Testing Surface Disinfection Agents for the Inactivation of Helminth Eggs (research in South Africa)

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  • DanicaNaidoo
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Testing Surface Disinfection Agents for the Inactivation of Helminth Eggs (research in South Africa)

The eThekwini Municipality (Durban, South Africa) has developed a programme that employs workers to empty pits, every 3-5 years. These workers then pose a risk to the health of their families due to cross-contamination, from the pits to their households. This study was therefore aimed at finding a cost-effective and easy way of disinfecting households and inactivating Ascaris (and other helminth) eggs using simple household disinfectants, such that disease transmission is minimised, when pits are emptied. Results of such a study could then be used as recommendations for workers in terms of how to maintain their PPE and as preventative measures of disease transmission in their own homes. Plastic and glass surfaces were inoculated with eggs, and were either immediately wiped (simulating cleaning) or soaked in a disinfectant solution of varying concentrations, for different periods of time. Disinfectants used in this study included Domestos, Jik, Jeyes Fluid and Miracle Mom Pine Gel (which was recommended for use by the municipality).The active ingredient in Domestos and Jik is sodium hypochlorite. The active ingredient in Jeyes Fluid is carbolic acid. The chemical composition of Miracle Mom Pine Gel is unknown.

Eggs were analysed via light microscopy, and categorized as follows:

1) Potentially viable eggs - undeveloped eggs (at different cell stages), embryonated eggs with a motile larva visible in the egg and embryonated eggs with an immotile larva within.

2) Non-viable eggs - mechanically broken eggs, dead eggs with globules at the centre of the egg, hatched eggs or larvae, embryonated eggs with a necrotic larva and infertile eggs

It was found that wiping of contaminated surfaces removed Ascaris eggs, only facilitating egg transfer but not egg inactivation. The eggs required a prolonged exposure to disinfectants, at concentrations of 50% and above, in order to reach significant inactivation numbers. Sodium hypochlorite-based disinfectants were the most successful for inactivation, as eggs were completely decorticated, allowing for easier access of the disinfectant to the larva within potentially viable eggs. Jeyes Fluid and Pine Gel appeared to have little to no effect on the viability of eggs. It was recommended that faecal sludge spills are soaked in Domestos or Jik solutions for an hour. Contaminated surfaces can be wiped with a disinfectant-saturated cloth, and should be soaked for an hour in either Domestos or Jik, at a dilution of 50% and above, to allow for inactivation of the transferred eggs.

I was contacted by Elisabeth von Muench (of the SuSanA Secretariat) for the microscopy images of the eggs from this study. Below are a few images of eggs, before and after treatment with the various disinfectants.

[img][url=https://flic.kr/p/tB972s][img]https://c2.staticflickr.com/8/7789/18119291260_9c277aa802_z.jpg[/url] Undeveloped Ascaris Egg, Post-Treatment by SuSanA Secretariat , on Flickr[/img][img][url=https://flic.kr/p/tTFd8T][img]https://c1.staticflickr.com/1/397/18306424225_1b9d6e333e_z.jpg[/url] Viable Ascaris Egg (Motile). by SuSanA Secretariat , on Flickr[/img][img][url=https://flic.kr/p/tB8Rmq][img]https://c2.staticflickr.com/8/7766/18119241900_4e14e3b40f_z.jpg[/url] Viable Ascaris Egg (Motile), Post-Treatment by SuSanA Secretariat , on Flickr[/img]

More photos are available at:

www.flickr.com/photos/gtzecosan/sets/72157653472663710
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  • joeturner
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Re: Testing Surface Disinfection Agents for the Inactivation of Helminth Eggs

Thanks, that is very interesting. I wonder if there was any effort to quantify how many of the helminths survived the wiping treatment (or maybe the implication is that they all survived..)

Pretty scary stuff.
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  • Elisabeth
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Re: Testing Surface Disinfection Agents for the Inactivation of Helminth Eggs

Dear Danica,

Thanks a lot for sharing these helminth egg photos from your research in South Africa! Much appreciated and a fascinating topic.

