Laboratory methods of identification of Entamoeba histolytica and its differentiation from look-alike Entamoeba spp.

Entamoeba histolytica, the causative agent of intestinal and extraintestinal amebiasis, is a common parasitic cause of significant morbidity and mortality in the developing countries. Hence, early detection and differentiation of pathogenic E. histolytica from nonpathogenic/commensal Entamoeba spp (Entamoeba dispar/Entamoeba moshkovskii/Entamoeba bangladeshi) plays a crucial role in clinical management of patients with amebiasis. Most diagnostic tests currently available do not reliably differentiate between the species of Entamoeba and are less sensitive, cumbersome to perform. Molecular-based methods are highly sensitive, easy to perform and differentiates the pathogenic Entamoeba from nonpathogenic species, serving the criteria for an ideal diagnostic test for amebiasis. Recently, microarray technology has been found to be a promising tool for the diagnostic and epidemiological evaluation of amebiasis.

INTRODUCTION

Entamoeba histolytica, the etiological agent of amebiasis, is a major parasitic cause of morbidity and death, particularly in developing countries. It is estimated that around 50 million symptomatic cases and 100,000 deaths worldwide/year.[1] In India, 15-20% of the population are affected by this parasite and in Bangladesh 1 in 30 children from an urban slum of Dhaka die of diarrhea or dysentery by his/her 5th year of age.[2] The clinical features of amebiasis has a broad spectrum ranging from asymptomatic colonization to extraintestinal invasive amebiasis. Although six species of Entamoeba have been identified to colonize the human gut, only E. histolytica is known to be pathogenic. Recent review of the literature also point Entamoeba moshkovskii as an agent causing diarrheal episodes in infants. For many decades, the laboratory diagnosis of intestinal amebiasis has been based upon the microscopic examination of stool samples and hence widely known as the 10% disease. However, the recent description of various nonpathogenic Entamoeba species like Entamoeba dispar, E. moshkovskii and the newly described Entamoeba bangladeshi as morphologically indistinguishable forms from E. histolytica has required the need for alternative diagnostic methods for differentiation.[3] Since the last decade, molecular methods have played a key role in accurate diagnosis of various infectious diseases, including amebiasis. Patients with dysentery and importantly 90% of individuals with asymptomatic infection with E. histolytica should be promptly diagnosed to prevent further transmission.

MICROSCOPIC EXAMINATION

For ages, amebiasis has been diagnosed based on the demonstration of cyst and/or trophozoite stages of E. histolytica. The techniques utilized for microscopic examination were 0.9% saline and Lugol's/Dobell's/D’Antoni iodine wet mount examination, concentration techniques and permanent stained smears of stool and pus aspirates. Microscopic examination of stool/pus specimen in saline wet mount is a less sensitive technique (sensitivity < 10%) even when viewed by an expert microscopist[4] and needs to be examined within a short period of collection time (usually within half an hour) for motile trophozoites. D’Antoni's iodine has been found to be better for detection of cysts and saline wet mount or buffered methylene blue solution for detection of trophozoites.[5] Patients with acute dysentery usually show motile trophozoites, which may contain ingested red blood cells (RBC's). Previously, it was thought that this microscopic feature is very specific for E. histolytica, but few reports have found that even nonpathogenic Entamoeba trophozoites can phagocytose RBC's.[67] Asymptomatic carriers usually shed only cyst in the faces and direct wet mount examination has been found to be less sensitive in the detection of these intermittent shedders. Concentration techniques like formol-ether/formol-acetone sedimentation techniques increase the sensitivity of detection of cyst stages of Entamoeba. Entamoeba trophozoites tend to degrade within few minutes of collection and hence stool samples need to be fixed to prevent degradation of the trophozoite morphology. Commonly utilized fixatives/preservatives are 5% or 10% formalin, merthiolate-iodine-formalin, polyvinyl alcohol, sodium acetate- acetic acid- formalin, etc., These fixed smears can be permanently stained using trichrome/iron-hematoxylin stains for future reference/academic purposes.

