1. Introduction and Literature Review

1. INTRODUCTION AND LITERATURE REVIEW

1.1 Introduction Parasites are the main causative agent of disease in man, where most of the human population are exposed to the risk and vast economic losses due to parasitic infection.(Ambrosio 1990) Intestinal protozoan infections are among the infections causing diarrhea and abdominal symptoms, including cramps, pain, bloating, or tenderness in humans. They have a cosmopolitan existence and cause a signiﬁcant morbidity and mortality (Rossignol 2001) . Before invasion, E. histolytica may inhabit the human intestine for a long time, extending two years, as asymptomatic infection (Blessmann 2002) . There are 75% of the world's population lives in developing countries where the amoebiasis spreads, and 70% of the world's livestock resources are in the developing countries according to the Food and Agriculture Organisation (FAO) which the situation is very critical (Ambrosio 1990). Entamoeba histolytica is an enteric protozoan parasite that infest half a billion people worldwide. Ten percent of infected individuals develop intestinal amoebiasis and extraintestinal amoebiasis, leading to about 70,000 deaths each year, making it the second disease to malaria as a cause of mortality due to a protozoan parasites .(Pillai and Kain 1999) The remainder being asymptomatic cyst passers. The numerical difference between the incidence of infection and expression of morbidity is based on the existence of pathogenic and nonpathogenic strains of E. histolytica. (Egbert Tannich and Gerd 1991; Dawn Britten 1997) E. histolytica is a pathogen or invasive parasite, whereas E. dispar and E. moshkovskii are nonpathogenic and noninvasive although they are morphologically identical to E. histolytica. There are at least eight amebas (E. histolytica, E. dispar, E. moshkovskii, E. coli, E. hartmanni, E. polecki, Iodamoeba bütschlii, and Endolimax nana) which inhabit the human intestinal lumen. However, these are generally accepted as commensal organisms except for E. histolytica. E. polecki, Dientamoeba fragilis, and I. bütschlii which have occasionally been implicated as causes of diarrheal illness in humans.(Tanyuksel and Petri 2003)

1.1.1 Historical Background : Amebiasis was earlier recognized as a deadly disease by Hippocrates (460 to 377 B.C.), who described a patient with fever and dysentery.(Tanyuksel and Petri 2003) The Milestones in the study of E. histolytica and amebiasis were its description by Fedor Lösch in 1873 when a case of a young farmer had been admitted to his clinic in St Petersburg, Russia for diagnosis. The patient was suffering from chronic dysentery and Lösch found very large numbers of amoebae in his faeces and he became convinced that those amoebae were responsible for his illness. The young farmer was the first person known to have died from amoebiasis. In 1875, Fedor Losch isolated E. histolytica from a stool specimen of a patient with dysentery. Afterwards Schaudinn renamed the organism Entamoeba histolytica.(Ackers 2002; Tanyuksel and Petri 2003) Leonard Rogers has chosen emetine as the first effective treatment for amebiasis in 1912. In the next year (1913), Walker and Sellards demonstrated the infective cyst form of E. histolytica. (Tanyuksel and Petri 2003) It was 1925 when Brumpt proposed that two morphologically identical species of Entamoeba produced tetra nucleated cysts measuring 10 mm or more in diameter, could infect humans. One of the species caused disease and the other did not, and he named the nonpathogenic species Entamoeba dispar. In 1961 Diamond performed axenic cultivation of E. histolytica which was considered as a major turning point in understanding the cell biology and biochemistry of E. histolytica.(Tanyuksel and Petri 2003) These studies were not sufficient to distinguish the two morphologically identical parasites until Sargeaunt and his colleagues demonstrated in 1978, that isoenzyme typing could be able to distinguish the two species of Entamoeba which have now been conventionally named Entamoeba histolytica (which is pathogenic, causes invasive amebiasis, and is still frequently referred to as the pathogenic strain of E. histolytica) and E. dispar (which is probably truly nonpathogenic and which is still found to be described as the nonpathogenic strain of E. histolytica). (Louis S. Diamond 1993; Rashidul Haque 1995; Dawn Britten 1997; Yury O. Nu´N˜Ez 2001)

1.1.2 Classification of Entamoeba Parasites: Entamoeba parasite was classified according to Garcia (1993) (Garcia 1993; Tanyuksel and Petri 2003) as follows:

Kingdom: Protozoa, ( single celled eukaryotic organism). Phylum: Sarcomastigophora, (Locomotion by flagella or pseudopodia or both). Subphylum: Sarcodina, (Locomotion by pseudopodia, temporary flagellated in some forms). Class: Lobosea, ( pseudopodia lobopodia) Order: Amoebida, ( No flagellated stage in life cycle) Family: Entamoebidae, Genus and Species:  Entamoeba histolytica  Entamoeba dispar  Entamoeba coli  Entamoeba polecki  Endolimax nana  Entamoeba moshkovski  Entamoeba hartmanni  Dientamoeba fragilis  Entamoeba gingivalis  Iodamoeba bütschlii  Blastocystis hominis The nomenclature Entamoeba histolytica is being used to indicate true pathogenic strain, while E. dispar is now being used to indicate nonpathogenic strain but actually the two organisms cannot be differentiated on the basis of morphological features seen in permanent stained faecal smears.(Garcia 1993)

1.1.3 Life Cycle, Morphology, and Biology of Entamoeba: The life cycle of Entamoeba histolytica is monogenic and humans act as its host. The infected individuals are the main source of transmission through contamination of fresh food or water and infection may be spread by arthropods such as cockroaches and ﬂies. It is thought that there is some sort of zoonotic transmission, but this is not clear. Experimental infections with E. histolytica have been produced in some animals such as dogs, cats, rats, monkeys, and some other laboratory animals.(Tanyuksel and Petri 2003)

There may be some animal reservoirs of E. histolytica, but they represent a very small source of human infection compared with humans themselves. However, there are no reports of occasional zoonotic transmission of cases between infected animals and humans, although E. histolytica is most commonly associated with animals such as cats, dogs and primates. (Tanyuksel and Petri 2003) The life cycle of E. histolytica is simple. It has two stages, an infective cyst stage and a vegetative multiplying trophozoite stage. Humans acquire infection by ingesting the infective stages, which pass through the lumen to the small intestine , where the process of excystation occurs giving eight daughter trophozoites from each cyst. The trophozoites are motile forms, which adhere to and invade intestinal mucosa. Trophozoites move by extending creeping projections of cytoplasm, the pseudopodia, which pull the parasite along resulting in amoeboid movement (Tanyuksel and Petri 2003). They also use these pseudopodia to surround and engulf food particles. The cytoplasm frequently contains many red blood cells (RBCs) that have been ingested. The trophozoites of E. histolytica always have a single nucleus. Trophozoites are easily destroyed in the outside environment, degenerating within minutes. The trophozoite of E. histolytica can convert to a precyst form with a nucleus (E. coli precysts have two nuclei), and this form matures into a tetranucleated cyst as it migrates down and out of the colon. The precyst contains aggregates of ribosomes, called chromatoid bodies, as well as food vacuoles that are extruded as the cell shrinks to become a mature cyst. It is the mature cyst that, when consumed in contaminated food or water, is infectious. In the process of becoming tetranucleated, the nucleus of the cyst divides twice. Chromatoid bodies and glycogen vacuoles cannot be seen at this stage.(Tanyuksel and Petri 2003) Cysts can remain viable in the external environment outside the host for weeks or months, especially under wet conditions, but are quickly destroyed at temperatures under 5°C and over 40°C. Cysts are not invasive, but trophozoites can invade the gastrointestinal mucosa and are able to migrate via blood to other organs, causing extraintestinal infections.(Tanyuksel and Petri 2003) Like other protozoa, E. histolytica appears unable of a fresh (de novo) purine synthesis. Biochemical analysis has indicated that glutathione is not present. For this reason, E. histolytica is different from higher eukaryotes. It also uses pyrophosphate instead of ATP. The cytoplasm of the cyst is vacuolated with numerous glycogen

4 deposits, visible by permanent stains such as ironhematoxylin, that decrease in size and number as the cyst matures. (Tanyuksel and Petri 2003) The gene organization of E. histolytica seems quite distinct from that of other eukaryotes. Although the structure of E. histolytica chromosomes is not yet known completely, electrokaryotypic analysis suggests that the chromosomes range in size from 0.3 to 2.2 Mb and give a total haploid genome size of approximately 20 Mb. A complete sequence map of the ribosomal DNA (rDNA) episome has been successfully completed, Sehgal et al (1994). and Bhattacharya et al (1998), found that E. histolytica circular DNA is 24.5 kb. This sequence has proved quite useful for the genotyping of the different enteric amoebae.(Tanyuksel and Petri 2003)

1.2 Review of Literature:

1.2.1 Redescription of E. histolytica and E. dispar: Entamoeba histolytica and Entamoeba dispar were first differentiated by characteristic isoenzyme patterns and recently, differences on the genomic level have also been described.(Katzwinkel-Wladarsch 1994) The pioneering work of Sargeaunt and his colleagues, concluded that the two strains of Entamoeba were morphologically identical but can be differentiated by isoenzyme analysis, typing with monoclonal antibodies (MAbs) to surface antigens, and restriction fragment length polymorphisms (RFLP). Entamoeba histolytica redescribed as the pathogenic species E. histolytica (formerly called the pathogenic zymodemes of E. histolytica) and the nonpathogenic species E. dispar (formerly called the nonpathogenic zymodemes of E. histolytica).(Rashidul Haque 1995) Clinically, E. histolytica is a cause of colitis and liver abscess but E. dispar is not. No cases have been documented where intestinal disease and colitis were caused by E. dispar. It cannot be forgotten that E. moshkovskii can colonize humans and is also identical in appearance to E. histolytica / E. dispar. Differentiation of E. histolytica and E. dispar in stool samples is not easy on the basis of microscopy alone. Diagnosis of most of the previous infections as E. histolytica infections based on microscopic examination only can be regarded as imperfect and misleading. In reality, many of these organisms were probably genetically distinct from E. dispar. Currently, there are many molecular tools available to allow the differentiation of E. histolytica from E. dispar, such as amoebic antigen and DNA detection, enzyme immunoassay (EIA) and PCR. Reclassiﬁcation of E. histolytica and E. dispar is of great importance because it allows the clinician to focus on early identiﬁcation and treatment of E. histolytica infection in the minority of patients who are at highest personal risk and pose a major public health problem.(Tanyuksel and Petri 2003)

1.2.2 Clinical Features of Amoebiasis: Symptoms of intestinal amoebiasis are varied. Clinical intestinal disease varies from acute dysentery with bloody mucoid stools, to mild abdominal discomfort with diarrhea containing blood or mucus, interchanging with periods of constipation or diminution of signs. Other symptoms include chronic abdominal pain due amoebic ulcers in the wall of the large intestine. Extra-intestinal disease is

6 disseminated via the blood stream producing abscesses of the liver or, less commonly, of the lung or brain. Intestinal amoebiasis is of significant epidemiological concern, as it is transmitted by the fecal-oral route and able to cause severe illness and possibly death. E. histolytica is characterized by its abnormal ability to penetrate and destroy human tissues. A number of molecules considered important for the tissue- damaging activity, including the galactose-inhibitable surface lectin, pore-forming peptides (amoebapores), and cysteine proteinases , have been identified.(Willhoeft 1999) The symptoms and signs are absent or very mild in up to 90% of E. histolytica infections.(Tanyuksel and Petri 2003) This low incidence of invasive amoebiasis (10%) in humans has occupied the attention of generations of clinical and nonclinical investigators, raising a question how to give an explanation that only a few of those humans infected with the parasite develop invasive disease? Modern studies estimate that less than 10% (36 million) of those infected with E. histolytica (480 million) develop clinical symptoms, of which at least 40,000 deaths occur annually.(Louis S. Diamond 1993; Clark 2001) Three major hypotheses have been claimed to explain this phenomenon as follows:  E. histolytica is a single pathogenic species which in all human hosts produces intestinal lesions that may or may not give rise to recognizable clinical symptoms.  E. histolytica is normally a commensal residing in the human colon which on occasion, for reasons poorly understood, converts into an invasive pathogen;  E. histolytica is comprised of two morphologically identical species, one an invasive pathogen exhibiting varying degrees of virulence and the other a noninvasive pathogen having the capacity of producing at most superficial erosion of the colonic mucosa.(Louis S. Diamond 1993) Recent studies have proved that the third hypothesis is the correct one, that infection in humans can occur by two morphologically identical species of Entamoeba producing tetradnucleate cysts measuring 10 mm or greater in diameter was proposed first by Brumpt in 1925. The invasive pathogen was identified as Entamoeba dysenteriae (Councilman and Lafleur Craig, 1905), which according to Dobell is a synonym for Entamoeba histolytica Schaudinn, 1903 (Emend. Walker,

1911). For the noninvasive amoeba, Brumpt chose the name Entamoeba dispar.(Louis S. Diamond 1993) Because Brumpt was unable to differentiate between the two parasites morphologically, and because there was a rising evidence obtained from experimental human and animal studies that amoebae obtained from asymptomatic carriers could produce disease, his explanation earned little support. It gained favor when Sargeaunt and colleagues reported distinguishing two groups of E. histolytica on the basis of isoenzyme typing one group isolated from asymptomatic individuals, the other from symptomatic patients.(Louis S. Diamond 1993; Yury O. Nu´N˜Ez 2001) Entamoeba histolytica is a pathogenic species displaying different degrees of virulence and able to invade a wide variety of tissues in the host including those of the colon and liver, and more rarely lung, skin, urogenital tract, brain, and spleen. This invasive feature separates it from E. dispar which, though it may be a pathogen producing erosion of the colonic mucosa, has not been identified with tissue invasion.(Louis S. Diamond 1993) Amoebiasis is an infection of the large intestine caused by the parasitic protozoon Entamoeba histolytica. Generally, a nonpathogenic form is differentiated from a pathogenic on the basis invasion.(Katzwinkel-Wladarsch 1994) However, it looks that many E. histolytica infections never progress to become invasive and bring out signs and symptoms and are spontaneously lost. This argument raises some important questions. Are the organisms that produce invasive, symptomatic disease genetically distinct from those that give rise to asymptomatic infections? Or do all E. histolytica isolates have the potential to become invasive? Do certain invasive isolates show response for specific organs, with some preferentially ending up in the intestinal wall while others reach extraintestinal areas?.(Clark 2001) It should be stressed that E. dispar and „nonpathogenic (NP) E. histolytica’ are synonymous. However, applying the term nonpathogenic to E. dispar may be inaccurate. The parasite appears capable of inducing focal intestinal lesions in experimental animals such as kittens, gerbils and guinea pigs, and thus would be, by definition, a pathogen. The possibility that E. dispar can also bring about pathological changes in the human colon, although not necessarily invasive lesions, cannot be excluded and have a right to further study.(Louis S. Diamond 1993) Cysts of E. histolytica have been found in the stool of asymptomatic cyst passers free of E. dispar and in patients with amebic liver abscess but patients are

8 free of intestinal symptoms. Therefore, asymptomatic cyst passers must not be assumed to carry E. dispar. Likewise, a positive serological test need not indicate E. histolytica infection since up to 20% of E. dispar infections may lead to seropositivity.(Sinchez-GuillCn 1990) The existence of two species within what was previously called E. histolytica has deep importance for interpretation of epidemiological data and the older literature, for clinical evaluation of carriers and for estimating the proportion of symptomatic infections. The numerous methods now available for distinguishing E. histolytica from E. dispar should allow more accurate diagnosis and data collection.(Louis S. Diamond 1993)

