Amoebic liver abscess in northern Sri Lanka: first report of immunological and molecular confirmation of aetiology
Kannathasan et al. Parasites & Vectors
Amoebic liver abscess in northern Sri Lanka: first report of immunological and molecular confirmation of aetiology
Selvam Kannathasan 0
Arumugam Murugananthan 0
Thirunavukarasu Kumanan 2
Devika Iddawala 1
Nilanthi Renuka de Silva 5
Nadarajah Rajeshkannan 4
Rashidul Haque 3
0 Department of Pathology, Faculty of Medicine, University of Jaffna , Jaffna , Sri Lanka
1 Department of Parasitology, Faculty of Medicine, University of Peradeniya , Peradeniya , Sri Lanka
2 Department of Medicine, Faculty of Medicine, University of Jaffna , Jaffna , Sri Lanka
3 International Centre for Diarrhoeal Disease Research , Dhaka , Bangladesh
4 Coomealla Health Aboriginal Corporation , Dareton, NSW , Australia
5 Department of Parasitology, Faculty of Medicine, University of Kelaniya , Ragama , Sri Lanka
Background: Since 1985, amoebic liver abscess (ALA) has been a public health problem in northern Sri Lanka. Clinicians arrive at a diagnosis based on clinical and ultrasonographic findings, which cannot differentiate pyogenic liver abscess (PLA) from ALA. As the treatment and outcome of the ALA and PLA differs, determining the etiological agent is crucial. Methods: All clinically diagnosed ALA patients admitted to the Teaching Hospital (TH) in Jaffna during the study period were included and the clinical features, haematological parameters, and ultrasound scanning findings were obtained. Aspirated pus, blood, and faecal samples from patients were also collected. Pus and faeces were examined microscopically for amoebae. Pus was cultured in Robinson's medium for amoebae, and MacConkey and blood agar for bacterial growth. ELISA kits were used for immunodiagnosis of Entamoeba histolytica infection. DNA was extracted from selected pus samples and amplified using nested PCR and the purified product was sequenced. Results: From July 2012 to July 2015, 346 of 367 clinically diagnosed ALA patients admitted to Jaffna Teaching Hospital were enrolled in this study. Almost all patients (98.6%) were males with a history of heavy alcohol consumption (100%). The main clinical features were fever (100%), right hypochodric pain (100%), tender hepatomegaly (90%) and intercostal tenderness (60%). Most patients had leukocytosis (86.7%), elevated ESR (85.8%) and elevated alkaline phosphatase (72.3%). Most of the abscesses were in the right lobe (85.3%) and solitary (76.3%) in nature. Among the 221 (63.87%) drained abscesses, 93.2% were chocolate brown in colour with the mean volume of 41.22 ± 1.16 ml. Only four pus samples (2%) were positive for amoeba by culture and the rest of the pus and faecal samples were negative microscopically and by culture. Furthermore, all pus samples were negative for bacterial growth. Antibody against E. histolytica (99.7%) and the E. histolytica antigen were detected in the pus samples (100%). Moreover, PCR and sequencing confirmed these results. Conclusion: To our knowledge, this is the first report from Sri Lanka that provides immunological and molecular confirmation that Entamoeba histolytica is a common cause of liver abscesses in the region.
Amoebic liver abscess; Entamoeba histolytica; Northern Sri Lanka
Amoebiasis caused by Entamoeba histolytica, is known
to affect at least 50 million people around the world and
is responsible for up to 100,000 deaths per annum [1, 2].
Amoebic liver abscess (ALA), the most common
extraintestinal manifestation of invasive amoebiasis, is
associated with high morbidity and mortality if the condition
is not diagnosed and treated promptly.
The first report of hepatic amoebiasis from Sri Lanka,
then Ceylon, dates to 1821 . Since then, reports
describing cases from all over the island have been
published [3–7]. After about 1975, while many cases of
clinically diagnosed ALA have been reported from the
northern part of Sri Lanka [8–11], no cases have yet
been reported from the rest of the island. Since 1985,
clinicians working at Jaffna Teaching Hospital have been
treating suspected ALA patients who were usually referred
either from private clinics or peripheral Government
hospitals . The diagnosis was mainly based on the patient’s
history, clinical features and other investigations such as
haematological parameters and ultrasonography.
