1 The geometric mean of seven samples must be comprised in these limits.

While class A biosolids are virtually pathogen free and do not pose a risk of infectious disease transmission through casual contact or ingestion, in class B biosolids pathogens are reduced to levels that are unlikely to pose a threat to public health and the environment under specified use conditions. Consequently, site restrictions are imposed to minimize the potential of human and animal contact with class B biosolids following their application to land. Furthermore, class B biosolids cannot be sold or given away in bags or other containers.

The degree of attractiveness of sewage sludge to vectors is the third parameter of biosolids quality. Vectors are animal and insects that can transmit pathogens to humans, animals or plants. Reducing the attractiveness of biosolids to vectors reduces the potential for spreading diseases from pathogens present in biosolids. The Part 503 rule contains 12 options to either reduce the attractiveness of biosolids to vectors (options 1 to 8 and option 12) or prevent vectors from coming in contact with the biosolids (options 9 to 11)(Table 1.3). One of these options must be met if biosolids are to be applied to land /34/.

Table 1.3.
The 12 options to reduce vector attraction in biosolids to be land applied /34/.

2 Pathogenic viruses in human faeces

2.1 Introduction

Over 130 different types of pathogenic viruses are shed in human faeces (Table 2.1). Based on their pathogenesis, such viruses can be classified as enteropathogenic viruses, for which the gastrointestinal system is the principal site of infection (e.g. astroviruses, caliciviruses and rotaviruses) and non-enteropathogenic viruses, which can occur in the intestinal tract but not in association with gastroenteritis (e.g. most adenoviruses, enteroviruses and hepatitis A/E viruses). Other viruses (e.g. enteric coronaviruses, certain "small round viruses" and "parvovirus-like" agents) have been detected in faeces of patients affected by gastroenteritis, but their pathogenicity has not been proved yet /36/.

Contamination with urine or blood may lead to the occurrence in faeces of other important human pathogenic viruses, like the human immunodeficiency virus (HIV) and the hepatitis B virus (HBV). However, such viruses are not of particular relevance in relation to composting of human faeces, due to their sporadic occurrence in human faeces, their poor survival in the environment, and their route of transmission (i.e. parenteral). These factors minimize the risks of human exposure to these viruses via the production and usage of composted human faeces.

This chapter describes the most important viral pathogens occurring in human faeces (section 2.2). Particular emphasis was given to review aspects relevant to composting of human faeces, like their occurrence in human faeces (section 2.3), resistance to physical-chemical factors (section 2.4) and infective dose for humans (section 2.5).

Table 2.1.
Main pathogenic viruses occurring in human faeces. Modified from Hurst /37/.

2.2 Major pathogenic viruses occurring in human faeces

This section reviews the basic morphological, biological, clinical and epidemiological traits of the principal viral pathogens occurring in human faeces. Data were obtained from relevant books /23,38), review articles in scientific journals /37/, websites /39,40/ and WHO documents /41,42/.

2.2.1 Adenovirus

Adenoviruses (family Adenoviridae) are non-enveloped double-stranded DNA viruses with an icosahedral shape, a diameter between 80 and 110 nm, and fiber appendages protruding from the vertices of the icosahedral viral capsid. These viruses are unusually stable to physical and chemical agents, and adverse pH conditions. They tolerate a pH range of 5.0-9.5 and temperatures between 4 and 36EC. Heating above 56^oC disrupts the virus capsid, causing inactivation. These resistance properties confer to adenoviruses the ability to survive for long periods outside of the host cells. Furthermore, these viruses are among the most persistent in sewage treatment systems.

Adenoviruses are divided into subgroups A-F, with members of the A-E subgroups causing respiratory infections and members of the F sub-group causing enteric infections. Infections are common and affect primarily children. Symptoms of infections and epidemiological patterns vary between sub-groups and even for different species. Although some adenoviruses can cause gastroenteritis in young children (i.e. serotypes 40 and 41) or genital infections (i.e. serotypes 19 and 37), these organisms are most frequently observed to cause respiratory and eye infections. The duration of the illness is generally 7-8 days. The predominant symptoms include fever, throat pain, headache, abdominal pain and conjunctivitis. Asymptomatic infections occur with long-term virus shedding from the respiratory or enteric tracts.

Adenoviruses are endemic worldwide. In temperate regions, they show a seasonal incidence, with highest incidences in the fall, winter and early spring. Transmission generally occurs by the respiratory route (inhalation of aerosols) and sometimes by the faecal-oral route. Transmission by recreational water (e.g. swimming-pools or other recreational waters) has been documented. An infected person can excrete the virus from the respiratory tract. However, the virus can disappear from respiratory secretions after a short time and can be found in faecal specimens, sometimes for extended periods.

2.2.2 Astrovirus

Members of the family Astroviridae are small (26-32 nm of diameter), non-enveloped RNA viruses. The name of this group of viruses derives from their star-like appearance observed by transmission electron microscopy after negative staining. These viruses are resistant to pH 3 and can survive at 60°C for 5 min.

