Detection of non-tuberculous mycobacteria in hospital water by culture and molecular methods.

Non-tuberculous mycobacteria (NTM) are emerging pathogens in immunocompromised patients. Therefore, it is important for hospitals to consider tap water as a source for these infections. Aim of the study was to establish highly sensitive and specific techniques to detect and identify NTM in hospital drinking water. A Mycobacterium genus-specific 16S rDNA-based real-time LightCycler PCR assay with internal inhibition control and a M. xenopi-specific PCR were developed. Ninety-three water samples from 53 taps from 4 hospitals were investigated. NTM were cultured from 21 of 49 (43%) cold and 32 of 44 (73%) warm water samples. M. chelonae, M. flavescens, M. frederiksbergense, M. gordonae, M. moriokaense, M. mucogenicum, M. vaccae, and M. xenopi were identified with molecular methods. All 93 water samples were positive in the genus-specific PCR. M. xenopi DNA was detected in 40 of 44 (91%) warm and 33 of 49 (67%) cold water samples including 45 of 65 (69%) M. xenopi culture-negative samples. In conclusion, selective culture followed by molecular identification methods enabled detection of rare species of NTM in hospital drinking water and may be used in further studies investigating the epidemiology of NTM in water samples. The real-time PCR assays have a high sensitivity and allow rapid quantification of mycobacterial DNA in water samples.

Rapid identification of Fusobacterium nucleatum and Fusobacterium necrophorum by fluorescence in situ hybridization.

Identification of clinically relevant Fusobacterium spp. is hampered by their slow growth, their frequent occurrence in polymicrobial culture, and the low reliability of biochemical differentiation methods. A newly developed fluorescence in situ hybridization (FISH) assay allowed reliable and rapid identification of Fusobacterium necrophorum and Fusobacterium nucleatum from culture. Preliminary results show that the method offers the perspective for direct detection of these pathogens in blood cultures and abscess aspirates.

Fluorescence in situ hybridization for rapid identification of Achromobacter xylosoxidans and Alcaligenes faecalis recovered from cystic fibrosis patients.

Achromobacter xylosoxidans is frequently isolated from the respiratory secretions of cystic fibrosis (CF) patients, but identification with biochemical tests is unreliable. We describe fluorescence in situ hybridization assays for the rapid identification of Achromobacter xylosoxidans and Alcaligenes faecalis. Both assays showed high sensitivities and high specificities with a collection of 155 nonfermenters from CF patients.

Superiority of molecular techniques for identification of gram-negative, oxidase-positive rods, including morphologically nontypical Pseudomonas aeruginosa, from patients with cystic fibrosis.

Phenotypic identification of gram-negative bacteria from Cystic Fibrosis (CF) patients carries a high risk of misidentification. Therefore, we compared the results of biochemical identification by API 20NE with 16S rRNA gene sequencing in 88 gram-negative, oxidase-positive rods, other than morphologically and biochemically typical P. aeruginosa, from respiratory secretions of CF patients. The API 20NE allowed correct identification of the bacterial species in 15 out of 88 (17%) isolates investigated. Agreement between the API and the 16S rRNA gene sequencing results was high only in isolates with an API result classified as "excellent identification". Even API results classified as "very good identification" or "good identification" showed a high rate of misidentification (67% and 84%). Fifty-two isolates of morphological and biochemical nontypical Pseudomonas aeruginosa, representing 59% of all isolates investigated, were not identifiable or misidentified in the API 20NE. Therefore, rapid molecular diagnostic techniques like real-time PCR and fluorescence in situ hybridization (FISH) were evaluated in this particular group of bacteria for identification of the clinically most relevant pathogen, P. aeruginosa. The LightCycler PCR assay with a P. aeruginosa-specific probe showed a sensitivity and specificity of 98.1% and 100%, respectively. For FISH analysis, a newly designed P. aeruginosa-specific probe had a sensitivity and specificity of 100%. In conclusion, molecular methods are superior over biochemical tests for identification of gram-negative, oxidase-positive rods in CF patients. In addition, real-time PCR and FISH allowed identification of morphologically nontypical isolates of P. aeruginosa within a few hours.

Rapid diagnosis of bacterial meningitis by real-time PCR and fluorescence in situ hybridization.

