Tropical Parasitology

Trop Parasitol. 4(2): 90-95

Laboratory methods of identification of

Entamoeba histolytica and its differentiation
from look-alike Entamoeba spp.
Subhash Chandra Parija, Jharna Mandal, Dinoop Korol Ponnambath

Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and

Research Gorimedu, Puducherry, India

Address for correspondence Prof. Subhash Chandra Parija, Dean- Research, Senior
Professor, Department of Microbiology, Jawaharlal Institute of Post Graduate Medical
Education and Research, Puducherry - 605 011, India. E-mail: subhashparija@yahoo.com

Copyright: © Tropical Parasitology

DOI: 10.4103/2229-5070.138535
Published in print: Jul-Dec2014

Abstract
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

th
Dhaka die of diarrhea or dysentery by his/her 5 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.

Footnotes

Source of Support: Nil

Conflict of Interest: None declared.

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