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Molecular-based diagnosis of Entamoeba histolytica infection

Christopher D. Huston, Rashidul Haque and William A. Petri, Jr

Infection with Entamoeba histolytica, the protozoan parasite that causes amoebic colitis and liver abscess, results in 34 million to 50 million symptomatic cases of amoebiasis (all illnesses caused by E. histolytica, including amoebic dysentery) worldwide each year, causing 40 thousand to 100 thousand deaths annually. As a result of accruing biochemical, genetic and immunological data, E. histolytica was re-defined in 1993 to recognise the existence of two morphologically identical but genetically distinct human parasites: E. histolytica, the aetiological agent of invasive intestinal and extraintestinal amoebiasis, and Entamoeba dispar, a non-pathogenic intestinal parasite. Because microscopy is unable to distinguish between these two organisms, it should no longer be relied upon to diagnose amoebiasis. Sensitive and specific molecular techniques that are able to distinguish E. histolytica from E. dispar have been developed recently; they include (1) the detection of an E. histolytica antigen using an enzyme-linked immunosorbent assay (ELISA), (2) the use of the polymerase chain reaction (PCR) to amplify amoebic DNA, and (3) the culture of stool samples followed by isoenzyme analysis. Of these three test methods, only antigen detection using ELISA can be performed rapidly and easily, making it the diagnostic test method of choice for clinical use in the developing world, where the morbidity and mortality caused by E. histolytica are greatest. However, the PCR method is a powerful tool for the genetic typing of different amoebic strains. Together these two methods should result in both improved clinical diagnosis and treatment of amoebiasis, and a greater understanding of the epidemiology of E. histolytica. Such knowledge will not only assist public health efforts to control amoebiasis, but also facilitate the careful testing of the anti-amoebic vaccines that are currently being developed.

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Entamoeba histolytica has recently been re-classified as two genetically distinct but morphologically identical intestinal parasites: one pathogenic and one benign. The name E. histolytica (first used by Schaudinn in 1903 and amended by Walker in 1911) has been retained for the protozoan parasite that causes invasive intestinal and extraintestinal amoebiasis, and the name Entamoeba dispar (designated by Brumpt in 1925) has been revived to refer to the non-pathogenic intestinal commensal organism that is visually indistinguishable from E. histolytica (Ref. 1).

Epidemiology
The true epidemiology of E. histolytica infection remains unknown, because much of the published epidemiological data fail to distinguish between E. histolytica and E. dispar. Fortunately, most of the individuals who were previously believed to have asymptomatic infection with E. histolytica actually carry E. dispar, which has never been shown to cause invasive human disease (Ref. 1). Furthermore, only ~10% of individuals who become infected with E. histolytica actually develop invasive disease (Ref. 2). The best current estimate is that E. histolytica causes between 34 million and 50 million symptomatic infections (i.e. amoebic colitis or liver abscess) each year (Ref. 3). The occurrence of amoebic liver abscess is probably 5–50 times less common than that of diarrhoea (Ref. 3). Although E. histolytica infection occurs worldwide, morbidity and mortality are greatest in Central and South America, Africa and the Indian subcontinent (Ref. 4). In Dhaka, Bangladesh, where diarrhoeal diseases are the leading cause of death in children younger than six years of age, ~50% of children have serological evidence of exposure to E. histolytica by five years of age (Ref. 5). An estimated 40 thousand to 100 thousand deaths from amoebiasis occur worldwide annually, which makes amoebiasis second only to malaria as a cause of death resulting from a protozoan parasite (Ref. 6).

Life cycle and pathogenesis
The life cycle of E. histolytica consists of two stages: an infective cyst and an invasive trophozoite (Fig. 1; fig001wpc). The trophozoites measure 10–50 mm in diameter and contain a single nucleus with a central karyosome. The cysts measure 10–15 mm in diameter and typically contain four nuclei. E. histolytica cysts, which are resistant to acidification, chlorination and desiccation, and capable of surviving in a moist environment for several weeks, are spread via the ingestion of faecally contaminated food or water. After ingestion of the cysts by a human host, excystation occurs within the lumen of the small intestine. During excystation, nuclear division is followed by cytoplasmic division, giving rise to eight trophozoites (Ref. 7). The trophozoites then reside in the lumen of the caecum and large intestine, where they adhere to the colonic mucus and epithelial layers; adherence is due to the interaction of a D-gal- or GalNAc- (D-galactose- or N-acetyl-D-galactosamine-) inhibitable amoebic lectin with host-derived glycoconjugates (Refs 8, 9, 10). Most of the individuals who become infected with E. histolytica are asymptomatically colonised (Ref. 2); re-encystation of the trophozoites occurs within the lumen of the colon, resulting in the excretion of cysts in the faeces and continuation of the life cycle. Alternatively, for reasons that remain unclear, E. histolytica trophozoites can invade the colonic epithelium, causing amoebic colitis. Tissue invasion is facilitated by the secretion of a family of amoebic cysteine proteinases, proteolytic enzymes that disrupt the intestinal mucus and epithelial barrier (Ref. 11). E. histolytica can spread in the bloodstream (haematogenously) after it has penetrated the colonic epithelium and can establish persistent extraintestinal infections (most commonly amoebic liver abscess).