Astroviruses are primarily associated with mild gastroenteritis in infants and young children, although elderly, hospital patients and immunocompromised individuals can also be affected. These viruses display many of the epidemiological and clinical features of rotaviruses, but are not as common and not as virulent. The illness has a normal duration of 2-3 days. Viral shedding may begin a day before symptoms are seen and continue for several days after the diarrhoea has stopped.

Astrovirus infection occurs worldwide and accounts for 2-8% of cases of diarrhoea in infants, second only to rotavirus as a cause of childhood diarrhoea. Like rotaviruses, astrovirus infections occur throughout the year with peaks in the winter months. Person to person spread by the faecal-oral route is the main route of transmission. Outbreaks tend to occur where children are in close contact, as in day-care centres, kindergartens and paediatric wards.

2.2.3 Enteroviruses

Enteroviruses form a genus within the family Picornaviridae, which includes important animal (e.g. foot and mouth disease) and human (e.g. hepatitis A) pathogenic viruses. The genus Enterovirus was traditionally divided into three groups (polioviruses, coxsackieviruses and echoviruses). Enteroviruses are small (22-30 nm), non-enveloped, RNA viruses that are highly resistant to the conditions prevailing in the gut, like acid pH, proteolytic enzymes and bile salts. They are stable at acid pH (3-5) for 1-3 hours.

The clinical syndromes caused by enteroviruses include neurological disease, cardiac and muscular disease, rash, respiratory disease, ocular disease and neonatal disease. For all members of the group, sub-clinical infection is far more common than clinically manifest disease. Certain serotypes are more frequently associated with epidemics involving a specific syndrome. However, the same serotypes may cause different clinical manifestations or produce no symptoms.

The most famous enteroviruses are the Polioviruses, of which there are 3 distinct types. These viruses cause poliomyelitis, an acute infection of the central nervous system, which can result in flaccid paralysis. The disease can have several forms: abortive poliomyelitis, a minor illness with fever, malaise, drowsiness, nausea, vomiting, constipation and sore throat, non-paralytic poliomyelitis or aseptic meningitis with the additional symptoms of a stiff neck and back, and paralytic poliomyelitis marked by flaccid paralysis. Non-paralytic illness is short-lived and patients recover without permanent damage. Paralytic disease occurs in about 1% of infections and symptoms may persist for months, with residual paralysis lasting years. Mortality among the paralytic cases varies between 4 and 10% depending on the virulence of the virus, the degree of medical assistance and the age of the patient. Recrudescence of paralysis and muscle wasting sometimes appears decades later in some persons who had paralytic poliomyelitis.

Vaccines against polioviruses have been available since the 1950s and efforts are now in progress to completely eradicate poliomyelitis and their causative viruses from the human population by the year 2005. Such global eradication seems achievable because regional eradication has already been achieved in the Western Hemisphere and Western Europe. However, there are no vaccines against the other enteroviruses.

Children are the prime targets of enteroviruses and serve as a vehicle for their spread. It has been calculated that more than 90% of children living under poor sanitary and socio-economic conditions experience infections with a number of the locally prevalent enteroviruses before they reach the age of 5 years. When infection is delayed to older childhood and adult young life, the incidence of paralytic poliomyelitis rises, together with the frequency of the most severe manifestations associated with other enteroviruses.

Almost all enteroviruses can be recovered from the oropharynx and intestine of individuals infected either clinically or sub-clinically. They are generally shed for a month or more in stool of infected individuals. Faecal contamination is the usual source of infections. However, droplets or aerosols from coughing or sneezing also can be a source of direct or indirect contamination for some enteroviruses.

Enteroviruses are found in all parts of the world. Climate is an important factor influencing the circulation and prevalence of these viruses. In tropical and semitropical regions, they are widely distributed throughout the year. In temperate climates, they are rarely present in the winter and are encountered far more commonly during summer and autumn.

Because enteroviruses are shed in faeces and respiratory secretions, and are relatively stable in sewage and water, it is assumed that they are transmitted by faecally contaminated water. Transmission by faecally contaminated water is likely to be one of the main routes for transmission under conditions of poor sanitation and crowding. However, the epidemiological evidence for waterborne transmission is weak despite years of surveillance for these viruses in populations.

2.2.4 Hepatitis A virus

The virus causing hepatitis A (HAV) is a small (27 nm), non-enveloped RNA virus with an icosahedral shape. HAV belongs to the same family of enteroviruses (Picornaviridae) and has similar morphological and biological characteristics to these viruses. HAV was previously classified as enterovirus 72, but it is genetically distinct from enteroviruses and is now in a separate genus called Hepatovirus. HAV is extremely resistant to degradation by environmental conditions, as demonstrated by its occurrence in freshwater, seawater, wastewater, soil, marine sediment and oysters. HAV has been found to be more resistant than some other enteric viruses to biosolids and wastewater treatment processes and to persist for as long as a 6 month in sewage-contaminated groundwater. The virus is highly resistant to heat (70° C for 10 min) and acid treatment (pH 1 for 2 h).

