title: Development of a new real-time PCR system for simultaneous detection of bacteria and fungi in pathological samples.
abstract: A novel system for simultaneous detection of pathogenic bacteria and fungi in pathological samples was developed using a real-time polymerase chain reaction (PCR) system. This system, designated the "multi-microbial real-time PCR", has the potential to simultaneously detect 68 bacterial and 9 fungal species in a 96-well plate format. All probe-primer sets were designed to produce amplicons smaller than 210 bp using formalin-fixed paraffin-embedded samples as input. The specificity and sensitivity of each probe-primer set were tested against DNA extracted from pure cultures of specific pathogens. The multi-microbial real-time PCR system revealed profiles of microorganism infection in lung samples collected at autopsy from 10 patients with acquired immunodeficiency syndrome. Staphylococcus aureus was the most common microbe detected (n=8), but with low copy numbers. High copy numbers of Pseudomonas aeruginosa were detected in the lung samples with abscess (n=6). Enterococcus faecium (n=6), Elizabethkingia meningoseptica (n=4), and Candida albicans (n=4) were also frequently detected. In addition, a latent infection of Mycobacterium tuberculosis was detected in one case of pneumonia. In conclusion, this multi-microbial real-time PCR system can be useful for detecting bacteria and fungi in pathological specimens from patients with uncertain diagnoses.
authors.affiliation: Department of Pathology, National Institute of Infectious Diseases1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan; Department of Dermatology, Nippon Medical School1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan.
authors.forename: Hitomi
authors.initials: H
authors.lastname: Fukumoto
authors.affiliation: Department of Pathology, National Institute of Infectious Diseases 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Sato
authors.affiliation: Department of Pathology, National Institute of Infectious Diseases 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Hasegawa
authors.affiliation: Department of Dermatology, Nippon Medical School 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan.
authors.forename: Hidehisa
authors.initials: H
authors.lastname: Saeki
authors.affiliation: Department of Pathology, National Institute of Infectious Diseases 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
authors.forename: Harutaka
authors.initials: H
authors.lastname: Katano
mesh_terms: D000328:Adult; D001344:Autopsy; D001431:Bacteriological Techniques; D004269:DNA, Bacterial / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D004271:DNA, Fungal / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D005260:Female; D006801:Humans; D008168:Lung / Q000382:microbiology*; D008297:Male; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction; D011237:Predictive Value of Tests; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D012141:Respiratory Tract Infections / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial; D004271:DNA, Fungal
keywords: FFPE sample; Real-time PCR; bacteria; detection; fungi; pathological sample
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