A Real-Time PCR Method for Quantifying Viable Ascaris Eggs Using the First Internally Transcribed Spacer Region of Ribosomal DNA

Abstract

Worldwide, 1.4 billion people are infected with the intestinal worm Ascaris lumbricoides. As a result, Ascaris eggs are commonly found in wastewater and sludges. The current microscopy method for detecting viable Ascaris eggs is time-and labor-intensive. The goal of this study was to develop a real-time quantitative PCR (qPCR) method to determine the levels of total and viable Ascaris eggs in laboratory solutions using the first internally transcribed spacer (ITS-1) region of ribosomal DNA (rDNA) and rRNA. ITS-1 rDNA levels were proportional to Ascaris egg cell numbers, increasing as eggs developed from single cells to mature larvae and ultimately reaching a constant level per egg. Treatments causing >99% inactivation (high heat, moderate heat, ammonia, and UV) eliminated this increase in ITS-1 rDNA levels and caused decreases that were dependent on the treatment type. By taking advantage of this difference in ITS-1 rDNA level between viable, larvated eggs and inactivated, single-celled eggs, qPCR results were used to develop inactivation profiles for the different treatments. No statistical difference from the standard microscopy method was found in 75% of the samples (12 of 16). ITS-1 rRNA was detected only in samples containing viable eggs, but the levels were more variable than rDNA levels and ITS-1 rRNA could not be used for quantification. The detection limit of the rDNA-based method was approximately one larvated egg or 90 single-celled eggs; the detection limit for the rRNA-based method was several orders of magnitude higher. The rDNA qPCR method is promising for both research and regulatory applications.

A Novel, Multi-Parallel, Real-Time Polymerase Chain Reaction Approach for Eight Gastrointestinal Parasites Provides Improved Diagnostic Capabilities to Resource-Limited At-Risk Populations

“…To the best of our knowledge, all primers and probes were novel except for A. lumbricoides (forward primer), A. duodenale, N. americanus, and S. stercoralis. 3,4 Each set of primers and probes was synthesized by Applied Biosystems.…”

“…[1][2][3] Classically, the diagnosis of intestinal parasites depends on the life cycle of the parasite and its egg/larvae shedding patterns. Although microscopy and quantitative real-time PCR (qPCR) are dependent on the presence of parasite material in the sample, 4 qPCR is less subjective and for several parasites (e.g., E. histolytica) more specific. 2 The ability of microscopy to detect parasites is directly related to the number of organisms in stool.…”

Enterobius vermicularis as a Novel Surrogate for the Presence of Helminth Ova in Tertiary Wastewater Treatment Plants

“…The presence of helminthic ova, particularly Ascaris lumbricoides (the traditional reference parasite) along with its surrogate Ascaris suum, has been assayed in biosolids and wastewater effluent by a variety of methods, including microscopic examination and various PCR and quantitative PCR (qPCR) methods (20)(21)(22)(23). Because Ascaris is a ubiquitous parasite (infecting ϳ1.4 billion people worldwide) and has a direct life cycle that facilitates transmission by water, food, and person-to-person contact, it remains a logical reference pathogen for worm presence in sewage sludge (24,25).…”

Detection of viable hookworm ova from wastewater and sludge

“…In addition, treated wastewater or sludge can have a high level of background debris which may provide a similar profile to helminths ova during detection/quantification and compromise the sensitivity of the method (Barbosa et al, 2008). (Bowman et al, 2003;Nocker et al, 2007b;McCarthy et al, 2012) Vital stain  Cheap and easy process  Assessment can be done in few hours  Less chemical and equipment  Can be done in small scale laboratory  Difficulty on viability assessment  Sensitivity depends on detection threshold of a microscope  Stain might not effective on recently inactivate ova  Lack specificity (Weber et al, 1991;Nelson and Darby, 2001;Cabaret et al, 2002;Victorica and Galvan, 2003;Verweij et al, 2007;McCarthy et al, 2012;Gyawali et al, 2016b) Flow cytometry  Quick and easy process  Higher sensitivity than microscopy  Automated process  Quantification can be done  Lack of specificity  Difficulty on viability assessment of ova  Difficulty distinguishing background debris from ova (Hammes et al, 2008;Barbosa et al, 2008;Wang et al, 2010) Molecular (PCR/qPCR)  Quick and easy process  Higher sensitivity and specificity  Viability could be possible  Automated process  a Multiple species can be identified from single sample  b Quantification is possible  Require advance laboratory and equipment  Genomic information is essential  Require right genomic target for viability  Possibility of providing false positive result by extracting DNA from inactivated ova  Possibility of false negative result via inhibitors present in the samples  a Multiple sets of primers require which can reduce the sensitivity  b Triplicate sample required (Pecson et al, 2006;Verweij et al, 2007;Traub et al, 2007;Traub et al, 2008;Janwan et al, 2011;Jonker et al, 2012, Ngui et al, 2012b a = Multiplex PCR, b =Quantitative PCR…”

“…There is, therefore, a need for a rapid, sensitive and specific detection method that can quantify viable helminth ova from wastewater and sludge. Polymerase chain reaction (PCR/qPCR) assays have been developed and used for rapid, sensitive and specific detection of helminths from faecal samples (Pecson et al, 2006;Verweij et al, 2007;Traub et al, 2008;Taniuchi et al, 2011;Ngui et al, 2012a;Ngui et al, 2012b). PCR/qPCR methods can detect pathogens in a one-step closed-tube reaction within 2-4 h with much higher sensitivity and specificity by directly amplifying a specific gene from a target microorganism (Botes et al, 2013;Schar et al, 2013), overcoming the limitations of the culture and vital stain methods.…”