Hepatitis A is an acute self-limited disease accounting for approximately 1.4 millions cases in the world per year. The actual burden of disease is probably much higher due to inadequate recognition and reporting. The predominant symptoms are anorexia, jaundice, nausea and vomiting. The symptoms are highly age dependent, with adults and children over 5 years being markedly more susceptible to jaundice compared with children less than 5 years. Duration and seriousness of the disease varies from 1-2 weeks of mild illness to 6-9 months of severely disabling. Mortality rates for hepatitis A are generally less than 1% and death occurs primarily in older people. HAV can be shed before the onset of symptoms and the shedding can continue up to 3 months after resolution of the symptoms.

HAV infections account for 20-25% of clinically apparent hepatitis cases worldwide. The virus is transmitted by the faecal-oral route, either directly by person-to-person or indirectly by ingestion of contaminated food (e.g. shellfish) and water. HAV can occur both sporadically and epidemically. Epidemics are uncommon in developing countries, where children are infected early in life and adults are generally immune. In developed countries Hepatitis A is still common and often occurs as common source outbreaks due to faecally contaminated food and water. The largest documented outbreak of Hepatitis A resulted in 300,000 cases of illness in Shanghai, China in 1988 and was caused by consumption of faecally contaminated clams.

Although not widely used, an inactivated vaccine against HAV has been available since 1995. Other prevention and control measures based on sanitation and hygiene continue to be the main barriers to transmission.

2.2.5 Hepatitis E virus

The agent causing hepatitis E (HEV) is a non-enveloped RNA virus of 32-34 nm. The virus is classified was previously within the family Caliciviridae, but because of genetic and replication differences, it is now unclassified and is likely to be placed in a unique virus family. Compared to HAV, HEV is less stable in harsh environmental conditions like high salt concentration or repeated freeze-thawing. The virus is more susceptible to heat than is HAV.

Hepatitis E has so far been observed almost exclusively in developing countries. Different strains of HEV occur in different parts of the world, with at least 4 main ones: (1) South-East, North and central Asian, Mexico, United States, and Taiwan. Individuals between 15 and 40 years of age are the most frequently affected. The disease closely resembles that described for hepatitis A, although bilirubin levels tend to be higher, and jaundice deeper and more prolonged. The mortality rate is 0.5-3%, but it can be extremely high for pregnant women (10-20%). HEV has been detected in stools 14 days after the onset of jaundice and persists for about 2 weeks. The infection is usually sub-clinical in children. As for hepatitis A, the disease does not progress to chronic hepatitis.

Outbreaks and sporadic cases of HEV have occurred over a large geographic area, most notably in regions with poor sanitation. Outbreaks of hepatitis E are more common in regions with hot climates and are rare in temperate climates. Most HEV outbreaks are due to faecally contaminated drinking water, but food-borne epidemics (raw or uncooked shellfish) have also been reported. Person-to-person transmission appears to be uncommon, perhaps because of the relatively low virus levels in faeces of infected persons.

Epidemic Hepatitis E was first identified in India, and it also occurs in the Middle and Far East, in northern and western Africa, the central Asian Republics of the former Soviet Union, in China and Hong Kong. Both epidemic and sporadic cases of HEV have been reported from southeast and central Asia, the Middle East, northern and western Africa and North America (Mexico). Sporadic cases of Hepatitis E occurring in non-endemic regions have been associated with travel to endemic regions. Recent evidence for the existence of HEV strains in animals (swine, rates, cattle, chickens, etc.) that resemble human HEV strains also raises the possibility of zoonotic transmission as the source of sporadic human cases in non-endemic areas. Experimentally, swine HEV infects primates and human HEV infects swine.

2.2.6 Norwalk virus and other human caliciviruses

The Norwalk virus is the prototype of a group of so-called "small round structured viruses" which are now classified as members of the family Caliciviridae on the basis of their nucleic sequence. Members of the family are non-enveloped contain single-stranded RNA surrounded by a capsid with cup-shaped surface structures. These viruses are generally associated with gastroenteritis in humans.

The human caliciviruses are genetically diverse. They are divided into three major groups, the Norwalk-Like Viruses (NLVs), which are subdivided into two major subgroups, GI and GII, and the Sapporo Viruses. There is genetic diversity within these groups and several different subgroups have been identified. Caliciviruses are endemic in human populations worldwide and there are distinct genetic subgroups that predominate regionally and over time. Norwalk Virus belongs to the GI group of human caliciviruses and these are no longer prevalent in Europe and North America. The GII NLVs are the predominant epidemic caliciviruses in these regions.

The Norwalk viruses are relatively stable. They can survive at pH 2.7 for 3 h, heat at 60°C for 30 minutes and drying on surfaces. The persistence Norwalk Viruses in water, wastewater and soil is similar to that of some other enteric viruses, such as poliovirus and MS2 (a male-specific RNA bacteriophage of E. coli or colipage). Information on the survival of human caliciviruses in faeces, sewage and other media are limited because these viruses infect only humans and no laboratory hosts, such as cell cultures or experimental animals are available.

The Norwalk group of viruses tends to infect older children and adults. The illness consists of an explosive episode of nausea, vomiting, diarrhoea and abdominal cramps, some times accompanied by headache, sore muscles and low-grade fever. Symptoms are usually last 12-48 hours for Norwalk virus and for 1-3 days for other human caliciviruses. The shedding of viruses declines after the onset of illness but can persist at declining levels for 1 to 2 weeks.

