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).

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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

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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.

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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).

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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).

Other subtypes of Blastocystis have been described as zoonotic strains, ST5 to 9 subtypes.

Unlike non human hosts, man is susceptible to infection by all the known nine subtypes

(ST1- 9) of the parasite, but the outcome of the infection may be dependent on the subtypes that exist in the locality (Jones et al., 2009).

Nevertheless, infection potentials in experimental animals were found to vary between the different subtypes, suggesting that ST1, ST5 and ST7 might be pathogenic to humans

(Kaneda et al., 2001, Iguchi et al., 2007). Other studies that focused on the symptomatic and asymptomatic individuals revealed that ST1 and ST3 predominated in human populations

(Hussein et al., 2008; Ozyurt et al., 2008; Jones et al., 2009; Rene et al., 2009).

In a previous study, among Danish patients suspected of enteropathogens ST4 was reported to have some strains that are pathogenic and non- pathogenic (Stensvold et al., 2009). The study noted the possibility that some phenotypic and genotypic traits could influence the pathogenic outcome in some Blastocystis infection. Similarly, another report, suggested amoeboid form observed in subtype4 Blastocystis demonstrated in cultures from

7 symptomatic individuals which could be suggestive of potential pathogenic status

(Stensvold et al., 2010).

More recently, Meloni, (2011) reported amoeboid Blastocystis in cultures of 91.6% or 11 symptomatic ST3 isolates, suggesting that subtype 3-symptomatic and asymptomatic strains exists, which implied a pathogenic role for the symptomatic ST3 strains.

Intestinal parasitic profile in Nigeria:

Surveys of intestinal parasitic organisms frequently carried out in several localities in

Nigeria, found scanty or no report of Blastocystis species detection or its infections

(Fagbenro-Beyioku and Oyerinde, 1987; Bello et al., 1997; Oyewole et al., 2003, Agbolade et al., 2007). An earlier survey of intestinal parasitic organisms conducted on 479 hospitalized patients in Abeokuta, Southwest Nigeria and reported Blastocystis hominis as a non-pathogenic organism with prevalence of 2.5% (Reinthaler et al., 1988). The study was based on microscopy of faecal samples, a less sensitive detection method for the parasite.

There was neither isolation nor characterization of the parasite genome to confirm the existence of Blastocystis in the region. Hence, there is lack of reports and awareness of the parasite in the western region and entire country. Meanwhile, previous study of Abeokuta area found high exposure of the residents to faecal contamination of drinking water, poor personal and environmental hygiene (Mascher and Reinthaler, 1987). The poor sanitary conditions inherent in Abeokuta were implicated as the factors supporting the transmission of faecal micro-organisms in the city (Mascher and Reinthaler, 1987).

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Table 1.1: The Molecular Epidemiology of Blastocystis subtypes (1-9) infection (Rene et al., 2009). Country Subtypes N (%)

No.of study subjects (N) (Reference) ST1 ST2 ST3 ST4 ST5 ST6 ST7 ST8 ST9 UST

Australia, Sydney N =91 28(29) 6(6) 41(43) 12(12) - 3(3) - - - - (Berger, 2007) Brazil, Tairape tribe N= 382 156(41) 122(32) 65(17) ------7(11) (Malheiros et al., 2011) China, Guanxi N=35 13(37.1) - 14(40) - - - 2(5.7) - - 6(17.2) (Yan et al., 2007) China, Yunnam N=78 16(20.5) 1(1.3) 55(70.5) 1(1.3) - - - - - 5(6.5) (Yan et al., 2007) Denmark N=29 1(3.4) 6(20.7) 15(51.7) 7(24.1) ------(Stensvold et al.,2006) N=28 5(17.9) 9(32.1) 13(46.6) 1(3.8) - - - - (Stensvold et al., 2007a) - - Denmark N=1000 20(17.4) 11(9.6) 39(33.9) 16(13.9) - 1(0.9) - 1(0.9) - 7(6.1) N=1390 92(6.6) 52(3.7) 277(19.9) 37(2.7) - 1(0.1) - 1(0.1) - 23(1.7) (Rene et al., 2009) Egypt, Ismaila N=44 8(18.2) - 24(54.4) - - 8(18.2) 4(9.1) - - - Hussein et al., 2008 Egypt, Cairo N=19 3(15.8) 4(20.1) 12(63.2) ------(Souppart et al., 2010)

Germany

N= 78 18(23.1) 1(1.3) 51(65.4) 5(6.4) - - - - - 3.0(3.8) (Bohm-Glonning et al., 1997) Japan N=55 11(20) 12(21.8) 24(43.6) 6(10.9) - - - - - 2(3.6) (Kenada et al., 2001) Turkey, Istanbul N=87 8(9.2) 12(13.8) 66(75.9) 1(1.1) ------(Ozyurt et al., 2008)

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Intestinal parasitic profile in Lagos Metropolis:

Curiously, reports of survey of intestinal parasitic profiles of studies conducted in Lagos metropolis, a nearby city to Abeokuta also in the south west of Nigeria, failed to show that infections due to Blastocystis or its subtypes existed in the locality (Alakpa et al., 2003;

Akujobi and Ogunshola, 2006; Fagbenro-Beyioku et al., 2007). The reason could be traced to the problem of diagnosis of Blastocystis by the use of microscopy alone, the dearth of information of the parasite in the locality and lack of awareness of the emerging pathogenic status of Blastocystis, particularly by the medical laboratory scientists, clinicians and researchers in Lagos.

1.2 STATEMENT OF PROBLEMS

The confusion of whether the genus Blastocystis is an artifact, commensal yeast or protozoa with pathogenic potentials in man has lingered for over a hundred years, following its description. As a result of the confusion, there is loss of interest by many clinicians and microbiologists and therefore, there is paucity of data of this emerging intestinal infection of man. With little or no awareness of this parasitic infection by care-givers in most developing communities, data of its existence and prevalence are lacking.

As a faecal pathogen, Blastocystis infection is believed to thrive in conditions where pockets of slum communities with poor personal hygienic practices and unsafe disposal of untreated fresh sewage in the environment is rampart. Oyerinde et al., (1999) highlighted the poor sanitary conditions in the locality of Lagos with the result of persistently high prevalence of the faecal protozoan pathogen, Entamoeba histolytica/dispar complex, in the city. Therefore, Lagos may be an ideal locality to investigate the occurrence of Blastocystis species infections.

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The location of tertiary health institutions for the specialist care of individuals with

HIV/AIDS conditions in urban centres such as Lagos may influence migration into the urban centers. There could be increase in the burden of overcrowding; meaning that some of these individuals could relocate from rural communities to the urban centers in other to have access to medicare. In these situations, the different community background from which these individuals come from may lead to an increase in the profile of pathogens that leads to infections in the locality. Some of the pathogens, even if opportunistic in immune competent subjects may have serious consequences in the immune compromised HIV/AIDS condition.

There could be debilitating symptoms such as profuse watery diarrhea; some of which could be life- threatening in the individual with the condition.

Diarrhea is one of the HIV/AIDS defining symptoms; and often of unknown aetiology. This raises the important question of the level of care given to these patients, where there is low level or total lack of awareness that some strains of Blastocystis species could be diahoerric and potentially pathogenic in the person HIV/AIDS condition.

Blastocystis sp. has been implicated as an emerging pathogen in some diarrhoea and intestinal diseases in patients from countries with reported high prevalence. The subtypes or strains of the parasite associated with pathogenicity in symptomatic individuals with

HIV/AIDS in other localities may be non- pathogenic in another local environment.There appear to be diversity in the geographical distribution of Blastocystis subtypes. More recently, Blastocystis subtype 4, a zoonotic subtype due to rodents and common in human populations, was not identified among the Tiarapé tribe of the Brazilian Amazon Basin locality (Malheirous et al., 2011). Nevertheless, how the intestinal diseases such as Irritable

Bowel Syndrome, IBS, and Chronic diarrhea disease, CDD; present clinically in the local populations and the strains of parasites causing the infection in the locality of Lagos is not known.

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1.3 RESEARCH QUESTIONS:

1 Is there Blastocystis species in Lagos metropolis, Nigeria?

2 Is it possible to associate the detection of Blastocystis species with the conditions

and symptoms reported by the individuals from whom the parasite was found?

3 What confirmation is needed to validate the existence of the parasite in the locality

4 Which genetic classes of Blastocystis species exist and molecular phylogeny do the

isolates in Lagos belong?

1.4 SIGNIFICANCE OF THE STUDY

The study will investigate the occurrence and prevalence of Blastocystis species among patients in Lagos and hopefully confirm the existence of the infection in the locality. This, it is hoped, will be a foundation to raise awareness for future cascade of investigations into all aspect of blastocystosis in the local population of Lagos and other communities in Nigeria.

It is expected that this work, will also help to define the role or possible association of this parasite with intestinal symptoms like diarrhoea and abdominal pain. This will improve knowledge of the parasite by care-givers including, laboratory scientists, who will be able to identify it, and report it to the clinicians, thereby enhancing good diagnosis and intervention in the locality.

Diarrhoea remains one of the HIV/AIDS defining conditions. Detection of a diarrheal pathogen in individuals of all ages and sexes could be vital in the choice of treatment that could be used in the management of the condition. Nevertheless, knowledge of Blastocystis induced–diarrhoea in the immune compromised, HIV/ AIDS individuals in Lagos are lacking presently. As an opportunistic parasite and a potential pathogen, its isolation in the

HIV/ AIDS individuals will become a useful and interesting aspect of clinical management with regard to expanding the aetiology of gastrointestinal illness in the community.

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The burden of HIV might be increasing in the city of Lagos, with an estimated population of

13.6 million people when it is considered that about 4.2% of these were HIV seropositive individuals (UNGASS, 2010). In view of the fact that Blastocystis species has several subtypes, some of which could be potential pathogens of diarrhea, the determination of the subtypes or strains with attributes of pathogenicity, therefore, will help to place Blastocystis

–induced infections in better perspectives among health workers in Lagos. Meanwhile, the subtypes of the parasite that exist in Lagos are not known.

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1.5 AIM AND OBJECTIVES OF THE STUDY

This work is aimed at investigating the prevalence of Blastocystis and characterization of the isolates in Lagos.

1.6 Objectives of the study

In order to achieve the aims, the specific objectives of the study were to:

1. Determine the prevalence of Blastocystis in Lagos metropolis.

2. Investigate the prevalence of Blastocystis among immune compromised, HIV/AIDS

individuals.

3. Isolate fecal Blastocystis spp. DNA directly from the stool sample.

4. Characterize the local isolates of the parasite and identify the molecular

phylogenetic class to which they belong.

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LITERATURE REVIEW

2.0 Brief history of the genus Blastocystis

The subject of Blastocystis species is an „enigma‟ which will be better appreciated from its history and questions about its taxonomy, phylogenetic classification, pathogenicity and laboratory demonstrations which were needful to fulfill the proof of its infective potentials.

2.0.1 Nomenclature:

Zierdt (1991) noted that the earliest descriptions, with the required drawings that purportedly were of the organism, Blastocystis hominis, were those shown as “cholera bodies” by Brittan in 1849 and as “annular cells” by Swayne, in the report of the London epidemics of 1849. He noted that the first description that fulfilled the requirement of nomenclature was given by Alexeieff in 1911; who proposed the name Blastocystis enterocola, a cyst of a flagellate and who also applied the same binomial to cells he observed in non- humans such as rats, guinea pigs, chickens, leeches and reptiles such as lizards and sea snakes (Zierdt, 1991).

However, Brumpt (1912) coined the name Blastocystis hominis because he worked with human faecal material; the name that became firmly entrenched and recognized in early studies of the parasite. The isolation of B. hominis only in humans, monkeys, apes, pigs and perhaps, guinea pigs, cast Alexeieff in a bad light as he was accused of creating his new genus by confusing pollen grains and helminth ova, such as Ascaris lumbricoides with a new organism. The accusation was also aimed at Brittan and Swayne, against whom it was more accurately made (Zierdt, 1991). As a result, the Brumpt‟s publication became most cited; although, in his classification he mistakenly assigned the organism to different genus,

Schizosaccharamyces and Saccharamyces.

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Nevertheless, the brief description of B. hominis as harmless, non-pathogenic intestinal yeast became accepted to workers in the field, and in other disciplines (Zierdt et al., 1967;

Tan et al., 2002).

By the mid 1960s, Zierdt et al., (1967) described some protozoan features of B. hominis namely; one or more nuclei smooth and rough endoplasmic reticulum, golgi complex and mitochondrion-like organelles. Moreover, the organism does not grow on fungal media and is not sensitive to antifungal drugs like amphotericin but is affected by the antiprotozoal drugs like metronidazole, which showed activity against the organism. Therefore, many protozoologists therefore allied Blastocystis with the protozoa. Furthermore, the findings on the parasite culture, its physiology and ultra structures revealed from electron microscopic studies all strengthened the need for change in its classification (Zierdt, 1973; Zaman, 1997;

Tan et al., 2002). Subsequently, (Zierdt and Tan, 1976) proposed the re-classification of

Blastocystis hominis as a protozoan, thereby renewing interest of protozoologists to continue further studies of Blastocystis spp.

2. 0.2 Taxanomy and Phylogenetic studies

There appear to be some characteristics that show that B. hominis may not be a protozoa or fungus as previously thought. The phylogenetic affinities were also studied and analyzed by comparism of several conserved gene sequences of Blastocystis with those from different eukaryotes such as the elfa-proteins (Johnson, 1989). Nevertheless, due to the unsettled taxanomy, the genus Blastocystis was not included in any of the systematic of the parasitic protist (Cox, 1991; Corliss, 1994) until, Silberman et al., (1996) analyzed the conserved

SSUrRNA gene of the Blastocystis in comparism with other eukaryotes to phylogenetically place the parasite among the informal group Stramenopile or Heterokont in the kingdom

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Chromista, defined in the new kingdom of life systematics (Cavalier-Smith, 1997, 1998).

Blastocystis therefore may be the only chromist known to infect humans (Tan et al., 2002).

Patterson (1994) defined Stramenopile or Heterokonts to include a complex group of free photosynthetic organism such as diatoms and non- photosynthetic taxa such as the plant

Phytophthora spp, unicellular and multi-cellular protistes such as brown algae, chrysophytes, Proteromonas, slime nets and water molds.

Further molecular studies employing other highly conserved genes and proteins from

Blastocystis sp. were further used to validate this classification and affirmed that the parasite is closely related to Proteromonas lacertae (Arisue et al., 2002; 2003; Noel et al.,

2005).

Rene et al., (2009) proposed that the taxonomic classification of Blastocystis species, therefore, may be based on a combination of the proposal of Jiang and He (1993) and the harmonized terminology for the parasite (Table 2.0).

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Table 2.0: Proposed taxonomic classification of Blastocystis species

Super Kingdom Protist

Subkingdom Protozoa

Phylum Sacromastigophora

Order Amoebida (or Blastocystea)

Family Blastocystidea

Genus Blastocystis

Species (subtype) STI-ST10 (based on SSUrDNA analysis)

Based on combination of proposal of Jiang and He (1993), and Stensvold et al., (2007) terminology for Blastocystis Source: Rene et al., (2009).

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2.0.3 Biology of the Parasite

The biology of Blastocystis is still emerging. It contains Mitochondria-like Organelles,

MLOs, (Zierdt and William, 1974; Zierdt and Swan, 1981). It is believed that the MLOs are anaerobic in nature due to inability to produce hydrogen (Wawrzykniak et al., 2008)

Blastocystis also grows well on pre-reduced parasitic medium: Jones medium (Jones, 1946),

Boeck and Drbohlav‟s insipissated egg medium (Zierdt and William, 1974), Döbell and

Laidlaw medium covered with Ringer solution containing 20% human serum and streptomycin sulphate (Silard, 1979), Iscove‟s modified Dulbecco‟s medium supplemented with 10% horse serum (Ho et al., 1993) and Loeffler‟s medium covered with Ringer solution supplemented with 20% human serum (Silard et al., 1983).

The isolates from other hosts show different culture requirements (Belova, 1992). A variety of other hosts also habour Blastocystis, but not including the bony fishes, Osteichthyes

(Jones et al., 2009).

Blastocystis specimens isolated from humans were reported to grow well at 37˚C incubation temperature and not at room and lower temperatures (Tan, 2008).

2.0.4 Life cycle

A number of proposals on the life cycle of the genus Blastocystis have been reported in literature (Zierdt et al., 1967; Singh et al., 1995; Stenzel and Boreham, 1996; Tan 2004,

2008).

First, following the conviction that this parasite is a protozoan protist, Zierdt et al., (1967) proposed a life cycle of binary divisions as the only mode of reproduction whereby; a mother cell divides to generate daughter cells, as seen in the unicellar amoebae. In this cycle, the classical vacuolar (central body) forms differentiate into granular forms, which produce daughter vacuolar cells, or amoeboid cell forms. Modes of the division by

19 plasmotogamy, schizogony, binary fission and budding were also proposed (Zierdt, 1973;

Singh et al., 2002). The mode of reproduction through binary fission, budding and sac- like pouches were supported by the protozoal nature of the Blastocystis and not true modes of reproduction (Govind et al., 2002). However, binary fission appears to be the only mode of division widely accepted for this organism and recently demonstrated in vitro (Tan, 2004;

2008).

A life cycle which involves the phenomenon of auto-infections, where the vacuolar form, differentiates into thin walled cyst form through the multivacuolar and precyst forms has been proposed; there occurs schizogony within the thin- walled cysts, leading to rupture, releasing daughter vacuolar forms (Singh et al., 1995).

Stenzel and Boreham, (1996) proposed that the multivacuolar forms differentiate into cysts which are transmitted to the host via the faeco-oral route. Cysts develop into avacuolar forms which may transform to amoeboid or multivacuolar forms and the cycle is repeated

(Stenzel and Boreham, 1996).

A previous proposal assumed that these avacuolar, multivacuolar, and amoeboid forms were only found in vivo (Stenzel et al., 1991). However, vacuolar forms have been found in vivo in an experimental animal study of Blastocystis infection (Moe et al., 1997). This development led to a revision of the proposal put forward on this premise (Tan, 2004).

The success of the animal study implicated Blastocystis as a zoonotic organism and taking into account the large reservoir of Blastocystis sp. among the animal population from which the parasite was detected, the life cycle was further revised, Figure 1 (Tan, 2008).

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Figure 1.0: Proposed life cycle of Blastocystis species (Source: Tan, 2008)

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2.0.5 Epidemiology

It has been difficult to define the epidemiology of Blastocystis spp. based on reports from different regions of the world. First, the pleomorphic nature of the parasite and lack of standardization of detection methods have made comparism of reports impossible. For instance, Stensvold et al., (2006) noted that microscopic methods of identification, which aided the recognition of the common central body or vacuolar forms of the organism in stool smears was ineffective in identifying other morphological forms.

Blastocystis species is believed to be ubiquitous in nature (Stenzel and Boreham, 1996). It is perhaps, the most common parasitic organism often isolated from fresh stool samples of man but the most neglected parasitic organism in surveys of entero-pathogens in most regions of the world (Amin, 2002, Windsor et al., 2002; Jones et al., 2008, 2009).

Nevertheless, prevalence of between 30% and 60% for Blastocystis have been seen in some communities in tropical, subtropical and developing countries (Taasmassri et al., 2000;

Khalifa et al., 2001; Cruz et al., 2003). An earlier report included a prevalence of 89% for the parasite in a Saudi Arabian community where serious sanitary problems were found

(Quadri et al., 1989).

Prevalence rates in most of the developed communities in Japan, United States of America and Germany were found in the range of 0.5% and 10% (Horiki et al., 1997; Cruz et al.,

2003). Some studies conducted in the USA reported Immigrants, refugees and adopted children to have higher incidence of Blastocystis than those raised in their new communities

(Geltman et al., 2003). Most of the infections seen in developed or highly industrialized nations were found in travelers and volunteer corp returnees from developing countries, believed to be endemic for intestinal parasites (Herwaldt et al., 2001; Chen et al., 2003).

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Nevertheless, exceptions do occour, where within some communities lower socioeconomic groups or those with lower standard of hygiene may be found with higher prevalence than the remainder of the community (Nimri and Boutchoun, 1994). For instance, variations in prevalence between urban and rural areas have been recognized (Reinthaler et al., 1988,

Fujita et al., 1992).

Nevertheless, Stensvold et al., (2006) also noted that the methodologies used in studying prevalence analysis of Blastocystis vary in terms of sensitivity. Most studies rely on data from analyzing samples from patients with gastrointestinal symptoms, submitted to parasitological laboratories, where details of patient information used in the studies are sometimes insufficient.

Moreover the detection methods and diagnosis by recognition of the vacuolar or central body forms of Blastocystis only have led to the difficulties in comparing data from such studies (Tan, 2004). Leeloyova et al., (2002) reported a comparative study of optical microscopy and culture method, using cultivation as the gold standard, noting that cultivation method was more sensitive. Similarly, Stensvold et al., (2007a) using PCR as a gold standard, demonstrated that the Formol Ether Concentration Technique (FECT) which is commonly used for ova and parasite was less sensitive than Xenic in vitro Culture

(XIVC) method and permanent staining of faecal smear fixed in sodium acetate-acetic acid formalin (SAF-PST) in the ratio of 50%, 89% and 82% respectively. Therefore the study concluded that it is difficult to define Blastocystis epidemiology worldwide by interpreting results from studies with different identification or analytical methods.

However, some importance could be derived from studies aiming to report the prevalence rate of a parasite in different communities using the same methods. Gamboa et al., (2003) observed that children in a sub-urban area in Argentina were less infected with Blastocystis

23 than their urban counterparts. In a similar study in Egypt, prevalence of 25% and 4% were observed in rural areas and cities respectively (El-Sharzy et al., 2005). Equal distribution of

Blastocystis infection between the sexes has been observed in Pitanga, Brazil and in

Bangkok city, Thailand (Nascimento and Moitnho, 2005; Yaicharoen et al., 2005).

Differences in age prevalence have also been reported, with adults having higher prevalence of Blastocystis than children (Doyle et al., 1990; Guimaeras et al., 1993; Logar et al., 1994).

Traveling to tropical or endemic countries and working closely with animals are known to increase the risk of being infected by Blastocystis (Jelinek et al., 1997, Salim et al., 1999;

Stensvold et al., 2009; Rene et al., 2009).

Moreover, seasonal variation in Blastocystis occurrence have been suggested, with infections occurring more frequent in hot climates (Amin, 2002, 2006), though this variation has not been observed in other studies (Reinthaler et al., 1988; Cegielski et al., 1993).

2.0.6 CLINICAL DISEASE AND PATHOLOGY

The clinical signs and symptoms of Blastocystis species infections are non-specific gastrointestinal symptoms but similar to those of infection with other pathogenic protozoa and include diarrhoea, abdominal cramps, flatulence, anorexia (Cruz et al., 2003).

Blastocystis was also found to occur in individuals suffering from intestinal disorders than in the asymptomatic groups (Craczyk et al., 2005; El-Sharzy et al., 2005; Kaya et al., 2007).

Its association with irritable bowel syndrome has continued to interest many clinicians

(Cirioni et al., 1997; Hussain et al., 1997, Yakoob et al., 2004).

This parasitic organism was also isolated from stool of some individuals that presented with urticaria or allergic skin disease and diarrhoea symptom (Gupta and Parsi, 2006; Ozcakir et al., 2007; Kartassauruo-Katrasara et al., 2008).

24

Non-invasive Blastocystis disease has been reported in some healthy individuals in whom symptoms correlated only to the large number of parasites seen (Garcia et al., 1984).

In an earlier study on German travelers to the tropics who developed intestinal disorders and diarrhoea after a stay in a developing country, believed to be endemic for intestinal

Blastocystis infection, it was found that all the individuals had the parasite detected as the only cause of their condition and these individuals‟ condition were relieved following treatment for Blastocystis (Jelinek et al., 1997).

Individuals living with HIV and AIDS conditions were reported as having higher incidence of harbouring Blastocystis species (Gassama et al., 2001; Florez et al., 2003; Kassu et al.,

2003, Hallematoun et al., 2004; Zali et al., 2004).

In some of the studies, the presence of the parasite was linked to symptoms such as abdominal pains, flatulence and diarrhea in the individuals with the HIV/AIDS condition

(Gassama et al., 2001; Zali et al., 2004). No significant difference was found in

Blastocystis infection in HIV-positive immunocompromised individuals (19.6%) and in

AIDS individuals (16%) in a case study (Junod, 1995). In a similar study, Albrecht et al.,

(1995) reported that 38% of HIV-positive German patients had Blastocystis in their stool, while 46% of the individuals living with AIDS demonstrated a greater tendency of habouring the parasites, but concluded that there was no association of Blastocystis to the clinical symptoms found in all the patients from the study.

In the immune suppressed individuals, it was seen that the gastrointestinal tract remained a major target organ and in some HIV and AIDS subject, an estimated 30-60% in developed countries and up to 90% in developing countries suffered from diarrhoea and other debilitating enteric diseases as a result of opportunistic parasites (Bartlett et al., 1992).