Since intermittent excretion of cysts is a typical feature of amebiasis particularly asymptomatic carriers, minimum of 3 stool samples collected over a period of 10 days is recommended by Centers for Disease Control and Prevention. This improves the sensitivity to 85-95%.[8] There are several factors affecting the detection of Entamoeba spp by microscopy which are lack of adequate training in microscopy, delay in delivery of the sample to the laboratory leading to degradation/death of active trophozoite forms, difficulty in differentiation of cyst with degenerated polymorphonuclear cells particularly the mature neutrophils, inadequate number of samples collected, presence of morphologically similar Entamoeba spp (E. dispar, E. moshkovskii and E. bangladeshi), inadequate collection conditions (contaminated with water/urine) since pH, and osmotic changes tends to kill the active trophozoites and start of antibiotics prior to collection of samples.[910]

CULTURE METHODS

Two types of culture media are available for the isolation of Entamoeba spp, xenic and axenic media. Xenic cultivation is cultivation of the parasite with undefined/unknown flora. Modified Boeck and Drbohlav egg diphasic medium, Balamuth's medium, Jones's medium and TYSGM-9 are examples of xenic medium utilized for culture of Entamoeba spp. Axenic cultivation is growth of parasites in the absence of any unknown/undefined flora other than the protozoa intended to be grown. Examples would be TP-S-1, TYI-S-33 etc., which are utilized for cultivation of E. histolytica. E. bangladeshi and E. moshkovskii have the ability to grow at 37°C and 25°C, and this feature helps in differentiation of these species from E. histolytica and E. dispar.[3] Culture of E. histolytica as a diagnostic procedure has poor sensitivity than microscopy, technically difficult, expensive and difficult to maintain.[11] Hence, currently culture methods has not been in the list of ideal diagnostic tests available for the diagnosis of amebiasis.

ISO-ENZYME/ZYMODEME ANALYSIS

When strains of Entamoeba have the same electrophoretic pattern for several enzymes, they are called as zymodemes. Enzymes studied in detecting and differentiating the various species of Entamoeba are hexokinase, malic enzyme, phosphoglucoisomerase etc., and 24 different zymodemes have been identified. These zymodeme pattern analyses clearly differentiate E. histolytica from E. dispar and hence it remained the gold standard for diagnosis of amebiasis in the premolecular era. The disadvantages of iso-enzyme analysis are its huge time consumption, difficulty to performing, dependent on culture methods and low sensitivity.[12] Currently, molecular techniques have superseded iso-enzyme analysis in the differential detection of Entamoeba species.

SEROLOGICAL TESTS

Antibody detection

Serological tests may be useful in the diagnosis of amebiasis in developed countries since E. histolytica infection is uncommon. Whereas in developing countries, infection due to E. histolytica remains endemic.[13] This makes definite diagnosis of amebiasis by antibody detection difficult because of the difficulty to corroborate the present from past infection.[14] In a study by Parija et al., 41 of 50 patients with amoebic liver abscess (ALA) were tested positive for antiamebic antibodies by indirect hemagglutination tests (IHA). Three (12%) patients with other parasitic infections gave false-positive reactions. The positive predictive value and negative predictive value were 93.1% and 83.9% respectively.[15] IHA is easy to perform and standardize compared to other in-house assays. Among the antibody detection assays, ELISA has been the most widely used test to study amebiasis. A study by Hira et al., reported a sensitivity and specificity of 97.9% and 94.8% respectively in ALA patients.[16]

IgG antibodies remains detectable for years after infection, whereas IgM antibodies clears from the circulation within a short period and can only be detected during current infection. Abd-Alla et al., reported an ELISA for detection of antilectin antibodies from serum in patients with acute amoebic colitis.[17] subsequently in the year 2000, they reported a test for demonstration of detection of antilectin antibodies from saliva and found it to be more sensitive and specific than serum IgG antibody detection.[18] ELISA remains an important diagnostic tool in patients with invasive amebiasis and has no cross-reactions with other nonpathogenic Entamoeba species contributing to high specificity. Indirect immmunofluorescence assays have been shown as a rapid, reliable and reproducible methodology of antibody detection to differentiate amebiasis from nonamebic diseases, and also past from current infection. Jackson et al., reported IgM level monitoring to be useful in the diagnosis of extraintestinal/invasive amebiasis.[19] In extraintestinal/invasive amebiasis, particularly ALA, the sensitivity and specificity of IFA has been reported as 93.6% and 96.7% respectively by Haque et al.,[20] The disadvantages of antibody detection is its low sensitivity in developing countries where infections are endemic.

