Development of a method for the detection of waterborne microsporidia
Rebecca M Hoffman1, Donna M Wolk, Susan K Spencer
1WI State Lab of Hygiene, University of WI-Madison, 2601 Agriculture Drive, Madison, WI 53718, USA. beckyh@mail.slh.wisc.edu
Abstract:
Microsporidia are obligate intracellular pathogens capable of infecting humans. There is credible evidence to suggest that microsporidial infections may be transmitted through consumption of spores in contaminated water; however, methods to detect this pathogen have not been standardized and microsporidia occurrence studies have not been conducted. Concentration of spores by continuous flow centrifugation (CFC), purification using immunomagnetic separation (IMS), and detection by either microscopy or real-time polymerase chain reaction (PCR) were evaluated for detection of Encephalitozoon intestinalis spores in seeded water samples. Recovery efficiency of CFC using microscopic detection ranged from 38.7-75.5% in filtered tap water. Using an indirect IMS method, 78.8-90.2% of seeded spores were recovered in ultrapure water (18 M Omega); however, the lack of a specific monoclonal antibody and the presence of other particulates interfered with the IMS assay in some turbid samples. Despite low recovery efficiencies and the detectable presence of PCR inhibitors in each of the samples, a combination of CFC concentration, indirect IMS, and real-time PCR produced a positive test result in six of ten natural water samples (turbidity 0.1-28.9 NTU) at a seeding level of 50 spores/L.
Insights
Detecting microsporidia in water is crucial for public health. This study evaluated methods for detecting Encephalitozoon intestinalis spores, finding a combination of continuous flow centrifugation, immunomagnetic separation, and real-time PCR effective in natural water samples.
Area of Science:
- Environmental microbiology
- Waterborne pathogens
- Parasitology
Background:
- Microsporidia are obligate intracellular pathogens infecting humans.
- Transmission via contaminated water is suspected, but detection methods are not standardized.
- Occurrence studies in water are lacking.
Purpose of the Study:
- To evaluate methods for detecting Encephalitozoon intestinalis spores in water.
- To assess the efficacy of continuous flow centrifugation (CFC), immunomagnetic separation (IMS), and real-time PCR.
Main Methods:
- Concentration of spores using continuous flow centrifugation (CFC).
- Purification using indirect immunomagnetic separation (IMS).
- Detection via microscopy and real-time polymerase chain reaction (PCR).
Main Results:
- CFC recovery efficiency ranged from 38.7-75.5% with microscopy.
- Indirect IMS achieved 78.8-90.2% recovery in ultrapure water, but faced interference in turbid samples.
- A combination of CFC, IMS, and real-time PCR detected spores in 60% of natural water samples at 50 spores/L.
Conclusions:
- The combined method (CFC, IMS, real-time PCR) shows promise for detecting microsporidia in natural water.
- Further optimization is needed to overcome limitations like low recovery and PCR inhibitors.
- Standardized methods are essential for microsporidia occurrence studies in water.
