Nucleic acid sequence-based amplification (NASBA) detection of medically important Candida species

Widjojoatmodjo, Myra N.; Borst, Annemarie; Schukkink, R; Box, A. T. A.; Tacken, N.; Gemen, Bob van; Verhoef, J.; Top, B.; Fluit, Ad C.

doi:10.1016/s0167-7012(99)00079-2

Cited by 40 publications

(28 citation statements)

References 31 publications

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“…and Aspergillus spp. (7,29,46), and no real-time NASBA methods had been reported for BSIs. Conversely, during the past decade, many PCR-based assays were developed and dominated the methods used for the identification of microorganisms and the diagnosis of infections.…”

Section: Vol 47 2009 Molecular Beacon Detection Of Bloodstream Infementioning

confidence: 99%

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Rapid Real-Time Nucleic Acid Sequence-Based Amplification-Molecular Beacon Platform To Detect Fungal and Bacterial Bloodstream Infections

Zhao¹,

Park²,

Kreiswirth³

et al. 2009

J Clin Microbiol

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Bloodstream infections (BSIs) are a significant cause of morbidity and mortality. Successful patient outcomes are diminished by a failure to rapidly diagnose these infections and initiate appropriate therapy. A rapid and reliable diagnostic platform of high sensitivity is needed for the management of patients with BSIs. The combination of an RNA-dependent nucleic acid sequence-based amplification and molecular beacon (NASBA-MB) detection system in multiplex format was developed to rapidly detect medically important BSI organisms. Probes and primers representing pan-gram-negative, pan-gram-positive, pan-fungal, pan-Candida, and panAspergillus organisms were established utilizing 16S and 28S rRNA targets for bacteria and fungi, respectively. Two multiplex panels were developed to rapidly discriminate bacterial or fungal infections at the subkingdom/ genus level with a sensitivity of 1 to 50 genomes. A clinical study was performed to evaluate the accuracy of this platform by evaluating 570 clinical samples from a tertiary-care hospital group using blood bottle samples.

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Section: Vol 47 2009 Molecular Beacon Detection Of Bloodstream Infementioning

confidence: 99%

“…The detection limits of pan-Candida and pan-Aspergillus assays are in agreement with the results reported by other studies that used traditional NASBA detection (electrochemiluminescence) for Candida spp. (28,46) or Aspergillus spp. (29).…”

Section: Vol 47 2009 Molecular Beacon Detection Of Bloodstream Infementioning

confidence: 99%

Rapid Real-Time Nucleic Acid Sequence-Based Amplification-Molecular Beacon Platform To Detect Fungal and Bacterial Bloodstream Infections

Zhao¹,

Park²,

Kreiswirth³

et al. 2009

J Clin Microbiol

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“…Previous reports on the application of "isothermal" nucleic acid amplification techniques to yeast identification (3,4,22,46) are all based on nucleic acid sequence-based amplification (6), but this method is rather unspecific due to the need to use a relatively low temperature (40°C) for amplification (33). We are interested in the development of a simple and user-friendly bench DNA-based diagnostic kit for the identification of clinically relevant yeasts.…”

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confidence: 99%

Efficient Identification of Clinically Relevant Candida Yeast Species by Use of an Assay Combining Panfungal Loop-Mediated Isothermal DNA Amplification with Hybridization to Species-Specific Oligonucleotide Probes

2008

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The occurrence of invasive mycoses has progressively increased in recent years. Yeasts of the genus Candida remain the leading etiologic agents of those infections. Early identification of opportunistic yeasts may contribute significantly to improved disease management and the selection of appropriate antifungal therapy. We developed a rapid and reliable molecular identification system for clinically relevant yeasts that makes use of nonspecific primers to amplify a region of the 26S rRNA gene, followed by reverse hybridization of the digoxigenin-labeled products to a panel of species-specific oligonucleotide probes arranged on a nylon membrane macroarray format. DNA amplification was achieved by the recently developed loop-mediated isothermal DNA amplification technology, a promising option for the development of improved laboratory diagnostic kits. The newly developed method was successful in distinguishing among the major clinically relevant yeasts associated with bloodstream infections by using simple, rapid, and cost-effective procedures and equipment.

