## Info

Calculation of the RR value involves the ratio of the exposed group as a proportion of the population examined to the unexposed group as a proportion of the population examined:

rr = a/(a+b) = 52/(52 + 32) _ 0.62 _ o c/(c + d) 21/(21+64) 0.24

The RR establishes that the relative risk of becoming ill for the group of people exposed to contaminated water as opposed to the group of people not exposed to contaminated water is 2.6.

Two types of information regarding disease in a population that can be helpful to an epidemiological study are incidence rate and prevalence rate. Incidence rate is defined as the number of new cases per unit of person-time at risk. For example, suppose the waterborne outbreak used in the previous examples occurred in a stable community of 10,000 people. Following the outbreak, the number of new cases occurring over a five-year period was 30 per 10,000 people. These new cases might have nothing to do with consuming water, but the waterborne incident might have established some carriers of the disease within the population that could contribute to the infection of others. In this example, the incidence rate of the disease in the community would be 6 cases per 10,000 people-years; the expression people-years arriving from the normalization of the 30 disease cases over a five-year period.

Prevalence rate is something different from incidence rate because prevalence rate concerns the actual number of disease cases in a community. In the case of the waterborne outbreak, there were 73 cases of the disease. Supposing that secondary infections occurred among the population to add another 43 cases of the disease bringing the total to 116 cases of the disease for the year. In the community of 10,000 people, the prevalence rate of the disease for the year of the outbreak would be 1 percent.

The incidence rate can be determined for both the exposed and unexposed individuals identified with the waterborne outbreak above. Looking at the data, we find that 52 people became sick out of 84 people that drank water and 21 people became sick out of 85 people that did not drink water. The incidence rate for the two subgroups of individuals is 62 percent and 25 percent, respectively. From these data, an attributable risk can be determined by subtracting the incidence rate of nondrinkers from drinkers of the water, which would be 37 percent.

Incidence measures reflect the level of infectivity of the causative agent of the disease. They do not establish the virulence of the causative agent because virulence relates to the damage produced as a result of the infection. Damage resulting from infection of an individual can range from a few mild symptoms to life-threatening symptoms, depending on many contributing factors (e.g., health and nutrition status, age, infectious dose of the pathogen received, how the pathogen was received, genetic disposition and others). In the study of an outbreak, a case is defined not by the severity of the infection but by the fact that an infection occurred.

The subject of risk assessment has advanced considerably in the last 20 years. Mathematical models have been constructed to estimate the probability of infection using databases of human exposure. Before models could be formulated it was necessary to ascertain the variables of the infection process. In the case of microbial risk assessment, such variables might include etiologic disease agent identification, human health effects manifested through infection, dose-response data relating dose received and probability of infection/disease in the target population, physiology of host-parasite relations, and epidemiological data.26

### Molecular Detection of Waterborne Pathogens

Water, especially drinking water, when under suspicion of the transmission of pathogens, requires laboratory examination for proof of contamination. Cultural methods may prove inadequate for the isolation of pathogens, may produce uncertain results, or may be too time-consuming to support ongoing epidemiological investigations. During the past three decades, environmental laboratories have exploited molecular-based protocols to gain insight into the presence of sundry infectious bacteria, viruses, and protozoa in aquatic environments and water supplies. These techniques can be useful to investigations of disease outbreak, especially, where no cultural evidence can be obtained to show the existence of an infectious agent. In fact, a fundamental challenge in proving the hypothesis that a disease outbreak has occurred is to establish conclusively that the suspected agent of disease existed at the suspected source of the disease. A broad range of sophisticated laboratory techniques, such as fluorescent antibody, enzyme-linked immunosorbent assay (ELISA), fluorescent in situ probe (FISH), flow cytometry, and the polymerase chain reaction (PCR), are available to provide answers not possible by classical measures. From these has emerged a branch of epidemiology called molecular epidemiology. Routine use of molecular tools is nonexistent in many health laboratories, however, owing to the requirement for relatively expensive equipment, need to employ technicians knowledgeable about molecular techniques, and the technical issues surrounding detection of specific genomes present in very low levels in water. Despite these apparent limitations to adopting molecular techniques for routine surveillance of pathogens in water-quality-control laboratories, molecular protocols have been used to detect a wide range of pathogenic agents in waters.

A brief introduction to molecular methods for microbiological investigation in the water environment is given based on descriptions by Rochelle and Schwab.141

Sample Collection Proper procedures for obtaining water samples are independent of the intended use of water. However, taking advantage of the sensitivity of molecular detection implies that the target organism is probably in very low in concentration, else it might be prudent to employ a cultural technique (assuming the target microorganism or virus is in a viable/recoverable state). Therefore, sample volumes earmarked for molecular applications are usually large and will require concentration of contents.

Sample Concentration Large water samples are processed by filtration procedures applicable to bacteria, protozoa, or viruses.