The Sapporo-like caliciviruses cause illness primarily in children and are endemic worldwide. Most children are infected with at least one calicivirus before leaving primary school. Outbreaks occur mainly in nursery schools and kindergartens, but also in day-care centres, orphanages, maternity hospitals and schools.

The Norwalk-like virus group is one of the major causes of gastroenteritis in adults worldwide. Approximately 40% of outbreaks of gastroenteritis in adults the USA have been attributed to this virus. Common-source outbreaks frequently occur via faecal contamination of water or food (e.g. shellfish and salads). Outbreaks frequently occur in camps, schools, nursing-homes and cruise ships. Because some caliciviruses of cattle and swine are genetically closely related to human caliciviruses, there is now suspicion that zooonotic transmission is possible and deserves consideration in elucidating the natural history of these viruses.

2.2.7 Rotavirus

Rotaviruses are members of the family Reoviridae. The family also includes reoviruses, which are commonly found in human stool, but are not associated with gastroenteritis or other illnesses. Rotaviruses are 60-80 nm in diameter, non-enveloped, contain 11 segments of double-stranded RNA viruses surrounded by a protein core and two capsid layers. This three-layered structure of virus proteins causes particles to have the typical appearance of a wheel with spikes. Rotavirus survives at 60° C for 30 minutes. The virus is stable at acid pH (3.0 - 3.5) and can survive for months outside of the host at temperatures between 4 and 20°C.

Rotaviruses are the major cause of infantile acute diarrhoea in children (95% of children worldwide are infected by age 3 to 5). The disease is a major cause for childhood mortality in Africa, Asia and South America. The infection occurs usually in children between the ages of 6 months and 24 months, with the peak around 12 months. Vomiting generally precedes diarrhoea, which lasts for 4-5 days and can lead to severe dehydration. Asymptomatic infection is the rule in newborns and is quite common in older children and adults, although outbreaks in adult populations have been reported. The virus is shed in stool for as long as 10 days after the onset of symptoms. The levels of rotavirus shedding can be as high as 10¹¹-10¹² virus particles per gram of stool.

Rotaviruses are major causes of disease worldwide. In temperate regions, infections are most frequent during the winter and early spring months, with high incidence in day-care settings. Rotaviruses are mainly transmitted by the faecal-oral route, although some authors have reported their presence in respiratory tract secretions and other body fluids. Because of their stability in the environment, transmission can occur through ingestion of contaminated water or food or contact with contaminated surfaces.

A rotavirus vaccine was released in the late 1990s, but serious complications of intestinal blockage were reported in enough immunized children that the vaccine was withdrawn from the market. Currently, rotavirus infection and illness must be prevented by adequate sanitation and hygiene and controlled in ill persons by adequate rehydration, supportive care and prevention of spread to other susceptible persons.

2.3 Occurrence in human faeces

Our literature search provided no data on the occurrence of pathogenic viruses in human faeces collected for composting. Only few data exist on the occurrence of pathogenic viruses in pit latrines. A study conducted in poor areas of Texas in 1953 reported the occurrence of both polioviruses (16 out of 220 samples) and coxsackieviruses (10 out of 63 samples) in pit latrines /43/. More recently, an investigation on faecal material stored in pit latrines in Botwsana reported concentrations of enteroviruses from 0.3 to 1.5 PFU per gram, and rotaviruses from 90 to 727 virions per gram /44/.

The concentration of enteric viruses in sewage may be indicative of their occurrence in human faeces collected for composting. Virus concentrations of 5.000 to 28.000 PFU per litre are commonly found in raw sewage /45/. Recent studies have demonstrated that adenoviruses, enteroviruses, HAV and Norwalk virus are common in raw sewage, as they can be detected in samples of 100 ml at frequencies varying between 10 to 100% of samples tested /46,47).

Epidemiological data on the incidence of enteric viruses in the population may be indicative of the occurrence of enteric viruses in human faeces. According to the incidence of human infections (see section 2.2), the distribution of enteric viruses in human faeces should undergo seasonal variations. Adenovirus, astrovirus and rotavirus are more frequent during autumn, winter and early spring, whereas enteroviruses are more common in the summer.

Geographical differences should be also taken into consideration. The HEV virus is likely to occur less frequently in faeces and sewage collected in Europe. However, recent studies have reported HEV detection in sewage in Barcelona, Spain and Washington, DC, which are non-endemic areas. Therefore, the presence of this virus in human wastes is not limited to developing countries. However, the occurrence of HEV and other enteric viruses, such as enteroviruses, HAV and rotaviruses is likely to be higher in developing countries than in industrialized countries due to less coverage of water, sanitation and protection against childhood diarrhoeal diseases in developing countries.

