Transport en horitzontal

emerged on the scene (1984) have been used for every waterborne pathogen; in fact, multiplexing, the detection of many pathogens simultaneously, is an approach that is now being explored rapidly. The study of pathogens in waters has become impera- tive: in particular, for applications toward discovery and environmental epidemiology, outbreak investigation, and risk assessment and control.

Yet while advances are being made, it is clear that further considerations must be given to the complete process for pathogen testing of waters. Pathogen detection in water must be considered as a search for a rare biological entity in a soup of biomes. This means that larger volumes of water must be collected, and generally, filtration has been used and remains the primary method for concentration. Extraction and purifi- cation and in many cases further concentration are needed, inhibition then becomes a big problem. Both quantification and determination of the infectious nature of the pathogen are of extreme interest. To undertake adequate risk assessment sensitivity, specificity along with potential viability must be determined.

The methods described in this chapter can be used for the detection and char- acterization of all waterborne pathogens and should be used to better understand the geographical distribution of these microorganisms in our global waters, given the threat of waterborne disease. To improve knowledge of waterborne pathogen fate and trans- port, concentrations and accumulation, and finally, spatial and temporal occurrences, these novel methods can now be used in water quality studies worldwide.

The latest methods used for the detection of viruses, bacteria, and protozoans in water samples are primarily molecular. Conventional methods are presented briefly in Table 3.1, and the newest detection methods for waterborne pathogens are described in Table 3.2. Effective detection of waterborne pathogens includes a series of steps, such as sample concentration, nucleic acid extraction, inhibition control, design of

Table 3.1 Waterborne Pathogen Detection: Conventional Detection Methods

Pathogen Method Outcome Characteristics

Viruses Cell culture and

infectivity

Presence/absence, infectivity determination

Infected cell cultures undergo morphological changes called cytopathic effects (CPEs) that are observed microscopically. The method is labor intensive and time consuming. Some viruses do not show CPEs.

Bacteria Bacterial culture Presence/absence

quantification

Based on selected media, various groups of bacteria can be detected.

Protozoans Microscopic methods along with fluorescent antibody tags

Counts of oo(cysts)

Antibody specific to the cell wall of the oo(cysts) tagged to fluorescence is used to stain the sample. Using morphometrics and fluorescence, the organisms are identified. No infectivity can be determined, and volumes that can be processed under the microscope are relatively small.

DISCOVERY, CHARACTERIZATION, AND MONITORING OF WATERBORNE PATHOGENS 59

Table 3.2 Waterborne Pathogen Detection: New Detection Methods

Method Outcome Characteristics

PCR Presence/absence Able to detect nonculturable pathogens. High sensitivity

and specificity. Requires design of primers that amplify specified DNA regions. Prone to environmental inhibition. Gel electrophoresis is required for visualization of results.

RT-PCR Presence/absence A reverse transcription step (RT) is required before PCR

amplification, for the conversion of RNA to cDNA. Following RT, the same steps are followed as with conventional PCR.

Nested PCR Presence/absence Requires two sets of primers. Inner primers amplify the

target sequence within the amplicon generated by outer primers. This technique has a higher sensitivity than that of conventional PCR.

Multiplex PCR Presence/absence Uses multiple primer sets in a single PCR reaction to

detect multiple targets simultaneously. It is time efficient and reduces the cost of reagents. The design and optimization of multiplex assays can be more challenging than those of conventional PCR assays.

Real-time PCR Quantitative This is the only quantitative method (except for the

MPN dilution method). No post-PCR handling step is required to view the results. It is a very target-specific and sensitive method. The costs of a thermocycler and reagents are higher than those used in conventional PCR.

ICC-PCR Presence/absence,

infectivity determination

This is a combination of the traditional cell culture/cell infectivity method with PCR. PCR is performed on cell culture supernatant. The method has higher sensitivity than that of conventional PCR.

Microarrays Presence/absence Able to detect multiple microbial agents simultaneously.

Microarrays provide rapid results and are an appropriate screening method.

appropriate primers, and confirmation of results. A flow schematic for the detection of waterborne viruses, indicating all these steps and processes, is also shown (Figure 3.1). Inhibition is one of the most common problems encountered environmental molec- ular laboratories. Wilson (1997) reviewed inhibition and facilitation of nucleic acid amplification. He grouped the mechanisms of inhibition into three categories: failure of lysis, nucleic acid degradation and capture, and polymerase inhibition. The inhibitor materials can be organic or inorganic compounds naturally present in the environment. All of these compounds may inhibit the enzymes involved in PCR. It is important to note that during sample collection and concentration, inhibition materials are also concentrated. Humic substances are the natural organic substances that have been most generally identified as inhibitors in environmental samples. Other organic compounds that may cause inhibition include fulvic acid, tannic acid, proteins, polysaccharides, and glycoproteins. Inhibitory inorganic compounds could be metals. Many studies have investigated the removal of PCR inhibitors. However, there is no single method that could remove all inhibitors. The humic acid removal techniques summarized by Wilson