Development of a Nested PCR for Detection of
            <i>Cryptococcus neoformans</i>
            in Cerebrospinal Fluid

Rappelli, Paola; Are, Riccardo; Casu, G; Fiori, Pier Luigi; Cappuccinelli, Pietro Antonio; Aceti, Antonio

doi:10.1128/jcm.36.11.3438-3440.1998

Cited by 63 publications

(38 citation statements)

References 14 publications

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“…A microtiter enzyme immunoassay (EIA) for the detection of Candida species in blood with a detection sensitivity of 2 cells/200 l of sample and a real-time fluorescent PCR-based assay for the detection of three Candida species in a single reaction tube with a detection sensitivity of 100 cells/ 200 l have previously been described (12,15). A nested PCR assay for the detection of C. neoformans in cerebrospinal fluid is reported to have a detection limit of 10 cells (29). Other researchers determined a detection level of 5 pg for A. fumigatus rDNA 18S gene amplicons in a microtiter plate EIA (10).…”

Section: Discussionmentioning

confidence: 99%

Development of a PCR-Based Line Probe Assay for Identification of Fungal Pathogens

Martin

Roberts

Weide³

et al. 2000

J Clin Microbiol

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We report on a reverse-hybridization line probe assay (LiPA) which when combined with PCR amplification detects and identifies clinically significant fungal pathogens including Candida,Aspergillus, and Cryptococcus species. DNA probes have been designed from the internal transcribed-spacer (ITS) regions of Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Candida dubliniensis, Cryptococcus neoformans,Aspergillus fumigatus, Aspergillus versicolor,Aspergillus nidulans and Aspergillus flavus. The probes were incorporated into a LiPA for detection of biotinylated ITS PCR products, and the specificity of the probes was evaluated. We established LiPA detection limits for ITS 1 and for full ITS amplicons for genomic DNA from C. albicans, A. fumigatus, and C. neoformans. Further evaluation of the LiPA was carried out on clinical fungal isolates. One hundred twenty-seven isolates consisting of dimorphic yeasts and dermatophytic and filamentous fungi were tested by the LiPA, which correctly identified 77 dimorphic yeasts and 23 of the filamentous isolates; the remaining 27 isolates represented species of fungi for which probes were not included in the LiPA. The fungal-PCR-LiPA technology was applied to blood samples inoculated with Candida cells which were pretreated by minibead beating to mechanically disrupt the cells, with the DNA extracted by either a previously described guanidium thiocyanate-silica method or the commercially available QIAmp tissue kit. PCR amplification of the extracted DNA and subsequent DNA probe hybridization in the LiPA assay yielded detection limits of 2 to 10 cells/ml. An internal standard control was included in the PCR amplification to monitor for PCR inhibition. This fungal PCR-LiPA assay is robust and sensitive and can easily be integrated into a clinical-testing laboratory with the potential for same-day diagnosis of fungal infection.

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Section: Discussionmentioning

confidence: 99%

Development of a PCR-Based Line Probe Assay for Identification of Fungal Pathogens

Martin

Roberts

Weide³

et al. 2000

J Clin Microbiol

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“…There are five studies using PCR for the diagnosis of CNS cryptococcosis, three with evidence class III and two evidence class IV. Another study reported similar findings [157]. The third class III study compared culture, India ink test, and PCR in CSF of 56 patients with cryptococcal meningitis and 16 patients with meningitis of other origin [158].…”

Section: Cryptococcosis (Cryptococcus Neoformans/gattii)mentioning

confidence: 79%

EFNS‐ENS guidelines for the use of PCR technology for the diagnosis of infections of the nervous system

Steiner

Schmutzhard

Sellner

et al. 2012

Euro J of Neurology

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Background: Polymerase chain reaction (PCR) as a means to amplify nucleic acids has become an essential element in diagnosis of infections. It has evolved into a simple and rapid, easy-to-use approach. At present there are no published guidelines for the usage of PCR technology for the diagnosis of infections of the nervous system. Methods: We reviewed the advantages and pitfalls of PCR in order to guide neurologists and infectious diseases experts in its application for the diagnosis of infections of the nervous system. Medical reference systems were searched, and original papers, meta-analyses, review papers, book chapters and guidelines recommendations were reviewed. The final literature search was performed in May 2012. Recommendations were reached by consensus. Recommendations: The reliability of PCR technology for the diagnosis of neurological infections is currently based on the pathogens. The main contribution of PCR is to the diagnosis of viral infections followed by bacterial CNS infections with the notable exception of tuberculous meningitis. Efficacy for the diagnosis of protozoal infections and helminthic infestations has also been established in many instances. Unfortunately, current molecular PCR technology is far from becoming routine in resource-poor countries where such infections are prevalent. Despite the importance of fungal infections in the context of the immune-compromised host, there is not enough data to recommend the routine use of PCR. Conclusions: PCR technology is currently a reliable method for the diagnosis of viral and bacterial (except tuberculosis) infections, and only for some protozoal infections and helminthic infestations.

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“…Unlike other pathogenic fungi, pathogenic Trichosporon species rapidly disseminate to multiple organs. There are many reports of PCR-based diagnosis methods in aspergillosis (I, 12), candidiasis (2,11), and cryptococcosis (9,16), but as far as we know only one paper has reported a PCR-based method for disseminated trichosporonosis. Nagai et al (8) attempted to detect the DNA of Trichosporon species in the sera of patients histologically diagnosed with disseminated trichosporonosis using oligonucleotide primers designed from 28S rDNA sequences.…”

Section: Discussionmentioning

confidence: 99%

A Nested PCR Assay to Detect DNA in Sera for the Diagnosis of Deep‐Seated Trichosporonosis

Sugita

Nakajima

Ikeda

et al. 2001

Microbiology and Immunology

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Deep-seated trichosporonosis caused by Trichosporon asahii has a high mortality rate and a very poor prognosis. New species-specific oligonucleotide primers for T. asahii were developed from a sequence analysis of rRNA genes that included the internal transcribed spacer regions. A nested PCR assay with specific primers was used to examine 11 serum samples from 7 patients, who were diagnosed with deep-seated trichosporonosis histologically at autopsy. In addition, Trichosporon cell wall polysaccharide (PS) was detected by a latex agglutination (LA) test. Of 11 samples, seven had a positive LA test, and T. asahii DNA was also detected with the nested PCR assay. Of the four samples in which PS antigen was not detected, the nested PCR of two samples was positive. Our new nested PCR assay may be used as an adjunct to conventional methods for diagnosing T. asahii infection.

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Development of a Nested PCR for Detection of Cryptococcus neoformans in Cerebrospinal Fluid

Cited by 63 publications

References 14 publications

Development of a PCR-Based Line Probe Assay for Identification of Fungal Pathogens

Development of a PCR-Based Line Probe Assay for Identification of Fungal Pathogens

EFNS‐ENS guidelines for the use of PCR technology for the diagnosis of infections of the nervous system

A Nested PCR Assay to Detect DNA in Sera for the Diagnosis of Deep‐Seated Trichosporonosis

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