We describe a rapid and reproducible PCR assay for quantitation of the Candida albicans ribosomal DNA (rDNA) in clinical blood samples based on the TaqMan principle (Applied Biosystems), in which a signal is generated by cleavage of a template-specific probe during amplification. We used two fluorogenic probes based on universal, fungus-specific primers, one for the detection of C. albicans species DNA and one for the detection of all Candida genus DNA. C. albicans blastoconidia mixed with whole blood in a titration experiment yielded a linear PCR signal over a range of 3 orders of magnitude. The TaqMan-based PCR assay for C. albicans exhibited a low limit of detection (5 CFU/ml of blood) and an excellent reproducibility (96 to 99%). While the C. albicans species-specific probe had 100% specificity for C. albicans, all Candida genus-specific probes cross-reacted with other organisms likely to coinfect patients with C. albicans infections. On the basis of these data, we determined the C. albicans loads with a species-specific probe from 122 blood samples from 61 hematology or oncology patients with clinically proven or suspected systemic Candida infections. Eleven positive samples exhibited a wide range of C. albicans loads, extending from 5 to 100,475 CFU/ml of blood. The sensitivity and specificity of the present assay were 100 and 97%, respectively, compared with the results of blood culture. These data indicate that the TaqMan-based PCR assay for quantitation of C. albicans with a species-specific probe provides an attractive alternative for the identification and quantitation of C. albicans rDNA in pure cultures and blood samples.Invasive candidiasis, a common cause of nosocomial infection, is a leading cause of infections among patients receiving bone marrow transplantations or those with leukemia or other cancers and is also associated with a high rate of morbidity and mortality (10,18,22). Given the rapidly fatal course of candidiasis, there is a need for improved methods for early diagnosis and subsequent initiation of antifungal therapy in order to have a significant impact on the death rate (1, 12). One such improvement is the application of rapid and sensitive methods based on PCR that enable the amplification and quantitation of a broad range of fungal pathogens directly from specimens and pure cultures.Two quantitative real-time PCR systems have been described for Candida detection. An assay based on the PCR LightCycler system (Roche Molecular Diagnostics Systems, Indianapolis, Ind.) was applied by Löeffler et al. (15) for quantification of Candida albicans DNA in blood to which known numbers of blastoconidia had been added. Those investigators also used the assay to determine the fungal burdens in a small number of blood samples taken from patients with hematological malignancies. More recently, Guiver et al. (7) and Bowman et al. (3) described the automated detection of fungal DNA by using the TaqMan assay (Applied Biosystems, Foster City, Calif.) with clinical isolates and murine tissues, respect...

Hemorrhagic cystitis that occurs late after bone marrow transplantation (BMT) in BMT recipients is often associated with adenovirus or polyomavirus BK infections. Intravesical instillation of cidofovir in a BMT recipient with intractable hemorrhagic cystitis resulted in clinical improvement. Local cidofovir therapy for viral hemorrhagic cystitis could be an alternative to intravenous administration of cidofovir.

In a recent study, Candida species in clinical blood samples were detected using a real-time PCR-based method (Maaroufi et al, J Clin Microbiol 2003, 41:3293-3298). For the present study, we evaluated the efficiency of this method as an adjunct to the BACTEC blood culture system to early detection of positivity and negativity of simulated low candidemias. We first established an in vitro correlation between the inoculum of the most frequently encountered Candida species and the time to positivity of these microorganisms. Then, aliquots from blood culture bottles infected with a final average candidal inoculum of 3.18 colony-forming units (CFU)/culture bottle (range, 1 to 6 CFU) were collected at increasing incubation times, and DNA was extracted and submitted to the TaqMan-based PCR assay. To optimize this assay, we evaluated the effect of adding 0.5% bovine serum albumin (BSA) to DNA extracts and found that it decreased the effects of inhibitors. Using specific probes for the tested Candida species, the PCR assay was positive on blood culture aliquots collected from the BACTEC system after a minimum culture turnaround time (TAT) of 3.11 +/- 1.24 hours. Addition of BSA to PCR reaction mixtures improves the TAT (1.84 +/- 0.41 hours). Hence, the combination of DNA "amplification" in the culture bottles by normal growth with an additional DNA amplification by PCR might be a reliable tool facilitating the early diagnosis of low candidemias.

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A major pitfall in most published genomic amplification methods for the detection and identification of human pathogens is that they do not include an internal amplification control in order to achieve an acceptable level of confidence for the absence of false-negative results. By applying composite primer technology, a single multiple internal amplification control DNA molecule was constructed to detect and quantify the hepatitis B virus, human polyomavirus, Epstein-Barr virus, Toxoplasma gondii and cytomegalovirus using real-time PCR. The multiple internal amplification control contains all forward and reverse primer binding regions targeted in the five distinct duplex PCRs, but with a unique probe hybridization site. Multiple internal amplification control detection sensitivity, assessed by Probit analysis, was 58 copies per PCR, associated with an extremely wide dynamic range (8 log 10 units). Moreover, in testing 614 patient samples, PCR inhibition occurred at a frequency of 0-8.8%. Similar multiple internal amplification controls for quantitative PCR-based assays could be designed to accommodate any infectious profiles in a particular institution as they are easy to make and inexpensive.