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

Abstract

Coccidioidomycosis is endemic to California, Arizona, and Mexico. In recent years, the reported cases of coccidioidomycosis have increased in nonendemic regions. Here, we reported a case of imported pulmonary coccidioidomycosis in a Chinese patient. A 63-year-old man presented with dry cough and fatigue for 6 months, and a computed tomography scan revealed a solitary nodule in the right lower lung and small nodules in both lungs. The diagnosis of coccidioidomycosis was initially confirmed by histopathologic examination. The pathogen Coccidioides spp. was identified by laser capture microdissection (LCM) combined with subsequent molecular techniques based on the positive histopathologic features. Additionally, we reviewed 47 reported cases of coccidioidomycosis in China. The number of reported cases is increasing, and the incidence of disseminated infection has exhibited a trend of shifting towards healthy young adults in China. Since clinical presentations and imaging findings lack specificity, a majority of domestic cases of coccidioidomycosis were initially misdiagnosed as tumours or tuberculosis. Moreover, the diagnosis of endemic mycoses may be challenging because of their rarity and the limited availability of diagnostic tests. The diagnosis was mainly confirmed by histopathological examination. The species involved were identified based on positive cultures in only 4 cases. To our knowledge, this is the first study to use LCM and molecular techniques to identify Coccidioides spp. in the histopathologically positive but uncultivable specimen. Comparing with previous reported studies, LCM combined with nucleic acid amplification techniques improve the ability of species identification for the timely diagnosis of coccidioidomycosis.

Keywords: Coccidioides spp; Coccidioidomycosis; laser capture microdissection; molecular diagnosis.

Figure 1.

Chest computed tomography (CT) scans. CT images of the lung window (A) and soft tissue window (B) revealed a 1.4 × 1.3 × 1.1 cm solitary solid nodule in the left lower lobe (arrowhead), mild ground-glass opacity and multiple small nodules in both lungs.

Figure 2.

Histopathological examination of biopsy tissue. PAS staining (A) and GMS staining (B) showing multiple thick-walled spherules and endospores (arrowhead) in the nodules.

Figure 3.

Isolation of fungi from formalin-fixed and paraffin-embedded (FFPE) tissue using LCM. (A) Small, round purpled-red fungal spores (arrowhead) scattered within alveoli were visible in the PAS-stained section before LCM. (B) Fungal spores were microdissected and collected after LCM.

Figure 4.

Electrophoresis of the nested-PCR amplification products in 2% agarose gels. (A) DNA products amplified in first-round PCR using the universal primers ITS1 and ITS4. Lane M, 2 kb DNA ladder. Lane 1, product amplified from Aspergillus fumigatus DNA as positive control. Lane 2, negative control, no amplification product by using DNA extracted from laser microdissected adjacent areas without fungal spores in the identical field of the same tissue section. Lane 3, products amplified from fungal DNA extracted from LCM-isolated FFPE tissue. Lane 4, no-template control. (B) Nested PCR products obtained by using the first-round PCR products as templates and ITSC1 and ITSC2 as the Coccidioides-specific primers. Lane M, 2 kb DNA ladder. Lane 1, nested PCR products obtained by using the first-round PCR products as templates. Lane 2, no product obtained by using first-round PCR products, amplified from A. fumigatus DNA, as templates.

Figure 5.

Amplification curves of TaqMan-based Real-time PCR assay using Coccidioides-specific TaqMan probes and fungal DNA extracted from LCM-isolated FFPE tissue as template.