Dear Blanca,

Thanks a lot for your help.

We want to test Ascaris and other pathogens in the “compost” we harvest from our prototypes (Dry Toilets). We have received a proposal from a local laboratory and they are proposing to use a RT-PCR (Reverse Transcription – Polimerase Chain Reaction). What’s your opinion about this methodology. They say that it is very precise since it is based on RNA.

If somebody else want to give his opinion… very welcome!

Marcos

Dear Marcos,

To identify, quantify and determine the nematode eggs’ viability, the suspension is incubated at 26°C for 20 days in a 0.1N solution of sulfuric acid. Each species is then identified and quantified using an optical microscope, and the presence/absence of the larvae are confirmed to determine viability (Maya et al., 2012). Cestode identification, quantification and viability are determined by directly observing the larvae hatching under the microscope. Hatching is induced using 0.4% sodium hypochlorite solution and a solution of 0.01% trypan blue (colorant). Live larvae are not stained (Wang et al., 1997).

In some case the nematode egg´s viability is determined by directly using of a solution of 0.01% trypan blue (colorant), but the problem is that sometimes false positives are observed. When the egg is colored it means that is nonviable, but after incubation period we can observe the development and even a larval stage.

References

Maya, C., Ortiz, M., Jiménez, B. (2010). Viability of Ascaris and other helminth genera non larval eggs in different conditions of temperature, lime (pH) and humidity. Water Science and Technology 62(11): 2616-2624.

Wang IC., Ma YX., Kuo C., Fan C. (1997). A comparative study on egg hatching methods and oncosphere viability determination for Taenia solium eggs. Int. J. Parasitol. 22(11): 1311-1314.

Dear Blanca, what methods are you using to test the existence and viability of helminth eggs? If you can give some recommendations from your experience, I will be very grateful.

FECES RECIPE (biological content)

We have been testing different ways to produce sludge/excreta to use it as model and follow up the inactivation of helminth eggs and different types or microorganisms. We work with samples of primary sludge to perform the biological experiments of fecal contamination and inactivation of pathogens solid matrix. This primary sludge has turned out suitable to obtain solids with high content of feces and with different types of particles and “interferences” for a disinfection/inactivation process. If the objective of the tests is merely rheological, this kind of sludge previously sterilized might be useful as diluted fecal matter. However, if biological or pathogenic tests are going to be conducted, we recommend the following procedure:

Collect and sterilize a primary sludge at 121°C and 1.05 kg/cm2 for 1 hour in autoclave. These conditions were assessed as effectively inactivating the original content of pathogens through experiments; with this procedure not all pathogenic structures (like helminth eggs) are completely destroyed, however, all of them are inactivated. Nevertheless, in sludge with a high content of pathogens, it is recommended to use a blank through your experimental work and proof viability of the remaining structures. Once the sludge is sterilized add a proper amount of the pathogens you want to test if applicable. The amount to be added is critical because pathogenic content is very variable among countries (see table below). By experience it is known that the standard biological parameters are covered using fecal coliforms and helminth eggs, especially for inactivation protocol tests. It is also considered that such parameters would be enough to simulate pathogen resistance in feces. However it would depend on the objectives of the study the kind of pathogens to be utilized.

Helminth eggs are one of the most resistant pathogenic structures that might be found in sludge and excreta. To perform inactivation tests with these organisms we have used different species (Ascaris and Taenia are recommended), in a mixture of helminth eggs (35 of each species) and sterilized sludge in a volume equivalent to 2g of total solids. A dryness of 10-20% would be equivalent to sludge from septic systems (Jiménez et al., 2006), where the total solids content oscillates between those values. If needed we can put you in contact with pathogenic inoculums providers.

Comparison of the biological pollutant content in wastewater from developing and developed countries, FROM: Jimenez Cisneros 2011

Organism Developed world Developing world
Enteric viruses, PFU 100 mL-1 (U,I) 10^2-10^4 10^4-10^6
Salmonella, MPN100 mL-1 (M, U, F, SA, IN, H) 10^0-10^4 10^5-10^7
Fecal streptococci , No. 100mL -1 (U, B, K) 10^4-10^6 10^6->10^7
Protozoan cysts, organisms L-1 (U, M) 10^1 10^3
Giardia lamblia, cysts L-1 (U, E, K) 1-10^3 10^2-10^3
Cryptosporidium parvum, oocysts L-1 (U, E) 1-10^3 ND
Helminth ova, egg L-1 1-9 6-800

Data from: England: E; Holland: H; India: In; Israel: I; Kenya: K; Mexico: M; South Africa: SA; USA: U; ND: No data.

REFERENCES

Jiménez B. and Wang L. (2006) Sludge Treatment and Management. Chapter 10 in Municipal Wastewater Management in Developing Countries: Principles and Engineering. Ujang Z. and Henze M. Editors, 237-292 pp. IWA Publishing, London, U.K.

Jiménez Cisneros B. (2011) Safe Sanitation in Low Economic Development Areas, Treatise M.Sc. 82. Chapter In Treatise on Water Science, vol. 4. Wilderer P. Editor, 147–201 pp. Oxford: Academic Press.