(2) VOL. 40, 2002. NOTES. 4305. of template. The PCR protocol was as follows: 1 cycle of denaturation at 95°C for 10 min (FastStart activation), 45 cycles of amplification (15 s of denaturation at 95°C, 8 s of annealing at 52°C, and 10 s of extension at 72°C). This step was followed by a melting curve analysis from 40 to 98°C and afterwards cooling to room temperature. All 17 ICU-relevant bacteria were extracted from biological fluids (water, plasma, urine, and sputum) and detected by real-time PCR. The fluorescence probes enabled a correct Gram stain classification. The detection limit was ⬍1 pg of bacterial DNA. As depicted in Fig. 1, all gram-negative bacteria examined showed a fluorescence signal on channel F2 (640 nm, labeling dye of the gram-negative probe). In contrast, the gram-positive bacteria showed no fluorescence signal on channel F2. It made no difference from which biological fluid the bacteria had been prepared. As depicted in Fig. 2, all gram-positive bacteria examined showed a fluorescence signal on channel F3 (705 nm, labeling dye of the gram-positive probe). The gram-negative bacteria showed no fluorescence signal on channel F3. It made no difference from which biological fluid the bacteria had been prepared. When simulating mixed infections by adding P. aeruginosa and S. epidermidis in one sample, the PCR yielded a gramnegative signal (channel F2) and a gram-positive signal (channel F3).. An early and adequate treatment of infection is a major goal in ICU therapy, but this is often not achieved. One important reason for inadequate treatment with specific antibiotics is the delay in routine microbiological detection systems, which require at least 24 to 48 h for detection (19, 28). Yet, early detection and adequate treatment of bacterial infections have a great impact on the outcome for the patient (12). A faster and more sensitive method for the detection and specification of bacteria or bacterial DNA would allow earlier treatment with a specific antibiotic. Moreover, it has been shown that rapid bacterial identification reduces costs by decreasing the length of hospitalization and conserving hospital resources (2, 11). PCR has been successfully used to detect bacterial DNA in clinical samples and has improved the sensitivity of microbiological diagnostics (5, 13). This study confirms that it is possible to detect bacterial DNA from different biological fluids and to achieve a fast and correct Gram stain classification in a one-run experiment. Traditionally, Gram stain classification of, e.g., sputum has been used to support the decision for an initial antibiotic therapy. Kalin et al. found Gram-stained smears to be a valuable aid in the diagnosis of bacterial pneumonia; the results were in agreement with those of the culture for about 75% of the purulent samples (16). On the other hand, Reed et al. found in a meta-analysis a high variability in sensitivity (15 to 100%) and also a high variability in specificity (11 to 100%) (27). The authors concluded that conventional Gram stain classification. Downloaded from http://jcm.asm.org/ on May 15, 2020 by guest. FIG. 1. Fluorescence signal of the gram-negative hybridization probes on channel F2 (640 nm). The fluorescence scale on the ordinate is adapted automatically by the real-time PCR machine and indicates the fluorescence of the selected probe..
