PCR and DNA sequencing in establishing the aetiology of bacterial infections in children.

The results of partial polymerase chain reaction (PCR) amplification of the bacterial 16S rRNA gene and subsequent DNA sequencing of clinical samples from children are described. In 13 out of 62 samples, DNA from bacteria likely to be the cause of infection was identified. In the vast majority (11/13) of samples with significant pathogen culture the specimen had been negative. Antibiotics had been given in all cases except for three prior to sampling. PCR and subsequent DNA sequencing is a valuable supplementary tool for establishing the cause of bacterial infections in children when culture is negative.

Ribosomal DNA sequencing: experiences from use in the Danish National Reference Laboratory for Identification of Bacteria.

Diagnostic tools for identification of bacteria have developed dramatically in the last decade. Sequencing of genes coding for rRNA has led to revolutionary insights into the phylogeny and taxonomy of bacteria, and to new demands on the service provided by national reference laboratories for identification of bacteria. At the Danish Reference Laboratory for Identification of Bacteria, partial 16S rDNA sequencing has been used since 2001 to identify "difficult" strains submitted for taxonomic elucidation. Experiences relating to phenotypic as well as 16S rDNA sequencing of the first 175 strains examined are presented. Approximately 2/3 of the strains were Gram-positive and 1/3 Gram-negative. One fifth of the strains were anaerobic, while 4/5 were either facultatively anaerobic or aerobic. Methodological agreement was seen for most strains at species and/or genus level. Methodological disagreement was relatively rare. In 1/6 of the strains valuable information was obtained from sequencing results, while for some strains identification was based primarily on the phenotypic results. Only a few strains could not be clearly identified by either method. A very large number of strains representing taxons ranging from facultatively anaerobic to aerobic and anaerobic species and genera, Gram-positive as well as Gram-negative, were successfully examined. Of the submitted strains many have only rarely been encountered as human pathogens. Thus, genotypic identification may result in recognition of hitherto seldom recognized or unrecognized bacteria as human pathogens, which will lead to a better understanding of the nature of human infections. It is self evident that we should focus on slowly growing, fastidious or 'difficult' organisms when using sequencing for national reference purposes. Short sequences (450-650 base pairs) seem sufficient for most identifications. Molecular bacterial identification is a powerful tool for national reference laboratories, enhancing both the speed and validity of examinations performed.

Advantages and limitations of ribosomal RNA PCR and DNA sequencing for identification of bacteria in cardiac valves of danish patients.

Studies on the value of culture-independent molecular identification of bacteria in cardiac valves are mostly restricted to comparing agreement of identification to what is obtained by culture to the number of identified bacteria in culture-negative cases. However, evaluation of the usefulness of direct molecular identification should also address weaknesses, their relevance in the given setting, and possible improvements. In this study cardiac valves from 56 Danish patients referred for surgery for infective endocarditis were analysed by microscopy and culture as well as by PCR targeting part of the bacterial 16S rRNA gene followed by DNA sequencing of the PCR product. PCR and DNA sequencing identified significant bacteria in 49 samples from 43 patients, including five out of 13 culture-negative cases. No rare, exotic, or intracellular bacteria were identified. There was a general agreement between bacterial identity obtained by ribosomal PCR and DNA sequencing from the valves and bacterial isolates from blood culture. However, DNA sequencing of the 16S rRNA gene did not discriminate well among non-haemolytic streptococci, especially within the Streptococcus mitis group. Ribosomal PCR with subsequent DNA sequencing is an efficient and reliable method of identifying the cause of IE, but exact species identification of some of the most common causes, i.e. non-haemolytic streptococci, may be improved with other molecular methods.

A program against bacterial bioterrorism: improved patient management and acquisition of new knowledge on infectious diseases.

In 2002 it was decided to establish laboratory facilities in Denmark for diagnosing agents associated with bioterrorism in order to make an immediate appropriate response to the release of such agents possible. Molecular assays for detection of specific agents and molecular and proteomic techniques for identification of bacteria were introduced as part of the program. All assays and techniques were made accessible for use in diagnosing patients, even when an intentional release was not suspected. Medical expertise on different diseases was established at the department as an integrated part of the program. The analyses included PCR assays for specific bacteria, identification of isolated bacteria by DNA sequencing, detection and identification of bacteria in clinical sample material by universal bacterial PCR and DNA sequencing, and identification of bacteria by mass spectrometry. The established analyses formed a basis on which a series of further developments was built. In addition to reducing the time for obtaining diagnoses and improving the accuracy of diagnosis of individual infected patients, the analyses provided new knowledge on the frequency and distribution of some bacterial infections, including Q fever, tularemia, trench fever, brucellosis, and melioidosis. The implementation of an antibioterrorism program in a clinical diagnostic setting improved the diagnostic possibilities for patients in Denmark and provided new epidemiologic information. It also introduced a number of diagnostic assays for bacterial infections not associated with bioterrorism that are difficult to culture or identify.

A Case of Helicobacter cinaedi Bacteraemia in a Previously Healthy Person with Cellulitis.

Helicobacter cinaedi is an infrequent, but well recognized cause of gastroenteritis in immunosuppressed patients. Here we report a case of an extra-intestinal infection in a previous healthy 61-year old heterosexual male. Focus for the infection was most likely cellulitis on the lower right leg. The bacterium was cultured from blood twice within one week. Electron microscopy of the isolate visualized bipolar flagella. Partial DNA sequencing of the 16S rRNA gene and phenotypic characterization of the isolate established the species diagnosis. The patient was treated with rifampicin. After end of treatment blood cultures were negative and the cellulitis had disappeared.

Infective Arthritis: Bacterial 23S rRNA Gene Sequencing as a Supplementary Diagnostic Method.

Consecutively collected synovial fluids were examined for presence of bacterial DNA (a 700-bp fragment of the bacterial 23S rRNA gene) followed by DNA sequencing of amplicons, and by conventional bacteriological methods. One or more microorganisms were identified in 22 of the 227 synovial fluids (9,7%) originating from 17 patients. Sixteen of the patients had clinical signs of arthritis. For 11 patients molecular and conventional bacterial examinations were in agreement. Staphylococcus aureus, Streptococcus dysgalactiae subspecies equisimilis and Streptococcus pneumoniae, were detected in synovial fluids from 6, 2 and 2 patients, respectively. In 3 patients only 23S rRNA analysis was positive; 2 synovial fluids contained S. dysgalactiae subspecies equisimilis and 1 S. pneumoniae). The present study indicates a significant contribution by PCR with subsequent DNA sequencing of the 23S rRNA gene analysis in recognizing and identification of microorganisms from synovial fluids.