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      Pathogen Surveillance Through Monitoring of Sewer Systems


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          I INTRODUCTION It is now possible to monitor sewage systems for pathogen occurrence in a community. An epidemiological approach to monitoring sewer systems is especially relevant for an early warning of pathogens used as biological weapons. In many situations, bioterrorist contamination events will result in the pathogen shedding to wastewater before a community level epidemic begins. Detecting the organism early allows the governments to respond on time and eliminate a potential catastrophe. A Monitoring for human pathogens in sewage Monitoring of human pathogens in sewage is possible because they may be excreted in a range of bodily fluids, skin, and hair during active infection (Feachem et al., 1983). All of these materials will find their way into sewage systems during the process of waste elimination (toilet flushing) and cleaning (e.g., bathing, hand washing). In addition to release during active infection, pathogens can be washed into sewage systems from cleaning of indoor (floor washing, kitchen sink use) and outdoor (auto washing, driveway cleaning, storm water collection) facilities. Thus, sewer systems collect pathogens from over a wide area to a common carrier, where they are transported to a central facility for processing. Wastewater presents a time dynamic collection point where many physical, chemical, and biological substances of our society are brought to a central location. Monitoring of centralized wastewater allows detection of intentional, natural, or accidental contamination events. Because of recent bioterrorism concerns in the U.S., routine monitoring is potentially useful since it can result in better preparedness of utilities and the public health response system (Meinhardt, 2005). The qualitative microbial risk assessment (QMRA) framework can be used as a tool to develop and interpret this type of wastewater monitoring system. Because the threat level drives the risk assessment analysis, a monitoring system should be coordinated with findings from modeling studies on the survival and dispersion of contaminants (Kim et al., 2007, Romero et al., 2008, Sinclair et al., 2008), the contaminant point of introduction (Danneels and Finley, 2004), the health risk (Haas et al., 1999), and the locations of early warning systems/sensors in wastewater and water treatment systems (Murray et al., 2004). A recent U.S. National Research Council study called for more resilient design/operation of wastewater and drinking water systems (USNRC, 2007) to improve response and recovery from adverse water quality events in collection systems, water distribution systems, and water/wastewater treatment systems. Monitoring programs for pathogens or surrogates could potentially aid in the accomplishment of these goals. The aim of this review of published literature and reports is to assess the feasibility of monitoring sewage systems as an early warning system for the release of pathogens from an intentional, natural, or accidental biological contamination event. We address issues from a QMRA perspective and explore methods to detect and monitor pathogens in wastewater. The review presents our conclusions on: (1) the potential biological agents that might be released into a sewage system, (2) the likely background level of those agents in sewage, (3) laboratory methods and detection, and (4) the probability of detecting select biological agents in sewage. II POTENTIAL BIOLOGICAL AGENTS IN SEWAGE A wide variety of pathogenic organisms pass through municipal waste-water treatment systems. One study found that a single toilet flush containing poliovirus was detectable at a nearby treatment plant for more than 4 days (Ranta et al., 2001). The toilet flush study was designed to replicate the number of virus released from an infected individual. Pathogenic microorganisms can also grow in the host but not produce sickness in the infected host. It is estimated that 50% or less of those individuals infected with enteric viruses or bacteria actually become ill (Haas et al., 1999). In the case of some respiratory pathogens, 90% or more of the persons infected will become ill (Belshe, 1991). During the growth of the organism in the host, the organism will be found in various organs and bodily fluids. Organisms transmitted by the fecal–oral route are usually excreted in large numbers in the feces, since the initial or primary site of replication is in the intestinal tract. However, this does not preclude their replication in other parts of the body. For example, enteroviruses (e.g., poliovirus) will replicate in nerve tissue causing paralytic disease, while Hepatitis A virus will replicate in the liver causing damage there (Belshe, 1991). Respiratory infections are usually the result of replication of the organism in the nose, throat, or lungs. Infection of other organs of the body often leads to the presence of the organisms in the blood and then the urine after their elimination by the kidneys. This explains the occurrence of insect-borne encephalitis viruses and enteric viruses in the urine (Pichichero et al., 1998). Any type of infection (Fig. 9.1 ) within a community is likely to lead to pathogen excretion in bodily fluids/substances and therefore, transported into the community sewage system. Figure 9.1 Outcomes of exposure to a microbial infection. This review considers biological agents prioritized by the Centers for Disease Control (CDC) as potential biological weapons that could be used by terrorists (Table 9.1 and 9.2 ). They are listed in three categories (i.e., A, B, and C) of decreasing concern. Category A agents require the most intensive public preparedness efforts due to the potential for mass causalities, public fear, and civil disruption. Category B agents are also moderately easy to spread, but have lower mortality rates. Category C agents do not present a high public health threat, but could emerge as future threats (Rotz et al., 2002). Many other pathogenic agents are present in sewage, but not on the CDC select agent list. Table 9.2 lists some common blood and respiratory agents and emerging pathogens, all of which could potentially be engineered for mass dissemination and detected through monitoring of wastewater. The methods described in this paper apply to many other pathogens and are not limited to those