**4. Diagnosis of amoebiasis**

Amoebiasis diagnosis rests on the demonstration of *E. histolytica* trophozoites or cysts in stool or colonic mucosa of patients. For many years a direct smear examined either as a wet mount or fixed and stained was done by microscopic examination of stool. Repeated stool sample examinations (at least three) may be needed. The presence of haematophagous amoebic trophozoites in a stool sample has always suggested *E. histolytica* infections (Gonzalez-Ruiz, A. et al 1994). Nonetheless, the specificity of this finding was further reduced when it was demonstrated that in some patients *E. dispar* also contains RBCs (Fotedar et al., 2007). Also, in view of the high frequency of *E. dispar* in many areas, dysentery due to entities such as shigellosis and campylobacter will probably be misdiagnosed as amoebic colitis if microscopy is the sole diagnostic criteria (Stanley 2003). However, in the absence of haematophagous trophozoites, the sensitivity of microscopy is limited by its ability to distinguish between samples infected with *E. histolytica* and the morphologically identical *E. dispar* and *E. moshkovskii*. Confusion between *E. histolytica*, other non-pathogenic amoeba and white blood cells such as macrophages and polymorphonuclear cells in feces frequently result in the overdiagnosis of amoebiasis. Delays in the processing of stool samples affect the sensitivity of light microscopy, which under the best circumstances is only 60% of that of the stool culture method followed by isoenzyme analysis (Krogstad et al., 1978).

Stool culture technique followed by isoenzyme analysis has been considered as the "gold standard" for many years. This method has been used to distinguish between *E. histolytica* and *E. dispar*. For more details on the culture technique the reader is advised to consult reference (Clark and Diamond, 2002). Culture of *E. histolytica* can be performed from fecal specimens, rectal biopsy specimens, or liver abscess aspirates. However, the process usually

occurring more often in HIV positive patients (Watanabe et al., 2011). In addition to HIV/AIDS, the increasing use of organ transplants and other immunosuppressed conditions such as neutropenia have been considered important risk factor for invasive amoebiasis in many countries. In Colombia for example, a study of organ transplant patients revealed that about 24.7% had detectable antiamoebic antibodies (Reyes et al., 2006) whereas in another study 14.3% neutropenic patients were found to have antiamoebic antibodies

Certain risk behaviors, such as homosexual relations and practicing oro-anal sex, can exacerbate the possibility of acquiring *E. histolytica* infections as well as other intestinal parasites such as *Cryptosporidium* spp., where the symptomatic pictures are more severe than those of immunocompetent individuals (Tatiana et al., 2008; Hung 2008). A recent study in Vietnam had indicated that socio-economic and personal hygiene factors determined infection with *E. histolytica*, rather than exposure to human and animal excreta in agricultural activities (Pham duc et al., 2011). In a study in Bangladesh, it was shown that wet environment is not the only factor that affects the detection curve of *E. histolytica*, but anti-Carbohydrate Recognition Domain IgA level in the gut is another determining factor for its occurrence in a closed population (Haque et al., 2006). Although, numerous seroprevalence studies suggest that HIV/AIDS individuals are at a higher risk of *E. histolytica* infections and are therefore more likely to develop symptomatic infections or severe forms of the disease, modest data exist to support these findings and further research