Clinical disease syndromes
Individuals who are infected with E. histolytica can either remain asymptomatic or present clinically with dysentery or extraintestinal disease (Fig. 1; fig001wpc). Approximately 10% of infected individuals develop invasive amoebiasis (Ref. 2).

Amoebic colitis
The diagnosis of amoebic dysentery should be considered when patients present with microscopically detectable (occultly) or grossly bloody diarrhoea. The diagnostic challenge is to distinguish amoebiasis from dysentery that is caused by bacteria [e.g. Shigella spp., Campylobacter spp., Salmonella spp. and enteroinvasive or enterohaemorraghic types of Escherichia coli] as well as from non-infectious causes of bloody diarrhoea including inflammatory bowel disease, ischaemic colitis, and gastrointestinal bleeding caused by diverticuli and arteriovenous malformations (Ref. 4). Typically, bacterial dysentery occurs suddenly, whereas amoebic dysentery occurs gradually, with symptoms [such as abdominal pain and tenderness, painful sudden bowel evacuation (tenesmus), and diarrhoea] developing over a period of one to several weeks (Ref. 12). Fever occurs in only a minority of patients with amoebic colitis, and only microscopically detectable blood is present in a majority. Because of the chronicity of the illness, weight loss is common (Ref. 4).

The occurrence of amoebic colitis with toxic megacolon is the most feared complication of intestinal amoebiasis. Toxic megacolon is an acute dilation of the colon, which occurs in ~0.5% of cases and, unless promptly recognised and treated surgically, is associated with a 40% mortality rate as a result of sepsis (Refs 13, 14). Other complications of intestinal amoebiasis include amoeboma (resulting from the formation of granular tissue extending into the lumen of the intestine), which can mimic carcinoma of the colon, and fistula formation.

Amoebic liver abscess
Liver abscess is overwhelmingly the most common manifestation of amoebiasis occurring outside the intestine (extraintestinal). This complication is 10 times more common in adult men than in adult women. Children rarely develop amoebic liver abscess, but interestingly the sex distribution of those who do is equal (Ref. 15). The typical patient with amoebic liver abscess is a 20–40-year-old male with a 1–2-week history of fever and diffuse or right, upper quadrant abdominal pain (Ref. 4). An amoebic liver abscess might be mistakenly diagnosed as a bacterial abscess, necrotic hepatoma (a type of liver neoplasm) or echinococcal cyst (a type of helminth infection of the liver; typically, an echinococcal cyst would be an incidental finding detected by imaging of the liver and unrelated to any abdominal symptoms). Most of the patients who present clinically with an amoebic liver abscess do not have coexistent dysentery, although a past history of dysentery is common.

Diagnosis of E. histolytica infection
Historically, light microscopy has been the method of choice to diagnose amoebiasis. Although the presence of haematophagous amoebic trophozoites (i.e. trophozoites that have ingested red blood cells) in a stool sample (faeces) strongly suggests E. histolytica infection, such a finding is rarely seen (Ref. 16). In the absence of haematophagous trophozoites, the sensitivity of microscopy is limited by its inability to distinguish between samples infected with E. histolytica and those infected with E. dispar (which is ~10 times more common). Confusion between E. histolytica, other non-pathogenic amoebae (such as Endolimax nana), and white blood cells (leucocytes) in the faeces frequently results in overdiagnosis of amoebiasis (Ref. 17). Delays in the processing of stool samples affect the sensitivity of light microscopy, which under the best of circumstances is only 60% of that of the stool culture method (which involves growing organisms that are found in the faeces in tissue culture; Refs 17, 18).