1.2.3 Asymptomatic Colonization: Clinical illness includes a wide spectrum of disease. However, asymptomatic shedders” are the most important group from a public health point of view. At the beginning of the 20th century it was very clear that many persons shedding cysts of E. histolytica were asymptomatic and in 1925 Emile Brumpt suggested that there were two undifferentiated strains of amoeba. However, because Walker and Sellards in 1913 had already demonstrated that cysts isolated from asymptomatic cyst passers, on occasion, cause disease when taken orally by volunteers, and because there was no way of distinguishing between the cysts of the pathogenic and non-pathogenic species, Brumpt‟s suggestion was largely ignored indeed, in 1969 WHO defined amoebiasis as “infection with Entamoeba histolytica, with or without clinical manifestations”, implying that all strains were potentially pathogenic.(Ackers 2002) Asymptomatic infections with E. dispar has no evidence of disease or a serum anti-amebic antibody response, while symptomatic E. histolytica intestinal infection does show a systemic immune response, this point created controversial debates for many years on whether the outcome of infection was due to differences in host or parasite, but pioneering observation on lectin-mediated agglutination has become more and more apparent that there are essential differences between the organisms recovered from symptomatic patients and asymptomatic cyst passers. Subsequently Sargeaunt and Williams demonstrated consistent differences between pathogenic and non-pathogenic isolates. (Ackers 2002; Tanyuksel and Petri 2003)

1.2.4 Extraintestinal Amebiasis: The clinical disease results from the capability of E. histolytica to invade the mucosa of the large bowel and to spread extraintestinally, this penetration of the gut wall may lead to ulceration and produce the classical signs and symptoms of amoebic dysentery. (Ackers 2002) Extraintestinal spread through blood stream involves the liver, causing hepatic amoebiasis or amoebic liver abscess and may spread to other distant organs such as the brain. (Ackers 2002) Liver abscess is the most common picture of extraintestinal amebiasis. The signs and symptoms of Amebic liver abscess (ALA) are fever and abdominal pain in most patients. In the acute phase ALA is associated with right upper abdominal pain or tenderness, while weight loss, fever, and more diffuse abdominal pain occur in the subacute phase. ALA occurs more commonly in adults than in children. E. histolytica has been identified microscopically in the stool samples of only a minority of patients. Clinical pathology parameters in many patients shows elevated peripheral white blood cell counts and alkaline phosphate levels. Unusual sites or complications of extraintestinal amebiasis include direct extension from the liver to the pleura and/or pericardium, brain abscess, and genitourinary amebiasis. (Tanyuksel and Petri 2003) The detection of liver abscess due to E. histolytica may be difficult because of the lack of a history of intestinal disease within 1 year in many patients, in addition to the lower sensitivity of serologic analysis and the inability to distinguish amebic abscess from pyogenic abscesses by imaging investigation. The definitive diagnosis of ALA is conﬁrmed by positive serological tests for antibodies to E. histolytica and demonstration of the hepatic lesion by imaging techniques such as computed tomography ultrasonography, magnetic resonance imaging, and technectium-99 liver scan. (Tanyuksel and Petri 2003)

1.2.5 Epidemiology: Amebiasis is responsible for approximately 100,000 deaths per year, as well as for considerable morbidity manifested as invasive intestinal or extraintestinal clinical features mainly in the tropics and subtropics. (Troll 1997) Worldwide, amebiasis is the third most common cause of death due to parasitic infection after malaria and schistosomiasis, as estimated by the World Health Organization

(WHO). The prevalence of amebiasis varies with the population of individuals affected, differing between countries and between areas with different socioeconomic conditions. In some tropical countries, antibody prevalence rates (reﬂecting past or recent infection) exceed 50%.(Tanyuksel and Petri 2003) Sometimes up to 50% of the population is affected in areas with poor sanitations, causing loss of manpower and subsequent economic damage. The overall prevalence of E. histolytica infection in industrialized countries such as the United States has been estimated to be 4% per year in spite of the presence of some high-risk groups.(Lowther 2000; Tanyuksel and Petri 2003) Epidemiological studies have shown that low socioeconomic status and poor sanitation conditions are signiﬁcant independent risk factors for infection. In addition, people living in developing countries have a higher risk and earlier age of infection than do those in developed regions . For example, in Mexico, 11% of the tested population aged 5 to 9 years were infected, with the prevalence of infection being higher in girls (9.34%).(Tanyuksel and Petri 2003) Serosurveys suggest that long term travelers residing in the developing regions where infection is endemic are at relatively increased risk of E. histolytica infection . (Tanyuksel and Petri 2003) Entamoeba histolytica causes amebic colitis and liver abscess in developing countries such as Mexico, India, and Bangladesh (Ghosh 2000) and the infection has been described in travelers to endemic areas such as parts of South America, Central America, Africa, and Asia.(Lowther 2000) The epidemiological implications of this disease situation are still to be verified, and studies to fill this gap will depend on the development of methods for differentiating between E. histolytica and E. dispar in large numbers of samples.(Dawn Britten 1997) The estimation of the load of infection with E. histolytica/E. dispar is not only important for the endemic tropical and subtropical regions, but also for the developed areas, in the light of the continuous increase in immigration and the number of travelers to developing countries. To determine the true prevalence of amebiasis in a hyperendemic area, such as the western part of South America, an epidemiologic survey using different copro methods for the diagnosis of infection by the E. histolytica/E. dispar complex was assessed in a rural area of northern Ecuador.

The results of the two antigen detection assays and three serologic methods (indirect haemagglutination, enzyme immunoassay, and indirect immunofluorescence) were compared with those of isoenzyme characterization of amebic isolates according to Sargeaunt and Williams gold standard technique. Gatti and his group concluded,(Simonetta Gatti 2002) that diagnostic methods more specific and sensitive than direct microscopy are needed to establish the true distribution of E. histolytica and E. dispar, and to determine the prevalence of asymptomatic carriers about which there is little current information and the biochemical identification of the different zymodemes of Entamoeba in any population leads to a better understanding of its epidemiologic status.(Simonetta Gatti 2002) Table 1 provides a summary of the prevalence studies reporting E. histolytica and/or E. histolytica / E. dispar infections in HIV positive individuals in different countries. The association of E. histolytica infections with HIV positive individuals in some studies is not clear. In a Mexican study no clear association between E. histolytica and HIV has been noted that the prevalence of E. histolytica in AIDS+ /HIV patients was 25.3% compared to 18.4% in HIV-group. Other studies in South American countries have shown no obvious association. However, a significant association between high levels of serum anti-E. histolytica antibodies and the presence of E. histolytica in the stool has been noted in studies from both Vietnam and Africa. In a South African study in the Vhembe district in the northern part of the country, a positive association between E. histolytica infection and HIV-positive individuals has been indicated. Among the HIV-positive individuals, those with CD4+ count less than 200 cells/µl, were relatively more likely to be seropositive for E. histolytica (AbuOdeh 2012). In a Chinese study, a higher seroprevalence of E. histolytica infections was also found in HIV-infected patients (Chen 2007). Furthermore, two studies conducted in Taiwan revealed a positive association as well (Windell L. R., Hiroshi T et al. 1999; Windell L. RIVERA 1999; Hung 2005; Tsai 2006) .

Table 1.1: Global prevalence of E. histolytica in HIV-infected and non-infected persons(AbuOdeh 2012). Prevalence of Entamoeba Country Reference Species Cuba 1.5% (E. histolytica/dispar) Escobedo, A. A. 1999 Bogota, Colombia 13% (E. histolytica) Florez et al., 2003 San Pedro Sula, Honduras 5.8%(E. histolytica) Lindo et al., 1998 Venezuela (Zulia state) 10.8%(E. histolytica) Rivero et al., 2009 Brazil 3.3% and 1% (E. histolytica/ Bachur et al., 2008 dispar before and after HAART) Mexico 25.3% in HIV+ and 18.5% in Moran et al., 2005 HIV-contacts (E. histolytica) Tajikistan 25.9% (E. histolytica/dispar non Matthys et al., 2011 HIV) Northern India 7.7% (E. histolytica) Prasad et al., 2000 Taiwan 5.8% (E. histolytica in HIV Hung et al., 2008 patients) Bangladesh 2.1% vs. 1.4% in diarrhea and Haque et al., 2009 control (E. histolytica) India (Kolkata) 3.6% (E. histolytica) Mukherjee et al., 2010 Sydney, Australia 3.2% (E. histolytica/E. dispar) Stark et al., 2007 Mazandaran province, Iran 1.6% (E. histolytica) Daryani et al., 2009 Uganda 1.4% (E. histolytica) Brink et al., 2002 Ethiopia Hailemariam et al., 10.3% (E. histolytica) 2004 Dakar, Senegal 5.1% (E. histolytica) Gassama et al., 2001 South Africa 12.4% (E. histolytica) Samie et al., 2006

Accurate differentiation of E. histolytica from its identical triplets E. dispar and E. moshkovskii is crucial to the control the disease. Current data shows that E. dispar is prevalent 10 times more common than E. histolytica worldwide, but the locally may vary signiﬁcantly in its prevalence, make the assessment of prevalence in different geographical regions is necessary.(R. Fotedar 2007) The epidemiology of Entamoeba in the Sudan is poorly understood because the methods that differentiate between pathogenic E. histolytica and non pathogenic E. dispar are not available in routine diagnostics.(Amir Saeed 2011) M. A Babiker et. al. (2009) in their studies on Food-handlers (n = 1500) attending the public health laboratory in Khartoum, Sudan, were screened for intestinal parasites by three

13 different techniques to evaluate the adequacy of annual screening showed that 29.4% of food-handlers were harboring intestinal protozoa in stool samples among which Entamoeba histolytica was 4.3%.(M.A. Babiker 2009) A study conducted on under five Sudanese children gave a prevalence rate of 44%. The most common infestations were Giardiasis (21.1%), Taeniasis (10.4%) and Enterobiasis (7.4%). Non pathogenic E. coli, E. histolytica and Taenia saginata were detected in 2.7%, 0.7% and 1.7% of stools specimen respectively.(Karrar ZA 1995) Regarding the development in amebiasis research, there is a great necessity to evaluate old reported prevalence data of E. histolytica infections in many parts of the world. Surveys that determine the prevalence of infection by copro examination of parasites estimate mostly E. dispar, since this species is more common,(Windell L. RIVERA 1999) while serologic surveys reflect the occurrence of E. histolytica infection because E. dispar does not bring out a positive serologic response in humans.(Windell L. RIVERA 1999) Also, seroepidemiological studies usually reflect seropositivity of samples even years after events of amebiasis infection.(Windell L. RIVERA 1999) These factors therefore, raise a problem on the preciseness of earlier time informed epidemiologic reports. Furthermore, the frequently quoted global prevalence of E. histolytica (500 million) is very deceptive. It is more likely that E. histolytica is responsible for only 10% of these infections (50 million) globally, while E. dispar accounts for the rest. Indeed, the recommendation of the WHO-Pan American Health Organization United Nations Educational, Scientific, and Cultural Organization to develop improved methods for the specific diagnosis of E. histolytica infection is very crucial for the establishment of accurate prevalence data of E. histolytica and E. dispar infections. These recommendations influence all aspects of amebiasis research, most notably epidemiology.(Windell L. RIVERA 1999; Mehreen Zaki 2002) The epidemiology of Entamoeba can be further studied by cultivation procedures and determination of isoenzyme patterns by gel electrophoresis. However, these techniques are laborious, expensive, and time-consuming, and are not practical for routine diagnostic laboratories.(Yury O. Nu´N˜Ez 2001) Detection of antibodies to amoeba in patients sera has been reported to indicate infection by E. histolytica. However, with serological testing, it may be difficult to differentiate between past and present infections in individuals who migrate from, or currently live in endemic areas.(Yury O. Nu´N˜Ez 2001)

1.2.6 Laboratory Diagnosis: Laboratory diagnostic tools available for diagnosis of amoebiasis include microscopy, serological tests and more advanced probes molecular techniques. The manual nature of routine parasitological diagnostic procedures took a lot of experienced technologists time to carry out and interpret the results. Meanwhile decreasing staff and cross-training individuals between laboratory disciplines in many laboratories lead to a decline in the skills of parasitology diagnostic procedures. This situation leads to a need to develop and improve new diagnostic testing methods for the presence of the most common pathogenic parasites.(Sharp 2001) Laboratory investigation is the primary step in the diagnosis of parasitic infections. However, failure to detect a parasite does not exclude the possibility of the infection. The demonstration of many of these parasites can be done by microscopic examination and requires considerable skill and practice.(Ambrosio 1990) The laboratory detection of E. histolytica in human feces has relied upon the microscopic examination of fresh or preserved stool samples.(Blessmann 2002; Trudel 2003) The diagnosis of E. histolytica infection has traditionally depended on microscopic examination of stool specimens. However, microscopy procedure has several limitations such as the inability to distinguish E. histolytica from E. dispar, because they are morphologically indistinguishable (Pillai 1999). So technically, however, differentiation is still a challenge. (Trudel 2003) A clinical diagnosis of amoebiasis can be confirmed by microscopic identification of characteristic cysts or trophozoites in the stool, in addition to the fact that multiple samples often have to be examined and the presence of cysts of different species such as Entamoeba, Iodamoeba, or Endolimax can make diagnosis difficult.(Yury O. NuŃ˜Ez 2001) According (Ackers 2002) the following biological characteristics are important to differentiate between E. histolytica and E. dispar  Isoenzyme patterns, particularly hexokinase.  Specific epitopes, recognized by reaction with several monoclonal antibodies.  Sequence differences in the rDNA episome.

 Significant sequence differences between homologous genes.  A small number of genes, like ariel and cp5 appear so far to be unique to E. histolytica.  It has been proved much easier to adapt E. histolytica to axenic growth while culture of E. dispar was proved extremely difficult and has so far been achieved in axenic medium for only one strain.  Scanning electron microscopic examination of axenic cultures of both species show significant differences particularly in the appearance of the surface. This may be linked to the clear lack of surface lipophosphoglycan (LPG) in E. dispar.

Recently, WHO (1997) convened to consider the implications of the separation of the two species. The definition of amoebiasis as “infection with Entamoeba histolytica, with or without clinical manifestations” was rectified but the name Entamoeba histolytica which is now to be used only for the pathogenic species, clearly separating away from E. dispar. The conclusions of the meeting included the following: 1. The criteria (such as size) used to form the classical taxonomic description of E. histolytica cannot distinguish between E. histolytica and E. dispar and, of particular importance in diagnostic microscopy, cysts of the two species are identical. 2. When diagnosis is made by light microscopy, since the cysts of the two species are indistinguishable, they should be reported as E. histolytica/E. dispar. 3. In asymptomatic individuals treatment is not appropriate when E. histolytica/E. dispar has been specifically detected. 4. Optimally, E. histolytica should be specifically identified and symptoms, if present, treated. This means, in practice, it would be ideal if every infected patient had a specific diagnosis of either E. histolytica or E. dispar made, the diagnostic procedure mainly employed in endemic areas (light microscopy) is unable to do this.(Ackers 2002)

A number of tests have been developed during recent years, such as protein and DNA detection systems, which are able to distinguish E. histolytica from E. dispar. However, most of these assays are not suitable for a rapid diagnosis directly from stool samples, in particular when large numbers of samples have to be processed such as screening and epidemiological studies.(Blessmann 2002) currently some reagents are available for identifying the E. histolytica/E. dispar group and for differentiating E. histolytica from E. dispar.(Garcia 1993) A critical question should be raised as for which improvement could be made in the performance of conventional or traditional diagnostic methods. For several years, researchers have been searching for methods that will allow an accurate and reliable assessment of amebiasis. Laboratory diagnosis of amebiasis is usually based on microscopy and serological methods including enzyme-linked immunosorbent assay (ELISA), indirect haemagglutination assay (IHA), and latex agglutination. During the last decade, there has been remarkable development in molecular biology-based diagnostic procedures to detect E. histolytica, to the point where today they are the preferred approach. Accurate diagnosis is important not just for patients with dysentery but also for the 90% of Entamoeba complex infections that are asymptomatic, because infection may easily be transmitted from person to person, especially in developing countries which have poor hygienic status .(Tanyuksel and Petri 2003)

