Enumeration of viable and non-viable larvated Ascaris eggs with quantitative PCR

Raynal, Maria; Villegas, Eric N.; Nelson, Kara L.

doi:10.2166/wh.2012.101

Cited by 16 publications

(18 citation statements)

References 16 publications

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“…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).…”

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confidence: 99%

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

Rudko

Ruecker²,

Ashbolt

et al. 2017

Appl Environ Microbiol

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Significant effort has gone into assessing the fate and removal of viruses, bacteria, and protozoan parasites during wastewater treatment to provide data addressing potential health risks associated with reuse options. Comparatively less is known about the fate of parasitic worm species ova in these complex systems. It is largely assumed that these helminths settle, are removed with the sludge, and consequently represent a relatively low risk for wastewater reuse applications. However, helminths are a highly diverse group of organisms that display a wide range of physical properties that complicate the application of a single treatment for helminth reduction during wastewater treatment. Moreover, their diverse biological and physical properties make some ova highly resistant to both disinfection (i.e., with chlorine or UV treatment) and physical removal (settling) through the wastewater treatment train, indicating that there may be reason to broaden the scope of our investigations into whether parasitic worm eggs can be identified in treated wastewater. The ubiquitous human parasitic nematode Enterobius vermicularis (pinworm) produces small, buoyant ova. Utilizing a novel diagnostic quantitative PCR (qPCR), this study monitored E. vermicularis presence at two full-scale wastewater treatment plants over the course of 8 months and demonstrated incomplete physical removal of E. vermicularis ova through tertiary treatment, with removal efficiencies approximating only 0.5 and 1.6 log 10 at the two wastewater treatment plants based on qPCR. These findings demonstrate the need for more-diverse surrogates of helminthic ova to fully assess treatment performance with respect to reclaimed wastewaters.IMPORTANCE Helminths, despite being a diverse and environmentally resistant class of pathogens, are often underestimated and ignored when treatment performance at modern wastewater treatment plants is considered. A one-size-fits-all surrogate for removal of helminth ova may be inappropriate to adequately assess risk and ensure public safety when treated and partially treated wastewaters are encountered. This study argues for the use of human pinworm as a conservative indicator of the presence of helminth ova due to its small size, buoyancy, prevalence in humans, and environmental resistance.KEYWORDS Enterobius, helminth, monitoring, ova, qPCR, wastewater W ater serves as a medium for the transmission of many species of parasitic protozoa and helminths (1, 2). Contamination of water sources by parasites (and other causative agents of gastrointestinal illness) often occurs when sewage containing human waste is improperly treated (2, 3). Drinking, irrigation, recreation, and numerous

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mentioning

confidence: 99%

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

Rudko

Ruecker²,

Ashbolt

et al. 2017

Appl Environ Microbiol

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“…However, the viability of pathogens including helminth ova in a sample cannot be determined using a standard DNA target and there is a risk of overestimating the infectious pathogens when qPCR is used for environmental samples (Byappanahalli et al, 2010;Srinivasan et al, 2011). A high number of rDNA/rRNA copies in the ITS-1 and ITS-2 region makes detection more sensitive (Pecson et al, 2006;Raynal et al, 2012), caution however, is required interpreting the qPCR data to minimise the overestimation of helminth ova in environmental samples.…”

Section: Molecular (Pcr/mpcr/qpcr) Methodsmentioning

confidence: 99%

“…The qPCR estimated gene copy numbers were then divided by corresponding ova numbers to obtain gene copy numbers per ovum. Based on the information available in the literature (Pecson et al, 2006;Raynal et al, 2012) that different cell staged ova produced different gene copy numbers and A.…”

Section: Estimation Of Gene Copy Numbers Per Ovamentioning

confidence: 99%

“…A handful of studies attempted to quantify Ascaris ova using qPCR in the laboratory setting (Pecson et al, 2006;Raynal et al, 2012). For instance,…”

Section: Introductionmentioning

confidence: 99%

“…In addition, it is highly likely that wastewater and sludge samples may contain mixed population (early to late cell staged) of hookworm ova. Therefore, estimating gene copy numbers according to the Pecson et al, (2006) and Raynal et al, (2012) may not yield an accurate result. Therefore, determining the average gene copy numbers for an ovum from a mixed population may be appropriate.…”

Section: Introductionmentioning

confidence: 99%

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Detection of viable hookworm ova from wastewater and sludge

