Validation of an rpoB gene PCR assay for detection of Tropheryma whipplei: 10 years' experience in a National Reference Laboratory.

The performance of a real-time PCR assay targeting the Tropheryma whipplei rpoB gene was evaluated using test strains and 1,236 clinical specimens in a national reference laboratory. The novel rpoB-PCR assay proved to be specific, revealed improved analytical sensitivity, and substantially accelerated detection of T. whipplei DNA in clinical specimens.

Molecular methods for diagnosis of infective endocarditis.

Infective endocarditis (IE) is a life-threatening disease associated with high mortality. Conventional microbiologic diagnosis is based mainly on culture-dependent methods that often fail because of previous antibiotic therapy or the involvement of fastidious or slowly growing microorganisms. In recent years, molecular techniques entered the field of routine diagnostics. Amplification-based methods proved useful for detection of microorganisms in heart valve tissue. More recently, they were applied to blood samples from patients with IE. Direct detection of microorganisms in valve specimens by fluorescence in situ hybridization allowed identification of the causative agent and simultaneous visualization of complex microbial communities. These techniques will gain more importance in the near future, provided that procedures are standardized and results are interpreted with caution. With this review, we intend to give an overview of the impact and limitations of molecular techniques for the diagnosis of IE, including a focus on recent developments.

Rapid and accurate diagnosis of human intestinal spirochetosis by fluorescence in situ hybridization.

Human intestinal spirochetosis (HIS) is associated with overgrowth of the large intestine by spirochetes of the genus Brachyspira. The microbiological diagnosis of HIS is hampered by the fastidious nature and slow growth of Brachyspira spp. In clinical practice, HIS is diagnosed histopathologically, and a significant portion of cases may be missed. Fluorescence in situ hybridization (FISH) is a molecular method that allows the visualization and identification of single bacteria within tissue sections. In this study, we analyzed intestinal biopsy samples from five patients with possible HIS. All specimens yielded positive results by histopathological techniques. PCR amplification and sequencing of the 16S rRNA gene were performed. Sequences of two isolates clustered in the group of Brachyspira aalborgi, whereas in three cases, the sequences were highly similar to that of Brachyspira pilosicoli. Three phylotypes showed mismatches at distinct nucleotide positions with Brachyspira sp. sequences published previously. In addition, culture for Brachyspira was successful in three cases. On the basis of these data, we designed and evaluated a Brachyspira genus-specific 16S rRNA-directed FISH probe that detects all of the Brachyspira spp. published to date. FISH of biopsy samples resulted in strong, unequivocal signals of brush-like formations at the crypt surfaces. This technique allowed simultaneous visualization of single spirochetes and their identification as Brachyspira spp. In conclusion, FISH provides a fast and accurate technique for the visualization and identification of intestinal spirochetes in tissue sections. It therefore represents a valuable tool for routine diagnosis of HIS.

A view on Bartonella quintana endocarditis--confirming the molecular diagnosis by specific fluorescence in situ hybridization.

Culture-negative endocarditis is a frequent problem in cardiology, especially if caused by fastidious organisms. Among these, the diagnostic tools for the detection of Bartonella quintana are still unsatisfactory. In a culture-negative case of suspected endocarditis undergoing aortic valve replacement, polymerase chain reaction amplification and sequencing of the 16S rRNA gene indicated B. quintana infection. To develop a new diagnostic tool, independent from culture and amplification techniques, we designed and optimized an oligonucleotide fluorescence in situ hybridization (FISH) probe specific for B. quintana and suitable for FISH. FISH succeeded in simultaneous visualization and identification of vital microorganisms directly within the aortic valve tissue and in fast and univocal diagnosis of B. quintana endocarditis.

Fluorescence in situ hybridisation (FISH) accelerates identification of Gram-positive cocci in positive blood cultures.

Sepsis is a life-threatening disease with a high mortality rate. Rapid identification of blood culture isolates plays a crucial role in adequate antimicrobial therapy in sepsis patients. To accelerate microbiological diagnosis, a comprehensive panel of oligonucleotide probes for fluorescence in situ hybridisation (FISH) targeting Gram-positive cocci was compiled and evaluated on 428 positive blood culture specimens. By combining genus-specific and species-specific probes, the assay allowed discrimination of staphylococci, streptococci and enterococci as well as differentiation of therapy-relevant pathogens such as Staphylococcus aureus and Enterococcus faecium/durans. Furthermore, the newly designed FISH probes STREP2, ENCO and GRANU targeted Streptococcus pneumoniae/mitis, Enterococcus spp. (except E. faecalis) and Granulicatella adiacens group, respectively. The FISH assay achieved an overall sensitivity of 98.65% and a specificity of 99.0% and therefore allowed rapid and reliable molecular identification of Gram-positive cocci in blood culture specimens.