agents listed in Tables 9.1 and 9.2. Table 9.1 The center for disease control select agents (Rotz et al., 2002) Category A Category B Category C Anthrax Bacillus anthracis Botulism Clostridium botulinum Plague Yersinia pestis Smallpox Variola majorTularemia Francisella  tularensis Hemorrhagic fever virusa  Arenaviridae  Bunyaviridae  Filoviridae  Flaviviridae  Lassa fever Hantavirus Dengue fever Ebola Marburg Brucellosis Brucella abortus Water and Food-borne agents Enteroviruses Poliovirus and Rotavirus Salmonellosis Salmonella  Caliciviruses Hepatitis A virus Protozoan parasites Cryptosporidium parvum  Giardia lamblia  Toxoplasma MicrosporidiumGlanders Burkholderia mallei Psittacosis Chlamydia psittaci Q fever Coxiella burnetii Typhus fever Rickettsia prowazekii Viral Encephalitis West Nile La Crosse Venezuelan equine encephalitis Japanese encephalitis Nipah virusTick-borne HFVCrimean-Congo HFVTick-borne encephalitis virusesYellow feverMultidrug resistant TBInfluenzaOther RickettsiasRabies a Hemorrhagic fever virus (HFV). Table 9.2 Nonenteric pathogens found in sewage and other emerging agents of concern Nonenteric agents Emerging agents Severe Acute Respiratory Syndrome (SARS) Parvoviruses John Cunningham virus (JC Virus) Picobirnaviruses Human Immuno-deficiency Virus (HIV) Enteroviruses types 78–100 Hepatitis B Virus (HBV) Torque teno virus (TTV) A Human pathogens secreted in bodily fluids A literature search was conducted to determine the occurrence of the agents in bodily fluids, feces, skin, and sewage. As indicated in Table 9.3 and a previous publication (Sinclair et al., 2008), many select agents may occur in bodily excretions or secretions even though this may not be their primary site of replication. It would appear that all of the viral agents are excreted in the urine and most of the bacterial agents in the feces or saliva. Since none of the organisms cause enteric infections they have seldom been sought in sewage, however, Bacillus anthracis and Yersina pestis (plague) have been detected in sewage. The source of B. anthracis spores in the sewage was believed to be from an African import tannery operation (Perone and Gelosa, 1982) and presumably not from enteric infections, which would normally result in the presence of spores in the feces. Category B agents differ in that, and they include many enteric pathogens which are excreted in large numbers in the feces (Table 9.3). All of the other agents in this category appear to be excreted in the feces; many of the viral agents are excreted in the urine. No studies were found that report examining sewage for their presence. The Category C viral agents appear to be excreted in the saliva and urine (Tables 9.3 and 9.4 ). No references for the presence of these agents in sewage could be found. Some typical blood-borne agents such as Hepatitis B virus (Alter et al., 1977) and Human Immuno-Deficiency virus (Levy, 1989) have been detected in sewage by molecular methods (Table 9.4). The coronavirus, which causes Severe Acute Respiratory Disease (SARS), is also excreted in the feces and other bodily fluids such as tears (Loon et al., 2004, Wang et al., 2005) (Table 9.4). Table 9.3 Select category B and C agents found in human bodily fluids and sewage Agent Category Urine Feces Saliva Sewage Reference C. psittaci B ? Yesa ? ? Midura and Arnon (1976); Anderson (1996); Smith et al. (2005) C. burnetii B Yes Yesb ? ? Tylewska-Wierzbanowska and Kruszewska (1993) Viral encephalitis B Yes Yesc ? ? Mathur et al. (1995) Nipah virus C Yes ? Yes ? Chua et al., (2002) Rabies C Yes ? Yes ? Wacharapluesadee and Hemachudha (2002) Influenza C ? Yesc Yes ? Buchy et al. (2007) a Nasal. b Semen. c Animals. Table 9.4 Occurrence of other agents of interest in bodily fluids Agents Urine Feces Saliva Sewage Reference Severe Acute Respiratory Syndrome ? Yes Yes ? He et al. (2007); Petrich et al. (2006) John Cunningham virus (poliovirus) Yes ? ? Yes Coleman et al. (1980) Human Immuno-deficiency Virus Yes Yes Yes Yes Levy (1989); Yolken et al. (1991) Hepatitis B virus ? ? Yes Yes Alter et al. (1977); Bancroft et al. (1977); Arvanitidou et al. (1998) B Duration of release and concentration in bodily fluids and skin The duration and concentration of pathogens released by a host during the course of an infection varies, with greater numbers being released in more severe infections. After infection, the number of organisms released usually rises rapidly reaching a peak when the symptoms appear in symptomatic infections. This is usually followed with a long decline in the amount of agent released by the host as long as death does not occur. For example, poliovirus appears in the throat and feces 7–10 days before clinical illness (fever) is apparent and may be excreted for more than 30 days after infection (Fig. 9.2 ). Poliovirus will also be detectable in the blood and urine during the course of infection (Pichichero et al., 1998). Hepatitis A virus appears in the stool of infected individuals 2–3 weeks before clinical illness (Belshe, 1991). Parainfluenza, a virus related to influenza, can be detected in nasal secretions in less than 24 h after infection and up to 2 weeks afterward (Belshe, 1991). In the case of SARS, the virus may still be present in the feces for 37 days after infection (Holmes, 2003). Variola major, the virus that causes smallpox, is released for up to 19 days after infection at concentrations of 102–105 per ml of urine (Table 9.5 ) (Sarkar et al., 1973). In many infections, the greatest concentrations are released during the first few days after the initial infection. Brucella abortus is excreted in concentrations as high as 106 per ml of urine for up to 12 weeks (Table 9.6 ). Marburg virus and flaviviruses are excreted in the urine of animals for 10–12 days. In summary, all of the nonenteric agents of interest (Categories A, B, and C) are released in the host for at least days to weeks in concentrations likely to be detectable in sewage systems (Table 9.6) (Sinclair et al., 2008). Figure 9.2 Occurrence of nonpolio enterovirus in bodily fluids and feces during the course of infection (interpreted from Pichichero et al., 1998). Table 9.5 Concentration of select agents in sewage and duration of agent release after infection of individuals. see Sinclair et al., (2008) for the following category A agents: Variola major, Hantavirus, Marburg virus, and Flavivirus Agent Category Fluid Concentrationa Duration Reference C. Botulinum A Feces 108 ? Paton et