Dear partner,

We did some research on PCR techniques for helminth eggs determination as described in our paper from 2006 but based on our results, such method is not adequate, neither for the identification nor the determination of viability of Ascaris eggs (I suggest you read the paper for more detailed information). As mentioned before and as is written in the powerpoint presentation that I uploaded few days ago (see here: forum.susana.org/forum/categories/97-ena...e-mexico-unam-mexico), we are currently testing a software to identify and quantify helminth eggs (so far from 8 general) to improve precision and reduce time and cost with respect to the conventional technique (US EPA).

This project is being funded by the Gates Foundation (Phase II) and we are working on a website to validate the software and will be willing to try it out with pictures from other forum members.

Best Regards, Blanca

Dear partner,

Taking advantage of the orientation you have provided us, I would like to share with you a paper related to PCR method for the identification of Ascaris. We think it would enrich the blog:

Brian M. Pecson, José Antonio Barrios, David R. Johnson and Kara L. Nelson (2006) A real-time PCR method for quantifying viable Ascaris eggs using the first internally transcribed spacer region of ribosomal DNA, Applied and Environmental Microbiology, 72(12):7864.

Abstract:

Worldwide, 1.4 billion people are infected with the intestinal worm Ascaris lumbricoides. As a result, Ascaris eggs are commonly found in wastewater and sludges. The current microscopy method for detecting viable Ascaris eggs is time- and labor-intensive. The goal of this study was to develop a real-time quantitative PCR (qPCR) method to determine the levels of total and viable Ascaris eggs in laboratory solutions using the first internally transcribed spacer (ITS-1) region of ribosomal DNA (rDNA) and rRNA. ITS-1 rDNA levels were proportional to Ascaris egg cell numbers, increasing as eggs developed from single cells to mature larvae and ultimately reaching a constant level per egg. Treatments causing >99% inactivation (high heat, moderate heat, ammonia, and UV) eliminated this increase in ITS-1 rDNA levels and caused decreases that were dependent on the treatment type. By taking advantage of this difference in ITS-1 rDNA level between viable, larvated eggs and inactivated, single-celled eggs, qPCR results were used to develop inactivation profiles for the different treatments. No statistical difference from the standard microscopy method was found in 75% of the samples (12 of 16). ITS-1 rRNA was detected only in samples containing viable eggs, but the levels were more variable than rDNA levels and ITS-1 rRNA could not be used for quantification. The detection limit of the rDNA-based method was approximately one larvated egg or 90 single-celled eggs; the detection limit for the rRNA-based method was several orders of magnitude higher. The rDNA qPCR method is promising for both research and regulatory applications.

These results are the unique experiences we had with PCR technique.

Blanca

For readers following this thread on "a better way to test pathogen inactivation", I would like to draw your attention to a related post by Blanca Jimenez from Mexico. She writes about the development of software to identify and quantify helminth eggs.

You can find it here on the forum:
forum.susana.org/forum/categories/97-ena...ico-unam-mexico#9351

Dear Ms. BJimenez

The work developed by your lab is very important for the rapid diagnosis of helminths, We are very interested in learning more about this work
In Bolivia we are proving new methods for producing safe human organic waste (free of pathogens Ascaris and Trichuris especially) we would like to establish a line of contact with you.

Raul Silveti
This email address is being protected from spambots. You need JavaScript enabled to view it.
Fundacion Sumja Huasi
www.sumaj.org

Dear Colleagues

I agree with many of you on the importance of monitoring helminth eggs. Bacteria, nor viruses nor protozoan are indicator of them. As the current internationally used methods is based on a simple but tedious and long technique to separate first the eggs from the detritus found in wastewater, excreta or sludge , to secondly, - once the eggs have been isolated - to identified and count them through an also time consuming step demanding skilled analysis.
As the methods is complicated, I think it is better to measure all the eggs and not only Ascaris, the additional effort is not very important. Moreover, even if Ascaris is the most abundant specie, its proportion is different in different regions. For practical purpose it is not that important to measure viability, given that this is linked to the integrity of the shell which is destroyed by most of the inactivating procedures. Nevertheless to assess the efficiency of a new treatment technology measuring viability is important.
I am attaching some papers on the comparison of helminth eggs analytical techniques and as well an alternative procedure to estimate the eggs number in wastewater based on the TSS content provided a calibration curve has been previously developed.
A last comment but critical, is that even if the part of the methodology to separate the eggs from the detritus is very time consuming, most of the errors on the quantification of the eggs come from the visual identification through the microscope.
Thanks to a grant from the Melinda and Gates Foundation, my lab has already develop an algorithm to recognized and count the eggs of several species found in wastewater, sludge and excreta from different regions of the world. We are now developing ‘a friendly interphase’. The idea behind is that routine laboratories will be separating the eggs from detritus using VERY simple material and that once the eggs are separated photos can be taken and sent to a central laboratory to do the counting. This will reduce costs and increase the reliability of the process
Blanca
Moderator note for attachment 2 and 3:

