Dear Elisabeth,

That was quite informative for me. Thanks. I would tend to go for your contents in the last para - post the file here for just a week.
You are right - not that many people would download it anyway as it's a niche topic - I also do not download all that pertains to wastewater.

Is it possible for Susana to establish working relationship (if that is the right term) with IWA? In another post, I got the impression that Susana has a working relationship with India Water Portal. If I have got it right, then, I would suggest collaboration with California-based Pacific Institute.

Regards,
F H Mughal

Dear Mughal,

Good question. My understanding is that we are not allowed to do that, and neither are the authors of the paper. That's because on the pdf file it says:
Please note that you are not permitted to post the IWA Publishing PDF version of your
paper on your own website or your institution’s website or repository.

Maybe, just maybe it's OK because I wouldn't be putting it up on a website but on the forum and people can access it only if they have a SuSanA login? But I doubt it, as it would mean that 9000 people would potentially have access to it and it would probably undermine the business model of IWAP.

Maybe it's OK if I post the file here for just a week and then take it down again before anyone notices (probably not that many people would download it anyway as it's a niche topic; so not much income lost for IWAP):

Edit on 1 June 2018: I have removed the attached pdf file now.

Regards,
Elisabeth

Dear Elisabeth,

Just for my information, and, probably, for the information of other forum users: Is it possible for Susana to purchase a paper officially, and post it on the Susana library, for use by the forum users - - any copyright issues problem?

Regards,
F H Mughal

Pradip Gyawali has published another paper on this topic which I would like to bring to your attention. He has posted in this thread before (scroll up).

This paper was published in Feb 2018 in the IWA journal Water, Science and Technology.

Title:
Infectious helminth ova in wastewater and sludge: A review on public health issues and current quantification practices

Abstract:

Raw and partially treated wastewater has been widely used to maintain the global water demand. Presence of viable helminth ova and larvae in the wastewater raised significant public health concern especially when used for agriculture and aquaculture. Depending on the prevalence of helminth infections in communities, up to 1.0 × 103 ova/larvae can be presented per litre of wastewater and 4 gm (dry weight) of sludge. Multi-barrier approaches including pathogen reduction, risk assessment, and exposure reduction have been suggested by health regulators to minimise the potential health risk. However, with a lack of a sensitive and specific method for the quantitative detection of viable helminth ova from wastewater, an accurate health risk assessment is difficult to achieve. As a result, helminth infections are difficult to control from the communities despite two decades of global effort (mass drug administration). Molecular methods can be more sensitive and specific than currently adapted culture-based and vital stain methods. The molecular methods, however, required more and thorough investigation for its ability with accurate quantification of viable helminth ova/larvae from wastewater and sludge samples. Understanding different cell stages and corresponding gene copy numbers is pivotal for accurate quantification of helminth ova/larvae in wastewater samples. Identifying specific genetic markers including protein, lipid, and metabolites using multiomics approach could be utilized for cheap, rapid, sensitive, specific and point of care detection tools for helminth ova and larva in the wastewater.

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The rest of the paper is behind a paywall but perhaps Pradap is allowed to share an earlier copy of the paper or a related powerpoint presentation. I will ask him.

Regards,
Elisabeth

Dear All,

Thank you all for your post. It was nice to read all of your comments regarding the topics. Detection limit of molecular method has limitations meeting WHO standards when applying the method in wastewater and sludge samples. Since helminth ova contain multiple gene gene copies depending on the development stage of the ova. Molecular method is so far best option to detect helminth ova from wastewater matrices. I have looked recovery rate of helminth ova from waste water matrices using qPCR and results indicated very low recovery rate. Focus should given to improve recovery rate, that can improve the detection limit of molecular method.

Comparison of concentration methods for rapid detection of hookworm ova in wastewater matrices using quantitative PCR.Exp Parasitol. 2015 Sep 8;159:160-167 (www.ncbi.nlm.nih.gov/pubmed/26358269).

Thank you

Dear All,

Our opinion, based on our experience with qPCR and some research papers of colleagues, is that the current detection limits are not suitable for testing samples that need to meet guidelines and regulations for wastewater reuse in agriculture (e.g. WHO) since the detection limit of molecular techniques is still above those values. Moreover, the number of template copies per egg depends on their stage of development and thus it is not easy to correlate those two numbers in actual samples. However, it is possible to determine viability of eggs but incubation is needed for this.

