How to measure helminth eggs (in a reuse context)

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What is Terra Preta Sanitation (TPS) all about? Hype or ingenious?

Thank you all for your postings yesterday.
Just a quick note back to Gina (and anyone else working with Ascaris eggs): I might have missed it but I couldn't find in your paper the exact method used to enumerate the Ascaris eggs?

There is an interesting publication from South Africa from 2008 on how to enumerate helminth eggs - did you follow a similar method as the one described there?

Moodley, P., Archer, A., Hawksworth, D. (2008). Standard Methods for the Recovery and Enumeration of Helminth Ova in Wastewater, Sludge, Compost and Urine-Diversion Waste in South Africa. Report to the Water Research Commission (WRC), WRC Report No. TT322/08, South Africa.
www.susana.org/lang-en/library?view=ccbktypeitem&type=2&id=420

I have never worked with Ascaris eggs myself. Has anyone from this community read and utilised this manual from South Africa? Is it good?

In the summary it says:

This manual has been developed to specify a standard analytical method for water and wastewater laboratories to recover and enumerate helminth ova in wastewater, sludge, compost and urine diversion waste. The method documented in this manual is an attempt at documenting a standard, simple and cost effective analytical method for South Africa for the recovery and enumeration of helminth ova.


Regards,
Elisabeth

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  • Florian
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Re: What is Terra Preta Sanitation (TPS) all about? Hype or ingenious?

muench wrote: I have never worked with Ascaris eggs myself. Has anyone from this community read and utilised this manual from South Africa? Is it good?


Hi Elisabeth, I had a brief look at the manual, the method looks similar to the one recommended in the WHO manual on helminth analysis in wastewater samples (which I used some time ago). But I can't tell what the advantages of the SA-method over other methods would be, and the manual itself also does not give any indications on how the method compares with others.

Regards, Florian

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

Thank you for your welcome Elisabeth. I realize I need to introduce myself a little bit :) . I am Gina S Itchon, a medical doctor with a master's degree in public health. I have been working with the Sustainable Sanitation Center of Xavier University in Cagayan de Oro City in Mindanao, Philippines for the last 5 years and we do a lot of studies on the health and hygiene aspects of sustainable sanitation.

The methods used to look for and enumerate Ascaris eggs is a standard method in hospital laboratories. 10 different high power fields are examined with a standard binocular microscope, then the number of parasite eggs seen is reported. If none are seen, this is reported as 'none seen.' I think this is a different method from what was used in the S African study. However, our laboratory people, most especially the parasitologists are more adept and comfortable with the method we used. Again, like many issues in sustainable sanitation, we need to come to a consensus even for methods which are used in the laboratory for investigation. This makes the field of sustainable sanitation all the more exciting for research.

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Re: What is Terra Preta Sanitation (TPS) all about? Hype or ingenious?

g_itchon wrote: The methods used to look for and enumerate Ascaris eggs is a standard method in hospital laboratories. 10 different high power fields are examined with a standard binocular microscope, then the number of parasite eggs seen is reported. If none are seen, this is reported as 'none seen.' I think this is a different method from what was used in the S African study. However, our laboratory people, most especially the parasitologists are more adept and comfortable with the method we used. Again, like many issues in sustainable sanitation, we need to come to a consensus even for methods which are used in the laboratory for investigation. This makes the field of sustainable sanitation all the more exciting for research.


Hi, as far as I know, the standard methods used in medical context are based on the direct microscopic examination of feaces samples (please correct me if I am wrong here). This works well, because in infected persons, there are a lot of eggs in the feaeces and these eggs are easy to find under the microscope.

In environmental samples (wastewater, sluge, soil) the egg concentrations are much lower than in fresh feaces. Direct microscopic examination of such samples is not really possible due to the low egg concentration. To detect the presence of eggs in environmental samples in the microscope, several sample preperation steps need to be undertaken, that aim at separating eggs from other particles (to allow easy observation under the microscope) and to concentrate the eggs in a small volume that can then be observed. The SA-method and the WHO method that I linked above do excactly this.

