**4. Detection methods in the environment**

Detection techniques in environmental samples are diverse. Molecular biology methods are used to differentiate genotypes by using hybridization DNA probe DNA and polymerase chain reaction (PCR) techniques, starting from diverse fragments of nucleic acids as ribosomal RNA, los genes *hsp* 70, and random amplification of polymorphic DNA fragments (RAPD). The advantages of PCR techniques include high sensitivity, rapid analysis of several samples, relatively low cost, simultaneous detection of several pathogens, and the capacity to discriminate among several species of strains. These techniques are used mainly to differentiate *Giardia* species and genotypes [40].

The techniques to number *Giardia* cysts are those that use fluorescent antibodies; they can also differentiate viable and nonviable cysts by phase-contrast imaging [53]. Immunofluorescent microscopy techniques are used to detect *G. intestinalis* in water. The methods endorsed by the United States Environmental Protection Agency (USEPA) are the Information Collection Request (ICR) method protozoans (1999) and the 1623 method (2005). For water samples, this method is based on elution and purification of a filter stained with fluorescent monoclonal antibodies to then count the structures in a brilliant apple green color in an epifluorescence microscope (**Figure 1**). Many of these methods have already been adapted to recover cysts in food [54].

Fluorescent staining as acridine orange, propidium iodide (PI), and 4,6-diamidino-phenylindole (DAPI) are prone to have false positives and have variable stain characteristics depending on the viability state of the microorganism; nonetheless, the use of these stains, especially DAPI, can be very useful in identification when used together with other microscopy techniques such as fluorescence and phase contrast and differential interference contrast (DIC) [53].

Immunomagnetic separation (IMS) methods and the 1623 method have been developed to concentrate bacteria and protozoan pathogens. These methods use specific antibodies on the surface of paramagnetic particles to link target pathogens, followed by a magnet used to separate them

**Figure 1.** *Giardia intestinalis* cysts stained with fluorescent antibodies.

(WHO) estimates that at least 10<sup>9</sup>

symptoms [1, 52].

152 Current Topics in Giardiasis

food [54].

trast (DIC) [53].

of the countries in the world, causing mortality of more than 5 × 106

**4. Detection methods in the environment**

*Giardia* species and genotypes [40].

economic costs of diseases are alarming and cause financial losses. For this reason, social institutions have decided to work in developing better techniques for researching and controlling parasites in such a way that water turns out to be a safe liquid. Knowing the relative importance of specific transmission routes of intestinal protozoa, including potential sources of environmental contamination, constitutes fundamental aspects that allow understanding the epidemiology of parasitic diseases. In this manner, corrective measures can be applied to minimize prevalence and incidence of these diseases in the population. In developed countries, giardiasis is an emerging infection because it plays an important role in diarrhea outbreaks linked to water and food consumption that affect the population in general. As to developing countries in Asia, Africa, and Latin America, approximately 200 million people experience giardiasis

Detection techniques in environmental samples are diverse. Molecular biology methods are used to differentiate genotypes by using hybridization DNA probe DNA and polymerase chain reaction (PCR) techniques, starting from diverse fragments of nucleic acids as ribosomal RNA, los genes *hsp* 70, and random amplification of polymorphic DNA fragments (RAPD). The advantages of PCR techniques include high sensitivity, rapid analysis of several samples, relatively low cost, simultaneous detection of several pathogens, and the capacity to discriminate among several species of strains. These techniques are used mainly to differentiate

The techniques to number *Giardia* cysts are those that use fluorescent antibodies; they can also differentiate viable and nonviable cysts by phase-contrast imaging [53]. Immunofluorescent microscopy techniques are used to detect *G. intestinalis* in water. The methods endorsed by the United States Environmental Protection Agency (USEPA) are the Information Collection Request (ICR) method protozoans (1999) and the 1623 method (2005). For water samples, this method is based on elution and purification of a filter stained with fluorescent monoclonal antibodies to then count the structures in a brilliant apple green color in an epifluorescence microscope (**Figure 1**). Many of these methods have already been adapted to recover cysts in

Fluorescent staining as acridine orange, propidium iodide (PI), and 4,6-diamidino-phenylindole (DAPI) are prone to have false positives and have variable stain characteristics depending on the viability state of the microorganism; nonetheless, the use of these stains, especially DAPI, can be very useful in identification when used together with other microscopy techniques such as fluorescence and phase contrast and differential interference con-

Immunomagnetic separation (IMS) methods and the 1623 method have been developed to concentrate bacteria and protozoan pathogens. These methods use specific antibodies on the surface of paramagnetic particles to link target pathogens, followed by a magnet used to separate them

cases of gastrointestinal diseases occur per year in one-third

persons at early age. The

from the matrix sample [55]. The assay method of immunoabsorption linked to enzymes (ELISA) is more sensitive than the microscopy techniques for (oo)cyst detection [56].

Flux cytometry (FC) is a method by means of fluorescent activators capable of classifying cells according to their fluorescence and size. Detection and selective enumeration of *Giardia* cysts that are applied in FC consists of separating and observing the stained particles with immunofluorescent antibodies by the dispersion process. This method can potentially turn out to be the most precise in detecting and quantifying cysts [57].

Knowing cyst concentration in environmental samples and the necessary dosage when giardiasis starts allows us to estimate pathogen exposure; with this information and using the appropriate mathematic model, it is possible to calculate health risk. This methodology called quantitative microbial risk assessment (QMRA) is based on a series of steps that convey predicting daily and annual risks. In developed countries, QMRA has been adapted to assess permissible risk limits for *Giardia* in drinking water samples where the accepted risk worldwide is one infection for each 10,000 individuals.

## **5. Quantitative microbial risk assessment**

The Codex Committee on Food Hygiene and the National Advisory Committee on Microbiological Criteria for Foods have proposed a framework for conducting QMRA. These guidelines also provide methods and approaches used to evaluate potential health effects and assess risks from contaminated source media, i.e., soil, air, and water. One of the key benefits of this method is the development of models describing the complex nature of pathogen populations in water or food supply [58].

Hazard identification involves pathogen detection in terms of concentration in water, for example. Next is exposure assessment where the quantity of water consumed for the people at risk is determined. In these two steps, one should take into account the recovery efficiency of the method, the characteristics of the people (age, sex, immune state, and customs), and pathogen survival. Then, the dose-response curve is calculated with the mathematical models described in the literature; finally, the integration of all the parameters provides the risk characterization that results in the likelihood of infection risk per day and year per person [59].

Quantitative microbial risk assessment has become a standard; the UK has pronounced a mandate that establishes that risk assessment be carried out by local government on many water supplies [60]. The US Environmental Protection Agency (EPA) handled permissible water *Giardia* risk values of <1:10,000 (10−4) in a yearly exposure [61]. In the UK, the Water Supply Regulations 1999, and The New Dutch Drinking Water Decree state that for pathogenic microorganisms, health risk should be less than 1 infection/10,000 consumer/ year [62]. These risk regulations are equal to those of EPA. Developed countries are in the position to provide guidance, training, information resources, and technical assistance to advance supports for water safety. Thus, greater cooperation and collaboration at all levels should be effective and ensure that QMRA, as a water safety tool, will be available to all countries.
