Detection of pathogenic fungi in human blood by the polymerase chain reaction

Polanco, Ana M.; Rodríguez-Tudela, Juan Luis; Martı́nez-Suárez, Joaquı́n V.

doi:10.1007/bf01690738

Cited by 24 publications

(21 citation statements)

References 14 publications

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“…For example, 18 different species of fungus were detected by a PCR method employing a universal primer that amplified a highly conserved region in the 18S ribosomal DNA (rDNA) (158), but this method cannot differentiate among these species. However, the subsequent probing of the amplicons with species-specific probes discriminated among individual fungal pathogens to species level (56,126,184).…”

Section: Species Identificationmentioning

confidence: 99%

Current Status of Nonculture Methods for Diagnosis of Invasive Fungal Infections

2002

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The incidence of invasive fungal infections has increased dramatically in recent decades, especially among immunocompromised patients. However, the diagnosis of these infections in a timely fashion is often very difficult. Conventional microbiologic and histopathologic approaches generally are neither sensitive nor specific, and they often do not detect invasive fungal infection until late in the course of disease. Since early diagnosis may guide appropriate treatment and prevent mortality, there has been considerable interest in developing nonculture approaches to diagnosing fungal infections. These approaches include detection of specific host immune responses to fungal antigens, detection of specific macromolecular antigens using immunologic reagents, amplification and detection of specific fungal nucleic acid sequences, and detection and quantitation of specific fungal metabolite products. This work reviews the current status and recent developments as well as problems in the design of nonculture diagnostic methods for invasive fungal infections

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Section: Species Identificationmentioning

confidence: 99%

Current Status of Nonculture Methods for Diagnosis of Invasive Fungal Infections

2002

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“…To shorten the time required for diagnosis, amplification reactions have been developed. Amplification targets have been genes coding for specific proteins (30,95,161,186), 18S rDNA (15,81,82,121,124,144), the 26S intergenic spacer region (180), or mitochondrial DNA (127). The last two represent repeated sequences, and thus their use increases the test sensitivity.…”

Section: Fungimentioning

confidence: 99%

Relevance of nucleic acid amplification techniques for the diagnosis of respiratory tract infections in the clinical laboratory

Ieven¹

1996

Journal of Microbiological Methods

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“…A method that would significantly improve detection by blood culture should be expected to detect 1 to 10 CFU/ml of blood (19). In our assays, we consistently could detect about three C. albicans cells in 0.5 ml of blood, which is in the range of sensitivity of or even better than most results obtained by PCR-based detection methods (5,7,9,13,18,23). Detection by fluorescent in situ hybridization is rapid; the whole procedure, from the time of blood sampling to the time of microscopic detection, can be performed within 1 working day, in contrast to methods requiring cultivation and phenotypic characterization.…”

Section: Discussionsupporting

confidence: 50%

“…Detection by fluorescent in situ hybridization is rapid; the whole procedure, from the time of blood sampling to the time of microscopic detection, can be performed within 1 working day, in contrast to methods requiring cultivation and phenotypic characterization. Many PCR protocols rely on further manipulations to enhance sensitivity, for example, Southern hybridization (7,9,13,18,23) or immunoassays (5), which increase the time until a positive result is obtained. Although the lysis-filtration assay and hybridization with fluorescencelabeled oligonucleotide probes require somewhat more hands-on time due to the hybridization and washing steps as well as the visual examination of the filters, the speed of the method is comparable to those of PCR-based methods.…”

Section: Discussionmentioning

confidence: 99%

Fluorescent in Situ Hybridization

Zheng¹,

Wu²

Encyclopedia of Genetics, Genomics, Proteomics and Informatics

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Two 18S rRNA-targeted oligonucleotide probes specific for Candida albicans and Candida parapsilosis were used to detect and identify by fluorescent in situ hybridization these medically important Candida species in deep organs of mice after experimental systemic infection. The C. albicans-specific probe detected fungal cells in kidney, spleen, and brain sections of a mouse infected with C. albicans but not in a mouse infected with the closely related species C. parapsilosis. Conversely, the C. parapsilosis-specific probe detected fungal cells in the deep organs of a mouse infected with C. parapsilosis but not in the deep organs of a C. albicans-infected mouse. In addition, the C. albicans-specific probe was used to detect this species in human blood spiked with yeast cells by a lysis-filtration assay and subsequent fluorescent in situ hybridization. By this assay, as few as three yeast cells per 0.5 ml of blood were consistently detected. Our results demonstrate that fluorescent in situ hybridization with species-specific rRNA-targeted oligonucleotide probes provides a novel, culture-independent method for the sensitive detection and identification of Candida species in clinically relevant material.The opportunistic fungal pathogen Candida albicans and other species of the genus Candida are able to cause superficial and often fatal disseminated infections in immunocompromised patients. A clear-cut diagnosis is often difficult to obtain, and deep-seated candidiasis is in most cases only recognized postmortem. Disseminated candidiasis is usually diagnosed by blood culture, although blood cultures from patients with histologically proven disseminated candidiasis are often negative. In addition, even with an optimal blood culture system such as the lysis-centrifugation method, it takes a minimum of 1 to 2 days for detection and even longer for species identification (8). To reduce mortality in patients suffering from invasive candidiasis, early initiation of proper antifungal drug therapy is critical (2, 4). Because Candida species differ in their susceptibilities to widely used systemic antifungal agents, such as azoles (20), identification of the infecting species is important for ensuring effective therapeutic strategies. Direct histologic detection of the fungus in biopsy material from deep tissue offers the relatively best diagnostic assurance (11). The infecting species, however, cannot be identified by microscopic examination alone. Among other nonculture methods, like the less reliable detection of antigens or specific antibodies (19), PCR-based approaches for the detection of Candida infections have also been developed. Identification of the Candida species, however, relies on the application of one of a number of techniques which in most cases require additional steps after PCR (6, 9, 14-16, 21, 24, 26). The diagnostic value of these methods, however, remains to be established (19).Fluorescent in situ hybridization of whole cells with rRNAtargeted oligonucleotide probes has increasingly become a valuable t...

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Detection of pathogenic fungi in human blood by the polymerase chain reaction

Cited by 24 publications

References 14 publications

Current Status of Nonculture Methods for Diagnosis of Invasive Fungal Infections

Current Status of Nonculture Methods for Diagnosis of Invasive Fungal Infections

Relevance of nucleic acid amplification techniques for the diagnosis of respiratory tract infections in the clinical laboratory

Fluorescent in Situ Hybridization

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