PCR Based Analysis of Fungal Infection in FFPE Specimens Using the Luminex Multiplex Panel
Jared D Barker, Cary Chisholm, Daniel A Smith, Kimberly Walker, Robert S Beissner, Mae Lopez, Arundhati Rao. Scott & White Memorial Hospital, Temple, TX
Background: Histopathological diagnosis of fungal infections can be limited by overlapping morphologic characteristics of species, presence of inflammatory infiltrate, and the number of fungal organisms. Nucleic acid amplification by PCR has been performed with varying degrees of success but has been shown to have some advantages including speciation, which is unavailable by routine histology. We investigated the new Luminex fungal ASR panel on FFPE tissue to determine the sensitivity and specificity for routine clinical use.
Design: 108 FFPE specimens positive by histology and cytochemical analysis with or without culture, along with 13 negative tissue sections, were selected and fungal elements or necrotic areas were isolated using the Arcturus Laser Capture Microscope and were incubated in the presence of proteinase K and lyticase enzyme. Tissue curls (10 sections of 5 micron thickness) were extracted with a modified protocol using the Qiagen DNA Mini kit. The Luminex ASRs in a Multiplex PCR format with a 24-plex primer mix was used to detect Candida species albicans, glabrata, lusitaniae, tropicalis, parapsilosis, guilliermondii and krusei, Aspergillus species terreus, fumigatus, flavus and niger, H. capsulatum, C. immitis, C. neoformans, B. dermatitidis, S. apiospermum, S. prolificans, Fusarium, R. microsporus, R. arrhizus, M. indicus, C. bertholletiae, and P. jirovecii on the MagPix system after nucleic acid extraction with the plate heated to 45°C; 100 beads/set were collected. Results were correlated with histology and culture results.
Results: Tissue curls were the specimen of choice and sensitivity was improved with the amount of specimen submitted. 12 of 13 necrotic specimens with no fungal elements identified by culture or histology were negative. 39 of 108 positive histology specimens were negative by Luminex fungal ASR panel. 66 were in agreement, and 3 specimens were positive for organisms not identified by histology. Organisms correctly identified included Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, Candida and Coccidioides with a specificity of 98%. Histologically undetected co-infections were also identified.
Conclusions: The Luminex ASR PCR panel has good specificity but the sensitivity of silver staining is better. Other Luminex PCR test advantages include speciation and detection of co-infections. The limiting factor appears to be the number of organisms since thick tissue curls worked better than laser capture specimens. PCR based detection is helpful for turn around time as compared to culture, accurate speciation, and prompt therapy of fungal infections.
Wednesday, March 21, 2012 9:30 AM
Poster Session V # 266, Wednesday Morning