1.2.7 Microscopy: The ability to perform the complete ova and parasite (O&P) examination should remain an option for those symptomatic patients with negative parasite results.(Garcia 2000) Diagnosis of E. histolytica has historically relied on microscopic examination of protozoan morphology. Current microscopy and histology-based identiﬁcation frameworks, however, are unable to differentiate between protozoa with similar morphological features. A separate problem is that the sensitivity and specificity of conventional microscopy on a single stool specimen for different species of Entamoeba have been shown in many studies to be less than optimal . A “poor man‟s” way to distinguish E. dispar from E. histolytica microscopically is erythrophagocytosis. Ingested RBCs in the cytoplasm may be visible; this finding is still considered diagnostic for E. histolytica in patients with dysentery. It may be

17 used to distinguish between E. histolytica and E. dispar. Mostly, E. histolytica used be diagnosed on the basis of protozoon morphology without the presence of RBCs . In fact, classical microscopy does not allow the invasive protozoon (E. histolytica) to be distinguished from the noninvasive one (E. dispar) unless erythrophagocytosis (the presence of ingested RBCs in trophozoites) is seen during microscopic examination. This classical feature has long been considered the definitive diagnostic criterion for E. histolytica. Also, it must be kept in mind that RBCs may be ingested but do not frequently appear in chronic amebic infections. In an in vitro study, E. histolytica was found to have a significantly higher phagocytic rate than do the nonpathogenic Entamoeba species (E. invadens and E. moshkovskii) which may be due to its virulence nature. Gonza´lez Ruiz et al. reported that the presence of E. histolytica organisms containing ingested RBCs is a diagnostic indication of active invasive amebiasis. However in some cases E. dispar is also observed to contain RBCs . Trophozoites are more frequently observed in fresh stool specimens that contain mucus, pus, and trace amounts of blood. In wet mounts, the trophozoite nuclei cannot easily be seen. Charcot-Leyden crystals (products of degenerated eosinophils) and clumped RBCs can be seen in a wet mount preparation . Definitive diagnosis of intestinal amebiasis requires high levels of skill and experience ; inadequate training and diagnostic testing may lead to misdiagnosis. Motility of E. histolytica in fresh preparations usually occurs in a linear (not random) fashion, with the clear hyaline ectoplasm flowing to form blunt-ended pseudopodia, which guide the endoplasm containing the nucleus . If a fresh stool specimen cannot be examined immediately, it should be preserved with a fixative such as polyvinyl alcohol or kept cool (4°C). Occasionally motile trophozoites are seen even after 4 h at this temperature, although the trophozoites generally disintegrate rapidly in unfixed stool specimens. Stool specimens can be examined either unstained or stained with Lugol‟s or D‟Antoni‟s iodine. Iodine stains make the nucleus perfectly visible. The appearance of chromatoid bodies is the same as in wet mount preparations. Although several other stains, including Giemsa, methylene blue, Chorazole black E, Wright‟s, and iodine-trichrome, may be used successfully. Wheatley‟s trichrome staining or one of the modified iron haematoxylin stains for permanent smears has been suggested for routine use in the diagnosis of E. histolytica/E. dispar . Shetty and Prabhu found that D‟Antoni‟s iodine was much better than saline or buffered

18 methylene blue for detection of E. histolytica cysts while saline and buffered methylene blue were equally good for detection of E. histolytica trophozoites. Microscopic examination of intestinal parasites is usually performed on ﬁxed stools and fresh specimens, but there are several factors that adversely affect the results of microscopy including the following:(Trudel 2003)  lack of well-trained microscopists with resulting difficulty in differentiation between non motile trophozoites and polymorphonuclear leukocytes, macrophages, and tissue cells.  Delayed delivery of the samples to the laboratory (motility can cease and trophozoites can lyse within 20 to 30 min).  Inadequate collection conditions (a clean, dry, wide-mouth plastic container not contaminated with urine and water is needed).  Interfering substances such as antibiotics (tetracyclines or sulfonamides), laxatives, antacids, cathartics (magnesium sulfate), antidiarrheal preparations, (kaolin or bismuth), or enemas (soap).  Inadequate number of specimens collected (at least three specimens are needed).  Lack of preservation of stool specimens with fixatives (polyvinyl alcohol, Schaudinn‟s fluid, merthiolate iodine formalin, sodium acetate-acetic acid- formalin, or 5 or 10% formalin is needed).  Presence of other amoebae (E. dispar and E. moshkovskii are identical and E. coli and E. hartmanni are similar in appearance to E. histolytica) . (Tanyuksel and Petri 2003)

1.2.8 Differential Diagnosis of E. histolytica From E. dispar:

1.2.8.1 Morphological detection: It is now believed that four, not three species of Entamoeba (E. histolytica, E. dispar, E. coli, E. hartmanni) may usually be found in the human intestine, only one of which is a pathogen, that is E. histolytica. There are also a few rare strains of E. histolytica, now known to be the normally free-living species ( E. moshkovski, E. polecki, E. chattoni and E. gingivalis). Microscopic examination which depends on certain morphological features is not sufficient for differential diagnosis between E. histolytica and E. dispar.

The important point to be highlighted is that the cysts of E. coli and E. hartmanni may be distinguished by light microscopy applying well established criteria from those of E. histolytica and E. dispar but that the latter two are indistinguishable from each other.(Ackers 2002)

1.2.8.2 Biochemical detection: In eukaryotic cells it is found that Calreticulin (CR) protein is widely distributed. Calreticulin is a highly conserved protein in both its amino acid sequence and molecular organization residing in the endoplasmic reticulum (ER) membrane protein that belongs to the family of KDEL proteins. It has been reported that partial sequencing of a 51kDa protein of Entamoeba histolytica is highly triggering the immune system in humans. This protein is recognized by IgA and IgG anti-amebic antibodies in the saliva and serum of patients with invasive amebiasis (> 91%), in contrast with the low recognition frequency obtained in asymptomatic cyst passers (<10%) and non-parasitized individuals.(Enrique Gonza´Lez 2002) In their work Enrique and his group(Enrique Gonza´Lez 2002) observed that after 6–12 months of follow-up, patients with invasive amebiasis have antibody levels against this protein that are decreased by nearly 70%. This makes the protein potentially useful in diagnosis and detection of new cases of invasive amebiasis in endemic areas.(Enrique Gonza´Lez 2002) The main virulence and the most prominent property of Entamoeba histolytica is its remarkable cytolytic capacity. Two decades ago, a protein termed amoebapore which is capable of forming ion channels or pores in lipid membranes and of depolarizing target cells, was discovered in E. histolytica. This protein was then extensively investigated and three isoforms of the amoebapore peptides (A, B, C in ratios of 21 : 9 : 1) were isolated and biochemically characterized. (RIVKA BRACHA 2002) Entamoeba histolytica can be discriminated from E. dispar on the basis of the migration of isoenzymes or separation of monoclonal antibodies against several proteins or genetically as follows according to (Louis S. Diamond 1993)table 1.2).

Table 1.2: Biochemical, Immunological and Genetic characters of E. histolytica (Louis S. Diamond 1993). Biochemical characters Immunological characters Genetic characters Migration of anyone of separation of monoclonal the six isoenzymes antibodies hexokinase EC 2.7.1.1 The Gal/GalNAc adherence Dot hybridization with probe P145. lectin. Phosphoglucomutase EC The 96-kda antigen. Fragment pattern comparison of 2.7.5.1 Southern-blotted restriction- digested genomic DNA probed with the actin, cysteine proteinase , 125-kda antigen, superoxide dismutase, or ribosomal, 331 genes. aldolase EC 4.1.2.13 The 29-kda/30-kda antigen. Restriction enzyme digestion of PCR amplification products of the small subunit ribosomal RNA, 29- kda/30-kda antigen or 125-kda antigen, 471 genes. acetyl-glucosaminidase The EDG antigen. PCR amplification of gene EC 3.2.1.30 fragments with oligonucleotides specific for the small subunit ribosomal RNA or 29-kda130-kda antigen genes. Peptidase and NADP- The 81/84-kda antigen. diaphorase Glucosephosphate an uncharacterized antigen. isomerase EC 5.3.1.9 * * Does not discriminate the two on starch gels, but does on polyacrylamide gels and cellulose acetate.

1.2.8.3 Differentiation of E. histolytica/E. dispar/E. moshkovskii from E. coli and E. hartmanni: The description of Entamoeba species has depended on the characteristic features of these parasites such as the size of the trophozoites and cysts, the number of nuclei in the mature cyst and the nuclear structure. E. histolytica is the only pathogenic Entamoeba species. It belongs to the subphylum Sarcodina, class Lobosea, and family Entamoebidae. E. histolytica exists in two morphologic forms: the tetranucleated hard infective cyst (10 to 15 mm in diameter) and the more fragile, motile, feeding and potentially pathogenic trophozoite (10 to 60 mm in diameter). The trophozoites of E. hartmanni do not have a spherical form and measured less than 12 mm in diameter, and considered the smallest of the Entamoeba trophozoites. The cyst is rounded, measuring less than 10 mm in diameter, and often contain only two nuclei. The cyst stage of E. hartmanni is characterized by a typical nuclear structure and many chromatoidal bars with rounded or squared ends in permanent stained smears of clinical specimens. The nuclear structure of stained E. hartmanni trophozoites is similar to but smaller than that of E. histolytica trophozoites. Previously, these parasites were known as a synonym of E. histolytica or small race E. histolytica. Now they are known to be separate commensal or nonpathogenic parasites, and their infections do not need to be treated. Trophozoites of E. coli have large, irregular, and eccentric karyosomes, along with nuclei with irregular clumps of peripheral chromatin. Cysts of E. coli are spherical and have eight nuclei, irregular karyosomes, and peripheral chromatin. Trophozoites of both E. coli and E. hartmanni may include ingested bacteria.(Tanyuksel and Petri 2003)

1.2.9 The detection of E. histolytica and E. dispar in faeces

1.2.9.1 Isoenzyme analysis: Isoenzyme analysis was between the earliest to imply that “pathogenic” and “non-pathogenic” E. histolytica were in fact two distinct species. Although it was considered as “gold standard” method but it has practiced disadvantages such as unsuccessful culture of microscopically cyst-positive faeces and its long period about fourteen days to grow enough trophozoites to prepare the lysates for the

22 analysis. The process itself is inconvenient and by now is rarely used in clinical diagnosis.(Ackers 2002) Recently, many studies have been dedicated to the development of modern techniques either based on monoclonal antibodies or molecular biology methods to successfully differentiate the two species in human feces.(Trudel 2003)

1.2.9.2 Antigenic detection: Although only very few genes specific to E. histolytica and absent from E. dispar have been discovered as yet, almost all homologous proteins contain amino-acid substitutions which result in the expression of species specific epitopes. Detection of these epitopes with monoclonal antibodies is the basis of a number of quick and convenient diagnostic methods. The target molecule which has been most intensively studied is the heavy subunit of the galactose/N-acetylgalactosamine inhibitable lectin; of six monoclonal antibodies raised against this molecule, only two reacted with E. histolytica and E. dispar while the other four reacted only with E. histolytica.(Louis S. Diamond 1993; Ackers 2002) These antibodies are the basis of two kits manufactured by TechLab Inc. (www.Techlab.com) one of which (based on one of the non- specific monoclonal antibodies) identifies E. histolytica / E. dispar while the other, based on a specific one can identify E. histolytica. Sequential application of these two kits can specifically identify both species although they cannot distinguish mixed infection with E. histolytica and E. dispar from infection with E. histolytica alone. Evaluation in Bangladesh shows clearly that these kits are more sensitive and specific than either wet film microscopy or culture.(Haque R 1998; Haque R 2000) Because the gold standard test requires cultivation of the organism which is known not to be 100% sensitive it is difficult to assess whether the kits produce false-positive results, but PCR suggests that most culture or microscopy negative but antigen positive samples are true positives. The speed and convenience of these kits is also a strong point in their favor. Other workers have used similar monoclonal antibodies with equal success for species specific diagnosis in Egypt(Abd-Alla M D 2000) . Other kits are available from Cellabs in Australia (http:// www.cellabs.com.au/) (detects E. histolytica and E. dispar); R-Biopharm in Germany (http://www.r-biopharm.com/ Human/HumanFrame.html) (E.

23 histolytica/E. dispar only) and Remel in the USA (http://www. remel.com/products/clinical/level2/MicrowellFormat.cfm) (E. histolytica only). A dipstick test for E. histolytica/E. dispar, G. intestinalis and Cryptosporidium parvum is also available commercially (http://www.biosite.com/ products/micro.asp).(Ackers 2002)

1.2.9.3 Molecular detection: The use of species-specific DNA probes to hybridize with unamplified DNA isolated from faecal samples has the great attraction of simplicity, particularly if non-radioactive labels are used. The method has been applied successfully but not widely taken up, probably because of doubts about its sensitivity. The polymerase chain reaction (PCR) method, however, has been as widely tested for amoebiasis as for most other diagnostic problems and forms the subject of much of the remainder of this review.(Ackers 2002)

1.2.9.4 PCR based methods: The ability of the PCR to specifically amplify minute amounts of pathogen DNA has revolutionised the diagnosis of many infectious diseases, and the numerous sequence differences between homologous genes in E. histolytica and E. dispar make it a natural candidate for identifying these two species. A number of methods have been published.(Tannich and Burchard 1991; Katzwinkel- Wladarsch 1994; Troll 1997) Most, but not all, rely on amplifying unique regions of the SSUrRNA episome, its high copy number providing increased sensitivity. Although in the original procedures the product was often detected by gel electrophoresis followed by ethidium bromide staining, colorimetric detection using specific probes is now frequently employed.(Ackers 2002) This provides the advantage of an easy-to read and familiar microtitre plate format. Light Cycler PCR has now been applied to the diagnosis of amoebiasis(Tannich and Burchard 1991). PCR has also proved capable of detecting E. histolytica DNA in liver abscess contents(Tachibana 1992). In invitro testing with cultured trophozoites PCR was about one hundred times more sensitive than antigen detection. It also has the advantage that it can be developed to provide strain (as well as species) identification. However, it is

24 important to be aware of the disadvantages of the method. Firstly, three separate steps are required – DNA extraction, amplification and product detection. While the necessary equipment is now widely available the process is not as quick or as simple as the use of an antigen detection kit. Secondly, as with all PCR-based methods, great care has to be taken to eliminate the risk of false positives due to contamination from product prepared earlier although the Light Cycler technique greatly reduces this danger. Neither PCR nor antigen detection kits are fully sensitive if the faeces have been frozen or preserved and neither method is affordable for routine use in most disease-endemic countries.(Ackers 2002) In practice, the theoretically higher sensitivity of PCR is balanced by the speed and convenience of a kit and the method chosen will usually depend on local resources and preferences.(Ackers 2002) Ampliﬁcation of amoeba DNA fragments by PCR has been verified to establish a sensitive and speciﬁc method to detect E. histolytica or E. dispar from human feces.(Blessmann 2002)

1.2.9.5 Molecular Biology Based Diagnostic Tests and PCR: An increasing number of differences between the two E. histolytica and E. dispar are now recognized and this information finally led to the formal separation of the two species with the name Entamoeba histolytica being retained for the pathogenic species and Brumpt‟s name E. dispar being restored for the non- pathogen.(Ackers 2002) It has been recently reported on the whole genome differences between pathogenic and nonpathogenic E. histolytica, and developed a rapid and reliable method to differentiate both forms on the basis of these differences.(Egbert Tannich and Gerd 1991) Nowadays, DNA technique assures without any doubt that amoebiasis is caused by two distinct species, Entamoeba histolytica (pathogenic) and Entamoeba dispar (nonpathogenic), respectively.(Troll 1997) To rectify the problems of microscopic or culture-based diagnosis and take advantage of the sensitivity, specificity, and simplicity of newer techniques, molecular biology-based technology has become commonly used. (Tanyuksel and Petri 2003) The PCR method offers sensitivity and specificity for the diagnosis of intestinal amebiasis that compete with that of antigen detection. However it will