However, it is well known that the clinical features of
ALA (acute onset of fever, abdominal pain and
hepatomegaly) are very similar to those of pyogenic liver
abscess (PLA), as are the ultrasonographic features. Thus,
it is difficult to differentiate ALA from PLA either
clinically or by ultrasound imaging . At the same time,
differentiating between PLA and ALA is important
because the treatment regimen is different.
Although antibody-based and molecular diagnostic
assays are very sensitive and specific for the confirmation
of ALA, the limited resources available at Jaffna
Teaching Hospital has meant that none of the patients
admitted there have had laboratory confirmation by a specific
diagnostic assay. The need to confirm the aetiological
agent causing clinically suspected ALA in Jaffna is
further highlighted by the fact that most patients had no
evidence of intestinal amoebiasis (dysentery).
Hence, this study was carried out to confirm the
aetiological agent causing clinically suspected ALA as
Entamoeba histolytica, with the aid of specific immunological
and molecular tools, for the first time in Sri Lanka.
Information on the clinical features, haematological
parameters such as full blood count, liver function tests,
erythrocyte sedimentation rate (ESR), co-morbid
conditions, and ultrasound findings were collected from the
bed head ticket (BHT) after obtaining permission from
the respective clinicians.
Collection of pus from the abscess
A pus sample was collected while ultrasound guided
aspiration was carried out as part of the treatment and
management procedure. The total volume and colour of the
pus were recorded. The last portion of the pus was
collected into a sterile bottle and immediately transported to
the Laboratory at the Division of Parasitology, Department
of Pathology, Faculty of Medicine, University of Jaffna for
the direct microscopic examination of the smear and
culture. An aliquot of pus was stored at -40 °C for further
immunological and molecular investigations.
Collection of blood from patients and controls
A sample of 3 ml venous blood was collected from all
patients under sterile conditions, before treatment
commenced. Control samples were collected from 100
randomly selected, healthy individuals (university students)
who had been living for more than 5 years in northern
Sri Lanka. Serum was separated from the clotted blood
sample by centrifugation at 3500× rpm /10 min and
stored at -20 °C for further serological investigations.
Collection of faecal samples
Faecal samples were collected from each patient in
sterile, dry containers and transported to the laboratory on
the same day. This procedure was repeated for three
consecutive days from each patient.
Basic laboratory diagnosis of ALA
Microscopic demonstration of the parasite
Both wet smears and permanent smears stained with
trichrome were examined for trophozoites or cysts of E.
histolytica in the pus, faecal sample, and in cultures.
Culture of Entamoeba spp.
Parasite culture was performed as per the method
described by Robinson . Briefly, freshly collected pus
and faecal samples were inoculated separately in Robinson
medium and incubated at 37 °C for 48–72 h.
Culture of bacteria
A loop of fresh pus sample was inoculated directly onto
the surface of pre-warmed MacConkey and blood agar
plates and streaked. The inoculated plates were incubated
aerobically at 37 °C. Plates were examined after 18–24 h
of incubation for the presence of bacterial growth.
Entamoeba histolytica IgG ELISA
Serum samples stored at -20 °C were thawed and
examined for circulating IgG antibody against E. histolytica
using an AccuDiag™ E. histolytica IgG (Amoebiasis)
ELISA kit (California, USA), as per the manufacturer’s
Entamoeba histolytica antigen detection ELISA
Pus and serum samples were examined for E. histolytica
antigen using an E. histolytica/E. dispar antigen
detection ELISA kit from Diagnostic Automation/Cortez
Diagnostics, Inc. (California, USA), as per the
DNA was extracted from each pus sample using a
QIAGEN stool mini kit (Maryland, USA), as per the
A nested PCR was performed based on the serine-rich
E. histolytica protein (SREHP) coding gene of the
parasite . Briefly, 2 μl of extracted genomic DNA was
used in the primary PCR in a 25 μl reaction using the
PCR master mix (Promega, Wisconsin, USA) and the
primers, SREHP-5 (5′-GCT AGT CCT GAA AAG CTT
GAA GAA GCT G-3′) and SREHP-3 (5′-GGA CTT
GAT GCA GCA TCA AGG T-3′). For the secondary
reaction, the same 25 μl reaction was prepared though
2 μl of a 1:50 dilution of the initial PCR product was
used as template. Also, a second set of nested primers;
nSREHP-5 (5′-TAT TAT TAT CGT TAT CTG AAC
TAC TTC CTG-3′) and nSREHP-3 (5′-GAA GAT AAT
GAA GAT GAT GAA GAT G-3′) was used.