25

Individuals with HIV/AIDS and diarrhea have greater degree of immunosuppression than those without diarrhea; thus predisposing their gastrointestinal tract to adverse actions of protozoa, bacterial, viral and fungal pathogens, some of which may lead to morbidity and death (Terra et al., 1998). Indeed, the association of diarrhoea as a disease condition in these immune compromised subjects can cause the patients to grow thin and lose weight tremendously (Terra et al., 1998).

However, the knowledge of Blastocystis-induced diarrhoeal conditions in HIV/AIDS is often highly limited by diagnostic difficulties, since optical microscopy, a less sensitive method of parasite detection, is more in use in most diagnostic centers in developing countries (Stensvold et al., 2006).

The mechanism of diarrhea due to Blastocystis sp. is not yet understood. Zierdt (1991) suggested that the presence of toxins found in fractions of culture medium could be the mechanism. Similar toxins in some protozoan induced diarrhoea, was demonstrated with toxins from Giardia lamblia which was shown to share some protein homology with sarafatoxins, a group of snake toxins from the burrowing Adder (Chen et al., 1995).

Oedema and inflammation of the large intestine and caecum mucosa is usually common in blastocystosis (Zukerman, 1994, Moe et al., 1997). In acute cases, mild invasion of the lamina propria of the colon has been indicated from endoscopies and biopsies (Zierdt, 1991,

Cirioni et al., 1999, Howard, 2007).

Dogruman –Al et al., (2008) demonstrated some Blastocystis spp., subtype 2 in individuals with intestinal disease, but who were asymptomatic. In another study, Blastocystis subtype,

ST1 and ST3 mixed subtypes were specifically associated as common in symptomatic individuals with some intestinal disorders of unknown cause, including Irritable Bowel

26

Syndrome (IBS) where ST2 and ST3 were isolated in chronic diarrhoeal disease and inflammatory bowel disease (Dogruman- Al et al., 2009a, b).

2.0.7 Blastocystis morphology

Blastocystis spp. is characterized as polymorphic with vacuolar or central body forms, granular, amoeboid and multivacuolar and avacuolar forms commonly identified (Belova,

1992). More recently, tiny cyst forms have been reported which were previously not recognized as Blastocystis sp. forms (Tan, 2004), (Figure2).

1. Vacuolar Forms

The central body or vacuolar forms of Blastocystis, have a diameter of about 3.5 µm

and up to14.6 µm but 200µm sizes have been seen in cultures; with 4- 15µm as the

average diameter (Stenzel and Boreham, 1996).The cell is spherical and surrounded

by a surface coat of varying thickness, with the plasma membrane showing coated

pits which function in endocytosis. The vacuolar forms are common in axenised

liquid cultures and fresh faecal samples as opposed to avacuolar forms, which have

not been isolated from the parasite cultures (Tan, 2004).

2. Grannular Forms

The granular form commonly arises from the vacuolar form. It therefore shares

many similarities with the vacuolar form but showing granules on the central body; a

transformation that comes up in the presence of factors such as increased serum

concentration in culture medium, transfer of cells to different culture medium and

axenisation (Stenzel and Boreham, 1996). The granular central vacuole is believed

to function in schizogony and endodyogeny with some reproductive structures

representing the progeny of Blastocystis (Zierdt et al., 1991; Suresh et al., 1993).

27

3. Amoeboid Forms

Amoeboid forms have been described as having diameter of 2.6- 7.8µm with

extended pseudopodia (Dunn et al., 1998). More recent description of this forms, as

oval- shaped with one or two large pseudopods and thin cell membrane (Tan, 2004).

It was characterized as appearing in old xenic culture, i.e. culture treated with

antibiotics and was also seen clinically in symptomatic individuals (Zierdt, 1991).

It was observed that the amoeboid form could be an intermediate between the

vacuolar and cystic forms, providing a nutritive role for the encystment stage by

phargocytising bacteria (Singh et al., 1995).

4. Avacuolar Forms

The avacuolar forms lack a central vacuole and a surface coat. Their diameter range

from 5-6µm and they have not been isolated from cultures (Stenzel et al., 1991).

5. Multivacuolar Forms

First, was reported in fresh human stool samples and have diameter of 5-8µm with a

thick surface coat. The multivacuolar and avacuolar forms are believed to represent

in vivo stages of Blastocystis while the vacuolar and granular forms occur in ‘in

vitro’ cultures (Tan et al., 2002).

6. Cyst Forms

Zaman et al., (1998) described the tiny cysts form, with an average diameter of 2-

5µm and thick walled cysts and thin walled cysts with diameter 12.5µm and 6.5µm

respectively. Cyst forms may be ovoid or spherical in shape with a protective

multilayer cyst wall which is refractile under phase contrast microscopy (Zaman et

al., 1995). Internal contents such as nuclei may be one to four, multiple vacuoles;

glycogen and lipid deposits have been reported (Moe et al., 1999). A loose fibrillar

layer has been described around the cyst forms, which appear to be shed as the cysts

28 mature (Zaman et al., 1997).Viability studies show that cysts do not lyse in water, can survive at room temperature for up to 19 days, but are fragile at extremes of temperature (cold and heat) and in common disinfectants (Zaman et al., 1995). The other forms of Blastocystis are sensitive to temperature changes, hypertonic and hypotonic environments and exposure to air (Moe et al., 1996). The cysts are the most resilient and the transmissible forms; believed to be the infective stage of

Blastocystis (Stenzel and Boreham, 1996). Table 3.0 shows a summary of morphological forms of Blastocystis species.

29

Table 2.1: Summary of Morphological forms of Blastocystis species.

Form Size (m) Source Central Surface Coat Number of Other remarks vacuole nuclei Vacuolar 2->200 Culture, faeces Present Present (thin) 1-4 Central vacuole (5-14 average) or absent occupies most of the cell volume Granular 6.5-80 Culture, faeces Present Present (thin) or 1-4 Granules in absent central vacuole; morphology similar to vacuolar form Multivacuolar 5-8 Faeces, culture Absent Present (thick) 1 or 2 Multiple small vacuoles ; too small to resolve by light microscopy Avacuolar ~5 Intestine, faeces Absent Absent 1 or 2 Rarely reported

Amoeboid 2.6-7.8 Faeces, culture Absent Absent 1 or 2 Rarely reported. Conflicting information on morphology Cyst 3-10 Faeces, culture Absent Present or 1-4 Cyst wall present

absent

(Source: Stenzel and Boreham, 1996).

30

Figure 2.0: Morphological forms of Blastocystis viewed under light microscopy.

Note: (A) Vacuolar form. (B) Granular form (C) Amoeboid form (D) Cyst forms Source: (Tan, 2004).

31

2.0.8 Diagnosis of Blastocystis infection

The following methods have been used in the diagnosis of Blastocystis, namely; smear microscopic identification, cultivation in parasitological medium, serological and

Polymerase Chain Reaction isolation methods.

1. Microscopy and Culture

Microscopy of smears of faeces in normal saline (0.9g/dl NaCl solution), and formol- ether or ethyl- acetate concentrate wet smears, aided with wet iodine staining solution or permanent stained smear mounts using Chromotrope trichome staining methods, which highlights the central body or the central vacuole of Blastocystis to stain reddish

(Leeleyoova et al., 2002, Suresh and Smith, 2004, Termmathurapoj et al., 2004, Windsor et al., 2006).

Other different staining methods applied to aid microscopic detection of Blastocystis include Iron haematoxylin stains, Kinyoun‟s acid fast stains and Giemsa stains of fixed stool smears (Stenzel and Boreham, 1996).

Cultivation of Blastocystis in culture medium such as in Jones medium (Jones, 1946) and other parasitological culture media such as axenic (using non- sterile media) „in vitro’ culture, has been used to detect Blastocystis in stool samples and to produce enough parasites for molecular studies (Clark, 1997, 2000; Leeleyoova et al., 2002; Stensvold et al.,

2006).

2. Serological Diagnosis

Serological diagnosis of Blastocystis has been carried out. Enzyme linked immunosorbent

Assay (ELISA), and Immunflouresence assay, IFA, have been used to detect serum antibodies to the parasite in only four patients (Garavelli et al., 1992).

32

In another separate study, threshold serum dilution titre for Blastocystis positive infections in patients was 1/50 dilution while attempting to use an ELISA to detect IgG antibodies in the sera of 28 patients (Zierdt et al., 1995).

However, Kaneda et al., (2000) by using IFA, observed that 70% of infected symptomatic individuals were serum positive for Blastocystis antibodies. Nevertheless, no monoclonal antibodies are currently avaliable for the clinical diagnosis of Blastocystis infection.

3. Molecular Diagnosis

PCR methods based analysis of SSUrRNA gene of Blastocystis followed by the Restriction

Fragment Length Polymorphyism (RLFP) analysis has been employed by some laboratories for the diagnosis of Blastocystis infection (Abe et al., 2003, 2004; Yoshikawa et al., 2004,

Yan et al., 2007, Tan, 2008). A variety of primers have been used for the PCR-RFLP analysis of the SSUrRNA gene and the method is mostly applied in prevalence studies

(Clark, 1997, Stensvold et al., 2007a).

Furthermore, PCR-nested amplification of intragenic regions has also been used to characterize Blastocystis isolates (Termmathrupoj et al., 2004). However lack of standardization of conditions of primers, mutation at restriction sites and difficulty in interpreting RFLP profiles from mixed infections were reported as some of the limitations of the method (Stensvold et al., 2006).

STS-PCR methods based analysis of the SSUrRNA gene using dideoxy sequencing was reported as more sensitive isolation method for Blastocystis (Stensvold et al., 2006, 2007a, c; Rivera et al., 2008).

More recent report of high sensitive pyrro-sequencing technique for the rapid sequencing of the SSUrRNA gene helped to genotype Blastocystis isolates within a short time, though mixed subtypes could be missed (Stensvold et al., 2007c). The methods have proved useful

33 for large-scale epidemiological studies and rapid identification of genotypes during outbreaks (Snowden et al., 2000; Scicluna et al, 2006; Tan, 2008).

2.0.9 Treatment of Blastocystosis

Over the years, the Center for Disease Control and Prevention, CDC, Atlanta, USA, has advised treatment for Blastocystis sp. infection in such patients where it is isolated and no other pathogen is found to explain conditions or symptoms found in the patient (CDC,

2001).

Treatment included the administration of Metronidazole, a common drug used for protozoan infections. Doses of 200mg to 800mg metronidazole (Flagyl) given orally, three times daily for 10days and up to 1500mg metronidazole orally may be applied in some cases.

Where there existed failure or resistance of Blastocystis hominis to this drug of choice further treatment choice included the use of aminoglycosides, paromomycin, cotrimoxazole, trimethoprim- sulfamethoazole and nitazoxanide (Rolstron et al., 1989, Diaz et al., 2003). A double dose of Trimethoprim (320 mg) and Sulphamethoxazole (800 mg) TMP-SMX twice daily for three weeks was used to eradicate Blastocystis among some Danish individuals investigated for enteropathogens (Stensvold et al., 2008). Trimethoprim-sulfamethoazole was reported used to stop outbreak of blastocystosis among primates at the San Diego Zoo in the USA (Zierdt, 1991).

Wolfe (1982) observed that removal of bacterial support essential to Blastocystis survival by the combination of antibiotics like tetracyclines and the imidazole drugs also helped in the treatment of some individuals with chronic cases.

Treatment failures may be attributed to a number of factors including drug resistance as seen in other antiprotozoal drugs (Tan et al., 2002) or to other factors such as; patients non- compliance, different pharmacokinetic properties, drug sensitivities of different subtypes or

34 strains of Blastocystis and inaccessibility of the organisms to the drugs or inactivation of drugs by concomitant organisms may occur (Rolstron et al., 1989).

2.1 Blastocystis subtype terminology

The extensive genetic heterogeneity of Blastocystis has been speculated which led to the assumption by some workers that parasite‟s pathogenic potentials might be genetically dependent (Yoshikawa et al., 1996; Bohm-Gloning et al., 1997). However, revelation of this features were variously reported (Arisue et al., 2003; Noel et al., 2005). The genetic distance between some isolates was reported to be 7.0 % (Clark, 1997). Nevertheless, various molecular approaches were used by different workers; with individual Blastocystis genetic strain terminology. This made it difficult to interpret and corroborate findings. For instance, Blastocystis terminology include clades (Arisue et al., 2003), subgroups (Bohm-

Gloning et al., 1997), ribodemes (Clark, 1997, Yoshikawa et al., 2000), and clusters

(Stensvold et al., 2006). Following the identification and characterization of isolates of

Blastocystis SSUrRNA gene analysis using DNA-based methods, a harmonized subtype terminology was proposed, (Table 4.0), where all human and animal isolates were designated Blastocystis species and subtypes 1 to 9 were assigned and also ST10 were recognized (Stensvold et al., 2007d, 2008). This present development of a consensus terminology for strains of this parasite has made it possible for workers to appreciate the emerging molecular epidemiology of the parasite.

The public health significance of intestinal Blastocystis infection is now beginning to emerge and understood by care-givers: clinicians, microbiologists and protozoologist, in most regions of the world due to ease of comparing and interpreting data based on the harmonized nomenclature.

35

Table 3.0: Hosts and Host-Specific subtype distribution of Blastocystis

Host/host Group Subtype (terminology: Stensvold et al, 2007d)

Primates STI, ST2, ST3, ST4, ST5, ST8, ST10

Ungulates and Dogs ST1, ST2, ST3, ST5, ST10

Rodents ST4

Birds ST6, ST7

Man ST1, ST2, ST3, ST4, ST6, ST7, ST8, ST9

Source: (Stensvold et al., 2008)

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3.0 GENERAL MATERIALS AND METHODS

3.0.1 Study Area: Lagos metropolitan city

Figure 2.1: Study area (insert) map of Lagos Source: Dr. M. Fasona, Department of Geography, University of Lagos (2012).

37

Lagos metropolitan city is located in Lagos state, south western region of Nigeria, west of

Africa. It is the largest city and chief port and former capital of Nigeria. The geographical location lies on latitude 6ᵒ 31‟N and longitude 3ᵒ18‟E, in the Bight of Benin; an arm of the

Atlantic ocean in the gulf of Guinea. The metropolis spreads over much of the state; 3345sq. km/1292 miles, which is located on four principal islands and adjacent parts of the Nigerian mainlands. The islands are connected to each other by bridges and landfills. Major sections of the city include the old city which now serves as the commercial district on western

Lagos Island; Ikoyi Island, situated just east of the Lagos Island and joined to it by a landfill;

Apapa, the chief port district is located on the mainland, residential Victoria Island and industralised Iddo Island.

Important mainland surburbs include: Ebute –metta, Yaba, Surulere, Ajegunle, Shomolu,

Agege, Mushin and Ikeja. Lagos has a diverse and fast growing population, resulting from heavy and on going migration to the city from all parts of Nigeria as well as neighbouring countries. The population of the metropolitan area was about10.1 million in 2003. The

Yoruba, an African people inhabiting southwest Nigeria constitute the city‟s principal ethnic group. English is the most commonly used language but Yoruba and other Nigerian languages are also spoken. The cities of Ibadan, Ijebo Ode, and Sagamu are nearby to Lagos metropolis. Lagos is an important center of Nigerian intellectual and cultural life. The

University of Lagos, with its teaching hospitals was established in 1962, College of

Technology, Yaba Lagos (1948), Lagos State University and its Teaching hospital (1983) and Lagos State Polytechnics (1977) are located in the city as are several research institutes.

Intense commercial and economic activities on the local and international fronts create an increase in travel and migration related activities in and across the metropolis. The attendant

38 problems of rapid urbanization, over-crowding and inadequate public utilities are encountered in the metropolis.

3.0.2 Sample Collection Centers:

The centers included Lagos University Teaching Hospital, LUTH and University of Lagos

College of Medicine, CMUL –PEPFAR/APIN clinics, Idi Araba in Lagos.

3.0.3 Sample size determination

Oyejide, (1992) enunciated the sample size determination using the formulae:

N =z².p (1-p)/D².

Where p = proportion of prevalence of 25%, and Z, define as 0.05 or 95% estimated from statistical table as 1.96. Therefore, sample size, N calculated from above, in each group selected was 288 samples and for the four groups targeted namely: Routine parasitological examination, HIV and AIDS-individuals, Diarrheal symptomatic gastrointestinal individuals and asymptomatic apparently healthy individuals or individuals for medical screening of fitness, amounts to a minimum of 1152 samples. However, a total of 1207 stool samples were used for the study and constitute the sample size.

3.0.4 Study Population

Participant’s inclusion criteria:

Participant‟s inclusion criteria included diarrhoea, passing at least 3 bowel movements within a day (WHO, 1998) and/or presenting with other gastrointestinal symptoms (colic or abdominal pains, dysentery, tenesmus, flatulence, persistent discomfort), as well as confirmed HIV/AIDS individuals attending clinics.

Control individuals were apparently healthy asymptomatic individuals on routine parasitological investigation or medical fitness purposes, with no prior illness

(gastrointestinal or diarrhoeal), 3 weeks to the time were interviewed and fresh stool

39 samples were collected (WHO, 1998) which served as negative controls. The age group of the participants range from 5years to 60years.

3.0.5 Study Design:

This was a cross sectional hospital and laboratory based study. The preliminary laboratory work was conducted in the Parasitology unit, of Department of Medical Microbiology and

Parasitology, College of Medicine IdiAraba, University of Lagos, Nigeria.

Molecular studies were done at the Parasitology unit, Dept. of Mycology, Bacteriology and

Parasitology of Statens Serum Institut, Copenhagen, Denmark.

3.0.6 Ethical Approval:

The study was approved by the Ethics and Experimentation Committee of Lagos University

Teaching Hospital, LUTH, and College of Medicine Uinersity of Lagos, IdiAraba, where patients were drawn, (Appendix 1 and 2). Informed consent was sought from participants.

Then research work followed the guidelines for good clinical and laboratory practices.

3.1 COLLECTION OF SAMPLES:

Stool samples were collected from the individuals attending the above mentioned health institutions during the period of study, between November 2005 and April 2009.

Stools were collected in waterproof, leak proof, screwed- capped plastic containers and were immediately transferred to the laboratory for processing. Bio-data of participants, whose stool samples were collected, were obtained from health records and by means of questionaires and oral interview.

40

3.2 PROCESSING OF STOOL SAMPLES:

3.2.1 Stool preservation:

Stool samples were processed immediately and were aliquoted into 3 cryoprecipitate tubes:

(i) Stored fresh with no additive with refrigeration at -20ºC to minimize deteriorations, when delay was expected

(ii) In 10% formol-saline, and

(iii) Preserved in 95% of ethanol solution for STS- PCR analysis (Stensvold et al.,

2006).

3.2.2 Macroscopic Examination

As soon as samples were received in the laboratory, macroscopic assessment of the characteristic of the stool sample was carried out before the addition of the preservatives.

Characteristics such as colour and consistency (semi-formed or soft, formed or hard, loose and watery), presence of blood, or mucus or both, as well as adult worms were noted.

3.2.3 Microscopic Examination

Routine Stool smears preparation for detection of ova of parasite by microscopy followed.

The stool specimens were first examined as saline and iodine wet smear preparations and followed by Formalin Ethyl-acetate Concentration Technique, FECT with a binocular microscope at X400 for the identification of Blastocystis cysts:

3.2.4. Microscopic Identification of Blastocystis in Stool Smears:

Blastocystis hominis forms including the size of cysts which varied from 3.5µm -14; and up to 200µm in culture (Zaman, 1997). Positive samples selected have ten and above cells per

X400, high power field. The common vacuolar forms have the large central vacuole, staining red and very thin cytoplasm staining green of Trichrome chromotrope staining reaction and malachite green counterstain, while the peripheral disposed cytoplasm

41 containing pockets of 3 to 5 dark nuclei (Plates1.6). In contrast to the size of the oocysts of

Cyclospora cyatanensis, of size between 8µm and 10µm in diameter; the shape, staining color varying from pink/red to colorless in the modified Kinyoun‟s acid fast staining reaction and Cryptosporidium but with sizes between 2.0µm and 3.5µm were also distinquished from Blastocystis cysts. Comparison with the positive control slides and pictures charts of the Blastocystis supplied by Dr. Niichiro Abe of Osaka School of Public

Health and Environmental Sciences, Osaka.

3.3. ISOLATION OF BLASTOCYSTIS DNA DIRECT FROM STOOL SAMPLE:

3.3.0 Sample selection Criteria:

The stool sample found positive with only Blastocystis were selected. The positive samples that had less than ten Blastocystis cells in each visual field, at X400 magnification were excluded in the study, since symptoms were consistent with the increased number of

Blastocystis cells.

On the basis of biodata and complaints, individuals were characterized as asymptomatic or symptomatic for diarrhoea condition. Twenty-four each of diarrhoeric symptomatic subjects and control asymptomatic individuals were selected from the HIV/AIDS participants were randomly selected from only Blastocystis -positive by smear microscopic method and further processed by PCR method.

3.3.1 Preservation of sample for PCR method

The stool aliquot collected in cryo- centrifuge tubes were preserved by proper mixing in

90% ethanol solution and stored for molecular studies using STS-PCR method.

42

3.3.2 Purification of Stool Samples from Preservative

From each stool sample, about 200mg of faeces was collected from the stool aliquot preserved in 90% ethanol was washed in three changes of phosphate-buffered saline, PBS

(pH 7.4) by centrifugation at 400g for 10 minutes, leaving clean Blastocystis organism at the end of the procedure as the concentrate.

The concentrate was overlaid on to 10ml of Ficoll-Paque column and centrifuged at 1200g for 10 minutes. Blastocystis separated into a band approximately 1 cm from the surface.

The layer of Ficoll-Paque-concentrated cysts were then aspirated into a clean tube, re- suspended in 8ml of PBS solution and centrifuged at 500g for 5minutes. The resultant pellet was stored at -20˚C until required for DNA extraction (Parkar et al., 2007)

3.3.3 Direct Blastocystis DNA Extraction:

DNA extraction was performed using QIAmp DNA stool mini kit (Qiagen, Hilden,

Germany), according to the manufacturer‟s protocol. The DNA extracts were eluted in

100µl AE buffer.

3.3.4 Blastocystis Genotyping by STS-PCR method:

Amplification in both rounds of PCR was performed using a Gene-Amp2400 thermocycler

(Applied Biosystems, Ca, USA) and employing nine sequence tagged site primers according to the harmonized nomenclature for Blastocystis (Stensvold et al., 2007). The PCR products of approximately 1.0kbp were electrophorized on 1.5 % Agarose gel and the PCR product of band 550- 590 basepair visualized under Ultra Violet-light after being staining with ethidium bromide in a UV- illuminator was considered specific of Blastocystis sp.

(Bohm-Glonning et al., 1997). The process was documented on high-density printing paper using a UV-save gel documentation system 1 (Uvitech, Cambridge, UK).

43

3.3.5 PCR Products Purification and Dedioxysequencing:

The UltraClean тм GelSpin DNA Purification sample kit (SANBIO, Uden, Netherlands) was used to purify the PCR products according to the manufacturer‟s instruction. The dedioxy sequencing was in one direction using the BHCRseq3 primer as the sequencing primer (Stensvold et al., 2007). For the PCR products of 1.0kbp, approximately, 20ng of

DNA was used for sequencing, and for the PCR product of 0.3kbp, approximately, 5-10ng was used as template and sequenced in both directions using the Big Dye Terminator system, version 3.1 (Applied Biosystems, Foster city, CA) on an ABI 3730 x1 capillary sequencer.

3.3.6 Subtype (ST) Identification:

For subtype identification, sequence chromatographs and pyrrograms were manually interpreted and nucleotide sequences checked and edited, using Sequencer version 4.0. The subsequent nucleotide sequences of the SSUrRNA gene of Blastocystis obtained in the study were multiple aligned, analyzed and interpreted using the software program BioEdit

Sequence Alignment Editor version 7.0 (Hall, 1999).

The high diagnostic specificity and sensitivity was secured by using a STS-PCR assay, and the internal primers amplifying the 170bp sequence were confirmed to be Blastocystis- specific when submitted to a Basic Local Alignment Search Tool (BLAST) trail

(http://w.w.w. ncbi. nlm. nih. gov/BLAST).

3.3.7 Molecular Phylogenetic classification

Phylogenetic analysis of the SSUrRNA was carried out using Mr BAYES version 3.1

(Huelsenbeck and Ronquist, 2001). Bayesian analysis of the SSUrRNA data set were performed using the general time reversible,GTR + Γ (gamma distribution of rates with four rate categories) +I (proportion of invariant sites) model of sequence evolution, with base

44 frequencies, the proportion of the invariant sites, and the shape parameter α of the Γ distribution estimated from the data.

In Bayesian analyses, the starting trees were random, four simultaneous Markov chains were run for 500,000 generations, burn- in values were set at 30,000 generations, and trees were sampled every 100 generations. Bayesian posterior probabilities were calculated using a Markov chain Monte Carlo sampling approach implemented in Mr BAYES version

3.1(Green, 1995). Distance- based analysis was conducted with MEGA 3.1 software,

(Kumar et al., 2004) using the Tamura-Nei distance estimates, a 2-parameter model, with reference sequences available in the GenBank used as reference sequences. Proteromonas larcertae (U37108) was used as the out-group and trees constructed using the neighborhood joining UPGMA algorithm. Figure 3.10, shows molecular phylogenetically tree constructed with subtypes generated from local Blastocystis isolates from study. The subtype, ST, was assigned to a given sequence based on the reference nucleotide sequence it clustered with

(Kumar et al., 2004). Subtype terminology according to Stensvold et al., (2007) was used in the study.