For equal or similar incidences in the human population, viruses faecally excreted at higher numbers and for longer periods are likely to be predominant in faeces collected for composting. HAV is excreted at densities of up to 10¹⁰ viral particles per gram and the shedding occur for at least 30 days after the onset of the disease and as long as three months /48-51/. Also rotaviruses are shed at even higher numbers (10¹⁰ to 10¹² viral particles per gram), sometimes for periods up to one month /52-55/. The Norwalk-like viruses have been estimated to occur at lower densities in faeces (typically 10⁴-10⁶ viral particles per gram) /56/ and the shedding lasts up to two weeks after infection /57,58/. Enteroviruses are generally excreted at densities of 10⁶ infectious units (corresponding to about 10⁸-10¹⁰ virus particles) per gram of faeces) and their excretion time is on average 7 weeks /23/.

2.4 Occurrence in Denmark

In Denmark, only the incidence of HAV is notifiable. In the year 2000, 81 patients were notified with hepatits A, corresponding to an incidence of 1.5 cases per 100.000 inhabitants /59/. According to preliminary data of the WHO Polio Eradication Certification Process, the occurrence of enteroviruses (Coxachie-, Echo, Polio- and Enterovirus 68-71) in faecal specimens from symptomatic patients was 15% in 1998, 13% in 1999 and 23% in 2000 (personal communication from Peter Henrik Andersen, Department of Epidemiology, Statens Serum Institut, Denmark).

The numbers of diagnosed rotavirus infections reported by the major laboratories in the country were 171 out of 2,770 patients tested in 2000 (6%), and 284 out of 10,222 patients tested in 2001 (3%) (personal communication from Francois-Xavier Hanon, Department of Epidemiology Research, Statens Serum Institute, Denmark). Caliciviruses (including the Norwalk virus) account for the vast majority of outbreaks of food-borne viruses and are estimated to be responsible for over 40% of total food-borne outbreaks in the country /60/. The contribution of other enteric viruses to food-borne outbreaks can be estimated to be approximately 2-3% (personal communication from Francois-Xavier Hanon, Department of Epidemiology Research, Statens Serum Institute, Denmark).

In another Scandinavian country (Finland), rotavirus appears to be the most common diarrhoea-causing virus in young children. Rotavirus was estimated to be responsible for 54% cases among children less than five years old that were hospitalised for acute diarrhoea in the period between 1985 and 1995 /61/. Caliciviruses and astroviruses were detected in 21% and 9% of episodes of acute gastroenteritis in children less than two years of age, respectively /62,63/. Also in Sweden, rotavirus is the predominant virus among children affected by gastroenteritis, being recovered from 53% of children attending hospitals with symptoms of gastroenteritis /64/.

In Denmark, the concentration of viruses in sewage has been estimated to be 10³ to 10⁵ PFU/100 ml for enteroviruses (including HAV) and 2 to 10² PFU/100 ml for rotaviruses /65/. Similar or more likely higher concentrations can be expected to occur in human faeces destined to composting, since the concentration of enteric viruses in sewage is reduced as a consequence of dilution.

2.5 Response to physical-chemical factors

The knowledge of the response of different viruses to physical-chemical factors is essential to evaluate the survival of viruses during storage of faeces and the efficiency of composting in viral inactivation. In particular, the survival of viruses during storage and composting of faeces is closely related to their resistance properties to changes of temperature, pH and moisture occurring during the different phases of the composting process.

2.5.1 Temperature

The principal factor affecting viral survival is temperature. In general, viruses better tolerate low temperatures than high temperatures. Surface proteins are denatured within a short time at temperatures of 55-60° C, with the result that viral particles are no longer able to bind to the host cell /66/. Enteroviruses seem to be more resistant than bacteria and parasites when exposed to heat for less than one day, whereas they are more susceptible than other microorganisms for prolonged exposure times (Fig. 2.1).

It is important to recognize that there are marked differences among different viruses in the temperatures and exposure times necessary for viral inactivation. Non-enveloped viruses, like all most important enteric viruses, are generally more heat-resistant than enveloped viruses /66/. Among enteric viruses, the most heat-resistant appears to be the HAV, for which temperatures of 60° C for 30 min or 70° C for 10 min are not sufficient for complete inactivation /41,67/. However, even HAV is inactivated by heat-treatment at 60° C for 10 hours /68/.

Fig. 2.1.
Effect of temperature and exposure time on enteroviruses, bacteria (Salmonella and Vibrio choleae) and parasites (Taenia and Ascaris). According to Feachem et al. /23/.

The effects of temperature on virus survival have also been extensively studied with respect to the inactivation of viruses in animal slurries /69-77/. Table 2.4 reports the data relative to animal viruses belonging to the same families of certain human pathogenic viruses. Also in this case, differences are evident between different types of viruses, with the porcine parvovirus being particularly heat-resistant compared with the other viruses tested.

Table 2.2.
Inactivation times for animal viruses in slurry at various temperatures. According to Bøtner /71/

* w, weeks; d, days; h, hours; m, minutes.