We agree that PCR is not available in many facilities around the world and this is a limitation for implementing the technique, however, this could change in the near future as the technology becomes affordable.

We suggest that people interested in this topic read the following papers:

• Raynal, M., Villegas, E.N., Nelson, K.L. (2012) Enumeration of viable and non-viable larvated Ascaris eggs with quantitative PCR, Journal of Water and Health, 10(4) 594-604.
• Pecson, B., Barrios, J.A., Johnson, D.R. and Nelson, K. (2006) A real-time PCR method for quantifying Ascaris eggs using the first internally transcribed spacer region of ribosomal DNA. Applied and Environmental Microbiology, 72 (12) 7864-7872.

Regards, TeamWTR

Hiii
I have tried to read all the inputs in this discussion so far and this is my take
(1) Methods used for the detection and quantitfication of helminth eggs in clinical samples cannot be applied effectively to environmental samples, due to so many factors, such as the concentration of these eggs in the environment, the type of sample, and the presence of materials such as soil particles that may affect the recovery of the eggs.
(2) Current methods used in the environmental setting mostly have two common steps; that is to separate the eggs from other particles, mostly employing the use of flotation solutions in an attempt to create differential gravity gradients allowing the eggs to float. This particular step however leads to differences in methods due to the type of solution used. e.g the US EPA makes use of magnesium sulphate, WHO uses zinc sulphate and then the SA method which features prominently in the discussions uses zinc sulphate as well. Other methods use NaCl, sucrose etc. In addition the specific gravity of the flotation solution used differs and this particular property is critical as well.
(3)The application of molecular techniques for the detection of helminths in environmental samples is one major advance in the field due to their specificity, however the challenge as it is is with the differentiation between viable and non-viable nucleic material. However there seem to be hope in this regard from the paper of Pecson et al., 2006

There is currently project funded by the Bill and Melinda Gates Foundation and Lead by Prof. T.A. Stenstrom of the Durban University of Technology, that is looking a the development of a uniform method for the detection and enumeration of helminth eggs in environmental samples, this is being done in collaboration with partner laboratories and researchers from Mexico, India, Senegal and South Africa (including the developers of the SA method as mentioned. As part of this project a review of current existing methods in use as well new and emerging techniques is almost complete and would be published in an open access journal so that all can have access to information on the current state, this review discusses the pros and cons of the current methods.

Ahh, that is a pretty interesting way to differentiate between free and encapsulated DNA. But does it really work on dead eggs that have their shell still unbroken?

In general PCR methods will probably become more common in the future, and relatively cheap and mobile equippment like this: www.bento.bio/ will help a lot.

Since a single ova contain high numbers of gene copy PCR is sensitive than other available methods. Yes it is expensive at the setup stage however, the method can provide species level detection within couple of hours that is fundamental for successful application of WASH program. Additionally, it is more automotive method than other methods.

Dear All,

Yes that is true; PCR/qPCR methods are unable to distinguish between viable and non-viable ova. To overcome the problem we have developed PMA-qPCR method. Some of you may already know, PMA (Propidium Monoazide) is a DNA intercalating dye that penetrates non-viable ova and makes a covalent bond to DNA upon exposure to light. This photoactivation process results in the formation of a stable DNA-PMA complex that makes the DNA undetectable during the PCR reaction. We were getting promising results. Our manuscript is currently under review on Water Research. I did present a poster containing preliminary data obtained from PMA-qPCR method on IWA conference at Lisbon (Portugal). We are currently attempting to quantitate hookworm ova using qPCR and compare that with US EPA (culture) and stain methods.

Please do not hesitate to contact me if you need further information. I will be available on This email address is being protected from spambots. You need JavaScript enabled to view it.

Thank you

Dear all,

I would like to come back to that PCR method to analyise for helminth eggs which Marcos asked about on 9 March 2013. PCR stands for Polymerase Chain Reaction (see also Wikipedia page: en.wikipedia.org/wiki/Polymerase_chain_reaction).

Kris had stated that:

The problem is that it is so sensitive that even DNA from dead ascaris will be picked up in very (very, very) low quantities (there are some workarounds for this: qPCR, but that one complicates the procedure further).
It also needs a quite well equipped and clean lab, relatively skilled lab-technicians and a good cooling supply chain as those primers need to be synthesized in a specialized lab and then be contiously frozen until they reach the test lab.

All in all: not a tech that is currently feasible (at larger scale) in most countries we are usually talking about .

