Real-time PCR method for detection of Encephalitozoon intestinalis from stool specimens

Abstract

The prevalence of microsporidiosis is likely underestimated due to the labor-intensive, insensitive, and nonspecific clinical laboratory methods used for the diagnosis of this disease. A real-time PCR assay was designed to assess DNA extraction methods and to detect three Encephalitozoon species in feces. Modifications of the MagNA Pure LC DNA isolation kit protocol (Roche Applied Sciences, Indianapolis, Ind.) were compared by using the automated MagNA Pure LC instrument (Roche) and fecal specimens spiked with Encephalitozoon intestinalis spores. Extracted DNA was amplified by the LightCycler (Roche) PCR assay. Assay sensitivity, reproducibility, and efficiency were assessed by comparing threshold crossover values achieved with different extraction and storage conditions (fresh, refrigerated, frozen, and preserved specimens). Optimal extraction conditions were achieved by using a commercial buffer, tissue lysis buffer (Roche), as the specimen diluent. LightCycler PCR results were compared to those obtained from routine stool microscopy with trichrome blue stain. The lower limit of detection for the LightCycler PCR assay varied by storage conditions from 10(2) to 10(4) spores/ml of feces, a value which represented a significant improvement over that achieved by staining (> or =1.0 x 10(6) spores/ml). Melting temperature analysis of the amplicons allowed for the differentiation of three Encephalitozoon species (E. intestinalis, E. cuniculi, and E. hellem). The assay is readily adaptable to the clinical laboratory and represents the first real-time PCR assay designed to detect Encephalitozoon species.

FIG. 1.

LC PCR assay linearity in water and stool samples. Spores were used to spike the specified matrix at the indicated concentrations, and then the samples were assayed immediately by the LC PCR method. The lines represent the linear regression analysis of each data set. The regression line for spores in stool samples is a combination of all data obtained for samples processed immediately, samples refrigerated at 4°C for 72 h, samples frozen at −70°C for 7 days, and samples preserved in ECOFIX for 14 days. CI, confidence interval. Spore concentrations are given as logarithmic dilutions of spores, i.e., 1.0 + E2 = 100 and 1.0 + E3 = 1,000.

FIG. 2.

Melting curve analysis of Enchephalitozoon species. Melting curves were obtained following the LC PCR assay for spiked stool samples (n = 3) containing E. hellem (63.7 ± 0.2°C), E. cuniculi (67.9 ± 0.4°C), and E. intestinalis (alveolar strain, 69.7 ± 0.8°C; duodenal strain, 69.7 ± 0.7°C) at 10⁵ spores/ml of feces. Although E. hellem and E. cuniculi are not expected to be found in fecal samples, the specificity testing demonstrates that E. intestinalis has a unique T_m even when the system is challenged by using organisms with very similar sequences. The melting curves for E. intestinalis at various spore dilutions are provided to demonstrate T_m stability as a function of spore concentration. The melting curve for a negative control specimen is also provided. −d(F2/F1), change in fluorescence; dT, change in temperature.