A method that involves the culture of stool samples followed by isoenzyme analysis can accurately distinguish E. histolytica from E. dispar, and is considered to be the 'gold standard' for diagnosis. However, this method takes between one and several weeks to carry out and requires special laboratory facilities, making it impractical for use in the developing world, where most cases of amoebiasis occur. Moreover, delays in the processing of stool samples and anti-microbial treatment before samples are obtained can lead to negative culture results (i.e. no detection of E. histolytica) even in those patients in whom cysts have been shown to be present by light microscopy (Ref. 19). There is confusion about the epidemiology of amoebiasis as a result of it not being possible to make an accurate diagnosis of amoebic infection in those countries where amoebiasis is most prevalent because the resources are not available. For this reason, the World Health Organisation (WHO), the Pan American Health Organization (PAHO) and the United Nations Educational, Scientific and Cultural Organization (UNESCO) published a joint statement in 1997, which stressed the urgent need to develop improved methods for the specific diagnosis of E. histolytica infection using technologies that are appropriate for developing countries (Ref. 6).

Molecular diagnostic tests for E. histolytica infection
Two new test methods that involve ELISAs to detect antigens in stool samples and PCR are able to distinguish accurately between infection with E. histolytica and E. dispar. They are replacing microscopy and stool culture followed by isoenzyme analysis for both clinical and research purposes. Serological test methods that detect the presence of anti-amoebic antibodies also remain useful.

ELISA methods
Several investigators have developed ELISAs that detect amoebic antigens in fresh stool samples with a sensitivity approaching that of the stool culture method. Furthermore, several ELISA kits are now commercially available (Refs 18, 19, 20, 21, 22, 23, 24, 25, 26). At the time of writing, however, only one, the TechLab E. histolytica test, can distinguish between E. histolytica and E. dispar infections (Ref. 27). This test uses a monoclonal antibody against an amoebic adherence lectin that is inhibitable by Gal or GalNAc. The lectin is conserved and highly immunogenic, and because of antigenic differences between the lectins of E. histolytica and E. dispar, it can be used to identify the pathogenic species. The sensitivity of this method for the detection of antigens in the stools of patients with amoebic colitis is >85%, and its specificity when compared with that of the stool culture method is >90% (Ref. 28). The ELISA method might also prove useful for the detection of E. histolytica lectin antigen in the sera of patients with amoebic colitis and liver abscess. In patients from Egypt, 42% of those with asymptomatic E. histolytica infection and 57% of patients with symptomatic colitis had detectable levels of lectin antigen in their serum samples (Ref. 20). In Dhaka, Bangladesh, 94% (15/16) of patients with amoebic liver abscess had detectable levels of lectin antigen in their serum samples before treatment. However, the sensitivity of this method was only 16% (3/19) after 7 days of treatment with metronidazole (R. Haque and W.A. Petri, unpublished).

PCR-based methods
Several PCR-based methods that amplify and detect E. histolytica DNA in stool samples have also been developed (Refs 29, 30, 31, 32, 33). The sensitivity and specificity of PCR-based methods for the diagnosis of E. histolytica infection both approach those of stool culture followed by isoenzyme analysis. When used to detect the presence of trophozoites in culture, PCR amplification and detection of small, ribosomal RNA (rRNA) genes is ~100 times more sensitive than the best currently available ELISA kit for the detection of E. histolytica antigens (Ref. 27). However, field studies that directly compare the rRNA PCR method with the stool culture or antigen detection methods for the diagnosis of E. histolytica infection suggest that these three different methods perform equally well. Single stool samples obtained from 98 patients with diarrhoea, in Dhaka, Bangladesh, were cultured for the presence of E. histolytica and also tested with both the PCR and antigen detection methods. 88 patients were shown to have stool cultures that were positive for E. histolytica and/or E. dispar, of which 53 were subsequently identified by isoenzyme analysis to be infected with E. histolytica. The PCR and antigen detection methods were equally sensitive when applied to fresh stool samples, identifying 87% (46/53) and 85% (45/53), respectively. Importantly, of the three diagnostic methods used in this study, only antigen detection using ELISA was both rapid and technically simple to perform, making it appropriate for use in the developing world, where amoebiasis is most prevalent (Ref. 28).

Serological methods
Serological methods can also be used to distinguish accurately between prior infection with E. histolytica and E. dispar. Patients infected with E. dispar do not develop serum anti-amoebic antibody titres, but 75–85% of patients with symptomatic E. histolytica infection develop detectable anti-amoebic antibodies acutely, and >90% of patients develop antibodies after they recover from infection (convalescent titres; Refs 34, 35, 36, 37). Levels of anti-amoebic antibodies remain elevated in the serum for years, however, which limits the usefulness of serological methods for the diagnosis of acute amoebiasis in endemic regions, where seroprevalence rates (indicating the frequency of prior amoebic infection) can be as high as 50% (Refs 5, 35, 37). Most of the epidemiological data available on E. histolytica were obtained with serological testing, using crude amoebic extracts as the target antigen. Future studies using highly purified amoebic antigens might provide more accurate information on the seroprevalence of disease in endemic regions.