The temperature cycling conditions for the primary
PCR amplification included an initial denaturing step,
carried out at 94 °C for 15 min. This was followed by 40 cycles
of 94 °C for 1 min, 50 °C for 1.5 min and 72 °C for 2 min,
with a final extension of 72 °C for 5 min. The secondary
PCR used the same temperature cycling conditions, with
one modification; an annealing temperature of 55 °C .
One representative PCR product was purified using
commercially purchased QIAquick PCR Purification
Kit (QIAGEN), according to the protocol of the
manufacturer. The purified PCR product was sequenced
bidirectionally using the ABI 3500 Dx capillary instrument
(Life Technologies, California, USA) at the Institute of
Biochemistry, Molecular Biology and Biotechnology
(IBMBB), University of Colombo, Sri Lanka.
Analysis of data
Data were analyzed using SPSS statistical software
(Version 16). Sequences generated were subjected to
BLAST searches against AmoebaDB
History and clinical presentation
A total of 346 suspected ALA patients were enrolled in
this study from July 2012 to July 2015. Among these
patients, almost all (98.6%) were males with a history of
consumption of alcohol (100%), especially a local drink,
consisting of the fermented sap of the Palmyrah palm
(toddy). The predominant clinical features observed
were fever, right hypochodric pain with or without
abdominal pain (Table 1). All the patients had high
swinging fever with the average temperature of 38.9 ± 0.5 °C
and the majority (98.26%) complained of fever for more
than 5 days. Further, all the patients complained of right
hypochodric pain, and a small number of patients (10%)
had pain at the right shoulder tip, whereas roughly half
of them reported abdominal pain (46.8%). A few of them
(27.5%) reported nausea and vomiting. A few reported
(7.8%) diarrhoea within the last 6 months, but the
diarrhoea they described was of the watery type. Other than
24 patients (6.9%) who identified themselves as diabetic,
there was no other significant co-morbidity (Table 1).
Most patients had leucocytosis (86.7%), elevated ESR
(85.8%) and elevated alkaline phosphatase (ALP) (72.3%).
Elevated aspartate transaminase (AST/SGOT) was
observed in some patients (37.8%) and slightly elevated in
very few (13.6%) (Table 1).
Most of the abscesses (85.3%) were found in the right
lobe of the liver whereas 51 were found in the left lobe
(Table 1). Most patients (76.3%) had a solitary abscess.
Details of abscess fluid
A total of 221 (63.87%) abscesses were drained within
this 3-year period. The pus drained from almost all
patients (93.2%) was chocolate brown in colour. The
volume of pus varied, but more than 50 ml was drained
from 133 abscesses. The average volume of pus drained
from all abscesses was 41.22 ± 1.16 ml.
Microscopic examination and the culture of pus samples
Trophozoites of E. histolytica were not observed in any
of 221 saline smears prepared from aspirated pus.
However, four pus samples (2%) cultured in Robinson’s media
were positive for E. histolytica. In addition, all 221
samples were negative for bacterial growth on MacConkey
and blood agar.
Microscopic examination and the culture of faecal samples
Only 207 (59.8%) faecal samples were obtained from the
patients. Among them, 30% provided three faecal
samples, 40% provided two, and remainder gave only one
Table 1 History, clinical presentation, haematological parameters and ultrasound findings of the clinically diagnosed amoebic liver
abscess patients admitted to the Teaching Hospital, Jaffna
Right hypochodric pain
Nausea and vomiting
Total white cell count
Erythrocyte sedimentation rate
Number of abscesses
Location of the abscesses
sample. All 207 faecal samples were negative for
Entamoeba spp. by microscopic examination and culture
in Robinson’s media.
Of 346 serum samples tested for E. histolytica
circulating antigen, 344 were positive (99.4%). All 221 pus
samples (100%) collected from patients who were
sero-positive for E. histolytica, were positive for E.
histolytica antigen (Table 2).
Among the 346 serum samples investigated, 345
were positive (99.7%) for IgG antibody against E.
histolytica. Although the one sample which was negative
for IgG antibody was positive for circulating
Table 2 Immunological findings of serum and pus samples of the ALA patient attended to the Teaching Hospital Jaffna
Moreover, E. histolytica IgG antibodies were negative in
100 serum samples collected from healthy individuals,
suggesting that the sensitivity of the test is 100% (345/345)
and the specificity is 99.01% (100/101). Further, the
positive and negative predictive values of the test were 99.7%
(345/346) and 100% (100/100), respectively (Table 3).