3.3.8 Statistical Analysis

Statistical analysis included testing for independence using Chi square, χ² Test with 95% confidence interval (CI) using the computer software package Coco (Bradsberg, 2001).

However, the t- test and ANOVA, analysis of variance were also used.

45

PREVALENCE OF BLASTOCYSTIS SPECIES AND INTESTINAL

PARASITES IN LAGOS, NIGERIA

4.0 INTRODUCTION

Human blastocystosis is an emerging enteric infection caused by the micro-eukaryotic protist of the genus Blastocystis which is associated with symptoms of abdominal pain, diarrhoea, constipation, fatigue, skin or anal itching, tenesmus, and other symptoms (Zierdt et al., 1991, Jones et al., 2009; Malheiros et al., 2011).

Although, Zierdt et al., (1967) gave detailed description of the protozoan characteristics of the parasite but the systematic classification of the parasitic protist did not accommodate the parasite, until Silberman et al., (1996) used the conserved SSUrRNA gene analysis of the

Blastocystis in comparism to other eukaryotes to phylogenetically place the parasite in the phylum Stramenopile or Heterokont, along with other organisms such as diatoms, brown algae, slime nets and water moulds.

Its potential for pathogenicity in humans was suspected following their implication as a cause of fatal watery diarrhoea reported among primates in a Zoo in Santiago, USA (Zierdt,

1991).

Infection in human populations are widely distributed, with high prevalence found in localities with poor personal hygiene and questionable environmental sanitation, particularly, sewer contamination of drinking water and food (Cruz et al., 2003, Suresh and

Tan, 2005). Several urban cities in the developing countries have been found to have high prevalence of faecally transmitted pathogens as a result of disposal of untreated faeces into the environment (Reinthaler et al., 1988, Bello et al., 1997, Oyerinde, 1999). It is curious that reports of blastocystosis in these cities are scanty or not reported at all.

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The burden of blastocystosis may be huge in the developing communities than presently suspected. An earlier report from WHO estimated that more than three thousand million people were exposed to sewer contamination in developing countries (WHO Press release,

1998) , where an average rate of Blastocystis infection approaches fifty percent, implying that there will be1500 million cases in any given time. This is three times the infection rate of 500million reported for Entamoeba histolytica/ dispar complex and more than the infection rate for Giardia lamblia of 1000 million cases (Eve et al., 1999). A more recent report estimated that one billion people in the world may be habouring this parasitic infection (Stensvold et al. 2012). Blastocystis spp. is believed to be the commonest parasitic pathogen detected in stool samples submitted for microbiological analysis (Windsor et al.,

2001; Windsor and Macfarlane, 2005; Stensvold et al., 2009; Malheiros et al., 2011).

Fortunately, human blastocystosis has been responsive to metronidazole treatment and treatment useful in the eradication of protozoan pathogens, but resistance is being recognized in some localities (Stensvold et al., 2008). Infection is probably of low infectivity which implies that outbreaks and epidemics in human populations which were believed to be more frequent during wars and civil disturbances, when sanitation was compromised, may still persist without informed interventions (Zierdt et al., 1991, Tan et al., 2002). Despite concerted efforts, investigations into all aspect of the parasite remains an enigma and difficult to establish the role of Blastocystis in gastrointestinal symptoms including diarrhea disease of unknown aetiology in which the parasite were often detected as the sole pathogen (Howard, 2007).

The transmission of Blastocystis species is believed to be faeco-oral through the ingestion of contaminated hand handling drinking water or food such as vegetables, raw water plants and unwashed fruits. Leeloyoova et al., (2002) demonstrated waterborne transmission

47 among Thailand military personnel. The WHO also recognizes the unsanitary water borne transmission of the parasite through over flowing of waste water for agriculture into the water for human consumption (CDC, 2003; Cruz et al., 2003; WHO, 2006).

The epidemiology and systematic classification of this parasite (i.e., the study of the diversification) are still emerging. There is an extensive genetic variability recognized among the Blastocystis sp. (Bohm-Gloning et al., 1997, 2001; Malheirous et al., 2011;

Menon et al., 2012, Stensvold et al., 2012), which explains variable outcome of infections in different individuals. The center for Disease Prevention and Control, Atlanta, advised that

Blastocystis should be treated when detected as the sole pathogen in the patient‟s clinical sample. The common imidazole drug, metronidazole (Flagyl) has been widely used (CDC,

2012). The Pan American Health Organization, PAHO, also recognized that the parasite could be opportunistic, with little or no pathogenic impact on the immune competent host, but may show serious consequences in the immune compromised individuals such as

HIV/AIDS condition (Stenzel and Boreham, 1996).It is imperative, therefore, to ascertain that Blastocystis exists in any area in order to help in making informed decision of intervention methods. Meanwhile, the prevalence of Blastocystis in the locality of Lagos is not known.

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4.1 MATERIALS AND METHODS

4.1.0 Study Area:

Lagos metropolitan city located in Lagos State, south western region of Nigeria.

4.1.1 Sample Collection Centers:

The centers included the Lagos University Teaching Hospital, LUTH and College of

Medicine, University of Lagos CMUL- PEPFAR clinics.

4.1.2 Sample size determination

Oyejide (1992) enunciated sample size determination by the formula:

N z².p (1 p)/D².

Where p = proportion of prevalence of 25%, and Z, define as C 0.05, 95% estimated from statistical table as 1.96. The sample size, N calculated from above, in each group selected was 288 samples; with a total of 1152 samples targeted the groups. However, a total of 1207 stool samples were used for the general population study.

4.1.3 Study Population

Participants were drawn from individuals presenting for routine parasitological laboratory investigations, Diarrheal symptomatic gastrointestinal subjects, and asymptomatic apparently healthy individuals who presented for medical screening fitness. The age groups were between 5 years and 60 years.

Inclusion criteria:

Study inclusion criteria included diarrhea individuals passing at least 3 bowel movements in a day (WHO, 1998) and/or presenting with other gastrointestinal symptoms: colic or abdominal pains, dysentery, tenesmus, flatulence, persistent discomfort (CDC, 2003) as well as confirmed HIV/AIDS individuals attending clinics, were recruited.

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Control subjects were apparently healthy asymptomatic individuals with no prior illness

(gastrointestinal or diarrhoeal) 3 weeks to the sampling time were interviewed and fresh stool samples were collected; which served as negative controls (WHO, 1998).

4.1.4 Study Design:

This was a cross sectional hospital and laboratory based study.

4.1.5 Ethical Approval:

The study was approved by the Ethics and Experimentation Committee of the institutions,

LUTH and CMUL- PEPFAR clinics from where patients that participated were drawn

(Appendix 2 and 3). Informed consent was ought from participants. The research work followed the guidelines for good clinical and laboratory practices.

4.1.6 COLLECTION OF STOOL SAMPLES:

A total of 1207 stool samples were collected during the period of study- between

November 2005 and April 2009.Stool specimens were collected in screwed-capped, leak proof, plastic containers and were immediately transferred to the laboratory for processing.

The bio-data of participants were collected personally, and from health records.

4.1.7 Processing of stool samples:

Stool samples were processed immediately and aliquot into 3 cryoprecipitate tubes:

(a) When there is delay, stool samples were stored fresh with no additive at -20ºC.

(b) Stored in 10% formalsaline

(c) Stored in 90% ethanol solution for use in PCR method.

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4.1.9 Macroscopic Examination

As soon as samples were received in the laboratory, characteristics such as stool colour and consistency (semi-formed or soft, formed or hard, loose and watery), presence of blood, or mucus or both and adult parasites were noted

4.1.10 Identification of Blastocystis by microscopy:

Routine Stool smears preparation for parasite microscopy was done. The stool specimens were first examined as saline and iodine wet smear preparations, followed by Formalin

Ethyl-acetate Concentration Technique (FECT). The concentrate smears were stained using

Kinyoun‟s modified acid fast Bacilli reaction to distinguish oocysts of Cryptosporidium and

Cyclospora spp. and Trichrome staining techniques for parasite detection which distinctly shows Blastocystis spp. vacuolar form, with a large central vacuole staining reddish colour

(Garcia and Bruckner, 2007).

4.1.11 Parasite identification by microscopy was based on the following criteria: -

A) The size (e.g. Cysts of Blastocystis 3.5-14µm).

B) Shape/morphology: oval/round central vacuoles, with peripheral tiny cytoplasm.

C) Consistence in staining color of reddish central vacuole

D) The peripheral positioning of bluish staining cytoplasm; containing, nuclei

numbering 3-5, and other cell organelles including a MLO, mitochondrion- like

organelle.

E) Comparison with Blastocystis positive slides supplied by Dr Niichiro Abe of the

School of Public Health and Environmental Sciences, Osaka, Japan.

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4.1.12 Confirmation of Positive samples:

The confirmation of suspected Blastocystis positive samples was done at the parasitology unit, Department of Microbiology, Statens Serum Institut in Copenhagen, Denmark.

4.1.13 Statistical Analysis:

Results were analyzed withChi square (χ²) test to compare the sensitivity of detection between the variables and Blastocystis in the stool samples at a probability value of 95% confidence level. Test for Independence were analysed with the χ²-test with the computer software Coco (Brasberg, 2001).

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4.2 RESULTS

4.2.1 Prevalence of parasitic infections in Lagos metropolis.

Out of the 1207 stool samples analyzed, 652 (54.0%) were found positive for 17 different intestinal parasites which were grouped as pathogenic and non- pathogenic protozoan and helminths (Table 4.0). Blastocystis hominis/spp. was detected in 232 of the samples and constituted 19.2%. Other pathogenic protozoa seen included Giardia lamblia 90(7.5%),

Entamoeba histolytica/dispar 48(4.0%). The coccidian, Cryptosporidium spp. and C. cayetanensis oocyst found in 26(2.2%) and 47(3.9%) of patient‟s stool respectively.

The prevalence of the Non- pathogenic protozoa were also isolated were; Entamoeba coli which were seen in 100(8.2%), Dientamoeba fragilis 74(6.2%). Other parasitic organisms of significance detected were Ascaris lumbricoides 36(3.0%), Hookworms 29(2.4%), Taenia species 36 (3.0%), Trichuris trichuria 12 (1.0%), Fasciola spp.10 (0.8%) and Strongyloides stercoralis12 (1.0%)

The mean age of patients whose samples were collected was 34.6±3 years for females and

35.9 ± 4 years for males.

4.2.2 Blastocystis detection in stool samples of individuals

Two hundred and thirty-two of the samples were positive for Blastocystis species, by microscopic analysis. Preliminary tests include saline and iodine wet smear which showed the various morphological forms: vacuolar, avacuolar, amoeboid, multivacuolar, and cyst forms with brownish large central body, and nuclei staining dark blue in peripheral cytoplasm (Plates 1- 8). The permanent trichrome stained slides show amoeboid form

Blastocystis species using methylene blue as counterstain (Plate7) and vacuolar forms showing dark reddish stained large central body or vacuole ,with the thin cytoplasm peripherally displaced taking the greenish colour of the counterstain, the malachite green dye (Plates 8).

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Table 4: Profile of Intestinal parasites encountered in Lagos metropolis.

Intestinal Parasites Positive (No) Percentage (%)

Pathogenic organisms

Blastocystis spp. 232 19.2

Giardia lamblia 90 7.5

Entamoeba histolytica/dispar 48 4.0

Cyclospora cyatanensis 47 3.9

Cryptosporidium species 26 2.2

Isospora belli 1 0.1

Non- pathogenic protozoa

Entamoeba coli 100 8.3

Dientamoeba fragilis 77 6.4

Chilomastix meilini 9 0.8

Trichomonas hominis 6 0.5

Helminths

Ascaris lumbricoides 40 3.3

Taenia species 36 3.0

Ancylostoma duodenale 29 2.4

Strongyloides stercoralis 12 1.0

Fasciola species 10 0.8

Hymenolepis nana 9 0.7

Trichuris trichuria 12 1.0

TOTAL 1207 100%

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TABLE 5: Frequency distribution of stool samples collected by Age groups.

Age group Frequency(f) Percentage

0-10 years 3 0.2

11-20 years 30 2.5

21-30 years 418 34.6

31-40 years 446 37

41-50 years 201 16.7

51-60years 88 7.3

>60years 21 1.7

TOTAL 1207 100

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Table 6: Frequency of diarrhoea condition among individuals in the study Frequency Percentage Diarrhoea Stool (No) Positive 380 31. 5

Negative 827 68.5

TOTAL 1207 100

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Table 7: Detection of Blastocystis with consistency of Stool sample

Nature of stool Blastocystis Blastocystis Positive Negative (No, %) (No, %)

Formed 68(5.63) 123(10.19)

Semi-formed 44(3.65) 213(10.11)

Watery 17(1.41) 100(8.29)

Loose 103(8.53) 539(44.66)

Total 232(19.22) 975(80.78)

χ²=38.09,

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Table 8: Detection of Blastocystis with Occurrence of diarrhoea condition in individuals

Occurrence of Blastocystis positive Blastocystis Negative Diarrhoea No (%) No (%)

Positive 76 (6.3) 304 (25.2)

Negative 156 (12.9) 671 (55.6)

Total 232 (19.2) 975 (80.8)

χ² 0.217

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Table 9: Detection of Blastocystis with reason for visiting the clinic.

Reason for visiting Clinic Blastocystis Blastocystis

Positive Negative Total

(No, %) (No, %) (No, %)

Routine parasitology 12 (0.99) 137(11.35) (149, 12.34%)

GIT complaints 25(2.07) 10(0.83) (35, 2.9%)

HIV/AIDS condition 124(10.27) 82 (6.79) (206, 17%)

Medical screen 9(0.75) 49(4.06) (58, 4.8%)

Healthy control 62(5.14) 697(57.75) (759, 62.9%)

Total 232 (19.22) 975(80.78) (1207, 100%)

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Table 10: Sex with detection of Blastocystis among individuals in the study

Frequency Percentage

Sex of Individuals (No) (%)

Female 134 57.8

Male 98 42.2

TOTAL 232 100

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Table 11: Diarrhoea condition with patients’ reason for visiting clinic.

Occurrence of Rou GIT HIV Chk Contl.

Diarrhoea No (%) No (%). No (%). No (%). No (%).

No (%). No (%). No (%). No (%).

Positive 46(3.81) 15(1.24) 55(4.56) 24(1.99) 221(18.31)

Negative 103(8.53) 20(1.66) 151(12.51) 34(2.82) 538(44.57)

TOTAL (1207) 149(12.35) 35(2.90) 206(17.07) 58(4.81) 759(62.88)

χ² =7.6, p<0.05

Key: ROU = routine parasitology work up;

GIT = diarrhoea/ gastrointestinal tract complain

HIV = human immunodeficiency virus test

CHK = Medical screen or checkup;

CTRL = control samples, no complaints.

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4.2.3 Frequency Distribution of the Patients by Age Groups:

The frequency distribution of the stools, showed that 37% of the stool samples collected came from individuals aged 31-40 years, 34.6% from age 21-30years, 16.7% from those aged 41-50years, 7.2% from those aged 51-60years, >60years 1.7% those in the 11-20years were 2.5% and children aged 0-10years were 0.3%.Seven hundred and forty-eight, 62% of the total stool samples screened came from females and 459 or 38% were from males Table

5.There is no statistical association of parasite detection with sex (P> 0.05).

Data showed that, of the 31.5% were confirmed diarrhoeal cases; passing at least 3 or more watery stool in 24 hours (WHO, 1998). Nevertheless, the majority, 68.5% of the stools, came from those without diarrhoea (Table 6).

The patients on routine parasitological screening accounted for 12.4% of the participants,

2.9% were from those with gastrointestinal (GIT) complaints, 17% were from HIV positive subjects, 4.8% were for medical fitness screening and 62.9% included healthy control and some conditions not clearly stated (Table7).

4.2.4 Analysis of Sex of Individuals who were positive for Blastocystis spp.

One hundred and thirty-four 134 (57.8%) of the Blastocystis-positive stools were from females, while ninety-eight 98 (42.2%) were from males (Table 10).

One hundred and twenty-four, (53.5%) positive Blastocystis were from HIV seropositive subjects and twenty-five, 25 (10.8%) were from non-diarrhoeal patients; whose stool was submitted following GIT complaints and the remaining, 83 (35.8%) were from diarrhoeic individuals (Table 8).

4.2.5 Consistency of the stool samples with Blastocystis Detection

By consistency of the stool samples, the loose stool were 53.2%, while 21 % were semi- formed, 16 % were formed, and 10 % was watery stool (Table 9).

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Two hundred and thirty-two individuals whose stool samples had Blastocystis, 37.5% were aged 31- 40 years. One hundred and thirty-four, 134 (57.8%) were females and Ninety- eight, 98(42.2%) were males (Table 10).

Some of the patients had loose or watery stools indicative of diarrhoea conditions were not positive for Blastocystis. One hundred and twenty-five (53.9%) of the stools had multiple infections with other intestinal parasites. Ascaris lumbricoides and Taenia spp. were the common helminthes while important protozoan detected included Entamoeba histolytica/dispar, Entamoeba coli. The coccidian parasites identified were

Cryptosporidium spp., and Cyclospora cyatanensis. Moreover, polyparasitism, where stool samples from the same individuals had 3 or more intestinal parasites, were common among the participants.

4.2.6 Reasons for Submission of Stool Samples

The bio-data of patients and their reasons for the submission of stool for laboratory investigation showed that 46(3.8%) came from those on routine parasitological investigation, 15(1.24%) were from those with gastroenteritis, with the rest comprising of

HIV/AIDS 55(4.6%) while control individuals of apparently healthy individuals with no complaint submitted 245(20.3%) diarrhea samples.

The study revealed a significant statistical relationship (p < 0.05) between the detection of

Blastocystis, diarrhoeal status and nature of stool (Table 11). However there was no statistical significant relationship between the detection of the Blastocystis cysts with age group, sex and reasons for visiting the hospital (p>0.05).

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Plate 1: Saline wet mount of Blastocystis sp. cysts (Magnification X400).

Plate 2: Iodine smear mounts of cyst of Blastocystis sp. microscopy (X800).

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Plate 3: Iodine smear mounts of cyst of Blastocystis (Magnification X400).

Plate 4: Iodine wet mount of Blastocystis sp. cell, vacuolar form (X800)

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Blastocystis

Plate 5: Vacuolar form of Blastocystis sp. in saline smears (X800)

Plate 6: Iodine wet mount of Blastocystis sp. cell, vacuolar form (X800)

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BH (Vacuolar form) Plate 7: Blastocystis sp. in trichrome staining with Methylene blue as counter stain (X1000 magnification).

Plate8: Permanent Trichrome stained Blastocystis with malachite green counterstain.Vacuolar forms (x1000 magnification).

NB: The dark reddish stained large central body or vacuole, with cytoplasm peripheral and very thin; taking the greenish counter stain of malachite green dye and interspersed with 3-5 pockets of nuclei darkish spots.

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4.3.0 DISCUSSION

The study found an overall prevalence of 19.2% in Lagos metropolis for Blastocystis in stool samples of individuals.. The prevalence rate in Lagos compares closely to the reported

Blastocystis prevalence from most developing countries of which ranged between 15% and

60% (Guimeras and Soyagar, 1993; Torres et al., 1992). For instance, a prevalence of

18.5% was found from the Quaylobia province of Egypt, following a survey of food vendors presenting for health clearance certificates (Sadek et al., 1997). In these countries, poor sanitation and contamination of water and environment with rampant disposal of untreated animal and human faecal waste were the common epidemiological factors

(Leeloyovaa et al., 2002; Suresh et al., 2005; Tan and Suresh, 2006). On the other hand, lower prevalence of 0.5% to 10% was reported for most other developed countries such as

Japan and Denmark where strict hygienic practices such as, treatment of sewage before disposal, were entrenched (Logar et al., 1994; Vickerman, 1994; Hoiriki et al., 1997;

Stensvold et al., 2008). The prevalence of 2.5% reported for Blastocystis infection of individuals in Abeokuta city, Southwest, Nigeria (Reinthaler et al., 1988) is lower than the present study. The degree of faecal contamination believed to predispose to endemicity of fecal intestinal parasites and Blastocystis in patients in Abeokuta city, may be low compared to Lagos. The occurrence of other endemic protozoa showed Giardia lamblia

7.4% and Entamoeba histolytica/ dispar complex 3.6% which were lower than Blastocystis parasite detected in the study. Similar observation was reported in the United States of

America, where the occurrence of Blastocystis species was 28.5 times higher than Giardia lamblia and about 10 times the incidence of Entamoeba histolytica/ dispar following three decades of neglect of the surveillance of Blastocystis parasite (Amin, 2002).

Residents of Lagos were reported to face high urbanization and over- crowding, with its attendant toll on infrastructure, especially public utilities for water provision with poor

68 personal hygiene, unsanitary environmental challenges and overcrowding (Oyerinde et al.,

1999). It is therefore possible that unsanitary personal habits and environmental contamination with heavy sewer contamination could occur in Lagos metropolis. This suggests that there may be sustained endemicity of faeco-oral protozoan parasites including

Blastocystis, which explains the high prevalence found in this study compared to the study in Abeokuta.

Nevertheless, the prevalence found in this study was based on microscope- positive smears of stool samples of individuals attending clinics, including those with defined gastrointestinal disorders, immune competent and immune- compromised conditions such as HIV/AIDS already receiving treatment at the clinics, as such, it may be a gross underestimation of the true infection rate, both in the population examined and in the Lagos area as a whole.

The relatively low sensitivity of the microscopic examination techniques used and the low level of awareness including recent description of tiny cysts forms of sizes of 3.5 to 4.6 µm that were not previously recognized as forms of Blastocystis species, inferred that some positive samples could have been missed by the study. It is also possible that the real prevalence of the parasite in Lagos might be higher than the present rate of 19.2% if a more sensitive method of analysis is used. Hence, there is need to validate the present hypothesis using a more sensitive molecular techniques such as nucleotide sequencing which offers species- specific DNA isolations (Zierdt et al., 1995; Tan and Suresh, 2006; Stensvold et al., 2006, 2007b; Hussein et al., 2008).

Another important finding from this study includes a statistical significant association of diarrhoeal symptoms with Blastocystis infection in this locality. Guptra and Parsi (2006) investigated skin rashes and urticaria presenting with diarrhoea in some patients, with indication that Blastocystis species was the only parasite identified and therefore associated

69 same with the condition. The eradication of the parasites following treatment with metronidazole, made the skin rashes and urticaria to resolve.

Other studies had also found statistical significance with diarrhoea and anal itching with

Blastocystis spp. isolated from the patients as the only parasitic organism that could account for the symptoms. Indeed, these patients‟s intestinal condition of diarrhoea and anal itching were normalized following treatment for the parasite (Kaya et al., 2007; Kartasssauro-

Katsara et al., 2008).

Although some stool samples were diarrhoea, others showed no consistency with diarrhoea or symptoms associated with gastrointestinal complaints in this study. It is possible that not all the diarrhoea conditions in the individuals were due to Blastocystis infection. Dogan,

(1998) correlated the presence of intestinal symptoms like abdominal pains, distention, lack of appetite and diarrhoea in 88 patients in whom only B. hominis was detected in the stool examination, but found no statistical significance with the symptoms. In other studies carried out in Denmark, symptoms reported were abdominal pain and diarrhoea, with stool samples showing about six characteristic natures or consistency such as, formed, soft, loose, mucoid, loose-watery and watery (Stensvold et al., 2008). In study of Danish subjects with enteropathogen, Rene et al., (2009) reported no significant association between the nature of stool samples submitted with Blastocystis detection.

From this study, there was no influence of gender on parasite detection. Quadri et al.,

(1989) in earlier study of a community of Saudi Arabia reported that more males were infected with Blastocystis hominis than females but this was attributed to religion where the males participated freely in the study but the women were in Padua and could not be accessed.

There was also no significant association between Blastocystis detection with age group investigated (p>0.05). However, the individuals from whose stool samples Blastocystis

70 were detected were all adults, aged between 21 and 60 years. The findings from this study also compared closely with reported isolation of Blastocystis in adult patients in Denmark in which the parasite was obtained from adults aged between 21 and 50 years (Stensvold et al.,

2007a). Furthermore, among city residents in Argentina, there appeared to be higher prevalence of the organism in those patients who were more than 14 years old (Minvielle et al., 2004). Fluctuations in the prevalence of blastocystosis with age have been reported in several studies, with adults having higher prevalence than children (Quadri, 1989; Doyle et al., 1990; Sanad et al., 1991; Ashford and Atkinston, 1992). Nevertheless, it is believed that, in the areas of endemicity, children are usually asymptomatic with mild diseases following Blastocystis infection (Nimri and Batchoun, 1994).

On the other hand, Logar et al., (1994) and Zuckerman et al., (1990) in a similar work using microscopic methods failed to show any significant difference between the age of patients and the prevalence of Blastocystis in both adults and children respectively.