al. (1983) C. Psittaci B Feces 102–103 28 daysb Takahashi et al. (1988) B. Abortus B Urine 102–106 8–12 weeks Bicknell and Bell (1979); Carmichael and Joubert (1988); Serikawa et al. (1981) Japanese encephalitis B Urine 1–4 3 days Mathur et al. (1995) Enteroviruses B Feces 108–1012 Weeks to Months Maier et al. (2000) Protozoa B Feces 106–107 Weeks to Months Maier et al. (2000) C. Burnetii B Feces 103–104 7 days Tylewska-Wierzbanowska and Kruszewska (1993) Influenza C Nasal 105–107 5 days to Weeks Belshe (1991) a per “milliliter” of volume or “gram” of solid. b Animals. Table 9.6 Titer of smallpox virus in urine (Sarkar et al., 1973) Days after infection Titer(mL) 3 103–105 4 102–105 5 102–104 6 101–104 7 101–103 8 101–102 10 101–102 15 101–102 19 101 20 0 Most of the existing data on the occurrence and concentration of pathogens was gathered using culture of viable or infective organisms. Molecular methods such as the polymerase chain reaction (PCR) or immuno-chemical methods (enzyme-linked immunoassays or ELISA) can detect both infectious and noninfectious organisms. These molecular techniques can detect concentrations from 1 to 10,000 greater than culture methods because some of the organisms may be inactivated (dead) or may not be able to grow on the selected media (bacteria) or cell culture (used for viruses). In the case of enteric viruses, the ratio of viruses detected by infectivity assay may be 100–50,000 times less than that detected by a molecular method (Ward et al., 1984). This is because cell culture methods have a low efficiency in virus quantification from clinical and environmental samples; however, they do provide a robust measure of viral activity not feasible with molecular methods. Agents causing enteric and respiratory infections are released in large numbers in feces and respiratory secretions (Table 9.4). Many of the enteric viruses such as the enteroviruses and adenoviruses may replicate both in the intestinal and respiratory tract. Using molecular methods the number of enteric viruses detected can approach peak concentrations of 1012 organisms per gram of stool while protozoa can approach 106-107 per gram. Cultivatable enteric bacterial pathogens such as Salmonella may also occur in concentrations as large as 1011 per gram (Feachem et al., 1983). By infectivity assays, the concentration of respiratory viruses ranges from 105 to 107 per ml of respiratory secretion. Even blood-borne viruses such as HIV will be found in the feces of infected persons (Ansari et al., 1992) and it appears that many viruses will occur in the urine during infection of the host (Table 9.6), although these excreted viruses may not be infectious. Little information is available on the concentration of pathogenic viruses or bacterial agents of interest in the urine. The total amount of virus released by a person is, of course, also related to the amount of feces, urine, respiratory secretion, and skin that is released by the person. On average, a person excretes between 100 to 400 g of feces and 700–2000 ml of urine per day (Table 9.7 ). Table 9.7 Factors that affect concentration of the biological agent in sewage Site of replication in the host GI, upper respiratory, nose, skin, internal organsDuration of release from the hostConcentration in the sourceIncidence of disease in the populationWater use per capitaSeasonSurvival in the sewer system A person with an enteric viral infection may excrete as many as 1014 viral particles per day and over 1015 during the course of an infection (Table 9.8 ). Nonenteric bacterial agents of interest appeared to be released in concentrations from 10° to 108 by viability assays (Boone and Gerba 2007). Respiratory pathogens end up in the feces from the swallowing of secretions. Table 9.8 Sources of biological agents in sewers (Feachem et al., 1983) Feces (100–400 g/person/day)Urine (700–2000 ml/day)Skin—from bath and hand washingSaliva, respiratory secretionsBloodFoodWash water (kitchen, drains)Storm watera a Some sewer systems are combined with the storm-water collection system. Ecological studies of bovine tuberculosis in badgers introduce the concept of “super-excretors,” which maintain the disease and pass infectious organisms in their stool or urine continuously. Super-excretors are individuals who excrete larger numbers than average of a pathogen during an infection. These super-excretors were almost exclusively animals with a progressive infection, which does not resolve and contributed to a higher mortality (Delahay et al., 2000). The occurrence of a similar “super-spreader” was also noted in a clinical epidemiological report of SARS in humans (Holmes, 2003). III CONCENTRATION OF BIOLOGICAL AGENTS IN SEWAGE The occurrence and concentration of pathogens in sewage is dependent upon a number of factors listed in Table 9.9 . One of the most important considerations is the amount of pathogen released by a person daily from bodily fluid, feces, skin, and urine. Because one infected individual typically produces at least 100 g of feces per day, a pathogen present at 108 per gram will introduce at least 1010 or more of the pathogen into the sewer system. Logically, pathogens excreted in urine and feces will be released several times during a 24-h period. Enteric and respiratory pathogens are almost always detected in sewage because of the long duration of release from the host during infection, the large concentrations released from the host, and the many infections that are asymptomatic. Table 9.9 Comparative occurrence of enteric agents (category B) in feces and sewage Agent(s) Feces (per gram) Stoola Sewage (100 ml) Enteric viruses (infectivity assay) 108 1010 102 Enteric viruses (PCR assay) 1010–1012 1012–1014 104–105 Giardia 106 108 10–102 Cryptosporidium 106 108 0.1-102 a 100 g stool (150 g average in the U.S.). Studies have shown that the types and concentration of enteric microorganisms in sewage is directly related to the incidence of disease in the community (Riordan, 1962, Sellwood et al., 1981). The concentration of enteric pathogens in sewage ranges from 0.1 to 100,000 per ml of sewage (Table 9.8). While many biological agents of interest have been detected in sewage (Table 9.3), the studies are limited and vary by location. IV LABORATORY METHODS AND DETECTION A Detection of pathogens Culture based methods can be used for the detection of pathogens in wastewater, but they may take days to weeks to perform. Alternative molecular methods, such as the PCR, have