Studies of Prevalence and Subtypes of Blastocystis Species in Lagos
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1.0 INTRODUCTION AND BACKGROUND OF THE STUDY The Genus Blastocystis was first described from the faecal contents of animals by Alexieff in 1911 and named Blastocystis enterocola, cyst of a flagellate and upon its isolation from the faeces of humans the following year by Brumpt; it was re-named Blastocystis hominis but classified as yeast and non- pathogenic in humans (Tan et al., 2002). As a result of the ensuing confusion of unsettled taxonomy, Blastocystis spp. was not included in any of the systematic classifications of the parasitic protist (Stenzel and Boreham, 1996). However, Silberman et al., (1996) analyzed the conserved SSUrRNA gene of the parasite in comparison to other eukaryotes to phylogenetically place Blastocystis in the informal group Stramenopiles or Heterokont. This single- celled eukaryote has been detected in faecal materials from various wild and domesticated animals, birds, fishes and recently cockroaches (Abe et al., 2002; Yoshikawa et al., 2007, 2009; Jones et al., 2009). Blastocystis is ubiquitous with a global distribution (Windsor and Macfalane, 2005; Malheiros et al., 2011). Human infection due to Blastocystis species is characterized by non-specific gastrointestinal symptoms which may include: chronic or intermittent diarrhoea and dysentery, abdominal pains or colic, anorexia, tenesmus, flatulence, malaise, bloating, urticaria and anal itching (Cruz et al., 2003). However, diarrhoea and abdominal cramps were the most commonly reported symptoms (Doyle et al., 1990; Tan et al., 2002). Blastocystis infection has been described both in immune competent individuals and immune compromised subjects such as individuals with HIV/AIDS (Gassama et al., 2001). It has also been frequently detected in stool samples of symptomatic patients who have chronic intestinal disorders such as Irritable Bowel Syndrome (IBS), a highly prevalent intestinal disorder of unknown cause, which is characterized by symptoms that include 1 intermittent diarrhoea, acute abdominal pains and constipation (Hussain et al., 1997; Yakoob et al., 2004). In most studies reported from Europe, between 30% - 40% of IBS patients were Blastocystis carriers (Boorom et al., 2008). However, earlier studies were not clear whether the parasite is the aetiological agent in this condition (Wilson et al., 2004; Yakoob et al., 2004). The transmission of this enteric parasite is believed to be faeco-oral and can be facilitated by the contamination of the environment, food such as fruits, raw water plants and vegetables, and water, with the excreted cysts of the parasite from a reservoir host (Leelayoova et al., 2002, 2008; Tan, 2008). Waterborne transmission has also been reported, through intake of contaminated drinking water, through untreated sewer polluting water for consumption and overflow of waste water from farm lands into homes have also been implicated (Geltman et al., 2001, Leelayoova et al., 2002, Suresh et al., 2006, WHO, 2006). Intestinal parasitic infections are believed to thrive in localities with poor personal and environmental hygiene, particularly in places where untreated sewage is disposed into the environment (Bello et al., 1997). The cyst form of Blastocystis species is reported to be the transmissible form of the parasite and evidence of its survival from sewer effluents and influents pose a huge threat to humans (Suresh et al., 2006). The cyst forms are the transmissible forms, in that they can survive in the environment long enough, when excreted, to be able to infect a new host. Cyst forms of sizes of less than 3.0µm were recently recognized as Blastocystis forms (Tan, 2004). Blastocystis is polymorphic; having a classical vacuolar or central body, avacuolar, amoeboid, multi-vacuolar and cyst forms (Tan, 2008). The vacuolar form is the most common form identifiable by microscopy. Other parasitological diagnostic methods such as serological diagnosis, employing ELISA and IFA assays, have not yielded satisfactory 2 results for Blastocystis detection and these methods are unavailable in most developing countries (Zierdt et al., 1991, Tan et al., 2002, Stensvold et al., 2006). The implication is that there is heavy reliance on microscopic diagnosis which has been shown to have low sensitivity and specificity for Blastocystis detection. The cultivation of the parasite using culture medium is reported as a sensitive diagnostic method but is laborious and time consuming (Leeleyoova et al., 2002). However, the recent adaptation of some molecular methods for routine parasitological diagnosis, such as the use of Polymerase Chain Reaction (PCR) methods including, the Restriction Fragment Length Polymorphism, (RFLP-PCR) and Sequence- Tagged Site (STS- PCR) methods, which offer direct isolation of specific Blastocystis DNA from clinical samples, have been employed for Blastocystis isolation. The method could also generate enough isolates for further studies without need for laborious and time consuming parasite cultivation assay and could be employed for rapid community diagnosis of blastocystosis (Tan, 2004; Stensvold et al., 2007c). Metronidazole has been the drug of choice for the treatment of protozoan parasitic infections and has been in common used for Blastocystis infection, but there have been increasing reports of resistance or failure of the drug in some localities; among patients and primates in San Diego in the United states of America and danish individuals with entropathogens in Denmark, in whom, Pyrimethamine- Sulfamethazole was used (Zierdt et al., 1978; Nasirudeen et al., 2004, Stensvold et al., 2009). Other forms of treatment include the use of Nitazoxanide, Pyrimethamine- Sulfamethazole and Paromoycin (Stensvold et al., 2008c). 