Gyawali¹

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Infectious helminths are a worldwide major public health problem. In terms of morbidity, approximately 3 × 10 9 people are infected by soil-transmitted helminths (STHs) influencing rates of malnutrition and failure to thrive. All of this increases the global disease burden (GDB) by up to 5.9 × 10 6 Daily Adjusted Life Years (DALYs). Helminth infections are more often seen as a major public health issue for developing countries however, concerns have been expressed in many of the developed nations because of the increased use of treated wastewater and sludge with no clear way of knowing the infection potential that can be attributed from such water.Guidelines have been established to minimise the potential public health risk associated with wastewater and sludge reuse. For example, the WHO guideline specified ≤ 1 ova (Ascaris lumbricoides) per L liquid (wastewater) or 4 g dry solid (sludge) for unrestricted use. Various wastewater treatment processes have been recommended to remove the helminths ova from wastewater depending upon its reuse. However, detection methods used to quantify viable helminths ova from wastewater has limitations. Therefore there is always a potential public health risk associated with reuse of wastewater and sludge.In this research, a real-time PCR method was developed. The new PCR method is rapid and specific. The method was found to be able to detect less than one ovum in one litre treated wastewater and approximately four ova in one litre raw wastewater and ~ 4 g sludge.The real-time PCR method was modified to a quantitative PCR (qPCR) method and further used to quantify hookworm ova from wastewater and sludge. The qPCR had estimated an average of 1.1, 8.6 and 67.3 ova for treated wastewater that was seeded with 1, 10 and 100 ova, respectively. The gene copy numbers obtained for 1, 10 and 100 ova by qPCR varied significantly (P < 0.05) within the tested samples indicating that absolute quantification of ova may not be accurate. Despite the difficulty quantifying accurate numbers of hookworm ova, the lower limit of quantification (LLOQ) of the qPCR method was 30 gene copies. This was a lot less than the gene copies produced by one ovum. Therefore, the qPCR method has potential to use for complying with wastewater guidelines.. iiAlthough the overall aim of this research was to develop a sensitive and specific method for quantitative detection of viable hookworm ova from wastewater, the importance of recovering the ova from wastewater and sludge samples for accurate detection was identified. While determining appropriate recovery rates was not an aim of this research, a suitable method that could be used to standardise research outcomes for further study was established. Therefore, ova recovery rate by different rapid methods was evaluated for further experiments. The result indicated that the ova recovery rate was higher for the treated wastewater (0.2 -50%) than the raw wastewater (0.3 -35%) and sludge (0.02 -4.7%) samples. A significant difference (P < 0.05) was observed between...

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Detection and quantification of soil-transmitted helminths in environmental samples: A review of current state-of-the-art and future perspectives

et al. 2017

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It is estimated that over a billion people are infected with soil-transmitted helminths (STHs) globally with majority occurring in tropical and subtropical regions of the world. The roundworm (Ascaris lumbricoides), whipworm (Trichuris trichiura), and hookworms (Ancylostoma duodenale and Necator americanus) are the main species infecting people. These infections are mostly gained through exposure to faecally contaminated water, soil or contaminated food and with an increase in the risk of infections due to wastewater and sludge reuse in agriculture. Different methods have been developed for the detection and quantification of STHs eggs in environmental samples. However, there is a lack of a universally accepted technique which creates a challenge for comparative assessments of helminths egg concentrations both in different samples matrices as well as between locations. This review presents a comparison of reported methodologies for the detection of STHs eggs, an assessment of the relative performance of available detection methods and a discussion of new emerging techniques that could be applied for detection and quantification. It is based on a literature search using PubMed and Science Direct considering all geographical locations. Original research articles were selected based on their methodology and results sections. Methods reported in these articles were grouped into conventional, molecular and emerging techniques, the main steps in each method were then compared and discussed. The inclusion of a dissociation step aimed at detaching helminth eggs from particulate matter was found to improve the recovery of eggs. Additionally the selection and application of flotation solutions that take into account the relative densities of the eggs of different species of STHs also results in higher egg recovery. Generally the use of conventional methods was shown to be laborious and time consuming and prone to human error. The alternate use of nucleic acid-based techniques has improved the sensitivity of detection and made species specific identification possible. However, these nucleic acid based methods are expensive and less suitable in regions with limited resources and skill. The loop mediated isothermal amplification method shows promise for application in these settings due to its simplicity and use of basic equipment. In addition, the development of imaging soft-ware for the detection and quantification of STHs shows promise to further reduce human error associated with the analysis of environmental samples. It may be concluded that there is a need to comparatively assess the performance of different methods to determine their applicability in different settings as well as for use with different sample matrices (wastewater, sludge, compost, soil, vegetables etc.).

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Enumeration of viable and non-viable larvated Ascaris eggs with quantitative PCR

Cited by 16 publications

References 16 publications

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

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

Detection of viable hookworm ova from wastewater and sludge

Detection and quantification of soil-transmitted helminths in environmental samples: A review of current state-of-the-art and future perspectives

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