been successful in detecting bacterial, viral, and protozoan pathogens in sewage without the need for cultivation (Gilbride et al., 2006). These new techniques detect live and dead organisms, have a high sensitivity for wastewater, and can reduce detection time to a few hours (He and Jiang, 2005, Holmes, 2003). Some promising new wastewater methods use nucleic acid microarrays or antibody/receptor technologies to detect multiple pathogens simultaneously (Boehm et al., 2007). Combining these multiplexed methods with fiberoptic sensors and lab-on-a-chip technology can allow utilities to rapidly screen, identify, and quantify multiple pathogens in real time. Because these technologies rely on PCR DNA techniques, the many interfering substances in raw sewage pose a problem. Without proper sample extraction, the sample analytes are exposed to many varying inhibitors, which can negatively impact the DNA isolation and amplification steps. These methods are also limited by their inability to differentiate between viable and nonviable or nonculturable organisms ( Josephson et al., 1993), a vital characteristic when assessing the microbial risk assessment for any given community. Certain methods are in development to automate the sample collection, sample processing, and concentration to separate analytes from inhibitors and deliver a suitable clean sample to a real-time detection microarray technology. These methods use latex beads, carbohydrates, anion exchange resins, or similar substances as part of sample collection and sample processing step (Straub and Chandler, 2003), but no fully automated method has been proposed for wastewater. A biosensor capable of identifying and quantifying a wide group of pathogens is necessary, but future development is needed in the areas of extraction from environmental samples, selection of a suitable target sequence of the pathogen (specificity), detection and differentiation of the signal from interfering sequences (sensitivity), and automation of all processes towards a functional real-time biosensor for wastewater (Gilbride et al., 2006). B Survival of pathogens in sewer systems A principal benefit of wastewater monitoring is that most pathogens of interest are expected to remain viable for at least several days in the sewerage environment (Table 9.10 ). Enteric and respiratory agents are particularly stable, while data is limited for viral encephalitis agents because transmission in water and other liquid media does not occur naturally. Using molecular methods, survival of the pathogens in the viable form is not necessary for their detection, thus increasing the length of time for which the pathogen may be detected. In the case of select agents, knowing the presence of the organism in the sewer system may be all that is needed to trigger further investigation regardless of viability. Table 9.10 The Helsinki poliovirus experiment (Ranta et al., 2001) Helsinki population = 740,000Sewage flow 2 × 108 l/dayContamination Event5 × 1010 TCID50 Poliovirus vaccineFlushed down toilet in one liter volume20 km from sewage treatment plantDetectionAutomatic sampler = 200 ml per 5 × 106 liters of sewage flowFour samples pooled per dayConcentrated from 400 to 1 ml before assayResultVirus was detected for the next 4 days (cell culture)Peak 24–48 h after flushVirus detected after passage of 800 million liters of sewage pass through systemConclusionMonitoring of sewage could detect 1 infected person in 10,000Assumes: 108 infectious virus excreted by child in 4 days C Lessons learned from poliovirus: Monitoring as an early warning system The benefits of pathogen monitoring in sewage have been recognized for poliovirus for more than 40 years. The relationship between the occurrence of poliovirus in sewage and clinical incidence of disease in a community was first noted in the late 1960s (Nelson et al., 1967). These early detection studies were designed as longitudinal epidemiological investigations to assess the success of polio vaccination campaigns (Riordan, 1962). The results of these studies demonstrated that a definite correlation exists among the isolation of enteroviruses in sewage, and the isolation of viruses in stools, and the number of recognized clinical cases within the community. Using cell culture assay techniques (which measure only infective viruses) and only grab samples (i.e., no steps to concentrate the sample) poliovirus could be detected when only 0.27–0.4% of the population was excreting the virus. It was also demonstrated that small outbreaks and epidemics of enterovirus and adenovirus disease within a community can be predicted by monitoring a community’s sewage. Virulent or wildtype (nonvaccine strain) poliovirus type 1 was detected in sewage 9 days before the first clinical case became evident (Kuwert et al., 1970). In an outbreak of Coxsackievirus B5, the virus was detected in the sewage 10 days before clinical cases were positive (Nelson et al., 1967). These studies make it clear that grab samples collected on a regular (weekly or every few days) basis could be used to assess the introduction of a new infectious agent in the community. This approach was later adapted to monitor the success of poliovirus vaccine campaigns internationally (WHO, 2003). To assess the sensitivity of poliovirus monitoring, one study (Ranta et al., 2001) flushed a one-time bolus of 11 containing 2 × 1010 infective poliovirus type 1 vaccine strain down a toilet 20 km (12 miles) from the sewage plant (Table 9.11 ). Samples were automatically collected and assayed for the next 4 days. Infectious poliovirus was still detected after 800 million liters had passed through the system. The authors concluded that their monitoring system could detect one infected person in 10,000 residents of the community, assuming that 108 infective viruses are excreted by a child over a 4-day period of time. The study showed that pathogens appear to be greatly retarded in sewage systems, where a onetime event resulted in a detection period over 4 days. The pathogen was also easily detected in 200-ml samples for every 5 × 1061 of sewage flow. Table 9.11 Survival time of pathogens in the environment (water, feces, urine, sewage) (Belanov et al., 1996, Belshe, 1991, Mitscherlich and Marth, 1984, Sinclair et al., 2008) Organism Days of survival B. anthracis Weeks to years C. botulinum Weeks Y. pestis Days Variola major (smallpox) Weeks to months F. tularensis 12–60 days Marburg virus (surfaces) 4–5 days Enteric