J.A. Barrios

A real-time PCR method for quantifying viable ascaris eggs using the first internally transcribed spacer region of ribosomal DNA.

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.

Identification and quantification of pathogenic helminth eggs using a digital image system.

A system was developed to identify and quantify up to seven species of helminth eggs (Ascaris lumbricoides -fertile and unfertile eggs-, Trichuris trichiura, Toxocara canis, Taenia saginata, Hymenolepis nana, Hymenolepis diminuta, and Schistosoma mansoni) in wastewater using different image processing tools and pattern recognition algorithms. The system was developed in three stages. Version one was used to explore the viability of the concept of identifying helminth eggs through an image processing system, while versions 2 and 3 were used to improve its efficiency. The system development was based on the analysis of different properties of helminth eggs in order to discriminate them from other objects in samples processed using the conventional United States Environmental Protection Agency (US EPA) technique to quantify helminth eggs. The system was tested, in its three stages, considering two parameters: specificity (capacity to discriminate between species of helminth eggs and other objects) and sensitivity (capacity to correctly classify and identify the different species of helminth eggs). The final version showed a specificity of 99% while the sensitivity varied between 80 and 90%, depending on the total suspended solids content of the wastewater samples. To achieve such values in samples with total suspended solids (TSS) above 150 mg/L, it is recommended to dilute the concentrated sediment just before taking the images under the microscope. The system allows the helminth eggs most commonly found in wastewater to be reliably and uniformly detected and quantified. In addition, it provides the total number of eggs as well as the individual number by species, and for Ascaris lumbricoides it differentiates whether or not the egg is fertile. The system only requires basically trained technicians to prepare the samples, as for visual identification there is no need for highly trained personnel. The time required to analyze each image is less than a minute. This system could be used in central analytical laboratories providing a remote analysis service.

Sludge electrooxidation as pre-treatment for anaerobic digestion

Anaerobic digestion of wastewater sludge is the preferred method for sludge treatment as it produces energy in the form of biogas as well as a stabilised product that may be land applied. Different pre-treatments have been proposed to solubilise organic matter and increase biogas production. Sludge electrooxidation with boron-doped diamond electrodes was used as pre-treatment for waste activated sludge (WAS) and its effect on physicochemical properties and biomethane potential (BMP) was evaluated. WAS with 2 and 3% total solids (TS) achieved 2.1 and 2.8% solubilisation, respectively, with higher solids requiring more energy. After pre-treatment, biodegradable chemical oxygen demand values were close to the maximum theoretical BMP, which makes sludge suitable for energy production. Anaerobic digestion reduced volatile solids (VS) by more than 30% in pre-treated sludge with a food to microorganism ratio of 0.15 g VSfed g-1 VSbiomass. Volatile fatty acids were lower than those for sludge without pre-treatment. Best pre-treatment conditions were 3% TS and 28.6 mA cm-2.

Helminths and their Role in Environmental Engineering

Helminth eggs represent an important challenge to environmental engineers as they are among the most difficult biological parasites to inactivate in wastewater and sludge. Even though no official data on helminthiasis exist, it is estimated that more than 2.6 billion people are affected. These parasites are of concern in developing countries, particularly in those areas where the reuse of wastewater and sludge for agriculture is common. With regard to this, the unrestricted use of wastewater for irrigation presents a serious health risk due to the dissemination of pathogens, particularly helminth eggs. Helminth eggs survive in water, soil, and crops for several months and over much longer periods than other microorganisms. Therefore, and in order to minimize risk, several guidelines and regulations exist which limit their content in wastewater and sludge. Risk assessment estimates that such regulations may be less strict in developing countries where higher concentrations of helminth eggs occur in wastewater and sludge. These eggs need to be removed from wastewater and inactivated in sludge using certain treatment processes, some of which are not feasible in developing countries. Adequate methods are needed to precisely identify and quantify helminth eggs in environmental samples. Therefore, a multidisciplinary approach is needed to address helminthiasis in environmental engineering issues.