INTRODUCTION AND LITERATURE REVIEW
1.1 The Global Burden of Human Schistosomiasis: Human schistosomiasis is an important and widespread infection in the tropics. It gives rise to a complex of acute and chronic diseases with widely differing signs and symptoms (WHO, 1993). It is the second most prevalent parasitic disease after malaria in the developing world with a huge impact on public health and socio-economic development (Doumenge et al., 1987; WHO 1993, 1998).Based on the latest extrapolations, it is estimated that 652 million people live at risk of infection and that 193 million people are actually infected with schistosomiasis, of whom 85% are concentrated in sub-Saharan Africa (WHO, 1999). It is believed that there are 120 million symptomatic cases, of who 20 million are suffering from severe disease (WHO, 1999). Recently, the concept of disability-adjusted life years (DALY) was developed in order to assess and refine estimates of the global burden of disease (World Bank, 1993). For sub-Saharan Africa, a morbidity burden due to schistosomiasis of 3.5 million DALYs has been estimated. In comparison with all other communicable diseases, schistosomiasis therefore ranks in position ten, after respiratory infections (31.6 million DALYs), malaria (31.5), diarrhoeal diseases (330.4), HIV infections (18.4), measles (16.1), tuberculosis (13.7), sexually transmitted diseases excluding HIV (7.5), tetanus (5.8) and pertussis (4.8), as reviewed by Murray et al.,(1994).
Although these numbers have to be cited with caution, as they were calculated after extrapolation of (often limited) prevalence survey data and were aggregated to country level, they clearly indicate the tremendous public health significance of schistosomiasis. Although several investigations have been performed to estimate the direct impact of infection with schistosomiasis on the school performance of children, their physical fitness and productivity, the results are inconclusive. Some studies revealed a clear negative impact due to the infection with schistosomiasis, while others did not (Gryseels, 1989; Tanner, 1989). This discrepancy was explained by the difficulty of devising a standardized methodology, and by other confounding factors, for example concurrent infection with another disease (Tanner, 1989). The recent advance in methods of calculating attributable risk allows the fraction of a particular morbidity indicator which was attributed to an infection with schistosomiasis to be calculated (Guyatt et al., 1995a; Booth, 1998).
There is general agreement that the global prevalence of schistosomiasis will likely increase due to the following three reasons: (i) increasing numbers of irrigation systems for agriculture and cattle breeding. (ii) construction of dams and man-made lakes for hydroelectric power production and (iii) civil strife and war contributing to additional human migration (Mott et al., 1995).
Despite the tremendous global burden of schistosomiasis, and the likelihood of the disease gaining more importance, its public health significance is often underestimated. There are two common explanations for this: (1) schistosomiasis is focally distributed and (2) severe disease only follows after many years of mildly symptomatic infections (Homeida et al., 1988, WHO, 1993). In most African countries, where resources are very limited and need to be allocated in the most effective way, primary health care delivery systems have to deal with many other and often more visible health problems, such as malaria and HIV (Gryseels, 1989; Gryseels & Poldeman, 1991). These reasons also explain why schistosomiases control is often given a low priority and many endemic countries have never set up national control programmes (Kusel & Hagan, 1999). However, the prospects of controlling schistosomiasis are good, as health managers now have sensitive but inexpensive diagnostic tools, and most importantly a safe, effective and cheap drug at their disposal (Kusel & Hagan, 1999).
In Nigeria, schistosomiasis constitutes a public health problem particularly in children. Both Schistosoma haematobium and S. mansoni are present with the former being more widespread (Awogu, 1990). Highest infection prevalence rates were observed among children aged 5-19 years who constitute 60-70% of all persons infected in the community (Cowper, 1973; Tayo, 1989). The distribution of schistosomiasis in the country is focal and at most times related to water development schemes, such as irrigation projects, rice/fish farming and dams.
1.2.0 Status of Schistosomiasis in Ebonyi State: Several works has been recorded and reviewed in the state. Anya and Okafor (1987), reported prevalence rates ranging from 1.7% to 70%.Their group worked in eight villages including Nkalagu, Ezillo and Ngbo with prevalence rates of 42.25%, 40.05% and 46.28% respectively. There were 25.81% males infected in the area while females had a higher infection rate 42.57%. The higher prevalence in females was as a result of their role in collecting water for domestic use and the part they play in agricultural production. They have more exposure to source of infection.
A research was carried out in Ebonyi State in 1998, in which children from 98 primary schools (representing 98 villages) including Ohaukwu, Ikwo, Ishielu and Izzi were examined. The prevalence of schistosomiasis ranged from 1.7% to 70% (Korve, 2002). This was 9 years after the National schistosomiasis control programme distributed praziquantel in the area. Another survey of Schistosoma haematobium infection was conducted among school children in Ohaukwu and Onicha in Ebonyi state, Nigeria using standard techniques. Of the combined total 876 pupils examined, 235 (26.8%) were infected with Schistosoma haematobium. A total of 129(27.01 %,) males and 106 (26.6 %,) females were infected. Individuals aged 5 – 10 years old were more infected and the intensity of infection was higher among the males (Uneke et al., 2007).
Anosike et al. (2001), working on endemicity of vesical schistosomiasis in Ebonyi river valley reported that out of 3, 296 persons examined from randomly selected 15 villages in the valley, 775 (23.5%) were excreting the eggs of Schistosoma haematobium in their urine. Of the 1, 238 females examined on the upland area, 234 (18.9%) were infected while 155 (29.9%) of the 519 males were infected. On the lowland area, 211 (21.9%) and 176 (30.6%) out of 964 females and 575 males examined were infected respectively.
Anosike et al, (2001) working on the epidemiological and bacteriological findings in some schistosomiasis endemic foci in Ebonyi State, Nigeria recorded that out of the 1, 284 persons, (21.5%) were infected with Schistosoma haematobium. Visible haematuria was their predominant presenting symptoms. Their work established that urinary schistosomiasis is endemic amongst the people of Ebonyi State, Nigeria. The disease has an overall prevalence of 55.7% which calls for priority attention in the health programme in Ebonyi State.
In another survey, Anosike et al., (2006), worked on vesical schistosomiasis among rural Ezza farmers in the south western border of Ebonyi State, Nigeria. Their work reported that out of the 2, 014 urine specimens examined in 10 communities, 466 (22.1%) comprising 305 (23.7%) men and 161 (19.7%) women were infected with visible haematuria as their predominant presenting symptoms. There was a gradual increase in the disease prevalence as the subjects age increased. About 78.3% of the infected persons are aged between 15 – 20 years.
Nigeria has a national schistosomiasis control programme that has the ultimate goal of eliminating schistosomiasis as a public health problem in the country. The age group 5-19 years has been defined as the target population for nationwide control through the school system. Considering the focal nature of the disease, the vast terrain of Nigeria, the large size of population at risk and the limited resources for control, it is vital that communities at highest risk be identified to ensure that available control resources are utilized in the most effective way. The rapid identification of disease pockets in the endemic states of the nation will greatly define the control programme’s actual needs and identify priority areas for intervention in a phased control programme. Donor agencies could also be attracted to make some of the responsibilities towards an effective and sustainable implementation of control measures.
1.3.0 The Role of Phenol in Schistosoma Egg Formation: The eggs of schistosomes are quinone-tanned to form sclerotins, which make eggshells resistant to enzymatic digestion. In quinone tanning, phenol, a major oxidized metabolite of benzene plays a key role. In general, all trematodes utilize a similar pattern of egg formation that results in the fertilized egg packed in a very resistant eggshell (Srivastra and Gupta, 1976). Thus, phenol has been shown to play a vital role in egg production in trematodes.
Individuals may be exposed to phenol through breathing contaminated air or through skin contact in the work place (ATSAR, 1998). Other exposures to phenol may occur through the use of phenol-containing media products (including mouth washes, toothache drops, throat lozenges, analgesic rubs and antiseptic lotions) or smoking tobacco.
Phenol is a constituent of coal tar and is formed during the natural decomposition of organic materials. Production and use of phenol and its products, especially phenolic resins, exhaust gases and residential wood burning are potential sources. Another potential source is the atmospheric degradation of benzene under the influence of light. Benzene and its phenol derivatives may by in-vivo conversion form a source of endogenous human phenol exposure.
Apart from environmental exposure, endogenous sources of phenol stem from dietary ingestion, catabolism of tyrosine and other substrate by gut bacteria (Bares et al., 1990; Smith, 1996).
Although the use if cigarettes and certain medicines can result in significantly elevated levels of phenol, diet is a significant source contributing to increased tissue level of phenol for all persons (McDonalds et al., 1994). Thus, the wide inter-individual variability in urinary levels of phenol may reflect the wide range of direct intake of phenolic containing foods and differences among individuals in the composition and chemistry of the gut flora (McDonald et al., 1993).
Urinary excretion is the major route of phenol excretion in animals and humans .This test can be used to determine whether a person has recently been exposed to phenol. However, no test will tell whether a person has been exposed only to phenol because many substances, like benzene are changed to phenol in the body (ATSDR, 1998).
It has been variously established that chemical agents from the environment plays a role in the process of man. Thus, a number of studies have assessed phenol levels in patients with different diseases including colon cancer, familial polyposis, and crohn’s disease (McDonald et al., 2001). It is hoped that this study using schistosomiasis patients will contribute some knowledge to the expanding field of the role of chemical substances in parasite physiology and pathogenicity, particularly trematode egg formation and fecundity; and the possible use of the variable excretion levels of phenol as a rapid diagnostic tool in cases of active infection.
1.4.0 Objectives: The general objectives of this study are:
- To study the correlation between the excretion of urinary total phenolic compounds and the egg output in patients with Schistosoma haematobium infection
- To compare the excretion levels of phenol in patients with intensity of infection.
- To assess the dietary habits of the children as a possible contributor to the variable urinary phenol levels.
- To assess the possible use of the variable excretion of urinary phenol as a diagnostic tool
1.5 Literature Review
1.5.1 The parasite and its life cycle: Schistosomiasis is caused by an infection with fluke worms (Trematoda, Platyhelminths) of the family Schistosomatidae, belonging to the genus Schistosoma. There are twenty-three species but only five species are able to infect man. These are Schistosoma haematobium, S. intercalatum, S.mansoni, S. japonicum and S. mekongi. These species can be subdivided into three groups characterized by the size, shape and appearance of the eggs produced by the female worm: (1) S. haematobium and S. intercalatum produce ovoid eggs with a size of 60X140-170 mm and a terminal spine, (2) eggs of S. mansoni have a similar shape and size but with lateral spine, and (3) S. japonicum and S. mekongi produce smaller eggs (size 50-90 mm) that are rounded and have only a rudimentary spine (WHO, 1994; Davis, 1996).
The species considered in this study is S. haematobium. Currently, S. haematobium is currently found in 53 countries in the Middle East and most of the African continent including the islands of Madagascar and Mauritius (WHO, 1999). Most of the results that will be presented in this work were obtained in a series of field studies carried out in Ebonyi State (Nigeria), a country where S. haematobium is endemic
The life cycle of Schistosoma is complex. It involves a phase of sexual reproduction by adult schistosomes in the definitive human host, and an asexual phase in the intermediate host, a freshwater snail. From the snail, cercariae are released into the surrounding water and can invade humans through the skin (Figure 1). Adult schistosomes are small white-grey worms varying in length from 6-26 mm and in width from 0.2-1.1 mm, depending on species and sex. The sexes are separate. The female worm is slender, and is longer than the male. They live in couples with the female permanently held by the male in a longitudinal ‘schist’ or gynaecophoric canal (Sturrock, 1993). Worm couples of the species S. mansoni inhabit the pericolonic venules within the portal venous system and S. haematobium worm pairs inhabit the terminal venules in the wall of the bladder, the genito-urinary system and the pelvic plexus within the distribution of the inferior vena cava of the definitive human host (Davis, 1996). Here, the worms feed on blood. It has been estimated that the average life span of S. mansoni worm is 3.3 years and that of S. haematobium is 3.4 years (Wilkins et al., 1979), but there is a confirmed report of viable S. mansoni eggs being discovered 37 years after a subject had left the endemic area (Chabasse et al., 1985). An adult S. mansoni female worm produces 100-300 eggs per day, and S. haematobium produces 20-300 eggs per adult female per day. It is assumed that approximately 50% of the eggs pass through the colon, the walls of the bladder or the genito-urinary apparatus and are excreted by feces or urine. The remaining 50% of the eggs are trapped within the tissues of these organs (Davis, 1996). In infections with S. haematobium, these eggs give rise to inflammation, haemorrhages and pseudopolyposis. Inflammation and in later stages calcification of the eggs can lead to stasis, hydroureter, hydronephrosis, kidney failure and even bladder carcinoma. In infections with S. mansoni, some of the eggs are trapped in the intestinal wall and lead to inflammation and psuedopolyposis, which may result in abdominal pain and blood in the faeces. Other eggs are
Fig. 1: The Life Cycle of Schistosoma Species (WHO 1998)
carried away and are finally trapped in the portal system of the liver and may cause hepatosplenomegaly. In a later stage these lesions may become fibrous. This condition is called “symmer’s pipe-stem fibrosis”.
Excreted eggs hatch after they have come into contact with a suitable aquatic environment, and they release a miracidium, which swims actively and is able to locate- most likely by chemotaxis – a compatible freshwater snail. Interestingly, there are only very few snail species that are compatible and these are Schistosoma species-specific.Both Bulinus globosus and B. truncatus act as intermediate snail hosts for transmission of S.haematobium in Nigeria (N’Goran et al., 1997a).
Miracidia penetrate into the intermediate snail host, predominantly via the foot of the snail (Jourdane & Theron, 1987). After penetration and close to the entry point, a small proportion of both male and female miracidia develop into mother sporocysts. They might produce daughter sporocysts which migrate to other parts of the snail body. Subsequently, there is an asexual multiplication within the mother and the daughter sporocysts and each forms many thousand cercariae, all of the same sex. According to biological environment and physical determinants, it takes between 4 and 6 weeks from the penetration of a miracidium to the production of mature cercariae (Webbe, 1982).
