Serum is more suitable than whole blood for diagnosis of systemic candidiasis by nested PCR

Abstract

PCR assays for the diagnosis of systemic candidiasis can be performed either on serum or on whole blood, but results obtained with the two kinds of samples have never been formally compared. Thus we designed a nested PCR assay in which five specific inner pairs of primers were used to amplify specific targets on the rRNA genes of Candida albicans, C. tropicalis, C. parapsilosis, C. krusei, and C. glabrata. In vitro, the lower limit of detection of each nested PCR assay was 1 fg of purified DNA from the corresponding Candida species. In rabbits with candidemia of 120 minutes' duration following intravenous (i.v.) injection of 10(8) CFU of C. albicans, the sensitivities of the PCR in serum and whole blood were not significantly different (93 versus 86%). In other rabbits, injected with only 10(5) CFU of C. albicans, detection of candidemia by culture was possible for only 1 min, whereas DNA could be detected by PCR in whole blood and in serum for 15 and 150 min, respectively. PCR was more often positive in serum than in whole blood in 40 culture-negative samples (27 versus 7%; P < 0.05%). Lastly, experiments with rabbits injected i.v. with 20 or 200 microgram of purified C. albicans DNA showed that PCRs were positive in serum from 30 to at least 120 min after injection, suggesting that the clearance of free DNA is slow. These results suggest that serum is the sample of choice, which should be used preferentially over whole blood for the diagnosis of systemic candidiasis by PCR.

FIG. 1

Sensitivities and specificities of DNA detection by species-specific nested PCRs of purified DNA from C. albicans, C. tropicalis, C. krusei, C. parapsilosis, and C. glabrata with ethidium bromide staining on agarose gel electrophoresis. Shown are nested PCR products obtained with each species-specific inner pair of primers from different amounts of template DNA from the corresponding Candida species (lane 1, 1 pg; lane 2, 100 fg; lane 3, 10 fg; lane 4, 1 fg; lane 5, 0.1 fg). The size of the specific amplified fragment is indicated on the left. The specificity of the C. albicans primers was tested on 100 ng of purified DNA from C. tropicalis (lane 6), C. glabrata (lane 7), C. krusei (lane 8), and C. parapsilosis (lane 9). The specificity of the C. tropicalis primers was tested on 100 ng of purified DNA from C. albicans (lane 6), C. glabrata (lane 7), C. krusei (lane 8), and C. parapsilosis (lane 9). The specificity of the C. krusei primers was tested on 100 ng of purified DNA from C. albicans (lane 6), C. tropicalis (lane 7), C. glabrata (lane 8), and C. parapsilosis (lane 9). The specificity of the C. parapsilosis primers was tested on 100 ng of purified DNA from C. albicans (lane 6), C. tropicalis (lane 7), C. glabrata (lane 8), and C. krusei (lane 9). The specificity of the C. glabrata primers was tested on 100 ng of purified DNA of C. albicans (lane 6), C. tropicalis (lane 7), C. krusei (lane 8), and C. parapsilosis (lane 9). In these experiments, the amplified products generated by the Candida universal primers (ITS1 and ITS4) in the first reaction are sometimes visible. M, molecular weight marker.

FIG. 2

Sensitivity of quantitative blood cultures compared to that of nested PCR performed on whole blood and on serum from five rabbits infected with 10⁸ CFU of C. albicans. Each rabbit is represented by a circle at each sampling time. ○, positive nested PCR in both whole blood and serum; ◒, positive nested PCR in whole blood and negative nested PCR in serum; ◑, negative nested PCR in whole blood and positive nested PCR in serum.

FIG. 3

Coamplification of internal PCR control and rabbit blood samples. Lanes 1, 2, 3, and 4, whole-blood samples; lanes 5 and 6, serum samples which exhibited a negative C. albicans nested PCR; lanes 7 and 8, two whole-blood samples for which this PCR was positive. Amplification of the internal control is indicated by the presence of a 491-bp fragment, and amplification of C. albicans DNA in blood is indicated by the presence of a 386-bp fragment. M, molecular weight marker.