ABSTRACT
Abstract Infective endocarditis (IE) remains a severe and potentially fatal disease demanding sophisticated diagnostic strategies for detection of the causative microorganisms. The aim of the present study was to develop a broad-range 16S ribosomal RNA gene polymerase chain reaction in the routine diagnostic of IE for the early diagnosis of fatal disease. A broad-range PCR technique was selected and evaluated in terms of its efficiency in the diagnosis of endocarditis using 19 heart valves from patients undergoing cardiovascular surgeries at the Habib Bourguiba Hospital of Sfax, Tunisia, on the grounds of suspected IE. The results demonstrated the efficiency of this technique particularly in cases involving a limited number of bacteria since it helped to increase detection sensitivity. The technique proved to be efficient, particularly, in the bacteriological diagnosis of IE in contexts involving negative results from conventional culture methods and other contexts involving bacterial species that were not amenable to identification by phenotypic investigations. Indeed, the sequencing of the partial 16S ribosomal RNA gene revealed the presence of Bartonella henselae, Enterobacter sp., and Streptococcus pyogenes in three heart valves with the negative culture. It should be noted that the results obtained from the polymerase chain reaction-sequencing identification applied to the heart valve and the strain isolated from the same tissue were not consistent with the ones found by the conventional microbiological methods in the case of IE caused by Gemella morbillorum. In fact, the results from the molecular identification revealed the presence of Lactobacillus jensenii. Overall, the results have revealed that the proposed method is sensitive, reliable and might open promising opportunities for the early diagnosis of IE.
Subject(s)
Humans , Male , Bacteria/isolation & purification , Polymerase Chain Reaction/methods , Endocarditis/microbiology , Endocarditis, Bacterial/microbiology , Phylogeny , Bacteria/classification , Bacteria/genetics , DNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Endocarditis/diagnosis , Endocarditis, Bacterial/diagnosis , Heart Valves/microbiology , Middle AgedABSTRACT
BACKGROUND: The aims of this study were to compare several DNA extraction methods and 16S rDNA primers and to evaluate the clinical utility of broad-range PCR in continuous ambulatory peritoneal dialysis (CAPD) culture fluids. METHODS: Six type strains were used as model organisms in dilutions from 10(8) to 100 colony-forming units (CFU)/mL for the evaluation of 5 DNA extraction methods and 5 PCR primer pairs. Broad-range PCR was applied to 100 CAPD culture fluids, and the results were compared with conventional culture results. RESULTS: There were some differences between the various DNA extraction methods and primer sets with regard to the detection limits. The InstaGene Matrix (Bio-Rad Laboratories, USA) and Exgene Clinic SV kits (GeneAll Biotechnology Co. Ltd, Korea) seem to have higher sensitivities than the others. The results of broad-range PCR were concordant with the results from culture in 97% of all cases (97/100). Two culture-positive cases that were broad-range PCR-negative were identified as Candida albicans, and 1 PCR-positive but culture-negative sample was identified as Bacillus circulans by sequencing. Two samples among 54 broad-range PCR-positive products could not be sequenced. CONCLUSIONS: There were differences in the analytical sensitivity of various DNA extraction methods and primers for broad-range PCR. The broad-range PCR assay can be used to detect bacterial pathogens in CAPD culture fluid as a supplement to culture methods.