Nested PCR was performed on selected DNA samples
extracted individually from the pus aspirated from 50 ALA
patients. The targeted Entamoeba species DNA sequence
was detected in all 50 pus samples (Fig. 1). The 450 bp
sequence obtained from the PCR amplicon was subjected to
NCBI BLASTn analysis (www.ncbi.nlm.nih.gov; GenBank
KX377525) and comparison to sequences available on the
Amoeba DB web site (http://amoebadb.org/amoeba/). The
sequences generated in this study were 99% similar to
corresponding gene sequences of serine-rich E. histolytica
This is the first report utilising immunological and
molecular testing to confirm that E. histolytica is the
aetiological agent of liver abscesses in northern Sri Lanka.
Currently at the Jaffna TH, ALA is mainly diagnosed
based on clinical findings (fever with right hypochondric
and/or abdominal pain), history of heavy consumption of
alcohol, followed by ultrasonography. This diagnosis is
also supported based on a positive response to treatment
In this study we examine 346 clinically diagnosed cases
of ALA admitted to Jaffna TH. All patients complained of
fever on admission. In addition, all had right hypochodric
pain with or without abdominal pain. Similar presenting
features have been reported from many other studies,
conducted in Sri Lanka and elsewhere [3, 8–12, 15–24].
Findings on clinical examination were similar to those
described previously in Sri Lanka and other Asian
countries [15–17]. Haematological findings such as leukocytosis,
elevated ESR and ALP were also comparable to those
described previously [16–19, 22–27]. To facilitate the
diagnosis, laboratory investigations play a major role. In our
study, only 2% (4/221) of pus samples were positive in
Robinson’s culture media. All pus samples and faecal
Table 3 Validity of the antibody test used for the serological
diagnosis in our local context
Healthy individuals (University students) 0
aSensitivity = 100%; bSpecificity = 99.01%; cPositive Predictive value = 99.7%;
dNegative Predictive Value = 100%
IgG antibody test a,b,c,d
Fig. 1 Results of nested PCR, amplified serine-rich protein coding
gene (450 bp) of Entamoeba histolytica. Lane M: 100 bp ladder; Lanes
1–6: liver abscess aspirate samples; Lane 7: negative control
samples were negative for the presence of E. histolytica
microscopically (wet smears in saline as well as trichrome
stained smears). Furthermore, all pus samples were
negative for bacterial growth indicating that they were sterile
and unlikely to be PLA. As the parasite resides in the
periphery of the abscess wall, the probability of obtaining the
amoeba in pus is very unlikely [28, 29]. Also, ALA may
occur many months after the original intestinal infection is
cleared. Hence, detecting the parasite in the faecal sample
is also unlikely in the absence of concurrent dysentery .
Haque et al. reported that the sensitivity of microscopy for
detecting amoebae in the pus of ALA as < 20% . Other
investigators report similar findings [12, 21, 23, 30].
Since the microscopic and culture methods available
for detecting E. histolytica are less sensitive,
immunological and molecular techniques are widely used. In Sri
Lanka, these techniques have not been used so far to
determine the causative agent of liver abscesses in
northern Sri Lanka.
Although the detection of specific antibodies to E.
histolytica in serum is thought to be highly sensitive
for the diagnosis of ALA [31, 32], people with amoebic
infection in endemic areas may have been repeatedly
exposed to E. histolytica and remain asymptomatic. This
situation makes the definitive diagnosis by antibody
detection difficult because of the inability to distinguish a
previous infection from a current infection. Combining clinical
presentation and antigen detection with antibody detection
will provide a definitive diagnosis of current infections.
In our study, 99.7% of serum samples were positive for
IgG antibody against E. histolytica. These findings are
comparable to those of others [33, 34]. Antigen
detection ELISA has several significant advantages compared
with other methods currently used for the diagnosis of
amoebiasis such as microscopy, culture and even
antibody detection tests . The presence of detectable
antigen in serum and pus indicates an ongoing infection
. In our study, 99.4% of serum and 100% of pus
samples were positive for E. histolytica antigen. This
provides immunological confirmation for the first time, that
the clinical diagnosis of ALA by clinicians at Jaffna TH
is well supported.
