Helminth recognition - Viability test using Live/Dead kit

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  • davidpereira
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Helminth recognition - Viability test using Live/Dead kit

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Hello all, I'm writing this post to find help on helminth recognition, please share this post with any expert that would give us references on this. We'll be glad to publish here the results when finished the experiment.

I'm David Pereira and I'm working on ecological sanitation in Ecuador, actually linked to Fundación In Terris and it's very interesting proposal of pedal operated UDDT.

We've been testing some methods for sanitation of the resulting material from the dry toilet. Now, the challenge is to recognize certain organisms we found in order to compare different states of the resulting byproducts (specially Ascaris l. as indicator).

Please take a look to the images attached which are the result of the lab analysis. Viability test has been done using LIVE/DEAD kit (see reference for the kit use in Nematodes viability; search for: AssessmentofviabilityofthenematodeeggsLIVEDEADkit.pdf).

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Any reference that you may have about what we've found in the samples will be very helpful. There is attached an excel sheet to register observations per picture.

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Thank you.

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  • JKMakowka
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Re: Helminth recognition - Viability test using Live/Dead kit

Interesting approach. Could you explain a bit what the requirements for the florescence microscope are? It will probably be difficult to find such equipment outside of research labs.
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  • davidpereira
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Re: Helminth recognition - Viability test using Live/Dead kit

Yes Krischan, we are using very specific lab equipment for this research. The samples are extracted by flotation method, and then using a Confocal microscopy and the fluorescent light let us to differentiate between live and dead organisms.

In this case, we are testing some sanitation methods, and the recognition of helminths is key to determinate which method can be used in field. With this, we would be sure that the resulting material from the UDDT is safe to be used or buried after treatment without human or natural risks of contamination.

The images shows our first results, and now we want to recognize the organisms, any help will be appreciated.

Regards,

David
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  • canaday
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Re: Helminth recognition - Viability test using Live/Dead kit

Dear David,

This is a very interesting technique and the photos you obtained are beautiful. At first glance, nothing looked like the classic Ascaris egg, but the lighting is different and the staining obviously affects how things look. Have you done trials with known, fresh Ascaris eggs?

How expensive are the reagents?
How toxic are they to the environment after their use?
How expensive is the equipment that is required?
Are you doing this work at a university in Guayaquil?
Has anyone looked at how long it takes dead eggs to decompose and no longer be recognizable?
What is the current In Terris protocol for treating the feces collected in UDDTs?

As I have mentioned in previous posts, it would be ideal to work out an efficient method for looking for Ascaris eggs that requires nothing more complicated than a standard microscope.

Good luck with this important work. Please keep us informed.

Best wishes,
Chris Canaday
Conservation Biologist and EcoSan Promoter
Omaere Ethnobotanical Park
Puyo, Pastaza, Ecuador, South America
inodoroseco.blogspot.com
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  • joeturner
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Re: Helminth recognition - Viability test using Live/Dead kit

Interesting stuff - I'd not heard of this technique before, I will ask some contacts to see if anyone knows more about how it might work with helminths.

There are a couple more references I've found which are on this topic:

Comparison of methodologies for enumerating and detecting the viability of Ascaris eggs in sewage sludge by standard incubation-microscopy, the BacLight Live/Dead viability assay and other vital dyes by Alaa Karkashana, Basma Khallafa and others

Water Research Volume 68, 1 January 2015, Pages 533–544 doi.org/10.1016/j.watres.2014.10.003

Abstract

The aim of this study was to evaluate the Live/Dead BacLight viability kit as a method for enumerating viable eggs of Ascaris suum in sewage sludge as a surrogate for the human roundworm. The number and viability status of eggs of A. suum were accurately measured directly in sewage sludge samples by the BacLight method, compared to the conventional incubation-microscopy procedure. BacLight stains were not toxic to A. suum eggs, in contrast to some conventional vital dyes which disrupted viable eggs. The method was effective for the direct examination of eggs in heavily contaminated samples or seeded sludge containing ∼200 eggs/g DS in sludge with 5% DS content. However, a recovery method would be necessary to examine samples with small numbers of eggs, for instance in sludge from regions where the prevalence of infection with Ascaris lumbricoides is low. The BacLight technique may therefore be an effective alternative to conventional incubation-microscopy for enumerating Ascaris eggs in contaminated field samples or to validate sludge treatment processes by examining decay rates of inoculated A. suum eggs in laboratory simulations. Most field samples would require recovery from an appropriate number of composite samples prior to vital staining.


