOGICAL AND MOLECULAR DIAGNOSIS OF CAMPYLOBACTER SP. IN CHILDREN WITH DIARRHEA ATTENDED AT OUTPATIENT CLINICS IN NORTHEASTERN BRAZIL Josiane da Silva Quetz (UFC); Ila Fernanda Nunes Lima (UFC); Renata de Sousa Alves (UFC); Alexandre Havt (UFC); Alberto Melo Soares (UFC); Luís Carlos Rey (UFC); Bernhard Weigl (PATH); Aldo Ângelo Moreira Lima (UFC)
ResumoINTRODUCTION: Campylobacter enteritis is a common cause of acute bacterial diarrhea worldwide. Polymerase chain rection (PCR) have been increasingly applied to diagnose several pathogenic bacteria. Our aim was to compare microbiological conventional diagnosis, PCR detection, and immunological identification of Campylobacter sp. directly from fecal samples.
MATERIAL AND METHODS: Stool samples from 432 children with diarrhea treated in outpatient clinics in Fortaleza, Ceara, Brazil were collected from May 2008 to April 2009. Specimens were processed by standard microbiological methods and 5.6% (24/432) presented a Campylobacter suggestive microbiologic phenotype (microaerophilic growth, characteristic colonial appearance, gull's wings morphology in Gram staining, and oxidase activity). Frozen aliquots of these 24 stool samples were used for DNA extraction (QIAamp DNA Stool Mini Kit, Qiagen, CA) and for Enzyme Linked Immuno Sorbent Assay (ELISA, ProSpect Campylobacter Microplate Assay, REMEL, TX). PCR amplification of hipO (hippurate hydrolase) and ask (aspartate kinase) genes were used for C. jejuni and C. coli molecular detection, respectively. We also evaluate the presence of cytolethal distending toxin (CDT) operon (cdtABC genes of C. jejuni) by Multiplex PCR.
RESULTS: Of the 24 samples tested, 23 (95.8%) were positive for one or both methods, where 19 (79.2%) were positive by PCR and 23 (95.8%) by ELISA. All 19 positive samples by PCR were identified as C. jejuni (hipO+) and none of them was C. coli (ask-). The negative samples for any PCR were submitted to a new extraction and PCR detection. No differences were observed between both data. The CDT genes were found in all hipO+ samples.
CONCLUSION: The results showed that PCR and immunological identifications can detect Campylobacter sp. directly from fecal samples as good as microbiological conventional diagnosis. However, the first two diagnostic methods can give results on the same day. Considering stool culture as gold standard method, ELISA results were more similar than PCR detection. The occurrence of immunological cross-reaction with other Campylobacter species must be considered. In addition, DNA extraction process could be involved in PCR results.
FINNANCIAL SUPPORT: CNPq (Brazil) and PATH (USA)
Palavras-chave: Campylobacter sp., CHILDHOOD DIARRHEA, DIAGNOSTIC |