How to measure helminth eggs (in a reuse context)

  • joeturner
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Ah ok. How do they know that they've incubated this long enough? Maybe it is a standard method. I guess in a field experiment you'd not exclude the prelarvals.
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  • Maria Magri
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Re: How to measure helminth eggs (in a reuse context)

Hello,

I have some considerations that might be useful:

I think that collecting samples from UDDTs or PIT, VIPs should be done really carefully, we should try to get a representative sample, I mean from different sites in the compartment and in good amounts, like kilos or liters (two at least)… This is what is working better for me.

The methods from WHO, SA and EPA, they all have the same principles, to clean up the samples, try to separate the eggs from the solid particles and concentrate them in smaller amounts to conduct the evaluation under the microscope. It is a lot of work but it works  and it is really important that you have the right sieves to separate the eggs from the solids. The hospital methods really don’t work well for this purpose since we can have really diluted samples as someone said, and they just evaluate the presence or absence of eggs, not if they are viable or not.

About evaluating the eggs viability, it is not enough to take the samples from the toilets or other samples, do the procedure and look in the microscope how they look like. They can look as one single cell not developed into the larvae stage, and still be viable. That’s why is part of the procedure to incubate them at 28ºC during approx.. 1 month. If viable the eggs in those conditions should develop into the larvae stage. That time is a standard.

And most treatment technologies that can inactivate ascaris eggs don’t destroy the eggs, since the structure of their membranes is so resistant, so they don’t disappear from the material, what happens is that they just lose the ability to develop into larvae stage. So it is crucial the incubation process, that’s the only way to know if the eggs can infect people or not.

Regards,

Maria Magri
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  • joeturner
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Re: How to measure helminth eggs (in a reuse context)

Thanks, that is very interesting.

What kind of method would you use for much more solid material - like a sludge or feces? Would you not also need some kind of cleaning, or would the hospital method be sufficient to accurately determine the viable Ascaris ova numbers?
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  • joeturner
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Re: How to measure helminth eggs (in a reuse context)

Friends, I thought this was interesting - a method just published looking at measuring Ascaris using an iphone

From what I've learned here about the method, I'm wondering how much this would help - as there needs to be equipment to decant and centrifuges etc.

I've not yet read the paper in detail, perhaps they cover this there.
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  • mtfioravanti
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Re: How to measure helminth eggs (in a reuse context)

Do anybody know the PCR method (Polymerase Chain Reaction)? Any experience, comment or critic about this method for Ascaris detection?

Thanks!

Marcos Fioravanti B.
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  • JKMakowka
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Re: How to measure helminth eggs (in a reuse context)

mtfioravanti wrote: Do anybody know the PCR method (Polymerase Chain Reaction)? Any experience, comment or critic about this method for Ascaris detection?

Thanks!


PCR is a standard molecular biologogy technology to amplify DNA. If you have the right "primers" you can thus multiply specific DNA for Ascaris and show it on a gel blot (basically the same way you would do a parent test or a genetic fingerprint ;) ). 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 .

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Re: How to measure helminth eggs (in a reuse context)

Thank you very much!

So finally, is a more complex method wich is also more expensive, but not necessarily gives more precision?

¿Does this PCR method have any contundent advantage considering that the main goal on ascaris is not only to enumerate, but identify viability?

So THE method is the microscope observation. Does anybody knows a field kit?

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  • joeturner
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Re: How to measure helminth eggs (in a reuse context)

From what we're heard here, a field kit is highly unlikely. The method requires a month of incubation plus centrifuges, reagents and careful cleaning of samples.
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  • Constanze
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Re: How to measure helminth eggs (in a reuse context)

Hello there,

This discussion is on HOW to measure Helminth eggs. But my problem lies with the starting point, the design of the Ascaris study. Is there any discussion where the actual method of Ascaris studies (not the measuring itself) is discussed? If yes, please ignore the below and just kindly send me the link.

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.
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?
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?
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.

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...

Cheers
Constanze
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  • joeturner
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Re: How to measure helminth eggs (in a reuse context)

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.
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  • muench
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Re: How to measure helminth eggs (in a reuse context)

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.

+++++++

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.

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  • Pradip
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Re: How to measure helminth eggs (in a reuse context)

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
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