I saw today a paper on the same topic (for wastewater samples) that came out of research in Australia. The paper is regrettabily not open or free access, so I only have the text of the abstract here:

++++++

www.ncbi.nlm.nih.gov/pubmed/26297680

Rapid Concentration and Sensitive Detection of Hookworm Ova from Wastewater Matrices Using a Real-Time PCR Method
Gyawali P, Sidhu JP, Ahmed W, Jagals P, Toze S.

The risk of human hookworm infections from land application of wastewater matrices could be high in regions with high hookworm prevalence. A rapid, sensitive and specific hookworm detection method from wastewater matrices is required in order to assess human health risks. Currently available methods used to identify hookworm ova to the species level are time consuming and lack accuracy. In this study, a real-time PCR method was developed for the rapid, sensitive and specific detection of canine hookworm (Ancylostoma caninum) ova from wastewater matrices. A. caninum was chosen because of its morphological similarity to the human hookworm (Ancylostoma duodenale and Necator americanus). The newly developed PCR method has low detection sensitivity with the ability to detect less than one A. caninum ova from 1 L of secondary treated wastewater at the mean threshold cycle (CT) values ranging from 30.1-34.3. The method is also able to detect four A. caninum ova from 1 L of raw wastewater and from ∼ 4 gm of treated sludge with mean CT values ranging from 35.6-39.8 and 39.8-39.9, respectively. The better detection sensitivity obtained for secondary treated wastewater compared to raw wastewater and sludge samples could be attributed to sample turbidity. The proposed method appears to be rapid, sensitive and specific compared to traditional methods and has potential to aid in the public health risk assessment associated with land application of wastewater matrices. Furthermore, the method can be adapted to detect other helminth ova of interest from wastewater matrices.

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I am going to try and encourage the authors as well as other experts in helminth egg analysis to give us their reactions regarding the points that Kris made that this technique may not be able to differentiate between viable and non-viable helminth eggs and that it would be difficult to apply it in a standard laboratory in a developing country.

If you know anything about this topic or know experts, please post here or encourage them to post here. thanks.

Regards,
Elisabeth

P.S. Those questions raised by Constanze and the answers offered by Joe were also very interesting.

Thanks for this post, Constanze. Good questions.

Constanze wrote: Hello there,


In my opinion you cannot conclude on the effectiveness of any system if you did not place the Ascaris eggs in yourself (tea bag method).
1) How can you justify comparing degradation rates in different systems if you do not even know how much was in the system to start with? The base load should be equal.

I think there is a difference between 'proving' a system and batch-testing a system. In the former, I think you do have to prove claims you are making (kills all Ascaris within 28 days, destroys all cholera etc). And I think the only way you can do this is to spike the samples and measure decay. On the other hand, I think you can form batch-testing protocols which prove that a system is working by showing that it has got to a particular standard - eg <1 Ascaris ova. For me, those are different protocols. As you say, until the system is proven with spiking, you can't compare effectiveness.

2) Soon as you establish the viability of your eggs, they are destroyed. Right? Meaning I can not take a baseline sample and see what will happen with these exact eggs during treatment. This means I have to take a new sample for the next Phase. If I do not use the “tea bag method”, the fact that there might be less Ascaris eggs in this sample does not necessarily mean eggs were destroyed through hygienization. I might just catch/sample less this time. Isn’t the number of Ascaris eggs sampled/detected completely by chance?

Well there is always going to be a sampling effect, hence the need to have good protocols and good statistics. It is true that >1 Ascaris ova might just be because they weren't there in the first place. But again, in a proven system, this might be all you need to know.

3) If you do not place the Ascaris eggs in yourself the health status of the community will be quite an important parameter. Only because one community might be healthier does not mean the system is more effective. Yes, in a healthy community/household I might catch less from the beginning. But maybe I just got lucky with the first sample. Also, how will I get data on the effectiveness of the system if the household just happened to be not affected at the time of the study?

That is to do with the protocols of proving the system.

4) Just sampling the material normally gives you small numbers of Ascaris eggs. The die off rates won’t allow any conclusion regarding the effectiveness of the system.

Using Ascaris studies to compare the effectiveness of different system is more tricky then some studies suggest.

I agree with this. But the whole thing is pretty complex already and those systems which are regularly tested for Ascaris seem fairly small, never mind those with an agreed protocol.

Regarding a field kit: I can not see how a field kit would solve the sampling issue to start with. And then talking about the analysis...

This needs a statistican to comment. Sampling highly heterogenous material is a problem.