From the preliminary investigation conducted on stool samples of children from two private primary schools located in Idi-Araba area, Mushin and Ikate areas and Surulere in Lagos, revealed no Blastocystis forms. The pupils were free of parasitic infections as a result of interventions of routine three monthly de-worming exercises, using albendazole drugs.

Albendazole and other imidazole class of drugs although common anti-helminthic drugs have been shown to eradicate protozoa and proved useful as the alternative drug of choice for Blastocystis treatment in localities where there is resistance to metronidazole therapy and its derivatives (Geltman et al., 2003).

From the findings, it might be right to suggest that all stool samples submitted for parasite detection in Lagos clinics should be screened for Blastocystis species and treatment recommended where no other agent is found to account for the patient‟s condition.

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As an important emerging parasite, its control should include identification of transmission routes, such as screening for Blastocystis in drinking water, bodies of water frequently used for agriculture and domesticated animals.

4.3.1 CONCLUSION

The detection of Blastocystis species in 19.2% of individuals that participated in the study implies that the parasite exists in Lagos metropolis.The finding of statistically significant association of the parasite detection with diarrhea, could mean that it is a diarrhoegenic pathogen and could be aetiologic in some diarrhea that occur in Lagos. However, the extent of diarrhea induced blastocystosis has not been determined. Nevertheless, the study is aimed at creating awareness of this emerging potential pathogen of man, through determining its prevalence in Lagos, which presently is not recognized as a pathogen in our clinics.

Finally, since eating of raw vegetables, fruits and water plants are common practice among the people, particularly, there is need to extend proper washing of raw food and routine stool screening exercise in suburban and rural area, in other to strengthen the knowledge of

Blastocystis in Lagos State.

Most important is the urgent need to train Medical laboratory scientists and other health practitioners to recognize and be able diagnose and report the infection, without which no reasonable interventions would be adopted against Blastocystis-induced conditions.

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BLASTOCYSTIS AND INTESTINAL PROFILE AMONG HIV/AIDS SUBJECTS

5.0 INTRODUCTION

The unicellular amoeba-like protozoan, Blastocystis spp. is one of the most common parasites in the intestinal tract of man and vertebrates and invertebrates such as pigs, poultry, and birds including more recently cockroaches (Abe et al., 2002; Windsor et al.,

2001; Yoshikawa et al., 2007; Jones et al., 2009).

Infection due to parasite, blastocystosis, is characterized by gastrointestinal discomforts such as diarrhea, abdominal colic, urticaria, bloating and excessive flatulence and malaise

(Doyle et al., 1990, Cruz et al., 2003). The parasite has also been implicated as causative agent in some intestinal disorders, such as chronic bowel disease, chronic diarrhoeal disease and colitis (Howard, 2007). Some report suggested that Irritable Bowel Syndrome, IBS, have strong association with Blastocystis infection (Hussain et al., 1997, Yakoob et al.,

2004; Macfarlane and Windsor, 2005).

More recent reports have shown that subtypes 3 of Blastocystis sp. occur in about 40% of individuals with irritable bowel syndrome in Europe (Drougman-Al et al., 2008, 2009).

Phylogenetic studies of several conserved genes and proteins in this eukaryotic parasite, in comparism with other eukaryotes revealed Blastocystis species is a Stramenopiles closely related to the Peteromonas larcaertae (Johnson et al., 1989; Silberman et al., 1996; Arisue et al., 2002, 2003; Noel et al., 2005).

The immunosuppressed individuals often have the gastrointestinal organ as a major target for microbes‟ invasion. In the HIV and AIDS conditions, an estimated 30% to 60% persons living with the condition in developed communities and up to 90% in developing countries suffer from diarrhea and other debilitating enteric parasitic infections as a result of opportunistic parasites (Bartlett et al., 1992).

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Individuals with HIV/AIDS and diarrhoea, which may be intermittent or profuse, have greater susceptibility to immunosuppression than those without diarrhea.

Although Blastocystosis is seen in healthy subjects, but individuals living with HIV and

AIDS were found with higher incidence of harbouring Blastocystis sp. (Gassama et al.,

2001; Florez et al., 2003; Kassu et al., 2003; Hallematoun et al., 2004; Zall et al., 2004).

In these studies, the presence of the parasite was linked to non- specific symptoms such as abdominal pains, flatulence and diarrhoea, (Gassama et al., 2001; Zall et al., 2004).

The knowledge of diarrhoeal pathogens in HIV/AIDS due to Blastocystis infection is often greatly limited by diagnostic difficulties, since optical microscopy is more in use in most diagnostic centers (Stensvold et al., 2006).

Over the years, the Center for Disease Control and Prevention, CDC, Atlanta, USA, advised treatment for Blastocystis sp. infection in several categories of patients where no other pathogen is detected to explain conditions found in a patient (CDC, 2001). Treatment includes the administration of 800mg metronidazole (Flagyl) for 3 weeks.

In localities where there existed failure or resistance of Blastocystis hominis to the common drug of choice, metronidazole (Flagyl), further treatment choice including the use of aminoglycosides, paromomycin, cotrimoxazole, and Trimethopim- sulfamethoazole, and

Nitazoxanide (Rolstron et. al.1989, Diaz et al., 2003). A double dose of Trimethoprim (320 mg), Sulphamethoxazole (800 mg) TMP-SMX twice daily for three weeks was used to control Blastocystis among some Danish patients (Stensvold et al., 2008, 2011).

Lack of awareness or ignorance of Blastocystis species as a potential diarrhoea pathogen and a possible cause of some unexplained chronic diarrhoea and cause of other symptoms such as abdominal pains, flatulence, anorexia and irritable bowel syndrome in HIV and

AIDS patients can no longer be justified.

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Despite over 100 years of the description of Blastocystis in humans its pathogenicity has been conflicting. Little wonder why its role and isolation in HIV/AIDS patients is quite scant (Albretch et al., 1995). In Africa, south of the Sahara, very scanty report of prevalence of Blastocystis were found among HIV/AIDS individuals in Zambia and Tanzania of 21% and 26.2% respectively (Atzori et al., 1992; Hunter et al., 1993). In the Southwestern city of Abeokuta, Nigeria, among hospitalized patients of undisclosed ailment, detection of

Blastocystis was reported in 2.5% (Reinthaler et al., 1988). This development may be attributed to lack of awareness among medical doctors, microbiologists, medical laboratory and research scientists of the milestone of knowledge revealed about Blastocystis sp. role in human health in the last decade.

Parasitic surveys are frequently carried out in Lagos, but few have targeted some of the

„newer‟ parasitic pathogens reported among HIV/AIDS patients in other countries, some of which include Cyclospora cyatanensis (Alakpa et al. 2003), Cryptosporidium sp., Isospora belli, and Microsporidium (Onuoha, 1997; Akujobi and Ogunshola, 2006; Fagbenro-

Beyioku et al., 2007).

There is no gainsaying the fact that inability to detect Blastocystis may be as a result of the common notion that the organism is an artifact and not worthy of recognition. It may also be as a result of the biology of the organism of polymorphic presentations which poses a challenge with microscopic detection methods. Presently, microscopic detection of

Blastocystis and validation by employing a more sensitive detection method as well as the molecular phylogenetic classification of the strains may be the only way to convince skeptics of the existence of this protistan pathogen in any locality.

This study therefore, aimed to primarily investigate the prevalence of Blastocystis species in

HIV seropositive and AIDS individuals with and without gastrointestinal symptoms in

Lagos using control of HIV seronegative healthy individuals.

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5.1 MATERIALS AND METHODS

5.1.1 Study Population

This study was carried out between April 2008 and April 2009 in the CMUL-PEPFAR

Clinics, and LUTH clinics all in Lagos metropolis, Nigeria in order to establish the prevalence of Blastocystis in HIV/AIDS individuals in Lagos. Stool samples were collected from 270 Human Immunodeficiency Virus seropositive individuals confirmed at any of

National HIV Screening and Confirmatory Centers in the Lagos metropolis and most of the individuals were also attending PEPFAR clinics. The individuals living with HIV/AIDS condition that participated in this study were engaged in active retroviral therapy at the clinics.

Ethical Approval

Ethical approval was given by the College of Medicine Ethics and Experimentation board, while good clinical and laboratory practices were adhered to through out the work.

5.1.2 Control individuals

Three hundred and fifteen HIV seronegative individuals, without diarrhoea and GI symptoms 3 weeks prior to the sampling period were obtained as control group.

Positive control slides: Positive control slides and charts of Blastocystis species from Dr

Abe Niichiro of School of Public Health and Environmental Sciences Osaka, Japan.

5.1.3 Samples and Sample Size:

Fresh stool specimens were collected, while observing all safety precautions. Samples were collected in transparent, plastic screwed capped universal containers. Sample size was determined using the formulae by: N=z².p (1-p)/D² (Oyejide, 1992); where N, is the calculated sample size for the group which equals a minimum of 288.

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However, 270 HIV seropositive stool samples and control of 315 HIV seronegative stool samples were collected and analyzed and formed the sample size for the study.

5.1.4 Processing of Stool Sample:

Stool examination using macroscopic and microscopic analysis of stool. Stool specimens were examined as saline and iodine wet smear mounts and concentrated by the formal- saline ethyl acetate sedimentation method for microscopy (Garcia and Bruckner, 2007).

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5.2 RESULTS

5.2.1 Analysis of stool smears of HIV/AIDS individuals

Two hundred and seventy stool samples from the HIV positive individuals were analyzed.

At least one parasite was found in 150(56%) of the samples. Forty eight or 17.8% were identified to have Blastocystis species among the HIV seropositive and AIDS individuals investigated.

Other parasitic pathogens were seen in 108(40%) stool samples, with the commonest ones being helminths namely, Ascaris lumbricoides 18 (6.7%), while Hookworms and Taenia species were found in 12(4.4%) of the samples each. Other pathogenic protozoan included

Giardia lamblia 6(2.2%) and Entamoeba histolytica/dispar 3(1.1%). The oocysts

Cryptosporidium spp. and Cyclospora cyatanensis were seen in 3(1.1%) each.

Non-pathogenic protozoa which were found in some of the stool samples examined included Chilomastix mesilini, 18(6.7%), while Entamoeba coli and Dientamoeba fragilis were found in 9 (3.3%) respectively. Trichomonas hominis was also seen in one stool sample (0.4%) from the HIV/AIDS patients. Mixed infections and co-infections with detection of helminths, protozoan and non- pathogenic species were also observed in some of the stool samples.

On the other hand, at least one parasite was isolated from 183(58.1%) of control stool samples, with pathogenic parasites occurring in 97(30.8%). Important helminths isolated from the study include, Ascaris lumbricoides 33(10.5%) and Taenia species in 21(6.6%), while Hookworms was isolated in 18(5.7%) of the apparently healthy individuals.

Blastocystis spp. was found in 30(9.5%) of the control individuals. Among the pathogenic protozoans, only Giardia lamblia cysts12 (3.8%) were seen in the healthy people. However, co-infections of Blastocystis with other non-pathogenic protozoa were common in these individuals, with Entamoeba coli found in 57(18.1%) and Dientamoeba fragilis in 21(6.7%)

78 of the stool samples screened. No Trichomonas hominis was found in the stool samples from the healthy control individuals (Table 12). Mixed infections were also seen in some of these individuals.

5.2.2 Sex and Age group of Individuals in the study:

One hundred and sixty-eight 168, (62.2%) females and one hundred and two, 102 males or

(37.8%) with clinically defined HIV and AIDS subjects were investigated.

The distribution of patients showed that out of 168 females, 89.3% were aged between 21-

50 years, with 81% were married. Eighty-five of the 102 (83.3%) males were in the 21-50 years age brackets, with majority married. Twenty-eight females (10.4%) and twenty (7.4%) males were positive for Blastocystis sp. The median age group was 31-40 years (Table13).

5.2.3: Marital status with Blastocystis detection:

Married individuals accounted for 80.7%, while 19.3% were single of the study participants

(Table14).

5.2.4 Diarrheal Status with Blastocystis Detection:

Confirmed diarrhoeal individuals were 60.4%, passing loose or watery stools for 3 weeks as at the time of sample collection. One hundred and sixty-two, (60.6%) HIV seropositive patients had diarrhoeal symptoms and of this number 59.9% were females and 33.1% males.

Twenty-three percent of these diarrhoeal patients had Blastocystis cysts detected in their stool (Table 15).

There was no statistical significance between diarrhoeal status and age, marital status or sex of these patients (p>0.05), although a significant proportion of stool samples were consistent with diarrhoeal status from where pathogen‟s cyst was detected, p<0.05

(Table16). It thus implies that not all diarrhoea condition seen in the patients was due to

Blastocystis infection.

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5.2.5 Data of patients who had Blastocystis spp. detected in their Stool.

The parasite cysts were detected from the stool of forty-eight (17.8%) HIV/AIDS individuals analyzed. Blastocystis positive individuals were aged between 21 to 60 years.

Thirty-eight (79.2%) were female and 20.8% were males. Forty, 81.6% of these positive patients were married. Three of those positive for Blastocystis hominis had co-infection with Entamoeba coli, with three other samples found with co- infections of

Cryptosporidium spp., Cyclospora cyatanensis and Entamoeba histolytica/dispar respectively. However, other co-infections which occurred included non- pathogenic protozoa namely Chilomastix mesilini and Dientamoeba fragilis that were seen in twelve individuals. The analysis of the nature of stool of the patients with detection of Blastocystis showed no statistical significance (Table 17).

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Table 12: Parasitic Profile of HIV/AIDS individuals and Control Non-HIV Group. Parasic Infection HIV/AIDS subjects Non-HIV Group

All parasites 150 (56%) 183(58.1%)

No parasites detected 120 (44%) 132(41.9%)

Pathogens isolated 108 (40%) 97(30.8%)

Helminths

Ascaris lumbricoides 18 (6.7%) 33(10.5%)

Trichuris trichuria 0 (0.0%) 3(0.95%)

Hookworms 12(4.4%) 18(5.7%)

Strongyloides stercoralis 3(1.1%) 3 (1.0%)

Taenia species 12(4.4%) 21(6.7%)

Pathogenic Protozoan

Entamoeba histolytica/ dispar 3(1. 1%) 0

Giardia lamblia 6(2.2%) 12(3.8%)

Cryptosporidium spp. 3 (1.1%) 0

Cyclospora cyatanensis 3(1.1%) 0

*Blastocystis species 48(18%) 30(9.5%)

Non Pathogenic Protozoan 42(16%) 132(42%)

Entamoeba coli 9 (3.3%) 57(18.1%)

Entamoeba hartmanii 0 3 (1.0%)

Dientamoeba fragilis 9 (3.3%) 21(6.7%)

Chilomastix mesilini 18(6.7%) 5(1.6%)

Trichomonas hominis 6 (2.2%) 0(0.0%) TOTAL 270(100%) 315(100%)

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Table 13: Age distribution of HIV/AIDS patients investigated for intestinal parasites

AGE GROUP Frequency (No.) %

P> 0.05

0-10 years 0 0

11-20 years 6 2.22

21-30 years 91 33.70

31-40 years 104 38.52

41-50 years 46 17.03

51-60years 20 7.41

>60years 3 1.11

TOTAL 270 100

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TABLE 14: Distribution of Blastocystis individuals investigated by Sex.

Occurence of Total Female Male p values

Blastocystis N N (%) N (%)

Positive (48) 28(16.7) 20(19.6) P<0.05

Negative (222) 140 (83.3) 82(80.4)

TOTAL (270) 168 (100) 102(100)

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TABLE 15: Distribution of Blastocystis individuals investigated by Marital Status.

Marital Status Female Male p values N (%) N (%)

Married 136(81.0) 82(80.4) P>0.05

Single 32 (19.0) 20(19.6) TOTAL 168 (100) 102(100)

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Table 16: Distribution of Blastocystis in HIV/AIDS Subjects with Diarrhoea status.

Occurrence of DiarrhoeaPos DiarrhoeaNeg

Blastocystis No (%) No (%)

POSITIVE 38(23.5) 10(9.3)

NEGATIVE 124 (76.5) 98(90.7)

TOTAL 162 (100.0) 108(100.0) P<0.05

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Table 17: Distribution of Blastocystis with Nature of stool samples in HIV/AIDS

Nature of stool Blastocystis Blastocystis Detected Not detected No (%) No (%)

Formed 0(0.0) 29 (13.1)

Semi-formed/soft 10(20.83) 40(18.0)

Watery 10(20.83) 63(28.4)

Loose 28(58.33) 90(40.5)

TOTAL 48(100) 222(100)

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5.2.6 DISCUSSION

The data in this study revealed that 17.8% of people living with HIV/AIDS had

Blastocystis.

Similar studies reported from HIV/AIDS subjects in Tanzania showed prevalence of 26.2%

(Atzori et al., 1992), and in Zambian patients with AIDS a prevalence of 21% occurred. In other regions of the world several individuals infected with HIV and AIDS were found to have higher incidence of harbouring Blastocystis sp. and other opportunistic infections

(Albretch et al., 1995; Gassama et. al., 2001; Florez et al., 2003). A number of similar studies have shown the detection of this organism in HIV/AIDS individuals who submitted stool samples for microbiological examination following intestinal symptoms including diarrhoea, abdominal pains, flatulence and other non-specific complaints (Gasamma et al.,

2001; Zali et al., 2004).

Few studies found no correlations between Blastocystis sp. infection and disease in individuals who are infected with HIV/AIDS (Albretch et al., 1995, Brandonisio et al.,

1999). Nevertheless, recent evidence links extensive genetic variability among and within the subtypes of Blastocystis spp., to the variability of symptoms of intestinal disorders

(Jones et al., 2009; Yoshikawa et al., 2009).

In the present study, all positive stool samples were from diarrhoeal symptomatic individuals and there was a statistically significant association between Blastocystis sp., detection and diarrhoeal status in this group of patients. One cannot however, from this study state categorically the exact involvement of this pathogen as a major diarrhoeal agent in HIV positive individuals in Lagos. It might serve as a pointer to a possible cause of diarrhoeal condition, which needs to be further investigated in some of the patients, employing more sensitive detection methods; particularly in tertiary healthcare laboratories.

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In the present study, detection of parasite was also correlated with some diarrhoea conditions; though there were no distinctions of the diarrhoea types, such as travel related or drug induced. It is possible that some of the infections could be travel-associated judging by the cosmopolitan way of living in Lagos metropolis.

Detection of Blastocystis was also statistically significant with loose and watery nature of stool sample of the individuals studied. On the contrary, Rene et al., (2009), reported that the presence of Blastocystis cysts in Danish patients investigated for enteropathogens, who were excreting cysts, was independent of stool consistency and that higher prevalence of

Blastocystis species occurred among patients with travel- associated diarrhoea than those with persistent diarrhoea. However, in this study, the immunocompromised status of the participants, the antiretroviral drugs being administered and their nutritional states might have contributed to the difference seen in this study. Prophylaxis for Blastocystis positive

HIV/AIDS individuals using antibiotic such as trimethoprim-sulfamethoxale and anti- parasitic for example metronidazole (Flagyl) medication have been reported effective

(Becker et al., 2007; Stensvold et al., 2011a,b).

There was no statistical significance between parasite detection with age and sex of the patients. The age range of 21 to 60 years were investigated in this study, and the median age group of 31 to 40 years was found to be consistent with other reported cases of Blastocystis sp. infection among HIV/AIDS individuals globally (Brites et al., 1997; Cirioni et al., 1999;

Zali et al., 2004; Stensvold et al., 2010). Adult populations are sexually active and ages of

18 to 60years appear more at risk of the HIV pandemic, these constitute the majority of the subjects investigated.

Infection with Blastocystis in this class of patients is likely to worsen the conditions through the exercebation of watery diarrhea common with people living with HIV.

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The growing involvement of Blastocystis and coccidian such as Microsporidia,

Cryptosporidium spp., Cyclorospora spp., all of which have been associated with watery diarrhoea could be life threatening in HIV/AIDS individuals (Ukegbu, 1999; Conclaves et al., 2000; Alakpa et al., 2003a, b, Fagbenro-Beyioku et al., 2007, Akujobi and Ogunsola,

2006). Not only is the chemotherapeutic eradication of infection with Blastocystis spp. challenging, but it shares a lot of similarity with some of these pathogenic protozoa (Stenzel and Boreham, 1986). As a result, diarrhoea in some HIV positive patients due to

Blastocystis may not be effectively managed without proper detection; as it could be taken as Cryptosporidium sp. or Cyclospora cyatanensis induced. The correct differential by knowledgeable microbiologists in the laboratory is therefore vital in diarrhoea management especially in the HIV/AIDS individuals.

This study also revealed that co-infection of Blastocystis sp., with other pathogenic and non- pathogenic parasites, such as Dientamoeba fragilis and Entamoeba histolytica/dispar may portends a very dangerous trend and needs to be further investigated in the immuno compromised subjects.

More recently, Johnson et al., (2004) observed that there are patients who harbour

Dientamoeba fragilis, with a growing number of case reports from many parts of the world which described patients whose clinical symptoms subsided only after therapeutic intervention and elimination of the organism, thereby, strongly suggesting that D. fragilis is now a bona fide pathogen. In view of the possibility of low awareness among health practitioners in the city of Lagos and elsewhere in Nigeria, there is urgent need for increased investigation for the presence of Blastocystis species in the stools of HIV positive individuals. This will not only help in determining the true prevalence of this emerging pathogen in HIV/AIDS individuals in Lagos state, it would also ensure that patients with

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Blastocystis-induced diarrhoea, and co- infecting pathogens are properly diagnosed and effective treatment instituted.

5.2.7 CONCLUSION:

The results from the study have identified and confirmed the existence of Blastocystis infection in Lagos and shown association with diarrhea condition. The extent and mechanism of parasite involvement is not yet known. Nevertheless, the finding of positive samples among the control group implies that Blastocystis carriers exist in the locality, which may be important in sustaining the persistence of the parasite in Lagos. The existence of blastocystosis in our mist suggests a significant association with diarrhea, one of HIV/AIDS defining conditions and lack of awareness and knowledge of this finding portrays an inadequacy in the management of this condition. Polyparasitism could be a serious complication in HIV /AIDS where another diarrhoeic organism occurs with

Blastocystis species.

It is recommended that proper surveillance or at least routine examination of stool samples of diarrhoeic individuals, for Blastocystis detection, where no other parasite is detected as the offending agent, be introduced.

Prophylaxis for diarrhea in Lagos should include antibiotics and anti-protozoan medication.

This finding will definitely impact on the level of care giving to some HIV/AIDS individuals who might have Blastocystis- induced diarrhea.

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ISOLATION OF BLASTOCYSTIS DNA DIRECT FROM STOOL SAMPLE

6.0 INTRODUCTION

The epidemiology, biology and clinical significance of Blastocystis are attracting a lot of interest in different regions of the world. Few morphological forms of the Blastocystis can be identified using microscopic method (Stensvold et al., 2006). Consequently, studies based on microscopy were limited and inadequate to give convincing proof to clinicians of the existence of this organism. As a result, debates on the biology and importance of

Blastocystis have lingered for about one hundred years of its description (Tan, 2004). It is also believed that the pleomorphism inherent among the Blastocystis made earlier studies of the parasite‟s biology confusing as data derived by microscopic analysis was conflicting and inadequate compared to the data derived using modern molecular methods such as PCR methods (Stensvold et al., 2006).

In most advanced countries, laboratories employ other sensitive parasitological detection methods such as cultivation, serological and molecular detection of parasites, which appear to be more sensitive than microscopic identification (Leeleyoova et al., 2002; Stensvold et al., 2006). However, in developing countries these methods were not only costly to use, but labourious for routine application and therefore, limited to research purposes (Zaman, 1998;

Leeleyoova et al., 2002; Sakirisampant et al., 2003).

Recent adaptation of the molecular methods for routine parasitological diagnosis, such as the use of sequence tagged site, restriction fragment length- Polymerase Chain Reaction techniques, which offers direct isolation of the Blastocystis DNA from stool samples of patients and will also validate the existence of the parasite in the community (Stensvold et al., 2007).

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The STS-PCR method therefore may be ideal for investigation of the Blastocystis sp. DNA directly in stool samples in Lagos, as it by passes the labourious cultivation and microscopic methods. And the method has the added advantage of specificity and sensitivity in confirming the Blastocystis genome; includng generating enough isolates that could be used for further study of the parasite. This work was aimed to isolating fecal Blastocystis DNA directly from stool in order to validate the microscopic detection of the parasite in Lagos metropolis.

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6.1 MATERIALS AND METHODS

6.1.1 Selection of Stool sample

A total of 48 stool samples, each of 24 diarrhoeric individuals and asymptomatic HIV/AIDS individuals were randomly selected from suspected only Blastocystis -positive by smear microscopy and further processed employing PCR method direct on the samples.

6.1.2 Stool Samples Preservation

The stool samples aliquot collected in cryo- centrifuge tubes were preserved by proper vortexes in 90% ethanol solution and shipped for fecal genome isolation and molecular phylogenetic studies employing, the sequence tagged sites -PCR method (Stensvold et al.,

2007).