Amoebiasis diagnosis rests on the demonstration of *E. histolytica* trophozoites or cysts in stool or colonic mucosa of patients. For many years a direct smear examined either as a wet mount or fixed and stained was done by microscopic examination of stool. Repeated stool sample examinations (at least three) may be needed. The presence of haematophagous amoebic trophozoites in a stool sample has always suggested *E. histolytica* infections (Gonzalez-Ruiz, A. et al 1994). Nonetheless, the specificity of this finding was further reduced when it was demonstrated that in some patients *E. dispar* also contains RBCs (Fotedar et al., 2007). Also, in view of the high frequency of *E. dispar* in many areas, dysentery due to entities such as shigellosis and campylobacter will probably be misdiagnosed as amoebic colitis if microscopy is the sole diagnostic criteria (Stanley 2003). However, in the absence of haematophagous trophozoites, the sensitivity of microscopy is limited by its ability to distinguish between samples infected with *E. histolytica* and the morphologically identical *E. dispar* and *E. moshkovskii*. Confusion between *E. histolytica*, other non-pathogenic amoeba and white blood cells such as macrophages and polymorphonuclear cells in feces frequently result in the overdiagnosis of amoebiasis. Delays in the processing of stool samples affect the sensitivity of light microscopy, which under the best circumstances is only 60% of that of the stool culture method followed by

Stool culture technique followed by isoenzyme analysis has been considered as the "gold standard" for many years. This method has been used to distinguish between *E. histolytica* and *E. dispar*. For more details on the culture technique the reader is advised to consult reference (Clark and Diamond, 2002). Culture of *E. histolytica* can be performed from fecal specimens, rectal biopsy specimens, or liver abscess aspirates. However, the process usually

(Cardona et al., 2004).

is needed to confirm this hypothesis.

isoenzyme analysis (Krogstad et al., 1978).

**4. Diagnosis of amoebiasis** 

takes between 1-4 weeks to perform and requires sophisticated laboratory equipment making it not feasible as a routine procedure especially in the developing world where *E. histolytica* is rampant. The rate of success of *E. histolytica* culture in reference laboratories has been reported to be between 50 and 70%. Moreover, isoenzyme (zymodeme) analysis is labor intensive, costly and often produces false-negative results for many microscopy positive stool specimens (Strachan et al., 1988).

Serological methods may be useful diagnostically to detect infections with *E. histolytica* in developed countries where infections are not as common as in endemic developing nations (Ohnishi et al., 1997). In developing countries individuals are constantly exposed to *E. histolytica* making serological tests unable to definitively distinguish past from current infections (Caballero et al., 1994). Amoebic serology is highly sensitive and specific for the diagnosis of ALA (Zengzhu et al., 1999). Conversely, a study of asymptomatic individuals living in an *E. histolytica* endemic area of Vietnam revealed that about 83% of those infected had detectable anti-amoebic antibodies (Blessmann et al., 2002). Several assays for the detection of antibodies to *E. histolytica* infections have been developed (Table 2). These include: indirect hemagglutination (IHA), latex agglutination, immunoelectrophoresis, counterimmunoelectrophoresis (CIE), the amebic gel diffusion test, immunodiffusion, complement fixation, indirect immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA). With the exception of ELISA, all the other tests have been either costly to perform (Complement fixation), less sensitive and nonspecific (IHA and Latex agglutination test), time consuming (immunodiffusion) or requires skills in culture and antigen preparation (IFA) (Fotedar et al., 2007).


Table 2. List of some of the commercially available antibody assays used for the diagnosis of amoebiasis.

ELISA is a reliable, easy to perform and rapid method for the diagnosis of *E. histolytica*  infections especially in developing countries. It has been used widely for the study of the epidemiology and diagnosis of symptomatic amoebiasis (intestinal and/or extraintestinal). An ELISA to detect antibodies to *E. histolytica* has been shown to be 97.9% sensitive and 94.8% specific for detection of *E. histolytica* antibodies in ALA patients in a non endemic country (Hira et al., 2001). Unlike IgG, immunoglobulin M (IgM) is short lived and does not

Amoebiasis in the Tropics: Epidemiology and Pathogenesis 211

1993; Katzwinkel-Wladarsch et al., 1994; Calderaro et al., 2006; Hamzah et al., 2006). The sensitivity and specificity of PCR-based methods for the diagnosis of *E. histolytica* infection approach those of stool culture followed by isoenzyme analysis. PCR methods can be used to detect *E. histolytica* in stool, tissues and liver lesion aspirates. Of all the different gene targets used to identify *E. histolytica*, the small-subunit rRNA gene (18SrDNA) is believed to be more sensitive than the best antigen detection method used and performs equally well

