Development and application of cell culture and molecular techniques for the diagnosis, identification, and viability testing of microsporidia

Abstract

The microsporidia are a group of organisms that have recently been implicated as the cause of a variety of human diseases. Because of their recent recognition as emerging pathogens, laboratory techniques for these organisms were neither optimized nor standardized. The work described in this document will detail the efforts toward that end. In vitro growth of human microsporidia species was optimized based on existing information about the biochemistry and biology of the microsporidia. Optimized systems were subsequently adapted for use in preliminary bench-top disinfection experiments with chlorine, ozone and pulsed UV light. The preliminary work suggested that microsporidia may be sensitive to chlorine and ozone. Further adaptation of optimized methods allowed for in vitro viability studies to be performed in multi-well cell culture plates for the determination of a tissue culture infectious dose 50% (TCID50). TCID50 comparisons enabled the calculation of a 3 log₁₀ reduction in microsporidia viability after 10 min. of exposure to sodium hypochlorite. Molecular diagnostics for microsporidia show promise both for detection and identification of various human species, but polymerase chain reaction (PCR) has not been designed for repetitive testing of human or environmental specimens. Methods described in this document are the first report of a non-nested PCR to incorporate uracil-N-glycosylase as a form of carry-over prevention (COP). It is also the first report of the use of microwave energy to release DNA for subsequent purification and amplification. Sequencing of PCR products confirm that the microwave spore lysis with subsequent PCR-COP is an accurate and sensitive method for identification of human microsporidia.