pathogens Days to months B. mallei 28–35 days Psittacosis (C. psittaci) Days Q fever (C. burnetti) 30–1000 days Typhus fever (Rickettsia typhi) Hours to days Influenza (surfaces) 3 days Surveillance of poliovirus in sewage has been used by several nations to assess the success of vaccination programs and to identify the potential need for vaccination to prevent outbreaks (Deshpande et al., 2003, Manor et al., 1999, Tambini et al., 1993). The World Health Organization has published guidelines for the environmental surveillance of poliovirus circulation (WHO, 2003). These guidelines assume that a single infected person will excrete 107 polioviruses per day and that one person infected in 100 could be detected using an infectivity assay without concentrating the sewage. However, if the tested sample is concentrated 100 fold then one infected person among 10,000 could be detected. The Public Health Laboratories of Israel have been conducting an environmental surveillance of sewage on a monthly basis since 1989 (Manor et al., 1999) to assess the spread of the wild type poliovirus strains capable of causing paralytic disease. This was done to determine the success and need for vaccination programs. Between 1989 and 1998, four “silent” separate episodes of wild-type poliovirus circulation were detected when no clinical cases were observed. The study described how surveillance of the sewage is much more effective than surveillance of clinical cases. The greater sensitivity of sewage surveillance was also validated in Mumbai, India where wild type poliovirus was detected 3 months before any clinical cases were observed (Deshpande et al., 2003). D Differentiation of vaccine and virulent strains In the poliovirus surveillance of sewage it is necessary to differentiate between vaccine strains and wild type strains of the virus. In the past this has been accomplished by using different cell lines or incubation conditions to limit the growth of the vaccine strains. However, today this can be accomplished by the use of molecular methods and sequence analysis. Sequences amplified directly from processed sewage samples by PCR using primer pairs specific for the indigenous type 1 genotype could be used to assess its occurrence in the presence of vaccine strains (Tambini et al., 1993). Vaccine strains have unique sequences from wild type strains of pathogens allowing easy differentiation. In addition, sequence analysis of sewage isolations has been shown useful in tracking the spread of wild type poliovirus from one country and community to another (Deshpande et al., 2003, Manor et al., 1999). This review of poliovirus is offered here as a case study and justification for the use of monitoring additional CDC select biological agents. With current molecular techniques and updated concentration methods, a much greater sensitivity and specificity can be achieved for poliovirus and many other CDC select agents. V CONCLUSIONS: THE PROBABILITY OF DETECTION Studies with poliovirus demonstrated the feasibility of how monitoring sewage for virulent pathogens can be used to assess the success of vaccine programs. This review identified three important benefits of developing a wastewater monitoring system. Sewage surveillance system has been shown to be more sensitive than reporting of clinical cases of serious illness in a community. It was also demonstrated that pathogens can be greatly retarded in a sewage systems allowing a detection time over many days for a one-time release into a sewage system. Finally, it was shown that infectivity assays have the ability to detect one infected person in 10,000 individuals. Sewage surveillance can detect the presence or increased amount of infections from enteric pathogens excreted in the feces or urine during infection. However, the success of such a surveillance system for nonenteric pathogens has not been demonstrated, although they have been found in sewage. The sensitivity of a sewage surveillance system will depend on several important factors including the amount and duration of the agent released into the sewers, the frequency of monitoring, and the sensitivity of the monitoring method. Nonenteric pathogens are released from the host for a minimum of several days. This has already been demonstrated for HIV, hepatitis B, and Y. pestis (see Tables 9.2 and IV). Given this fact and the expected several day retardation in sewer systems, all or most of the nonenteric category agents will be present in the sewer system if there is an infection in the population served by the sewer system. Based upon the conclusions of the Helsinki experiment, which measured infectious poliovirus (Table 9.11), one individual excreting 108 infectious virus per gram of feces for a period of 4 days could be identified in a population of 10,000. If we consider the concentration and amount of infectious agent in the fluid or feces released during infection, this same sensitivity should be achieved with the agents of smallpox, Brucella, botulism and perhaps influenza. Based on existing information in Table 9.5 at least one person in 100 could be detected for most of the agents for which information is available. Because many of the agents take several days to detect by conventional culture methods the preferred detection system would be by a rapid, but highly specific method such as the quantitative real time PCR or other similar molecular detection system. Because PCR can detect both culturable and nonculturable organisms, it can be expected to be more sensitive than methods that have been used in the past for sewage surveillance. Use of PCR should increase sensitivity by as much as 50,000 over cultivation methods (Ward et al., 1984). Also, when using PCR to detect viruses in sewage, a 10-fold loss in sensitivity is likely with current methods. This loss is due to interfering substances present in the sewage, but still leaves a method that may be 5000 times more sensitive than conventional culture methods. Increasing the volume of wastewater that is tested may also increase the sensitivity of current methods. Technology is available (Hurst and Crawford, 2002) which allows for the concentration of bacteria and viruses from up to 10 l of raw sewage. Thus, increasing the volume analyzed from 400 to 4000 ml could increase the sensitivity of detection another 10-fold. Surveillance of pathogens in wastewater has several advantages over aerosol and other monitoring methods. Longer survival times in soil, water, and