2.5.2 pH

Viruses are generally best preserved at physiologic pH, although some viruses tolerate a wide pH range. While most enveloped viruses are rapidly inactivated at pH 5-6, non-enveloped enteric viruses are able survive gastric acidity (pH=3)/65/, or even lower pH levels (e.g. HAV is stable at pH 1 for 2 h). Hence, low pH levels (3 to 5) are not deleterious for enteric virus survival. Most enteric viruses also are relatively stable at moderately high than high pH levels, or at least up to pH 9.5. At pH 10 and higher, the rates of enteric virus inactivation vary among the different viruses. Some viruses are not stable to pH 10 and become inactivated by >99.99% within 1 day. Other enteric viruses are stable at pH 10 for periods of weeks to months. However, most enteric viruses are inactivated by 99.99% at pH 11 or higher within 1 day or less. The low stability of enteric viruses at high pH (pH >11) is used for viral inactivation in sewage sludge by lime treatment /37/.

2.5.3 Moisture

Enteric viruses are susceptible to dry conditions, but only if very low moisture levels are achieved (<5%). Dewatering of raw sludge was shown to effectively inactivate human polioviruses /78/. Inactivation was due to disruption of the viral capsid with consequent release of nucleic acids. However, other studies have shown that viruses can persist in relatively dry soils for long time periods. Therefore, other factors in the suspending medium or matrix besides loss of water also may contribute to virus inactivation. Insufficient data are available for comparison of the survival of different types of viruses under dry conditions in different media, as only some virus groups have been studied in certain media. However, some enteric viruses, such as HAV, have been shown to persist for long periods of time under drying or dried conditions.

2.6 Human infectivity and dose-response

The risks associated with survival of pathogenic viruses in human composted faeces are dependent on their human infectivity. Infectivity of enteric viruses and other enteric pathogens can be described quantitatively as a dose-response relationship, which is the relationship between number of virus particles ingested and the probability of resulting infection and disease in humans. As shown in Figure 2.2, the risk or probability of infection increases as the ingested dose of rotavirus to human volunteers increases.

The models that best describe such dose-response relationships are the exponential model and the Beta-Poisson model. The exponential model assumes a random distribution of organisms in the doses to which humans are exposed and that each organism has an independent and identical probability of surviving to initiate infection. The Beta-Poisson assumes that the probability of the microbe surviving to initiate infection is not a constant value but instead varies and is described by a probability distribution, the Beta-Poisson distribution.

Fig. 2.2.
Dose-response relationship for infection by a human rotavirus in human volunteers with fitted curves for exponential and Beta-Poisson models.

Most of the available data on the dose-response relationships of enteric virus infectivity for humans have been obtained from human volunteer studies. Only some enteric viruses have been studied because of the difficulties of conducting such human infection studies. However, the available data clearly indicate that most enteric viruses have a high probability of causing infection at relatively low virus doses.

The doses at which viruses have a high probability of causing infection are markedly lower than those for bacteria. For some enteric viruses, such as rotavirus and Norwalk virus, only a few viral particles need to be ingested to cause a high probability of producing disease in humans (Table 2.3). The 50% infective doses of viruses (dose at which the probability of infection is 50%) may vary between 1 and 12,000 viral particles, depending on the virus type and the state of the human exposed to the virus.

Based on dose-response relationships for infection and illness, survival of low numbers of pathogenic viruses (and parasites) in human composted faeces appears to pose a greater hazard in comparison with bacterial pathogens. However, it should be considered that, in contrast to viruses, bacteria can multiply outside of the host and their numbers can therefore increase during storage of faeces and after application of composted faeces to soil.

Table 2.3
Doses of enteric microbes infecting 50% of exposed humans (ID₅₀ based on excretion). Adapted from Teunis et al /79,80/.

3 Virus survival in composted human faeces

3.1 Introduction

In Denmark, there is an increased interest in using human faeces and urine for agricultural and other purposes. This interest is seen especially when new urban areas and houses are established. Traditional sewerage-based sanitation systems can cause environmental pollution and often do not utilize the nutrients contained in human excreta. An alternative to these systems is the direct recycling of human excreta using treatment systems designed to regard and employ human excreta as a resource to be used rather than as a waste to be disposed. By such systems, the nutrients contained in urine and faeces are returned to soils and plants, thereby contributing to a circular flow of nutrients (Fig. 3.1).

Fig. 3.1.
The circular flow of nutrients associated with recycling of human excreta.

An essential prerequisite for safe reuse of human faeces is the removal or destruction of pathogens by composting or other sanitation methods. This chapter describes how and to what extent viruses are inactivated during storage, production and utilization of composted faeces. The mechanisms and the efficiency of viral inactivation by composting are reviewed and evaluated on the basis of the existing literature.

3.2 Virus survival during storage of faeces

Virus survival in composted faeces depends not only on the conditions used for composting, but also on the storage conditions. Storage should therefore be viewed as part of the composting process. Faeces are collected by toilets separating faeces from urine (i.e. urine-diverting toilets) and stored into a vault beneath the toilet seat. During storage there will normally be no or very little composting effect because of anaerobic conditions and limited temperature increase. Other human waste management systems collect faeces and urine from homes either manually or by automated systems (such as vacuum tankers) and then treat it in bulk by composting or other processes /81/.

Viruses decrease in numbers during storage of faeces, due to their incapability to replicate outside of the host and susceptibility to a number of adverse environmental conditions. However, the die-off rate of viruses present in human faeces may show large variations depending on both the type of virus (see section 2.4) and the storage conditions. High pH, low moisture, microbial activity, free ammonia and high temperature are among the most unfavourable conditions for virus survival during storage of faeces.