Antigen detection

Antigen detection methods have numerous advantages when compared to the previously mentioned modalities, like specific identification of E. histolytica from E. dispar/moshkovskii infection, better sensitivity and specificity and less expertise requirement and large scale screening tools. The antigens which are reported and utilized in the diagnosis of amebiasis are Gal/Gal-NAc lectin, lipophosphoglycan, and 29 kDa surface antigen. Among these antigens, Gal/Gal-NAc lectin has been extensively studied since this antigen is highly conserved and immunogenic allowing species-specific identification. Haque et al., (1995, 1996 and 1998) studied the utility of this antigen detection in stool specimens of asymptomatic individuals and patients with acute colitis. These studies reported were found to be highly sensitive (80-94%) and specific (94-100%) with good correlation with molecular methods.[7] Haque et al., reported that 96% and 100% of patients with ALA had demonstrable levels of Gal/Gal-NAc lectin antigen in serum and liver abscess pus respectively prior to treatment with metronidazole, whereas only 33% and 41% were detected after few days of metronidazole therapy.[20] Parija et al., utilized counter-current immunoelectrophoresis test for amebic antigen detection in serum of ALA patients. 38 (76%) of 50 ALA patients was detected by this assay.[15] Co-agglutination assay used by Karki and Parija detected amebic antigen from serum samples in 45 (90%) of 50 ALA patients.[21] An immunochromatographic card assay had been developed to detect 29 kDa surface antigen for E. histolytica/E. dispar. This assay was also designed to detect other stool pathogens like Giardia and Cryptosporidium spp from stool samples. Controversial reports exists on the sensitivity of this assay.[22] The major disadvantages encountered were the inability of the test to differentiate E. histolytica, E. dispar and E. moshkovskii and the requirement for fresh, unfixed stool specimens. Several vaccine candidate antigens have been identified – 25 kDa serine-rich E. histolytica protein (SREHP), 260 kDa Gal/GalNAc inhibitable lectin, lipophosphoglycans, cysteine proteases and peroxiredoxins. Studies in animal models like gerbils, and severe combined immunodeficiency mice reveal SREHP and Gal/Gal NAc lectin as potential and promising vaccine candidates for prevention of invasive amebic diseases.[23] These antigen targets are yet to be studied for their diagnostic role in amebiasis.

Molecular methods

In the last decade, molecular biology-based diagnostic tests have gained importance in the diagnosis of various infectious diseases including amebiasis to circumvent problems of older conventional techniques with the advantages of increased sensitivity, specificity and simplicity.

Accurate identification of pathogenic E. histolytica from nonpathogenic Entamoeba spp is crucial in the management of patients and epidemiological study of amebiasis outbreaks. Molecular-based techniques have been proven to be adequate to satisfy these needs and hence has emerged as the gold standard diagnostic tests in the current era.

DNA extraction procedure standardization from fecal samples is the most important step in the diagnosis of intestinal amebiasis. Difficulty in DNA isolation from fecal samples exists due to of the presence of various polymerase chain reaction (PCR) inhibitors like heme, bilirubin, bile salts, and complex polysaccharides.[24] There are many in-house methods of DNA extraction from feces published, the most common being phenol-chloroform method. The disadvantages of these in-house methods are huge time-consumption and laborious to perform. Commercial kits like QIA amp DNA stool kit has been used successfully and reliably in extraction of DNA from stool samples overcoming the disadvantages of in-house/conventional extraction methods.[25]

Transportation and storage of fecal or liver abscess pus samples at room temperature leads to rapid degeneration of the target DNA. The sensitivity of PCR performed on unpreserved samples falls with improper storage temperature and the duration of storage and hence the specimens need to be preserved at −20°C. Stool fixatives were also used previously, but studies revealed that freezing a fresh specimen at −20°C prior to DNA extraction is a better strategy due to reproducible and sensitive results.[2627]

CONVENTIONAL POLYMERASE CHAIN REACTION

There is a wide spectrum of PCR methods described for the detection and to differentiate the Entamoeba species. The genes that are well-studied are 18S rRNA (small subunit rRNA gene), extra-chromosomal DNA, cysteine proteinase, SREHP gene, 30 kDa protein, etc., PCR assays targeting the 18S rRNA have been widely used since they are present in multiple copies of extrachromosomal plasmids, making it to be more easily detected than a DNA copy.[28] Studies report that PCR targetting 18S rRNA have very high sensitivity than the best ELISA available in the market.[29] Discorrelation between microscopy and PCR results (microscopy positive for Entamoeba cysts and PCR negative) can arise if primers of narrow specificity are used. Hence, primers with broader specificity (e.g. small subunit rRNA) are to be utilizsed to reduce discorrelation results. Moreover, such utilization of primers with broad specificity had led to the detection of newer species of Entamoeba like E. bangladeshi.[3] Two separate nested PCR studies, one reported by International Centre for Diarrheal Diseases and Research, Dhaka, Bangladesh[30] and another from JIPMER, Puducherry, India on stool samples targeting 16S-like rRNA were found to distinguish accurately between the infections caused by E. histolytica, E. dispar and E. moshkovskii with 100% specificity.[31] Khairnar and Parija (2007, 2008) utilized conventional PCR assay in detection of E. histolytica 16 S rRNA gene from urine and saliva in ALA patients. The assay demonstrated E. histolytica DNA in 4 (17.4%) of 23 urine specimens collected prior to administration of metronidazole, but the DNA was demonstrated in 17 (56.7%) of 30 urine specimens collected subsequent to metronidazole therapy. Hence, detection of E. histolytica gene from urine can be used as a prognostic marker to evaluate treatment of invasive/extraintestinal amebiasis.[32] Similar results were also obtained in saliva samples from invasive cases of amebiasis.[33]