When cercariae emerge from the snails, they are directly released into the freshwater environment. It is suggested that the main stimulus for their release is light intensity; therefore emergence follows a circadian rhythm. Recently, the cercarial shedding patterns of S. haematobium were analyzed in Côte d’Ivoire along a North – South transect. Peak intensity was observed around noon. However, the mean shedding time in the forest zone in the South (11.00 hours) was significantly earlier than the one in the savannah zones in the North (13:40 hours) (N’Goran et al., 1997a).
Cercariae are non-feeding organisms and they have a relatively short life span between 36 and 48 hours. If they find a human host within this time, they are able to penetrate the skin while the human is in contact with the infested water during occupational and/or recreational activities. It is remarkable that the process of penetration only takes a few minutes and is coupled with the transition of the cercaria from a freshwater to a saltwater (isotonic) environment in the human body. After penetration, the cercariae lose their tails and are subsequently called schistosomula. They traverse the subcutaneous tissue within 2 days and penetrate into the venous or lymphatic channels, when they are transported to the right side of the heart and lungs. Then, schistosomula leave the lungs and are distributed passively with the blood flow to the systemic organs. Most of the parasites are trapped in the liver. Sexual maturity is reached in the liver with a schistosome – specific duration. For S. mansoni, it takes between 25 and 30 days (Clegg, 1965). For S. haematobium, sexual maturation is reached at day 31; however the pre-patent period is much longer, approximately 70 days (Smith et al., 1998). During the development from schistosomula to adult worm, a feverish syndrome called “Katayama fever” may develop, usually in previously non-exposed individuals. After the worms have paired, they remain attached together and actively migrate against the blood flow in the hepatic portal vein to finally inhabiting the two venous branches around the intestine (S. mansoni) or the vesicule plexus (S. haematobium). In these locations, they begin to lay eggs, which are detected in faeces and urine, respectively, some 6 to 10 days later.
1.5.2 The epidemiology of human schistosomiasis: For a comprehensive understanding of the epidemiology of human schistosomiasis, five features of the diseases are of central importance: (a) it is complicated, (b) it is heterogeneous in time, (c) it is heterogeneous in space, (d) it shows aggregation and (e) is sensitive to environmental alteration.
1.5.3 Complicated epidemiology: The epidemiology of schistosomiasis involves humans as the definitive host, various but very specific aquatic or amphibious snails acting as intermediate hosts and freshwater as the environment where the disease is transmitted. However, ttransimission only occurs when schistosome eggs reach the freshwater environment, as a result of the absence of appropriate sanitary facilities, or insanitary behavior of humans, directly contaminating the freshwater environment with their excreta. Humans can then acquire the disease by (repeated) contact with infested water, by means of recreational and/or occupational activities (Davis, 1996).
1.5.4 Heterogeneity in time: In most, if not all areas where it is endemic, schistosomiasis is characterized by seasonal transmission patterns. In numerous studies it has been both shown that the distribution and density of the intermediate snail host is an important determinant, accounting to a large extent for the observed variability in rates of schistosomiasis infection (Babiker et al., 1985; Woolhouse & Chandiwana , 1989, 1990a, b, Woolhouse, 1992). There is firm evidence that in lotic environments, water current velocity is the key determinant influencing the distribution of snails, and that in lentic environment, water temperature plays the key role for snail abundance (Appleton, 1978). Both water current velocity and temperature vary over time and show a seasonal pattern, and therefore are the most important factors explaining the heterogeneity of epidemiology patterns in time.
1.5.5 Heterogeneity in space It is widely accepted that schistosomiasis has a focal distribution ( Webbe and Jordan, 1993; WHO, 1993). It is assumed that the focal nature of the disease is the result of the complex interrelationship between the distribution and the density of infected persons and of compatible intermediate snail hosts, the distance between infected persons and suitable freshwater environments that are contaminated and acts as transmission sites, and the frequency and duration of water contacts of humans. The focality of schistosomiasis is well documented world-wide, with the disease currently being endemic iin 76 counttries and territories over several continents (WHO, 1999). The spatial heterogeneity is also well illustrated within countries, with specific foci of schistosomiasis being restricted to only those areas where all components of the schistosomiasis complex also converge in time ((Doumenge et al., 1987). Focal distribution on a regional scale has been observed in many studies (Hunter et al., 1993; Red Urine Study Group, 1995; Malone et al., 1997). However, the micro-geographical distribution of schistosomiasis within a community has received far less attention because its complexity requires more detailed assessment. Nonetheless, it has been shown convincingly that the mean frequency and the mean duration of water contacts per person, as well as the mean number of sites frequented per person, correlate with the intensity of intestinal schistosomiasis (Kloos et al., 1997) and urinary schistosomiasis (Useh and Ejezie, 1999). The distribution of intermediate snail hosts has been studied, and preferences for particular habitats and specific habitat features could be shown (Ndifon and Ukoli, 1989; Woolhouse and Chandiwana, 1989; Odermatt, 1994). In a recent study attention was drawn to the microhabitat level, by showing that snails showed spatial microhabitat preferences within a single river system (Utzinger et al., 1997).
1.5.6 Aggregation: It is well established that the prevalence and intensity of schistosome infections both peak in specific age groups (Woolhouse, 1992). The likely explanation for this pattern is the fact that these age-groups are almost frequently exposed to schistosome-infected water. It is also widely acknowledged that the distribution of schistosome worm pairs per person is extremely uneven, resulting in a great variation in the number of excreted eggs per person. The majority of the population only excrete a small proportion of schistosome eggs, while a small proportion of people are responsible for the greater part of the egg excretion (Bradley, 1972; Polderman, 1979; Anderson and May, 1985; Guyatt et al., 1995b). Furthermore, there is individual day-by-day variation in schistosome egg output, which was comprehensively reviewed by Hall (1982). There is also intra-stool variation in the number of schistosome eggs, but this is less important than the day-to-day variation (Engels et al., 2002). The aggregation of worms in individual human hosts is of considerable importance for understanding the transmission dynamic of schistosomiasis, and ultimately for the control of disease.
1.5.7 Dynamic epidemiology: Areas confirmed to be free of schistosomiasis at a particular point in time can rapidly become important disease foci and may challenge previously unexposed populations. Often, environmental alterations, for example caused by water resource development projects (damming and irrigation), are the cause of an onset of schistosomiasis transmission (Wen and Chu ,1984; Hunter et al., 1993). There is accumulated evidence from several countries that the completion of dam constructions may also be followed by a changing pattern of schistosomiasis with a shift in predominance from urinary to intestinal schistosomiasis (Abdel-Wahab et al., 1979; Mott et al., 1995). The latest example is reported from Senegal, where an outbreak of intestinal schistosomiasis was observed only three years after the completion of the Diama dam (Picquet et al., 1998; Southgate, 1997). However, though the construction of two large dams in Côte d’Ivoire was followed by a significant increase in the prevalence of S. haematobium, S. mansoni remained at a very low prevalence; therefore no shift was observed so far (N’Goran et al., 1997b). It is interesting to note that in the Kilombero district in Tanzania S. haematobium was the predominant schistosome species for many years (Zumstein, 1983), but recent studies suggest that S. mansoni is spreading rapidly (Odermatt, 1994; Pervilhac et al., 1998) and will eventually replace S. haematobium.
1.5.8 Diagnosis of human schistosomiasis: Diagnosis is of pivotal importance for all aspects of human schistosomiasis (Feldmeier, 1993). The decision to treat an individual with an antischistosomal drug, the assessment of morbidity due to schistosomiasis, the rapid identification of communities at highest risk of infection, studies of the regression and reappearance of pathology after chemotherapy, or the evaluation and monitoring of control programmes, are all based on the results of diagnostic tests. There are many different techniques and approaches that may be used both at the individual and community level. Their selection and application depends not only on the type of information sought but also the resources available. From a public health perspective, simple and robust techniques are requested, which can be performed with supplies and equipment that are readily available (WHO, 1993). In addition, they should be inexpensive and easily applicable in the field (WHO, 1999).
The diagnostic technique that used in this study is the urine filtration .The Kato-Katz smear is used in the diagnosis of S. mansoni. Both methods are discussed in detail below. Both methods are used to directly demonstrate the presence of schistosome eggs in urine and faeces, respectively and are the most commonly used techniques in epidemiological surveys (WHO, 1999). Indirect methods also exist, which rely on perceived symptoms, clinical examinations, or biochemical or immunology disease markers.
It is interesting to note that over the last 10 years remarkable progress has been made with immunological disease marker (WHO, 1998). The existence of detectable amounts of circulating antigens (circulating anodic antigen and circulating cathodic antigen) in schistosome-infected people has prompted research into their potential for immunodiagnosis. Subsequently, a variety of assay methods have been developed (Deelder et al., 1989; De Jonge et al., 1989; Gunderson et al., 1992; Van Lieshout et al., 1995a, b; Van Etten et al., 1997). Schistosome infections can also be indirectly detected by the presence of antibodies (Hamilton et al., 1998).
Diagnoses using imaging techniques to detect pathology due to schistosomiasis have also been increasingly used over the last 15 years. With the exception of ultrasound, these rather sophisticated techniques are performed in hospital settings. The advent of portable ultrasound devices allowed the technique to be carried into the field. The first experience was reported by Degrémont et al., (1985). This safe and non-invasive method has since proved to be feasible, relatively rapid for assessing pathology resulting from schistosome infections in surveys (Hatz et al., 1992a, b). It proved to be especially useful in investigating the resolution (Hatz et al., 1990) and reappearance of pathology following treatment (Hatz et al., 1998; Wagatsuma et al., 1999).
1.5.9 Urine filtration: Usually 10 ml of urine is filterd through a membrane (consisting of paper, polyamide (Nytrel) or polycarbon). Depending on the filter material, the membrane needs to be stained with one or two drops of a coloring solution (Nile-blue, lugol, eosin, hematoxylin or carbol-fuchsin). Then the membrane is scanned under a light microscope. The use of Nytrel filters (Plourier et al., 1975) has been recommended for large-scale community-based schistosomiaisis control programmes, as their cost is low and they can be re-used (Mott, 1983; WHO, 1985). However, in subsequent studies it was reported that a significant proportion of filter retained S. haematobium eggs even after thorough washing, which led to false positive results (Rohde et al., 1985,). Although this was questioned by Mott et al., (1995), alternative washing procedures were evaluated and boiling of filters for at least 5 minutes in tap water prior to washing was found to be a reliable method to remove all eggs, so that filters could be re-used (Mshinda et al., 1989). Many years ago, it was established that the excretion of S. haematobium eggs follows a circadian rhythm, with a peak and lowest variability being observed around noon (Bradley, 1963). Therefore urine specimens should be collected between 10am and 2pm. Infected individuals are classified as having light (<50 eggs/10 ml of urine) or heavy (≥50 eggs/10 ml of urine) infections (WHO, 1993).
1.5.10 Kato-katz thick smear: In the mid 1950s, Kato and Miura (1954) introduced the idea of faecal thick-smear examinations. The use of glycerol for the clearing of faecal material was described, which allowed to analyze larger stool samples than before. Some 20 years later, Katz and collaborators describe a modification of this initial method for the quantification of S. mansoni eggs in stool (Katz et al., 1972). It has since become the most widely used technique for the diagnosis of S. mansoni (WHO, 1998) and is also used in studies with S. japonicum (Yu et al., 1998) and S. mekongi (Stich et al., 1999). The procedure is relatively simple and can be summarized as follows: a small amount of fresh stool is sieved through a fine screen and filled into a hole of defined dimensions in a plastic template of standard thickness, placed on the center of a microscope slide. This provides a stool sample of known volume. After carefully removing the template, the faecal material is covered with a strip of cellophane, previously soaked in glycerol and malachite green. The microscope slide is inverted and firmly pressed against a hard surface, so that the sample is evenly spread. After clarification (time depending on the amount of faeces), the slide can be scanned under a microscope at low magnification (WHO, 1994). With the templates used by Katz et al., (1972), the measured amount of stool is approximately 43.7 mg. Subsequently, modifications were proposed with the aim of simplifying the preparation. These included using a thick glass cover slip instead of the cellophane and reducing the clearing time by using templates giving only 20mg samples (Peters et al., 1980). At present, different commercial kits are in the market, most commonly with 43.7mg or 25mg slides (Polderman et al., 1985). Egg counts can easily be converted into eggs per gram stool (epg) by multiplication by a factor of 24 or 40, respectively. According to the WHO classification (1993) there are three intensity levels: (i) light infection: 1-100 epg, (ii) moderate infection: 101-400 epg, and (iii) heavy infection: > 400epg. In population surveys, the geometric mean output is also frequently calculated and often used for the evaluation and monitoring of control proggrammes.
These two direct parasitological techniques – urine filtration and Kat-Katz thick smear –are considered ‘gold standard’ for diagnosis, especially when repeated specimens are analyzed, and the results obtained by alternative techniques are tested against them. The main advantages of both methods are their very high specificities, approaching 100%, their relative ease in execution, their direct applicability in the field and the fact that they provide quantitative results. In addition, faecal thick smears also allow the identification and quantification of concurrent helminth infections, such as hookworms, Ascaris lumbricoides, Trichuris trichiura and Taenia species.
Like all other diagnostic techniques, both filtration and Kat-Katz thick smear have also limitations. Firstly, the collection of specimens is tedious and may not be well accepted in certain cultures (especially the collection of stool). Secondly, only a very small amount of excreta is analyzed. Thirdly, there is intra-specimen variation in egg counts and even more importantly, day-to-day variation in egg-output (Engels et al., 2002). Therefore, analysis of a single specimen fails to estimate the true proportion of infected individuals, with light infections most likely to be missed (De Vlas and Gryseels, 1992, De Vlas et al., 1992, 1997). When emphasis is placed on individual diagnosis, analysis of several stool specimens over consecutive days is recommended (De Vlas & Gryseels, 1992). However, it is well acknowledged that such procedures fatigue study subjects, resulting in low compliance (WHO, 1998).