The detection of immunoglobulin M (IgM) antibodies might also prove to be useful for the diagnosis of acute disease because, unlike IgG antibodies, IgM antibodies do not persist in the serum. Recently, the use of an ELISA for the detection of serum IgM antibodies to the amoebic Gal or GalNAc-inhibitable adherence lectin has been reported. In this study, conducted in Egypt, anti-lectin IgM antibodies in the serum were detected in 45% of patients who had been suffering from acute colitis for <1 week, whereas <6% of patients had detectable levels of IgG antibodies after 1 week. The lectin antigen was detectable using the ELISA method in the sera of 65% of patients (Ref. 38).

Clinical implications/applications
A summary of the available tests that are used to diagnose amoebic colitis and liver abscess is presented in Table 1 (tab001wpc). Currently, stool culture followed by isoenzyme analysis and the PCR-based methods for the diagnosis of E. histolytica infection are research tools only. These methods, though labour-intensive, are extremely useful in epidemiological studies because they enable distinction between different strains of E. histolytica, as well as distinction between E. histolytica and E. dispar.

Amoebic colitis
Clinicians who are presented with a case of amoebic dysentery must distinguish it from the other causes of grossly or occultly bloody diarrhoea, as discussed earlier. The simplest initial approach to diagnose amoebic dysentery is to test stool samples for the presence of E. histolytica antigens (as discussed above); this should be performed in conjunction with testing stool samples for the presence of bacteria and bacterial toxins (such as Clostridium difficile toxin) to rule out bacterial disease. Because of its poor sensitivity and specificity, microscopy to identify E. histolytica cysts and haematophagous trophozoites should be used only to diagnose amoebiasis when the stool antigen test is not available. Although the presence of haematophagous amoebic trophozoites on stool examination strongly correlates with E. histolytica infection, this finding is infrequent (Ref. 16). The primary reason for examining a stool sample of a patient presenting with diarrhoea is to check for the presence of other parasitic infections that cause gastrointestinal disease (such as Cryptosporidium spp. and Giardia lamblia).

As discussed above, 75–85% of patients who present with amoebic colitis have measurable levels of anti-amoebic IgG antibodies in their serum, and >90% develop convalescent titres (Refs 34, 35, 36, 37). Thus, a negative serological test result for E. histolytica helps to rule out amoebic infection. However, the persistence of detectable levels of anti-amoebic IgG antibodies in the serum for many years makes the measurement of serum titres for the diagnosis of acute amoebiasis unreliable in regions where infection is endemic (Refs 35, 37). In the case of patients who have not spent an extended period of time in the developing world, a positive serological test result for the presence of IgG strongly suggests E. histolytica infection.

Visual examination of the colon using colonoscopy remains a very useful diagnostic method to eliminate non-infectious causes of bloody diarrhoea, and to confirm uncertain diagnoses of amoebic colitis. Examination of the sigmoid colon using sigmoidoscopy cannot be used to rule out amoebic colitis, because the disease tends to be localised higher up the intestine in the caecum of the large intestine. When colonic biopsies are stained with Periodic-Acid-Schiff (PAS), amoebic trophozoites appear magenta in colour and are clearly visible at the base of ulcerations (Refs 39, 40).

Amoebic liver abscess
Bacterial liver abscess, necrotic hepatoma and echinococcal cyst (typically, an incidental finding and unrelated to any abdominal symptoms a patient might have) must all be considered in the differential diagnosis of a patient presenting with amoebic liver abscess. Microscopy of stool samples is not helpful for diagnosis, because only ~35% of patients with amoebic liver abscess have a history of dysentery symptoms and <10% have identifiable amoebae in stool specimens on presentation (Ref. 41). A curative response to anti-amoebic therapy confirms indirectly the diagnosis of amoebic liver abscess in a patient with a positive serological test result for E. histolytica, a liver abscess and a history of recently (within 1 year) spending time in an endemic region. Although the precise role of the ELISA method, which can detect the Gal- or GalNAc-inhibitable amoebic lectin antigen in the serum of patients with amoebic liver disease, in the diagnosis of amoebic liver abscess remains unclear, this method appears to be quite sensitive (>90%) when used before treatment. Its sensitivity falls rapidly when used after anti-amoebic therapy has begun (R. Haque and W.A. Petri, unpublished).