I have a few small questions:
(1)
What does "motile" mean in this context? Does it mean something that moves?

(2)
Can an experienced person distinguish a dead (non-viable) helminth egg or larvae under the microscope from a live/viable one? I guess for the larvae/worm under the microscope they wriggle around, right? (do you have any videos by the way?) But on a photo is there some indicators that show the alive or dead state? For the eggs is the only way to find out to give them time to hatch and if there is no hatching after x days then it means the egg was not viable? Sorry if these are really basic questions for a worm expert. ;-)

(3)
You said:

It was found that wiping of contaminated surfaces removed Ascaris eggs, only facilitating egg transfer but not egg inactivation.


Perhaps the strategy should be to focus on egg transfer, not necessarily egg inactivation: if all the eggs were transferred from an area where they can do harm to an area where they can do no harm, then this would already stop disease transmission. Question is what is the area or space where they can do no harm and how do we get them there...

That's also similar to how we wrote it here on Wikipedia (we = Catalina Maya Rendón from Mexico and myself):

Thus, in conventional wastewater treatment processes the helminth ova are not inactivated but only removed from the wastewater. This is done by processes that remove particles through sedimentation or filtration such as in waste stabilization ponds (lagoons), storage bassins, constructed wetlands, coagulation-flocculation, rapid filtration and upflow anaerobic sludge blanket (UASB) reactors.

en.wikipedia.org/wiki/Helminths#Removal_in_wastewater_treatment

Would you agree with that? (of course it only shifts the issue from wastewater to sewage sludge)

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Elisabeth
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  • DanicaNaidoo
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Re: Testing Surface Disinfection Agents for the Inactivation of Helminth Eggs

Hi All,

This study was carried out towards my honours degree at the University of KwaZulu-Natal, and is currently in the process of being published. I therefore cannot upload a copy of my thesis right now, but thank you for taking interest and posting questions.

This study fell under a much larger project conducted by Partners in Development (PID), whom are based in Pietermaritzburg, South Africa and was supported by the WRC. The link to the PID website with their contact details are as follows:

www.pid.co.za/contact-us.html

In response to your questions Joe:

Yes, the percentage of viable helminth eggs that survived the wiping treatment (and the soaking treatment) were quantified. The results are as follows:

Wiping experiment:

a) Domestos: At the recommended dilution on a glass surface - 81.2%
At the recommended dilution on a plastic surface - 85.8%
At a 50% dilution on a glass surface - 54%
At a 50% dilution on a plastic surface - 71%

b) Jik: At the recommended dilution on a glass surface - 87.8%
At the recommended dilution on a plastic surface - 88.7%
At a 50% dilution on a glass surface - 77.5%
At a 50% dilution on a plastic surface - 77.7%

c) Jeyes Fluid: At the recommended dilution on a glass surface - 42.5%
At the recommended dilution on a plastic surface - 60.7%
At a 50% dilution on a glass surface - 28.4%
At a 50% dilution on a plastic surface - 38.2%

d) Miracle Mom Pine Gel: At the recommended dilution on a glass surface - 81.8%%
At the recommended dilution on a plastic surface - 80.1%

It should be noted that the reduction in the percentage viable eggs recovered for Jeyes Fluid is not attributed to efficient inactivation, but instead the inability of the disinfectant to lift eggs off either surface. When the Petri dishes were inspected under a microscope, a lot of residual eggs were found, indicating that Jeyes Fluid was not a suitable disinfectant. It is indeed quite scary - such little things showing such resilience.

In response to your questions Elisabeth:

1) Yes, motile referred to movement. The larvae within treated eggs seemed quite agitated by the exposure to disinfectants, thus moving about quite rapidly within the egg (depending on the concentration if disinfectant it was exposed to).