Reprinted from Water Science and Technology, volume 53, issue number 7, pages 43-49, with permission from the copyright holders, IWA Publishing.
www.iwaponline.com/wst/05405/wst054050169.htm

Reprinted from Water Science and Technology, volume 54, issue number 5, pages 169-177, with permission from the copyright holders, IWA Publishing.
www.iwaponline.com/wst/05307/wst053070043.htm

[Moderator note made by Sebastian Klos]

A bit of a tangent, but has anyone heard about this paper microscope?

www.scidev.net/global/biotechnology/news...aper-microscope.html
www.foldscope.com/

My first thought when I heard about it was that it might be a good thing to use for counting Ascaris.

joeturner wrote: It seems to me to be a bit of a myth that one can monitor the temperature of a compost windrow with a single temperature probe.

I think it is still easier to do muliple temperature probes in different points of a windrow at various time intevalls, than a single helminth egg analysis. In particular if this is meant as rountine process control.

Florian wrote:
This is true for this type of treatment (but simply measuring temperature would be more practical), however, as I said above, in some treatment systems, especially for wastewater, helminth eggs may be much easier retained as bacteria.

I actually think temperature is more difficult to measure - because to meet standards you'd have to be able to prove that every part of your compost has reached the required temperature for the required time and there is no possibility of reinoculation. Which would be hard to prove without using thermocouples and a data recorder. It seems to me to be a bit of a myth that one can monitor the temperature of a compost windrow with a single temperature probe.

Florian wrote:

JKMakowka wrote: That said, the reason for taking Ascaris as far as I know, is that in heat disinfection processes (and maybe composting ones?) those are about the most durable, i.e. if none of those survive, pretty much all pathogenic bacteria and viruses can be assumed to be dead too.

This is true for this type of treatment (but simply measuring temperature would be more practical), however, as I said above, in some treatment systems, especially for wastewater, helminth eggs may be much easier retained as bacteria.

True, but the problem with measuring heat is that that is only an very indirect measure and can be easily messed up by measuring at the wrong point or if the material is not properly mixed.

JKMakowka wrote: That said, the reason for taking Ascaris as far as I know, is that in heat disinfection processes (and maybe composting ones?) those are about the most durable, i.e. if none of those survive, pretty much all pathogenic bacteria and viruses can be assumed to be dead too.

This is also my understanding - if we are to choose a single indicator, it seems best to choose the one which is hardest to destroy.

I can agree it isn't perfect, but it seems to me to be the best available option.

Ok, I keep out of the discussion on bacteria, I feel I'd need to do some more reading before that...

JKMakowka wrote: That said, the reason for taking Ascaris as far as I know, is that in heat disinfection processes (and maybe composting ones?) those are about the most durable, i.e. if none of those survive, pretty much all pathogenic bacteria and viruses can be assumed to be dead too.

This is true for this type of treatment (but simply measuring temperature would be more practical), however, as I said above, in some treatment systems, especially for wastewater, helminth eggs may be much easier retained as bacteria.

As mentioned before: in disinfection processes the usual measure is full/heterotrophic plate count reduction. When talking about water supplies, one usually has to artificially spike the count prior to get sensible log-reduction counts, but for fecal sludge treatment that is obviously not necessary in most cases.

The problem is though that you can't know the range before so you have to do several dilutions, and to be statistically correct even multiples of those. All of which is problematic in a non full-lab setting.

That said, the reason for taking Ascaris as far as I know, is that in heat disinfection processes (and maybe composting ones?) those are about the most durable, i.e. if none of those survive, pretty much all pathogenic bacteria and viruses can be assumed to be dead too.