6.1.3. Purification ethanol preserved Stool Samples

First, 200mg of stool preserved in 90% ethanol, collected in a cryo-centrifuge tube, were washed in phosphate-buffered saline, PBS (pH 7.4) by centrifugation at 400g for 10 minutes for three changes. Each time, the supernatant was discarded and the pellet was treated with fresh PBS to remove debris and impurities in the faeces leaving clean Blastocystis organism at the end of the procedure as the concentrate.

The concentrate was overlaid on to 10ml of Ficoll-Paque column and centrifuged at 1200g for 10 minutes. Blastocystis separated into a band approximately 1 cm from the surface.

The layer of Ficoll-Paque-concentrated cysts were then aspirated into a clean tube, re- suspended in 8ml of PBS solution and centrifuged at 500g for 5minutes.

The resultant pellet was stored at -20˚C until required for DNA extraction (Parkar et al.,

2007).

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6.1.4. Direct DNA Extraction

DNA extraction was performed using QIAmp DNA stool mini kit (Qiagen, Hilden,

Germany), according to the manufacturer‟s protocol. DNA extracts were eluted in 100µl AE buffer.

In a PCR tube, mix (counting for one sample) the following:

12.5µl Miracle mix

8.5µl Dnase-Rnase free water

1.0 µl Forward F1 primer (1400) RD3 5‟-GGG ATC CTG ATC CTT CCG CAG

GTT CAC CTAC-3‟

1.0 µl Reverse R1 primer (1700) RD5 5‟GGA AGC TTA TCT GGT TGA TCC

TGCCAG TA-3‟ (Clark, 1997), amplifying a 1.8kbp region, (2.5 of 10X PCR

buffer, 2.0 of 25Mm MgCl², 1.0 of 20Mm dNTPs).

In a 25µl DNA mixer, mix the 23µl reaction with 2µl of template DNA. In addition, positive 10³ Blastocystis sp. concentration and a negative sample of Dnase free water were made. The PCR tubes with added DNA were then run on the PCR machine employing a program 1417.

The primers F1 and the sequenced primer, BHCRseq3 (Stensvold et al., 2007) were also included in cases of negative results, where the former primers may be less specific.

PCR Program 1417 Involves:

The denaturing of the DNA followed initial activation of the Taq DNA polymerase (Biotech

International, Perth, Australia) at 95ºC for 15minutes. Heating to 94ºC for 5mins and repeating the following cycle 35 times, with Temperature at 94ºC for 1min to denaturating;

Cooling to 60ºC for 1 min to annealing and heating to 72ºC for 1min to extending.

Thereafter, temperature was kept at 72ºC for 10minutes for elongation, and Cooling to 8ºC

94 to avoid annealing of the DNA strands, until the DNA in the tube was required from the machine. Amplification in both rounds of PCR was performed using a Gene-Amp2400 thermocycler (Applied Biosystems, CA, USA). The PCR products of approximately 1.0kbp, was electrophorized on 1.5 % agarose gel and the PCR product of band 550- 590 bp visualized after being stained with ethidium bromide in a Ultra Violet illuminator was considered specific of Blastocystis sp. DNA (Bohm-Glonning et al., 1997), Figures 3.

6.2 RESULTS

Forty-eight suspected Blastocystis-positive stool samples comprising 24 each of the symptomatic (diarrhea) individuals and asymptomatic control samples using microscopy were randomly selected and subjected to Polymerase chain reaction analysis using sequence tagged site primers, STS- PCR techniques. Primers specific for all the known Blastocystis species subtypes 1- 9 were employed throughout the work.

Of these, Blastocystis DNA was directly isolated in 24(50%) of the faecal samples investigated made up of 13 positive seen in the symptomatic diarrhoeic HIV/AIDS individuals, with 11 positive parasite DNA isolated in the asymptomatic HIV/AIDS control group.

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Table 18: Direct Blastocystis DNA isolation from individuals in Lagos metropolis.

Diarrhoeric individual Asymptomatic Individuals S/No Sample ID Blastocystis DNA S/No Sample ID Blastocystis DNA 1 2701 Positive 1 SL3 Negative 2 2702 Positive 2 SL4 Negative 3 2703 Negative 3 SL5 Positive 4 3001 Positive 4 SL6 Negative 5 3002 Positive 5 SL7 Negative 6 3003 Negative 6 SL8 Negative 7 3004 Positive 7 SL9 Negative 8 3005 Negative 8 SL10 Negative 9 3006 Positive 9 SL11 Negative 10 3007 Positive 10 R1 Positive 11 SP1 Negative 11 R2 Positive 12 SP2 Positive 12 R3 Negative 13 SL1 Positive 13 R4 Positive 14 SL2 Positive 14 R5 Negative 15 1E Negative 15 R6 Negative 16 3E Positive 16 R7 Positive 17 4E Negative 17 R8 Positive 18 7E Negative 18 R9 Negative 19 8E Negative 19 R10 Positive 20 10E Negative 20 R11 Positive 21 25E Positive 21 R12 Positive 22 27E Positive 22 R13 Positive 23 33E Negative 23 R14 Positive 24 38E Negative 24 R15 Negative Total 13Positives Total 11Positives

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M 1 2 3 4 5 6 7 8 9 Figure 3.0: Ethidium stained gel electrophoreses identifying Blastocystis DNA at 3, 4, and 5 which aligned with known positive BlastocystisDNA control at position 7 identical base pair, Negative control of Dnase free water at position 8. M represent 100 basepair ladder

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6.3 DISCUSSION

The isolation of Blastocystis DNA directly from faecal samples was vital to validate the occurrence of Blastocystis and confirm the microscopic detection in the locality of Lagos. The specificity of employing BIastocystis primers that target the parasite‟s conserved SSUrRNA gene also meant that the confusion of microscopic detection has been dispensed with. STS-

PCR is very sensitive method for the isolation of this parasite as up to 94%-98% sensitivity compared to microscopy of 45%-50% could be achieved (Leeloyoohova et al., 2002;

Stensvold et al., 2007). Although short term cultivation of Blastocystis species in parasitic culture methods was adjudged quite sensitive for isolation of the parasite, however, it is labourious and time consuming (Stensvold et al., 2009). In the locality of Lagos, the public electricity supply is under- developed and cannot support long hours of cultivation methods.

Even if for short time culture, the maintenance of parasite culture will be challenging, hence, the need to adapt STS-PCR methods for direct isolation and confirmation of Blastocystis in the locality.

6.4 CONCLUSION

In conclusion the work emphasized the preliminary microscopic detection of Blastocystis in

stool samples with diarrhoeic presentation. However, there is need to confirm the positive by

microscopic methods samples, using a more sensitive and specific parasitological method

such as the STS-PCR method. In this way, doubt could be erased, while the right intervention

against the parasite would be initiated for the good of the individual with the condition.

The importance of effective diagnosis of any enteric infections before treatment cannot be

over emphasized. Therefore, it is recommended that sensitive parasitological diagnostic

method should be provided in the health laboratory to support and confirm the parasites

detected by microscopy such as Blastocystis, even if it is only in our tertiary health

institutions laboratories.

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DNA SEQUENCING AND MOLECULAR PHYLOGENETIC CLASSIFICATION

7.0 INTRODUCTION

Blastocystis is widely distributed throughout the world. The parasite was first described in

1912 as a yeast and non pathogenic in man by Brumpt, who named the human isolates,

Blastocystis hominis (Tan et al., 2002).This single- celled, micro-eukaryotic protist is believed to be anaerobic and causes blastocystosis which may be mild or moderate infection in the immune competent individuals (Gassama et al., 2001; Stensvold et al., 2011b).

In the immune compromised conditions, such as HIV/AIDS, the infection outcome could be serious. Fatality has not been seen in humans, though epidemiologic data remain sparse.

Fatal diarrhoea due to Blastocystis infection was earlier reported as the cause of death of some primates in a Santiago Zoo in the USA, with the parasite implicated as the sole aetiologic agent (Zierdt et al., 1991). The public health implication of such diarrhoea in primates, a closely related mammal to humans, is better imagined. Human infection is characterized by non- specific gastrointestinal infections, namely: diarrhea, abdominal colics or pain, tenesmus, flatulence, malaise and bloating and enteric symptoms (Stenzel and

Boreham, 1996; Cruz et al., 2003). The pathogen has been detected in vertebrates such as birds, pigs and non vertebrate animals, such as reptiles and recently cockroaches (Yoshikawa et al., 2004, 2007; Jones et al., 2009). Its transmission, vectors and reservoir hosts have remained speculative. However, earlier definition of the parasite as a protozoan gave impetus to assumption of the characteristics of protozoan. Nevertheless, taxonomy and systematic classification of the protist did not place Blastocystis in any of the phylum, until Silberman et al., (1996) using the SSUrRNA gene of the parasite in comparism to other eukaryotes to molecular phylogenetically the classify Blastocystis as Stramenopiles, in the infra-kingdom chromista (Cavalier-Smith, 1997). As a result, the parasite may be the only chromista known to infect man, with potential for pathogenicity.

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Infections in humans were variable, which has been explained by the extensive genetic variability in and within the organism (Clark, 1997, 2000; Bohm-Gloning et al., 1998).

Serotypes were lacking, as a result of the genetic variability, hence the inability to develop good serological assay for Blastocystis (Kaneda et al., 2001). Other diagnostic assays include the use of microscopic method which shows low sensitivity as it detects only few morphological forms of the parasite (Stensvold et al., 2009). The effect and prevalence of

Blastocystis in many parts of sub-Saharan Africa are largely unknown because awareness and diagnostic laboratories are lacking and epidemiologic data remain sparse.

The burden of blastocystosis is not known, but appears to be high. Considering the fact that

3000 million people in developing countries are exposed to high sewer contamination

(WHO, 1998) and where the average prevalence rate of this infection approaches 50%,

implying that there may be over 1500 million cases of blastocystosis at any given point in

time. Stensvold et al., (2012) reported an estimated over one billion people to be infected by

Blastocystis in the world. Recently, the report of pathogenicity of the Blastocystis species

were believed to be related to the subtypes of the parasite found in the locality

(Dogroundman-Al et al., 2008, 2009a; Hussein et al., 2008). More recently some studies

have shown geographical variations, in the Blastocystis sp., inherent in a particular locality.

Among the Tiarape ethnic group of the Brazillian Amazon basin, subtypes1, 2, 3 were

identified but there was no Blastocystis subtype 4 (Malherious et al., 2011). In most

European cities and communities, all known subtypes of the parasite have been reported,

with ST3 as the most predominant subtypes (Souppart et al., 2010, Stensvold et al., 2008,

2009, 2011). Elsewhere, in China the molecular epidemiology appear to suggest that

subtype 5 was prevalent in the study area of Yaunan (Yan et al., 2007).

The objective of the study, therefore, was to investigate the Blastocystis subtypes in both symptomatic and asymptomatic diarrhoeal HIV/AIDS subjects in Lagos metropolis.

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7.1 MATERIALS AND METHODS

7.1.1 Selection of Stool sample

A total of 48 stool samples, each of 24 diarrhoeric individuals and asymptomatic individuals were randomly selected from suspected Blastocystis positive individuals by smear microscopy and further processed for confirmation employing PCR method.

7.1.2 Stool Samples Preservation

The stool samples aliquot collected in cryo- centrifuge tubes were preserved by proper vortexes in 90% ethanol solution and shipped for molecular phylogenetically studies employing, the Sequence tagged sites -PCR method (Stensvold et al., 2007).

7.1.3 Purification of Stool Samples

First, from the stool aliquot in 90% ethanol, about 200mg of faeces collected in an

Eppendorf or cryo-centrifuge tube, were washed in phosphate-buffered saline, PBS (pH 7.4) by centrifugation at 400g for 10 minutes for three changes.

Each time, the supernatant was discarded and the pellet was treated with fresh PBS to remove debris and impurities in the faeces leaving clean Blastocystis organism at the end of the procedure as the concentrate.

The concentrate was overlaid on to 10ml of Ficoll-Paque column and centrifuged at 1200g for 10 minutes. Blastocystis separated into a band approximately 1 cm from the surface.

The layer of Ficoll-Paque-concentrated cysts were then aspirated into a clean tube, re- suspended in 8ml of PBS solution and centrifuged at 500g for 5minutes.

The resultant pellet was stored at -20˚C until required for DNA extraction (Parkar et al.,

2007).

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7.1.4 Direct DNA Extraction

DNA extraction was performed using QIAmp DNA stool mini kit (Qiagen, Hilden,

Germany), according to the manufacturer‟s protocol. DNA extracts were eluted in 100µl AE buffer.

In a PCR tube, mix (counting for one sample) the following:

12.5µl Miracle mix

8.5µl Dnase-Rnase free water

1.0 µl Forward F1 primer (1400) RD3 5‟-GGG ATC CTG ATC CTT CCG CAG

GTT CAC CTAC-3‟

1.0 µl Reverse R1 primer (1700) RD5 5‟GGA AGC TTA TCT GGT TGA TCC

TGC CAG TA-3‟ (Clark, 1997), amplifying a 1.8kbp region, (2.5 of 10X PCR

buffer, 2.0 of 25Mm MgCl², 1.0 of 20Mm dNTPs).

In a 25µl DNA mixer, mix the 23µl reaction with 2µl of template DNA. In addition, positive 10³ Blastocystis sp. concentration and a negative sample of Dnase free water were made. The PCR tubes with added DNA were then run on the PCR machine employing a program 1417. The primers F1 and the sequenced primer, BHCRseq3 (Stensvold et al.,

2007) were also included in cases of negative results, where the former primers may be less specific.

PCR Program 1417 Involves:

The denaturing of the DNA followed initial activation of the Taq DNA polymerase (Biotech

International, Perth, Australia) at 95ºC for 15minutes. Heating to 94ºC for 5mins and repeating the following cycle 35 times, with Temperature at 94ºC for 1min to denaturating;

Cooling to 60ºC for 1 min to annealing and heating to 72ºC for 1min to extending the DNA strands. Thereafter, temperature was kept at 72ºC for 10minutes for elongation, and Cooling

102 to 8ºC to avoid annealing of the DNA strands, until the DNA in the tube was required from the machine.

Amplification

Amplification in both rounds of PCR was performed using a Gene-Amp2400 thermocycler

(Applied Biosystems, CA, USA). The PCR products of approximately 1.0kbp, was electrophorized on 1.5 % agarose gel and the PCR product of band 550- 590 bp visualized after being stained with ethidium bromide in a UV- illuminator was considered specific of

Blastocystis sp. DNA (Bohm-Glonning et al., 1997).

7.2. Blastocystis Genome Purification and Dedioxysequencing

The PCR products were gel- purified. The UltraClean тм Gel Spin DNA Purification sample kit (SANBIO, Uden, Netherlands) was used to purify the PCR products according to the manufacturer‟s instruction.

The dedioxy sequencing was in one direction using the BHCRseq3 primer as the sequencing primer (Stensvold et al., 2007). For the PCR products of 1.0kbp, approximately, 20ng of

DNA was used for sequencing, and for the PCR product of 0.3kbp, approximately, 5-10ng was used as template and sequenced in both directions using the Big Dye Terminator system, version 3.1 (Applied Biosystems, Foster city, CA) on an ABI 3730 x1 capillary sequence

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7.3 RESULT

Twenty-three isolates or 47.9% of the local Blastocystis DNA genomes were successfully amplified and sequenced while only one sample coded R11(4.2%), failed the amplification process (Table 5.6 and 5.7). The amplification and successfully sequencing of 23(98.5%) isolates from the 24 extracted DNA indicated high sensitivity.

The genotypes or subtypes of Blastocystis species obtained from the local isolates from individuals in Lagos were subtypes ST1, ST3 and ST4 (figures.3.1, to 3.9).

The result also indicated 41.6% equal occurrence for ST1 and ST3each and 12.6% for ST 4

Blastocystis sp. genotypes were found among individuals that participated in the study.

7.3.1: Distribution of the Blastocystis subtypes between the symptomatic gastrointestinal individuals and the asymptomatic control group.

From the symptomatic diarrhoea individuals, 13 of the Blastocystis genome were successfully sequenced and the subtypes distribution were ST1 53.9%, ST3 and ST4 were

23.08% respectively. On the other hand, 10 isolates were sequenced in the asymptomatic control group with ST1 seen in 27.3% and ST3 in 63.6% of the group, while one, 9.1% failed the sequencing (Table 20). Subtype 1 Blastocystis was more in the symptomatic individuals with 7/13 or 53.9% of the individuals, compared to 3/10 or 30% of the asymptomatic control.

Subtype 3 Blastocystis was more frequently seen in the asymptomatic group with 63.6% than in the symptomatic individuals with 23.08% (Table 19 and 20). The subtype 4 genotypes were only seen in the symptomatic group constituting 23.08%.

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Table 19: The subtypes of Blastocystis spp. from Symptomatic HIV/AIDS individuals

S/No SAMPLE ID SUBTYPE IDENTIFIED 1. 27E 4

2. 2702 1

3. 3001 3

4. 3006 3

5. 3007 1

6. 3E 1

7. 25E 1

8. 3002 1

9. 3004 1

10. 2701 1

11. SL1 4

12. SL2 4

13 SP 2 3

(Note that the Primer sets were BHCRseq3, 1400, and 1700).

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Table 20: The subtypes of Blastocystis species isolated from control of asymptomatic HIV/AIDS individuals in Lagos metropolitan city

S/No SAMPLE ID SUBTYPE IDENTIFIED

1. R1 3

2. R2 3

5. R8 3

6. R10 3

7. R11 Failed

8. R12 1

9. R13 1

10. R14 3

11. SL5 3

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Table 21: Subtypes of Blastocystis isolated from all study participants in Lagos.

Subtypes

Condition of Total ST1 ST2 ST3 ST 4 MST

Participants No (%) No (%) No (%) No (%) No (%) No (%)

Diarrhea = 13(56.5) 7(53.9) 0(0) 3(27.08) 3(27.08) 0(0)

Asymptomatic=11(43.5) 3(27.3) 0(0) 7(63.6) 0(0) 1(4.2)

Total = 24 (100) 10 (41.6) 0 (0) 10 (41.6) 3(12.6) 1 (4.2 )

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Table 22: The subtypes of Blastocystis species isolated from individuals in Lagos

S/No SAMPLE ID SUBTYPE DETECTED 1. 27E 4 2. 2702 1 3. 3001 3 4. 3006 3 5. 3007 1 6. 3E 1 7. 25E 1 8. 3002 1 9. 3004 1 10. 2701 1 11. R1 3 12. R2 3 13. R4 1 14. R7 3 15. R8 3 16. R10 3 17. R11 Failed 18. R12 1 19. R13 1 20. R14 3 21. SL 1 4 22. SL2 4 23 SL5 3 24 SP 2 3

Note: Primer sets BHCRseq3, 1400, and 1700 as sequencing primers.

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M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Figure 5.0: The PCR amplification of 8 Blastocystis isolates with the sequenced- tagged site(STS) from18 patients stool samples using primers 1400/1700 and BHCRSeq3, as sequencing primer, M is the molecular marker (100bp ladder) Lane 18, Blastocystis species control.

M 1 2 3 4 5 6

Figure 5.1 The PCR amplification of 3 Blastocystis isolates with the sequence tagged site (STS) from 5 stool samples, using primers 1400/1700 and BHCRSeq3, M is the molecular marker (100bp ladder). Lane 6, Blastocystis control sample

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M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Figure 5.2: Blastocystis subtypes of isolates from patients in Lagos using PCR methods; M is the DNA ladder of 100bp (base pairs). Subtype1 (-351bp) in Lane 4, 6, 7, 8 and 10(Samples2701, 3002, 3004,3E, and 25E) Blastocystis DNA positive control, Lane 13; Negative control lane 19.

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Figure 5.3: Figure 3.2 Blastocystis subtypes of isolates from Nigerian patients in Lagos using PCR methods; M is the DNA ladder of 100bp (base pairs).Subtype3-(526bp); Lane2, 5 and 6(Samples coded 3001, 3006 and 3007) respectively.Blastocystis positive control, Lane 18.

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M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Figure 5.4: Blastocystis subtypes of isolates from individuals in Lagos using PCR methods M is the DNA ladder of 100bp (base pairs). Subtype 1-351bp: Lane1, (2702; 2 bands) Subtype3-(526bp); Lane2, 5, and 6 (Samples 3001, 3006, and 3007) respectively. Subtype 4-338bp): Lanes 9, 11(7E, 27E); positive control, Lane 16

M 1 2 3 4 5 6 7 8 9 10 11 12 13 141516171819 20 21 22 23 24 25 26

Figure 5.5: Blastocystis subtypes of isolates from Nigerian patients in Lagos using PCR methods M is the DNA ladder of 100bp (base pairs).Subtype 1-351bp: Lane 17 (Sample R4),Subtype3-(526bp); Lane2, 7, 14, 15, 20, 21 and 23 (Samples SP2, SL5, R1, R2, R7, R8, R10) respectively. Subtype 4-338bp): Lanes 2, 3(sample SL1, SL2), Blastocystis positive control, Lane 26.

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M 1 2 3 4 5 6 # Figure 5.6: Blastocystis subtypes of isolates from Nigerian patients in Lagos using PCR methods M is the DNA ladder of 100bp (base pairs), Subtype 1-351bp: Lane2,3(samples R11 ,R12) Subtype3-(526bp) and Lane4 (Samples R14) respectively. Blastocystis positive control, lane 6.

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Figure 5.7: Blastocystis hominis subtypes of isolates from Nigerian patients in Lagos using PCR methods M is the DNA ladder of 100bp (base pairs).Subtype3-(526bp); Lane14, 15, 20 21 and 27(SamplesR1, R2, R7, R8, R14) respectively. Subtype 4-338bp): Lanes 3 4(samples SL1, SL2), Blastocystis control, Lane

20.

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M 1 2 3 4 5 6 “ Figure 5.8: M is the DNA ladder of 100bp (base pairs). Subtype 1, 351bp: Lane1 and 2(samples R11, R12) Subtype3 (526bp), Lane4 (Samples R14), Blastocystis positive control, lane 6.

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Figure 5.9: Entamoeba histolytica in mixed infection with STI and ST3 Blastocystis in patient sample 2702 identified by further sequencing employing primer sets EM500 –Marianne Percard (F1=E.coli 500F and R= E.coli 500R), P63 on lane 2 and Positive and Negativecontrol on lane20 and 21;M = Molecular base ladder of 100bp.

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38 R12

75 R13 3002 99 2701 ST1 (Ab070993)

95 99 3004 99 3E

100 R4 100 25E 83 S T2(AB070997)

41 S T5(AB091249)

S T 8(A B107971)

88 SL 1 SL 2 27E ST4(AY244619) 51 3006 R10 SP2 R1 3001 R8 ST3(AB091234) 61 R2 R7 R14 S L5 S T6(AB091242) ST9(AY135410)

99 S T 7(A B091245)

U37 108 P. Lacertae(outgroup)

0.05

Fig 6.0: Phylogenetic tree constructed with identified subtypes from Lagos isolates.

Note: Subtype classification was according to the recent consensus terminology for Blastocystis sp. based on the SSUrRNA gene analysis (Stensvold et al., 2007). 7.3.2 Subtype (ST) Molecular phylogenetical Analysis

The sequence chromatographs were edited and analyzed using the software programs Finch

TV, version 4.0 (© Geospiza Inc.), aligned and manually interpreted using the software

115 program BioEdit Sequence Alignment Editor (Hall, 1999). The identified subtypes of isolates of Blastocystis from symptomatic individuals and control individuals in Lagos,

Nigeria are shown (Table 21, 22).

7.3.3 Tree construction from isolates identified in Lagos:

Distance- based analysis was conducted with MEGA 3.1 software, (Kumar et al., 2004) using the Tamura-Nei distance estimates, a 2-parameter model and trees constructed using the neighborhood joining UPGMA algorithm. Proteromonas larcertae (U37108) was used as the out-group. Figure 6.0, shows molecular phylogenetical analysis and tree construction using subtypes generated from 21 Blastocystis isolates from individuals in Lagos.

7.3.4 Reference Sequences

Reference sequences available in the GenBank (National center for Biotechnology

Information (NCBI) database (http://www.ncbi.nlm.nih.gov/BLAST/) were used. The subtype, ST, was assigned to a given sequence based on the reference nucleotide sequence it clustered with (Kumar et al., 2004; Noel et al., 2005). Subtype terminology according to

Stensvold et al., (2007) was used throughout. Sequences that were >400bp long were submitted to the EMBL Nucleotide Sequence Database and were given accension numbers.

Table 23 shows the Blastocystis isolates representing subtypes 1(1) - V11 (2) used to identify an 18S small subunit rRNA gene segment for nucleotide sequence analysis of

Blastocystis species. The bold characters were in Phylogenetic alignment with Blastocystis isolated from Lagos, employing Proteromonas larcertae as the out group (Stensvold et al.,

2007).

7.3.5 Statistical Analysis

Statistical analysis included Chi Square (χ²) was used to compare the sensitivity of detection between the variables and direct microscopic detection of Blastocystis sp. in stool samples.