Besides urine filtration and Kat-Katz thick smears, there are some alternative approaches for the direct parasitological demonstration of eggs in excreta. The more traditional diagnosis of S. haematobium was urine centrifugation/ sedimentation. Although the method has a high sensitivity, which is even superior to that of filtrarion of 10ml urine samples in low intensity infections (Richards et al., 1984), it is not suitable for large-scale community screening because it is too labor-intensive and requires well- equipped laboratories.
Direct faecal smear are an alternative to Kato-Katz thick smears. The method is widely used in health facilities; smears are very easy to prepare and the slides can be read immediately under a microscope. Unfortunately, the amount of stool analyzed with a direct faecal smear is 10-20 times smaller than with a Kato-Katz thick smears and therefore the sensitivity for detection of S. mansoni eggs is considerably lower (Engels et al., 2002). It is often important to look for concurrent infections. Direct faecal smears are a good means to detect hookworm eggs.
Finally, there exist also some rather sophisticated concentration techniques. They are mainly used in specialized laboratories with skilled personnel. The sodium acetate-acetic acid-formalin method has a relatively low sensitivity for S. mansoni and geohelminths, but is reliable for diagnosing intestinal protozoa such as amoebiasis and giardiasis
1.5.11 Perceived disease markers: Over the last decade there has been a considerable amount of research on perceived morbidity indicators of schistosomiasis, with the aim of developing a procedure that would allow the rapid identification of communities at high risk of schistosomiasis (WHO, 1998). Excellent progress has been made with rapid screening of S. haematobium using simple questionnaires administered through schools. Based on these results, it was hoped that a similar questionnaire procedure might also be developed for S. mansoni.
1.5.12 Questionnaire for Schistosoma haematobium: Although blood in urine (haematuria) has been associated with S. haematobium for a very long time, it was only 15 years ago when Mott et al., (1995) assessed the usefulness of asking children in Ghana and Zambia about the presence of blood in urine as an indirect diagnosis of infection. However interviews bias and considerable variation in results between the two epidemiological settings raised concern about the reliability of this approach. Four years later a comprehensive school- questionnaire was presented for the first time which allowed the rapid identification of high risk communities for S. haematobium in a rural district in Tanzania (Lengeler, 1989; Lengeler et al., 1991a). The idea of asking school children about the presence of blood in urine was also used on Pemba Island, and it was found that this question was a sensitive indicator for heavily infected boys (Savioli et al., 1989). Consequently, the method was validated with success in a neighbouring district (Lengeler et al., 1991b), and also in seven other African countries in the framework of a WHO/TDR-supported multi-country study in Cameroon, Congo, Democratic Republic of Congo (formerly Zaïre), Ethiopia, Malawi, Zambia and Zimbabwe (Red Urine Study Group 1995). So far, with only one exception, Ethiopia (Jemaneh et al., 1996), highly significant correlations were found between the proportion of children reporting blood in urine, and the proportion of S. haematobium infected children.
It was concluded that school-questionnaires provide rapid and reliable results, are non-intrusive and highly cost-effective in the screening of S. haematobium at the community level. However, it was pointed out that further validation is mandatory, when significant changes have been made to the questionnaire or where strong arguments are required to convince health authorities about the usefulness of this method for a particular place.
In the case of Côte d’Ivoire, both these reasons applied. When the original questionnaire (Lengeler et al., 1991a, b) was presented to health authorities and school teachers, it was felt that it needed considerable modification before application an a larger scale. Furthermore, the national coo-ordinator for schistosomiasis control was interested in using questionnaires as a first step of the programme to identify priority areas, and a thorough validation of questionnaires was therefore requested to provide a solid foundation for later application on a national scale
1.5.13 Questionnaires for Schistosoma mansoni: There has been considerable diiscusion about the development of a similar questionnaire method for rapid identification of individuals and/or communities at highest risk of S. mansoni. It is widely acknowledged that this is a complex issue, because the signs and symptoms associated with infection and/or morbidity generally shows low sensitivities and specificities.
Several hospital and community-based studies have been conducted to assess the morbidity due to S. mansoni. They also provide information on the diagnostic value of signs and symptoms associated with infection of S. mansoni. These studies clearly revealed significant association between infections with S. mansoni and the presence of diarrhoea (especially bloody diarrhoea) and/or blood in the stool for most areas of sub-Sahara Africa (Arap Siongok et al., 1976; Hiatt, 1976; Omer et al., Haitt and Gebre-MEdhin ,1977; Abdel-Wahab et al., 1980; Sukwa et al., 1985; Gryseels and Polderman, 1987; Gryseels, 1988; Gryseels and Nkulikyinka, 1990; Kardorff et al., 1997). In addition to the blood in stool and (bloody) diarrhea, abdominal pain and colicky cramps were correlated with an S. mansoni infection.
However, another study in Zambia could not demonstrate any association between any one of the above symptoms and an infection with S. mansoni (Mungomba and Kalumba, 1995). No significant differences between infected and non-infected individuals were found in Brazil (Proietti and Antunes ,1989). Patra (1982) concluded for a variety of abdominal symptoms that they may be, but are not necessarily associated with S. mansoni.
Two recent studies carried out in Ethiopia (Hailu et al., 1995) and Tanzania (Booth et al., 1998) gave promising results for the use of reported blood in stool for rapid screening of S. mansoni. Unfortunately, these studies followed slightly different protocols, so care is needed in the comparison of the results. In view of all these findings, there was a clear need to design a series of studies that would elucidate whether questionnaires would be useful and could be recommended for the rapid screening of S. mansoni.
1.5.14 Indirect methods: As widely used for S. haematobium is the detection of blood and protein in urine using reagent stick. It is based on the fact that passing of eggs through the bladder wall causes damage, and due to these lesions small amount of blood and proteins are also released into the urine (Wilkins et al., 1979; Doehring et al., 1985). Reagent sticks are able to detect these small amounts of blood and protein in urine; therefore they can be used as an indirect indicator for an infection with S. haematobium. It has been shown that the amount of blood and protein in urine correlates with the intensity of egg excretion (Wilkins et al., 1979), so reagent sticks provide semi-quantitative results. Over the last 20 years, numerous studies have compared reagent sticks with urine filtration and their diagnostic performance was estimated. The use of reagent sticks has been shown to be reliable, but sensitivity and specificity values varied considerably from one endemic area to another (Wilkins et al., 1979; Mott et al., 1985; Savioli et al., 1989; Mtasiwa et al., 1996; Mafe, 1997). In view of these observations it was stressed that the diagnostic performance of reagent sticks needs to be assessed in every epidemiological setting before using this approach for large-scale community diagnosis (Feldmeier, 1993).
1.5.15 Prevention and control of human schistosomiasis: It has been described above that the epidemiology of schistosomiasis is characterized by five important features: complexity, heterogeneity in time and space, aggregation and sensitivity to environmental alteration. With these features in mind, it can be assumed that preventing and controlling the disease is a complicated issue and requires multifaceted approaches depending on an epidemiologically-specific mixture of interventions. Ideally, interventions should be specific to an endemic area with its observed transmission pattern, the predominant schistome-species and intermediate snail host(s), the sanitary facilities available to the population and their water contact patterns, as well as their socio-economic status. The overall aims of prevention and control of schistosomiasis can be summarized in five points.
1.5.16 Reduction of the number of schistosome eggs that are excreted by infected people: This is mainly achieved by chemotherapy with effective antischistosomal drugs. At present, this is the recommended strategy for schistosomiasis control, as it is directly targeted towards morbidity control (WHO, 1993). This strategy only became conceivable with the advent of safe and effective drugs, and at present praziquantel is the drug of choice. It is active against all schistosome species, can be administered in single oral dose, and has high cure and egg reduction rates, and more or only mild side effects (King et al., 1989; Kumar and Gryseels, 1994). Furthermore, it is relatively inexpensive. A series of field studies conducted in Senegal, in a community recently exposed to an intense focus of intestinal schistosomiasis (Gryseels et al., 1994; Stellma et al., 1995; Guissé et al., 1997) and laboratory data (Fallon and Doenhoff, 1994) raised considerable concern that praziquantel-tolerance and/or resistance may be developed. There are two effective alternative drugs – oxamniquine and metrifonate – but they have become difficult to obtain in some African countries (Cioli, 1998).
1.5.17 Reduction in the number of schistosome eggs reaching freshwater environment that harbour intermediate snail host(s): Health education with the ultimate aim of changing human behaviour is the key issue with regard to this objective (WHO, 1993). Specific teaching materials have been developed and evaluated in the field. It is of importance that these materials are well adapted to a particular epidemiological setting; otherwise it is likely that the message will not be understood. An example from Tanzania illustrates such a failure: children did not understand the cycle of schistosomiasis transmission, as the freshwater environment discussed was a pond, whereas the infested water source in their village was a small perennial river. Therefore, they continue to frequent the river, as they perceive no danger from this water source. It has been emphasized that teachers should play a key role in providing health education and they also showed great commitment in contributing to schistosomiasis control campaigns (Lengeler et al., 1991a, b; Red Urine Study Group ,1995; Magnussen et al., 1997). The provision and use of safe and adequate sanitary facilities should also be mentioned here, as it is an important contributor towards schistosome eggs not reaching water bodies.
1.5.18 Reduction of contact between miracidia and intermediate snail host(s): This depends on all the factors mentioned above, and also to a large extent on the reduction of the density of the intermediate snail host. For about 60 years, the snail was the main target for schistosomiasis control, and reducing the snail population is still an important component of control programmes. It was clear that transmission could be halted without the snail, and it was believed that the snail was the easiest link in the cycle to break (Fenwick, 1987). Several compounds toxic to the intermediate snail host have been discovered, but only niclosamide has emerged and remained commercially available in the market as a widely applied molluscicide (McCullough, 1986; Fenwick, 1987). In the 1950s and 60s, large-scale schistosomiasis control programmes were launched in Sudan (Sharaf el Din and El Nagar, 1955) and Egypt (Chu, 1976) with the use of molluscicides. Despite impressive reductions in incidence rates, the large-scale application of molluscicides was claimed to be expensive and of limited effectiveness (recolonization of snail immediately after application). It led to the killing of non-target organisms and was only applicable through skilled personnel (Klumpp and Chu, 1987). However, extensive experiences from several African countries revealed that focal mollusciciding is effective in most habitats (Klumpp and Chu, 1987). Efforts have also been put into the discovery and field validation of molluscicides derived from plant extracts. These include Phytolacca dodecandra (endod) in Ethiopia (Goll et al., 1983), Swartzia madagascariensis in Tanzania (Suter et al., 1986), and recently also with Jatropha curcas, so far only in the laboratory (Liu et al., 1997).
In view of the limitations associated with molluscicides, ecologically less risky approaches have attempted to reduce snail densities by environmental management or by means of biological control (Madsen 1990, 1992). Although methods of biological control of intermediate snail hosts are still under development, some promising results were obtained with the introduction of competitor snails (Pointier and McCullough, 1989; Pointier, 1993). Predators and parasites of intermediate snail hosts may become suitable bio-control agents; however as yet, virtually no research is focused on this topic (Madsen, 1990).
1.5.19 Reduction of the probability of human encountering cercariae: All three of the measures discussed above should contribute to this goal. In addition, it is of great importance for human to decrease the frequency and duration of their contacts with infested freshwater bodies (Kloos et al., 1997; Useh and Ejezie, 1999). This can only be achieved by changing human behavior, for example through increasing awareness about the mode of transmission and the impact of the disease in health education campaigns (Useh and Ejezie, 1999). Furthermore, the development of more satisfactory water supply schemes for domestic use should be promoted.
1.5.20 Damaging or killing of schistosomula: This is a novel approach which has only recently been discussed in the literature. The basis is given by the fact that after cercariae have penetrated the human skin, they become schistosomula. According to the schistosome species, it takes 4-8 weeks before they develop to adult egg-laying schistosomes (Clegg, 1965; Smit, 1976). Pathological changes due to schistosomiasis are caused by the excretion of eggs. Therefore, if schistosomula are prevented from developing into egg-laying adults, the human host will be protected. Theterm ‘chemoprophylaxis’ has been used to describe this concept. The drug artemether was found in a series of laboratory experiments with rabbits and dogs infected with S. japonicum to kill schistosomula more effectively than adult worm (Xiao et al., 1995, 1998). Artemether was already in wide use against malaria and was already known to have no or only few side effects (Klayman, 1985; White, 1994), so trial could safely be started in humans. In seven randomized control trials in China with more than 4,500 individuals exposed to S .japonicum, the prophylactic effect of artemether has been confirmed convincingly (Xiao et al., 2000a). To see whether similar effects were produced with S. mansoni, laboratory experiments with mice and hamsters were conducted and the prophylactic effect of artemether against this schistosome species could be confirmed (Xiao et al., 2000b). In view of these findings it was recommended to conduct a randomized double-blind placebo-controlled trial to assess the prophylactic effect of oral artemether to prevent Schistosoma mansoni infections.
1.5.21 Efficacy of praziquantel against schistosoma haematobium infection in children: Praziquantel (PZQ) is currently the drug of choice for the treatment of schistosomiasis and is rapidly becoming the only commercially available antischistosomal drug. It is highly effective against all five schistosome species that infect humans. With the advent of this safe drug, morbidity control has become the main stay of schistosomiasis control (WHO, 1993). Praziquantel has been extensively and successfully used in national control programmes in Brazil, China, Egypt and the Philippines, and there is little evidence of the development of clinically relevant resistance. The renewed impetus for extending schistosomiasis control throughout sub-Saharan Africa will probably result in greater use of PZQ than ever before. This welcomes development is set against the background of limited knowledge on many aspects of this drug.
