Background Because the success of deworming programs targeting soil-transmitted helminths (STHs) is evaluated through the periodically assessment of prevalence and infection intensities, the use of the correct diagnostic method is of utmost importance. The STH community has recently published for each phase of a deworming program the minimal criteria that a potential diagnostic method needs to meet, the so-called target product profiles (TPPs). Methodology We compared the diagnostic performance of a single Kato-Katz (reference method) with that of other microscopy-based methods (duplicate Kato-Katz, Mini-FLOTAC and FECPAK G2 ) and one DNA-based method (qPCR) for the detection and quantification of STH infections in three drug efficacy trials in Ethiopia, Lao PDR, and Tanzania. Furthermore, we evaluated a selection of minimal diagnostic criteria of the TPPs. Principal findings All diagnostic methods showed a clinical sensitivity of ≥90% for all STH infections of moderate-to-heavy intensities. For infections of very low intensity, only qPCR resulted in a sensitivity that was superior to a single Kato-Katz for all STHs. Compared to the reference method, both Mini-FLOTAC and FECPAK G2 resulted in significantly lower fecal egg counts for some STHs, leading to a substantial underestimation of the infection intensity. For qPCR, there was a positive significant correlation between the egg counts of a single Kato-Katz and the DNA concentration. Conclusions/Significance Our results indicate that the diagnostic performance of a single Kato-Katz is underestimated by the community and that diagnostic specific thresholds to classify intensity of infection are warranted for Mini-FLOTAC, FECPAK G2 and qPCR. When we strictly apply the TPPs, Kato-Katz is the only microscopy-based method that meets the minimal diagnostic criteria for application in the planning, monitoring and evaluation phase of an STH program. qPCR is the only method that could be considered in the phase that aims to seek confirmation for cessation of program. Trial registration ClinicalTrials.gov NCT03465488

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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.

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.

In Mexico, physicochemical sludge contains high levels of pathogens; and alkaline stabilization is an alternative for their control. However, the odours caused mainly by ammonia generation represent a disadvantage. On the other hand, the ammonia is known as an effective disinfectant. The aim of this study was to evaluate the disinfectant properties of ammonia in sludge, and use it in a closed alkaline stabilization system, which, not only copes with odours but also increases the efficiency of the process. Raw sludge from a municipal wastewater treatment plant using a physicochemical process was used. Ammonia was applied in doses from 10 to 50% w/w; also, doses from 5 to 40% of CaO were applied in open and closed systems and raw and treated sludge quality was evaluated. Results showed that ammonia removed 6 and 5 logs of faecal coliforms and Salmonella spp., respectively and up to 94% of viable helminth ova. The closed system was more efficient than the open system when applying doses from 5 to 20% of CaO. Finally, the results indicate that the ammonia represents an alternative to disinfecting wastewater sludge and it can be used to enhance alkaline stabilization processes.

Worldwide, unsanitary conditions are responsible for more than three million deaths annually. One of the reasons is the low level of sanitation in developing countries. Particularly, sludge from these regions has a high parasite concentration and low heavy metal content even though the available information is limited. Different issues needed to achieve a sustainable sludge management in developing nations are analysed. Based on this analysis some conclusions arise: sludge management plays an important role in sanitation programs by helping reduce health problems and associated risks; investments in sanitation should consider sludge management within the overall projects; the main restriction for reusing sludge is the high microbial concentration, which requires a science-based decision on the treatment process, while heavy metals are generally low; adequate sludge management needs the commitment of those sectors involved in the development and enforcement of the regulations as well as those that are directly related to its generation, treatment, reuse or disposal; current regulations have followed different approaches, based mainly on local conditions, but they favour sludge reuse to fight problems like soil degradation, reduced crop production, and the increased use of inorganic fertilizers. This paper summarises an overview of these issues.

The parasites present in the residual sludge generate by the diverse treatment processes of residual water vary, depending on the socioeconomic and epidemiological conditions of different communities. And although data exist at the global level regarding the microbiological quality of sludge, these data are practically nonexistent in developing countries. Therefore, this project compares the content of the four principal groups of parasites in residual sludge from the United States and Mexico. The marked differences between the orders of magnitude found in the two countries (coliphages 10(3)-10(6) and 1.3 x 10(5) PFU/gTS, fecal coliforms 10(7)-10(10) and 10(7), Salmonella typhi 10(6)-10(8) and 10(3) MPN/gTS, Giardia lamblia 10(2)-10(4) and 10(2) cysts/gTS and helminth ova from 73-177 and <1 to 10 viable HO/gTS, respectively) will permit proposing real, effective and economical stabilization processes (undoubtedly different from those of industrialized countries), and will thus take advantage of the benefits represented by the reuse and disposal of sludge, in addition to developing regulations in agreement with the conditions of each region.

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