Many laboratories experience difficulties in diagnosing
ALA using microscopy, faecal concentrates or
culturebased methods and even using antibody detection in the
serum in endemic areas. Hence, more sensitive and
specific DNA-based detection methods have become popular
as a solution to overcome these constraints [31, 35, 36].
Though successful use of PCR for the diagnosis of E.
histolytica has been reported in many countries [37–40] no
report has been published to date from Sri Lanka.
In our study, we targeted the serine-rich protein-coding
gene which has been widely used elsewhere [14, 41–43].
By applying PCR and sequencing PCR amplicons in this
study, we confirm Entamoeba histolytica as a common
cause of liver abscesses in northern Sri Lanka.
To our knowledge, this is the first study from Sri Lanka
that provides immunological and molecular
confirmation that Entamoeba histolytica is a common cause of
clinically diagnosed liver abscess in northern Sri Lanka.
ALA: Amoebic liver abscess; ALP: Alkaline phosphatase; AST: Aspartate
transaminase; BHT: Bed head ticket; ELISA: Enzyme-linked immunosorbent
assay; ESR: Erythrocyte sedimentation rate; IgG: Immunoglobulin G;
PLA: Pyogenic liver abscess; SREHP: Serine-rich Entamoeba histolytica protein
The authors thank the participants (patients and volunteers) of this study, the
Director, all the consultants, clinicians, Medical Officers, and nursing staff
attached to the Teaching hospital, Jaffna for their permission and assistance to
carry out this study. Further, they wish to thank Dr. S. Ketheeswary and Dr. S.
Pathmini, Radiologists, Teaching Hospital, Jaffna who provided the aspirated
Availability of data and material
Data supporting the conclusions of this article are included within the article.
The sequence is submitted in the GenBank database under accession
SK, AM, NRS, DI and RH conceived the study. SK and AM performed the
laboratory investigations, SK and TK involved in the data and sample collection.
NR did analysis. SK, AM and NRS wrote the manuscript. All authors read and
approved the final manuscript.
Consent for publication
Ethics approval and consent to participate
Ethical clearance was obtained from the Ethical Review Committee of the
Faculty of Medicine, University of Jaffna. Permission to carry out this study
was obtained from the Director, TH, Jaffna and written consent was
obtained from the participants after explaining the purpose and the nature
of the study.
1. World Health Organization. World Health Organization/Pan American Health Organization/UNESCO report of a Consultation of Expert on Amoebiasis . Wkly Epidemiol Rec . 1997 ; 72 : 97 - 9 .
2. Ximenez C , Moran P , Rojas L , Valadez A , Gomez A. Reassessment of the epidemiology of amebiasis: state of the art . Infect Genet Evol . 2009 ; 9 : 1023 - 32 .
3. Rajasuriya K , Nagaratnam N. Hepatic amoebiasis in Ceylon . J Trop Med Hyg . 1962 ; 65 ( 7 ): 165 - 78 .
4. Ramachandran S , Sivalingam S , Perumal JRA. Hepatic amoebiais in Ceylon . J Trop Med Hyg . 1972 ; 75 : 23 - 33 .
5. Jayewardene L , Wijaratnam Y. The serological diagnosis of amoebiasis in Ceylon . Pt I. The indirect haemagglutination test (IHAT) . Ceylon J Med Sci . 1973 ; 22 ( 1 ): 1 - 5 .
6. Canagaretna C. Amoebic liver abscess - a clinical study . Ceylon Med J . 1974 ; 19 : 18 - 27 .
7. Ramachandran S. The Marcus Fernando memorial oration 1974 . Ceylon Med J . 1975 ; 20 : 69 - 81 .
8. Sreeharan NMD , Yogeswaran P , Puthrasingam S , Ranjadayalan K , Ganeshamoorthy J. Hepatic amoebiasis in Northern Sri Lanka: a retrospective study . Jaffna Med J . 1985 ; 20 ( 2 ): 69 - 74 .
9. Fernando K , Fernando R , Kandasami A , Jude R , Fernando N , Tennakoon S. SP6-3 Fermented sap of spiky Palmyra toddy (Borassus flabellifer) suggested as a vehicle of transportation of amoebiasis in the district of Mannar, Sri Lanka: 50 cases of amoebic liver abscess within 15 months . J Epidemiol Commun H . 2011 ; 65 ( 1 ): 455 .