A critical aspect in the assessment of PZQ efficacy is its activity against the different parasite developmental stages. Indeed, experimental laboratory studies have shown that the activity of PZQ is stage dependent. This drug is active primarily against the adult worm stages, whereas immature schistosomes (2-4 weeks old) are less susceptible (Gonner and Andres, 1977). Thus doses of drug that are curative against mature adult infections are sub curative against developing worms. In high transmission areas, the removal of adult worms by treatment will not result in normal levels of cure rate due to the development of immature worms into egg- producing adults by the time of the follow-up assessment of cure, as observed in Senegal for S. mansoni (Gryseels et al., 1994; Stelma et al., 1995; Picquet et al., 1998)
Despite the extensive use of PZQ against S. haematobium, which accounts for 67% of the schistosome infection in sub-Saharan Africa (WHO, 1998), we know very little on its efficacy against the immature stages of this species. A recent study of efficacy of PZQ against S. haematobium showed a low worm reduction at 4-weeks post-infection, i.e. 56% whereas high reductions were obtained at 8 and 12 weeks post-infection. A key point yet to be clarified concerns the activity of PZQ during the very long pre-patent period of this species, i.e. approximately 10-12 weeks compared with 5-7 weeks for S. mansoni. Therefore a study was conducted to compare a single treatment with PZQ with two or three treatment regimens at three weeks intervals. The objective of the repeated treatments was to determine the efficacy of PZQ against all development stages of S. haematobium. The children were expected to be infected with all stages because they were in contact with active transmission site before and during the study period. The cure rate and reduction in egg counts were monitored every three weeks over a nine-week period, i.e. within the pre-patent period of S. haematobium.
1.5.22 Antischistosomal properties of praziquantel: Praziquantel (2 – cyclohexylcarbonyl – 1,2,3,6,7,11b,- hexahydro – 4H – prazino [2,1 – a] Isoquinalin – 4 – one) was synthesized in the 1970s . While undergoing initial veterinary screening, it showed efficacy against Cestodes. With regard to schistosomes, efficacy was first demonstrated against S. mansoni in different host animals (Gonnert and Andrew, 1977). These findings were confirmed for all other schistosome parasites pathogenic for humans. The stage specific susceptibility of a Pueto Rican strain of S mansoni harbored in mice treated with three oral doses of Praziquantel on alternated day is depicted. Although only few replicates were used, these studies revealed that the invasive stages (cercariae and very young schistosomula) and the mature worms were affected more than the liver stages of the parasites. These findings were confirmed for S. japonicum. However, the underlying causes of the stage – specific susceptibility is far from being fully understood (Cioli, 1998).
Despite considerable efforts, the mechanism of action of praziquantel has yet to be fully elucidated (Andrew, 1985; Cioli, 1998). However, three central features are observed in schistosomes following the administration of praziquantel, and these effects are directly or indirectly associated with Ca2+ redistributions between worm tissues and the surrounding environments. First, worm motor activity is immediately stimulated followed by strong muscular contraction. Secondly, praziquantel induces extensive tegumental damage, commencing 5 min post-treatment. Third, treatment is accompanied by metabolic changes altering glycogen content and energy metabolism (Cioli, 1998). The drug includes a rapid shift of worms from the mesenteric veins to the liver, known as hepatic shift. In the mouse model, praziquantel efficacy depends on host antibody responses, and treatment increases the exposure of schistosome antigens at the worm surface. In humans, however, it proved difficult to identify the host related factors that influence the efficacy of praziquantel (Van Lieshout et al., 1995a). Pharmacokinetic studies found that orally administered praziquantel is rapidly and almost completely absorbed (Maximum concentrations serum are reached 1 – 2h – post-treatment) but undergoes extensive first pass liver clearance and prompt metabolic processing into inactive metabolites. The half – life of the unchanged drug in plasma is about 1 – 2h. Up to 80% praziquantel is reversibly bound to proteins. Praziquantel elimination is primarily through urine with about 80% of the drug cleared within 24h post-treatment (Cioli, 1998). The very encoourageing laboratory findings of the broad – spectrum anti-schistosomal activity of praziquantel were consistently confirmed in clinical trials designated to test the effect of the drug against the major human schistosome parasites and carried out in different epidemiological settings. Consistently, a single oral dose of 4. 0 mg of praziquantel per kg of body weight was safe, showed no or only a few but transient side effects and resulted in high parasitological cure and egg reduction rates. In view of these operational and therapeutic properties and gradually decreasing costs, it is not surprising that millions of people have been treated with praziquantel over the past 20 years. Many millions more patients suffering from schistosomiasis will be treated with this drug over the next several years (WHO, 1998)
Awareness about the possible development of praziquantel resistance is growing because of laboratory data and field observations. Experiments with mice revealed the possibility of selecting praziquantel tolerant schistosome strains after repeated administration of sub curative doses of praziquantel (Fallon and Doenhoff, 1994). Among other possible explanations, it has been speculated that antimicrobial resistance is responsible for the usually low cure rate in S. mansoni – infected patients from Senegal. In Egypt, patients failed to be completely cured of S. mansoni infections even after praziquantel had been administered three times, which is the most compelling evidence of praziquantel resistance to date (Fallon and Doenhoff, 1994).
1.5.23 Combination chemotherapy of schistosomiasis: In areas where schistosomiasis is highly endemic, the present goal to mitigate the burden of the disease is to control morbidity. Chemotherapy with Praziquantel is the main stay for morbidity control; e.g. it prevents chronic liver disease or bladder cancer (WHO, 1985). Praziquantel will certainly remain the drug of choice over the next several years since the 54 World Health Assemble recently put forth a target to treat at least 75% of school-age children in areas with high burdens of schistosomiasis with Praziquantel by 2010 . Metrifonate, a drug that exhibits activity against S. haematobium singly, has recently been withdrawn from the market because of medical, operational and economic criteria (Feldmeier, 1993). Oxamniquine is the only alternative antischistosomal drug, but its use is declining. Derivatives of artemisinin (e.g. artemether and artesunate), best known for their anti-malarial properties, also display activity against schistosomiasis, hence strategic discussions are under way on how this evidence base can be translated into second public health action (Utzinger et al., 1997; Xiao et al., 2000b).
Against this background of virtually relying on a single drug for the treatment and control of schistosomiasis (Cioli, 1998), and considerable concern about the development of praziquantel resistance (Fallon and Doenholf , 1994), it is timely to critically review potential alternatives. Research and development of novel antischistosomal drugs are warranted (Engels et al., 2002), but this will become feasible only through the creation of innovative and committed public – private partnerships, including academic and the pharmaceutical industry. In the interim, present morbidity control approaches need to be improved and optimized alongside concerted efforts to prolong the useful life span of praziquantel. In this regard, the combination of Praziquantel with Oxamniquine or an artemisinin derivative might be an obvious and appealing option.
Below is a brief summary of the results from laboratory studies and field trials of combination chemotherapy for the treatment of schistosomiasis by Engels et al., (2002) The properties of Praziquantel, Oxamniqunine, and artemisinin derivatives including species and stage specific susceptibilities, possible mechanisms of action, pharmacokinetics and pharmacodynamics, toxicologic parameters, and experience from clinical use are reviewed. The evidence obtained thus far from in vivo experiments and clinical trials that used praziquantel together with Oxamniquine or an artemisinin derivative are also summarized. The most urgent needs for new tools and approaches for schistosomiasis chemotherapy that will be critical as part of an integrated and sustainable control approach to alleviate the present intolerable burden of schistosomiasis are highlighted.
1.5.24 Praziquantel and oxamniquine combination: The first series of laboratory experiments with adult S. mansoni worms harbored in mice treated simultaneously with praziquantel and oxamniquine reported encouraging results, since treatment outcomes were superior to these expected by simply adding the effects of each drug used singly . Additionally, laboratory testing with combined low doses of praziquantel and oxamniquine (one – third of the curative dose) against different developmental stages of S. mansoni showed only slightly higher worm burden reductions than those achieved with praziquntel administered alone at curative doses. The combination regimen showed the highest efficacy when it was administered 4h post – infection.
Two field trials with praziquantel and oxamniquine combinations were carried out shortly after the encouraging laboratory results were published .Both trials were randomized and non-blinded and were designed as dose – ranging studies, with the dose used being considerably lower than those recommended if either drug is used alone. No direct comparisons were made with praziquantel or oxamniquine mono – therapy as most of the study participants were concurrently infected with S. mansoni and S. haematobium, and the therapeutic efficacies were evaluated 1, 3 and 6 months post – treatment for both parasites separately. The first study was carried out with school children from Malawi aged 6 to 20 years. Complete data records were obtained for 102 children with S. mansoni infection and 56 children with S. haematobium infection. For those with S. manssoni infections, there were a clear trend toward higher egg reduction rates with increasing doses, egg count reductions of 99% or more were found after concurrent administration of 15 to 20 mg of praziquantel per kg and 7.5 to 10 mg of oxamniquine per kg. The combined treatment also resulted in very high S. haematobium egg count reduction. It was safe and there were only a few side effects, which were self limiting. The investigators concluded that they had demonstrated a synergistic effect of praziquantel and oxamniquine. Care in the acceptance of this conclusion is needed because (i) the sample sizes in the different treatment groups were small (22 to 23 subjects per group). (ii) about half of the study participants were concurrently infested with S. mansoni and S. haematobium. (iii) egg count with reductions were used as the only end point, (iv) infection intensities were high prior to drug administration, and (v) only a single stool or urine specimen was examined 1 month post-treatment. In light of the recent findings obatained in different epidemiological settings, one can argue that some ligh infections were missed hence; therapeutic efficacies were over estimated (Engels et al., 2002).
The other study was carried out among 58 schoolchildren from Zimbabwe aged 7 to 16 years. All children were concurrently infected with S. mansoni (> 100 eggs/g of stool) and S. haematobium (> 500 eggs/10 ml of urine). Three different dose combinations were investigated while a high cure rate of 89% for those with S. mansoni infections was achieved with the highest doses (20 mg of praziquantel per kg and 10 mg of oxamniquine per kg), combination chemotherapy failed to cure S. haematobium infections. High egg count reductions were found for both parasite species. It was concluded that the combination of praziquantel and oxamniquine for the treatment of schistosome infections in Zimbabwean schoolchildren has no curative advantage over praziquantel alone. The design of this study has limitations similar to those of the investigation carried out in Malawi, namely (i) the sample sizes in the different treatment groups were small (10 to 30 subjects per group). (ii) the study participants were concurrently infected with two schistosome species and (iii) the infection intensities were high prior to treatment. On the other hand, study endpoints included both cure and egg reduction rates, and the therapeutic efficacy was assessed by screening multiple stool or urine specimens.
1.5.25 Combinations with praziquantel and artemisinin derivatives: Since praziquantel and artemether display broad spectrum antischistosomal activities and the susceptibilities of the different stages to the two drugs are distinctively different,. It has been suggested that use of these two compounds in combination might be beneficial for the treatment of infections caused by all human schistosome species. Hence, use of the combination may increase worm burden reductions and, as a consequence, augments cure and egg reduction rates.
Two laboratory studies have been published to date that comparatively assessed worm burden reduction after administration of praziquantel and artemether singly or in combination. The two series of experiments used different host-parasite models and found consistently higher worm burden reductions following treatment with the combination regimen compared to those achieved with the monotherapies. In the first set of experiments, rabbits simultaneously infected with juvenile and adult S. japonicum worms were treated with 50 mg of praziquantel per kg and 15 mg of artemether per 1kg 1 day apart. The treatment resulted in a worm burden reduction of 82%. This was significantly higher than the reductions achieved after the administration of praziquantel (66%) or artemether (44%) singly at the same dose (Xiao et al., 2000a). These results were confirmed with rabbits infected only with adult S. japonicum (Utzinger et al., 1997). In the second set of experiments, hamsters with a mixed infection with juvenile and adult S. mansoni worm were simultaneously treated with 75 mg of praziquantel per kg and 150 mg of artemether per kg. The worm burden reduction of 77% was significantly higher than the 2% reduction achieved following praziquantel monotherapy (P < 0.01) but was not significantly different from the 66% reduction obtained with artemether against S. haematobium. There is also a need for comparative appraisal of schistosome worm burden reductions following treatment with praziquantel in combination with different derivatives of artemisinin. So far, the praziquantel and artemether combinations have not undergone clinical trials. However, a combination of praziquantel and artesunate was used in a non -blinded open label treatment trial with S. mansoni in Senegal and a randomized controlled clinical trial with S. haematobium in Gabon. The study in Senegal enrolled a total of 110 S. mansoni infected patients aged 1 to 60 years. The patients were treated with either praziquantel (single oral dose of 40 mg/kg), artesunate (recommended malarial treatment regimen, total oral dose of 12 mg/kg, and initial dose of 4 mg/kg, followed by four daily doses of 2 mg/kg), or a combination of these two drugs at the doses mentioned above. Cure and egg reduction rates were evaluated 5, 12 and 24 weeks post-treatment by examination of two Kato- Katz thick smears derived from a single stool specimen. It is widely acknowledged that Northern Senegal is one of the most intense S. mansoni transmission zones; thus, reinfections must occur rapidly. Therefore only the therapeutic efficacies achieved at 5 weeks post-treatment are considered here. The praziquantel and artesunate combination resulted in cure and egg reduction rates of 69% and 89% respectively. While this cure rate was significantly higher than the ones observed after praziquantel and artesunate monotherapies, the egg reduction rate was similar to the one achieved following praziquantel treatment (84%) (Xiao et al., 2000b).