Several groups have developed a variety of excellent conventional PCR assays, targeting different genes, for the direct detection and differentiation of *E. histolytica*, *E. dispar*, and *E. moshkovskii* DNA in clinical specimens such as stool and liver abscess samples (Tanyuksel and Petri Jr., 2003; Paul et al., 2007). Of all the targeted genes, assays amplifying the 18SrDNA genes are the ones in wide use as they are present in multiple copies on extrachromosomal plasmids thus making them easily detectable than single copy genes (Battacharya et al., 1989). Other gene targets used in PCR to study the epidemiology of *E. histolytica* include: the serine-rich *E. histolytica* protein (SREPH) gene (Stanley et al., 1990), cysteine proteinases gene and actin genes (Freitas et al., 2004). The SREHP is also used to study the genotypes of *E. histolytica* in human populations. However, it is now being replaced by the use of PCR amplification of tRNA gene-linked short tandem repeats which in addition to providing details of the epidemiology of *E. histolytica*, it also provides a tool to

A nested multiplex PCR was developed by many groups. This method has the added advantage of increasing the sensitivity and specificity of the test whilst simultaneously detecting and differentiating *E. histolytica* and *E. dispar* from DNA extracted from microscopy-positive stool specimens (Evangelopoulos et al., 2000; Hung et al., 2005; Nunez et al., 2001). A nested PCR method for the identification of *E. moshkovskii* in fecal samples was developed as a nested 18S rDNA PCR followed by restriction endonuclease digestion

Real time PCR is another type of PCR which is more sensitive than the conventional PCR. It is faster than the conventional PCR and characterized by the elimination of gel analysis and other post-PCR analysis, thus reducing the risk of contamination and cost (Klein 2002). However, its application in developing countries is limited to research only. Real-time PCR allows specific detection of the PCR product by binding to one or two fluorescence-labeled probes during PCR, thereby enabling continuous monitoring of the PCR product formation throughout the reaction. Furthermore, real-time PCR is a quantitative method and allows the determination of the number of parasites in various samples (Fotedar et al., 2007). Despite being used for the successful identification of *E. histolytica*, *E. dispar* and *E. moshkovskii,* the various PCR methods use is still confined to research institutes in the developing world where amoebiasis is endemic. PCR-based methods application in routine clinical diagnostic laboratories in low income societies is hindered by difficulties such as

A new platform for the detection of pathogens has been developed known as loop-mediated isothermal amplification (LAMP) and was developed in 2000 by Notomi and colleagues. This method uses a set of two specifically designed inner primers and two outer primers that recognize six distinct regions of the targeted DNA. The reaction is performed under isothermal conditions and simple incubators, such as a water bath or heat block, are

(Ali et al., 2003). The method exhibited a high sensitivity and specificity (100%).

compared to stool culture (Mirelman et al., 1997).

predict the outcome of the infection (Ali et al., 2005).

cost, and time to perform the test.

remain in the serum for longer periods making it a very useful marker for the detection of present or current *E. histolytica* infections. 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 (Abd-Alla et al., 1998). Since there is no cross-reaction with other non-*E. histolytica* parasites (Goncalves et al., 2004), the use of ELISA thus seems to be an excellent choice for the routine laboratory diagnosis as well as the surveillance and control of amoebiasis in the developing world.

