Specific Detection of Aspergillus and Penicillium Species From Respiratory Specimens by Polymerase Chain Reaction (Pcr)

Makimura, Koichi; Murayama, Somay Yamagata; Yamaguchi, Hideyo

doi:10.7883/yoken1952.47.141

Cited by 24 publications

(10 citation statements)

References 21 publications

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“…Previous studies evaluating PCR-mediated detection of Aspergillus species showed significantly high rates of sensitivity but involved assays with different methods and objectives, partly for optimization of culture assays (37) and partly for typing in epidemiological studies (3,4).…”

mentioning

confidence: 99%

Development of a LightCycler PCR Assay for Detection and Quantification ofAspergillus fumigatusDNA in Clinical Samples from Neutropenic Patients

et al. 2003

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The increasing incidence of invasive aspergillosis, a life-threatening infection in immunocompromised patients, emphasizes the need to improve the diagnostic tools for this disease. We established a LightCyclerbased real-time PCR assay to detect and quantify rapidly, specifically, and sensitively Aspergillus fumigatus DNA in both bronchoalveolar lavage (BAL) and blood samples from high-risk patients. The primers and hybridization probes were derived from an A. fumigatus-specific sequence of the mitochondrial cytochrome b gene. The assay is linear in the range between 13.2 fg and 1.3 ng of A. fumigatus DNA, corresponding to 3 to 300,000 CFU per ml of BAL fluid or blood. No cross-amplification was observed with human DNA or with the DNA of fungal or bacterial pathogens. For clinical evaluation we investigated 10 BAL samples from nine neutropenic patients with malignant hematological diseases and 12 blood samples from seven neutropenic patients with malignant hematological diseases. Additionally, we tested one blood sample and one BAL sample from each of two neutropenic patients. In order to characterize the validity of the novel PCR assay, only samples that had shown positive results by a previously described sensitive and specific nested PCR assay were tested. Twelve of 12 BAL samples and 6 of 14 blood samples gave positive results by the LightCycler PCR assay. Eight of 14 blood samples gave negative results by the novel method. The LightCycler PCR-mediated quantification of the fungal burden showed 15 to 269,018 CFU per ml of BAL sample and 298 to 104,114 CFU per ml of blood sample. Twenty of 20 BAL samples and 50 of 50 blood samples from subjects without evidence of invasive pulmonary aspergillosis (IPA) were PCR negative. Compared to a previously described nested PCR assay, these preliminary data for the novel real-time PCR assay indicate a less sensitive rate of detection of IPA in high-risk patients, but the assay may be valuable for quantification of the fungal burden in individual clinical samples.

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mentioning

confidence: 99%

Development of a LightCycler PCR Assay for Detection and Quantification ofAspergillus fumigatusDNA in Clinical Samples from Neutropenic Patients

et al. 2003

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“…but also Penicillium spp. (5). However, this does not hamper the interpretation of our results, because Penicillium spp.…”

Section: Discussionmentioning

confidence: 71%

“…PCRlabeling probe for identification A 385 bp sequence of 1 8S ribosomal RNAfrom A.fumigatus was selected for probe synthesis (5,6). Multiple digoxigenin molecules-bearing probes were produced by PCRaccording to a method reported previously with modification (7).…”

Section: Methodsmentioning

confidence: 99%

In Situ Detection of Aspergillus 18S Ribosomal RNA in Invasive Pulmonary Apergillosis.