wastewater (Sinclair et al., 2008) facilitate a retardation of pathogens in sewage which allows a longer sampling window than aerosols where organisms are much more susceptible to factors such as settling, desiccation, and relative humidity. Additionally, wastewater is collected in a central location, allowing monitoring to be defined or subdivided to specific areas. Lastly, wastewater systems can include many pathogens originating in aerosol, surface water, tap water, or fomites as storm-water and watersheds will often flow into sewerage systems. Of course background levels and alert levels of the agents of interest would have to be established. Most agents of interest are likely to occur at one time or another in wastewater or at least have some normal range of background. Further research would be needed to determine what these levels might be and normal variation of concentrations of the agents in wastewater. The types and concentration would be expected to vary by location and the size of the population, area served and type of connections (e.g., the presence of a slaughterhouse may increase the likelihood of finding animal pathogens). In summary, given the potential enhanced sensitivity of molecular methods and current abilities to test larger volumes of all of the CDC select agents of interest (enteric and nonenteric), it is possible to detect if an infected individual enters a monitored population. Although the concentration and duration of release of all of the agents of interest are not known, it is still possible to detect at least one infection in populations of 1000 or more. This monitoring is especially useful when combined with other components of the QMRA framework such as modeling of sewage dispersion, back calculation of contaminant point of introduction, and calculations of the health risk. ACKNOWLEDGMENTS This study was supported by the United States Defense Advanced Research Projects Agency, the Center for Advancing Microbial Risk Assessment funded by the United States Environmental Protection Agency Science to Achieve Results, and the United States Department of Homeland Security University Programs grant number R3236201. Ryan Sinclair was supported through the National Research Council’s Research Associate Program with funding from the United Sates Department of Homeland Security.

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          Public Health Assessment of Potential Biological Terrorism Agents

          As part of a Congressional initiative begun in 1999 to upgrade national public health capabilities for response to acts of biological terrorism, the Centers for Disease Control and Prevention (CDC) was designated the lead agency for overall public health planning. A Bioterrorism Preparedness and Response Office has been formed to help target several areas for initial preparedness activities, including planning, improved surveillance and epidemiologic capabilities, rapid laboratory diagnostics, enhanced communications, and medical therapeutics stockpiling (1). To focus these preparedness efforts, however, the biological agents towards which the efforts should be targeted had to first be formally identified and placed in priority order. Many biological agents can cause illness in humans, but not all are capable of affecting public health and medical infrastructures on a large scale. The military has formally assessed multiple agents for their strategic usefulness on the battlefield (2). In addition, the Working Group on Civilian Biodefense, using an expert panel consensus-based process, has identified several biological agents as potential high-impact agents against civilian populations ( 3 – 7 ). To guide national public health bioterrorism preparedness and response efforts, a method was sought for assessing potential biological threat agents that would provide a reviewable, reproducible means for standardized evaluations of these threats. In June 1999, a meeting of national experts was convened to 1) review potential general criteria for selecting the biological agents that pose the greatest threats to civilians and 2) review lists of previously identified biological threat agents and apply these criteria to identify which should be evaluated further and prioritized for public health preparedness efforts. This report outlines the overall selection and prioritization process used to determine the biological agents for public health preparedness activities. Identifying these priority agents will help facilitate coordinated planning efforts among federal agencies, state and local emergency response and public health agencies, and the medical community. Overview of Agent Selection and Prioritization Process On June 3-4, 1999, academic infectious disease experts, national public health experts, Department of Health and Human Services agency representatives, civilian and military intelligence experts, and law enforcement officials (see footnote) met to review and comment on the threat potential of various agents to civilian populations. The following general areas were used as criteria: 1) public health impact based on illness and death; 2) delivery potential to large populations based on stability of the agent, ability to mass produce and distribute a virulent agent, and potential for person-to-person transmission of the agent; 3) public perception as related to public fear and potential civil disruption; and 4) special public health preparedness needs based on stockpile requirements, enhanced surveillance, or diagnostic needs. Participants reviewed lists of biological warfare or potential biological threat agents and selected those they felt posed the greatest threat to civilian populations. The following unclassified documents containing potential biological threat agents were reviewed: 1) the Select Agent Rule list, 2) the Australian Group List for Biological Agents for Export Control, 3) the unclassified military list of biological warfare agents, 4) the Biological Weapons Convention list, and 5) the World Health Organization Biological Weapons list ( 8 – 12 ). Participants with appropriate clearance levels reviewed intelligence information regarding classified suspected biological agent threats to civilian populations. Genetically engineered or recombinant biological agents were considered but not included for final prioritization because of the inability to predict the nature of these agents and thus identify specific preparedness activities for public