The newer methods available to distinguish between *E. dispar* and *E. histolytica* have thrown into question the commonly accepted figure of 500 million infections worldwide suggesting that the actual number may be closer to 50 million. PCR and monoclonal antibody techniques are now available to distinguish between these three species in fresh and preserved stool samples, including those with mixed infections. Several investigators have developed ELISAs that detect antigens in fresh stool samples with sensitivity closer to that of stool culture methods and PCR. These ELISAs are usually easy and rapid to perform. Copro-antigen based ELISA kits specific for *E. histolytica* exploit monoclonal antibodies against the Gal/GalNAc-specific lectin of *E. histolytica* (*E. histolytica* II; TechLab, Blacksburg, VA) or against serine-rich antigen of *E. histolytica* (Optimum S kit; Merlin Diagnostika, Bornheim-Hersel, Germany). Other ELISA kits include the Entamoeba CELISA PATH kit (Cellabs, Brookvale, Australia) and the ProSpecT EIA (Remel Inc.; previously manufactured by Alexon-Trend, Inc., Sunnyvale, CA) (Fotedar et al., 2007). The early nineties of the 20th century have witnessed the introduction by TechLab of an ELISA kit for the specific detection of *E. histolytica* in feces. This antigen detection test captures and detects the parasite's Gal/GalNAc lectin in stool samples. It can also be used for the detection of the lectin antigen in the serum and liver abscesses in patients with invasive intestinal amoebiasis and ALA (Haque et al., 2000). However, the diagnosis of ALA normally relies on the identification of liver lesions and positive anti-*E. histolytica* serology. Yet neither provides conclusive results for ALA. The Gal/GalNAc lectin is conserved and highly immunogenic, and because of the epitopic differences in the lectins of *E. histolytica* and *E. dispar*, the test enables specific identification *E. histolytica* (Haque et al., 1993; Mirelman 1997). Because of some disadvantages observed with the TechLab ELISA kit, a newer more sensitive and specific version, TechLab *E. histolytica* II kit, was produced. This second – generation *E. histolytica* II kit has demonstrated good sensitivities and specificities when compared to real-time PCR (71 to 79% and 96 to 100%, respectively) (Roy et al., 2005; Visser et al., 2006). Other studies however, have reported a lesser sensitivity (14.3%) and specificity (98.4%) in comparison to stool culture and isoenzyme analysis (Gatti et al., 2002). Cross reactivity is another concern with the use of the assay, since it seems that *E. dispar* positive samples by means of PCR may sometimes give false-positive outcomes (Furrows et al., 2004). Accordingly, accurate detection of *E. histolytica*, *E. dispar* and *E. moshkovskii* could be helpful for diagnostic and epidemiological studies in places where it is impractical and expensive to use molecular assays and where amoebiasis is most prevalent, such as in the developing countries. An antigen detection kit for the specific identification of *E. dispar* and *E. moshkovskii* is yet to be developed.

Several PCR-based techniques that amplify and detect *E. histolytica* DNA are currently used for the clinical and epidemiological studies in non-endemic rich countries (Acuna-Soto et al.,

remain in the serum for longer periods making it a very useful marker for the detection of present or current *E. histolytica* infections. 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 (Abd-Alla et al., 1998). Since there is no cross-reaction with other non-*E. histolytica* parasites (Goncalves et al., 2004), the use of ELISA thus seems to be an excellent choice for the routine laboratory diagnosis as