2) Yes it is possible to do so. If you look at the following pictures:

[img][url=https://flic.kr/p/tRnaR7][img]https://farm1.staticflickr.com/517/18280271516_754dcf8ce5_b.jpg[/url] Viable Ascaris Egg (Immotile) by SuSanA Secretariat , on Flickr[/img]

The above image is of a potentially viable Ascaris egg. The reason I say potentially viable, is that there is no guarantee that the egg will develop further - Ascaris eggs will only develop further and hatch in the small intestine of the host. Depending on the room temperature, and state of the egg (whether it has or has not been treated), the larva within might just move around within the egg. Also, the larva is fat, well-defined and well-developed, as it fills the space within the egg quite well. These characteristics indicate a potentially viable Ascaris egg.

[img][url=https://flic.kr/p/tB8dFQ][img]https://farm1.staticflickr.com/307/18119118558_eee933f807_z.jpg[/url] Dead Ascaris Egg by SuSanA Secretariat , on Flickr[/img]

The above image is of a dead/inactivated Ascaris egg. You can see that the larva within has sort of disintegrated to form globules - it is no longer a well-defined larva, which is characteristic of a dead egg.

[img][url=https://flic.kr/p/tRkXXj][img]https://farm8.staticflickr.com/7760/18280036396_40d8c86b9c_z.jpg[/url] Ascaris Egg with Necrotic Larva by SuSanA Secretariat , on Flickr[/img]

The above image is of a necrotic Ascaris egg. This is the stage between potentially viable and dead, where the larva within the egg is slowly dying off/disintegrating. You can see how the larva pulls away from the walls of the egg, and appears shriveled.

In terms of determining viability, I carried out an incubation process after treating eggs. This was done for the eggs that were soaked in disinfectant for an hour. This comes back to the life-cycle of Asacris. The eggs will only develop to a certain stage outside of the host body, thus monitoring the amount of undeveloped eggs in a given sample of eggs can give a good indication if treatment was successful. If the number of undeveloped eggs has dropped after incubation as compared to before incubation, then it is evident that the eggs were viable and continued to develop, even after being treated.

3) I agree, to a certain extent. I mean yes, it is effective to transfer eggs from an area that could cause harm to one that doesn't would help hinder disease transmission, but at the end of the day, something has to be done to destroy these pathogens. Inevitably, someone has to at some point handle them, thus propelling the pathogen-human contact interface. If a surface is wiped with a disinfectant-saturated cloth, that cloth has to be dealt with. even if discarded, someone somewhere along the line, will have to deal with that specific piece of cloth. Also, lack of proper sanitation is directly linked with socio-economic status. If people find it difficult to purchase and use hand soap (practicing proper hygiene), then what are the chances that they are able to continuously discard sponges or clothes that can actually be reused?

I hope this has sufficiently answered all questions asked.

Best Regards,
Danica
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  • SudhirPillay
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Re: Testing Surface Disinfection Agents for the Inactivation of Helminth Eggs

I think the take-home message here is that simple cleaning measures may not be enough. The concentrations required to disinfect are quiet high. In context of pit emptiers, care must be taken when washing their clothes / taking out protective wear and probably have designated areas for that.
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  • davidpereira
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Re: Testing Surface Disinfection Agents for the Inactivation of Helminth Eggs

Hello Danica,

Thank you for the post and the nice images that you're showing. I hope you've gathered nice results for your thesis; I will also be interested in reading it when ready.

I've been in some research about dry sanitation methods here in Ecuador, while doing so we use alcohol and in extreme cases diluted chlorine to disinfect ourselves and the equipment. I have not measured how efficient is this cleaning method, but according to literature it will be enough to prevent any cross contamination by the use of the equipment and to protect the research team.

I would also like you to take a look to this post: forum.susana.org/forum/categories/207-de...g-livedead-kit#15808 .

If you, or any colleague, has experience in helminth recognition I will be very excited to have your opinion about the kind of organisms we've found. Until now we have doubts about which one of these images reveals Ascaris or Thricuris or any other indicator.

Please, share your thoughts about it if possible.

Regards,

David
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