The temperature of faeces stored in latrines generally does not significantly differ from ambient temperature /82/. Thus, the role played by temperature in the inactivation of viruses during storage of faeces appears mainly dependent on climate. Studies on sewage applied to soil indicate that viruses can persist for 23 weeks during the winter season in Denmark /83/ but for only 2-4 weeks during the summer or fall in Florida /84/. Similarly, the survival of animal pathogenic viruses in faeces has been demonstrated to be longer under winter conditions rather than summer conditions /85/.

Recent studies have demonstrated that some animal enteric viruses, including enteroviruses, can persist in faeces for a longer time, especially at low ambient temperatures. The inactivation of a bovine enterovirus in liquid cattle manure stored at 20^oC was found to be only 2 log₁₀ after 26 weeks /86/. The porcine rotavirus was demonstrated to maintain its infectivity after 32 months of storage at 10° C in original stool specimens /87/.

Among human pathogenic viruses occurring in faeces, HAV is a relatively heat-resistant virus and is inactivated more slowly than enteroviruses in a variety of media, including faeces. At lower temperatures, HAV, like other enteric viruses, is very persistent in faeces and other media. For example, HAV is inactivated very slowly in human faeces and manure kept at 5° C, with an observed reduction of only 1-2 log units after 70 days of storage in manure /88/ and about 625-1250 days in faeces /89/. At 25^oC, HAV is relatively persistent in stored faeces (about 139 days for 4 log₁₀ inactivation) when compared to poliovirus and male-specific (F+) coliphages (about 83-84 day). At 40^oC, times for 4 log₁₀ inactivation in faeces are about 20-21 days for poliovirus and F+ coliphages, and 29 days for HAV /89/.

A number of studies have investigated the survival of viruses in latrines, including urine-diverting vault latrines intended for recovery and potential reuse of both urine and faecal solids. High temperature and high pH levels are the most important physico-chemical factors affecting virus survival (see section 2.5). However, in many latrines, neither high temperature nor high pH levels are achieved. Therefore, extensive enteric microbe reductions are not achieved unless storage times are very long.

The bacteriophage Salmonella typhimurium 28B was used as an indicator to study the behaviour of human enteric viruses during storage of faeces collected by urine-diverting toilets /82/. The die-off of the phage was slow, with a reduction from 10⁸ to 10⁶ during 6 weeks of storage at temperatures between 20 and 35° C (Fig. 3.2). The long survival of the phage in faeces was attributed to the neutral pH conditions (7.8-8.5). Under these pH conditions, the loss of moisture observed during the study (dry weight increased from 85.2 to 96.5%) was not great enough to affect virus survival. On the contrary, it could have reduced viral inactivation by slowing down the biological process of degradation /82/.

Fig. 3.2.
Mean die-off rate of the bacteriophage S. typhimurium 28B during storage of human faeces in urine-diverting toilets. Adapted from Franzen and Scott /82/.

A similar study conducted in Vietnam suggested that the combination of high pH and low moisture have a high impact on virus survival during storage of faeces in urine-diverting latrines /90/. After 3 weeks of storage, the bacteriophage S. typhimurium 28B was reduced 8 log units in faeces with high pH (10.0 to 10.3) and low moisture content (24 to 29%). In contrast, the bacteriophage persisted for 7 weeks in faeces with lower pH (8.5 to 9.4) and higher moisture content (27 to 55%), with a reduction of 2-3 log units only. The high pH of faeces reported in this study was due to the addition of ash originating from wood and leaves /90/.

According to a summary report of various studies, Stenstrom /91/ stated that enteric microbe reduction in the material of dry latrines was mainly governed by time and high pH. Complete inactivation of indicator viruses was achieved in 6 months or less in association with the use of ash, lime or similar additives for pH elevation. Addition of only moisture-absorbing materials was shown not to ensure efficient microbe reduction.

Austin /92/ studied enteric microbe reductions in wood ash-supplemented faecal matter of urine diversion toilets in Eastern Cape province, South Africa. After 10 months of storage in a separate plastic container in the latrine, faeces contained log₁₀ concentrations of 0-3 coliphages per gram. In additional studies, it was shown that microbial reductions were more rapid and extensive in faeces stored on the concrete floor of the latrine vault and turned weekly for aeration than in faeces stored in a closed plastic container. Coliphages were not detected after 2 months of storage at mild to cold conditions (moisture 4-8 % and pH 8.4-8.6) and when latrine faeces were subjected to a temperature of 50^oC for 48 hours, while keeping the moisture content approximately the same /92/.

Moe and colleagues /93/ studied the performance and stored fecal waste quality of urine-diverting double vault latrines and solar toilets in seven communities in El Salvador. Latrine wastes ranged widely in microbial quality, probably due to high variability in the storage conditions (pH ranged from 5.1 to 12.8 and the percentage of solids ranged from 2 to 98%). Somatic coliphage concentrations varied widely and were as high as 8 log₁₀ per gram. Temperatures of stored latrine material were 20-37.5^oC (mean of 27^oC), indicating that these toilets are not "true" composting systems because they do not achieve the high internal temperatures (>50^oC) typical of aerobic composting. No single physical factor (pH, temperature, moisture content, or storage time) could predict microbial indicator concentration, suggesting that microbial quality is the net result of the effects of a multiple factors /93/.