25 potentially become the “gold standard” by which other diagnostic techniques (microscopy, antibody detection, etc.) are measured. Many investigators have reported successful application of PCR to the diagnosis of amebiasis. Some investigators have improved the PCR solution hybridization enzyme-linked immunoassay technique and have suggested that it is more practical in the study of the complex ecology of amebiasis. PCR is also very helpful for Amoebic Liver Abscess (ALA) diagnosis when aspirated pus is available, since it appears not to require protease treatment for DNA isolation. Methods of DNA extraction from stool specimens and specific primers are the key to successful PCR diagnosis. A commercially available DNA isolation kit (Qiagen, Hilden, Germany) is recommended due to its ease and success . One major advantage seems to be that formalin-fixed stool specimens can be used for DNA extraction. This has the benefits of safe handling, storage and transportation. With this technique, one E. histolytica trophozoite/mg of stool can be detected. Fixation with 1 to 10% formalin is very important in the storage, transportation, and fixation of stool specimen. No reduction in the ability to perform PCR amplifications of E. histolytica DNA fixed in 1 to 10% formalin was noted for 7 days. Nunez et al.(Yury O. NuŃ˜Ez 2001) described multiplex PCR amplification for the detection and characterization of both E. histolytica and E. dispar in stool samples by using two pairs of specific primers combined in a single reaction mixture. This novel approach had 94% sensitivity and 100% specificity. It showed an E. histolytica and E. dispar co infection rate of 24.5% in the Mexican children studied. (Tanyuksel and Petri 2003) Riboprinting, the restriction site polymorphism analysis method involving amplification followed by restriction fragment length polymorphism analyses of the small- and large subunit rDNA, is a very useful tool to evaluate different Entamoeba species. In this method, fragments can be seen in agarose gels after amplified rDNA is digested with restriction enzymes. Riboprints of E. histolytica can be easily distinguished from those of other amoebas, especially E. dispar, by using the restriction enzymes XbaI, RsaI, TaqI, Sau96I, and DdeI . Ribotyping has been of great value in understanding the epidemiology of Entamoeba species and in investigating disease outbreaks; however, the process of ribotyping is difficult and time-consuming.(Tanyuksel and Petri 2003)

Numerous groups have tried, with variable success, molecular methods on this type of sample. Recently, the effect of formalin ﬁxation on PCR was further investigated. It was shown that even if its effect on DNA is indirect, concentrations of formalin higher than 1% seemed to inhibit PCR amplification starting at 4 days of ﬁxation. This is corroborated by the work of (Troll 1997) who showed that sensitivity of PCR usually decreased within 2 days in feces.(Trudel 2003)

1.2.10 Prevention and control of Amoebiasis: To discontinue transmission of the parasite and to prevent further development of infected individuals to invasive disease, treatment of E. histolytica carriers has been recommended.(Blessmann 2002) E. histolytica is anaerobic and is sensitive to metronidazole when in the trophozoite form. Metronidazole is not effective against the cyst form of the organism, and therefore is followed up with iodoquinol or paromomycin to target the cysts. Dehydroemetine, a treatment that requires hospitalization due to the need for close supervision, and Diloxanide furoate, which is used in conjunction with other treatments in systemic cases, are only available through the Center for Disease Control and Prevention. If liver infection occurs Cholorquine may be used, in the event that Metronidazole is ineffective(Upcroft and Upcroft 2001). Entamoeba histolytica is a typical example of diseases that impact poor populations in developing countries. This makes it far more difficult to fund research and development for new treatments or vaccines, in spite of the fact that research has shown some possibilities for vaccines (Ivory and Chadee 2007). The presence of IgA antibodies against E. histolytica indicates that a vaccine that brings about a mucosal immune response could be effective. However, the lack of projected profit limits the interest of pharmaceutical and biotechnology companies. Sanitation and hygiene are effective controls but often cannot be applied in many poor nations. Until a vaccine is created and distributed, Entamoeba histolytica will remain an important disease in mortality rates, especially among children in developing countries.

1.3 Rationale and Study Objectives

1.3.1 Rationale:  E. histolytica is the causative agent of amoebiasis. Non-pathogenic E. dispar and E. moshkovskii are morphologically identical species to E. histolytica.  Within the genus Entamoeba commensal amoebae other than E. dispar and E. moshkovskii as E. polecki, E. chattoni, E. coli, and E. hartmanni can colonize the lumen of the human large intestine but only E. histolytica is able to invade the large intestinal mucosa causing a disease.  Diagnosis of E. histolytica infections based on microscopic examination only can be regarded as defective and misleading.  The identification and differentiation of pathogenic Entamoeba, E. histolytica from morphologically identical non-pathogenic species is an important goal of accurate laboratory diagnosis of amoebiasis and has highlighted the need for non-microscopic detection methods able to differentiate among them to avoid unnecessary and possibly harmful therapies and to determine the true prevalence and epidemiology of E. histolytica.  The prevalence of Entamoeba histolytica infection in Sudan and regionally was unclear and very few data was found due to limited number of researches conducted.

1.3.2 Study Objectives:

1.3.2.1 General Objective: The present study aimed to determine the prevalence of infections caused by E. histolytica and the non-pathogenic Entamoeba, using conventional microscopical examination and molecular assays as well as analyzing the existing methods of diagnosis in the main public laboratories in the area.

1.3.2.2 Specific Objectives 1. To determine the copro-prevalence of Entamoeba complex using microscopy among study population. 2. To identify and determine the molecular prevalence of pathogenic E. histolytica, non-pathogenic E. dispar and E. moshkovskii using PCR assays among study population.

3. To validate the effectiveness of the used diagnostic methods. 4. To analyze associated variables as the estimated risk for E. histolytica infections. 5. To correlate retrospective data regarding Entamoeba complex with study data. 6. To evaluate the performance and practice of Wad Medani town laboratory personnel towards amoebiasis detection.

2. MATERIALS AND METHODS 2.1. Study Area: This study was carried out in Wad-Medani Town and some surrounding villages in the Gezira State in the period from May 2011 to February 2013.

Figure 2-1: Diagrammatic map of Gezira State and Wad Medani town (Maps from: Statistic Information Center, Ministry of Health, Gezira State).

2.2. Ethical consideration: The present study was ethically approved by the General Administration of Planning and Research committee at the State Ministry of Health, Gezira State, Sudan. Permission was also obtained from the health school directorates in the Ministries of Health and Education. The project was explained to the teachers of the different schools and health centres which have been visited for the survey and their consent was obtained verbally.

2.3. Study type: The study was carried out in two phases to achieve the proposed objectives. First, a cross sectional study was carried out to detect the prevalence of infections caused by Entamoeba complex using microscopy i.e. E. histolytica and the nonpathogenic; E. dispar and E. moshkovskii, using Multiplex Polymerase Chain Reaction (mPCR) assays. Also, to evaluate the used diagnostic tests. Second is to analyse the data as reported from the two main laboratories (retrospective study) regarding the diagnosis of dysentery including methods used as an outcome (dependent variable).

2.4. Study population: This study was conducted on 2009 individuals, clinically suspected for Entamoeba infection suffering from diarrhoea/dysentery or abdominal pain/cramp of both gender and including all age groups. They were divided into 2 groups: Group I: including a total of 271 individuals attending Wad-Medani teaching hospital central laboratory and other selected laboratories in addition to different Basic Primary schools in different areas of Wad-Medani town. Their faecal samples were collected in the period from May 2012 to February 2013. Study population related demographic, environmental and clinical data were recorded in a designed questionnaire adapted from (Ngindu, Kamar et al. 2002; Beltramino 2009). Group II: including a total of 1738 individuals results attending the central laboratory in Wad-Medani teaching hospital as reference laboratory (1120) and Arkaweet health center laboratory in East of Wad-Medani as general service laboratory (618). The stool examinations results and their related demographic, environmental and clinical data were retrieved retrospectively from records of statistic information center, Ministry of Health, Gezira State about amoebiasis situation. Also, at the same time the performance and practice of laboratory personnel in different laboratories in Wad-Medani town towards amoebiasis detection were recorded in a designed questionnaire(Ngindu, Kamar et al. 2002)(Ngindu et al., 2002)(Ngindu et al., 2002)(Ngindu, Kamar et al. 2002)(Ngindu, Kamar et al. 2002)(Ngindu, Kamar et al. 2002)(Ngindu, Kamar et al. 2002)(Ngindu, Kamar et al. 2002)

2.5. Sample size: The sample was deliberately selected in a systematic way according to the following simple formula of Daniel (n = Z2P(1-P) / d2) where n = sample size, Z = Z statistic for a level of confidence, P = expected prevalence or proportion (in proportion of one; if 20%, P = 0.2), and d = precision (in proportion of one; if 5%, d = 0.05). Z statistic (Z): For the level of confidence of 95%, which is conventional, Z value is 1.96(L. Naing 2006).

Regarding the above mentioned formula the actual prevalence and incidences of intestinal amoebiasis is unknown for most parts of under developing countries

31 including Sudan except some studies done on common intestinal parasites in Southern Sudan and northern Sudan and West Africa which considered the prevalence rate of intestinal parasites along with Entamoeba as 30-40%(Karrar ZA 1995; Dolo A 1996; M.A. Babiker 2009). The sample size was calculated accordingly and 2009 samples were involved (271 individuals, their stool samples were examined and 1738 individuals their recorded results of stool examination collected retrospectively) of both sexes and all age groups.

2.6. Sample collection and processing: An adequate stool sample was collected from each participant of group I in dry clean wide screw capped containers. The study participants were advised on the healthy attitudes and hygiene, besides stressing the importance of the study research and were given instructions to avoid urine and water contaminations of stool samples. For safety, containers were put in plastic bags and carefully transferred at the shortest time as possible after collection to the laboratory.

The gross appearance of each collected stool samples was recorded immediately then divided into five parts as follow: (i) Part one freshly (unpreserved), was microscopically examined immediately. (ii) Part two was preserved in 5% formalin for faecal concentration. (iii) Part three was preserved in polyvinyl alcohol fixative (PVA) for trichrome staining for confirmation of morphology. (iv) Part four was collected in Eppendrof tubes and immediately frozen at -20°C for further molecular assays. (v) Part five was fixed in 70% ethanol for further molecular assays in case frozen samples were spoiled. Each collected stool sample was subjected for:  Parasitological examination  Molecular assays Parasitological examination was carried out in the Faculty of Medical Laboratory Sciences, Department of Medical Parasitology, Wad-Medani, the Gezira State, Sudan. Molecular studies were carried out at the Diagnostic and Research Unit of Parasitic Diseases (DRUP) and Lab Molecular Medical Parasitology (LMMP), Department of

Medical Parasitology, Kasr Al-Ainy School of Medicine, Cairo University, Cairo, Egypt.

2.6.1. Parasitological coproscopic examination: Fixative-free fresh stool samples were subjected to macroscopic examination. Stool consistency was recorded, where consistency was classified as liquid or soft, also, presence of mucous and/or visible blood and presence of macroscopically identifiable parasite elements were recorded. Then samples were subjected for microscopic examination. Binocular microscope was calibrated for the purpose of measuring the parasites observed under the microscope as a crucial step in the parasitic diagnosis. A single fresh stool specimen, collected from each patient, was microscopically examined by direct wet smear under low (×10) and high (x40) power using a drop of normal saline to aid visualization of motile trophozoites and another drop of lugol's iodine to detect protozoa trophozoites and/or cysts or any other parasitic objects. All formalin preserved stool samples were concentrated (Ehsan Nazemalhosseini Mojarad 2010) and thin smears were prepared from the sediment and examined with and without iodine under low (×10) and high (x40) power to aid visualization of parasitic stages as it maximizes the numbers of detected organisms. Stool samples positive microscopically for Entamoeba complex trophozoites and /or cysts their PVA preserved part were stained using trichrome to confirm the morphological details of protozoa(Garcia, Brewer et al. 1979; Jensen, Kepley et al. 2000). 2.6.1.1. Direct wet mount Normal saline preparation: One or two drops of 0.85% physiological saline was dispensed on glass microscopic slide and a small portion from the stool sample (about 1 mg) was added by a wooden stick and mixed well, then a 22 mm X 22 mm cover slip was put on the specimen and systemically examined under low (×10) and high (x40) power.

Iodine preparation: Lugol's iodine solution, was prepared by adding 20g potassium iodide, l0g iodine in 100 ml distilled water stored in a brown bottle container. One or two drops from lugol's iodine solution was put on the slide and a small portion of stool sample mixed and systemically examined under low (×10) and high (x40) power.

2.6.1.2. Formal Ether Concentration technique: Procedure: 1. Using wooden stick a quantity of faeces (approximately 1g or pea size) to include external and internal portions was put in a centrifuge tube and 7 ml of 10% formalin was added and mixed thoroughly. 2. The faeces was emulsified in the formalin and filtered through a brass/nylon filter into the dish. 3. the filtrate was transferred to a another centrifuged tube and 3 ml of ether/ethyl acetate was added and mixed well on a vortex mixer for 15 seconds or by hand for 1 minute. 4. The mixture was centrifuged at 3,000 rpm. for 2 minute. 5. The fatty plug was loosened with wooden stick and the supernatant was discarded by quickly inverting the tube. 6. The deposit was mix well and a drop was transferred to a slide for examination under a coverslip using the x10 and x40 objectives to examine the whole of the deposit for cysts and ova.

2.6.1.3. Trichrome staining: The stool sample for trichrome staining technique was done on the samples preserved in PVA. PVA preservative preparation consisted of Schaudinn‟s solution 93.5 ml, glycerol l.5 ml, glacial acetic 5 ml, and 5 g PVA powder. The PVA powder was added by constant stirring, after the other ingredients had been heated to 75C to give 100 ml mixture. One part of a well mixed faecal sample was added to 3 parts of PVA fixative in a small glass container, mixed well and closed tightly.

Procedure: At a convenient time a smear was prepared form PVA preserved sample(Garcia, Brewer et al. 1979). After the smears were made and dried they were treated in 70% ethanol containing 4 to 5 drops of lugol‟s iodine to remove the excess mercury residue for 2 to 5 mints, then the slides were transferred to two steps of 70 % ethanol for 2 to 5 mints. The slides were transferred to trichrome stain for 10 minutes. Trichrome stain was prepared by mixing 0.6 g chromotrope 2 R, 0.3 g light green SF. 0.7 g phosphotungestic acid, 1.0 ml glacial acetic acid added to 100 ml distilled water. The slides were then transferred to acidified alcohol which contains 99 ml 90 % ethanol to which 1 ml glacial acetic acid was added for three seconds for differentiation. Then the slides were transferred to absolute ethanol for one dip and 5 mints respectively for dehydration and were then cleared in xylene for 5 mints and mounted with DPX. The slides were examined under x100 oil-immersion lens for identification of Entamoeba species.

2.6.2. Copro-mPCR assay: Out of 271 collected samples, 40 samples had Entamoeba complex microscopically, each divided in to three parts (32 samples were preserved in 5% formalin, 28 samples were frozen and 30 samples were preserved in 70% ethanol) and 10 freezed samples negative for Entamoeba complex microscopically were taken to (DRUP), (LMMP) at Kasr Al-Ainy School of Medicine, Cairo University, Cairo, Egypt for molecular studied. The 50 samples were processed for detection of E. histolytica, E. dispar and E. moshkovskii copro-DNA targeting 18S rRNA gene using mPCR as follows:

2.6.2.1. Extraction of genomic DNA from stool samples: This was done using FavorPrepTM stool DNA isolation Mini Kit (Cat. No. FASTI 001, Favorgen Biotech corporation ping-Tung 908, Taiwan).

Principle: The Principle of Extraction and purification of DNA procedure involves digestion of proteins, binding DNA to the silica membrane, washing away impurities, and elution of pure DNA from the spin column. Proteins were digested and degraded under denaturing conditions during 70°C incubation with proteinase K. Buffering conditions are then adjusted to allow optimal binding of DNA to the membrane, and the sample is loaded onto the spin column.

Figure 2-2: FavorPrepTM stool DNA isolation Mini Kit

DNA is adsorbed onto the silica membrane during a brief centrifugation step. Optimized salt concentrations and pH conditions in the lysate ensure that remains of digested proteins and other impurities, which can inhibit PCR and other downstream enzymatic reactions, are not retained on the membrane. DNA bound to the membrane is washed in two centrifugation steps. Optimized wash conditions ensure complete removal of any residual impurities without affecting DNA binding.