The study in Gabon was randomized, double-blind, placebo-controlled trial and enrolled 296 S. haematobium infected children aged 5 – 13 years. Four different treatment were administered: (i) a single oral dose of 40 mg of praziquantel per kg plus three daily oral doses of 4 mg of artesunate per kg (total dose 12 mg/kg corresponding to the recommended treatment for malaria). (ii) Praziquantel plus a placebo (iii) artesunate plus a placebo, and (iv) a double placebo. Therapeutic efficacy was evaluated 8 weeks post-treatment by determination of quantitative egg counts from two consecutive urine specimens. The highest cure rate of 81% (95% confidence interval [Cl], 72% to 89%) was observed among those children who received both praziquantel and artesunate. However this was not significantly different from the cure rate obtained after praziquantel monotherapy (73%: 95% Cl, 64% to 82%). Furthermore, the cure rate of 27% (95% Cl, 18% to 36%) in the artesunate monotherapy group was not significantly different from that in the placebo group (20%; 95% Cl, 5% to 35 %). The investigators acknowledged that their finding of a 20% cure rate among the placebo recipients is difficult to explain and might simply be attributable to day to day variation in S. haematobium egg out put. In terms of egg count reductions, the praziquantel and artesunate combination was beneficial and the 99% reduction was significantly higher than that for all other groups (Engels et al., 2001). This is an important finding, since morbidity generally correlates with infection intensities.
1.5.26 Other combinations: A recent laboratory study investigated a possible additive or synergistic effect of the combination of praziquantel and RO 15 – 5458 (a relatively new antischistosomal drug developed by Hoffman La Roche, Basel, Switzerland) against two different strains of Schistosoma mansoni in mice. The treatment outcomes achieved with a single curative dose of praziquantel or RO15 – 5458 were compared with those achieved with the drugs used in combination at doses that were one – third of the curative doses of the drug used singly. The investigators found that treatment with the combination at these low doses was beneficial with regard to worm burden reduction and hepatic shift. These findings warrant further laboratory investigations, including investigations of mutagenicity and carcinogenicity, as a basis for possible clinical trials with human (Cioli, 1998).
1.5.27 Antischistosomal properties of oxamniquine: Oxamniquine (6 – hydroxymethyl – 2 – isopropyl – aminomethyl – 7 – nitro – 1,2,3,4 – tetrahydroquinoline) was first described in the late 1960s. In contrast to praziquantel which displays activity against all human schistosome species, the activity of oxamniquine is confined to S. mansoni (Cioli, 1998,). The stage – specific susceptibility of S. mansoni to oxamniquine exhibits a pattern very similar to that for susceptibility to praziquantel. The invasive stages and the adult worms are significantly more affected than the liver stage . Adult male worms are considerably more affected by oxamniquine than the adult females (Foster et al., 1971), and central and East African strains of S. mansoni are significantly less susceptible than South American strains.
The mechanism of action of oxamniquine is reasonably well understood and the body of evidence suggests that it is closely associated with an irreversible inhibition on the nucleic acid metabolism of the parasites. The following working hypothesis has been put forth: the drug is activated by a single step, in which a schistosome enzyme converts oxamniquine into an ester (probably acetate, phosphate or sulphate). Subsequently, the ester spontaneously dissociates, while the resulting electrophilic reactant is capable of alkylation of schistosome DNA (Cioli et al., 1995). In contrast to treatment with praziquantel, treatment with oxamniquine results in less specific morphological alteration, and the hepatic shift occurs much more slowly and is completed only 6 days post-treatment. Pharmacokinetic studies with human administration of oxamniquine revealed many similarities to praziquantel. Oxamniquine is rapidly adsorbed (Peak concentrations in Plasma are reached 1 – 4h post-treatment), and the half – life is 1.5 – 2h. (Homeida et al., 1988). Oxamniquine is extensively metabolized through oxidation processes. Metabolites are inactive and are almost exclusively excreted through urine (Cioli et al., 1995).
Oxamniquine at a single oral dose of 15 – 20mg/kg shows high degree of efficacy in South America, the Caribean Islands, and West Africa, while higher doses (up to 60 mg/kg given over 2 – 3 days) are required to obtain the desired therapeutic efficacies in Central and East Africa and the Arabian Peninsula. Oxamniquine is safe and side effects are limited to mild but transient dizziness (Foster, 1987). Large scale application of oxamniquine, sustained over the past two decades, has shown great success in S. mansoni control in Brazil (Katz 1998). Although S. mansoni – infected patients resistant to oxamniquine have been described repeatedly these findings are of no public health significance thus far. Despite the success of oxamniquine for schistosomiasis control in Brazil, efforts are under way to replace it with praziquantel, primarily because of the lower price of praziquantel (Becck et al., 2001; Peich and Fennick, 2001).
1.5.28 Antischistosomal properties of artemisinin derivatives: Artemisinin, a sesquiterpene llactone with a peroxide group is the active principle derived from the leaves of Artemisia annua and is best known for its anti-malarial properties. Several semi-synthetic derivatives with even higher anti-malarial activities were developed, namely artecther, artemether, artesunate, and dudydroartemisinin. The initial work was done in the early 1970s and over the last decade, derivatives of atemisinin have gained tremendously in importance for the treatment and control of malaria. It is anticipated that their popularity will further increase, particularly also in combination with other anti-malaria drugs with unrelated mechanisms of action (White, 1994; Nosten and Brasseur, 2002).
The anti – schistosomal activites of artemisinin, artemether, and artesunate were discovered in the early 1980s with the initial experiments focusing on S. japonicum. More recent studies confirmed that arteether and sihydroartemisinin also display antischistosomal properties (Xiao et al., 1995). Laboratory experiment conducted so far in different animal models found that artemether is active against the three major human schistosome parasites (Xiao et al., 2000b). The stage – specific susceptibility of a Liberian strain of S. mansoni harbored in mice treated with a single oral dose of artemether or very similar stage – specific susceptibilities have also been reported for S. japonicum (Xiao et al., 1995). In contrast, praziquantel and oxamniquine, exhibits the highest level of activity against 1 -3 weeks old liver stage while the invasive stage and the adult worms are less susceptible. Adult female worms are somewhat more susceptible to artemether than the male worm (Utzinger et al., 2001), which is opposite the activity of oxamniquine. There is a need for additional in vivo experiments to assess the stage – species and stage-specific susceptibilities of schistosomes to other artemisinin derivatives. A first comparative appraisal revealed that artemether exhibits consistently higher level of activity against S. mansoni parasites of different ages than artesunate (Utzinger et al., 2002).
The exact mechanism of action of artemether against schistosomes remains elusive, but progress has been made in recent years. A typical biochemical feature is that following artemether treatment, adult worms showed significant reductions in their glycogen content (Xiao et al., 2000a). As with praziquantel, artemether also induce severe and extensive tegumental damage; however, the onset of tegumental alterations is considerably slower (Utzinger et al., 2001). Another important finding is that in vitro exposure of schistosomes to a medium containing artemether plus hemin results in parasite death, while exposure to artemether or hemin alone showed no effect. Therefore, it has been suggested that artemether might be activated by hemin and sequentially cleaves the endoperoxide bridge and generates free radicals that might form covalent bonds with schistosome – specific proteins.The hepatic shift commences within 8h after artemether administration and is completed within 7 days (Xiao and Catto 1989); hence, it is much slower than after praziquantel treatment but only somewhat slower than that after oxamniquine ttreatment.Oral formulations of artemisinin derivates are absorbed rapidly but incompletely (the peak concentrations of most artemisinins in plasma are reached 1 to 3h post-treatment) and have short half-lives in plasma of 1 to 3h before undergoing hepatic metabolism. Dihydroartemisinin is the principal active metabolite (Mordi, 1997). In vivo studies in different animal models revealed brain stem neurotoxicity after repeated treatment with high doses of some artemisinin derivatives over at least 7 days (Genorese, 2000). However, repeated treatment with high doses of artemether once every 2 weeks, the recommended dosees schedule for the prevention of S. japanicum infection, revealed no nneurotoxicity. Most importantly, there is no clinical evidence of neurological lesions, although several million people have been treated with artemisinin derivatives for malaria (Price et al., 1999). To date, clinical testing of artemether for the prevention of schistosomiasis included 2,670 people who received oral artemether at a dose of 6 mg/kg once every 2 to 4 weeks for period of up to 6 months. Artemether was safe, showed no or only a few but transient side effects, and was efficacious in reducing the incidence and intensity of infection (Xiao et al., 2000b).
The application of drugs in combination is not a new concept. In fact, the rationale for the use of combination chemotherapy was first developed in the treatment of bacterial infections such as tuberculosis and has subsequently been adapted for chemotherapy for cancer and human immunodeficiency, virus infection and AIDS, primarily to delay the emergence of drug resistance. Additional momentum has recently been gained in the field of malaria chemotherapy. Experience from South-East Asia suggests that the combination of an artemisinin derivative with another anti-malarial drug with an unrelated mechanism of action as an effective strategy for prevention of the emergence and spread of drug resistance and might have contributed to the interruption of the transmission of falciparum malaria (Nostern and Brasseur, 2002). It has been recommended that this kind of combination be evaluated in Sub – Saharan Africa, where it might become the mainstay of malaria chemotherapy. With regard to combination chemotherapy for schistosomiasis, the partner drugs should have different mechanisms of action to reduce the likelihood of resistance development and/or target different developmental stages of the parasite to enhance cure and egg reduction rates. In case the two drugs exhibits synergism, it might be possible to achieve the same or even higher levels of efficacy by using smaller doses of either agent, which might also result in fewer or milder side effects. In view of the present rmamentarium against schistosomiasis, praziquantel could be combined with either oxamniquine or an artemisinin derivative. Interestingly, initial laboratory experiments and preliminary clinical trials with praziquantel and oxamniquine combinations were already launched two decades ago. There is new interest in the combinations and the UNDP/World Bank/WHO special programme for Research and Training in Tropical Diseases has recently called for proposals to evaluate its tolerability and efficacy against S. mansoni.
1.5.29 Immune response in schistosomiasis: Most chronic morbidity in schistosomiasis is not due to the adult worms but is related to the T-cell-dependent immune response of the host, which is directed against schistosome eggs trapped in tissues, mainly in the liver and intestines in the case of the intestinal forms (S. japonicum and S. mansoni) and in the bladder in the case of S. haematobium. The trapped eggs secrete a range of molecules leading to a marked CD4+ T-cell programmed granulomatous inflammation involving eosinophils, monocytes, and lymphocytes, akin to a form of delayed-type hypersensitivity. Granulomas are also characterized by collagen deposition, and with the intestinal schistosomes, severe hepatic periportal (Symmer’s) fibrosis occurs. Much of the morbidity and mortality associated with this disease is attributable directly to the deposition of connective tissue elements in affected tissues. In mice, a predominantly T-helper 1 (Th1) reaction in the early stages of infection shifts to an egg-induced Th2-biased profile, and imbalances between these responses lead to severe lesions (Wilson et al., 2007). A notable accomplishment in the past few years was the identification of interleukin-13 (IL-13) and the IL-13 receptor complex as central regulators of disease progression in schistosomiasis (Reiman et al., 2006,). Similar regulatory control could be at the basis of fibrotic pathology in humans (Abath, 2006), although this has not yet been established.
1.5.30 Effector mechanisms and expression of immunity in animal models of schistosomiasis: A number of recent reviews have considered the immunobiology of schistosomiasis, including the nature of the host innate and adaptive responses to schistosomes and strategies used by the parasites to manipulate such responses (Abath, 2006, Capron et al., 2005; Mentink-kane and Wynn, 2004; Pearce, 2005; Pearce and Macdonald, 2002; Stadecker et al., 2004; Wynn et al., 2004). Much of our understanding of the mammalian immune response to schistosomes is based on the use of gene-disrupted (knockout) mice (Freeman et al 2006; Layland et al., 2005; Reiman et al., 2006; Taylor et al., 2006; Wynn et al., 2004) and the immunization of mice, nonhuman primates, or other mammalian hosts with UV- or -irradiated cercarial vaccines, with or without a subsequent challenge infection with non-attenuated cercariae (Bickle et al., 2001; Eberi et al., 2001, Hewitson et al., 2005; Kariuki and Farah 2005; Kariuki et al., 2006; Sato and Kamiya, 2001; Torben and Hailu, 2007). The attenuated larvae fail to mature into adult worms and do not produce eggs, so any results obtained are not confounded by egg-induced liver pathology. An even greater effect of triggering high-level resistance against schistosome reinfection has been shown for mice treated with artemether, a methyl ether derivative of dihydroartemesinin, followed by challenge (Bergquist et al., 2004). This model may provide an alternative approachto irradiated vaccines for dissecting different immune responsesas putative effector mechanisms during schistosome infectionand protective responses against reinfection.
In general, these studies have established that T-cell-mediated immunity is fundamental to acquired resistance to schistosomes in mice. Much of this protection was shown to be mediated by activated macrophages and, together with studies of cytokines, suggested that a vaccine that induced macrophage-activating Th1 cytokines (gamma interferon [IFN- ] and IL-2) may be beneficial in preventing schistosomiasis. However, repeated vaccination with irradiated cercariae produced incremental increases in Th2-mediated (IL-4 and IL-5 predominance) protection, which was transferable to non-vaccinated animals. Studies using B-cell-deficient and cytokine-deficient mice demonstrated that successful antischistosome vaccination required induction of strong Th1 and Th2 responses. Following infection by normal or radiation-attenuated cercariae, the predominant early immune response was Th1 mediated and aimed at the adult worm. Following egg deposition in tissues (at 6 weeks post infection for S. mansoni and 4 to 5 weeks post infection for S. japonicum), the Th1 response was diminished, being replaced by a prominent Th2-mediated phase. Indeed, it appears that egg antigens are able to directly suppress the Th1 response (Pearce et al., 1991, Pearce and Macdonald, 2002), a phenomenon which may also occur in humans. The Th2 response results in an increase in serum IL-5, massive bone and blood eosinophilia, and a granulomatous response aimed at the egg, resulting in collagen deposition, tissue fibrosis, and the disease manifestations of schistosomiasis. The precise role of eosinophils in the disease process in the mouse model of infection remains undetermined (Swartz et al., 2006). The complexity of immune regulation and T-cell regulation in schistosome infection in mice is well recognized (Mckee and Pearce ,2004; Pearce, 2005; Taylor and Pearce, 2006; Wilson et al., 2007), and this was further illustrated by a recent study by Walsh et al.,(2007), who highlighted a specific role for CTLA-4+ but not CD25+ cells in the regulation of Th2 responses in helminth infection. Furthermore, whereas the cytokine interplay during the development of protective immunity to the radiation-attenuated (RA) schistosome vaccine has been characterized extensively over recent years, the role of co- stimulatory molecules in the development of cell-mediated immunity is much less well understood. The importance of CD40/CD154 in vaccine-induced immunity was recently demonstrated (Hewitson et al., 2007), as it was shown that CD154–/– mice exposed to RA schistosomes developed no protection to challengeinfection, suggesting that protective immunity to the RA schistosomevaccine is CD154 dependent but is independent of (IL-12 orchestrated)cellular immune mechanisms in the lungs.
