Detection of candidemia by polymerase chain reaction.
Current blood culture methods may not identify all patients with candidemia. We attempted to develop a polymerase chain reaction (PCR)-based method for the detection of candidemia. Several major problems were encountered: use of primers based on single copy or low-copy-number genes lacked sensitivity, while those based on the 18S ribosomal RNA gene often crossreacted with human DNA. One primer set with an internal primer was found to be suitable in a hemi-nested PCR. Sensitivity was in the range of 1 colony forming unit of C. albicans per ml blood in reconstruction experiments. Using stored blood which had been obtained within 12 h of the time a positive blood culture was obtained, PCR was positive in 7/15 (46.7%) patients with culture proven C. albicans candidemia (44% of samples). However, only 1-3 ml blood was available for PCR, while culture results were based on 5-10 ml or more. Two out of 34 (5.9%) unselected outpatient blood samples stored under identical conditions were positive, but both samples were negative when the original DNA was retested. Blood collection and PCR processing equipment appears to be free of contaminating fungal DNA and organisms; however, human skin contains DNA amplifiable by these primers. At present the major limitation is the need for a simple method to recover Candida from 5-10 ml blood while removing haemoglobin and excess white blood cell DNA in a volume suitable for PCR.
Department of Pathology, University of Florida, Gainesville 32611.
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Rand
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Houck
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Wolff
D001483:Base Sequence; D002176:Candida albicans / Q000235:genetics; D002177:Candidiasis / Q000097:blood* / Q000175:diagnosis* / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*
D004271:DNA, Fungal