Purified, concentrated DNA is eluted from the Mini spin column in low-salt buffer equilibrated to room temperature. DNA yield is typically 15–60 μg but, depending on the individual stool sample and the way it was stored, may range from 5–100 μg. DNA concentration is typically 75–300 ng/μl. The eluted DNA is up to 20 kb long and is suitable for direct use in PCR and other enzymatic reactions.

Materials: 1. FavorPrepTM stool DNA isolation Mini Kit (figure 2-2) including the following reagents:  Glass beads  Proteinase K  SDE1 Buffer  SDE2 Buffer  SDE3 Buffer  SDE4 Buffer  Elution buffer  Washing buffer  Elution tube  SDE column  Bead tubes 2. Micro centrifuge. 3. Vortex. 4. Incubator. 5. Absolute Ethanol. 6. Water bath. 7. Micropipette (1 μl -1000 μl). 8. Pipette tips (10, 100 μl-1000 μl). 9. Ice cubes. 10. Disposable gloves. 11. 1.5 ml micro-centrifuge tubes. Procedure: 1. 200 mg of glass beads were added into 2.0 ml bead tube and 200µl of sample was transferred into bead tube then placed on ice. 2. 300µl of SDE1 Buffer and 20µl of proteinase K was added to the sample then vortexed for 5 minutes and incubated at 95°C for 60 minutes. 3. The samples were cooled down then 100µl of SDE2 Buffer were added, and the samples were incubated on ice for 5 minutes. 4. The samples were centrifuged for 5 min at full speed (14000 rpm).

5. The supernatant was transferred to 1.5 ml micro centrifuge and the stool pellet was discarded 6. 200 µl of SDE3 Buffer were added to the sample then vortexed. 7. The samples were incubated at room temperature for 2 minutes. 8. The samples were centrifuged for 2 min at full speed. 9. 250 µl of supernatant were transferred to 1.5 ml micro centrifuge and the stool pellet was discarded. 10. 250 µl of SDE4 Buffer and 250µl of ethanol (100%) were added to the sample then vortexed. 11. SDE column was placed into a collection tube, and all of the sample mixture was transferred to SDE column. Centrifugation for 1 min at full speed was done then the flow through was discarded. SDE column was placed into new collection tube. 12. 750 µl of washing buffer (ethanol added) were added to SDE column. Centrifugation for 1 min at full speed was done then the flow – through was discarded. This step was repeated once. 13. Centrifugation for 3 min) at full speed was done to dry the SDE column. 14. SDE column was placed into a respectively labeled elution tube, 100µl of preheated elution buffer was added to the membrane center of SDE column. Centrifugation for 1 min at full speed was done to elute DNA and this step was repeated for one more time. 15. The eluted DNA was measured for concentration and purity then used immediately or stored at -200C till used.

2.6.2.2. Copro-DNA amplification using mPCR:

Figure 2-3: Thermal cycler.

Principle: Extension of the primers by thermo stable DNA polymerase enzyme is done using deoxynucleotide triphosphates (dNTPs) at 72 °C. Repeating these steps many times results in exponential increase in the amount of target DNA. The multiplex PCR process is a new development of PCR based approach for the detection and characterization of the species of the Entamoeba complex. In which the method used to amplify simultaneously two or more species-specific DNA fragments using two pairs or more of specific primers combined in a single reaction mixture. This procedure therefore saves considerable time and resources. This PCR method was originally developed by Chamberlain and others to detect human genes, and later Bej and others modified the method to detect gene sequences associated with different groups of bacteria in environmental samples. Even though this approach, has not been frequently applied in novel PCR-based research and diagnosis. The principle behind this modified method is the design of primers that all have a similar annealing temperature (Ta) and similar target DNA sizes (Yury O. NuŃ˜Ez 2001).

Materials: o Sample: Extracted genomic DNA. o Equipments and supplies: 1. Thermal cycler (Figure 2-3) (Professional thermocycler, Biometra, Applied Biosystem, California, USA). 2. Vortex 3. Centrifuge 4. Automatic Micropipette (1 μl -1000 μl). 5. Pipette tips (10, 100 and1000 μl) 6. PCR 0.2ml tubes. 7. Spinner. 8. Gloves 9. Water, nuclease free 10. PCR master mix kit (#K1081 – Fermentas UAB, V. Graiciuno 8,LT- 02241Vilnius, Lithuania):  PCR master mix (2 x 1.25ml) composition:

 Taq DNA polymerase (recombinant) in reaction buffer: 5units/ul.  MgCl2: 4mM.  dNTPs: dATP, dCTP, dGTP, dTTP: 0.4mM of each.  Water, nuclease free (2 x 1.25ml). 11. PCR Primer.

Table 2-1: The used primers and their sequences (Hamzah 2006).

Primers Sequence Expected product size (bp) Ta Entamoeba Enta 5'-ATGCACGAGAGCGAAAGCAT-3' forward primer F E. histolytica, E.hR 5'-GATCTAGAAACAATGCTTCTCT-3' 167 reverse primer 58o E. dispar C E.dR 5'-CACCACTTACTATCCCTACC-3' 753 reverse primer E. moshkovskii E.mR 5'-TGACCGGAGCCAGAGACAT-3' 579 reverse primer

PCR Target gene: The highly sensitive primer sets specific to the 18S tRNA gene that were used for detection of E. histolytica, E. dispar and E. moschkovskii together with the mPCR procedure described. Blasting the used primers sets using NCBI further enhance the specificity in addition to sensitivity of PCR (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Procedure: - To optimize PCR, Primers were titrated using primer concentration 50, 100, 200 and 400 nM each with different annealing gradient temperature (45 to 60 °C) and concentration from DNA template (1, 3, 5 and 7 µl) - The optimized PCR conditions for PCR assays reaction components are shown in table 2-2.

Table 2-2: The optimized 1ry and 2ry PCR assays reaction components. Reaction components Amount

ReactionMixture Master Master Mix 2X 12.5µl EntaF 1 µl (200 nM) E.hR 1 µl (200 nM)

(Rx) E.dR 1 µl (200 nM)

E.mR 1 µl (200 nM) Taq Polymerase (5units/µl)* 0.1 µl (5 U/µL)

ddH2O 5.4 µl Genomic extracted DNA 3 µl *as an initiator for MM - Sterile distilled water was added to bring the final volume to 25µl (i.e. 5.4 µl). - The reaction master mixture (Rx) was prepared for each PCR set as one reaction, performed in a volume of 22μl (without genomic DNA) and multiplied by the number of samples. - 22 µl of the master mixture was added to each PCR tube (Rx). - 3 µl of DNA sample was added to each PCR tube (Rx) then the PCR tubes were inserted in the thermocycler. -The thermal profile was adjusted and the thermocycler program was run (table 4-3).

Table 2-3: The thermocycler program. Step Temperature (Co) Time No. of Cycles Step 1 Initial Denaturation 94 3 s 1 Step 2 Denaturation 94 1 m Step 3 Annealing 58 1 m 35 Step 4 Extension 7 1 m Step 5 Final Extension 72 7 m 1

Controls: - Negative controls: The extraction of DNA as well as the PCR was monitored strictly for possible contamination. Two negative extraction controls were routinely used for every

41 extraction series. The PCR itself was monitored by one reaction in every PCR, which contained only the reagents and no template.

- Positive controls: DNA of E. histolytica, E. dispar and E. moschkovskii (provided by Prof. Dr. Ayman A. El-Badry, Department of Medical Parasitology, Faculty of Medicine, Cairo University), were included during PCR to ensure reliability, validity and to check for possible contaminations of the amplification reaction.

- Inhibition control: In order to detect possible inhibitors, PCR inhibition control that contained both the sample DNA and reference Entamoeba DNA was run along the sample.

2.6.2.3. Detection of PCR products using gel electrophoresis and UV light transillumination: The amplified samples were then run in parallel on 2% agarose gel using gel electrophoresis and visualized on a UV transilluminator to detect presence of amplified material (Smith 1993).

Materials: 1. Vortex. 2. A conical flask 3. Tape 4. Metal foil 5. Gel electrophoresis and power supply 6. UV transilluminator 7. Hot plate 8. Automatic micro-pipette 9. Pipette tips (1-50 μl) and (10-50 μl) 10. Scale 11. Thermometer 12. Disposable gloves. 13. Water, nuclease free

A B C

Figure 2-4: Hot plate stir (A), Automatic micro-pipette (B) and Vortex (C).

13. 10X TBE buffer (Tris-borate EDTA buffer): 0.44 mol / L Tris-borate, 0.01 mol / L EDTA, pH 8. 1X TBE buffer working solution (89 mmol/L Tris borate, 2mmol/L EDTA pH 8.0) prepared by mixing 1 volume of 10X TBE buffer with 9 volume water and stored at room temperature. 14. Agarose (Promega Corporation: 2800 Woods Hollow Road-Madison, WI 53711- USA, cat No. V 3121) stored at room temperature. Its melting point is at 87-89ºC and its gelling point is at 36-39ºC. 15. 6x loading buffer: ready to use, stored at 4º 16. Ethidium bromide 10mg/ml: ready to use, stored at room temperature. 17. PCR marker (50 and 100 base pair ladder) (fermentas, Cat#SM0371) Pack size: 50μg. Conc. (0.5μg/μl) used for sizing the PCR products.

Figure 2-5: Gel electrophoresis chamber and power supply.

Procedure: i) Preparation of 2% agarose gel: a. The gel tray was cleaned and dried before use. The ends of gel tray were sealed with tape which was pressed firmly to the edges to ensure a tight seal. b. The gel comb (11 teeth, 3x1 mm) was placed in position in the gel tray and the tray was placed on the horizontal surface. c. Two g agarose were added to 100ml of the 1X TBE buffer in a conical flask which was then covered with metal foil. d. This agarose was dissolved by heating using hot plate (Dylatherm) for 4 minutes. e. The agarose was then left to cool between 50-60ºC followed by addition of 10µl ethidium bromide (10mg/ml) with proper mixing. f. This solution was then poured into the gel tray and left to dry at room temperature. g. The gel comb was carefully removed. This was facilitated by gently rocking the comb backwards and forwards by few millimeters while pulling upwards. h. The tape was then removed from the sides of the tray and the tray was placed in the gel electrophoresis apparatus. i. Enough 1X TBE buffer to cover the gel surface to the depth of 1mm was poured into the apparatus (submarine gel electrophoresis). The apparatus was ready for loading of samples and performance of the electrophoresis. ii) Sample loading: Five μl from each sample (PCR product) were slowly loaded into the sample wells using an automatic micro-pipette with caution not to damage the wells with pipetting device. The PCR marker was also loaded well.

Figure 2-6: Sample loading.

44 iii) Performing the electrophoresis: a. The lid was carefully placed on the gel electrophoresis apparatus and the free ends of the power cables were connected to the power supply (Promega, Thermo Hybaid electrophoresis). b. The gel apparatus was connected so that the negative electrode (cathode) is the nearest to the sample wells at the start of electrophoresis (as at the pH used for the electrophoresis, nucleic acids have a net negative charge and will therefore migrated towards the positive electrode (anode) during electrophoresis). c. The power supply was programmed to give a constant voltage of 130V and 150 milli- amperes for 30 minutes. d. After electrophoresis, the power supply was switched off, the lid was removed and the gel was taken for viewing on an UV transilluminator.

A B

Figure 2-7: UV Transilluminator (A) and Visualization (B).

2.7. Statistical Analysis: Data were coded and entered using the statistical package SPSS version 17 (Chicago, IL, USA) for statistical analysis. Data were tabulated, described using mean and standard deviation (SD) for quantitative variables and rate (percentage) for qualitative variables. Comparisons between groups were done using Chi square test for qualitative variables and data were considered statistically significant if P- values < 0.05. To identify the estimated risk factors among study variables, association between the variables with the prevalence of Entamoeba infection was done by Chi square test. For significantly associated variables an Odds Ratio (OR) and 95% confidence interval (CI) were computed by the univariate model and were included in logistic regression analyses.

Sensitivity (100 x [a/(a+c)]), specificity (100 x [d/(b+d)]), positive predictive value (100 x [a/(a+b)]), negative predictive value (100 x [d/(c+d]), accuracy (100 x [a+d/(a+b+c+d)]) were calculated to test the diagnostic yield and kappa agreement was done to test the validity of microscopy in relation to (mPCR) results, where a: true positive, b: false positive, c: false negative and d: true negative samples.

3. RESULTS The present study was conducted on 2009 individuals suffering from diarrhea/dysentery which is clinically suspected for Entamoeba infection of both gender and including all age groups in the period from May 2011 to February 2013. A total of 271 individuals, faecal samples and related data were collected during attending the central laboratory in Wad-Medani teaching hospital, and pupils from some schools selected in Wad-Medani town and other localities is Gezira State. Recorded results of stool examination and related data of 1738 individuals were retrospectively obtained from the central laboratory in Wad-Medani teaching hospital, Arkaweet health center. And other retrieved data regarding amoebiasis were obtained from the center of health information of the Ministry of Health.

All collected faecal samples (271) were examined parasitologically and (50) samples were diagnosed molecularly for detection of Entamoeba. Collected data were analysed and data regarding laboratorians diagnostic procedures and the way they perform them were also collected and analyzed.

3.1. Group I

3.1.1. Demographic, environmental and clinical data of studied subjects: The study population involved all age groups and it ranged from one year to 72 years with a mean of 18 years (table 3-1).

Table 3-1: Mean age of group I studied individuals.

Minimum Maximum Mean SD* Age (Years) 1 72 18.29 12.99 *SD: Standard deviation

Study population was classified into different age groups according to the classification of the ministry of health reports.

Table 3-2: Distribution of age groups and gender among study individuals (n,271). Group I study individuals (n= 271)

n. % - 1 Year 2 0.74 1-4 Years 12 4.43 5-14 Years 152 50.09 Age group 15-24 Years 33 12.18 25-44 Years 55 20.30 + 45 Years 17 6.27 Male 140 51.66 Gender Female 131 48.34 Total 271 100 Data presented as n. (number) and % (percent) Most of the population studied resided in Wad Madani Alkobra locality where the main hospital and University of Gezira are situated.

Table 3-3: Distribution of study individuals according to their Locality (n,271). Group I study individuals (n= 271)

n. % Wad Madani Alkobra 208 76.75 Ganoub Algezira 4 1.48 Sharg Algezira 53 19.56 Locality Um Algura 5 1.85 Alhasaheesa 1 0.37 Almanagil 0 0 Alkamleen 0 0 Total 271 100 Data presented as n. (number) and % (percent)

The study population also were divided according to their residence areas; town, village and kampu which differe in the community hygenic status and health services provided.

166

64 61.3% 41 23.6% 15.1%

Kampu Village City

Figure 3-1: Distribution of study population according to residence.

3.1.2. Results and diagnostic effectiveness of used diagnostic test: 3.1.2.1. Results of parasitological methods:

Table 3-4: Yield of macroscopic examination of stool collected from study individuals (n,271). Group I study individuals (n= 271)

n. % Liquid 49 18.1 Stool consistency Soft 221 81.5 Formed 1 0.4 Mucus 53 19.6 Blood 2 0.7 Stool contents Mixed 3 1.1 Nil 213 78.6 Total 271 100 Data presented as n. & %.

Table 3-5: Results of microscopic examination of faecal samples for Pus cells and RBCs of group I study individuals (n,271). Group I study individuals (n= 271)

n. % Pus cells 31 11.4 RBCs 45 16.6 Mixed 8 3.0 Nil 187 69.0 Total 271 100

79 83 63

33 29.2% 31.4% 23.2% 21.2%

Normal saline Iodine Concentration Trichrome smear

Figure 3-2: Results of detection of Entamoeba complex by conventional methods among study individuals.