Imaging using ultrasound, computed tomography and magnetic resonance all have excellent sensitivity for the detection of liver abscesses arising from any cause; however, they cannot distinguish amoebic abscesses from pyogenic (bacterial pus-forming) abscesses or necrotic tumours. Widening (dilatation) of the biliary tree (between the liver and gall bladder), especially in a patient with diabetes, suggests bacterial involvement rather than an amoebic liver abscess (Ref. 42). When an amoebic liver abscess is suspected, the aspiration of abscess fluid for diagnostic purposes should generally be avoided, because it carries the risk of spillage of pus into the peritoneal cavity and bacterial super-infection of the liver abscess. Because the vast majority of patients with amoebic liver abscess respond to treatment without drainage of the abscess, abscess aspiration and drainage for therapeutic purposes specifically should be reserved for patients with very large abscesses (>6 cm, which might pose a high risk of rupture) or for abscesses that do not respond to non-invasive treatment (Refs 43, 44). If it is not possible to wait for serological test results, aspiration of fluid from the liver can assist in the differentiation between amoebic and bacterial abscesses. The sensitivity of microscopy for identifying amoebic trophozoites in liver abscess pus is only ~25% at best (Ref. 45). The use of commercially available ELISA kits for detecting amoebic lectin antigen in pus that has been aspirated from liver abscesses has not yet been fully evaluated, but this method has yielded positive results in every patient tested thus far (R. Haque and W.A. Petri, unpublished).

Research in progress and outstanding questions
In recent years, extraordinary advances in the development of molecular techniques for the diagnosis of E. histolytica infection have been made. Stool culture followed by isoenzyme analysis, PCR-based amplification and detection of amoebic DNA (particularly rRNA), and stool antigen detection using ELISA are all sensitive methods that are capable of accurately distinguishing infection by E. histolytica from infection by the non-pathogenic parasite E. dispar (Refs 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33). Detection of the amoebic lectin antigen in stool samples using the ELISA method is the most suitable method for the diagnosis of amoebiasis in the developing world. It is rapid and simple to perform, and if appropriate antibodies are used, it can distinguish E. histolytica from E. dispar (Ref. 28). The detection of lectin antigen is replacing light microscopy (which is insensitive and nonspecific) as the initial test method for diagnosing amoebic colitis. Serological test methods for the detection of anti-amoebic antibodies remain extremely useful for the diagnosis of amoebic liver abscess. The potential role of the detection of antigens in the serum for the diagnosis of amoebiasis both within and outside the intestine remains uncertain, but this method also appears promising (Refs 22, 38).

It has been estimated that E. histolytica causes between 34 million and 50 million cases of invasive amoebiasis (i.e. amoebic colitis or liver abscess) each year, and between 40 thousand and 100 thousand deaths annually (Ref. 3). Much of the published data on the epidemiology of E. histolytica infection are based on methods that are unable to distinguish E. histolytica from the recently recognised, non-pathogenic parasite E. dispar; therefore, the true burden and distribution of amoebiasis worldwide remains unknown. Studies that use the new molecular-based techniques to clarify the epidemiology of E. histolytica are ongoing. The primary advantages of using antigen detection by ELISA in epidemiological studies are its low cost and convenience. However, PCR-based methods are extremely powerful tools for genetically typing different amoebic strains. Together, these two methods should help us gain an accurate understanding of the local and global epidemiology of E. histolytica infection. Knowledge of the epidemiology of amoebiasis will not only be useful for public health control programmes, but also facilitate the reliable testing of the anti-amoebic vaccines that are currently being developed in the laboratory (Ref. 46).

Acknowledgements and funding
The writing of this review was supported by National Institutes of Health/National Institute of Allergy and Infectious Diseases grants R01:AI-43596 (W.A. Petri), R03:TW-00848 (R. Haque) and T32:AI-07046 (Infectious Diseases Training Grant, University of Virginia Department of Infectious Diseases). David Lyerly (Techlab, Blacksburg, Virginia, USA) and Kris Chadee (Institute of Parasitology, McGill University, Montreal, Canada) critically reviewed the manuscript.

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Citation details for this article Christopher D. Huston, Rashidul Haque and William A. Petri, Jr (1999) Molecular-based diagnosis of Entamoeba histolytica infection. Exp. Rev. Mol. Med. 22 March, http://www.expertreviews.org/99000599h.htm

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