Application of laser capture microdissection and polymerase chain reaction in the diagnosis of Trichoderma longibrachiatum infection: a promising diagnostic tool for ‘fungal contaminants’ infection

Abstract

Although Trichoderma species are usually considered to be culture contaminants, an increasing number of case reports have demonstrated their pathogenicity. Current diagnostic tools, including fungal culture, radiology, histopathology, and direct microscopy examination, are often unable to differentiate the pathogenicity of ‘fungal contaminants’ such as Trichoderma species in patients. Accurate diagnostic tools for ‘fungal contaminants’ infection have become the urgent needs. To that end, we applicated laser capture microdissection (LCM) and polymerase chain reaction (PCR) to confirm T. longibrachiatum infection for the first time. A 57-year-old man presented with a cough and hemoptysis lasting for more than 40 days. Computed tomography scan revealed a mass at the left hilum. In addition to pulmonary spindle cell carcinoma, fungal hyphae were also detected in histopathological examination. The cultured fungus was identified as T. longibrachiatum using molecular procedures. The results from DNA sequencing of DNA obtained by LCM revealed the identical result. Antifungal susceptibility testing revealed resistance to itraconazole, fluconazole and flucytosine. The patient was managed with oral voriconazole for 4 months. No relapse of Trichoderma infection was observed at a year follow-up visit. Although there are potential disadvantages, LCM-based molecular biology technology is a promising diagnostic tool for ‘fungal contaminants’ infection.

Fatal Rhinofacial Mycosis Due to Aspergillus nomiae: Case Report and Review of Published Literature

“…Genomic DNA of the fungal isolate was extracted using the Biospin Fungus Genomic DNA Extraction kit (Bioer Technology, Hubei, China) in accordance with manufacturer's instructions. The internal transcribed spacer (ITS) region was amplified using the ITS5 and ITS4 primers (Zhou et al, 2019), a segment of the β-tubulin gene (BenA) was amplified with βtub1 and βtub2 primers (Balajee et al, 2005), as well as a segment of the calmodulin gene (CaM) with cmd5 and cmd6 primers (Hong et al, 2005). Each PCR mixture contained 1 µL extracted fungal DNA, 0.08 µM each of the primers, and 12.5 µL 2 × Taq PCR MasterMix (Tiangen Biotech, Beijing, China) in a 25 µL reaction volume.…”

Application of laser capture microdissection and PCR sequencing in the diagnosis of Coccidioides spp. infection: A case report and literature review in China

“…The predominant etiologic agents, including the species of Aspergillus, Alternaria, Cladosporium, Penicillium, and Fusarium were also identified in FFPE tissue from this patient, which could cause asthma in this patient for up to 20 years since these fungi were speculated colonizing the respiratory tract and triggering IgE-mediated allergic reactions [57,58]. [6]. This method and subsequent molecular techniques have been used to isolate pure tumor tissues and detect the expression of target genes, which was initially applied for analyzing cancers [6].…”

“…Pure cells or tissues on the slides were dissected with a 337 nm pulsed ultraviolet laser and collected in a sample tube. As a negative control, an adjacent nonfungal tissue sample of similar size was excised from the same specimen and processed in parallel [6].…”

“…[6]. This method and subsequent molecular techniques have been used to isolate pure tumor tissues and detect the expression of target genes, which was initially applied for analyzing cancers [6]. In a previous study, Trichoderma DNA in FFPE specimens was detected using the combined LCM and PCR technique, resulting in the clinical diagnosis of rare Trichoderma infections [8].…”

“…In recent years, laser capture microdissection (LCM)-based analytic techniques have been applied for the microbiological diagnosis of infectious diseases [6,7]. LCM is a novel and powerful technique for selectively harvesting cells of interest directly from histological sections under direct microscopy [8].…”