Table 3-6: Results of used microscopic diagnostic methods for detection of parasites other than Entamoeba complex among group I study individuals (n,271). Group I study individuals (n= 271) Before conc. Wet mount faecal smears After Conc. N.S I

Parasitic stage other G. lamblia 42 (15.5%) 36 (13.3%) 66 (24.4%) Protozoa E. coli 32 (11.8%) 26 (9.6%) 59 (21.8%) E. complexE. Blastocytis hominis 3 (1.1%) 1(0.4%) 0

than chilomastic msenilii 1(0.4%) 2 (0.7%) 2 (0.7%)

Iodamoeba bütschlii 0 1(0.4%) 2 (0.7%) Endolimax nana 0 0 1(0.4%)

Helmi

nthes H. nana eggs 11(4.1%) 11(4.1%) 22 (8.1%) E. vermicularis eggs 1 (0.4%) 0 2 (0.7%)

Total 271 Data presented as n. & %.

Table 3-7: Results of used diagnostic methods for detection of Entamoeba among study individuals correlated with Concentration method (n,271). Entamoeba Complex Detected by Concentration Method (n= 271) Positive Negative Total Positive 57 6 63 Normal Saline Negative 22 186 208

Total 79 192 271 Positive 32 1 33 Lugol's Iodine Negative 47 191 238

Total 79 192 271 Positive 58 27 85 Trichrome staining Negative 21 165 186 method Total 79 192 271

Table 3-8: Diagnostic yield and Kappa agreement of the used diagnostic tests for detection of E. histolytica among group I study individuals using Concentration method as a reference standard (http://www.quantitativeskills.com/sisa/statistics/diagnos.htm).

Normal Saline Lugol's Iodine Trichrome staining method Sensitivity 72.2 40.5 73.4 Specificity 96.9 99.5 85.9 PPV 90.5 97.0 68.2 NPV 89.4 80.3 88.9 Accuracy 89.7 82.3 82.3 Kappa ()* 0.884 0.614 0.732

()* Interpretation < 0 : Poor agreement 0.01 – 0.20: Slight agreement 0.21 – 0.40: Fair agreement 0.41 – 0.60: Moderate agreement 0.61 – 0.80: Substantial agreement 0.81 – 1.00: Almost perfect agreement

3.1.2.2. Results of Molecular assays: Table 3-9: Results of mPCR methods for detection of Entamoeba species among study individuals (n,50). All study individuals PCR results (n= 50) n. % E. histolytica 18 36 E. dispar 2 4 Positive E. moschkovskii 2 4 Mixed E. histolytica & E. dispar 1 2 Total 23 46 Negative 27 54 Total 50 100

Figure 3-3: Showing Agarose gel electrophoresis for the products of the mPCR targeting 18s tRNA gene of Entamoeba at 167, 579 and 753 bp.

L: 100 bp DNA molecular weight marker. Lanes 1 and 2: Positive E. histolytica samples. Lane 3: Negative sample. Lanes 4 and 5: Positive E. moshkovskii samples. Lane 6: Positive E. dispar sample.

Table 3-10: Results of used diagnostic methods for detection of Entamoeba among study individuals (2x2) (n,50). mPCR (n= 50)

Positive Negative Total Positive 16 18 34 Normal Saline Negative 7 9 16

Total 23 27 50 Positive 6 6 12 Lugol's Iodine Negative 17 21 38

Total 23 27 50 Positive 20 18 38 Concentration Negative 3 9 12 Total 23 27 50 Positive 21 11 32 Trichrome staining method Negative 2 16 18 Total 23 27 50

Table 3-11: Diagnostic yield, accuracy and Kappa agreement of the used diagnostic tests for detection of Entamoeba histolytica among group I study individuals using mPCR as a reference standard (http://www.quantitativeskills.com/sisa/statistics/diagnos.htm). Trichrome Normal Lugol's Concentration staining Saline % Iodine % method % method % Sensitivity 69.6 26.1 87.0 91.3 Specificity 33.3 77.8 33.3 59.3 PPV 47.1 50.0 52.6 65.6 NPV 56.3 55.3 75.0 88.9 Accuracy 50.0 54.0 58.0 74.0 Kappa ()* 0.35 0.35 0.48 0.82 ()* Interpretation < 0 : Poor agreement 0.01 – 0.20: Slight agreement 0.21 – 0.40: Fair agreement 0.41 – 0.60: Moderate agreement 0.61 – 0.80: Substantial agreement 0.81 – 1.00: Almost perfect agreement

3.1.3.Description of data variables of parasitologically positive cases. Table 3-12: Results of microscopic diagnostic methods for detection of Entamoeba complex among study individuals correlated with gender. Normal Concentration Trichrome Iodine Saline technique stain male 41(15.1%) 20 (7.4%) 50 (18.5%) 58 (21.4%) female 22 (8.1%) 13 (4.8%) 29 (10.7%) 27 (10.0%) Data presented as n. & %.

Table 3-13: Results of microscopic diagnostic methods for detection of Entamoeba complex among study individuals correlated with Age groups. Normal Concentration Trichrome Iodine Saline Technique Stain - 1-y 0 1 (0.4%) 1(0.4%) 1 (0.4%) 1-4y 5 (1.8%) 1 (0.4%) 3 (1.1%) 2 (0.7%) 5-14y 34 (12.5%) 18 (6.6%) 43 (15.9%) 39 (14.4%) 15-24y 8 (3.0%) 4 (1.5%) 8 (3.0%) 9 (3.3%) 25-44y 11 (4.1%) 8 (3.0%) 18 (6.6%) 20 (7.4%) + 45y 5 (1.8%) 1 (0.4%) 6 (2.2%) 10 (3.7%) Data presented as n. & %.

Table 3-14: Results of microscopic diagnostic methods for detection of Entamoeba complex among study individuals correlated with locality. Normal Concentration Trichrome Locality \ Method Iodine Mount Saline Technique Stain Wad Madani Alkobra 40 (14.8%) 20 (7.4%) 50 (18.5%) 55 (20.3%) Ganoub Algezira 1 (0.4%) 1 (0.4%) 2 (0.8%) 3 (1.1%) Sharg Algezira 20 (7.4%) 12 (4.4%) 24 (8.9%) 24 (8.9%) Um Algura 2 (0.8%) 0 2 (0.8%) 2 (0.8%) Alhasaheesa 0 0 0 1 (0.4%) Data presented as n. & %.

Table 3-15: Results of microscopic diagnostic methods for detection of Entamoeba complex among study individuals correlated with Residency. Normal Concentration Trichrome Iodine Mount Saline Technique Stain Town (166) 40 (24.1%) 19 (11.5%) 49 (29.5%) 49 (29.5%) Village (64) 11 (17.2%) 6 (9.4%) 15 (23.4%) 21 (7.7%) Kampu (41) 12 (29.3%) 8 (19.5%) 15 (36.6%) 15 (36.6%) Data presented as n. & %.

Table 3-16: Results of microscopic diagnostic methods for detection of Entamoeba complex among study individuals correlated with Stool Consistency. Normal Concentration Trichrome Iodine Mount Saline Technique Stain Liquid (49) 17 (34.7%) 12 (24.5%) 23 (46.9%) 24 (42.9%) Soft (221) 46 (20.8%) 21 (9.5%) 56 (25.3%) 61 (27.6%) P value 0.032 0.004 0.002 0.003 Data presented as n. & %. Table 3-17: Results of microscopic diagnostic methods for detection of Entamoeba complex among study individuals correlated with mucus and blood contents. Normal Concentration Trichrome Iodine Mount Saline Technique Stain Mucus (53) 21 (39.6%) 16 (30.2%) 26 (49.1%) 24 (45.3%) Blood (2) 0 0 0 1 (50%) Mixed (3) 1 (33.3%) 0 1 (33.3%) 0 Nil (213) 41 (19.2%) 17 (8.0%) 52 (24.4%) 60 (28.2%) P value 0.003 0.000 0.001 0.009 Data presented as n. & %.

Table 3-18: Results of microscopic diagnostic methods for detection of Entamoeba complex among study individuals correlated with pus cells and RBCs contents. Normal Concentration Trichrome Iodine Mount Saline Technique Stain Pus cells (31) 8 (25.8%) 9 (29.0%) 13 (41.9%) 11 (35.5%) RBCs (45) 23 (51.1%) 11 (24.4%) 22 (48.9%) 34 (75.6%) Mixed (8) 1 (12.5%) 0 1 (12.5%) 0 Nil (187) 31 (16.6%) 13 (7.0%) 43 (23.0%) 40 (21.4%) P value 0.003 0.000 0.001 0.000 Data presented as n. & %.

3.2. Group II 3.2.1. Description of data variables: Regarding the retrospective study, the data was collected at the same period time of that cross sectional study from laboratories records. The records of Wad Medani teaching hospital laboratory (1120) and Arkaweet center (618) contains was showing only the final result of detection of parasite without mentioning of detailed other variables such as age, residence, and other risk factors and microscopic contents examination (pus cells and RBCs).

Table 3-19: Distribution of gender among study Data from the laboratory of Wad Medani teaching hospital. Study individuals (n= 1120)

n. % Male 563 50.27 Gender Female 557 49.73 Total 1120 100

Table 3.20: Results of microscopic examination method for detection of parasites in the laboratory of Wad Medani teaching hospital: Study individuals (n= 1120)

n. %

Protozoa Entamoeba complex 137 12.2

Parasitic stages G. lamblia 128 11.4 E. coli 0 0

Helminthes H. nana eggs 1 0.1 Strogyloides stercoralis 1 0.1 Mixed E. complex/Giardia 6 0.5

Negative 847 75.6 Total 1120 100 Data presented as n. & %. Table 3.21: Distribution of gender among study Data from Arkaweet health center laboratory. Study individuals (n=618)

n. % Male 354 57.3 Gender Female 264 42.7 Total 618 100

Table 3.22: Results of microscopic examination method for detection of parasites in Arkaweet health center laboratory: Study individuals (n=618)

n. %

Protozo Entamoeba complex 172 27.8

Parasitic

stages G. lamblia 175 28.3 E. coli 0 0

Helmint

hes H. nana eggs 8 1.3

Mixed E. complex/Giardia 8 1.3

Negative 255 41.3 Total 618 100 Data presented as n. & %.

Table 3.23: Stool examination results in the laboratories of the main hospitals in Wad Madani town According the Center of Statistic information in the years 2011and 2012: Laboratory activity Laboratory activity 2011 2012 Hospital name Stool Stool Entamoeba Entamoeba samples samples cases cases Wad Madani Teaching 21662 11912 4685 (39.3%) 6200 (28.6%) hospital Pediatric Teaching hospital 3186 3669 295 (8.0%) 286 (9.0%) Obstetrics and gynecology 2749 1165 24 (2.1%) 52 (1.9%) teaching hospital Renal hospital 1392 1227 380 (31.0%) 441 (31.7%) Police hospital 8439 4555 443 (9.7%) 412 (4.9%) Alia'a hospital 114 0 0 5 (4.4%) Total 37542 22528 5827 (25.9%) 7396 (19.7%)

Table 3.24: Outpatient Cases of Amoebiasis in Gezira State Hospitals According the Center of Statistic information in the years 2011 and 2012:

Amoebiasis cases Amoebiasis cases Age and Gender 2011 2012 Male 206 198 - 1 Year Female 216 195 Male 628 620 1-4 Years Female 580 562 Male 884 1159 5-14 Years Female 934 1124 Male 1040 1126 15-24 Years Female 1186 1273 Male 1120 1186 25-44 Years Female 1239 1212 Male 919 917 + 45 Years Female 991 869 Male 4797 (48.2%) 5206 (49.9%) Total Female 5146 (51.8%) 5235 (50.1%) Overall total 9943 10441

Table 3.25: Inpatient Cases of Amoebiasis in Gezira State Hospitals According the Center of Statistic information in the years 2011 and 2012:

Amoebiasis cases Amoebiasis cases Age and Gender 2011 2012 Male 23 6 - 1 Year Female 14 10 Male 56 24 1-4 Years Female 54 15 Male 23 23 5-14 Years Female 26 21 Male 33 11 15-24 Years Female 51 29 Male 45 31 25-44 Years Female 101 49 Male 58 36 + 45 Years Female 74 56 Male 238 (42.7%) 131 (42.1%) Total Female 320 (57.3%) 180 (57.9%) Overall total 558 311

Table 3.26: Patient Cases of Amoebiasis in Gezira State health units According the Center of Statistic information in the years 2011and 2012:

Amoebiasis cases Amoebiasis cases Age and Gender 2011 2012 Male 671 436 - 1 Year Female 722 574 Male 2116 2289 1-4 Years Female 2512 2508 Male 2856 3004 5-14 Years Female 2863 3117 Male 2754 2950 15-24 Years Female 2951 3072 Male 2505 2695 25-44 Years Female 2635 2734 Male 2149 2206 + 45 Years Female 1975 2295 Male 13051(48.9%) 13580(48.7%) Total Female 13658(51.1%) 14300(51.3%) Overall total 26709 27880

Figure 3.4: Overall Cases of Amoebiasis in Gezira State hospitals, health centers and health units According the Center of Statistic information in the years 2011 and 2012: 3.3. Laboratories diagnostic procedures for Amoebiasis The questionnaire data has been collected from different laboratories in wad medani town regarding laboratories diagnostic procedures and the way they perform them was analyzed .

Table 3.27: The Questionnaire data collected from different laboratories regarding their diagnostic procedures for Amoebiasis:

Question Answer 1 Answer 2 Answer 3 Answer 4 Laboratory Numbers 36

No. of stools / week (Mean) 31

Request of the test by.. (Doctor) 15 (Patient) 4 (Both) 17

Tentative diagnosis (Done) 7 (Not done) 23 (Sometimes) 5 (Not Filled) 1 Instruction given to patient (Yes) 29 (No) 7 sample container (screw cap) 34 (Paper box) 2 sample size (Adequate) 18 (Little) 18 collection time labeling (Yes) 21 (No) 12 (Not Filled) 3

Gross exam (Done )33 (Not Done) 2 (Not Filled) 1

Sample processing (Immediate) 34 (Delayed) 2

Type Wet prep (N.S) 33 (Iodine) 1 (N.S & Iod) 2 concentration tech. Perform (Formal ether) 3 (Kato) 1 (Non) 31 (Not Filled) 1 Sample preservation (Yes) 2 (No) 31 ( Sometimes) 1

Type of fixative (Formalin10%)1 1 34

Amoebic dysentery stage (E. histolytica)36

36 34 40

20 94.4% 100.0% 11.1% 4 5.5% 2 0 All Labs. Considered NS + I+ Conc. NS + Iodine Normal Saline E complex as E.h Method Prep. Prep.

Figure 3.5: Diagnostic Procedures Of Amoebiasis In Different Laboratories

4. DISCUSSION A parasitic infection represents a major problem for human health world wide. The World Health Organisation considered that amoebiasis is among the six major killing parasitic diseases of which 50% are of protozoan nature (Noor Azian 2006).

Amoebiasis is defined as infection with E. histolytica, regardless of associated symptoms and signs ranging from asymptomatic colonization to amebic colitis and life-threatening abscesses. It is endemic in developing countries and is seen primarily in travelers to and emigrants from endemic areas (Pritt B. S. 2008).

The clinical diagnosis of amoebiasis remains difficult in most of the cases due to different illness course in different communities and different clinical presentations. However, diagnosis of amoebiasis is usually performed on clinical grounds alone in most of the endemic countries. A fact which makes the actual prevalence and epidemiology of amoebiasis obscure (Ramana K V 2012).

Detection of E. histolytica, the causative agent of amoebiasis and realization that E. histolytica, E. dispar and E. moschkoviskii are distinct but morphologically identical species had a major impact on all aspects of amoebiasis diagnosis and research, so it is an important goal of the clinical parasitology laboratory. The identification of pathogenic Entamoeba from a morphologically identical but non-pathogenic species has highlighted the need for non-microscopic detection methods able to differentiate among them (S J Furrows 2004).