(3) 4306. NOTES. J. CLIN. MICROBIOL.. may yield misleading results. Furthermore, Cooper et al. found an excessive variability when the same samples where examined by different technologists; so there is a substantial degree of subjectivity involved in reading Gram stains (7). Nagendra et al. found a similar variability when different microscopists examined the same Gram stains (23). This leads to the conclusion that conventional Gram stains, especially for sputa and tracheal aspirates, is of limited value in providing information for an efficient antibiotic therapy. Other authors have performed Gram stain classification successfully by Gram stain-specific PCR (18), by nested PCR (5), and by PCR followed by probe hybridization (1, 13, 30), but all these methods are time consuming (because of the probe hybridization in blotting techniques) or needed more than one reaction vial per clinical sample (for specific primers and nested PCR). The method introduced here enables analysis in less than 40 min by real-time PCR in a single-tube assay. An important issue is the amount of time required between the deposit of a clinical sample and a definite result. The hands-on time necessary for preparing DNA and performing and evaluating the PCR is less than 3 h. Real-time PCR is a promising tool for the detection of bacterial DNA from biological fluids. Fluorescence hybridization probes allow a fast detection of low amounts of bacterial DNA and a correct Gram stain classification. This may accelerate therapeutic decisions and enable an earlier adequate. antibiotic treatment. In the future, new approaches for an even more specific differentiation of microbes are conceivable. REFERENCES 1. Anand, A. R., H. N. Madhavan, and K. L. Therese. 2000. Use of polymerase chain reaction (PCR) and DNA probe hybridization to determine the Gram reaction of the infecting bacterium in the intraocular fluids of patients with endophthalmitis. J. Infect. 41:221–226. 2. Barenfanger, J., C. Drake, and G. Kacich. 1999. Clinical and financial benefits of rapid bacterial identification and antimicrobial susceptibility testing. J. Clin. Microbiol. 37:1415–1418. 3. Brakstad, O. G., K. Aasbakk, and J. A. Maeland. 1992. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J. Clin. Microbiol. 30:1654–1660. 4. Brisson-Noel, A., B. Gicquel, D. Lecossier, V. Levy-Frebault, X. Nassif, and A. J. Hance. 1989. Rapid diagnosis of tuberculosis by amplification of mycobacterial DNA in clinical samples. Lancet ii:1069–1071. 5. Carroll, N. M., E. E. Jaeger, S. Choudhury, A. A. Dunlop, M. M. Matheson, P. Adamson, N. Okhravi, and S. Lightman. 2000. Detection of and discrimination between gram-positive and gram-negative bacteria in intraocular samples by using nested PCR. J. Clin. Microbiol. 38:1753–1757. 6. Chen, K., H. Neimark, P. Rumore, and C. R. Steinman. 1989. Broad range DNA probes for detecting and amplifying eubacterial nucleic acids. FEMS Microbiol. Lett. 48:19–24. 7. Cooper, G. M., J. J. Jones, J. C. Arbique, G. J. Flowerdew, and K. R. Forward. 2000. Intra and inter technologist variability in the quality assessment of respiratory tract specimens. Diagn. Microbiol. Infect. Dis. 37:231– 235. 8. Cursons, R. T., E. Jeyerajah, and J. W. Sleigh. 1999. The use of polymerase chain reaction to detect septicemia in critically ill patients. Crit. Care Med. 27:937–940. 9. Dagan, R., O. Shriker, I. Hazan, E. Leibovitz, D. Greenberg, F. Schlaeffer, and R. Levy. 1998. Prospective study to determine clinical relevance of detection of pneumococcal DNA in sera of children by PCR. J. Clin. Microbiol. 36:669–673. 10. da Silva, F., J. E. Levi, C. N. Oda Bento, S. R. da Silva Ramos, and T. Rozov.. Downloaded from http://jcm.asm.org/ on May 15, 2020 by guest. FIG. 2. Fluorescence signal of the gram-positive hybridization probes on channel F3 (705 nm). The fluorescence scale on the ordinate is adapted automatically by the real-time PCR machine and indicates the fluorescence of the selected probe..
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(5) JOURNAL OF CLINICAL MICROBIOLOGY, June 2003, p. 2799 0095-1137/03/$08.00⫹0 DOI: 10.1128/JCM.41.6.2799.2003. Vol. 41, No. 6. ERRATUM Real-Time PCR for Detection and Differentiation of Gram-Positive and Gram-Negative Bacteria Sven Klaschik, Lutz E. Lehmann, Ansgar Raadts, Malte Book, Andreas Hoeft, and Frank Stuber Department of Anesthesiology and Intensive Care Medicine, University of Bonn, Bonn, Germany Vol. 40, no. 11, p. 4304–4307, 2002. Page 4304, column 2, lines 37 and 38: “FastStart DNA Master SYBR Green I” should read “FastStart DNA Master hybridization probes.” Page 4304, column 2, lines 40 and 41: “SYBR Green I dye,” should be deleted.. 2799.