3 The prevalence of between 15% and 60% were reported for most developing countries and in localities with poor hygiene (Stenzel and Boreham, 1996). On the other hand, low prevalence of between 0.5% and 10% have been reported in developed and highly industrialized countries, where personal and environmental hygiene were entrenched. In Asia, the prevalence of Blastocystis in Japan was found to be between 0.5 -0.8% (Horiki et al., 1997). A prevalence of 7% was seen in a day care centre in Ontario, Canada (Koustalis et al., 2001). In the USA, prevalence of Blastocystis infection rose from 2.3% in 1987 to 23% in the year 2000, from a survey reported for the forty-nine states including the district of Colombia, a development that was attributed to three decades of neglect of surveillance for the parasite, increased migration of people from developing nations into the USA and Volunteer Corps travels to these countries by American citizens (CDC, 1987; Amin, 2002). In Europe, prevalence of 6.7% was reported in Wales, England (Windsor et al., 2002) and 4.7% in Switzerland (Nguyen and Krech, 1989). In the developing communities of South and Central America, a prevalence of 41.7% was reported for Blastocystis among food vendors in Xilimilco market in Mexico (Cruz et al., 2003), while a prevalence of 34 % and 14% were reported in Sao Paulo, Brazil and Buenos Aires, Argentina respectively among day care centers investigated for Blastocystis infection (Guimaeres and Soyagar, 1993; Minieville et al., 2004). In Africa, a prevalence of 18.5% was reported by Sadek et al., (1997) in the Quaylobia Governorate of Egypt; 23% among HIV/AIDS patients in Tanzania (Atzori et al., 1993), 21.6% among Zambian study participants investigated for intestinal parasites (Hunter et al., 1992) and a prevalence of 2.5% among hospitalized patients in Abeokuta, a city in the Southwest of Nigeria (Reinthaler et al., 1988). The global prevalence of Blastocystis infection found across the regions of the world is shown (Table 4 1.0). The public health significance of determining the prevalence of Blastocystis infection in Nigeria will be to raise awareness to personal and environmental hygiene, particularly to safe sewage disposal. The implication for health promotion approaches in public health will entail good laboratory evidence of parasite; exposure and disease monitoring that will help trigger information for action through policy development for the disease surveillance and control. 5 Table 1.0: Prevalence of Blastocystis spp. infection in countries across the globe. Region Country Prevalence Reference North America America(USA) 2.3% CDC,1987 America(USA) 23% Amin, 2002 Canada 7% Koulstalis et al., 2001 Central and South Brazil 34.7% Guimaeres and Soyagar, 1993 America Guatemela 31-32% Babcock et al., 1985 Mexico, Mexico city 29.2% Devera et al.,1998 Xilimchico market 41.7% Cruz et al., 2003 Venezuela 16% Caincross et al., 2002 Asia Hong Kong 7.0- 17% Rajah-Salim et al., 1999 Japan 0.5- 0.8% Horiki et al., 1997 Malaysia 4% Menon et al., 2000 Nepal 33% Taylor et al., 1998 Australia Australia, Sydney Control 11% Berger (2010) MSM(HIVPOS) 18.5% MSM (Non-HIV) 21% Europe and United England, Wales 6.9% Windsor et al., 2002 Kingdom Switzerland 4.7% Nguyen and Krech,1989 Middle East Jordan 47% Nimri,1993 Turkey 7.7% Koltas et al., 1999 Turkey 13% Tasova et al., 2000 Saudi Arabia 89% Quadri et al., 1989 Africa Egypt 18.5% Sadek et al., 1997 Tanzania 26.2% Atzori et al., 1992 Nigeria :(Abeokuta) 2.5% Reinthaler et al., 1988 Note that MSM denotes men having sex with men (Homosexual men). 6 1.1 The Molecular Epidemiology of Blastocystis species across the globe The molecular epidemiology of Blastocystis is still emerging. Over the past ten years, the development of subtype (ST) specific primers for differentiating human and animal isolates have increased understanding of the parasite‟s transmission viz a viz human to human, human to animal and animal to human (Abe et al., 2003, Iguchi et al., 2007, Yoshikawa et al., 2007, 2009). The molecular epidemiology of Blastocystis based on the nine known Blastocystis subtypes (ST 1-9), and mixed or unknown subtype (MSI/UST), infecting humans have been reported in some studies (Table 1.1). Blastocystis subtypes 1- 4 were reported to be the common strain isolated from humans, with ST3 having the greatest occurrence in some populations (Yan et al., 2007; Li et al., 2007b; Yoshikawa et al., 2009).