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

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.

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?

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 .

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

Thanks!

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.

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?

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

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.

Joe, thanks for the link. I had a look at the paper, the sentence before the part you quoted explains why they exclude prelarval stages:

Sampling and analysis. At sampling, an egg bag from each replicate was collected and rinsed in 0.9% (wt/vol) NaCl solution, followed by incubation at room temperature in 0.1 N sulfuric acid for 28 to 35 days to allow larval development (2). Viability counts were performed under the microscope. Unfertilized eggs, identified by their incomplete egg shells, were excluded from further counting. Eggs developing to the larval stage were considered viable, while prelarval stages were not.

They incubate the eggs before examination under the microscope. All healthy eggs should then have developed larvae, and the ones that did not can be discounted.

joeturner wrote: Thanks, that is very interesting.

They didn't discuss how to identify viable eggs, or the life stage, which seems important given that one of the Nordin papers suggests that prelarval stages are not counted.

Edit above because I'd missed out a word. I am refering to this paper

Viability counts were performed under the microscope. Unfertilized eggs, identified by their incomplete egg shells, were excluded from further counting. Eggs developing to the larval stage were considered viable, while prelarval stages were not.

Excellent, thanks! Wish I'd have had this 10 years ago The method shown in the Video is the one of the WHO manual.

Joe: identification of viable eggs is done visually. With a little experience, for Ascaris eggs this is not too difficult. A bit simplified: viable eggs are intact with a larvae visible inside the egg (sometimes one can even see it moving), dead eggs are emtpy and may have a broken shell.

Both discussed manuals have some photos of different stages of Ascaris eggs.

which seems important given that one of the Nordin suggests that prelarval stages are not counted.

Why one would not count the pre-larval stages, I am not sure. These still can pass to the larval stage and become infective. Clearly one does not count infertile eggs and dead eggs (in older sluge, the latter is the most common type of Ascaris eggs found).