In this study different parasitological techniques were used for the detection of Entamoeba complex, gave collectively good results, Normal saline was superior in detecting motile trophozoite stages in 63 case (23.2%), whereas iodine preparation stained the cystic stages but killed the motile stages and that is why the detected parasites decreased to 33 cases (12.2%), concentration techniques detected more parasites than normal saline because of the increased amount of samples about ten folds condencing the number of parasites to 79 cases (29.2%), it considered the gold standard method nevertheless it is poor in differential diagnosis o species level. Regarding trichrome staining techniques it was superior to other techniques in

61 giving clear details of the morphological features of parasites so it can differentiate them on genus and species levels detecting 85 of E. complex (31.4%) nevertheless it is time consuming. This result agreed with that of (Simonetta Gatti 2002). who showed that microscopy yeilded about 27% of Entamoeba complex detection. The finding (29.2%) is also in accordance with (Magambo JK 1998) in their study on children in southren Sudan in which they found a prevalence of (28.4%) among intestinal parasites .

These results (23.2%, 21.2% and 29.2%) are comparable to similar percentages reported in a study made by (El Shazly AM 2006) on 3180 patients attending Mansoura University Hospital Clinics. Patients were subjected to stool examination by direct wet smear and formal-ether concentration. Prevalence of Entamoeba infection was found to be 20%. Similar prevalence findings were recorded in developed countries, Kaminsky in 1991 performed a study on 266 children in two rural villages in the Honduras. He found that 20% of the children had cysts of Entamoeba in their stools (Kaminsky 1991).

However, Romano and his group detected the prevalence 17.6% as of Entamoeba complex. The findings of his study confirmed a trend of high risk of infection with Entamoeba species among the rural population as shown by other local studies, where widespread poverty, poor socioeconomic condition, low standards of sanitation and hygiene and lack of education attainment may contribute to high prevalence of Entamoeba infection (Romano Ngui 2012). On the other hand lower results were obtained in a study conducted in Egypt by (Makhlouf S A 1994) who studied 100 children living in Ain-Shams, Cairo. 20 children living under appropriate health conditions were studied as controls. Entamoeba prevalence was 9% in his study group compared to 10% in the control group.

Also, the present results (21.2% and 31.4%) disagree with that of (E Malatyalı 2011) who reported that a total of 1449 stool samples were examined by native-Lugol and Trichrome staining, 1.5% (n=22) stool samples were found to be positive for the presence of E. histolytica / E. dispar cysts and these samples were further examined by E .histolytica specific antigen based ELISA. The data reveals that E. histolytica prevalence may be lower than estimated.

Also the study results disagreed with those of Babiker and Karrar who indicated that the prevalence of Entamoeba infection was 4.3% and 2.7% respectively. Thier study was conducted on food handlers attending public health laboratory in Kartoum and under five yeasr children in Kartoum Sudan respectively.

These variations need accurate differentiation of E. histolytica from its identical triplets E. dispar and E. moshkovskii as the current data show that E. dispar is 10 times more prevalent than E. histolytica worldwide, this percentage may vary signiﬁcantly in some regions, this differences make the assessment of prevalence in different geographical regions is necessary (R. Fotedar 2007). In the contrary a higher prevalence rate than obtained in the previously mentioned studies and the present study were reported. Abdalla 2002, examined 84 patients presented with diarrhoea at an outpatient tropical medicine clinic in Cairo. Entamoeba infection was found to be present in (57.1%) of samples by direct method (M. D. Abd-Alla 2002). Also a high prevalence, up to 50% or 80%, have been reported, in some regions. The global prevalence of E. histolytica is around 10% on average (Türkan Toka Özer 2011).

Comparison of concentration method with normal saline mount showed 72.2% sensitivity, 96.9% specificity and a P. value = 0.000, with iodine mount showed 40.5% sensitivity, 99.5% specificity and a P. value = 0.000 and while with trichrome staining method showed 73.4% sensitivity, 85.9% specificity and a P. value = 0.000. in this result also showed that concentration method was considered the gold standard method due to its sensitivity, specificity and reliability over other conventional methods (table 3-7 and 3-8).

In the earlier reports about the prevalence of amoebiasis interpretation is very difficult because older data did not differentiate between morphologically identical species, the non-pathogenic E. dispar morphologically identical to the pathogenic E. histolytica. It is very important to keep in mind that according to the older data, many E.histolytica infections were most probably confused with E.dispar due to limited data obtained from microscopic examinations (Tanyuksel and Petri 2003).This has remarkably affected the estimates of global prevalence of amoebiasis

62 due to E. histolytica. The prevalence and the true epidemiology of amoebiasis are still unclear and need to be clarified.

Other parasites beside Entamoeba complex, were found to be prevalent in this study also, such as Giardia lamblia, Entamoeba coli, Blastocytis hominis, chilomastic mesnilii, Iodamoeba butschulii, Hymenolepis nana and Enterobious vermicularis; which may point to the problems of sanitation and hygiene in the study area.

Molecular diagnostic techniques were performed to resolve the problem of the identical triplets of pathogenic and non pathogenic strains of Entamoeba complex by using mutiplex PCR for 50 samples from which 18 (36%) were positive for Entamoeba histolytica, 2 (4%) for Entamoeba dispar, 2 (4%) for Entamoeba moschkovskii and 1(2%) for mixed Entamoeba histolytica and E. dispar. This result showed more sensitivity in mPCR than convetional microscopic examination.

Comparison of mPCR method as gold standard method with normal saline mount showed 69.6% sensitivity, 33.3% specificity, kappa agreement measures 0.348 and insignificant P. value = 0.829. Comparison with iodine mount showed 26.1% sensitivity, 77.8% specificity, kappa agreement measures 0.354 and insignificant P. value = 0.752. Comparison with concentration method showed 87% sensitivity, 33.3% specificity, kappa agreement measures 0.483 and insignificant P. value = 0.097. And comparison with trichrome staining method showed 91.3% sensitivity, 59.3% specificity, kappa agreement measures 0.822 and significant P. value = 0.000. In this study mPCR method was taken the gold standard method on species level differentiation due to its sensitivity over other conventional methods (table 3- 10 and 3-11).

These results indicate that molcular diagnostic techniques are very important to differentiate pathogenic from non pathogenic strains of Entamoeba complex. The results obtained agreed with Hamza's results in which he indicated the difficulty facing the technicians in morphologically differenting the cysts of Entamoeba and other species using microscopy for routine diagnosis (Hamzah 2006), but the molecular method is very costly and it is unlikely to be adopted in routine use in endemic areas in the near futrure (Ackers 2002).

Although E. histolytica was previously thought to infect 10% of the world‟s population, two morphologically identical but genetically distinct and apparently nonpathogenic Entamoeba species are now recognized as causing most asymptomatic cases. To avoid unnecessary and possibly harmful therapies, clinicians should follow the diagnostic and treatment guidelines of the WHO (B. S. Pritt and C. G. Clark 2008).

Regarding the other variables, this cross sectional study population was represented almost equally gender wise 140 (51.7%) for males and 131(48.8%) for females with Entamoeba complex prevalence 18.5 % for males and 10.7% for females which is in agreement with the global literature (Simonetta Gatti 2002; Ehsan Nazemalhosseini Mojarad 2010).

The results obtained in this study regarding the prevalence of infection in the age group, the relation was found proportional. The lowest prevalence of Entamoeba complex was in young age group less than 5 years (3, 1.1%) which may be due to the fact that they less active in taking food from vendors which make them less exposed to infections than older group, also this group is dependent on their parents or other in their cleaning and washing habits. In the age group 5-14 years (43, 15.9%), these are almost children and pupils who are exposed mostly to infection mostly due to their habit of sharing food and drinks, low immunity and active mode of life. The age group 15-24 years, 25-44 years and more than 45 years were having low prevalence rate of infection (8, 3.0%), (18, 6.6%) and (6, 2.2%) respectively, these groups are more mature than the other age groups and may be caring for hygienic practices, but may be they were more exposed to contamination due to out side home food consuming and out door mode of life.

Most of the study population was living in Wad Madani Town 166 (61.3%), the place where the research was conducted. The villagers were 64 (23.6%) who may have some relatives and some other interests leading them to come down to the town especially for medical care. Kampu poeples 41 (15.1%) were living at outskirts of the town on temporary settelment.

It was very clear that the hygienic status of the community affects the prevalence of Entamoeba complex very much in the study group as follows: in the town 49 (29.5%) were psitive for Entamoeba complex, village 15 (23.4%) and Kampu 15 (36.6%) respectively. The high incidence in Kampu residence (36.6%) may be due to the very poor hygienic status, as well as that they in least socioeconomic situation.

Regarding the stool consistency gross examination, 49 (18.1%) stool samples were liquid, 23 (46.9%) were positive for Entamoeba complex. The rest of the samples 221 (81.5%) were soft in appearance yeilded 56 (25.3%) Entamoeba complex. Although all samples were taken from symptomatic individuals (Diarrhea or abdomainal pain/cramp as stated in inclusion criteria in the study population) but 213 (78.6%) of stool samples look normal stools without blood or mucus contents which reflect the nature of asymptomatic infetion of Entamoeba complex, (24.4%) (Blessmann 2002).

Among all samples (271), microscopic contents of the stool such as pus cells were 53 samples, among which 26 (49.0%) Entamoeba complex were detected by conventional methods. RBCs were in 45 samples which yeilded 22 (48.9%) Entamoeba complex. This result shows that the presence of microscopic contents in stool such as Pus cells and RBCs can be considered as indicator for infection (table 3-18 to 3-20). The rest of the samples 187 (69%) showed neither pus cells nor RBCs.

These results disagreed with that of Saeed et al in which the infection rate in symptomatic and asymptomatic samples was very high (54%) (Amir Saeed 2011).

Results of microscopic diagnostic methods for detection of Entamoeba complex among study individuals when correlated with gender revealed that the prevalence of infection among males was higher than in females, (18.5 % for males and 10.7% for females) this may be due to the mode of life such as contact with cotaminated sources out side homes which is more likely in males than females.

When comparing the 23.2% detection of Entamoeba complex in the cross sectional study with the recorded study prevalence of Wad Medani teaching hospital central laboratory and Arkaweet health center Laboratory 12.2% and 27.8% respectively in the same period they look comparable. The two laboratories use only normal saline method and all cases of Entamoeba complex considered as Entamoeba histolytica only. Also other factors of variability of the labs staff whom were having a high turnover and there are no written standards operating procedure (SOPs) in the two laboratories.

In comparison of prevalence results obtained from Wad Medani hospital central laboratory (12.2%), with that prevalence obtainedfor the same laboratory from the Center of Health Information in the Ministry of Health, Gezira State (34%), these two figures are completely different and difficult to explain. One possibility is manner that the laboratory staff reporting results to the center and not keeping proper records.

Based on the retrospective data collected from the center of health information, the Ministry of Health, Gezira State about stool examination in the main laboratories in Wad Medani town, the records showed that prevalence of Entamoeba complex in Gezira Hospital for renal diseases and surgery laboratory was (31%), which is the second to Wad Medani hospital central laboratory.

This result was different from the present cross sectional study result which indicated the nature of how the diagnostic procedures were performed and the dependency on a single method in diagnosis, (normal saline) which is not sufficient. The records contain only the final result of detection of parasite without mentioning the detailed gross and microscopic examination. This situation needs to be improved by training (Gonin and Trudel 2003) so as to reduce reporting of non pathogenic amoebae as pathogenic and hence their percentages.

The renal hospital laboratory high incidence result may be justified due to the fact that renal failure patients are usually immuno compromised and (also some other parasites such as coccidia may be found).

Although the Ministry of Health center of health information records showed pediatric teaching hospital has 9.0% of Entamoeba cases but the laboratory has no kept recorded data for these results. This proves the importance of proper registration system in the laboratory, and keeping of information and data.

The overall cases of amoebiasis (Entamoeba histolytica infection) in outpatients and inpatients wards in addition to patients attending health units in Gezira State according to the center of statistic information for the last two years, showed higher prevalence in the outpatient, about (72.0%) indicating a serious situation.

The frequently quoted estimated total 500 million global cases of E. histolytica is very misleading and it is more likely that E. histolytica is responsible for only 10% of these infections (50 million) worldwide, while E. dispar is responsible for the rest (Windell L. RIVERA 1999). These comparisons should encourage health decision makers and other concerned parties to address this problem especially registration, documentation and recording system.

The data collected from different laboratories for the diagnostic procedures followed for amoebiasis, were analyzed and showed that 33 out 36 (91.7%) laboratories were using normal saline preparation only as the sole diagnostic procedure for amoebiasis and only 3 (8.3%) were performing concentration technique for stool analysis.

All laboratories in the study area consider the detected Entamoeba, whatever species, as pathogenic Entamoeba histolytica and there was no differentiation between species even from Entamoeba coli or others.

The recommendation of the WHO-Pan American Health Organization United Nations Educational, Scientific, and Cultural Organization (UNISCO) to develop improved methods for the specific diagnosis of E. histolytica infection is very important for the establishment of accurate prevalence data of E. histolytica and E. dispar infections worldwide (Windell L. RIVERA 1999).

4.1. Limitation of the study: - Difficulty in searching the laboratories records. - No proper recording system in many Laboratories. - The stools collected in the laboratories are from symptomatic patients. - No proper clinical data were taken in these laboratories.

4.2. CONCLUSION AND RECOMMENDATIONS

4.2.1. Conclusion.

This study, is the first study conducted in the Sudan to focus on all most aspects of Entamoeba parasites diagnosis to evaluate conventional methods for improving the diagnosis of Entamoeba parasites in contrast with molecular based techniques and retrospective data records. It is hoped that it will contribute to the development and improvement of laboratory diagnosis of parasitic infections.

`The study concluded that up to date, there are still wide gaps in our knowledge of species prevalence rates in different regions of the world particularly in Africa and very few studies were conducted using molecular methods.

In order to address this limitation, there is need to execute species-specific diagnosis of E. histolytica, E. dispar and E. moshkovskii, particularly in countries where these organisms are endemic and construct molecular parasitology unit to look after such work.

PCR based techniques are the most recent advance in technology, however conventional methods of diagnosis are still important as they are the easiest and cheapest to perform, regarding proper and correct way of use.

The reported result must be written as Entamoeba complex because of the identity of the triple Entamoeba and the impossibility to differentiate them from each other unless by PCR.

Based on the limited information available to date it appears that molecular and genomic studies are still needed to make our students and researchers aware about these parasites concerns and the disease they cause.

4.2.2. Recommendations.

1. Normal saline preparation is recommended to demonstrate motility of Entamoeba complex trophozoites. 2. Iodine mount preparation is recommended to confirm the finding made in the saline preparation especially cysts. 3. The confirmation of any wet preparation by permanent stained smears is recommended and calibration of the microscope is essential for parasitic measurements. 4. Combination of conventional methods is recommended as diagnostic procedure of Entamoeba complex. 5. Polyvinyl alcohol ( PVA) is recommended, as fixative for any stool sample prepared to be preserved and stained with Gomori,s trichrome stain for differential diagnosis and teaching purposes. 6. Standard Operating Procedures (SOPs) in all laboratories and well prepared registration and documentation system must be followed. 7. Where it is possible PCR techniques must be used especially in controversial infections to avoid useless treatment. 8. Further researches and collaborations between scientists is essential in answering questions on the epidemiology of amoebiasis and to fill in the gaps in our understanding about Entamoeba complex.