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“…NASBA-based assays have been described for the detection of Candida spp. (19), Salmonella spp. (15), human immunodeficiency virus (14), cytomegalovirus (1), and hepatitis C virus (4) RNA in clinical specimens.…”

mentioning

confidence: 99%

“…In contrast, RT-PCR (13) and NASBA-based assays (1) proved to be highly specific methods for the detection of viral infection as it directly reflects transcriptional activity. Widjojoatmodjo et al demonstrated that NASBA is a good alternative to PCR for the detection of candidemia (19). Candida RNA was extracted according to a modified protocol from van Deventer et al (18), which allows RNA isolation within 3 h. However, for Aspergillus RNA extraction, a protocol including immersion of the fungal cells in liquid nitrogen and incorporation with ␤1,3-glucanase is mandatory because of the complexity of its cell wall.…”

mentioning

confidence: 99%

Nucleic Acid Sequence-Based Amplification of Aspergillus RNA in Blood Samples

et al. 2001

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Nucleic acid sequence-based amplification (NASBA), an isothermal amplification technique, was established and evaluated for the detection of Aspergillus RNA and compared with a previously published, well-defined real-time PCR assay amplifying a region of the Aspergillus 18S rRNA gene. NASBA showed a lower detection limit of 1 CFU and detected RNA from five different clinically relevant Aspergillus species, including Aspergillus fumigatus. All 77 blood samples tested by PCR and NASBA showed identical results in both assays. Results with the NASBA technique were obtained within 6 h. Thus, the NASBA technique provided a valuable tool for sensitive, specific, fast, and reliable detection of Aspergillus RNA with potential for routine diagnosis, including the possibility to test the viability of cells.Aspergillus species cause life-threatening acute invasive disease in immunocompromised patients. The incidence of invasive aspergillosis has been steadily increasing over the last few decades due to an increasing number of patients undergoing chemotherapy or bone marrow and solid organ transplantation and to an improved survival rate among patients with AIDS. The incidence is estimated to be up to 25% and the mortality is up to 90% in patients with acute leukemia (5). Early diagnosis is essential for appropriate and successful antifungal therapy. However, conventional tests for the detection of Aspergillus spp., such as blood culture and serology, have limited sensitivity and specificity (5). Diagnosis by biopsy or histopathology is difficult due to the severity of illness in these patients. Recently developed methods such as antigen detection and PCR-based assays may provide sensitive tools for the diagnosis of invasive aspergillosis.Nucleic acid sequence-based amplification (NASBA) is an isothermal amplification technology which specifically amplifies RNA sequences in a DNA background by using T7 RNA polymerase (2). NASBA-based assays have been described for the detection of Candida spp. (19), Salmonella spp. (15), human immunodeficiency virus (14), cytomegalovirus (1), and hepatitis C virus (4) RNA in clinical specimens. However, to our knowledge, NASBA protocols have not yet been applied to the detection of Aspergillus RNA.The objective of our study was to establish and to evaluate a NASBA-based assay for a sensitive and specific extraction, amplification, and detection of RNA from different Aspergillus species in blood and to compare these data with a previously published, well-defined real-time PCR assay amplifying a region of the Aspergillus 18S rRNA gene (11).For the determination of the lower detection limit of the NASBA assay, as well as for RNA degradation experiments, blood from healthy volunteers was spiked with Aspergillus fumigatus conidia (10 6 to 10 0 /ml, in serial dilution). Additionally, 77 blood specimens from neutropenic patients after allogeneic bone marrow transplantation (n ϭ 20) were analyzed in parallel by real-time PCR and NASBA-based assays for the presence of Aspergillus nucleic acid.RNA was...

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Nucleic acid sequence-based amplification (NASBA) detection of medically important Candida species

Cited by 40 publications

References 31 publications

Rapid Real-Time Nucleic Acid Sequence-Based Amplification-Molecular Beacon Platform To Detect Fungal and Bacterial Bloodstream Infections

Rapid Real-Time Nucleic Acid Sequence-Based Amplification-Molecular Beacon Platform To Detect Fungal and Bacterial Bloodstream Infections

Efficient Identification of Clinically Relevant Candida Yeast Species by Use of an Assay Combining Panfungal Loop-Mediated Isothermal DNA Amplification with Hybridization to Species-Specific Oligonucleotide Probes

Nucleic Acid Sequence-Based Amplification of Aspergillus RNA in Blood Samples

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