Nucleic Acid Extraction The material of interest to be assayed by molecular techniques is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Extraction of nucleic acids from filtered/centrifuged biomass containing the target organism of interest may take place directly or following repeated elution and centrifugation steps (principally required for virus recovery). Ideally, the extraction step will be minimally time consuming, produce a high yield of intact nucleic acid, and preclude carryover of inhibitory substances inimical to the polymerase chain reaction (PCR) analysis. Special procedures can be introduced prior to nucleic acid extraction for removal of inhibitors. Published protocols and commercial kits may be used for postextraction purification of nucleic acids to eliminate inhibitors.

Methods of Detection The basic approach to assaying purified target nucleic acid is the application of PCR. The purpose of PCR is to amplify the nucleic acid of the target organism so that workable quantities of product become available for subsequent sequence analysis. It is important that the PCR procedure be sensitive and specific. PCR assays are typically operated in three cycles of temperature to accommodate three steps:

1. Denaturation of the double-stranded, target DNA (92°-94°C)

2. Annealing of specific primers to the single-strand form (denatured) of the target DNA at some prescribed or trial-and-error temperature (45°-55°C)

3. Complementary strand synthesis by primer extension of each of the single strands produced by step 1 at a temperature of 75°-80°C

The three-step procedure is repeated usually 30 to 40 times in order to obtain exponential copies of PCR product. The two important variables in successful use of PCR as a tool are primer synthesis or selection and PCR operating conditions. These two factors dictate the level of specificity and sensitivity that can be obtained by PCR and are instrumental in facilitating the detection of target nucleic acids at refined taxonomic levels.

Evaluation of PCR Products The purpose of amplifying target nucleic acids present in the environmental sample is to be able to subject a sufficient quantity of the representative material (PCR product) to a laboratory procedure for the determination of the microbial agent that it represents. Classic procedures for this purpose involve application of a series of concentrations of the PCR products to an agarose gel electrophoresis slab along with a molecular marker. Various amplified gene fragments migrate through the gels in proportion to their molecular weights. The separated gene fragments can then be confronted with an oligonucleotide probe specific for the organism of interest in relation to its possible presence in the original water sample. Oligonucleotide probes are conjugated with a reporter molecule (typically a fluorogenic compound) that under appropriate conditions (fluorescent lighting) signals hybridization with a complementary (target) nucleic acid fragment.

Two areas of interest in connection with molecular detection of specific micro-bial agents in environmental samples are robustness of the detection effort and the level or density of the target microbe in the representative environmental sample. In the former, since molecular detection is a gene-based exercise, it stands to reason that the more types of gene fragments that are available as probes, the more information that can be learned about the genome of the target organism. The technique that makes use of the multiple probe approach is the microarray. The microarray is a glass microscope slide that serves as a solid support for the spotting of literally thousands of genes or gene fragments—in this example, oligonucleotide probes—that serve to test hybridization potential with amplified gene fragments (PCR products) of unknown identity. The nucleotide sequence of the probe is known and representative of specific microbes. The location of each of the probes on the glass slide is carefully recorded, so when hybridization with unknown PCR products (amplicons) is indicated by reporter signals, the strain, species, and genus identity of the unknown amplicon can be learned.

Quantification of the target microbe in the environment with the aid of a PCR instrument must involve procedural modifications and special equipment in order to measure the level of production of PCR products. Fluorogenic probes and a fluorescence detection device are used to track the formation of PCR product formation. Quantitative PCR (qPCR) is still relatively new, and advances are being made to increase its utility. The following brief description is based on methodology described by Grove.142 In the qPCR process, two fluorogenic probes anneal to the template nucleic acid between the primers. As the nucleic acid polymerase extends the primer, the probe is displaced, and the polymerase cleaves the fluo-rogenic dye. Released dye is freed from the quencher and a fluorescent signal is produced. The detection device consists of a multiwell thermal cycler connected to a laser and a charge-coupled optics system. A fiber optic inserted through a lens is positioned over each of the wells, and a laser beam is directed through the fiber to excite fluorochrome in the PCR fluid present in wells. Fluorescence emissions are sent through the fiber to the CCD camera, mathematically analyzed by the system software, and the data are computerized.

Obtaining quantitative data on the original sample requires construction of a calibration curve. This is done by preparing dilutions of a known quantity of nucleic acid and performing PCR. Emissions data are obtained for each dilution of the nucleic acid and plotted against thermal cycle numbers. A series of curves result, and a line is drawn through the curves parallel to the thermal cycle numbers (x axis) at a height just above the background fluorescence (Figure 1.8). Another line is drawn perpendicular to the thermal cycles (x axis) at the intersection of the parallel line and each of the curves representing the nucleic acid dilutions. The thermal cycle number corresponding to each curve is the threshold cycle (Ct). The calibration curve is a plot of each C t value against the corresponding nucleic acid concentration in the dilution series. The Ct is inversely proportional to the copy number (concentration) of nucleic acids in the dilution series, so a straight line should result. The actual concentration of nucleic acid in the unknown sample is determined by obtaining a C t value under identical conditions of PCR operation

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