The question is now: when is the classical hospital method (direct examination of samples) still approriate, and when the more complex "washing and concentration before examination" methods need to be applied? I am not sure if there are any studies on this. My feeling is that as long as feaces remain undiluted, the hospital method could still work ok, e.g. for the content of pit latrines or of peepoo bags. But as soon as feaces are diluted with lots of materials, as it happens in UDDTs or in TPS, I would start to doubt the efficiency of the classical hospital method.

Best, Florian

Florian Klingel
Water and Sanitation Specialist at Skat Consulting Ltd.
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  • joeturner
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Re: What is Terra Preta Sanitation (TPS) all about? Hype or ingenious?

Thanks both, that is interesting information.

I'm guessing that the hospital method is used to identify if an individual has an infection, whereas the other method is to give an indication of potential infection from a sludge. Are you saying that the hospital method is more of a presence/absence qualitative method whereas the Hawksworth method is more qualitative, Florian? Or am I misunderstanding what you are saying?

Surely in a sense, if the material in a latrine is more dilute, it could be argued that fewer directly measured eggs adequately indicate the risk of the material, no?
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Re: What is Terra Preta Sanitation (TPS) all about? Hype or ingenious?

joeturner wrote: Are you saying that the hospital method is more of a presence/absence qualitative method whereas the Hawksworth method is more qualitative, Florian? Or am I misunderstanding what you are saying?


The WHO method that I used my self is quantitative (result will be number of eggs / volume or mass of sample), the Hawksworth method also states that it is quantitative (I did not read it in all details). For the hospital method I do not know. I guess the main aim is to detect presence/absence, but as it is called "enumeration" I assume that also quantiative results can be obtained from these methods.

However the main difference is the sensitivity. For the "hospital" method, a tiny bit of fresh feaces is directly placed under the microscope and examined (at least that's how I understand it so far, perhaps someone with experience in this can give more info), whereas in the Hawksworth or the WHO methods, the eggs contained in a larger volume of sample are concentrated to a tiny volume that can be placed under the microscope and examined .

Following considerations may help to visualise this a bit:
- An Ascaris worm can produce more than 200.000 eggs per day. So from thousends to tens of thousends of eggs in a sample of feaces, you need to find some in order to detect if a person is infected or not.
- The WHO recommends that for unrestricted irrigation with treated wastewater, helminth egg concentration should be at maximum 1 egg/Litre. So you need to be able to find that 1 egg in the liter to know whether your water is safe or not.

Material from dry toilets, be it VIP, UDDT or PTS, is of course less diluted than wastewater. However concentration will be much lower than in fresh feces because of dilution with added materials and because of eggs dieing off; and secondly there are a lot of particles that make microscopic examination difficult. Difficult to find eggs under the microscipe without prior seperation of eggs from other particles and concentration.

Florian

Florian Klingel
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Re: What is Terra Preta Sanitation (TPS) all about? Hype or ingenious?

Thanks, yes I was asking whether the hospital is qualitative and the other method quantitative. Sorry for my typo.

What you are saying sounds like sense - otherwise if you directly measured 1L of material, you have a good chance of missing one ova when you sample it. If it is cleaned and concentrated, you can be reasonably sure you are seeing all the ova and can back-calculate to work out concentrations in the original volume.
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Re: What is Terra Preta Sanitation (TPS) all about? Hype or ingenious?



Video from Prof. Duncan Mara on "Analysis Of Wastewater For Reuse In Agriculture" that might give people a better idea how the concentration talked about above is done.

P.S.: Most of the other video lectures by him are recommendable too.

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Re: What is Terra Preta Sanitation (TPS) all about? Hype or ingenious?

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 suggests that prelarval stages are not counted.
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Re: What is Terra Preta Sanitation (TPS) all about? Hype or ingenious?

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).

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Re: What is Terra Preta Sanitation (TPS) all about? Hype or ingenious?

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.

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Re: What is Terra Preta Sanitation (TPS) all about? Hype or ingenious?

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.

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