The use of combined PCR, fluorescence in situ hybridisation and immunohistochemical staining to diagnose mucormycosis from formalin‐fixed paraffin‐embedded tissues

Abstract

Objective: To develop a comprehensive diagnostic system for mucormycosis from formalin-fixed paraffin-embedded tissues, consisting of own-designed real-time polymerase chain reaction (PCR) assays, fluorescence in situ hybridisation, and immunohistochemical staining. Methods: We designed 11 primers and probes for specific real-time PCR assays based on genome sequences, and validated the specificity by Aspergillus, Fusarium, Scedosporium, Lomentospora, Cryptococcus and Candida species. Formalin-fixed paraffin-embedded (FFPE) tissues from forty-four mouse model infected by above fungi were collected and extracted DNA by laser capture microdissection (LCM) and direct extraction methods for real-time PCR assays. In addition, seventeen clinical specimens histopathologically proven for mucormycosis were included for specific detection with the new diagnostic system. Results: The real-time PCR assays allowed detection of a minimum of 10 CFU/ml equivalent gDNA of each species. No cross-reaction with gDNA among species was noted. From mouse model specimens, the sensitivity of real-time PCR in samples extracted with LCM versus direct extraction method was 100% versus 91.43% at Mucorales level and 80% versus 45.71% at species level, respectively. The specificity was 100%. From clinical samples, LCM combined with real-time PCR can test 88.24% (15/17) of Mucorales. Sensitivities of fluorescence in situ hybridisation (FISH) and immunohistochemical staining (IHC) were 70.59% and 41.18%, respectively. Combined LCM-RT-PCR, FISH and IHC yielded positive results in all samples.Conclusions: The combination diagnostic system we developed is a cultureindependent and robust method which enables rapid species identification from FFPE tissues for timely diagnosis of mucormycosis.

Clinical impact of metagenomic next-generation sequencing of peripheral blood for the diagnosis of invasive mucormycosis: a single-center retrospective study

“…Although mucorales PCR has not yet been included in these standards due to the lack of standardization or test availability, it has significantly improved mucormycosis diagnosis in recent years. Fluorescence in situ hybridization methods for promptly identifying Mucorales from formalin-fixed, paraffin-embedded tissues, along with real-time quantitative PCR systems for precise genus- and species-level identification, have been developed to accurately detect pathogenic Mucorales in tissues ( 12 ). A French study, conducted at multiple centers, assessed the usefulness of qPCR in diagnosing invasive trichothecosis through serum samples.…”

A rare case of pediatric gastrointestinal mucormycosis with a review of the literature

“…Early diagnosis of mucormycosis is difficult given its relatively nonspecific initial symptoms [4] , [5] ; the diagnostic dilemma is compounded by inconsistent staining patterns of Mucormycetes on commonly used fungal stains and the challenges and slow timeline of culturing mucormycosis in the routine lab setting [13] , [18] . If mucormycosis is on the differential diagnosis, nucleic acid based diagnostic modalities offer high sensitivity and specificity with a much faster turn-around time: results can be available within hours of sample collection [18] , [19] . A wide array of samples are suitable for testing, including fresh and frozen tissue, formalin-fixed paraffin-embedded tissue, blood, urine, and bronchoalveolar lavage fluid [18] , [20] .…”

Serum sphingolipids aid in diagnosing adult HIV-negative patients with pulmonary cryptococcosis: a clinical cohort study

“…The combination of various diagnostic methods has emerged as a prominent trend and has proven advantageous in augmenting the diagnostic accuracy for different diseases. For example, previous study has shown that the combination of polymerase chain reaction (PCR), fluorescence in situ hybridization, and immunohistochemical staining can significantly enhance the diagnostic accuracy for mucormycosis ( 33 ). Moreover, PCR in combination with galactomannan (GM) exhibits superior diagnostic performance compared to GM alone in identifying Aspergillus infections ( 34 ).…”