10. Janani T , Pushpana P , Surenthirakumaran R , Kumanan T , Kannanthasan S. Amoebic liver abscess: An emerging threat in northern Sri Lanka . EMBO global lecture course and symposium on amoebiasis: Exploring the biology and pathogenesis of Entamoeba . Khajuraho, India; 2011 . p 80 .
11. Kannathasan S , Iddawala WMDR , De Silva NR , Haque R. Knowledge , attitude and practice towards liver abscess among patients admitted to the teaching hospitals, Jaffna . In: Proceedings of the Peradeniya Univ. International Research Sessions, Sri Lanka , vol. 18 . 2014 . p. 355 .
12. Salles JM , Moraes LA , Salles MC . Hepatic amebiasis . Braz J Infect Dis . 2003 ; 7 ( 2 ): 96 - 110 .
13. Robinson GL . Laboratory diagnosis of some human parasitic amoeba . J Gen Microbiol . 1968 ; 53 : 69 - 79 .
14. Ayeh-Kumi PF , Ali IM , Lockhart LA , Gilchrist CA , Petri WA , Haque R. Entamoeba histolytica: genetic diversity of clinical isolates from Bangladesh as demonstrated by polymorphisms in the serine-rich gene . Exp Parasitol . 2001 ; 99 ( 2 ): 80 - 8 .
15. Alam F , Salam MA , Hassan P , Mahmood I , Kabir M , Haque R. Amebic liver abscess in northern region of Bangladesh: sociodemographic determinants and clinical outcomes . BMC Res Notes . 2014 ; 7 ( 1 ): 625 .
16. Bhatti AB , Ali F , Satti SA , Satti TM . Clinical and pathological comparison of pyogenic and amoebic liver abscesses . Adv Infect Dis . 2014 ; 4 : 117 - 23 .
17. Chaudhary S , Noor MT , Jain S , Kumar R , Thakur BS . Amoebic liver abscess: a report from central India . Trop Doct . 2015 . doi:10.1177/ 0049475515592283.
18. Siddiqui MA , Ahad MA , Ekram AS , Islam QT , Hoque MA , Masum QAAI . Clinico-pathological profile of liver abscess in a teaching hospital . TAJ: J Teachers Assoc . 2008 ; 21 ( 1 ): 44 - 9 .
19. Vallois D , Epelboin L , Touafek F , Magne D , Thellier M , Bricaire F , ALA-PCR Working Group. Amebic liver abscess diagnosed by polymerase chain reaction in 14 returning travelers . Am J Trop Med Hyg . 2012 ; 87 ( 6 ): 1041 - 5 .
20. Wuerz T , Kane JB , Boggild AK , Krajden S , Keystone JS , Fuksa M , Anderson J. A review of amoebic liver abscess for clinicians in a nonendemic setting . Can J Gastroenterol Hepatol . 2012 ; 26 ( 10 ): 729 - 33 .
21. van Hal SJ , Stark DJ , Fotedar R , Marriott D. Amoebiasis: current status in Australia . Med J Aust . 2007 ; 186 ( 8 ): 412 .
22. Cordel H , Prendki V , Madec Y , Houze S , Paris L , Caumes E , et al. Imported amoebic liver abscess in France. PLoS Negl Trop Dis . 2013 ; 7 ( 8 ): e2333 .
23. Nari GA , Ceballos ER , Carrera LDGS , Preciado VJ , Cruz VJ , Briones RJ , Góngora OJ . Amebic liver abscess . Three years' experience. Rev Esp Enferm Dig . 2008 ; 100 ( 5 ): 268 - 72 .
24. Albenmousa A , Sanai FM , Singhal A , Babatin MA , AlZanbagi AA , Al-Otaibi MM , Bzeizi KI . Liver abscess presentation and management in Saudi Arabia and the United Kingdom . Ann of Saudi Med . 2011 ; 31 ( 5 ): 528 .
25. Cosme A , Ojeda E , Zamarreño I , Bujanda L , Garmendia G , Echeverría MJ , Benavente J. Pyogenic versus amoebic liver abscesses. A comparative clinical study in a series of 58 patients . Rev Esp Enferm Dig . 2010 ; 102 ( 2 ): 90 - 5 .
26. Lodhi S , Sarwari AR , Muzammil M , Salam A , Smego RA . Features distinguishing amoebic from pyogenic liver abscess: a review of 577 adult cases . Trop Med Int Health . 2004 ; 9 ( 6 ): 718 - 23 .