In the case of S. japonicum, zoonotic transmission adds to the complexity of S. japonicum control programs but provides a unique opportunity to develop a transmission-blocking veterinary vaccine to help prevent human infection and disease. However, studies of protective immunity in bovine schistosome infections are few (Mcmanus and Dalton, 2006), and consequently, our knowledge of the immunology of schistosome infections in buffaloes and cattle is extremely limited. This is particularly the case for water buffaloes, for which immunological reagents for studying immune responses are scarce. Recent PZQ treatment and reinfection studies of bovines infected with S. japonicum in China have indicated that age-related resistance occurs in buffaloes but not cattle (Wang et al., 2006). Whether this self-cure phenomenon has an immunological basis has yet to be determined. Additional studies on the immunology of buffaloes and cattle represent an important area for future research and will be essential in selecting S. japonicum vaccineantigens and in defining the optimum route of immunization.
1.5.31 Effector mechanisms and clinical expression of immunity in human schistosomiasis: Numerous longitudinal cohort studies of reinfection rates following curative drug treatment have shown that people living in areas where schistosomes are endemic acquire some form of protective immunity after years of exposure to S. mansoni, S. haematobium, or S. japonicum (Ross et al., 2001; Capron et al., 2002; Ross et al., 2002; Mountford, 2005; Gryseels et al., 2006). However, age-related innate resistance mechanisms may also play an important part in the epidemiology of schistosomiasis (Capron, 2005; Gryseels et al., 2006). Immune correlative studies in various parts of the world suggest that acquired antischistosome protective immunity after curative drug therapy is mediated (although not exclusively) by a Th2 response, orchestrated by immunoglobulin E (IgE), against adult and larval antigens which stimulate eosinophils to release cytotoxins targeting schistosomula (Capron et al., 2002; Capron et al., 2005; Gryseels et al., 2006). Despite the protective role of IgE, high levels of IgG4 are also produced during infection, potentially blocking the protective effects of other immunoglobulins (Caldas et al., 2000). Subsequently, it was shown that immunity to reinfection is more closely related to the IgE/IgG4 balance than to the absolute level of each isotype (Caldas et al., 2000). The opposing effects of IgE and IgG4 could not be dissociated in the analysis, indicating that these isotypes probably antagonize each other in terms of protection (Caldas et al., 2000). Although both IgE and IgG4 responses initially depend on IL-4 and IL-13 production, the production of IgG4 antibodies is regulated in an antigen-specific context by IL-10 and IFN- produced by Th0 cells (Caldas et al., 2000). This supports the view that IgE and IgG4 can be dissociated, in spite of their reported dependence on IL-4. The putative role of IL-10 in the preferential induction of an IgG4 response should be placed in the broader perspective of the general properties of this cytokine. Indeed, it is now well established that IL-10 prevents antigen-presenting cell-dependent IgE synthesis and that IgE-dependent cytokine release from host cells causes activation of eosinophils as well as IL-5 release (Caldas et al., 2000). The clinical expression of immunity to schistosome infection is obviously not determined simply by the mere balance between IgE and IgG4 antibodies. One cannot exclude the participation of additional mechanisms, such as a potential protective role of IgA antibodies in human schistosomiasis, supported by a series of correlation studies from several parts of the world; the effector functions of IgA antibodies may be associated with a decrease in female worm fecundity and egg viability (Caldas et al., 2000). In our opinion, the development of a vaccine for schistosomiasis that is dependent on IgE would potentially be problematic and would likely be impeded by regulatory and safety issues due to potential anaphylaxis induced by vaccination. Therefore, looking to the immune responses of chronically infected individuals, and even those who become refractory by producing IgE after drug treatment, should be approached with caution. Perhaps the most important clue of all towards understanding protective immunity to schistosomiasis is the naturally acquired immunity displayed by some individuals in Brazil in the absence of prior drug treatment (Coirrea-Oliveira et al., 1989, Viana et al., 1994, Viana et al., 1995 Capron et al., 2002). This small but well-defined cohort is referred to as endemic normals (Correa-Oliveira et al., 2000) or, more recently, putative resistant (PR) individuals (Tran et al., 2006). These individuals are resistant to infection despite years of exposure to S. mansoni and are defined as follows: (i) negative for over 5 years for S. mansoni infection based on fecal egg counts, (ii) never treated with antihelminthic drugs, (iii) continually exposed to infection, and (iv) have maintained vigorous cellular and humoral immune responses to crude schistosome antigen preparations (Correa-Oliveira et al., 1989; Viana et al., 1994; Viana et al., 1995; Correa-Oliveira et al., 2000 ). PR individuals mount vigorous but very different (compared to those of chronically infected patients) immune responses to crude S. mansoni extracts from schistosomula (using detergent to solubilize the tegument) and adult worms (Viana et al., 1995, Viana et al., 1999, Caldas et al., 2000). In response to stimulation with these antigens, peripheral blood mononuclear cells from PR individuals secrete both Th1- and Th2-type cytokine responses (Bahia-Oliveira et al., 1996, Caldas et al., 2000), while chronically infected individuals make a Th2-type response (Roberts et al., 1993). It is the Th1 response (particularly IFN- ) to schistosomulum antigens that is thought to be the key to resistance to schistosomiasis in these subjects (Correa-Oliveira et al., 2000). Indeed, recent studies described the use of PR individuals to select two new vaccine antigens that are expressed in the tegument membrane of S. mansoni, namely, SmTSP-2 (Tran et al., 2006) and Sm29 (Carados et al., 2006). Both proteins were preferentially recognized by sera from PR individuals as opposed to sera from chronically infected patients, supporting the potential of the PR immune response to guide discovery of tegument plasma membrane proteins as recombinant vaccines (Loukas et al., 2001).
Although the immune responses of resistant cohorts have been characterized, very little is still known about the protective mechanisms required to engineer an efficacious recombinant vaccine for human schistosomiasis. Contrasting and conflicting data have been presented from the mouse model and from human field studies. For example, activation of predominantly Th1 cells by schistosomulum antigens correlates with naturally acquired protection of PR individuals (who are exposed to the parasite but are not infected and have never been treated with PZQ) (Correa-Oliveira et al., 2000). On the other hand, partial resistance can be induced in some adult individuals with repeated PZQ treatment, and this correlates with a predominantly Th2 response (Walter et al., 2006). In mice, recombinant vaccines conferring various levels of protection induce different immune response phenotypes. This is influenced at least in part by the properties of the adjuvant used or the intrinsic immunogenicity of the respective proteins, but a general consensus is lacking. Studies using the RA cercaria model in mice suggest that protection can be induced with either a mixed Th1/Th2 response, a polarized Th1 response, or even a polarized Th2 response (Hewitson et al., 2005). Given that antibodies alone can confer protection in this model (Jankovic et al., 1999), perhaps the phenotype of the response and even the isotype/subclass of antibody produced, is not of prime importance. Most commercially available vaccines rely specifically on the induction of neutralizing antibodies that block the function of their target protein(s). This appears to also be the case for other helminth vaccines that are showing promise in preclinical studies, where neutralizing antibodies block proteins that have pivotal roles in tissue migration or digestion of the blood meal (Loukas et al., 2006).
An understanding of immune regulation in human schistosomiasis is essential if schistosome vaccines are to be delivered to previously infected individuals. As emphasized above, experimental schistosome infections of laboratory animals, particularly mice, have contributed significantly to our understanding of the immunobiology of infection, particularly the mechanisms associated with egg-induced granuloma formation and subsequent fibrosis (Abath et al., 2006). Immune mechanisms elucidated in experimental models of schistosomiasis are not easily investigated in humans for ethical and logistical reasons, so available knowledge on human responses to schistosomes falls far short of what is known for mice (Abath et al., 2006). Furthermore, caution is required in extrapolating and interpreting results from murine experiments because, in many respects, the infection is dissimilar to the clinical situation, where there are a number of potentially confounding factors relating to exposure, infection/reinfection, coinfections, host and parasite genetics, nutritional status, and environmental modifiers that cannot be controlled, or even adequately assessed (Abath et al., 2006). Studies undertaken with experimental models of schistosome infection, need to be validated fully in humans, which will prove challenging, as the immune regulatory mechanisms operating are clearly so complex. Nevertheless, there is accumulating evidence indicating that at least some features of the immune response evoked in infected humans are similar to those in mice (Abath et al., 2006).
1.5.32 Strategies for antischistosome vaccine development: Schistosomes do not replicate within their mammalian hosts. Consequently, a nonsterilizing naturally or vaccine-acquired immunity could significantly decrease human pathology and disease transmission. Vaccination against schistosomes can be targeted towards the prevention of infection and/or to the reduction of parasite fecundity. A reduction in worm numbers is the “gold standard” for antischistosome vaccine development, with the migrating schistosomulum stage likely to be the major vaccine target of protective immune responses (McManus ,2005; Wilson and Coulson, 2006). However, as schistosome eggs are responsible for both pathology and transmission, a vaccine targeted at parasite fecundity and egg viability also appears entirely appropriate. While they regularly induce 50 to 70% (over 90% in some cases) protection in experimental animals and additional immunizations boost this level further, it may be premature to pursue RA schistosome vaccines for human use, but their development for veterinary application is feasible. Although technically challenging, there is a case for promoting the development of a live, attenuated, cryopreserved schistosomulum vaccine for use against S. japonicum in buffaloes to reducezoonotic transmission to humans in China (Mcmanus 2005). If successful,the veterinary vaccine could provide a paradigm for the developmentof antischistosome vaccines for human use.
In addition, while the S. mansoni RA vaccine model has enabled the dissection of different immune responses as putative effector mechanisms (Hewitson et al., 2005) and raised hopes for the development of molecular vaccines, this has not equated to advances in the development of recombinant vaccines. Independent testing of six candidate S. mansoni antigens (glutathione S-transferase 28 [Sm28-GST], paramyosin, Ir-V5, triose-phosphate isomerase, Sm23, and Sm14) in the mid-1990s, orchestrated by a UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR/WHO) committee, resulted in protective responses being recorded, but the stated goal of consistent induction of 40% protection or better was not reached with any of the antigens tested, highlighting the possible negative influence of insufficient antigen stability and the need for standardized and effective adjuvant formulations (Bergquist et al., 2004). Furthermore, of these six antigens, only one (Sm23) is exposed on the apical membrane surface of the parasite (Wright et al., 1991), although it is not one of the more abundant apical membrane proteins on the parasite surface (Braschi et al., 2006). Also, the failure to develop an efficacious schistosome vaccine can be attributed in part to the complex immunoevasive strategies used by schistosomes to avoid elimination from their intravascular environment (Pearce and MacDonald 2002). Nevertheless, convincing arguments still support the likelihood that effective vaccines against the various schistosome species can be developed (Bergquist et al., 2004); first, as discussed above, irradiated cercariae regularly induce high levels of protection in experimental animals, and additional immunizations boost this level further; second, as has been emphasized, endemic human populations develop various degrees of resistance, both naturally and drug-induced; and third, veterinary antihelminth recombinant vaccines against cestode platyhelminths have been developed successfully and applied in practice (Craig et al., 2007). The optimism sparked by these arguments has resulted in the discovery of a large number of schistosome antigens (utilizing the almost-complete genome sequence), and additional candidates are now being found through proteonic approaches (Braschi et al., .2006, Braschi and Wilson 2006). However, antigen identification and successful protective results are of little value if recombinant proteins cannot be produced easily (and cheaply) with good manufacturing practice (GMP). Even the best protective results are no guarantee for any immunological investigation. This was underscored when several of the frontline candidates chosen by the TDR/WHO committee discussed above had to be abandoned because, in addition to the low independent testing efficacy recorded, hurdles in consistent protein production could not be overcome. Nevertheless, there is still considerable interest in developing these and other molecules as antischistosome vaccines. Compromises may be necessary, however, because as emphasized by Bergquist and colleagues, “we might eventually have to settle for moderately effective antigens, but where the scaled-up GMP versions do not pose a problem. Despite the extra costs of scaling-up production of all antigens under consideration, it is a selection criterion in assessing vaccine candidacy that cannot be avoided” (Bergquist et al., 2004).
A number of recent studies, particularly on S. japonicum, have utilized plasmid DNA vaccines to deliver protective antigens. DNA vaccines generate both T-cell and B-cell (or antibody-mediated) immune responses and are thus particularly appealing for schistosome vaccine development. The preparation and production of DNA vaccines are convenient and cost-effective, and they can even be used in the field without a cold chain. Another advantage of applying DNA vaccines compared to other approaches is the possibility of targeting the in vivo expressed recombinant antigen to different cell compartments. Furthermore, methods such as prime-boost regimens and the use of adjuvants (such as IL-12) in combination with a DNA vaccine can enhance its protective effectiveness. The advantages and disadvantages of plasmid DNA vaccination, the strategies employed for DNA vaccine delivery, and technological and clinical advances in the area have been reviewed recently (Ulmer et al., 2006, Brave et al., 2007).