The newer methods available to distinguish between *E. dispar* and *E. histolytica* have thrown into question the commonly accepted figure of 500 million infections worldwide suggesting that the actual number may be closer to 50 million. PCR and monoclonal antibody techniques are now available to distinguish between these three species in fresh and preserved stool samples, including those with mixed infections. Several investigators have developed ELISAs that detect antigens in fresh stool samples with sensitivity closer to that of stool culture methods and PCR. These ELISAs are usually easy and rapid to perform. Copro-antigen based ELISA kits specific for *E. histolytica* exploit monoclonal antibodies against the Gal/GalNAc-specific lectin of *E. histolytica* (*E. histolytica* II; TechLab, Blacksburg, VA) or against serine-rich antigen of *E. histolytica* (Optimum S kit; Merlin Diagnostika, Bornheim-Hersel, Germany). Other ELISA kits include the Entamoeba CELISA PATH kit (Cellabs, Brookvale, Australia) and the ProSpecT EIA (Remel Inc.; previously manufactured by Alexon-Trend, Inc., Sunnyvale, CA) (Fotedar et al., 2007). The early nineties of the 20th century have witnessed the introduction by TechLab of an ELISA kit for the specific detection of *E. histolytica* in feces. This antigen detection test captures and detects the parasite's Gal/GalNAc lectin in stool samples. It can also be used for the detection of the lectin antigen in the serum and liver abscesses in patients with invasive intestinal amoebiasis and ALA (Haque et al., 2000). However, the diagnosis of ALA normally relies on the identification of liver lesions and positive anti-*E. histolytica* serology. Yet neither provides conclusive results for ALA. The Gal/GalNAc lectin is conserved and highly immunogenic, and because of the epitopic differences in the lectins of *E. histolytica* and *E. dispar*, the test enables specific identification *E. histolytica* (Haque et al., 1993; Mirelman 1997). Because of some disadvantages observed with the TechLab ELISA kit, a newer more sensitive and specific version, TechLab *E. histolytica* II kit, was produced. This second – generation *E. histolytica* II kit has demonstrated good sensitivities and specificities when compared to real-time PCR (71 to 79% and 96 to 100%, respectively) (Roy et al., 2005; Visser et al., 2006). Other studies however, have reported a lesser sensitivity (14.3%) and specificity (98.4%) in comparison to stool culture and isoenzyme analysis (Gatti et al., 2002). Cross reactivity is another concern with the use of the assay, since it seems that *E. dispar* positive samples by means of PCR may sometimes give false-positive outcomes (Furrows et al., 2004). Accordingly, accurate detection of *E. histolytica*, *E. dispar* and *E. moshkovskii* could be helpful for diagnostic and epidemiological studies in places where it is impractical and expensive to use molecular assays and where amoebiasis is most prevalent, such as in the developing countries. An antigen detection kit for the specific identification of *E. dispar* and

Several PCR-based techniques that amplify and detect *E. histolytica* DNA are currently used for the clinical and epidemiological studies in non-endemic rich countries (Acuna-Soto et al.,

well as the surveillance and control of amoebiasis in the developing world.

*E. moshkovskii* is yet to be developed.

1993; Katzwinkel-Wladarsch et al., 1994; Calderaro et al., 2006; Hamzah et al., 2006). The sensitivity and specificity of PCR-based methods for the diagnosis of *E. histolytica* infection approach those of stool culture followed by isoenzyme analysis. PCR methods can be used to detect *E. histolytica* in stool, tissues and liver lesion aspirates. Of all the different gene targets used to identify *E. histolytica*, the small-subunit rRNA gene (18SrDNA) is believed to be more sensitive than the best antigen detection method used and performs equally well compared to stool culture (Mirelman et al., 1997).

Several groups have developed a variety of excellent conventional PCR assays, targeting different genes, for the direct detection and differentiation of *E. histolytica*, *E. dispar*, and *E. moshkovskii* DNA in clinical specimens such as stool and liver abscess samples (Tanyuksel and Petri Jr., 2003; Paul et al., 2007). Of all the targeted genes, assays amplifying the 18SrDNA genes are the ones in wide use as they are present in multiple copies on extrachromosomal plasmids thus making them easily detectable than single copy genes (Battacharya et al., 1989). Other gene targets used in PCR to study the epidemiology of *E. histolytica* include: the serine-rich *E. histolytica* protein (SREPH) gene (Stanley et al., 1990), cysteine proteinases gene and actin genes (Freitas et al., 2004). The SREHP is also used to study the genotypes of *E. histolytica* in human populations. However, it is now being replaced by the use of PCR amplification of tRNA gene-linked short tandem repeats which in addition to providing details of the epidemiology of *E. histolytica*, it also provides a tool to predict the outcome of the infection (Ali et al., 2005).