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A probability value of less than 0.05 was considered statistically significant. Moreover, test for independence using the Pearson‟s Chi square test with the computer software package

Coco (Bradsberg, 2001). Also the t-test and Fisher‟s exact test were used.

Table 23: Blastocystis species reference isolates representing subtypes 1(1)-VII (2) (In bold were the subtypes used for the molecular phylogenetically tree construction).

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Subtype Accession numbers of the reference isolates from NCBI Bank

Subtype I (1) AB107966, AB107968, AB107961, AB107962, AY135406, AY135403

AY135405, AB023578, AB023499, AF439782, AB091241, AB070993,

AY135404, AB107967, AB070989, AB091240, U51151, AM275342,

AM275343, AM275344, AM275345, AM275346,AM275347,AM275348,

AM275349, AM275350, AM275351, AM275352, AM275353, AM275354,

AM275355, AM275355, AM275356, AM275357, AM275358, AM275359,

AM275360, AM275361.

Subtype II(5) AB107969, AB070987, AB070997, AM275367, AM275368, AM275369,

AM275370, AM275371, AM275372, AM275373, AM275374, AM275375,

AM275376, AM275377, AM275378, AM275379, AM275380, AM275381,

AM275382, AM275383, AM275384, AM275385, AM275386, AM275387,

AM275388.

Subtype III (3) AB107963, AB107965, AY135402, AB070986, AB070988, AB070992,

AB091233, AB091234, AB091235, AM275365.

Subtype IV (7) U51152, AY244621, AY244620, AY244619, AY135407, U26177, AY135408,

AB091251, AB071000, AM275389, AM275390, AM275391, AM275392, AM275393.

SubtypeV (6) AB107964, AB107966, AB091249, AB070998, AB091248, AB091250, AB070999.

Subtype V1 (4) AB091236, AB107972, AB070994, AB091242, AB070990, AB091243,

AB070995, AB091236, AB091237, AY135411, AM275366.

Subtype VII (2) AY135412, AB091247, AB091246, AB091245, AB091239, AB070991, AB107973,

AB070996, AB091244, AF408427, AM275362, AM275363, AM275364.

ST8 AY107971

ST9 AY135410

Proteromonas U 37108

7.4 DISCUSSION

Blastocystis subtypes identified from the study were ST1, ST3 and ST4 from overall study participants. These subtypes were common in human population as found in reports from

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Africa, north of the Sahara, but differed in that ST2 was not isolated. In Egypt, the reported human subtypes were ST 1, 2, 3, 4, and ST6, with subtype 3 being the most prevalent

(Hussein et al., 2008; Souppart et al., 2010).

Other molecular epidemiological investigations from Denmark, Germany, Japan, Turkey and USA (Oregon) showed that Blastocystis spp. infected individuals haboured either genotypes ST1, ST2, ST3 ST4 or some unknown or mixed subtypes (MSI) Subtypes 1 and

2 were quite common; with subtype 3 being the most predominant in all the countries

(Kenada et al., 2001; Stensvold et al., 2007; Ozyurt et al., 2008; Jones et al., 2009;

Yoshikawa et al., 2009). In a study of suspected Danish patients with entero-pathogens,

Rene et al., (2009) reported that ST1, 2, 3, in addition to zoonotic strains; ST4, 6, 8 and

MSI were most common among Blastocystis cyst excreting patients in Copenhagen,

Denmark. In another study, Blastocystis ST1, ST2, ST3, ST4, ST6 ST7, ST9 were found to occur as mono-infections in the majority of the patients with suspected parasitic gastrointestinal infections, with some mixed subtype infections in Denmark (Stensvold et al., 2009)

Secondly, in this present study, ST1 and ST3 were predominant with occurrence of 41.6% each, whereas ST3 was more prevalent in human populations in America, Europe and the

Middle East countries of Turkey. Genetic variability of Blastocystis isolated in China showed that ST1 genotype was more predominant 18 (51.4%), with a possible relationship between ST1 and a pathogenic potential of the parasite existing (Yan et al., 2007). In addition to the first four subtypes, subtype 5 was found among farmers in China, while the

ST1 genotype was the most prevalent (Li et al., 2007). The most prevalent genotypes among these four populations, except for all four isolates from Thailand were subtype 3, and they varied from 41.7% to 92.3%, while the second most common genotype among the four populations was either subtype 1 (7.7% -25%), or subtype 4 (10-22.9%).

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Blastocystis species ST4 was identified in 3 (12.6%) of symptomatic samples in the present study. While the subtype4 is common in human population, recent report among the

Indigenous Tapirape Ethnic Group from the Brazilian Amazon Region, Brazil, showed isolation of subtypes ST1, 2 and 3, and mixed infections but ST4 was not detected, and ST3 was not the predminant subtype (Malheiros et al., 2011). There appear to be a geographical distribution of some of the Blastocystis subtypes but the implications of this diversity in the subtypes in human populations are unknown.

The ST4 was associated with pathogenicity in experimental animals and could be important in high risk group such as people who work closely with animals as well as the immune compromised HIV/AIDS individuals.

Among the asymptomatic control study participants, ST3 subtypes, were more prevalent, which compares favourably with reports in some countries such as Egypt, Turkey, France and United States of America (Hussein et al., 2008; Ozyurt et al., 2008; Jones et al., 2009;

Souppart et al., 2010) . On the other hand, from the symptomatic diarrhoea subjects, the most common genotypes found was ST1 while ST3 and ST4 were seen in low proportions of 23.1% each . It could be inferred that all the subtypes were potentially pathogenic, but

ST1 was the most pathogenic strain in the locality.

In Oregon community, USA, three symptomatic Blastocystis sp. infected- patients harboured predominantly subtype 3, 75% of patients that participated in the study, with one patient having ST1, and the remaining two patients showing unknown subtypes U-ST or mixed subtype, MSI infections (Jones et al., 2009) . In a more definitive study, Hussein et al., (2008) described similar genotypes ST1, 2, 3, and 4 genotypes from Egyptian isolates with symptomatic ST1 found to be clinically and statistically highly relevant to the pathogenicity of the parasite while non pathogenic strains could occur among subtypes 3

120 and 4. Furthermore, a study of symptomatic patients in Greece, with gastrointestinal illness and rashes were shown to have predominantly ST3 Blastocystis infection (Katsaraou-

Katsari et. al., 2008). Another report from two groups of isolates from 15 symptomatic and

11 asymptomatic patients in Bangladesh showed only two distinct genotypes of ST1 or ST3, with no statistically significant difference were observed between the two groups.

However, the study could not show that the specific genotype correlated with the pathogenic potential of B. hominis (Yoshikawa et al., 2009). Other studies also isolated predominantly STs 1 and 3 which were clinically and statistically reported as highly relevant to the pathogenicity of the organism (Hussein et al., 2008; Li et al., 2007; Parkar et al., 2007).

7.4.1 CONCLUSION

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Blastocystis show extensive genetic variability, even within a subtype. The isolation of the classical parasite subtypes in human populations that have been variously described as potential pathogen calls for caution.

The findings from this study shows a significant association of the Blastocystis subtype 1 and diarrhea symptom; a potentially pathogenic strain. Moreover, the subtype 4, was only isolated from the diarrhoeic symptomatic individuals. The implication of ST4 strains in intestinal disorders is well documented in literature.

From the fore going, therefore, a wide collaboration is needed in order to have an indepth study of the parasite in Lagos and in deed the entire country. This will form the basis of informed interventions. The provision of sensitive and specific isolation methods at least in the tertiary health system will help improve knowledge and awareness of this parasite within the state and region.

GENERAL DISCUSSION AND CONCLUSION

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8.0 GENERAL DISCUSSION

8.0.1 The Prevalence of Blastocystis species in Lagos, Nigeria.

A prevalence of 19.2% for Blastocystis was found among the study participants who attended clinics in tertiary hospital facilities in Lagos metropolis. Similar studies reported prevalence of 18.5% from the Quaylobia province of Egypt, following a survey of food vendors presenting for health clearance certificates (Sadek et al., 1997). Also among participants in a study in Zambia, a prevalence of 21% was found for the infection (Hunter et al., 1993). In Tanzania, a prevalence of 26.2% was also reported for Blastocystis infection of study participants (Atzori et al., 1992).

The prevalence of other endemic pathogenic protozoa showed Giardia lamblia to be 7.4% and Entamoeba histolytica/ dispar complex, 3.6%, both were lower than Blastocystis. A similar observation was reported in the United States of America, where the occurrence of

Blastocystis sp. was twenty eight times higher than Giardia lamblia and about ten times higher than the incidence of Entamoeba histolytica/ dispar, following three decades of neglect of the surveillance of Blastocystis spp. (Amin, 2002).

Diarrhoea was observed to be significantly associated with Blastocystis infection in this study (p <0.05). Several reports found skin rashes and urticaria presenting with diarrhoea in some individuals and associated these with the Blastocystis infection which also resolved following treatment for Blastocystis (Guptra and Parsi, 2006; Kaya et al., 2007;

Kartasssauro-Katsara et al., 2008). Although some stool samples were diarrhoeric, other positive samples from the healthy controls showed no consistence with diarrhoea or symptoms associated with gastrointestinal complaints, in both asymptomatic and symptomatic study participants. Therefore, it is possible that not all the diarrhoea conditions in the study participants were due to Blastocystis infection.

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There was no influence of gender (Zierdt et al., 1991and nature of stool samples submitted with Blastocystis detection in this study (p > 0.05) and this finding agrees with report of similar studies reported in Denmark (Rene et al., 2009). Some reports that showed gender diffrences were believed to be influenced by the religious and cultural settings in the study area (Nimri, 1993; Tan et al., 2002). There was also no significant association between

Blastocystis detection and age groups investigated, however, the stool samples with

Blastocystis were from adults aged between 21 and 60 years. The findings from this study also compared favourably with reported isolation of Blastocystis in adult patients in

Denmark where the parasite was detected from adults aged between 21 and 50 years

(Stensvold et al., 2007a). Among city residents in Argentina, there appeared to be higher prevalence of the organism in those patients who were more than 14 years old (Minvielle et al., 2004).

8.0.2 Prevalence of Blastocystis among HIV/AIDS study participants

Blastocystis detection in HIV seropositive and AIDS subjects showed a prevalence of

17.8%, and in the HIV seronegative control samples constituted 9.5% positive stool samples.

Studies that used the same methodology as the present study showed that several individuals infected with HIV and AIDS have high incidence of harbouring Blastocystis sp. and other opportunistic infections than non-HIV positive individuals (Gassama et al., 2001;

Florez et al., 2003; Zali et al., 2004). A few studies found no correlations between

Blastocystis sp. infection and intestinal disease in individuals who were infected with

HIV/AIDS (Brandonisio et al., 1999). The discrepancies were probably due to extensive genetic variability among the subtypes of Blastocystis spp. (Bohm-Glonning et al., 1997;

Jones et al., 2009, Yoshikawa et al., 2009).

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There was a statistically significant association between Blastocystis sp., detection and diarrhoeal status in the HIV/AIDS participants (p < 0.05). One cannot however, from this study state categorically the exact involvement of this pathogen as a major diarrhoeal agent in HIV positive individuals in Lagos. However, it may serve as a pointer and a possible cause of diarrhoeal condition, which needs to be fully investigated in a standard healthcare laboratory.

There was no statistical significance between parasite detection with age of participants (p >

0.05). The age range of 21 to 60 years was investigated in this study, and the median age group of 31 to 40 years was found to be consistent with other reported cases of Blastocystis infection among adult Danish subjects with HIV/AIDS (Stensvold et al., 2010) and in

Northern India (Prasad et al., 2000) and other regions of the globe (Brites et al., 1997;

Cirioni et al., 1999; Zali et al., 2004).

8.0.3 DNA sequences and phylogeny of local isolates

Subtypes, ST1, ST3 and ST4 of Blastocystis were common among the study participants.

These subtypes were common, in other human populations, but this present study differed in that ST2 was not found in Lagos. This could be the first report of a locality where the subtype two was not seen in a locality. Recent report among the Indigenous Tapirape Ethnic

Group from the Brazilian Amazon Region, Brazil, showed isolation of subtypes ST1, 2 and

3, and mixed infections but ST4 was not detected while ST3 was not the predominant subtype (Malheirous et al., 2011). There is growing evidence of the geographical distribution of the subtypes of Blastocystis sp, but its clinical implication is yet to be seen.

The ST4 was associated with pathogenicity in experimental animals, and could be important in high risk groups such as people who work closely with animals as well as the immune compromised HIV/AIDS individuals.

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Secondly, ST1 and ST3 were more predominant with occurrence of 41.6% each.

Blastocystis species ST4 was seen in 3 (12.6%) of sequenced samples, while an isolate

(4.2%) that failed the sequencing was denoted as unknown UST.

Other molecular epidemiological investigations from Denmark, Germany, Japan, Turkey and USA (Oregon) showed that Blastocystis sp. infected individuals‟ haboured either genotypes ST1, ST2, ST3 ST4 or some unknown or mixed subtypes (MSI) Subtypes 1 and

2 were quite common; with subtype 3 being the most predominant in all the countries

(Kenada et al., 2001, Stensvold et al., 2007, Ozyurt et al., 2008; Jones et al., 2009,

Yoshikawa et al., 2009).

In Egypt, the reported human subtypes were ST 1, 2, 3, 4, and ST6; with subtype 3 being the most prevalent (Hussein et al., 2008; Souppart et al., 2010).

Genetic variability of Blastocystis sp. isolated in China showed that ST1 genotype was more predominant (18 or 51.4%), with a possible relationship between ST 1 and a pathogenic potential of the parasite existing (Yan et al., 2007). In addition to the first four subtypes, subtype 5 was found among farmers in China, while the ST1 genotype was the most prevalent (Li et al., 2007). The most prevalent genotypes among these four populations, except for all four isolates from Thailand was subtype 3 and it varied from 41.7% to 92.3%, while the second most common genotype among the four populations was either subtype 1

(7.7%-25%), or subtype 4 (10-22.9%). In a study of suspected Danish patients with entero- pathogens, Rene et al., (2009) reported that ST1, 2, 3, in addition to zoonotic strains; ST4,

6, 8 and MSI were most common among Blastocystis cyst excreting patients in

Copenhagen, Denmark. In another study, Blastocystis ST1, ST2, ST3, ST4, ST6 ST7, ST9 were found to occur as mono-infections in the majority of the patients with suspected parasitic gastrointestinal infections, with some mixed subtype infections in Denmark

(Stensvold et. al., 2009)

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Among the asymptomatic control study participants ST3 subtypes, were more prevalent, which compares favourably with reports in some countries such as Egypt, Turkey, France and United States of America (Hussein et al., 2008; Ozyurt et al., 2008; Jones et al., 2009;

Souppart et al., 2010) . On the other hand, from the symptomatic diarrhoea subjects, the most common genotypes found was ST1 while ST3 and ST4 were seen in low proportions of 23.1% each . It could be inferred that all the subtypes were potentially pathogenic, but

ST1 was the most pathogenic strain in the locality.

In Oregon community, USA, three symptomatic Blastocystis spp infected individuals harboured predominantly subtype 3, 75% of patients that participated in the study, with one patient having ST1, and the remaining two patients showing unknown subtypes U-ST or mixed subtype, MSI infections (Jones et al., 2009) . In a more definitive study, Hussein et al., (2008) described similar genotypes ST1, 2, 3, and 4 genotypes from Egyptian isolates with symptomatic ST1 found to be clinically and statistically relevant to the pathogenicity of the parasite while non pathogenic strains could occur among subtypes 3 and 4.

Furthermore, a study of symptomatic patients in Greece, with gastrointestinal illness and rashes were shown to have predominantly ST3 Blastocystis infection (Katsaraou-Katsari et. al., 2008). Another report from two groups of isolates from 15 symptomatic and 11 asymptomatic patients in Bangladesh showed only two distinct genotypes of ST1 or ST3, with no statistically significant difference were observed between the two groups. However, the study could not show that the specific genotype correlated with the pathogenic potential of B. hominis (Yoshikawa et al., 2009).

Other studies also isolated predominantly STs 1 and 3 which were clinically and statistically reported as highly relevant to the pathogenicity of the organism (Hussein et al., 2008; Li et al., 2007; Parkar et al., 2007).

127

8.0.4 SUMMARY

In summary, the findings from the study were:

1. The overall prevalence of 19.2% of Blastocystis infection was found among the

participants in the study.

2. In the HIV/AIDS study participants a prevalence of 17.8% was found compared to a

control group of non-HIV individuals, 9.5% while Diarrhoea was significantly

associated with Blastocystosis.

3. Direct isolation of fecal Blastocystis DNA confirmed the existence of the parasite in

the locality of Lagos.

4. Subtypes (ST) 1, 3, and 4 were the common strains found among participants in

Lagos with ST1 and ST3 predominating, while ST1 was the most prevalent strain in

the symptomatic diarrhoeal HIV/AIDS individual.

5. Twenty-two of the local Blastocystis isolates aligned phylogenetically with other

Blastocystis nucleotide sequences in the GenBank and Peteromonas larcertae as the

out group.

8.0.5 GENERAL CONCLUSION

In conclusion, the main aim of the study was to investigate the occurrence and prevalence of the emerging diarrhea pathogen, Blastocystis species in Lagos. The findings show that the parasite exists with an overall prevalence of 19.2% and diarrhea was statistically associated with the parasite. It could be inferred that Blastocystis induced diarrhea might be prevalent and persistent, since the awareness of the emerging parasitic organism may be low among the health practitioners in the locality of Lagos.

128

There is a high prevalence of 17.8% of Blastocystis infection among the HIV/AIDS individuals compared to the rate found in the control of apparently healthy individuals who had an occurrence of 9.5%. Although the role of the parasite in the immunocompromised condition of HIV/AIDS is not fully known, however, it is important for care-givers to be aware that the parasite could be an opportunistic in the condition.

The statistically significant association of diarrhoea condition and Blastocystis detection in the HIV/AIDS individuals investigated also implies that this opportunistic parasitic organism could contribute to gastrointestinal disorders, including watery diarrhoea syndromes, some of which may be life threatening to people living with HIV and AIDS.

Nevertheless, the detection of the cysts from the control group of non- diarrhoeic subjects indicated the possible existence of ‟carrier status‟in some apparently healthy individuals in

Lagos.

The isolation of faecal Blastocystis sp. DNAs direct from stool was also another objective of the study, a procedure that provided confirmation that Blastocystis exists in Lagos.

Moreover, all the identified subtypes of Blastocystis were aligned phylogenetically with known parasite stored sequences and as such the work has contributed to the molecular epidemiology of Blastocystis.

The subtypes, ST1, ST3 and ST4 of Blastocystis species were identified in Lagos and all three subtypes were seen in the symptomatic individuals. These strains might have pathogenic capability considering the association of parasite with diarrhoea and more prevalence in loose and watery stool samples. Therefore, some diarrhoea conditions and gastrointestinal symptoms may be good indicator for testing of stool samples for

Blastocystis sp. Subtype 3 was more predominant in the asymptomatic individuals, and is the human subtype. Due to the extensive genetic variability among and within subtypes,

129 there could be potentially pathogenic strains among the ST3. On the other hand, the subtype1 occurred as the most prevalent pathogenic subtype among the symptomatic diarrhea subjects.

The fact is that pathogenic Blastocystis subtypes may abound in Lagos metropolis gives cause for worry. Considering the global human travel activities of which Lagos is a major station, it is not surprising that this pathogenic agent is now endemic in this locality.

The route of spread of the infection in Lagos has not been elucidiated; though faeco-oral and waterborne routes have been reported in Thailand (Tan et al., 2002, Leeleyohova et al.,

2004). The epidemiology, transmission, the extent of involvement in diarrhea cases and reliable intervention methods has not been settled for Blastocystis species and will be the focus of future studies.

Moreover, mixed infections and other subtypes might exist considering that one isolate

(unknown subtype) failed the dedioxy-sequencing process using the 10 avaliable primers.

Therefore, further surveys for other subtypes of Blastocystis in high risk individuals and groups in the rural, sub-urban and urban populations of Lagos and other regions might be necessary.

130

8.0.6 CONTRIBUTIONS TO KNOWLEDGE:

The contributions of this study to the generation of knowledge were:

1. Blastocystis spp. ocour in Lagos with overall prevalence of 19.2% and in HIV/AIDS

individuals with prevalence of 17.8% and the parasite could be an important

emerging parasitic diarrhea pathogen in Lagos.

2. The isolation of Blastocystis DNA directly from stool samples is a valid

confirmation of occurrence of the parasite in Lagos.

3. Blastocystis subtypes identified were (ST) 1, 3, and 4; with ST1 and ST3

predominating at 41.6% each and ST1 found to be the most common subtype among

symptomatic diarrhea HIV/AIDS subjects in Lagos metropolis.

131

9.0 RECOMMENDATIONS

Blastocystis species, a potential diarrhoegenic parasite occurs in Lagos, and the following recommendation will be necessary in order to attempt the control of the parasite.

1. There is need to create awareness, through workshops and symposia; first, among care-givers: Researchers, Medical Doctors, Laboratory Scientists, Nurses and general public especially those at high risk of infection.

2. The updating of current facts known of the parasite in curriculum of medical education.

3. More studies should be conducted in order to provide information on the epidemiology, prevalence, transmission routes and the biology of Blastocystis in Nigeria.

4. It is also vital to improve the awareness of personal and environmental hygiene of Lagos resident through mass media communication systems.

5. Treatment of sewage, in order to immobilize faecal pathogens, before the disposal or use of the product in farms as manure could be a good control of transmission of this parasitic infection.

6. Proper screening of food such as vegetables and fruits and water as well as the food- vendors for intestinal parasites.

7. There should be frequent surveillance and inspection of imported fruits and vegetables into the country for Blastocystis species indeed other diarrhoeic pathogens.

8. Preliminary use of the microscope method is emphasized in this study to differentiate one aetiological agent of enteric parasitic infection such as Blastocystis from another parasite before an effective treatment can be given.

9. Moreover, the provision of very sensitive diagnostic method for parasite isolation such as the STS- PCR- method, if only, in our tertiary health Laboratories, will help in the proper diagnosis of Blastocystis species infection.

132

REFERENCES

Abe, N., M. Nagoshi, K. Takami, Y. Sawano, H. Yoshikawa.(2002). A survey of

Blastocystis sp., in livestocks, pets, and zoo animals in Japan. Veterinary Parasitology. 106:

203-212.

Abe, N., Z. Wu, H. Yoshikawa. (2003). Zoonotic genotypes of Blastocystis hominis detected in cattle and pigs by PCR with diagnostic primers and restriction fragment length polymorphism analysis of the small subunit ribosomal RNA gene. Parasitology Research.

90:124-128.

Abe, N. (2004). Molecular and Phylogenetic analysis of Blastocystis isolates from various hosts. Veterinary Parasitology. 120: 235-24

Agbolade, O. M, Agu, C.N., Adesanya, O.O., Odejayi, AO, Adigun, A.A. (2007). Intestinal helminthiases and schistosomiasis among school children in an urban center and some rural communities in southwest Nigeria. Korean Journal of Parasitology 45 (3): 233-238.

Akujobi, C. N and F.T. Ogunshola. (2006). Prevalence of Cryptosporidium Species In

Patients With Human Immunodeficiency Virus (HIV)/ Acquired Immunodeficiency

Syndrome (AIDS) Presenting With Diarrhoea In Lagos. Nigerian Quarterly Journal of

Hospital Medicine. vol. 16 (2):49- 52.

Alakpa, G.E., Clarke, S.C., Fagbenro-Beyioku, A.F. (2003a). Cyclospora cyatanensis:

Vegetable and water as possible vehicle for its transmission in Lagos. British Journal of

Biomedical Science. 60(2):113-114.

Alakpa, G.E., Clarke, S.C., Fagbenro-Beyioku, A.F. (2003b). Cyclospora cyatanensis in

Lagos, Nigeria. Clinical Microbiology and Infection. 9 (7): 731-732.

133

Albretch, H., H. J. Stellbrink, K. Koperski, H. Greten. (1995). Blastocystis hominis in human immunodeficiency virus- related diarrhoea. Scandanavian Journal of

Gastroenterology. 30: 909 - 914.

Alexieff, A. (1911). Sur la nature des formations dues “kystes de Trichomonas intestinalis.” Clinics Royal Society Biology. 71: 296-8.

Allison, M. J., Tamera B, and Enrique G. (1999). Further studies on fecal parasites in

Antiquity. America Journal of Clinical Pathology. 112: 605-606.

Amin, O.M. (2002). Seasonal prevalence of intestinal parasites in the United States during

2000.American Journal of Tropical Medicine Hygiene. 66: 799-803.

Amin, O.M. (2006). Epidemiology of Blastocystis hominis in the United States of America, USA. Research Journal of Parasitology. 1 (1): 1-10.

Arisue, N., T Hashimoto, H. Yoshikawa, Y. Nakamura, G. Nakamura, F. Nakamura, T.A.

Yano, and M. Hagasewa. (2002). Phylogenetic position of Blastocystis hominis and of

Stramenopiles inferred from multiple molecular sequence data. Journal Eukaryotic

Microbiology. 49: 42-53.