health and medical response to them. In addition, no information was available about the likelihood for use of one biological agent over another. This aspect, therefore, could not be considered in the final evaluation of the potential biological threat agents. Participants discussed and identified agents they felt had the potential for high impact based on subjective assessments in the four general categories. After the meeting, CDC personnel then attempted to identify objective indicators in each category that could be used to further define and prioritize the identified high-impact agents and provide a framework for an objective risk-matrix analysis process for any potential agent. The agents were evaluated in each of the general areas according to the objective parameters and were characterized by the rating schemes outlined in the Appendix. Final category assignments (A, B, or C) of agents for public health preparedness efforts were then based on an overall evaluation of the ratings the agents received in each of the four areas. Results Based on the overall criteria and weighting, agents were placed in one of three priority categories for initial public health preparedness efforts: A, B, or C (Table 1). Agents in Category A have the greatest potential for adverse public health impact with mass casualties, and most require broad-based public health preparedness efforts (e.g., improved surveillance and laboratory diagnosis and stockpiling of specific medications). Category A agents also have a moderate to high potential for large-scale dissemination or a heightened general public awareness that could cause mass public fear and civil disruption. Table 1 Critical biological agent categories for public health preparedness Biological agent(s) Disease Category A Variola major Smallpox Bacillus anthracis Anthrax Yersinia pestis Plague Clostridium botulinum (botulinum toxins) Botulism Francisella tularensis Tularemia Filoviruses and Arenaviruses (e.g., Ebola virus, Lassa virus) Viral hemorrhagic fevers Category B Coxiella burnetii Q fever Brucella spp. Brucellosis Burkholderia mallei Glanders Burkholderia pseudomallei Melioidosis Alphaviruses (VEE, EEE, WEEa) Encephalitis Rickettsia prowazekii Typhus fever Toxins (e.g., Ricin, Staphylococcal enterotoxin B) Toxic syndromes Chlamydia psittaci Psittacosis Food safety threats (e.g., Salmonella spp., Escherichia coli O157:H7) Water safety threats (e.g., Vibrio cholerae, Cryptosporidium parvum) Category C Emerging threat agents (e.g., Nipah virus, hantavirus) aVenezuelan equine (VEE), eastern equine (EEE), and western equine encephalomyelitis (WEE) viruses Most Category B agents also have some potential for large-scale dissemination with resultant illness, but generally cause less illness and death and therefore would be expected to have lower medical and public health impact. These agents also have lower general public awareness than Category A agents and require fewer special public health preparedness efforts. Agents in this category require some improvement in public health and medical awareness, surveillance, or laboratory diagnostic capabilities, but presented limited additional requirements for stockpiled therapeutics beyond those identified for Category A agents. Biological agents that have undergone some development for widespread dissemination but do not otherwise meet the criteria for Category A, as well as several biological agents of concern for food and water safety, are included in this category. Biological agents that are currently not believed to present a high bioterrorism risk to public health but which could emerge as future threats (as scientific understanding of these agents improves) were placed in Category C. These agents will be addressed nonspecifically through overall bioterrorism preparedness efforts to improve the detection of unexplained illnesses and ongoing public health infrastructure development for detecting and addressing emerging infectious diseases (13). Agents were categorized based on the overall evaluation of the different areas considered. Table 2 shows the evaluation schemes as applied to agents in Categories A and B. For example, smallpox would rank higher than brucellosis in the public health impact criterion because of its higher untreated mortality (approximately 30% for smallpox and ≤2% for brucellosis); smallpox has a higher dissemination potential because of its capability for person-to-person transmission. Smallpox also ranks higher for special public health preparedness needs, as additional vaccine must be manufactured and enhanced surveillance, educational, and diagnostic efforts must be undertaken. Inhalational anthrax and plague also have higher public health impact ratings than brucellosis because of their higher morbidity and mortality. Although mass production of Vibrio cholera (the biological cause of cholera) and Shigella spp. (the cause of shigellosis) would be easier than the mass production of anthrax spores, the public health impact of widespread dissemination would be less because of the lower morbidity and mortality associated with these agents. Although the infectious doses of these bacteria are generally low, the total amount of bacteria that would be required and current water purification and food-processing methods would limit the effectiveness of intentional large-scale water or food contamination with these agents. Table 2 Criteria and weightinga used to evaluate potential biological threat agents Disease Public health impact Dissemination
potential Public
perception Special preparation Category Disease Death P-Db P - Pc Smallpox + ++ + +++ +++ +++ A Anthrax ++ +++ +++ 0 +++ +++ A Plagued ++ +++ ++ ++ ++ +++ A Botulism ++ +++ ++ 0 ++ +++ A Tularemia ++ ++ ++ 0 + +++ A VHFe ++ +++ + + +++ ++ A VEf ++ + + 0 ++ ++ B Q Fever + + ++ 0 + ++ B Brucellosis + + ++ 0 + ++ B Glanders ++ +++ ++ 0 0 ++ B Melioidosis + + ++ 0 0 ++ B Psittacosis + + ++ 0 0 + B Ricin toxin ++ ++ ++ 0 0 ++ B Typhus + + ++ 0 0 + B Cholerag + + ++ +/- +++ + B Shigellosisg + + ++ + + + B aAgents were ranked from highest threat (+++) to lowest (0).
bPotential for production and dissemination in quantities that would affect a large population, based on availability, BSL requirements, most effective route of infection, and environmental stability.
cPerson-to-person transmissibility.
dPneumonic plague.
eViral hemorrhagic fevers due to Filoviruses (Ebola, Marburg) or Arenaviruses (e.g., Lassa, Machupo).
fViral encephalitis.