1.5.33 Current status of vaccine development for schistosoma mansoni and Sschistosoma haematobium: Given the enormous burden of disease related to schistosomes, relying solely on existing disease control methods, i.e., mass and repeated treatment of exposed populations with the antihelminthic PZQ, is unlikely to be feasible. Vaccines in combination with other control strategies, including the use of new drugs, are needed to make elimination of schistosomiasis possible (Todd and Colley, 2002). Despite the discovery and publication of numerous potentially promising vaccine antigens from S. mansoni and, to a lesser extent, S. haematobium, only one vaccine, namely, BILHVAX, or the 28-kDa GST from S. haematobium, has entered clinical trials (Capron et al., 2002). Published data are not available on the clinical efficacy of this vaccine, but nonetheless, it is disappointing that other vaccines have not progressed to this stage.
Below are summarized, some of the most promising S. mansoni vaccine antigens discovered in the last 10 years, as well as those that were independently tested under the umbrella of the TDR/WHO committee in the mid-1990s (Bergquist and Colley, 1998).
1.5.34 Tetraspanins: Tetraspanins are four-transmembrane-domain proteins found on the surfaces of eukaryotic cells, including B and T cells. They have two extracellular loops, including a short loop 1 of 17 to 22 residues (EC-1) with little tertiary structure and a larger, 70- to 90-residue loop 2 (EC-2), which has four or six cysteines that form two or three disulfide bonds. In general, the extracellular loops mediate specific protein-protein interactions with laterally associated proteins or, in some cases, known ligands. The four tetrastability bonds are needed during biosynthesis and are crucial for assembly and maintenance of the tetraspanin web, a scaffold by which many membrane proteins are laterally organized (Andre et al., 2006). Although their functions are unknown, it is now apparent from proteomic studies that a family of tetraspanins is expressed in the schistosome tegument (Braschi et al., 2006), and at least three of these show promise as vaccines. Sm23 is a tetraspanin (Wright et al., 1991) expressed in the tegument of S. mansoni and is one of the independently tested WHO/TDR vaccine candidates (Bergquist and Colley, 1998). Sm23 is most efficacious when delivered as a DNA vaccine (Da-Dara et al., 2003) and does not confer protection as a recombinant protein when formulated with alum. More recently, a reporter-based signal sequence capture technique was used to identify two new S. mansoni tetraspanins (SmTSP-1 and SmTSP-2) (Smyth et al., 2003). Both proteins are expressed in the tegument membrane of Schistosoma mansoni (Tran et al., 2006), and TSP-2 was identified as one of only a subset of proteins that were bio-tinylated on the surfaces of live worms and subsequently identified using tandem mass spectrometry (Braschi and Wilson 2006). TSP-2 in particular provided high levels of protection as a recombinant vaccine in the mouse model of schistosomiasis, and both proteins were strongly recognized by IgG1 and IgG3 from PR individuals but not from chronically infected people (Tran et al., 2006). In addition to TSP 2, two more tetraspanins were identified from the outer teguments of biotinylated Schistosoma mansoni adults (Loukas et al., 2001).And both are clearly now vaccine targets (Braschi and Wilson, 2006).
1.5.35 Other membrane proteins: The next few years will hopefully see the assessment of some of the newly identified tegument plasma membrane proteins from S. mansoni (Braschi and Wilson, 2006). Other than the tetraspanins (Sm23 and SmTSP-2), only one of these membrane-spanning proteins, Sm29, has been assessed as a vaccine. Like TSP-2 (Tran et al., 2006), Sm29 is preferentially recognized by antibodies from PR compared with chronically infected individuals (Cardoso et al., 2006), although the extent of selectivity is not as great as that reported for TSP-2. Moreover, preliminary trials in mice suggested that this protein is an efficacious recombinant vaccine (Loukas et al., 2007), lendingfurther support to its development as a recombinant vaccine.Other apical membrane proteins from the tegument (Braschi and Wilson 2006) that warrantattention as vaccines include the structural membrane proteinswith large extracellular regions, such as annexin and dysferlin,and other accessible (to antibodies) proteins with no homologuesof known function, such as Sm200.
1.5.36 Sm28/Sh28 GST: Sm28-GST has GST properties and is expressed in subtegumental tissues of most developmental stages of the parasite (Porchet et al., 1994). Vaccination of semipermissive rats and permissive hamsters with recombinant Sm28-GST resulted in significant reductions of worms (Balloul et al., 1987), kick-starting a 20-year program on Sm28- and Sh28-GSTs as vaccine antigens. Primate trials were conducted and showed an anti-fecundity effect (Boulanger et al., 1999), and an anti-Sm28 monoclonal antibody showed anti-fecundity and anti-egg embryonation effects (Xu et al., 1993). This led to the clinical testing of Sh28-GST in people and the description of its immunogenicity and induction of antibodies capable of neutralizing the enzymatic activity of the recombinant protein (Capron et al., 2005). Unfortunately, there are no data availableon the efficacy of this vaccine in phase II clinical trials.
1.5.37 Smp80 Calpain: Calpain is a calcium-activated neutral cysteine protease. The calpain large subunit was first discovered from S. mansoni by immunoscreening of a lambda phage cDNA library with sera from infected humans (Andresen et al., 1991). Calpain was immune-localized to the tegument and underlying musculature of adult worms and was shown to be involved in surface membrane turnover (Siddiqui et al., 2003) and to be associated with the inner tegument membrane (Braschi and Wilson , 2006 ). Calpain was shown to be the target of a protective CD4+ T-cell clone that induced peritoneal macrophages from syngeneric recipients to kill schistosomula in vitro (Jankovic et al., .1996). In addition, mouse recipients of this T-cell clone displayed significant resistance against cercarial challenge, making calpain the first vaccine antigen identified on the basis of T-cell reactivity. The large subunit of calpain, called Sm-p80, was expressed in baculovirus, and the semipurified protein induced 29 to 39% reductions in worm burdens (Hota-Mitchell et al., 1997). Subsequent efforts to improve the efficacy of this vaccine have focused on DNA vaccine constructs, with and without Th1-type cytokine cDNAs, in mice and baboons (Siddiqui et al., 2003).
1.5.38 Superoxide dismutase (SOD): Granulocytes release oxygen radicals that are toxic for S. mansoni, and exogenous superoxide dismutase (SOD) inhibited granulocyte toxicity for egg metabolic activity and hatching (Kazura et al., 1985). A cDNA encoding a SOD with a signal peptide was cloned from S. mansoni, and its protein product was recognized by sera from infected humans (Simurda et al. 1988). A cDNA encoding a cytosolic SOD (CT-SOD) was then identified (Hong et al., 1992), and both SODs were immune-localized to the tegument and subtegumental tissues (Hong et al., 1993,). Proteomic studies have since shown that SOD is localized below the tegument plasma membrane (Braschi et al., 2006.). Vaccination experiments using the recombinant SOD proteins have not been reported, but CT-SOD and a partial sequence encoding the structural protein filamin showed promise as DNA vaccines, resulting in significant reductions in adult S. mansoni in a murine challenge model (Shalaby et al., 2003).
1.5.39 Paramyosin: Paramyosin is a 97-kDa myofibrillar protein with a coiled-coil structure and is found exclusively in invertebrates. It is expressed on the surface tegument of lung-stage schistosomula in the penetration glands of cercariae (Gobert and McManus, 2005) and may function as a receptor for Fc (Loukas et al., 2001). The vaccine efficacy of paramyosin against S. mansoni was first described in the 1980s; mice immunized intradermally with S. mansoni extracts and Mycobacterium bovis BCG adjuvant were significantly protected against subsequent infection, and antibodies predominantly recognized paramyosin (Lanar et al., 1986). Vaccination of mice with native and recombinant paramyosin was then shown to provide modest (26 to 33%) but significant protection against challenge infection with S. mansoni (Pearce et al., 1991).
1.5.40 Fatty acid binding proteins (FABPs) : The S. mansoni fatty acid binding protein (FABP), Sm14, is a cytosolic protein expressed in the basal lamella of the tegument and the gut epithelium (Brito et. al., 2002). Sm14 has been assessed thoroughly as a recombinant protein vaccine and, to a lesser extent, as a DNA vaccine. Despite a high efficacy of recombinant Sm14 protein in mouse vaccine trials (Tendler et al., 1996), Sm14 failed to induce protection levels of >40% when tested in other laboratories (Fonseca et al., 2004) and as part of the WHO/TDR-sponsored trials (Bergquist and Colley ,1998). Co-administration of recombinant Sm14 protein with either IL-12 (Fonseca et al., 2004) or tetanus toxin fragment C (Abreu et al., 2004) boosted protection. Immunization of mice with recombinant Sm14 expressed in Mycobacterium bovis BCG showed no induction of specific antibodies to Sm14, but splenocytes from vaccinated mice produced IFN- upon stimulation with recombinant Sm14. Moreover, mice that were vaccinated once with Sm14-BCG and then challenged with Schistosoma mansoni cercariae showed a 48% reduction in worm burden, which was comparable to that obtained by immunization with three doses of recombinant Sm14 protein (Varaldo et. al., 2004).
1.5.41 Phenol and hydroquinone play an important role in benzene-induced
leukemia: Benzene and ionizing radiation are among the few established environmental causes of leukemia in humans. Although the precise mechanism by which benzene induces leukemia is not known, the prevailing hypothesis is as follows (Smith, 1996 and Rose, 1996):. Benzene is metabolized in the liver primarily by cytochrome P4502E1 (CYP2E1), first to benzene oxide and then to phenol, hydroquinone and other polyphenolic metabolites. These phenolic metabolites can be detoxified by conjugation with sulfate, glutathione or glucuronide. Sulfation may not be a potent detoxification mechanism, because the bone marrow contains high levels of sulfatase that can break down the sulfur conjugates to free phenols (Low et al., 1995). The phenolic metabolites also travel to the marrow where they are converted to highly reactive quinones by peroxidases, such as myeloperoxidase, or through autooxidation (Smith, 1996 and Rose, 1996). The major defenses against these toxic quinone products include reduction via NAD (P)H: quinone oxidoreductase (NQO1) or conjugation with glutathione (Twerdok and Trush, 1990,1991). Quinone oxidation products form DNA adducts and induce a wide-range of DNA damage both directly and indirectly. These quinone metabolites also increase oxidative stress (Subrahmanyam et al., 1991) and alter differentiation and cell growth in the myeloid compartment (Irons and Stillman, 1996; Hazel and Kalf, 1996). This combination of genetic and epigenetic effects on bone marrow progenitor cells leads to the production of leukemia in some exposed individuals.
When phenol and hydroquinone are administered together, they reproduce benzene’s myelotoxic effects (Eastmond et al., 1987, Guy et al., 1990). Phenol and phenol-derived metabolites (eg 4,4′-diphenoquinone) inhibit topoisomerase II and enhance the genotoxic effects of hydroquinone (Chen and Eastmond, 1995a and b). Many chemotherapeutic drugs that inhibit topoisomerase II also induce leukemias (Felix, 1998). The co-administration of phenol and hydroquinone to mice induces micronuclei and oxidative DNA damage in the bone marrow in a manner similar to benzene (Barale et al., 1990 and Kolachan et al., 1993). Further, combinations of the phenolic metabolites of benzene enhance DNA adduct formation (Levay and Bodell, 1992). It has therefore been suggested that all the phenolic metabolites of benzene play a role in benzene-induced toxicity, and that it is the combination that is most toxic ( Eastmond et al., 1987; Zhang et al., 1994)
Quinones derived from phenol, catechol, hydroquinone and 1, 2,4-benzenetriol cause various forms of genetic damage including chromosome breakage and aneuploidy (Chen and Eastmond, 1995; DeCaprio, 1999). Aneuploidy is the loss or gain of whole chromosomes and is a frequent clonal aberration in leukemia. Common aneuploidies include trisomy of chromosome 8 and monosomy of chromosomes 5 and 7 (Rowley, 1999 and Harrison, 2000). The phenolic metabolites of benzene have been shown to induce trisomy 8 and monosomy 5 and 7 in cultured human cells in vitro, including CD34+ progenitor cells (Smith and Zhang, 1998, Zhang et al., 1998 and Smith et al., 2000). Workers exposed to benzene also have higher levels of these aneuploidies in their peripheral blood (Zhang et al., 1998). Through their inhibitory action on topoisomerase II, binding to other DNA-associated proteins and formation of DNA adducts, phenol and hydroquinone cause double-strand breaks and chromosome breakage (Chen and Eastmond, 1995; Bodell et al., 1996). This has been shown in vitro to lead to deletions and translocations commonly found in leukemia, including del (5q) and del (7q) (Zhang, et al., 1998). Again, workers exposed to benzene have been shown to have higher levels of del (5q), del (7q) and translocation (8; 21) in their blood cells (Zhang et al., 1998, Smith et al., 1998). Mice treated with phenol and hydroquinones also show elevated levels of aneuploidy and chromosome breakage in their bone marrow (Chen and Eastmond, 1995.)
1.5.42 Hydroquinone produces epigenetic changes that increase the risk of developing leukemia: Other research has focused on the possible epigenetic mechanisms involved in benzene induced leukemia. Hydroquinone has been shown to alter hematopoietic stem cell proliferation and differentiation in the myeloid compartment (Iron and Stillman, 1996; Hazel and Kalf, 1996). Related effects may include alteration of apoptosis and alteration of clonal expansion of blood progenitor cells. These perturbations may proceed through a variety of means including alteration of inflammatory mediators, growth factors and other cellular messengers (MacEachern and Laskin, 1994; Snyder and Kalf, 1994). Irons and coworkers demonstrated that treatment of mouse of human marrow progenitor cells of the granulocyte-macrophage lineage with hydroquinone increased the number of colonies dependent on granulocyte-macrophage colony-stimulating-factor (Iron and Stillman, 1996). In theory, this increase provides more targets for the genotoxic effects of phenol, hydroquinone and related species (Smith, 1996).