Chien et al. /94/ studied the survival S. typhimurium phage in various designs of urine-diverting, double-vault latrines in Viet Nam. Phage survival was 23-154 days, with temperatures of 30-40^oC, moisture contents of 25.4-58.8% and pH of 8.4-10.3. In general, microbial survival was most influenced by pH. Both pH and moisture content influenced bacteriophage when temperature was below 40^oC. To achieve acceptably low levels of microbial risk, 6 months of retention for faecal materials were needed in test toilets /94/.

The available data on viral persistence in stored faecal material suggest that in temperate countries the impact of temperature on virus survival is limited to the summer months. At temperatures below 20° C, high pH values (>9.0) in combination with low moisture contents are the most important virucidal factors. Accordingly, wood ash and other substances increasing the pH levels (e.g. lime) can be used to reduce the content of viral pathogens in faeces collected for composting. Soil may be added to faeces for reducing their moisture content, although this practice does not seem to be particularly effective in viral inactivation /82/.

Aeration is another factor enhancing viral inactivation during storage of faeces. According to a study on faeces from urine-diverting toilets /91/, the die-off of viruses is higher if faeces are collected and stored in heaps and turned weekly, rather than in closed compartments. Similar evidence was provided by a study on animal waste /85/, in which a large variety of viruses (i.e. coliphage f2, picorna-, rota-, parvo-, adeno- and herpesvirus) were shown to persist for longer periods under non-aerated conditions. Consequently, the effect of aeration conditions on viral inactivation may have important implications in the design and construction of urine-diverting toilets.

3.3 Factors affecting viral survival during composting

The inactivation of viruses during composting is determined by a combination of chemical, physical and biological factors. The most important cause of viral inactivation is the heat generated during the thermophilic phase of composting. To a lesser extent, viruses are also inactivated by microbial degradation and ammonia. It should be noted that these mechanisms operate at the same time during composting. Thus, viral inactivation should be attributed to a synergistic interaction between them, rather than to each mechanism taken individually.

Environmental factors such as moisture content and pH play a secondary role in viral inactivation during composting. The optimal conditions for composting are given by pH levels of 5.5 to 8.0 and moisture contents of 40 to 60% /8,9/. These conditions are not harmful for enteric viruses by themselves (see section 2.5). Accordingly, pH and moisture can affect virus survival only by influencing the metabolic activity of bacteria and fungi and the production of ammonia during composting.

The temperature increase seen during the first phase of an optimal compost process exceeds the temperature levels needed for viral inactivation. Feachem et al. /23/ studied the effects of heat and exposure time on enterovirus survival with respect to composting of sewage sludge. According to these authors, exposure at 30° C for 3 months, at 40° C for 2 weeks, at 50° C for 1 day or at 60° C for 2 hours are sufficient to assure complete inactivation of enteroviruses, adenoviruses and reoviruses. The zone of safety described in Fig. 3.3 indicates the possible combinations of temperature and time necessary for complete elimination of enteroviruses in composted sludge.

Look here!

Fig. 3.3.
The influence of temperature and exposure time on survival of enteroviruses. According to Feachem et al. /23/.

Due to their remarkable resistance to heat (see section 2.5.1), some enteric viruses (e.g. HAV and parvoviruses) are likely to represent an exception to the safety zone described by Feachem et al. /23/. A recent study demonstrated that the bacteriophage S. typhimurium 28B is only reduced 1-2 log units after composting at 55° C for 24 hours /95/. The inactivation of certain animal enteric viruses (i.e. parvovirus) by composting requires up to 8 days at 55° C /70/. However, heat-resistant viruses are appreciably inactivated when compost facilities are operated at times and temperatures specified in accordance with current legislation (Table 3.1).

Problems may occur in the control of temperature during composting. Strauch et al. /96/ reported that it is nearly impossible in daily practical operations to constantly maintain the necessary parameters and control them. The authors proposed that composting of sewage sludge should be operated as a two-stage system, with a minimum hydraulic retention time in the system of 5 days and minimal retention time of one day in each reactor. The recommended temperatures and exposure times were either 48 hours at more than 50° C or 24 hours at 58° C. These temperature and time requirements are largely fulfilled by the current legislation on composting in different countries (Table 3.1).

Among the different systems of composting, enclosed systems ensure the best control of temperature, with horizontal reactors being preferable to vertical reactors /97/. Static aerated piles guarantee a better temperature control and therefore also a more efficient pathogen removal compared with windrow systems. The control of temperature is particularly critical in the windrow system because the temperature in the outer part of the windrow is influenced by ambient temperature. This is also true for static compost piles. Thus, the techniques used for turning must ensure that also the outer material is transported into the interior of the windrow and exposed to the temperature and time necessary for inactivation of viruses and other pathogenic organisms.

Table 3.1.
Minimum temperature and time requirements for composting in various countries. According to Stentiford /9/ and Strauch /28/.