Although conventional PCR assays has been increasingly used for detection and differentiation of Entamoeba species, the disadvantages it faced were time consumption when large-scale sample processing was required, cost, inability to produce quantitative results and false positive results due to carry-over contamination.

REAL-TIME POLYMERASE CHAIN REACTION

Real-time PCR (RT-PCR) assay, the successor of conventional PCR has gained attraction for the laboratory diagnosis of infections because of its features of enhanced sensitivity, eliminating post-PCR manipulation leading to short turn-around times, minimized laboratory environment contamination with the amplicons and quantitative analysis. Although estimation of parasite burden may not be relevant in cases of amebiasis (parasite content varies between, and even within specimens from the same patient), it can be used in environmental sampling.[34] Various chemistries are available to detect the amplicon in RT, which involve fluorescent labeled oligonucleotides and probes. The three chemistries which are widely used are the hydrolysis probes (Taqman chemistry), hybridization probes (molecular beacons and FRET probes) and SYBR Green assay.

Real-time polymerase chain reaction has been studied in various clinical samples for the diagnosis of intestinal and extraintestinal amebiasis. Most of them target either the 18S rRNA (small-subunit rRNA) gene or species-specific episomal DNA repeat genes. Qvarnstrom et al., compared the RT-PCR assays performed in stool specimens, which were published till the year 2004.[35] They were Light cycler assay utilizing hybridization probes to amplify 18S rRNA gene); Taqman assay targeting 18S rRNA gene; Taqman assay targeting the episomal repeats of SREHPgene (Verweig et al., 2004) and SYBR Green assay adapted from conventional PCR.[3637383940] Among the above assays, Taqman assay targeting the 18S rRNA gene had a higher sensitivity than other RT-PCR assay for differential detection of Entamoeba spp. SYBR Green assay can be used as a good alternative in laboratories already performing conventional PCR for Entamoeba.[35] Rahman et al., had used SYBR Green assay targeting the SREHP gene in genotyping of E. histolytica.[41] (Haque et al., 2005) had utilized molecular-beacon assay to amplify 18S rRNA in stool and liver abscess pus samples and compared them with antigen detection and conventional PCR. The analytical sensitivity of this test was around 1 parasite/specimen.-[42] (Haque et al., 2010) reported detection of E. histolytica DNA from blood, urine or saliva in 97% (92/95) of ALA cases by Taqman based assay and had commented that the sensitivity was less than that of antigen detection or RT-PCR assay performed on stool samples, but had the advantage of samples being obtained noninvasively.[43] Although RT-PCR assay is highly sensitive, specificity is lost when multiplexing is tried utilizing different probes in the same tube. The major disadvantage of RT-PCR assay is its high cost, limiting its utility in developing countries. However as time progresses, these modalities will become affordable in developing nations where it can be utilized as a routine diagnostic modality. Currently, RT-PCR has evolved as the “gold standard” test in differential detection of Entamoeba spp and epidemiologic study of amebiasis.

Since the WHO redefinition of amebiasis, various diagnostic techniques are being introduced for accurate detection and differentiation of Entamoeba. Molecular methods, particularly the newly introduced RT-PCR has assisted in accurate diagnosis and specific selection of patients for antiamebic therapy. Recently, DNA microarray technology had been studied in the diagnosis of amebiasis. (Wang et al., 2004) had reported utilization of microarray technology for detection of E. histolytica, E. dispar, Giardia lamblia and Cryptosporidium parvum with high sensitivity and specificity.[44] (Shah et al., 2005) also had developed a microarray-based genotyping assay using sequence DNA clones of E. histolytica HM1:IMSS strain which revealed capability to distinguish E. histolytica from nonpathogenic E. dispar, to detect genes genes present only in virulent strains of Entamoeba virulent strains and to find potential phenotypic-genotypic associations.[45] These recent tools are promising, and their scope of utilization in the field of amebiasis as diagnostic techniques needs to be further evaluated.