A nested multiplex PCR was developed by many groups. This method has the added advantage of increasing the sensitivity and specificity of the test whilst simultaneously detecting and differentiating *E. histolytica* and *E. dispar* from DNA extracted from microscopy-positive stool specimens (Evangelopoulos et al., 2000; Hung et al., 2005; Nunez et al., 2001). A nested PCR method for the identification of *E. moshkovskii* in fecal samples was developed as a nested 18S rDNA PCR followed by restriction endonuclease digestion (Ali et al., 2003). The method exhibited a high sensitivity and specificity (100%).

Real time PCR is another type of PCR which is more sensitive than the conventional PCR. It is faster than the conventional PCR and characterized by the elimination of gel analysis and other post-PCR analysis, thus reducing the risk of contamination and cost (Klein 2002). However, its application in developing countries is limited to research only. Real-time PCR allows specific detection of the PCR product by binding to one or two fluorescence-labeled probes during PCR, thereby enabling continuous monitoring of the PCR product formation throughout the reaction. Furthermore, real-time PCR is a quantitative method and allows the determination of the number of parasites in various samples (Fotedar et al., 2007). Despite being used for the successful identification of *E. histolytica*, *E. dispar* and *E. moshkovskii,* the various PCR methods use is still confined to research institutes in the developing world where amoebiasis is endemic. PCR-based methods application in routine clinical diagnostic laboratories in low income societies is hindered by difficulties such as cost, and time to perform the test.

A new platform for the detection of pathogens has been developed known as loop-mediated isothermal amplification (LAMP) and was developed in 2000 by Notomi and colleagues. This method uses a set of two specifically designed inner primers and two outer primers that recognize six distinct regions of the targeted DNA. The reaction is performed under isothermal conditions and simple incubators, such as a water bath or heat block, are

Amoebiasis in the Tropics: Epidemiology and Pathogenesis 213

inflammatory responses following contact of the trophozoites to the gut wall induces a massive neutrophil infiltration across the epithelium into the underlying tissues resulting in weakening of epithelial cells and the mucous layer and allowing trophozoites to invade the intestinal epithelium and disseminating to other bodily sites (Ackers and Mirelman, 2006). The ulcers formed may be generalized involving the whole length of the large intestine or they may be localized in the ileo-caecal or sigmoido-rectal regions. Ulcers are normally disconnected with sizes varying from pin-head size to more than 2.5 cm in diameter. They may be deep or superficial. Base of the deep ulcers is generally formed by the muscularis layer. Nonetheless, superficial ulcers do not extend beyond the muscularis layer. A large number of fatalities results from perforated colons with concomitant peritonitis. *E. histolytica* also causes amoebomas. These are pseudotumoural lesions, whose formation is associated with necrosis, inflammation and oedema of the mucosa and submucosa of the colon. These

Fig. 3. "Flask-shaped" ulcer of invasive intestinal amebiasis (hematoxylin-eosin, original magnification ×50). Source: **Pritt B S , Clark C G Mayo Clin Proc. 2008;83:1154-1160: Mayo** 

While the serine rich *E. histolytica* protein (SREHP) have been shown to promote adhesion of the trophozoites to host cells, cysteine proteases (CP), are known for their virulence in other protozoa as well as in tumour metastasis. Five *E. histolytica* proteins (EhCP1, 2, 3, 5 and 112) have been identified. All are alleged to play a role in the destruction of host cells, phagocytosis, together with the recruitment of neutrophils and macrophages and the induction of intestinal

granulomatous masses may obstruct the bowel.

**Clinic Proceedings** 

adequate for the specific amplification of the desired genetic material. Considering these advantages, the LAMP assay could be a useful and valuable diagnostic tool particularly in developing countries where most of the infections are common as well as in hospital laboratories. Recently this method was developed specifically for the detection of *E. histolytica* (Liang et al., 2009). The efficiency of the developed method was compared to that of existing PCR methodology and was similar in terms of sensitivity and specificity. This method needs further evaluations to be used in local conditions in Africa in order to improve the understanding of amebiasis in the continent as well as elsewhere.
