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

Wellinghausen, Nele; Wirths, Beate; Franz, Axel R.; Károlyi, Lothar; Marre, R.; Reischl, Udo

doi:10.1016/j.diagmicrobio.2003.11.005

Cited by 77 publications

(57 citation statements)

References 22 publications

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“…Previously published hybridization probe LightCycler PCR assays (30) were, in addition, evaluated in terms of their utility to detect bacterial pathogens in CSF samples for the first time (Tables 1 and 3). The analytical sensitivity of the specific PCR assays in CSF was comparable to that previously published for DNA preparations in physiological saline (30). Among all assays, the Staphylococcus spp.-specific PCR and the S aureus-specific PCR had the lowest analytical sensitivities.…”

Section: Discussionmentioning

confidence: 99%

“…This algorithm also considers the presence of gram-negative rods, although there were no samples with gram-negative rods included in this study. The PCR assays for the detection of E. coli, Enterobacteriaceae, Haemophilus influenzae, and Pseudomonas aeruginosa were published previously (30). Amplification mixtures consisted of 2 l of 10ϫ reaction mix (LightCycler Fast-Start master DNA hybridization probes; Roche Diagnostics), 4 mM MgCl 2 , 0.5 M concentrations of each primer, 0.2 M concentrations of each oligonucleotide probe, and 2 l of template DNA in a final volume of 20 l. Samples were amplified as described above but with an annealing temperature of 54°C for the N. meningitidis-specific assay.…”

Section: Bacterialmentioning

confidence: 99%

“…For identification of meningeal pathogens in microscopy-positive samples, specific hybridization probe LightCycler PCR assays were run (Table 1) using primers and probes published previously (20,30). Identification of enterococci and streptococci was achieved in the same PCR by melting curve analysis, since streptococci show a melting temperature of Ͻ60°C, E. faecalis shows a melting temperature of 70°C, and E. faecium shows a melting temperature of 63°C in the PCR assay (30). PCR assays to be run on each sample were chosen according to the result of microscopy (morphology and Gram stain characteristics) by following an algorithm based on the Gram stain (see Table 3).…”

Section: Bacterialmentioning

confidence: 99%

“…Therefore, in this study, a broad-range real-time eubacterial LightCycler PCR assay for the detection of all relevant bacteria causing meningitis was evaluated. In addition, a panel of previously published speciesand genus-specific real-time LightCycler PCR assays (30) was evaluated by use of CSF samples for the first time.…”

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confidence: 99%

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Rapid Diagnosis of Bacterial Meningitis by Real-Time PCR and Fluorescence In Situ Hybridization

et al. 2005

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Bacterialmentioning

confidence: 99%

Section: Bacterialmentioning

confidence: 99%

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Rapid Diagnosis of Bacterial Meningitis by Real-Time PCR and Fluorescence In Situ Hybridization

et al. 2005

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“…The diagnostic sensitivities and specificities of these tests for the investigation of most positive blood cultures are extremely high, but some PCR assays could fail to identify gram-negative rods including P. luteola and no specific PCR assay for P. luteola had been developed (25).…”

Section: Discussionmentioning

confidence: 99%

Bacteremia Caused by Pseudomonas luteola in Pediatric Patients

Bayhan¹,

Şenel²,

Tanır³

et al. 2015

Jpn J Infect Dis

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SUMMARY: Pseudomonas luteola has rarely been reported as a human pathogen. The clinical manifestations of P. luteola bacteremia and its susceptibility to antibiotics have not been characterized. This retrospective study was conducted at a 382-bed tertiary care center in Turkey. During the 9-year study period, 7 patients (5 females and 2 males) were diagnosed with P. luteola bacteremia. Six of these patients had hospital-acquired bacteremia, whereas 1 patient had community-acquired P. luteola infection. All patients had monomicrobial bacteremia. Antimicrobial susceptibility testing revealed that all strains of P. luteola were sensitive to amikacin, gentamicin, trimethoprim-sulfamethoxazole, and meropenem, and that all strains were resistant to piperacillin-tazobactam, aztreonam, and colistin. In conclusion, we believe that P. luteola can cause both community-and hospital-acquired bacteremia. Amikacin, gentamicin, trimethoprim-sulfamethoxazole, and meropenem were effective against P. luteola in the present study.

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