gExamples of food- and waterborne diseases. Discussion Although use of conventional weapons such as explosives or firearms is still considered the most likely means by which terrorists could harm civilians (14), multiple recent reports cite an increasing risk and probability for the use of biological or chemical weapons ( 15 – 18 ). Indeed, the use of biological and chemical agents as small- and large-scale weapons has been actively explored by many nations and terrorist groups ( 19 , 20 ). Although small-scale bioterrorism events may actually be more likely in light of the lesser degrees of complexity to be overcome, public health agencies must prepare for the still-possible large-scale incident that would undoubtedly lead to catastrophic public health consequences. The selection and prioritization of the potential biological terrorism agents described in this report were not based on the likelihood of their use, but on the probability that their use would result in an overwhelming adverse impact on public health. Most evaluations of potential risk agents for biological warfare or terrorism have historically been based on military concerns and criteria for troop protection. However, several characteristics of civilian populations differ from those of military populations, including a wider range of age groups and health conditions, so that lists of military biological threats cannot simply be adopted for civilian use. These differences and others may greatly increase the consequences of a biological attack on a civilian population. Civilians may also be more vulnerable to food- or waterborne terrorism, as was seen in the intentional Salmonella contamination of salad bars in The Dalles, Oregon, in 1984 (21). Although food and water systems in the United States are among the safest in the world, the occurrence of nationwide outbreaks due to unintentional food or water contamination demonstrates the ongoing need for vigilance in protecting food and water supplies ( 22 , 23 ). Overall, many other factors must be considered in defining and focusing multiagency efforts to protect civilian populations against bioterrorism. Category A agents are being given the highest priority for preparedness. For Category B, public health preparedness efforts will focus on identified deficiencies, such as improving awareness and enhancing surveillance or laboratory diagnostic capabilities. Category C agents will be further assessed for their potential to threaten large populations as additional information becomes available on the epidemiology and pathogenicity of these agents. In addition, special epidemiologic and laboratory surge capacity will be maintained to assist in the investigation of naturally occurring outbreaks due to Category C “emerging” agents. Linkages established with established programs for food safety, emerging infections diseases, and unexplained illnesses will augment the overall bioterrorism preparedness efforts for many Category B and C agents. The above categories of agents should not be considered definitive. The prioritization of biological agents for preparedness efforts should continue. Agents in each category may change as new information is obtained or new assessment methods are established. Disease elimination and eradication efforts may result in new agents being added to the list as populations lose their natural or vaccine-induced immunity to these agents. Conversely, the priority status of certain agents may be reduced as the identified public health and medical deficiencies related to these agents are addressed (e.g., once adequate stores of smallpox vaccine and improved diagnostic capabilities are established, its rating within the special preparedness needs category would be reduced, as would its overall rating within the risk-matrix evaluation process). To meet the ever-changing response and preparedness challenges presented by bioterrorism, a standardized and reproducible evaluation process similar to the one outlined above will continue to be used to evaluate and prioritize currently identified biological critical agents, as well as new agents that may emerge as threats to civilian populations or national security. Supplementary Material Appendix Risk-Matrix Analysis Process Used to Evaluate Potential Biological Threat Agents
            • Record: found
            • Abstract: found
            • Article: not found

            Isolation of Nipah virus from Malaysian Island flying-foxes.

            In late 1998, Nipah virus emerged in peninsular Malaysia and caused fatal disease in domestic pigs and humans and substantial economic loss to the local pig industry. Surveillance of wildlife species during the outbreak showed neutralizing antibodies to Nipah virus mainly in Island flying-foxes (Pteropus hypomelanus) and Malayan flying-foxes (Pteropus vampyrus) but no virus reactive with anti-Nipah virus antibodies was isolated. We adopted a novel approach of collecting urine from these Island flying-foxes and swabs of their partially eaten fruits. Three viral isolates (two from urine and one from a partially eaten fruit swab) that caused Nipah virus-like syncytial cytopathic effect in Vero cells and stained strongly with Nipah- and Hendra-specific antibodies were isolated. Molecular sequencing and analysis of the 11,200-nucleotide fragment representing the beginning of the nucleocapsid gene to the end of the glycoprotein gene of one isolate confirmed the isolate to be Nipah virus with a sequence deviation of five to six nucleotides from Nipah virus isolated from humans. The isolation of Nipah virus from the Island flying-fox corroborates the serological evidence that it is one of the natural hosts of the virus.
              • Record: found
              • Abstract: found
              • Article: not found

              The severe acute respiratory syndrome coronavirus in tears.

              Severe acute respiratory syndrome (SARS) is a new infectious disease that caused a global outbreak in 2003. Research has shown that it is caused by a novel coronavirus. A series of cases is reported where polymerase chain reaction (PCR) testing on tears had demonstrated the presence of the virus. Detection of ocular infection from tears using the PCR technique has been widely used by ophthalmologists to diagnose infections for other viruses. This is a case series report from cases classified as probable or suspect SARS cases. Tear samples were collected from 36 consecutive patients who were suspected of having SARS in Singapore over a period of 12 days (7-18 April 2003), and analysed by PCR using protocols developed by the WHO network of laboratories. Three patients with probable SARS (one female and two male patients) had positive results from their tear samples. Tear samples were used to confirm SARS in the female patient, who was positive only from her tears. The positive specimens were found in cases sampled early in their course of infection. This is the first case series reported with the detection of the SARS coronavirus from tears, and has important implications for the practice of ophthalmology and medicine. The ability to detect and isolate the virus in the early phase of the disease may be an important diagnostic tool for future patients and tear sampling is both simple and easily repeatable. Many healthcare workers are in close proximity to the eyes of patients and this may be a source of spread among healthcare workers and inoculating patients. Ophthalmic practices may need to change as more stringent barrier methods, appropriate quarantine, and isolation measures are vital when managing patients with SARS.

                Author and article information

                Adv Appl Microbiol
                Adv. Appl. Microbiol
                Advances in Applied Microbiology
                Elsevier Inc.
                20 November 2008
                20 November 2008
                : 65
                : 249-269
                [* ]Department of Soil, Water and Environmental Science, University of Arizona, Tucson, Arizona 85721
                []Department of Agricultural and Biosystems Engineering, University of Arizona, Tucson, Arizona 85721
                Author notes
                [1 ]Corresponding author: University of Arizona, 2601 E. Airport Drive, Tucson, AZ 85706
                Copyright © 2008 Elsevier Inc. All rights reserved.

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