Arisue, N., T. Hashimoto and H. Yoshikawa. (2003). Sequence heterogeneity of the small subunit ribosomal RNA genes among Blastocystis isolates. Parasitology. 126:1-9.

Ashford, R.W and Atkinson E.A. (1992). Epidemiology of Blastocystis hominis infection in

Papua, New Guinea: Age-prevalence and association with other parasites. Annal Tropical

Medicine Parasitology.86:129-136.

134

Atzori, C. A.Bruno, G. Chichino, C. Cevini, A.M. Bernuzzi, S. Gatti, G. Conolli, M.

Scaglia.(1993). HIV-1 and Parasitic infections in rural Tanzania. Annals Tropical Medicine and Parasitology. 87(6): 585-593.

Awogun, I.A. (1984). The prevalence of intestinal parasites in children living in Ilorin,

Kwara state. Nigerian Medical Practitioners.7 (6):176-178.

Babcock, D, R. Houston, D. Kumaki, D. Shlim. (1985). Blastocystis in Kathamandu, Nepal.

New England Journal Medicine. 313: 1419(Letter).

Bartlett , J. G., Bellisos P.C., Sears C.L.(1992). AIDS enteropathy. Clinical Infectious

Disease. 16: 726-735.

Becker, M.L., C. R.Cohen, M. Cheang, R.G.Washington, J.F. Blanchard, S. Moses. (2007).

Diarrheal Disease among HIV-Infected Adults in Karnataka, India: Evaluation of Risk

Factors and Etiology. American Journal of Tropical Medicine and Hygiene.76; (4): 718 -

722.

Bello, C.S.S, Tanyigna, K.B., C.O. Olotu, (1997). Intestinal Parasites in Jos: A Four –Year

Review. Nigerian Medical Practice. 34, 1/2; 11-13.

Belova, L. M. (1992). On the occurrence of Blastocystis galli (Rhizopoda, Lobosea) in

Turkey. Parazitologia. 26: 166-168.

Berger S. (2007). Infectious Disease of Australia. American Journal Tropical Medical

Hygiene.76; 3: 549-552.

Bohm-Glonning, B, J. Knobloch, B. Walderich. (1997). Five subgroups of Blastocystis hominis isolates from symptomatic and asymptomatic patients revealed by restriction site

135 analysis of PCR- amplified 16S- like rDNA. Tropical Medical International Health. 2: 771-

778.

Boorom, K.F., Smith, H., Nimri L., Viscogliosi E., Spanakos, G, Parkar U, Li, L.H., Zhou,

X. N, Ok U.Z., Leeleyoova, S., Jones, M.S. (2008). Oh my aching gut: Irritable Bowel

Syndrome, Blastocystis and asymptomatic infection. Parasitology Vectors. 1: 40-56.

Boreham, P. F.L., and D. J. Stenzel. (1993). Blastocystis in humans and animals: morphology, biology and epizootiology. Advances in Parasitology. 32: 1-70

Bradsberg, J. H. (2001). A Guide to Coco. Journal of Statistical Software, Vol. 6 : 4.

Brandonisio, O., P.Maggi, M.A. Panaro, S. Lisi, A. Andriola, A.Acquafredda, G. Angarano.

(1999). Intestinal protozoa in HIV- infected patients in Apulia, South Italy. Epidemiology of

Infection. 123: 457- 462.

Brites C., M. G. Barberino, M. A, Bastos, M.Sampiao SA, N. Silva. (1997). Blastocystis hominis as a potential cause of diarrhoea in AIDS patients: a report of six cases in Bahir,

Brazil. Brazil Journal Infectious Disease.1:91- 94

Brumpt, E. (1912). Blastocystis hominis N. sp. Et formes voisines. Bulettin of Society of

Pathology Experiment. 5: 725-30.

Cavalier-Smith, T. (1997). Sagenista and Bigyrra, two phyla of heterotrophic heterokont chromists. Achive fur Protisten. 148: 253-267.

Cegielski, J.P., E. A. Msengi, C.S. Dukes, R. Mbise, R. Redding-Lallinger, J.N.Minjas,

M.L.Wilson, J. Shao, D.T. Durack. (1993). Intestinal parasites and HIV infection in

Tanzanian children with chronic diarrhoea. AIDS. 7:213-221

136

Center for Disease Control and Prevention, CDC. (1987). Atlanta, USA; 12/4/2003.

Center for Disease Control and Prevention, CDC. (2001). Atlanta, USA; 3/8/2003.

Center for Disease Control and Prevention, CDC. (2012). Atlanta, USA;16/8/2012

Cheesborough, M. (2001). Processing of Clinical samples. Medical Laboratory Manual for

Tropical Countries. 14th. Edition: 178-198. Butterworth and Co., United Kingdom

Chen T, Chan C., Chen H, Fung C., Chan W., Liu, C. (2003). Clinical characteristics and endoscopic findings associated with Blastocystis hominis in healthy adults. America Journal

Tropical Medicine and Hygiene. 69: 213- 216.

Cirioni, O., A. Giacometti, D. Drenaggi, F. Ancarani, G. Scalise. (1999). Prevalence and

Clinical relevance of Blastocystis hominis. European Journal of Epidemiology. 15:389-393.

Clark, G.C., (1997). Extensive genetic diversity in Blastocystis hominis.

Molecular Biochemistry and Parasitology. 87: 79-83.

Clark, G.C. (2000). Cryptic genetic variation in parasitic protozoa. Journal of

Medical Microbiology. 49: 489-491.

Corliss J.O. (1994). An interim Utilitarian (user friendly) hierarchical classification and characterization of the protists. Acta Protozoology. 20; 33:1-

51

Cox, F.E. (1991). Systematics of parasitic protozoa.In J.F. Krieger and J.R Baker (ed.):

Parasitic Protozoa.1: 55-80. 2nd ed. Academic Press Incoporated. San Diego.Califonia,

USA.

137

Cruz, V. C., A.P. Crespo, C.M. Alvarez, S.V. Rojas, G.R. Sanchez, L.V. Franco.(2003).

Blastocystis hominis among food vendors in Xochimilco markets. Review Latinoam

Microbiology. 45(1-2):12-15.

Devera , R.A., V.J. Velaxquez, M. Vaquez. (1998). Blastocystosis in preschool children from Bolivar city, Venezuela. Cad. Saude Publication.14: 401-407.

Diaz, E, Mondragon I, Ramitrez E, Bemal R.(2003). Short report of epidemiology and control of intestinal parasites with Nitazoxanide in children in Mexico. American Journal

Tropical Medicine Hygiene. 68: 384- 385.

Dogan, N. (1998). Prevalence of Blastocystis hominis in Bozan region. Turkiye

Paracitologie Derg. 22 (3): 247-250.

Dogrundman-Al, F., H. Dagci, H. Yoshikawa, O. Kurt, M. Demineh.(2008). A possible link between subtype 2 and asymptomatic infection of Blastocystis hominis. Parasitology

Research. 103:685-689.

Dogrundman-Al, F., Kustimur, S, Yoshikawa H, Tuncer, C, Simsek, Z, Tanyuksel, M,

Araz, E., Boorom, K.(2009a). Blastocystis subtypes in Irritable Bowel Syndrome and

Inflammatory bowel disease in Ankara, Turkey. Memoriam Instituta Oswaldo Cruz 104:

724-727.

Dogrundman-Al, F, Yoshikawa, H, Kustimur, S. Balaban, N.(2009b). PCR-based subtyping of Blastocystis isolates from symptomatic and asymptomatic individuals in a major hospital in Ankara, Turkey. Parasitology Research. 106:263-268

138

Dominquez-Marquez, M. V., Guma, R., Munoz, C., Gomez-Munoz, M.T., Borras,

R.,(2009).High prevalence of subtype 4 among isolates of Blastocystis hominis from symptomatic patients of a health district of Valencia(spain).Parasitology Research. 105:

949 - 955.

Doyle, P.W., M.M. Helgason, R. G. Mathias and E.M. Proctor. (1990). Epidemiology and pathogenicity of Blastocystis hominis. Journal Clinical Microbiology. 28:116-121.

Dunn, L.A., P.F.Boreham and D.J. Stenzel. (1989). Ultrastructural variation of Blastocystis hominis stock in culture. International Journal of Parasitology. 19:43-56

El-Sharzy, A. M., A. A.Abdel-Magied, S. N. El- Beshibishi, H. A.El- Nahas, M. A Fouad, and M.S. Monib. (2005). Blastocystis hominis among symptomatic and asymptomatic individuals in Talkha center, Dakahlia goveronat. Journal of Egypt Society of Parasitology.

35: 653-666.

Eve, E, E. Ferraz, and E. Thatcher. (1998). Parasitic infections in Villages from three districts of the Brazillian Amazon. Annals of Tropical Medicine and Parasitology. 92; 1: 79-

87.

Fagbenro-Beyioku, A. F and J.P.O. Oyerinde, (1987). Parasitic Intestinal Infections of

Children in Lagos. Nigeria Journal of Paediatrics. 14: 89-95.

Fagbenro-Beyioku, A. F., Oyibo, W.A, Oladosu, O.O. (2005). Prevalence of intestinal parasites in HIV and Non- HIV patients in Metropolitan Lagos, Nigeria. Amsul Digest.4:

24-26.

Fagbenro-Beyioku, A.F., Oyibo, W.A, Onuoha, E.S.U., Ukaegbu, C.B., Ojuromi, O.T.,

(2007). Evaluation of Microscopic Staining Techniques for the Diagnosis of Opportunistic

139

Protozoan infections in a developing country. The internet Journal of Tropical Medicine. 3:

1-6.

Florez A.C., D. A.Garcia, L. Moncada, M. Betran. (2003). Prevalence of Microsporidia and other intestinal parasites in patients with HIV infections, Bogota, 2001. Biomedica. 23:

274-278.

Fujita, O., M. Ohnishi, V. Diaz, l. Martinez, M. Kamiya. (1993). Epidemiological investigation for intestinal parasitic infection in children in rural communities in Paraguay.

Japan Journal of Parasitolology. 42: 409- 414.

Galev, A, M. Kadurinaz, J. Dimova, P.Chrbarnova, B. Dimitrov. (2004). Blastocystis hominis as a causative agent in acute and chronic urticaria? 40 (4): 53-58.

Gamboa, M.I., J.A. Basualdo, M.A. Cordoba, B.C. Pezzani, M.C. Minvielle, H. B. Lahitte.

(2003). Distribution of intestinal parasitosis in relation to environmental and socio- cultural parameters in La Plata, Argentina. Journal of Helminthology. 77: 15-20

Garavelli, P. L and L. Scaglione. (1998). Blastocystis hominis. Review of Parasitology. 5:1-

15.

Garcia, L.S., D.A. Bruckner, and M.N.Clancy.(1984). Clinical Relevance of Blastocystis hominis. Lancet 1: 1233-1234.

Garcia, L. S., and Bruckner, D.A. (2007). Diagnostic Medical Parasitology.3rd. ed. ASM

Press.Washington D.C: 54- 83.

Gassama A., P.S.Sow, F. Fall, P. Camara, A. GueyeNdiaye, R. Seng. (2001). Ordinary and

Opportunistic entero-pathogens associated with diarrhoea in Senegalese adults in relation to

140 the human immunodeficiency virus serostatus. International Journal of Infecious. Dieases.

5: 192-198.

Geltman, P.L., Cochran, J. C. Hedgecock, (2003). Intestinal parasites among African refugees resettled in Massachuseettes and the impact of an overseas pre-treatment program.

American Journal of Tropical Medicine and Hyieneg. 69; (6): 657-662.

Gilbert D.N., Moellering (Jr), B.C., Sander M.A. (1998). The Sanford Guide to

Antimicrobial Therapy. Antimicrobial Therapy Inc.: 44-50 USA.

Govind, S.K., A. A. Khairul, H. V. Smith. (2002). Multiple reproductive processes in

Blastocystis. Trends Parasitology. 18: 528.

Graczyk, T. K., C.K. Shiff, L. Tamang, F.Munsaka, A. M. Beitin, W.J. Moss.(2005). The

Association of Blastocystis hominis, and Endolimax nana with diarrhoeal stools in

Zambian school-age children. Parasitology Research. 98: 38-43.

Green, P.J. (1995). Reversible jump Markov chain Monte Carlo computation and Bayesian model determination. Biomerika. 82: 711-732.

Guimeraes, S. and M.I.L. Soyagar. (1993). Blastocystis hominis: Occurrence in children and staff members of municipal day care center from Botucatu, Sao Paulo state, Brazil.

Memoriam Institut Oswaldo Cruz Rio de Janeiro. 88:427-429.

Gupta, R., and Parsi K. (2006). Chronic urticaria due to Blastocystis hominis. Australasia.

Journal of Dermatology. 47(2):117-9.

141

Hailemariam, G, A. Kassu, G. Abebe, E. Abate, D. Damte, E. Mckonnen, F. Ota. (2004).

Intestinal parasitic infections in HIV/ AIDS and HIV seronegative individuals in a teaching

Hospital in Ethopia. Japan Journal of Infectious Diseases. 57:41-43.

Hall, T.A. (1999). Bioedit: a user friendly biological sequence alignment editor and analysis program for Windows (95, 98 NT.) Nucleic acid symposium series.41: 95-98.

Herwaldt, B. L., deArroyave, K. R., Wahlgrust, S.P., deMerrida, A.M., Lopez, A.S.,

Juranek, D.D. (2003). Multi-year Prospective study of intestinal parasitism in a cohort of

Peace Corps volunteers in Guatemela. Journal Clinical Microbiology. 39; 34-42.

Ho, L. C. ., M. Singh, G. Suresh, G.C. Ng, E.H. Yap. (1993). Axenic culture of Blastocystis hominis in Iscove‟s modified Dulbecco‟s medium. Parasitology Research. 79:614-616.

Horiki, N., Maruyama, M., Fujita, Y., Yone-Kura, T., Minato, S., Kaneda, Y. (1997).

Epidemiologic Survey of Blastocystis hominis infection in Japan. American Journal of

Tropical Medicine Hygiene. 56:370-4.

Howard, S.W. (2007). Blastocystis hominis and colorectal cancer.www.horwardsteel.co. uk/papers/008.

Huelsenbeck J. P., Ronquist F. (2001). Mr. BAYES: Bayesian inference of phylogenetically trees. Bioinformatics. 17:754-755.

Hunter, G.A.F Bagshawe, K.S. Baboo, R. Luke, P. Provic.(1992). Intestinal Parasites in

Zambian patients with AIDS. Transactions of Royal Society of Tropical Medicine and

Hygiene. 86:301-305.

142

Hussain, R. W. Jaferi, S. Zuberi, R. Bagai, W. Abrar, A. Ahmed, V. Zaman. (1997).

Significantly increased IgG² subclass antibody levels to Blastocystis hominis in patients with Irritable Bowel Syndrome. American Journal Tropical Medicine and Hygiene. 56:301-305.

Hussein, E.M, Hussein, A.M., Eida, M.M., Atwa, M.M, (2008). Pathophysiological variability of different genotypes of human Blastocystis hominis Egyptian isolates in experimentally infected rats. Parasitology Research. 102: 853-860.

Iguchi, A, Ebisu, A, Nagata, S, Saitou, Y, Yoshikawa, H, Iwatani, S., Kimata, I. (2007).

Infectivity of the different genotypes of human Blastocystis hominis isolates in chickens and rats. Parasitology International. 56: 107-112.

Johnson, A.M., A. Thanou, P.F. Boreham, P.R. Baverstock. (1989). Blastocystis hominis:

Phylogenetic affinities determined by rRNA sequence comparism. Experimental

Parasitology. 68: 283- 288.

Johnson, E. H., J. J. Windsor, C.G. Clark. (2004). Emerging from obscurity: Biological,

Clinical and Diagnostic Aspects of Dientamoeba fragilis. Clinical Microbiology Review. 17

(3): 553-570.

Jones, W. R. (1946). The experimental infection of rats with Entamoeba histolytica. Annals of Tropical Medicine and Parasitology. 40:130

Jones, M. S., H.R. D. Ganac, G. Hiser, N.R. Hudson, A. Le, C.M. Whipps. (2008).

Detection of Blastocystis from stool samples using real time PCR. Parasitology Research.

103: 551-557

143

Jones, M. S., C. M. Whipps, R.D. Gannes, Nn.r. Hudson, K. Boroom. (2009). Association of Blastocystis subtype 3 and 1 with patients from an Oregon Community presenting with chronic gastrointestinal illness. Parasitology Research.104: 341- 345.

Kain, K. C, M. A. Noble, H. J. Freeman, R. I. Bartcluk. (1987). Epidemiology and clinical features associated with Blastocystis hominis infection. Diagnostic Microbiology Infectious

Disease.8: 235-244.

Kaneda, Y, N. Horiki, X. J. Cheng, H. Tachibama, Y. Tsutsumi. (2000). Serologic response to Blastocystis hominis infection in asymptomatic individuals. Tokai Journal of

Experimental Clinical Medicine. 25:51-56

Kaneda, Y, N. Horiki, X. J. Cheng, Y. Fujita, M. Maruyama, H. Tachibama. (2001).

Ribodemes of Blastocystis hominis isolated in Japan. American Journal Tropical Medicine

Hygiene. 65: 393-396.

Kassu, A., A. Tregaye, D Wolday, B Petros, M. Aklilu, E.J. Sanders, A.L. Fontanet, D. Van

Baarle, D. Hamann, T. F. Rinke De Wit (2003). Role of incidental and/or cured intestinal parasitic infections on the profile of CD4 and CD8 T-cell activation status in HIV-1 infected and uninfected adult Ethopians. Clinical Experimental Immunology. 132: 113-119.

Katsaraou-Katsari, A., C.M. Vassalos, K. Tzanctou, G. Spanakos, C. Papadopoulou, N.

Vakalis. (2008). Acute urticaria associated with the amoeboid forms of Blastocystis species subtype3. Acta. Dermatologie Venerenologie 88 : 80-81.

Kaya, S., E. Seslicetin, B. Cicioglu-Aridogan, S. Arikan, M. Demirci. (2007). Pathogenicity of Blastocystis hominis, A Clinical Re evaluation. Turkiye Parazitoloji Dergisi. 31 (3):

184-187

144

Khalifa, A.M., M.M. El-Temsahy, E. N.I. Abou. (2001). Effect of ozone on the viability of some protozoa in drinking water. Journal of Egypt Society of Parasitology. 31: 603-616

Koustalvis, A.T., Valiquette, L., Allard R, J. Soto. (2001). Blastocystis hominis: A new pathogen in Day care center? Canadian Communicable Disease Report, CCDR: 27 (9):

1243-5.

Kumar, S., Tamura, K., Nei, M. (2004). MEGA 3: Integrated software for molecular evolutionary genetics analysis and sequence alignment.Bioinformatics 5: 150-163.

Leelayoova, S., P. Taamasri, R., Rangsin, T. Naaglor, U. Thathaisong, M. Mungthin.

(2002). In vitro cultivation: a sensitive method for detecting Blastocystis hominis. Annal of

Tropical Medicine and Parasitology. 96: 803-807.

Li, L-H, Zhang X.P., S. Lv, L. Zhang, H. Yoshikawa, Z. Wu, P. Steinmann, J. Utzinger, X.

M. Tong, S.H. Chen, X. N. Zhou. (2007a). Cross sectional surveys and subtype classification of human Blastocystis isolates from four epidemiological settings in China.

Parasitology Research.102: 83-90.

Li, L-H, .Zhou X.N, Z. W. Du, Zhang X.P ,.X.J. Wang, L.W Wang, J.Y.Jiang H.

Yoshikawa P. Steinmann, J. Utzinger, Z. Wu, J.X. Chen, S.H.Chen, L. Zhang. (2007b).

Molecular epidemiology of human Blastocystis in a village in the Yuannan province China.

Parasitology International. 56:281-286.

Logar, J., A. Andlovic, M. Poljsak-Prijatelj. (1994). Incidence of Blastocystis hominis in patients with diarrhea.Journal Infection. 28: 151-154.

Malheiros, A.F, Stensvold C.R, Clark C.G,Guilherme B.B, Shaw J.J.(2011).Molecular

Characterization of Blastocystis Obtained from Members of the Indigenous Tapirape Ethnic

145

Group from the Brazilian Amazon Region. American Journal of Tropical Medicine

Hygiene. 85; 6: 1050-1053.

Marshall, M .M, Naumavitz D, Ortega Y., Sterling C. R. (1997). Waterborne protozoan pathogens. Clinical Microbiology Review. 10 (1): 67- 85

Mascher, F. and Reinthaler, F.F. (1987). Zur Trinkwasserproblematik in tropischen

Klimaten am Beispiel von Abeokuta, Nigerien. Zentralblatt far Bakteriologie,

Microbiologie and Hygiene. B184: 297-303.

Meloni, D, Sanciu G, Pourier P, Alaoui H, Chabe M, Delhaes L, Dei Cas E, Delbac

F.(2011). Subtypes of Blastocystis among symptomatic patients in Italy. Parasitology

Research. 109: 613-619.

Menon, B.S, M. S. Abdullah, F. Mahamud, M. Singh.(2000). Intestinal parasites in

Malaysian children with cancer. Journal of Tropical Paediatrics. 45(4): 241-242

Minvielle, M. C., B.C. Pezzani, M. A. Cordoba, M .M. De-Luca, M. C. Apeztegul, J.A.

Basualdo. (2004). Epidemiological survey of Giardia spp. and Blastocystis hominis in an

Argentine rural community. The Korean Journal Parasitology. 42 (3): 121-127.

Moe, K. T., M. Singh, J. Howe, l.c. Ho, S.W. Tan, G.C. Ng, X. Q. Chen, E.H. Yap.(1996).

Observations on the ultrastructure and viability of the cystic stage of Blastocystis hominis from human feces. Parasitology Research. 82: 439- 444.

Moe, K. T., M. Singh, J. Howe, l.C. Ho, S.W. Tan, G.C. Ng, X. Q. Chen, E.H. Yap.(1999).

Development of Blastocystis hominis cysts into vacuolar forms in vitro. Parasitology

Research. 85: 103-108

146

Nascimento, S.A and M. D. L. R. Moitinho. (2005). Blastocystis hominis and other intestinal parasites in a community of Pitanga City, Parana state, Brazil. Review Institute of

Medicine Tropical Sao Paulo. 47: 213-217.

Nasurideen, A.M.A., Eu-Hian, Y, Singh, M., Tan, T.S.W. (2004). Metronidazole induces programmed cell death in the protozoan parasite Blastocystis hominis. Microbiology.

150:33- 43.

Nimri, L. F. (1993). Evidence of an epidemic of Blastocystis hominis infection in pre-school children in northern Jordan..Journal Clinical Microbiology. 31: 2706- 2708.

Nimri L.and R. Batchoun. (1994). Intestinal colonization of symptomatic and asymptomatic school children with Blastocystis hominis. Journal Clinical Microbioogy. .32: 2865-2866.

Noel, C., Peyronment c, Gerbod D, Egdecomb V, Delgardo-Viscogliosis P, Sogin L,

Capron M, Viscogliosis E, Zenner I. (2003). Phylogenetic analysis of Blastocystis isolates from different hosts based on the comparism of small subunit rRNA gene sequences.

Molecular Biochemistry Parasitology.126:119-123

Noel, C., G. Dufernez, V. P. Edgecomb, P. Delgado-Viscogliori, H. Lipchuen, M. Singh, R.

Wintjens, M. L. Sogin, M. Capron, R. Pierce, L. Lenner, E. Viscoglosi. (2005). Molecular

Phylogenies of Blastocystis isolates from different hosts: implication for genetic diversity, identification of species, and zoonosis. Journal Clinical Microbiology. 43:348-355.

Obiamiwe, B.A. (1977).The pattern of parasitic infection in human gut at the specialist

Hospital, Benin city, Nigeria. Annals of Tropical Medicine and Parasitology. 71 : 35-40.

147

Odugbemi, T. O., J .P.O. Oyerinde, Isaac-Sodeye, J.O, Roberts, J. I. K. (1981). Parasitic and

Bacterial aetiology of childhood enteritis in the under 5years. West African Medical

Journal 2 : 19- 24.

Odujurin, O. (1987).Prevalence, treatment, and average re-infection of Ascaris among

Primary school entrants in a peri-urban area of Lagos. Nigerian Medical Journal. 17 (12):

93-96.

Ofosu-Barko, K. (1998). Emerging and re- emerging infectious diseases: Acquired immunodeficiency syndrome (AIDS). Nigerian Medical Journal. 1: 1-9

Ogunba, E.O. (1976). The prevalence of human intestinal protozoa in Ibadan, Nigeria.

Journal of Tropical Medicine and. Hygiene. 80:187-191.

Ogunbi, O. (1971). Intestinal microbial and helminthic infections in Lagos University

Teaching Hospital, LUTH, Lagos (1965-1968). Journal of Nigerian Medical Association.1:

53-58.

Okeke, I. N., Oladipupo, O., Lamikanra, A., Kaper J. B. (2003). Etiology of Acute Diarrhea in Southwestern, Nigeria. Journal Clinical Microbiology.41 (10): 4525-4530.