Real-time PCR and fluorescence in situ hybridization (FISH) were evaluated as rapid methods for the diagnosis of bacterial meningitis and compared to standard diagnostic procedures. For PCR, a LightCycler approach was chosen, implementing eubacterial and specific PCR assays for the most relevant bacteria. For FISH, a similar probe set containing eubacterial and specific probes was composed of published and newly designed probes. Both methods were evaluated by use of cerebrospinal fluid (CSF) samples from patients with suspected bacterial meningitis. For all microscopy- and culture-positive samples (n = 28), the eubacterial PCR was positive. In addition, all identifiable pathogens were detected with specific PCR assays, according to an algorithm based on the Gram stain. The FISH method detected the pathogen in 13 of 18 positive samples. While the FISH method remained negative for all microscopy- and culture-negative samples (n = 113), the eubacterial PCR was positive for five of these samples. Sequencing of the amplicon revealed the presence of Neisseria meningitidis, Streptococcus agalactiae, and Haemophilus influenzae in three of these five samples. In addition, samples with discordant results by culture and microscopy were successfully investigated by PCR (10 samples) and FISH (5 samples). In conclusion, PCR is a highly sensitive tool for rapid diagnosis of bacterial meningitis. FISH is less sensitive but is useful for the identification of CSF samples showing bacteria in the Gram stain. Based on our results, an approach for laboratory diagnosis of meningitis including PCR and FISH is discussed.

Evaluation of the Hyplex BloodScreen Multiplex PCR-Enzyme-linked immunosorbent assay system for direct identification of gram-positive cocci and gram-negative bacilli from positive blood cultures.

We evaluated the Hyplex BloodScreen PCR-enzyme-linked immunosorbent assay (ELISA) system (BAG, Lich, Germany), a new diagnostic test for the direct identification of gram-negative bacilli and gram-positive cocci from positive blood cultures, with 482 positive BACTEC 9240 blood culture bottles. The test involves amplification of the bacterial DNA by multiplex PCR and subsequent hybridization of the PCR product to specific oligonucleotide probes in an ELISA-based format. The available probes allow the separate detection of Escherichia coli, Pseudomonas aeruginosa, Enterobacter aerogenes, Klebsiella spp., Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis/Enterococcus faecium, Streptococcus pyogenes, and Streptococcus pneumoniae and the staphylococcal mecA gene. The Hyplex BloodScreen test showed an overall sensitivity of 100% for the identification of gram-negative bacilli and 96.6 to 100% for the identification of gram-positive cocci (S. aureus, 100%; S. epidermidis, 97.2%; Enterococcus faecalis/Enterococcus faecium, 96.6%; and Streptococcus pneumoniae, 100%). The specificities of the test modules ranged from 92.5 to 100% for gram-negative bacilli and 97.7 to 100% for gram-positive cocci (Escherichia coli, 92.5%; Pseudomonas aeruginosa, 98.5%; Klebsiella spp., 100%; Enterobacter aerogenes, 100%; S. aureus, 100%, S. epidermidis, 97.7%; Enterococcus faecalis/Enterococcus faecium, 99.6%; Streptococcus pyogenes, 100%; and Streptococcus pneumoniae, 99.3%). The result of the mecA gene detection module correlated with the result of the phenotypic oxacillin resistance testing in all 38 isolates of Staphylococcus aureus investigated. In conclusion, the Hyplex BloodScreen PCR-ELISA system is well suited for the direct and specific identification of the most common pathogenic bacteria and the direct detection of the mecA gene of Staphylococcus aureus in positive blood cultures.

Algorithm for the identification of bacterial pathogens in positive blood cultures by real-time LightCycler polymerase chain reaction (PCR) with sequence-specific probes.

We developed real-time polymerase chain reaction (PCR) assays for rapid detection of the most common and clinically relevant bacteria in positive blood culture bottles, including Staphylococcus spp., S. epidermidis, S. aureus, Enterococcus spp. (including differentiation of E. faecalis and E. faecium), Streptococcus spp., Streptococcus agalactiae, Enterobacteriaceae, E. coli, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Acinetobacter spp., Bacteroides spp., Haemophilus influenzae, and Neisseria meningitidis. A total of 507 positive blood cultures were investigated according to a specific PCR algorithm based on the microscopic result of the blood culture, and the PCR results were compared to the culture results. Apart from-cross reactions between E. coli and Chryseomonas luteola and Enterococcus faecium and E. durans, the PCR assay correctly identified all bacteria in the blood cultures and did not show any false-positive results. Regarding blood cultures positive with a single species of bacteria (n = 474), 98.3% of all bacteria were correctly detected by the PCR algorithm within a few hours. However, in mixed infections, the sensitivity was lower. The PCR algorithm is well suited for rapid identification of the most common bacteria in positive blood cultures.