Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

Abstract

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori's modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking γδ T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Immunogenicity and protective efficacy of a pan-fungal vaccine in preclinical models of aspergillosis, candidiasis, and pneumocystosis

“…At study termination, right lungs were preserved in 10% neutral buffer formalin, and subsequently the fixed lung tissue was embedded in paraffin, cut, and stained with Grocott’s modified methenamine silver (GMS) stain. Images of five distinct fields were taken and fungal burden quantified as previously described ( 33 , 38 ). Percent weight loss was calculated from the baseline body weight measured at time of challenge.…”

Vaccine-Induced Protection in Two Murine Models of Invasive Pulmonary Aspergillosis

“…Five random fields were imaged and fungal burden quantified according to the guidelines provided by Stolz et al. ( 12 ).…”

“…The fixed lung tissue was embedded in paraffin, cut, and stained with Gomori's modified methenamine silver stain. Five random fields were imaged and fungal burden quantified according to the guidelines provided by Stolz et al (12).…”

The Lysine Demethylases KdmA and KdmB Differently Regulate Asexual Development, Stress Response, and Virulence in Aspergillus fumigatus

“…Kaplan–Meier survival curves were analyzed using the log-rank (Mantel–Cox) test for significance. Histological quantification of fungal burden was determined using the Image J 1.52k software (NIH, Bethesda, MD, USA) as described previously [ 37 ].…”