Okodua, M., Adeyeba O. A., Taffeng, Y.M., Okpala, H.O. (2003). Age and Sex Distribution of Intestinal Parasitic Infection among infected patients in Abeokuta, Nigeria. Online

Journal of Health and Allied Sciences; 4: 3.

Onah, D.N., and Chiejina, S.N. (1995). Taenia solium cystercosis and human taeniasis in the Nsukka area of Enugu State, Nigeria. Annals of Tropical Medicine and Parasitology.

89; 4: 399- 407.

148

Onuoha, E, S.U. (1997). Intestinal and Haemo-parasites in HIV seropositive, AIDS, and

AIDS related complex disease in metropolitan Lagos, Nigeria. M.Sc. Dissertation.:

University of Lagos, Nigeria. 23- 24.

Oyedeji, C. O. (1992). Health research methods for developing country scientist: 59- 63.

Codat publication, Ibadan Nigeria.

Oyerinde, J.P.O., Ogunbi, O., Alonge, A. A. (1977). Age and Sex distribution of infections with Entamoeba histolytica and Giardia intestinalis in Lagos population. International

Journal of Epidemiology. 6 : 231-234.

Oyerinde, J.P.O., Alonge, A.A., Adegbite-Hollist, A.F., Ogunbi, O. (1979). The epidemiology of Entamoeba histolytica in a Nigerian urban population. International

Journal of Epidemiology. 8: 55-59.

Oyerinde, J. P.O, A.F. Adegbiti-Hollist, O. Ogunbi (1981). The Prevalence of Intestinal

Parasites of Man in Metropolitan Lagos. Nigerian Journal of Natural Sciences. 3: 147-155.

Oyerinde, J.P.O. (1999).Control of parasites: which way forward? A paper presented at a scientific seminar organized by the Nigerian Society for Parasitology, Lagos branch.

9th September.

Oyewole, F.F. Ariya, A, Sanyaolu, W.A Oyibo,T. Fayewa, P. Monye, M. Ukpong, C.

Okoro. (2002). Intestinal Helminthiases and their control with Albendazole among primary school children in Riverrine communities of Ondo State, Nigeria. Southeast Asian Journal

Tropical Medicine and Public Health. 33; 2: 214- 217.

149

Ozcakir O, S. Gureser, S. Eguven, Y.A. Yilmaz, R. Topaloglu, G. Hascelik. (2007).

Characterestics of Blastocystis hominis infection in a Turkish University Hospital. Turkiye

Parazitolojizia Dergesie.31:277-282.

Ozyurt, M., O. Kurt, K. Molbak, H.V.Nielsen, T. Haznedaroglu, C.R.Stensvold. (2008).

Molecular epidemiology of Blastocystis infections in Turkey. Parasitology. 10: 1-7.

Parker, U., Traub R. J., Kumar, S., Mungthin, M., Vitalis, S., Leelayoova, S., Morris, K,

Thompson, R.C.A. (2007). Direct characterization of Blastocystis from faeces and evidence of zoonotic potential. Parasitology.134:359-367.

Patterson, D.J. (1994). http:// tolweb.org/note_id= 53

Prasad, K.N., V.L. Nag, T.N Dholes, A. Ayyagari. (2000). Identification of enteric pathogens in HIV-positive patients with diarrhoea in Northern India. Journal Health

Population and Nutrition. 18: 23- 26.

Quadri, S. M. H., G. A. Al-Okaili, F. Al-Dayel. (1989). Clinical significance of

Blastocystis hominis. Journal of Clinical Microbiology. 27 : 2407-2409.

Reinthaler, F. F., Mascher, G. Klen, W. Sixl (1988). A survey of gastrointestinal parasite in

Ogun State, Southwest, Nigeria. Annal of Tropical Medicine and Parasitology. 82:181-184.

Rene, B.A., Stensvold, C.R., Badsberg, J. H. and Nielsen, H.V. (2009). Subtype analysis of Blastocystis Isolates from Blastocystis Cyst Excreting Patients. American Journal of

Tropical Medicine and Hygiene. 80 (4): 588-592.

Riveira, W.L. (2008). Phylogenetic analysis of Blastocystis isolates in the philipines.

Vetrinary Parasitology. 156: 178-182

150

Rolstron, V.I., R. Winans, S. Rodriguez. (1989). Blastocystis hominis, Pathogen or not?

Journal of Clinical Microbiology. 27 : 2407- 2409.

Rolstron, V. I, R. Winans, S. Rodriguez (2001). Blastocystis hominis. Pathogen or not? Rev.

Infectious Disease. 11: 661- 662.

Sadek, Y, EL-Fukahany, A.F, Lashin A.H. (1997). Intestinal parasites among food-handlers in Qualyobia governorate with respect to the pathogenic parasites, Blastocystis hominis.

Journal of Egypt Society of Parasitology. 27:471- 8

Saksirisampant, W., Nuchprayoon S. Wiwanitkit V. Yenthakam S, Ampavasiri A. (2003).

Intestinal parasitic infestations among children in an orphanage in PathumThani province.

Journal of Medical Association of Thailand. 86; 2: 263-270.

Salim, H., Suresh, K.G, Vellayan, S., Mak, J.W.,Khairul-Anura, A., Init, I., Vennik, G.D.,

Saminathan, R, Ramakrishnan, K. (1999). Blastocystis in Animal handlers. Parasitology

Research. 85: 1032-1033.

Sanad, M. M., R. M. Darwish, S.M.Yousef, N.E.Nasser. (1991). Blastocystis hominis: laboratory identification and clinical relevance. Journal of Tropical Medicine. 1:61-70.

Scicluna, S.M., Tawari B, Clark C. G. (2006). DNA Barcoding of Blastocystis. Protist.

157: 77-85

Sheehan, D.J, B.G. Raucher, J.C.Mekitrick. (1986). Association of Blastocystis hominis with signs and symptoms of human disease. Journal of Clinical Microbiology. 24: 548-550.

Silard, R. D. (1979). Contribution to Blastocystis hominis studies. Aspect of degenerescence Archieve Rourn Pathology and Experimental Microbiology. 38: 105- 114.

151

Silberman, J. D., Sogin, M.L., Leipe, D.D. Clark, C.G. (1996). Human parasite finds taxanomic home. Nature. 380: 105-114.

Singh, M., K. Suresh, L.C. Ho, G.C. Ng, E. Yap. (1995). Elucidiation of the life cycle of the intestinal protozoa Blastocystis hominis. Parasitology Research. 81: 446- 450.

Snowden, K., K. Logan, C.Blozinski, J. Hoevers, P. Holman. (2000). Restriction- fragment length polymorphism analysis of small- subunit rRNA genes of Blastocystis isolates from animal hosts. Parasitology Research. 86: 62- 66

Souppart,L. Sanciu, G. Cian, A.Wawrzyniak I, Delbac, F. Capron M, Dei-Cas E, Boorom

K, Delhaes L,Viscogliosi E.(2009). Molecular epidemiology of human Blastocystis isolates in France. Parasitology Research. 105(2):413-21.

Souppart, L., Sanciu, G, Moussa H., Cian A., Poirier P, El-Alaoui, Delbac, F, Boorom, K,

Delhaes, L., Dei-Cas, E., Viscogliosi, E.(2010). Subtype analysis of Blastocystis isolates from symptomatic patients in Egypt. Parasitology Research.106: 505-511.

Stensvold, C.R., A. Brillowska-Dabrowska, H.V. Nielsen, M.C. Arendrup. (2006).

Detection of Blastocystis in unpreserved stool specimen using polymerase chain reaction.

Journal of Parasitology. 92: 1081-1087.

Stensvold, C.R., M.C. Arendrup. C. Jaspersgaard, K. Molbak, H.V. Nielsen (2007a).

Detecting Blastocystis by parasitological and DNA-based methods: A comparative study.

Diagnostic Microbiol. Infectious. Disease. 59: 303- 307.

Stensvold, C.R., M.C. Arendrup. C. K. Molbak, H.V. Nielsen (2007b). The prevalence of

Dientamoeba fragilis in patients with suspected enteroparasitic diseases in a metropolitan area in Denmark. Clincal Microbiology Infection. 13: 839-842.

152

Stensvold, C.R., R.J. Traub, G, von Samson-Himmelstjerna, C. Jaspersgaard, H.V. Nielsen,

R.C.A.Thompson. (2007c). Blastocystis: subtyping isolates using pyrrosequencing TM technology. Experimental Parasitology. 116: 111-119.

Stensvold, C.R., G.K. Suresh, K.SW. Tan, R.C.A. Thompson, R.J. Traub, E. Viscogliosi, H.

Yoshikawa,, C.G. Clark. (2007d). Terminology for Blastocystis subtypes-a consensus.

Trends in Parasitology. 23: 93-96.

Stensvold, C.R., M.A Alfellani, S. Norskov-Lauritsen, K. Prip, E.L. Victory, C. Maddox,

H.V. Nielsen, C.G. Clark. (2008a). Subtype distribution of Blastocystis isolates from synanthropic and zoo animals and identification of a new Blastocystis sp. subtype.

International Journal of Parasitology. 39: 473- 479.

Stensvold, C.R., A., H.V. Nielsen, K. Molbak, H. V. Smith. (2008b). Pursuing the clinical significance of Blastocystis -diagnostic limitations. Trends in Parasitology. 25: 23-29.

Stensvold, C.R., M.C. Arendrup, H.V. Nielsen, S. Thorsen. (2008c). Symptomatic

Blastocystis infection successfully treated with Trimethoprim/sulfamethoxazole, Annals of

Tropical Medicine and Parasitology. 102: 271-274.

Stensvold, C.R., H.C. Lewis, A.M .Hammerum, L.J. Porsbo, S.S. Nielsen,, K.E.P. Olsen,

M.C. Arendrup, H.V. Nielsen, K. Molbak. (2009). Blastocystis: unraveling potential risk factors and clinical significance of a common but neglected parasite. Epidemiology of

Infection. 137: 1655 -1663.

Stensvold, C.R, Christansen, D.B. Olsen, K.E, Nielsen, H.V. (2011a). Blastocystis sp.

Subtype 4 is common in Danish Blastocystis positive patients presenting with acute diarrhea. American Journal of Tropical Medicine and Hygiene. 84(6): 883-885.

153

Stensvold, C.R, Nielsen, S.D, Badsberg, J.H, Engberg, J, Friss- Moller, N, Nielsen, S.S.

Nielsen, H.V. Friss- Moller, A. (2011b). The prevalence and clinical significance of intestinal parasites in HIV-infected patients in Denmark. Scandanavian Journal of

Infectious Disease. 43: 129-135.

Stenzel, D.J., P. F. L. Boreham, R. McDougall. (1991). Ultrastructure of Blastocystis hominis in human stool samples. International Journal Parasitology. 21: 807-812.

Stenzel, D.J. and P.F. Boreham. (1996). Blastocystis hominis revisited. Clinical

Microbiology. 9: 563-584.

Suresh, K.,G.C. Ng, N.P. Ramachandran, L. C. Ho, E. H. Yap, M. Singh. (1993). In vitro encystment and experimental infections of Blastocystis hominis. Parasitology Research. 79:

456-4609

Suresh, K and H. Smith. (2004). Comparism of methods for detecting Blastocystis hominis.

European Journal of Clinical Microbiology Infectious Diseases. 23: 509-511.

Suresh, K., H. Smith, T.C. Tan. (2005). Viable Blastocystis cysts in Scottish and Malaysian sewage samples. Applied Environmental Microbiology. 71: 5619- 20.

Taasmari, P., M.Mungthin, R. Rangsin, B. Tongupprakarn, W. Areekul S. Leeleyoova.

(2000). Transmisssion of intestinal blastocystosis related to the quality of drinking water.

Southeast Asian Journal of Tropical Medicine and Public Health. 31: 112-117.

Tan, K.S.W., M. Singh, E. H. Yap. (2002). Recent advances in Blastocystis hominis research: hot spots in terra incognita. International Journal of Parasitology. 32: 789-804.

154

Tan, K.S.W. (2004). Blastocystis in humans and animals: new insights using modern technologies. Veterinary Parasitology. 126 :121-144.

Tan, K.S.W. (2008). New insights on Classification, Identification and Clinical relevance of

Blastocystis spp. Clinical Microbiology Review. 21(4): 639- 665.

Tan T.C and K.G Suresh, (2006). Predominance of amoeboid forms of Blastocystis hominis in isolates from symptomatic patients. Parasitology Research. 98: 189-193.

Tasova, Y., B. Sahin, S. Koltas, S.Paydas. (2000). Clinical significance and frequency of

Blastocystis hominis in Turkish patients with haematological malignancy. Acta Medica

Okayama. 54: 133-136

Taylor, D. R. Houston, D.R Shlim,M. Bhaibulaya, B.L.Ungar, P. Echeverria. (1998).

Etiology of diarrhea among travelers and foreign residents in Nepal. Journal of American

Medical Association. 260:1245-1248

Termmathurapoj, S S. Leeleyoova, P. Aimpun, U. Thathiasong, T. Nimmmanon, P.

Taasmari, M. Mungthin. (2004). The usefulness of short term in vitro cultivation for the detection and molecular study of Blastocystis hominis in stool specimens. Parasitology

Research. 93: 445-447.

Terra, L. Pellicano S, Milano M.(1998). Laboratory diagnosis of intestinal micro-parasitosis in AIDS patients: State of the art. Minerva Medicine. 89 (1-2): 23- 27.

Torres, P., J.C. Miranda, L. Flores, J. Riqueline, R. Franjola, J. Perez, S. Auad, C.

Hermosilla, S. Riquelme. (1992).Blastocystis and other intestinal protozoan infections in human riverside communities from Valdivia River Basin, Chile. Revista Institut Medicine

Tropica Sao Paulo. 34: 557-564.

155

Ukegbu, C.B. (1999). The prevalence of Cryptosporidium, Isospora and Microsporidium in

HIV and AIDS patients; comparative staining techniques for these parasites (MSc.

Dissertation. University of Lagos, Nigeria. 32-33

UNGASS. (2010). Report on the global AIDS pandemic. United Nations, New York

Vickerman, K. (1994). Playing at being Pasteur. International Journal of Parasitology. 24:

779-786.

Wawrzykniak, I., Roussel M, Diogon M, Couloux A, Texier C, Tan, K.S., Vivares, C.P,

Delbac F., Wincker P,El Alaoui, H. (2008). Complete circular DNA in the mitochondria- like organelles of Blastocystis hominis. International Journal of Parasitology. 12:1377-

1382.

Wilson, M.L. (1999). Emerging infections: The once and future diseases. American Journal of Clinical Pathology. 112:595-596

Wilson, S., Roberts L., Roalfe A, Bridge, P., Singh S. (2004). Prevalence of irritable bowel syndrome: a community survey. British Journal General Practitioner. 54: 495-502.

Windsor, J. J., L. Macfarlane, G. Hughes-Thapa, S.K.Jones, T.M. Whiteside (2002).

Incidence of Blastocystis hominis in faecal samples submitted for routine microbiological analysis. British Journal of Biomedical Sciences. 59: 154-157.

Windsor, J. J., and L. Macfarlane. (2005). Irritable Bowel Syndrome: The need to exclude

Dientamoeba fragilis. American Journal Tropical Medicine and Hygiene. 72: 501-502.

156

Windsor, J. J., A. I. Bamber, L. Macfarlane (2006). Detection of Dientamoeba fragilis and

Blastocystis hominis using simple staining method. British Journal Biomedical Sciences.

63: 27-28

Windsor, J. J. (2007). Blastocystis hominis and Dientamoeba fragilis: neglected human protozoa. British Journal of Biomedical Sciences. 64: 524-527.

WHO. (1998). The state of world health. Life in the 21st century. A vision for all. WHO,

Geneva, Switzerland. 57-58.

World Health Organization, WHO. (2006). Plan of work of the rolling revision of the WHO

Guidelines for Drinking–water Quality. WHO, Geneva.

Wolfe, M.S. (1982). The treatment of intestinal protozoan infection. Medical Clinics of

North America. 66:707-720.

Yaicharen, R., S. Sripochang, S. Bunguorn, P Pidetcha. (2005). Prevalence of Blastocystis hominis infection in asymptomatic individuals from Bangkok, Thailand South East Asian

Journal of Tropical Medical Public Health. 36: 17- 20.

Yakoob, J., W. Jafri, N. Jafri, R. Khan, Mohammed-Islam M, Beg A, Zaman V. (2004).

Irritable Bowel Syndrome, IBS: In search of aetiokogy; the role of Blastocystis hominis.

American Journal Tropical Medicine and Hygiene. 70: 383-385.

Yan, Y., S. Su, J. Ye, X. Lai, R. Lai, H. Liao, G. Chen, R. Zhang, Z. Hou,and X. Luo.

(2007). Blastocystis sp. subtype 5: possibly zoonotic genotype. Parasitology Research. 101

: 1527-1532.

157

Yoshikawa, H., Abe, N., Iwasawa, M., Kitano S, Nagano I., Wu Z., Takahashi Y. (2000).

Genomic analysis of Blastocystis hominis strains isolated from two long- term health care facilities. Journal of Clinical Microbiology. 38: 1324-1330.

Yoshikawa, H., Wu Z, Nagano I, Takahashi Y. (2003). Molecular comparative studies among Blastocystis isolates obtained from humans and animals. Journal of Parasitology.89:

585-594.

Yoshikawa, H., Wu Z, Kimata I, Iseki M, Ali IK, Hossain M. B, Zaman V, Haque R,

Takahashi Y. (2004a). Polymerase chain reaction-based genotype classification among human Blastocystis hominis populations isolated from different countries. Parasitology

Research. 92: 22-29.

Yoshikawa, H., Abe N, Wu Z. (2004b). PCR- based identification of Zoonotic isolates of

Blastocystis from animals and birds. Microbiology. 150: 1147-1151.

Yoshikawa, H., Yoshida K, Nakajima A, Yamanari K, Iwatani S, Kimata I. (2004c). Fecal- oral transmission of the cyst form of Blastocystis hominis in rats. Parasitology Research.

94: 391-396.

Yoshikawa, H, Wu Z, Howe J, Hashimoto T, Geok-Choo, N.G., Tan, K.S.W. (2007).

Ultrastructural and Phylogenetic studies on Blastocystis isolates from Cockroaches. Journal of Eukaryotic Microbiology. 54: 33-37.

Yoshikawa, H. Wu Z, Pandey K., Pandey, B.D.,Sherchand J.B, Yanagi T, Kanbara H

(2009). Molecular characterization of Blastocystis isolates from children and rhesus monkeys in Kathamandu, Nepal. Veterinary Parasitolology. 16 : 295-300.

158

Zali M.R, A.J.Mehr, M. Rezaian, A. R.Meamar, S. Vaziri, M. Mohraz. (2004). Prevalence of intestinal parasitic pathogens among HIV positive individuals in Iran. Japan Journal of

Infectious Disease. 57: 268-270.

Zaman V., J. Howe, M. Ng. (1995). Ultrastructure of Blastocystis hominis cysts.

Parasitology Research. 81:465-469.

Zaman V, (1996). The diagnosis of Blastocystis hominis cysts from human feces. Journal of Infection. 33 : 15.16.

Zaman V., J. Howe, and M. Ng. (1997) Observations on the surface coat of Blastocystis hominis. Parasitology Research. 83:731-733.

Zaman, V. (1998). The differential identification of Blastocystis hominis cysts. Annal of

Tropical Medicine and Parasitology. 92: 233-235.

Zaman V, Howe Y., Ng M.L. (1999). Scanning electron microscopy of Blastocystis hominis cysts. Parasitology Research. 84: 476-7.

Zierdt, C. H., W. A. S. Rude, B. S. Bull. (1967). Protozoan characteristics of B. hominis.

American Journal of Clinical Pathology. 48: 495- 501.

Zierdt, C.H. (1973). Studies of Blastocystis hominis. Journal of Protozoology. 20: 114-121

Zierdt C. H. and R. L. Williams. (1974). Blastocystis: Axenic cultivation. Experimental.

Parasitology. 36: 233-243

Zierdt C.H. (1978). Blastocystis hominis, an intestinal protozoan parasite of man. Public

Health Laboratory. 36: 233-243.

159

Zierdt, C. H., and L. Swan (1981). Generation time and growth rate of the human intestinal parasite Blastocystis hominis. 28:483-485.

Zierdt C.H. (1988). Blastocystis hominis- a long misunderstood intestinal parasite.

Parasitology Today. 4: 15-17.

Zierdt, C.H. (1991). Blastocystis hominis: Past and Future. Clinical Microbiology Review.

4: 65-71

Zierdt, C.H., W.S.Zierdt, B. Nagy. (1995). Enzyme linked Immunosorbent assay for the detection of serum antibody to Blastocystis hominis in symptomatic infections. Journal

Parasitology. 81:127- 129

Zuckerman, M. J., H. Ho, L. Hooper, B. Anderson, S. M. Polly. (1990). Frequency of

Recovery of Blastocystis hominis in clinical practice. Journal clinical Gastroenterology. 12:

525-532.

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APPENDICES

1.0 Routine Microscopic methods

The routine parasitological methods were found helpful in microscopic identification of

Strongyloides stercoralis, Hookworm larva and protozoa and of vacuolar forms of

Blastocystis species.

A. Direct wet smear preparations

Materials

Binocular Microscope and microscope glass slides

Glass cover slips

Applicator sticks or broom sticks

Markers for labeling

Lab. Reagents in bottles:

Isotonic or normal saline (0.9gNaCl/100ml d.w) and 1% Lugol‟s iodine solution

Procedure

On labeled microscope glass slides with the patient‟s identification number was placed a drop of normal saline on the center of half of the slide and a drop of Lugol‟s iodine on the other center half on the slide. Using an applicator stick, a small portion of the stool sample is emulsified in the saline and iodine drops to make wet smear preparations. The smears are thereafter covered with cover slips. Slide with wet smears was mounted on the microscope stage and each field examined until the entire smear is covered.

Use was made of first, 0.25 or (X10) low power objective lens for scanning, while the high power Objective lens 0.65 or (X40) to observe diagnostic features in the parasites.

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B. Formalin Ethyl-acetate Concentration Technique, FECT:

Materials:

10% Formalin

Ethyl –acetate solution

10ml Bucket centrifuge tubes

Surgical guaze

Applicator sticks

Microscope slides and glass cover slips

Funnel and Pipettes with rubber bulbs

Procedure

Stool fixed in 10% formal saline was concentrated by the formal-saline ethyl -acetate concentration techniques or sedimentation method which involved shaking approximately

2g of each faecal specimen in 8.5ml of formalin fixative, with 1.5ml ethyl-acaetate solution before centrifuging at 2000 rpm for 5minutes. The debris plugged coat was loosened and the supernatant discarded by quikly inverting the tubes. Tubes were placed upright and the remnants of the supernatant fluid were allowed to drain down to the sediment. The sediment and fluid were reconstituted by gentle tapping of the tubes.

Faecal deposits obtained were smeared on clean glass slides and examined with the X10 and

X40 Objective lens. Thin faecal smears were also made with the concentrates, air dried and permanent slide preparations made by using the Trichrome staining techniques and Kinyoun modified carbolfuchsin acid-fast (Ziehl-Neelsen) procedures respectively, to distinguish cysts of Blastocystis species and oocyst of coccidian parasites respectively (Chesborough,

2001, Garcia and Bruckner, 2007).

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C The Trichrome staining technique

Materials:

Schaundin‟s fixative

Iodine-alcohol solution

Grades of ethanol solution: 70%, 80% 90%, 95%

Trichrome stain solution

Carbol-xylene solution

Acetic acid- alcohol destaining solution

Xylene

Canada balsam mounting solution

Procedure:

The air dried thin faecal smears were made onto labeled clean glass slides from each of the stool samples and placed into Schaundin‟s solution for 1 hour. Thereafter, slides were picked with foceps and excess fluid drained with paper towel. Slides were transferred to the iodine- alcohol solution for one minute, removed and drained of fluid. Slides were replaced in 70% ethanol solution for one minute, removed, drained of fluid and the process repeated in another change of 70% ethanol; drained and placed in trichrome staining solution for

8minutes., de-stained in an acetic acid- alcohol solution by dipping twice for 5seconds, drained and placed once into a 90% ethanol. Thereafter, two changes of 95% ethanol solution were made for 3seconds each and the slide drained of fluid. The drained slides were dipped into Carbol-Xylene solution for 1 minute, drained and placed into Xylene solution for 3 minutes and drained. Stained slides were examined microscopically by adding mounting medium unto the stained smear and viewed using theX10 and X40 lens.

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D Kinyoun’s modified, carbol-fucsin acid-fast staining technique

Materials:

Carbol fuchsin solution

1% hydrochloric acid in ethanol

Methanol solution (Absolute)

0.25% Glycerol-Malachite Green solution

Water

Procedure

Similarly the air dried thin faecal smears were fixed in absolute methanol for 3 minutes and thereafter stained with Carbol fuchsin for 5minutes. The stained slides were then decolourised in 1% hydrochloric acid- ethanol solution until the colour ceased to flow out.

Slides were rinsed in tap water and counterstained with 0.25% Malachite Green solution for

30seconds and rinsed in tap water before drying.

The slide preparation was examined using the high dry objective and oil immersion lens

(x100).

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