27. Ghosh S , Sharma S , Gadpayle AK , Gupta HK , Mahajan RK , Sahoo R , Kumar N. Clinical , laboratory, and management profile in patients of liver abscess from northern India . J Trop Med . 2014 . http://dx.doi.org/10.1155/2014/ 142382.
28. Bruckner DA . Amebiasis . Clin Microbiol Rev . 1992 ; 5 ( 4 ): 356 - 69 .
29. Haque R , Huston CD , Hughes M , Houpt E , Petri Jr WA. Amebiasis . N Eng J Med . 2003 ; 348 ( 16 ): 1565 - 73 .
30. Blessmann J , Van Pham L , Anthon Nu P , Duong Thi H , Muller-Myhsok B , et al. Epidemiology of amoebiasis in a region of high incidence of amoebic liver abscess in central Vietnam . Am J Trop Med Hyg . 2002 ; 66 ( 5 ): 578 - 83 .
31. Tanyuksel M , William Jr AP . Laboratory diagnosis of amoebiasis . Clin Microbiol Rev . 2003 ; 16 ( 4 ): 713 - 29 .
32. Knobloch J , Mannweiler E. Development and persistence of antibodies to Entamoeba histolytica in patients with amebic liver abscess: Analysis of 216 cases . Am J Trop Med Hyg . 1983 ; 32 : 727 - 32 .
33. Kraoul LH , Adjmi V , Lavarde JF , Pays C , Tourte-Schaefer C , Hennequin C. Evaluation of a rapid enzyme immunoassay for diagnosis of hepatic amoebiasis . J Clin Microbiol . 1997 ; 35 : 1530 - 2 .
34. Mathur S , Gehlot RS , Mohta A , Bhargava N. Clinical profile of amoebic liver abscess . J Indian Acad Clin Med . 2002 ; 3 : 367 - 73 .
35. Paul J , Srivastava S , Bhattacharya S. Molecular methods for diagnosis of Entamoeba histolytica in a clinical setting: An overview . Exp Parasitol . 2007 ; 116 : 35 - 43 .
36. Stensvold CR , Lebbad M , Verweij JJ . The impact of genetic diversity in protozoa on molecular diagnostics . Trends Parasitol . 2011 ; 27 ( 2 ): 53 - 8 .
37. Acuna-Soto R , Samuelson J , De Girolami P , Zarate L , Millan-Velasco F , Schoolnick G , Wirth D. Application of the polymerase chain reaction to the epidemiology of pathogenic and nonpathogenic Entamoeba histolytica . Am J Trop Med Hyg . 1993 ; 48 ( 1 ): 58 - 70 .
38. Britten D , Wilson SM , McNerney R , Moody AH , Chiodini PL , Ackers JP . An improved colorimetric PCR-based method for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in feces . J Clin Microbiol . 1997 ; 35 ( 5 ): 1108 - 11 .
39. Zaman S , Khoo J , Ahmed SW , Ng R , Khan MA , Hussain R , Zaman V. Direct amplification of Entamoeba histolytica DNA from amoebic liver abscess pus using polymerase chain reaction . Parasitol Res . 2000 ; 86 : 724 - 8 .
40. Khan U , Mirdha BR , Samantaray JC , Sharma MP . Detection of Entamoeba histolytica using polymerase chain reaction in pus samples from amoebic liver abscess . Indian J Gastroenterol . 2006 ; 25 ( 2 ): 55 - 7 .
41. Fotedar R , Stark D , Beebe N , Marriott D , Ellis J , Harkness J. Laboratory diagnostic techniques for Entamoeba species . Clin Microbiol Rev . 2007 ; 20 ( 3 ): 511 - 32 .
42. Haghighi A , Kobayashi S , Takeuchi T , Masuda G , Nozaki T. Remarkable genetic polymorphism among Entamoeba histolytica isolates from a limited geographic area . J Clin Microbiol . 2002 ; 40 ( 11 ): 4081 - 90 .
43. Simonishvili S , Tsanava S , Sanadze K , Chlikadze R , Miskalishvili A , Lomkatsi N , et al. Entamoeba histolytica: the serine-rich gene polymorphism-based genetic variability of clinical isolates from Georgia . Exp Parasitol . 2005 ; 110 ( 3 ): 313 - 7 .