et al. 1999

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Objects Weattempted to evaluate the usefulness of in situ hybridization (ISH) in the specific diagnosis ofAspergillus pulmonary infection. Methods Weused an ISH technique using a multiple digoxigenin-incorporating probe, which was constructed by means of the polymerase chain reaction (PCR) from the 18S ribosomal RNAofAspergillusfumigatus.Materials Westudied twelve formalin-fixed, paraffin-embedded lung tissue sections from autopsy-confirmed invasive pulmonary aspergillosis (IPA) (5 acute myelocytic leukemias, 2 acute lymphocytic leukemias, 2 chronic myelocytic leukemias, 1 adult T-cell leukemia, 1 non-Hodgkin's lymphoma and 1 chronic obstructive pulmonary disease.), and 18 sections from other pulmonary infections as control. Results ISH using the probe and a low-viscosity hybridization buffer solution (LV) positively stained hyphal elements in 12 of 12 autopsy lung tissue specimens from subjects with IPA, while ISH using the probe and a high viscosity hybridization buffer solution (HV) positively stained the hyphal elements in 6 of 12. Specifically, ISH (LV) demonstrates hyphal elements of Aspergillus spp. in the center ofAspergillus abscess. While, ISH (HV) can detect hyphal elements located in the periphery of a suppurative abscess as well as those in the blood vessel. Conversely, ISH did not showpositive results for any of the autopsy tissue specimens from subjects with other fungal pneumonia infections (Candida n=5, Mucorn=2, Cryptococcus n=2, and Pseudallescheria n=l), Pneumocystis carinii pneumonia (n=5), and cytomegalovirus pneumonia (n=3). Dual staining by means of ISH and immunohistochemistry (IHC) using anti-neutrophil elastase (NE) and antiCD68monoclonal antibodies showed that NEpositive cells were localized at the edge of the radial growth of the organism, but CD68positive cells were located around the center of the abscess. The accumulation of NEpositive cells was rarely seen in half of the cases (6/12). In contrast, CD68 positive cells were routinely present in the center of the abscess (12/12). Conclusion ISH in conjunction with IHC is a useful tool for differentiating Aspergillus spp. from other fungal genera in tissue sections from patients with IPA and mayhave a certain role in the evaluation of the interactions between organisms and recruiting inflammatory cells.

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“…As realized successfully for other pathogenic organisms difficult to detect (for example cytomegalovirus [39], Borrelia burgdorferi [40] and Toxoplasma gondii [41]), more sensitive and rapid detection assays have been established by the use of the polymerase chain reaction method (PCR). PCR-based detection methods for fungi have been developed successfully since multicopy gene targets were identified and sequenced [42][43][44][45][46]. Previous studies for the evaluation of PCR-mediated detection of fungi showed significantly improved sensitivity, but were performed with methodically different assays and different objectives, partly to optimize culture assays [47], partly for typing in epidemiological studies [48,49], mainly in nonclinical settings [50].…”

Section: Noncultural Methods To Detect Candida Speciesmentioning

confidence: 99%

“…5 of 7 BAL samples of patients, who had radiological evidence of invasive pulmonary aspergillosis, showed positive PCR signals; in their investigation the galactomannan ELISA showed less false-positive results with BAL samples. With another assay, Makimura et al [42,43] obtained PCR products from BAL of immunocompromised and neutropenic patients, but no amplicons were obtained from immunocompetent individuals. Bretagne et al [73] pointed to the high risk of BAL specimen contamination by Aspergillus conidia resulting in approximately 23% of false-positive results.…”

Section: Noncultural Methods To Detect Aspergillus Speciesmentioning

confidence: 99%

Systemic Infections with <i>Candida</i> sp. and <i>Aspergillus</i> sp. in Immunocompromised Patients with Hematological Malignancies: Current Serological and Molecular Diagnostic Methods

Buchheidt

Skladny²,

Baust³

et al. 2000

Chemotherapy

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Within recent years, novel serological and molecular methods have been developed to improve the early diagnosis of invasive fungal infections especially in patients with malignant hematological diseases being at highest risk of developing these life-threatening infections. Early diagnosis is essential for adequate therapeutic management, which, however, often remains difficult since the diagnostic tools clinically used either lack specificity or sensitivity, or both at worst. The clinical value, the advantages and remaining problems of recently developed, more sensitive and specific serological assays and molecular methods are reviewed.

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Specific Detection of Aspergillus and Penicillium Species From Respiratory Specimens by Polymerase Chain Reaction (Pcr)

Cited by 24 publications

References 21 publications

Development of a LightCycler PCR Assay for Detection and Quantification ofAspergillus fumigatusDNA in Clinical Samples from Neutropenic Patients

Development of a LightCycler PCR Assay for Detection and Quantification ofAspergillus fumigatusDNA in Clinical Samples from Neutropenic Patients

In Situ Detection of Aspergillus 18S Ribosomal RNA in Invasive Pulmonary Apergillosis.

Systemic Infections with <i>Candida</i> sp. and <i>Aspergillus</i> sp. in Immunocompromised Patients with Hematological Malignancies: Current Serological and Molecular Diagnostic Methods

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