They also say in the conclusions that the dye Methylene blue was found to be equally effective. I don't know anything about that dye - but it might be more available than the branded dye?

This is also an interesting overview: Methods for Quantification of Soil-Transmitted Helminths in Environmental Media: Current Techniques and Recent Advances by Philip A. Collender Amy E. Kirby and others - Trends in Parasitology (in press) dx.doi.org/10.1016/j.pt.2015.08.007

As that latter reference suggests, the issue with counting helminth ova is complex due to the problems with extracting them from the faeces and then assessing their viability.
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  • davidpereira
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Re: Helminth recognition - Viability test using Live/Dead kit

Hi Chris,

Thanks for your interest.

It has been hard to find local labs with appropriate methods and enough experience in Helminths identification. We are now working in combination with a local private lab and a local University. We are also having collaboration of two international universities. Considering this mentioned challenge, now we are not focused in finding the best and more cost-effective method for Heminths identification, even when we consider that as a key aspect as well as you do. Believe me, it has been a challenge getting what we have now in terms of methods and lab capabilities. I can give you more details on cost and protocols by e-mail or in a separate conversation, but I really need help on identifying the organisms in these pics, so I prefer to focus this conversation on the aspects directly related to that subject.

This experiment is an isolated process where we have selected "compost" harvested from UDD Composting Toilets and we are assessing 3 treatments including a Control. I don't want to go deeper in this details in order to keep the conversation in the subject: identifying the organisms in the pictures. But I will be happy to share those details later.

In my first post you can find:
1- The pics we have from the samples (File helmitsrecognition_1stSampling.pdf)
2- An Excel file to be filled with comments about each pics. It will be great to have comments either in the Excel file (Dataregistry_HelminthsRecognition.xlsx) or directly in the post.

Thanks, David.
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  • davidpereira
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Re: Helminth recognition - Viability test using Live/Dead kit

Hello Joe,

Those references you've shared are very interesting, the unique difference is that we are taking samples from a UDDT, which are almost dried samples. The extraction of the helminth ova is harder because they have less opportunities to live on dried conditions than the sludge ones.

Please let us know if any of your contacts can recognize the images shown below. This is our main task to do right now, we're looking for the appearance of indicators for this first phase of the experiment, then we will compare treatments.

Thank you.
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  • Pradip
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Re: Helminth recognition - Viability test using Live/Dead kit

Hi David,

Thank you for the nice and interesting pictures. The major disadvantage of microscopy (ordanary or flurescence)is to identify eggs. if you want I can test your samples using molecular method. I have methods ready for most of the nematodes. form there you can narrow down to identification.

Thank you
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  • Elisabeth
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Re: Helminth recognition - Viability test using Live/Dead kit

Dear David,

I was just wondering why the exact identification of the helminth type is important in your case? Isn't the only thing that matters whether the larvae in the eggs are viable or not? The anthelminthic drugs ( en.wikipedia.org/wiki/Anthelmintic )* that people would take against the helminths are also not really helminth specific but "one drug kills all"? Or is it more of a fundamental science interest?

I actually wonder if anthelmintic resistance is something we need to worry about in future (like antibiotics resistance); this bit says very little about it: en.wikipedia.org/wiki/Anthelmintic#Anthelmintic_resistance

But that could be the topic of a separate thread.

Regards,
Elisabeth

* Actually here ( en.wikipedia.org/wiki/Mass_deworming#Pills ) it says:

Deworming programmes usually give children a pill of an of anthelmintic drug. The treatment of choice for STH is mebendazole and albendazole.[3] and praziquantel for schistosomiasis.[5]

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