1.5.43 Phenol and hydroquinone levels in benzene-exposed individuals: Historically, when occupational exposures to benzene were high (e.g. >50 p.p.m.), urinary phenol levels were seen as a good biomarker for exposure to benzene (Walkley et al., 1961, Van Haaften and Sie, 1965; Docter and Zielhuis, 1967). Studies, such as those conducted by Inoue et al., 1986 andInoue et al., 1988 observed a significant correlation between air concentration of benzene in breathing zone and phenol or hydroquinone concentrations in urine (corrected for creatinine and specific gravity). The authors reported a significant difference between urinary phenol levels in a group exposed to 10 p.p.m. benzene compared to unexposed workers. Similarly, a dose-related increase in urinary phenol levels was observed among groups of Chinese workers exposed to benzene (Rothman et al. 1998). Workers exposed to 0, 1 to 31 p.p.m., or >31 p.p.m. exhibited urinary phenol concentrations of 4 to 55 p.p.m., 16 to 487 p.p.m., or 28 to 517 p.p.m., respectively. These findings indicate that high benzene exposure significantly affects the levels of excreted phenol. However, in these and other studies, urinary phenol and hydroquinone were found to be poor predictors of lower benzene exposures (Docter and Zielhuis, 1967; Inoue et al., 1986; Inoue et al., 1988; Ong et al., 1995 Rothman et al., 1998). For example, urinary phenol levels of workers exposed to 1 to 5 p.p.m. benzene were indistinguishable from levels in unexposed individuals (Inoue et al., 1986). Likewise, the distributions of urinary hydroquinone levels of unexposed individuals overlapped with urinary levels from female workers exposed to benzene between 9 and 14 p.p.m. (7 h, time-weighted average) (Inoue et al., 1988).
1.5.44 Phenol and hydroquinone levels in unexposed individuals: High background concentrations of phenol, hydroquinone, catechol and 1,2,4-benzenetriol have been measured in the blood, urine and intestines of presumably unexposed humans (Ong et al., 1995, Smith and Macfarlane, 1996) and in the blood and urine of rodents (Bakke, 1969; Bechtold et al., 1996). Representing measurements of hundreds of individuals in total, these studies showed that mean urinary phenol and hydroquinone levels were about 10 p.p.m. (13 g/mg creatinine) and 4 p.p.m. (5 g/mg creatinine), respectively. The urinary concentrations of phenol and hydroquinone varied widely (ie 5- to 25-fold) within a given population (Inoue et al., 1986, Inoue et al., 1986, Rothman et al., 1998). Some studies have reported differences in phenol levels between sexes, whereas others have not (Docter and Zielhuis, 1967). Variability of phenol levels among individuals appears to be greater than the difference among males and females (Docter and Zielhuis, 1967). Urinary concentrations of phenol have been observed to be approximately 10-fold higher than concentrations in serum from the same individuals, (Ling and Hanninen, 1992), which may reflect the rapid removal of phenol from the blood and excretion via urine. Based on mean 24-h urinary levels in control individuals (Inoue et al., 1986, Inoue et al., 1988) and assuming a daily fluid intake of 2 liters, we estimate that humans produce endogenously or ingest each day roughly 0.2 mg/kg of phenol, 0.1 mg/kg hydroquinone, and 0.3 mg/kg of catechol, with considerable inter-individual variability expected.
Interestingly, high backgrounds levels of covalent macromolecular adduct of benzoquinone (the reactive metabolite of hydroquinone) were observed in the blood of humans (McDonald et al., 1993; Waidyanatha et al., 1998) and in the blood and bone marrow of rodents,( Reddy et al., 1994). The adduct levels were equivalent to those resulting from a relatively high exposure to benzene (Waidyanatha et al., 1998). Several investigators have estimated that exposures to benzene far in excess of the current workplace standard would be required to significantly increase the adduct levels over background levels (Bechtold et al., 1996). These observations prompted speculations on the pathological consequences that background levels of phenol and hydroquinone may have for the general population.
A number of studies have also assessed phenol levels in patients with different diseases, including colon cancer, familial polyposis, and Crohn’s disease (Bone et al., 1976). Patients with diverticular disease or polyposis with ileorectal anastamosis appeared to have elevated urinary phenol levels (about 2-fold) relative to normal individuals. The most noticeable increase in phenol levels was observed among patients with Crohn’s disease, which is a common type of inflammatory bowel disease. Phenol levels among Crohn’s patients were approximately 4- to 30-fold higher than levels among normal subjects. Interestingly, in agreement with our hypothesis, there are studies suggesting that Crohn’s patients have an increased risk of leukemia (Orii et al., 1991).
Thus, based on the strong evidence implicating phenol and hydroquinone in benzene-induced leukemia coupled with the observations of high background levels of these compounds in the general population, it is hypothesized that these background concentrations and associated adducts may play a causal role in de novo leukemia.
1.5.45 Sources of phenol and hydroquinone in “unexposed” people: Investigation of the available literature has uncovered a surprising number of potential sources of the background levels of phenol and hydroquinone. These include over-the-counter medicines, smoking, numerous foods and beverages, and the catabolism of protein (tyrosine) and other substrates by the gut flora. These sources are discussed below as well as the host and environmental factors that affect the variability of the background levels of the phenolic compounds
Phenol is used in some relatively unusual medications and treatments, which result in sharp but transitory increases in blood phenol levels (Rahn and Lazar, 1997, Puig et al., 1998,.Guliaev et al., 1998; Yoon and Ahn, 1999). Of more importance to the general population is the use of some over-the-counter medications such as Pepto-Bismol and Chloraseptic lozenges (Fishbeck et al., 1975). In a trial of Pepto-Bismol, urinary phenol concentration increased over 40-fold, reaching a concentration of 260 p.p.m. in a volunteer who ingested 1 oz of Pepto-Bismol every hour for 8 h. Additional experiments indicated that phenyl salicylate in Pepto-Bismol was responsible for the increase in urinary phenol levels. Fishbeck et al (1975) also observed increases in phenol levels in human subjects after ingestion of Chloraseptic lozenges. The total urinary phenol rose to a maximum of 270 p.p.m. These studies show that high urinary phenol levels can be achieved without any significant exposure to benzene.
Cigarette and wood smoke contain phenol, catechol, hydroquinone as well as benzene (Carmella et al., 1982; Hoffmann and Wynder, 1986; Hawthorne et al., 1989). Amounts received from non-filtered mainstream cigarette smoke were estimated to be 60-140 g phenol per cigarette, 140-500 g catechol per cigarette, and approximately 150-430 g hydroquinone per cigarette (Hoffmann and Wynder, 1986). Haufroid et al, (1997) observed statistically significant dose-related increases in the urinary concentrations of phenol, hydroquinone and catechol associated with the number of cigarettes smoked. Cigarette smoking is associated with an approximately 50% increase in leukemia risk (Mclaughlin et al., 1989; Garfinkel and Boffetta, 1990).
Although the use of cigarettes and certain medicines can result in significantly elevated levels of phenol and hydroquinone in some groups of individuals, diet is a significant source contributing to increased tissue levels of phenol and hydroquinone for all persons. Phenol and hydroquinone are derived from the diet both directly and indirectly. Many common foods and beverages contain phenol and hydroquinone. A major source of phenol stems from catabolism of protein and other compounds by gut bacteria, which appears to depend highly on the metabolic activity of the intestinal bacterial microflora (Bures et al., 1990; Smith and Macfarlane, 1996). A potentially significant source of hydroquinone comes from ingestion of foods containing arbutin, a naturally occurring plant product that is converted to hydroquinone by stomach acids. The wide inter-individual variability in urinary levels of phenol noted above may reflect the wide range of direct intake of phenolic-containing foods and differences among individuals in the composition and chemistry of the gut flora.
Phenol, hydroquinone, catechol, and 1, 2, 4-benzenetriol are found in a wide variety of foods and beverages. Fairly high concentrations of phenol, hydroquinone, catechol and benzenetriol are found in coffee. Levels of hydroquinone in some herbal teas were estimated as high as 1%. Lower concentrations of phenol or hydroquinone have been measured in numerous vegetable-based products, alcoholic beverages, dairy products, green and black teas, fruits, roasted nuts, honey, molasses, beef, and spices.
1.5.46 Production of phenol and other phenolics by the gut flora: Gut microflora may play an important role in the biotransformation of chemical and dietary components into procarcinogenic compounds in the gut. Smith and Mcfarlane (1996) have shown that simple phenols are major products of tyrosine metabolism by gut microflora in the distal colon. Tyrosine is an aromatic amino acid that may be ingested through the diet or formed from the essential amino acid, phenylalanine, through an irreversible reaction catalyzed by phenylalanine hydroxylase. Tyrosine and phenylalanine are found in high concentrations in milk, beef and eggs. Phenylalanine is also commonly used as an artificial sweetener in a number of sugarless foods and beverages. Similarly, some commonly occurring plant phenolics are efficiently metabolized by the gut flora of rodents to phenol, catechol and other simple phenols (Scheline, 1966a and b). Escherichia coli can convert glucose to catechol through a minor metabolic pathway (Draths and Frost, 1991). However, the amount of catechol that might be generated by gut flora from sugars is unknown.
Endogenous bacterial strains that convert tyrosine to phenol include the obligate anaerobes, Bacteroides fragilis, Peptostreptococcus asaccharolyticus, and the facultative anaerobes, Escherichia coli, Proteus sp., Staphylococcus faecalis and Staphylococcus albus (Bone et al., 1976; Smith and Mcfarlane, 1996; Marteau et al., 1993). Studies of human colonic contents in sudden death victims in the UK showed that anaerobic bacteria outnumber facultative microorganisms in the human gut by two to three orders of magnitude with Bacteroides fragilis being the most numerous (McBain and Macfarlane, 1998). However, the composition of intestinal bacteria varies considerably among different populations, particularly in Western vs other non-industrialized societies. For example, the microfloras from individuals in developed countries have higher levels of Bacteroides, whereas the flora from individuals from undeveloped countries exhibit higher counts of beneficial Lactobacilli (Bennet et al., 1991; Sepp et al., 1997). Several adduced reasons for these differences include the sterility of processed western foods, and high meat intake in western diets which results in a substitution of Lactobacilli (which do not produce phenol) with high levels of Clostridia and Bacteroides populations (which produce phenol) (Sepp et al.,1997)
Epidemiological studies have identified several risk factors for leukemia including consumption of high meat diets, (Hursting et al., 1993; Peters et al., 1994), use of antibiotics, and absence of breastfeeding (Adlerberth et al., 1991, Bennet et al., 1991; Sepp et al., 1997; Shu et al., 1999). Each of these risk factors is associated with the potential to alter colonic microflora activity, composition, and resultant phenol production. It is interesting to speculate that phenol production by the gut flora may be a contributing factor in these epidemiological associations.
Individuals consuming high-beef diets have increased populations of phenol-producing anaerobic Bacteroides compared to individuals on vegetarian diets (Maier et al., 1974; Hentges, 1980). Comparisons of western and eastern populations indicated that individuals who consume a typical British and American high-meat diet have a higher ratio of anaerobic to aerobic micro flora than persons from Japan, Uganda or India, whose diets are largely vegetarian (Hill et al., 1971). These findings were confirmed in a study of volunteers who adopted a strictly vegan diet which led to a decrease in serum and urinary phenol and p-cresol concentrations (Ling and Hanninen, 1992). Upon returning to a conventional diet that included the addition of meat and eggs, the volunteers’ urinary phenol and p-cresol concentrations correspondingly increased (Ling and Hanninen, 1992). Substitution of carbohydrate for protein may not be the only factor responsible for the reduction in phenol levels seen in vegetarians. Carbohydrate fermentation interferes with bacterial protein degradation (MacNeil, 1988) and would, therefore, reduce the level of tyrosine metabolism in the gut.
Antibiotic treatment is known to change intestinal microflora composition (Bennet et al., 1991) by suppressing the growth of some strains of bacteria while promoting the growth of others. For example, the administration of antibiotics is a factor responsible for the lower colonization rate of Lactobacillus in infants (Hall et al., 1990), while studies by Snydman and colleagues (Snydman et al., 1999) have shown the antimicrobial resistance of Bacteroides fragilis. This resistance could lead to disturbances in the intestinal flora, encouraging the overgrowth of these phenol-producing bacteria. Since the sequence in which bacteria populate the colon influences subsequent species’ colonization and proliferation, it has been hypothesized that early colonization by Lactobacillus serves as a barrier against potentially pathogenic bacteria (Bennet et al., 1991). In the absence of this barrier, other bacteria may flourish.
Interestingly, breastfeeding also serves as another source of Lactobacillus (Orrhage and Nord, 1999), which do not produce phenol, and breastfeeding has recently been associated with a reduction in risk of childhood leukemia (Shu et al., 1999). More study into this possible link is needed.
1.5.47 Hydroquinone from arbutin: Arbutin, a glucose conjugate of hydroquinone (4-hydroxy- -D-glucopyranoside), occurs naturally in many plant foods. Arbutin is readily hydrolyzed in the stomach to free hydroquinone, which is extensively absorbed through the gastrointestinal tract. Studies by Deisinger et al. (1996) showed that wheat products and pears contain high levels of arbutin while the concentration of free hydroquinone is quite low. They identified the highest levels of total hydroquinone in wheat germ (10.65 p.p.m.) and d’Anjou pears (15.1 p.p.m.). While lesser amounts of total hydroquinone are found in beverages such as coffee, tea, and red wine (0.1 to 0.4 p.p.m.), these beverages may contribute significant amounts of hydroquinone to the diet, particularly in those who drink more than one cup (>200 ml) per day. Within 2 h of ingestion, volunteers fed a high arbutin-containing diet (784 to 1279 g total hydroquinone) exhibited a five-fold elevation in plasma hydroquinone levels, increasing from a mean background level of 0.028 p.p.m. to 0.14 p.p.m. (Deisinger et al., 1996). Likewise, significant increases in urinary hydroquinone levels were observed. Background levels of hydroquinone in the range of 0 to 100 g/h surged to 700-1200 g/h for up to 6 h following treatment ((Deisinger et al., 1996).
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