4.3. REFERENCES

Abd-Alla M D, W. A. A., Ravdin J I (2000). "Comparison of antigen-capture ELISA to stool-culture methods for the detection of asymptomatic entamoeba species infection in Kafer Daoud, Egypt;." Am. J. Trop. Med. Hyg. 62: 579–582. AbuOdeh, A. S. A. E. R. (2012). "Amoebiasis in the Tropics: Epidemiology and Pathogenesis." Current Topics in Tropical Medicine , InTech,. Ackers, J. P. (2002). "The diagnostic implications of the separation of Entamoeba histolytica and Entamoeba dispar " J Biosci 27: 573-578. Ambrosio, R. E. d. W., D. T. (1990). "Diagnosis of parasitic disease." Rev Sci Tech 9(3): 759- 778. Amir Saeed, H. A., Birgitta Evengard. , Gunnar Sandstrom (2011). "Epidemiology of entamoeba infection in Sudan " African Journal of Microbiology Research 5(22): 3702-3705. B. S. Pritt and C. G. Clark (2008). "Amebiasis." Mayo Clin Proc 83(10): 1154-1160. Beltramino, J. S., H. Gamba, N. Busquets, N. Navarro, L. Virgolini, S. Ricardo, O. (2009). "Overdiagnosis of amebiasis in children with dysentery." Arch Argent Pediatr 107(6): 510-514. Blessmann, J. B., H. Nu, P. A. Dinh, B. T. Ngo, Q. T. Van, A. L. Alla, M. D. Jackson, T. F. Ravdin, J. I. Tannich, E. (2002). "Real-time PCR for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in fecal samples." J Clin Microbiol 40(12): 4413-4417. Chen, Y. Z., Y. Yang, B. Qi, T. Lu, H. Cheng, X. Tachibana, H. (2007). "Seroprevalence of Entamoeba histolytica infection in HIV-infected patients in China." Am J Trop Med Hyg 77(5): 825-828. Clark, M. Z. A. C. G. (2001). "Isolation and Characterization of Polymorphic DNA from Entamoeba histolytica." Journal Of Clinical Microbiology 39(3): 897±905. Dawn Britten, S. M. W., Peter L. Chiodini, Ruth Mcnerney,Anthony H. Moody, And John P. Ackers (1997). "An Improved Colorimetric PCR-Based Method for Detection and Differentiation of Entamoeba histolytica and Entamoeba dispar in Feces." Journal Of Clinical Microbiology 35(5): 1108-1111. Dolo A, C. G., Traoré F, Traoré S, Kassambara L, Diakité M, Camara F, Doumbo O. (1996). "Protozoan infections and intestinal helminthiasis among the population of a village in the northern Sudan savannah area of Mali (West Africa)." Parasitologia 38(3): 585-589. E Malatyalı, S. Ö., AÇeliksöz (2111). "The Investigation of Entamoeba histolytica Prevalence in Some Villages of Sivas by ELISA Method." Turkiye Parazitol Derg 35: 6-9. Egbert Tannich and Gerd, D. B. (1991). "Differentiation of Pathogenic from Nonpathogenic Entamoeba histolytica by Restriction Enzyme Fragment Analysis of a Single Gene Amplified in Vitro." Journalof Clinical Microbiology 29(2): 250-255. Ehsan Nazemalhosseini Mojarad, Z. N., Navid Sahebekhtiari, Mohammad Rostami Nejad, Hossein Dabiri ali Ali Haghighi (2010). "Characterization of Entamoeba histolytica and Entamoeba dispar in fresh stool by PCR " Gastroenterology and Hepatology From Bed to Bench 3(1): 37-41. El Shazly AM, A. S., Sultan DM, Sadek GS, Khalil HH, Morsy TA (2006). "Intestinal parasites in Dakahlia governorate, with different techniques in diagnosing protozoa." Journal of the Egyptian Society of Parasitology 36(3): 1023-1034. Enrique Gonza´Lez, G. R., Guillermo Mendoza, Fernando Ramos, Gabriela Garci´A, Patricia Mora´N, Alicia Valadez, Emma I. Melendro, And Cecilia Xime (2002). "Calreticulin- Like Molecule In Trophozoites Of Entamoeba Histolytica HM1:IMSS (SWISSPROT: ACCESSION P83003)." Am. J. Trop. Med. Hyg. 67(6): 636-639.

Garcia, L. S. (1993). "Classification of human parasites, vectors, and similar organisms." Clin Infect Dis 16(5): 614-615. Garcia, L. S., T. C. Brewer, et al. (1979). "A comparison of the formalin-ether concentration and trichrome-stained smear methods for the recovery and identification of intestinal protozoa." Am J Med Technol 45(11): 932-935. Garcia, L. S. S., R. Y. Bernard, C. N. (2000). "Detection of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum antigens in human fecal specimens using the triage parasite panel enzyme immunoassay." J Clin Microbiol 38(9): 3337-3340. Ghosh, S. F., M. Ramirez-Avila, L. Descoteaux, S. Sturm-Ramirez, K. Newton-Sanchez, O. A. Santos-Preciado, J. I. Ganguly, C. Lohia, A. Reed, S. Samuelson, J. (2000). "Molecular epidemiology of Entamoeba spp.: evidence of a bottleneck (Demographic sweep) and transcontinental spread of diploid parasites." J Clin Microbiol 38(10): 3815-3821. Gonin, P. and L. Trudel (2003). "Detection and differentiation of Entamoeba histolytica and Entamoeba dispar isolates in clinical samples by PCR and enzyme-linked immunosorbent assay." J Clin Microbiol 41(1): 237-241. Hamzah, Z. P., S. Mungthin, M. Leelayoova, S. Chavalitshewinkoon-Petmitr, P. (2006). "Differential detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii by a single-round PCR assay." J Clin Microbiol 44(9): 3196- 3200. Haque R, A. I. K., Akther S and Petri W A Jr (1998). "Comparison of PCR, isoenzyme analysis, and antigen detection for diagnosis of Entamoeba histolytica infection." J. Clin. Microbiol. 36: 449-452. Haque R, M. N. U., Ali I K, Alam K, Eubanks A, Lyerly D and Petri W A Jr (2000). "Diagnosis of amoebic liver abscess and intestinal infection with the TechLab Entamoeba histolytica II antigen detection and antibody tests." J. Clin. Micro- biol. 38( 3235– 3239). Hung, C. C. D., H. Y. Hsiao, W. H. Hsieh, S. M. Hsiao, C. F. Chen, M. Y. Chang, S. C. Su, K. E. (2005). "Invasive amebiasis as an emerging parasitic disease in patients with human immunodeficiency virus type 1 infection in Taiwan." Arch Intern Med 165(4): 409-415. Ivory, C. P. and K. Chadee (2007). "Intranasal immunization with Gal-inhibitable lectin plus an adjuvant of CpG oligodeoxynucleotides protects against Entamoeba histolytica challenge." Infect Immun 75(10): 4917-4922. Jensen, B., W. Kepley, et al. (2000). "Comparison of polyvinyl alcohol fixative with three less hazardous fixatives for detection and identification of intestinal parasites." J Clin Microbiol 38(4): 1592-1598. Kaminsky, R. G. (1991). " Parasitism and diarrhoea in children from two rural communities and marginal barrio in Honduras." Trans R Soc Trop Med Hyg. 85(1): 70-73. Karrar ZA, R. F. (1995). "Prevalence and risk factors of parasitic infections among under-five Sudanese children: a community based study." East Afr Med J 72(2): 103-109. Katzwinkel-Wladarsch, S. L., T. Rinder, H. (1994). "Direct amplification and differentiation of pathogenic and nonpathogenic Entamoeba histolytica DNA from stool specimens." Am J Trop Med Hyg 51(1): 115-118. L. Naing, T. W., B.N. Rusli (2006). "Practical Issues in Calculating the Sample Size for Prevalence Studies " Archives of Orofacial Sciences 1: 9-14. Louis S. Diamond, G. C. C. (1993). "A Redescription of Entamoeba histolytica Schaudinn, 1903 (Emended Walker, 1911) Separating It from Entarnoeba &par Brumpt, 1925." J. Euk. Microbiol. 40(3): 340-344.

Lowther, S. A. D., M. S. Hanson, D. L. (2000). "Entamoeba histolytica/Entamoeba dispar infections in human immunodeficiency virus-infected patients in the United States." Clin Infect Dis 30(6): 955-959. M. D. Abd-Alla, J. I. R. (2002). "Diagnosis of amoebic colitis by antigen capture ELISA in patients presenting with acute diarrhoea in Cairo, Egypt." Tropical Medicine and International Health 7(4): 365-370 M.A. Babiker, M. S. M. A. a. E. S. A. (2009). "Frequency of intestinal parasites among food- handlers in Khartoum, Sudan." Eastern Mediterranean Health Journal 15(5): 1098- 1103. Magambo JK, Z. E., Wachira TM (1998). "Prevalence of intestinal parasites among children in southern Sudan." East African Medical Journal 75 (5): 288-290. Makhlouf S A, S. M. A., Mahmoud D M, Mohamad A A. (1994). "Parasitic infection among children living in two orphanages in Cairo." J Egypt Soc Parasitol. 24(1): 137-145. Mehreen Zaki, P. M., Wei Sun and C. Graham Clark (2002). " "Simultaneous Differentiation and Typing of Entamoeba histolytica and Entamoeba dispar." " J. Clin. Microbiol. 40(4): 1271-1276. Ngindu, A., K. Kamar, et al. (2002). "Survey of faecal parasites in patients from western Kenya." J Egypt Soc Parasitol 32(1): 1-7. Noor Azian, M. Y., Lokman Hakim, S and Maslawaty, M.N (2006). " Use of molecular tools to distinguish Entamoeba histolytica and Entamoeba dispar infection among the aborigines in Cameron Highlands." Tropical Biomedicine 23(1): 31-36. Pillai, D. R. and K. C. Kain (1999). "Immunochromatographic strip-based detection of Entamoeba histolytica-E. dispar and Giardia lamblia coproantigen." J Clin Microbiol 37(9): 3017-3019. Pillai, D. R. K., J. S. Sheppard, D. C. MacLean, J. D. MacPherson, D. W. Kain, K. C. (1999). "Entamoeba histolytica and Entamoeba dispar: epidemiology and comparison of diagnostic methods in a setting of nonendemicity." Clin Infect Dis 29(5): 1315- 1318. Pritt B. S., C. C. G. (2008). "Amebiasis." Mayo Clin Proc 83(10): 1154-1160. R. Fotedar, D. S., N. Beebe, D. Marriott, J. Ellis, and J. Harkness (2007). "PCR Detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in Stool Samples from Sydney, Australia." Journal of Clinical Microbiology 45(3): 1035-1037. Ramana K V , K. P. G. (2012). "Conventional Microscopy Versus Molecular and Immunological Methods in the Diagnosis of Amoebiasis." Annals of Medical and Health Sciences Research 2(2). Rashidul Haque, L. M. N., Pauline Hahn, And William A. Petri, Jr. (1995). "Rapid Diagnosis of Entamoeba Infection by Using Entamoeba and Entamoeba histolytica Stool Antigen Detection Kits." Journal Of Clinical Microbiology 33(10): 2558-2561. RIVKA BRACHA, Y. N. a. D. M. (2002). "Amoebapore is an important virulence factor of Entamoeba histolytica." J. Biosci. 27(6): 579-587. Romano Ngui, L., A. Siti Aminah Fakhrurrazi, Yvonne AL Lim, Ling Y Lau, Jamaiah Ibrahim and Rohela Mahmud (2012). "Differentiating Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii using nested polymerase chain reaction (PCR) in rural communities in Malaysia." Parasites & Vectors 5: 187. Rossignol, J. F. A., A. Ayers, M. S. (2001). "Treatment of diarrhea caused by Giardia intestinalis and Entamoeba histolytica or E. dispar: a randomized, double-blind, placebo-controlled study of nitazoxanide." J Infect Dis 184(3): 381-384. S J Furrows, A. H. M., P L Chiodini (2004). "Comparison of PCR and antigen detection methods for diagnosis of Entamoeba histolytica infection." J Clin Pathol 57: 1264- 1266.

Sharp, S. E. S., C. A. Duran, Y. Poppiti, R. J. (2001). "Evaluation of the Triage Micro Parasite Panel for detection of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum in patient stool specimens." J Clin Microbiol 39(1): 332-334. Simonetta Gatti, G. S., Francisco Robinson, Mariella Anselmi, Javier Corrales, Juan Moreira, Gregorio Montalvo, Antonella Bruno, Roberta Maserati, Zeno Bisoffi, And Massimo Scaglia (2112). "Amebic Infections Due To The Entamoeba histolytica−Entamoeba dispar Complex: A Study Of The Incidence In A Remote Rural Area Of Ecuador." Am. J. Trop. Med. Hyg. 67(1): 123-127. Sinchez-GuillCn, M. C., Arguello-Garcia, R., Garduiio, G., Valadez-Salazar, A., Martinez- Garcia, M. C., Muiioz, 0. & Ortega-Pierres, M. G. (1990). "Study of human Entamoeba histolytica infection by zymodeme, indirect hemagglutination and electroimmunotransfer blot assays." Arch. Invest. Med. (Mex.) 21((Suppl. 1)): 209- 215. Smith, L. C. (1993). "Membrane and intracellular effects of ultraviolet irradiation with Hoechst 33342 on bovine secondary oocytes matured in vitro." J Reprod Fertil 99(1): 39-44. Tachibana, H. K., S. Okuzawa, E. and Masuda, G. (1992). "Detection of pathogenic Entamoeba histolytica DNA in liver abscess fluid by polymerase chain reaction." Int. J. Parasitol. 22: 1193–1196. Tannich, E. and G. D. Burchard (1991). "Differentiation of pathogenic from nonpathogenic Entamoeba histolytica by restriction fragment analysis of a single gene amplified in vitro." J Clin Microbiol 29(2): 250-255. Tanyuksel, M. and W. A. Petri, Jr. (2003). "Laboratory diagnosis of amebiasis." Clin Microbiol Rev 16(4): 713-729. Troll, H. M., H. Weiss, N. (1997). "Simple differential detection of Entamoeba histolytica and Entamoeba dispar in fresh stool specimens by sodium acetate-acetic acid- formalin concentration and PCR." J Clin Microbiol 35(7): 1701-1705. Trudel, P. G. a. L. (2003). "Detection and Differentiation of Entamoeba histolytica and Entamoeba dispar Isolates in Clinical Samples by PCR and Enzyme-Linked Immunosorbent Assay." JOURNAL OF CLINICAL MICROBIOLOGY 41(1): 237–241 Tsai, J. J. S., H. Y. Ke, L. Y. Tsai, K. S. Chang, S. Y. Hsieh, S. M. Hsiao, C. F. Yen, J. H. Hung, C. C. Chang, S. C. (2006). "Higher seroprevalence of Entamoeba histolytica infection is associated with human immunodeficiency virus type 1 infection in Taiwan." Am J Trop Med Hyg 74(6): 1016-1019. Türkan Toka Özer, E. Y., Özcan Deveci, Alicem Tekin, Süleyman Durmaz, Keramettin Yanık (2011). "Investigation of Entamoeba histolytica in stool specimens by direct microscopic examination and ELISA in a hospital." Dicle Medical Journal 38 (3): 294-297. Upcroft, J. A. and P. Upcroft (2001). "Drug susceptibility testing of anaerobic protozoa." Antimicrob Agents Chemother 45(6): 1810-1814. Willhoeft, U. H., L. Tannich, E. (1999). "A DNA sequence corresponding to the gene encoding cysteine proteinase 5 in Entamoeba histolytica is present and positionally conserved but highly degenerated in Entamoeba dispar." Infect Immun 67(11): 5925-5929. Windell L. R., Hiroshi T, et al. (1999). "Application of the Polymerase Chain Reaction (PCR) in the Epidemiology of Entamoeba histolytica and Entamoeba dispar Infections." Tokai J Exp Clin Med. 23(6): 413-415. Windell L. RIVERA, H. T. a. H. K. (1999). "Application of the Polymerase Chain Reaction (PCR) in the Epidemiology of Entamoeba histolytica and Entamoeba dispar Infections

" Tokai J Exp Clin Med. 23(6): 413-415. Yury O. Nu´N˜Ez, M. A. F. N., Diamela Torres-Nu´Nez, Jose A. Silva, Ivon Montano, Jorge L. Maestre, And Luis Fonte (2001). "Multiplex Polymerase Chain Reaction Amplification And Differentiation Of Entamoeba Histolytica And Entamoeba Dispar Dna From Stool Samples." Am. J. Trop. Med. Hyg. 64(5, 6): 293-297.