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.

Six cases of Aerococcus sanguinicola infection: clinical relevance and bacterial identification.

Aerococcus sanguinicola is a Gram-positive coccus first described in 2001. Infections in humans are rare but the use of 16S rRNA gene sequencing and improved phenotypic methods has facilitated the identification of A. sanguinicola. We report here 6 cases of A. sanguinicola bacteraemia, 2 of which were associated with infective endocarditis. Most patients were elderly (median age 70 y) and had underlying neurological disorders including dementia, cerebral degeneration, and myelomeningocele. The primary focus of infection was the urinary tract in 3 cases and the gallbladder in 1; no focus was detected in 2 cases. Long-term prognosis was poor reflecting the frailty of the patients. All strains were susceptible to penicillin, ampicillin, cefuroxime, vancomycin, erythromycin, and rifampicin. The optimal treatment of infection with A. sanguinicola has yet to be determined.

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.

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.

Infections with beta-haemolytic streptococci: detection by a universal PCR for bacterial DNA and DNA sequencing.

PCR using universal bacterial primers and subsequent DNA sequence analysis of the PCR product was used to detect beta-haemolytic streptococci in clinical samples. Streptococcus pyogenes, Streptococcus agalactiae and Streptococcus dysgalactiae were identified in 19 primary culture-negative cases of infections from 203 patient samples with detectable bacterial DNA.

Granulicatella elegans bacteraemia in patients with abdominal infections.

Bloodstream infections with Granulicatella (previous Abiotrophia) elegans are rare. A few reported cases were associated with infective endocarditis. Three cases of bacteraemia with G. elegans in patients who were operated for acute abdominal diseases are described. Abdominal foci should be considered when G. elegans is recovered from blood.

[Chancroid]. / Ulcus molle.

Chancroid is caused by the bacterium Haemophilus ducreyi. It is a sexually transmitted disease causing a soft chancre with a necrotic base and purulent exudate. The incidence of this illness is very low in Denmark and is probably underestimated. The bacterium is very fragile in transport, and culture is often negative. The chance of demonstrating the bacterium is greatly enhanced by the use of molecular techniques. In this case, we report on a specific PCR test for H. ducreyi that was used to establish the diagnosis in a 40-year-old male.

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.

Ten cases of Actinobaculum schaalii infection: clinical relevance, bacterial identification, and antibiotic susceptibility.

Nine of 10 strains of Actinobaculum schaalii caused urinary tract infections in predisposed individuals. Identification included 16S rRNA gene sequence analysis and use of the API Coryne and Rapid ID 32 A test systems. A. schaalii is easily overlooked due to its slow growth in ambient air and its resemblance to the normal bacterial flora on skin and mucosa.

[Botulinum and ricin toxins]. / Botulinum- og ricintoksiner.

Due to their acute toxicity, accessibility and history of use, ricin and botulinum toxins are at the present time the most relevant bioterror toxins. We give a brief review of their toxicity and possible uses in bioterror attacks and describe two cases in Denmark in which immunochemical, PCR and mass spectrometric assays developed in-house were used to confirm a foodborne botulinum toxin E case and to identify bovine serum albumin as the powder enclosed in a letter sent to the U.S. Embassy in Copenhagen.

[New diagnostic methods for bacterial infections after the introduction of increased bioterrorism preparedness]. / Ny diagnostik af bakterielle infektioner efter indførelse af øget biologisk beredskab.

Bacillus anthracis, Francicella tularensis, Yersinia pestis, Burkholderia pseudomallei and Brucella melitensis are the bacteria most often mentioned in connection with possible biological terrorist acts. It is known that these microorganisms can be cultured and weaponised for such a purpose. This survey provides examples of the use of diagnostic methods for those microorganisms, further detailed in relation to establishing a national center for biological preparedness in Denmark.