Parasitology International 63 (2014) 308–314

Contents lists available at ScienceDirect

Parasitology International journal homepage: www.elsevier.com/locate/parint

Multilocus sequence typing system (MLST) reveals a significant association of Entamoeba histolytica genetic patterns with disease outcome Koushik Das a,1, Avik K. Mukherjee a,1, Punam Chowdhury a, Rakesh Sehgal b, Mihir K. Bhattacharya c, Tetsuo Hashimoto d, Tomoyoshi Nozaki d,e, Sandipan Ganguly a,⁎ a

Division of Parasitology, National Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme XM, Beliaghata, Kolkata 700010, India Department of Parasitology, Postgraduate Institute of Medical Education & Research, Chandigarh 160012, India c Division of Clinical Medicine, National Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme XM, Beliaghata, Kolkata 700010, India d Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8572, Japan e Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan b

a r t i c l e

i n f o

Article history: Received 17 July 2013 Received in revised form 9 November 2013 Accepted 28 November 2013 Available online 7 December 2013 Keywords: Entamoeba histolytica Amoebic outcome Genotyping Chitinase tRNA linked STR Phylogeny

a b s t r a c t The relationship between parasite genotypes and outcome of amoebic infection is still a paradox and needed to be explored. Proper identification and genetic characterization of Entamoeba histolytica clinical isolates is an effective tool for exploring this relation. Along with conventional polymorphic marker (Chitinase), tRNA linked short tandem repeat (STR) loci has been employed as multilocus genotyping tool due to its better resolution and evolutionary significance. Some common as well as exclusive repeat patterns showing significant relation with disease outcome have been identified. Phylogenetic analysis revealed that repeat patterns exclusively found in asymptomatic and amoebic liver abscess derived isolates are placed in a common lineage and has similar association pattern with the disease outcome. Assumption can be made that isolates of E. histolytica remaining asymptomatic is genetically closer siblings of those causing liver abscess rather than the diarrheal isolates. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Amoebiasis is one of the major enteric diseases in human caused by the protozoan parasite Entamoeba histolytica (Eh). It, being one of the major parasitic diseases after malaria is responsible for approximately 100,000 human deaths per annum [1]. E. histolytica infection develops differential clinical manifestation. Most infections remain asymptomatic while some develop diarrhea, dysentery and very rarely causes extraintestinal complication like amoebic liver abscess [2,3]. Specific determinants for this differential clinical outcome are mostly unknown; however, few rely on the fact that host genetics and parasite genotypes possibly play important roles [4,5]. Proper identification and genetic characterization of clinical isolates became obligatory to explore the relationship between parasite genotypes and outcome of amoebic infection. Although some PCR based genotyping method utilize single

⁎ Corresponding author. E-mail addresses: [email protected] (K. Das), [email protected] (A.K. Mukherjee), [email protected] (P. Chowdhury), [email protected] (T. Hashimoto), [email protected] (T. Nozaki), [email protected] (S. Ganguly). 1 Authors contributed equally to this work. 1383-5769/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.parint.2013.11.014

polymorphic markers like- Serine rich Entamoeba histolytica protein (SREHP), Chitinase (CHI) gene etc. [6,7], but it has now become obsolete due to its low resolution. In contrast, multilocus genotyping systems based on highly polymorphic non-coding regions like tRNA linked STR loci have been greatly employed to obtain a much better resolution [8]. tRNA genes are “hotspot” for recombination and mutation due to their unique telomeric location and genetic organization and has been greatly preferred in evolutionary study of E. histolytica [9]. In the present study genotyping of E. histolytica clinical isolates obtained from the individual with different disease outcome was performed using both protein coding Chitinase loci (CHI) as well as non-coding tRNA linked STR loci like DA-H, N-K2 and R-R as genotyping marker. The goal of our study was to determine the genetic pattern and the diversity of E. histolytica isolates based on specific genetic markers and to identify any significant association of parasite genotypes with the disease outcomes. Efforts were also made to determine a possible phylogenetic relation among STR patterns obtained from the genetic analysis of our study isolates. High degree of genetic diversity was observed among E. histolytica isolates and repeat patterns exhibiting significant association with a particular disease outcome were also identified. STR patterns from asymptomatic (AS) and liver abscess (LA) derived isolates were found to have close phylogenetic association.

K. Das et al. / Parasitology International 63 (2014) 308–314

2. Material and methods 2.1. Sample collection and detection of Entamoeba histolytica A total of 51 Indian E. histolytica isolates were included in our present study, among them 26 were from diarrheal cases, 20 from liver abscess cases and 5 from asymptomatic cases. The diarrheal fecal samples were collected from the patients admitted in Infectious Diseases Hospital, Kolkata with sole diarrheal complications and asymptomatic samples were collected through an on-going field project for studying the parasite burden in Kolkata. In both cases, the samples were mostly obtained from a population of low socioeconomic status [10]. Liver aspirates of Amoebic Liver Abscess (ALA) patients were obtained from Dr. Sehgal's lab from IPGMER, Chandigarh. Since, STR patterns have a wide geographic distribution [9,11] and differ between the E. histolytica isolates from liver aspirates and enteric samples of the same patient [12], so geographical origin of clinical samples could not influence the STR pattern. Amoebic Liver Abscess (ALA) was more common among adult male compared to diarrheal and asymptomatic outcomes. The individuals with different amoebic symptoms were screened based on the predetermined inclusion and exclusion criteria. Information regarding age and sex of the study populations was provided in Supplementary Table 1. The study received an ethical clearance from the NICED IEC (i.e. National Institute of Cholera and Enteric Diseases Institutional Ethical Committee). Informed consent was obtained from the patients (in case of children, consent was obtained from their parents). The parasites were primarily detected by conventional microscopy and Antigen capture ELISA (E. histolytica II, TECHLAB, USA) followed by genetic identification by PCR amplification of E. histolytica SSU rRNA gene [10]. DNA was isolated directly from human stool using DNA Stool Mini Kit (QIAGEN, USA) as per manufacturer's protocol.

309

and CHI). All the sequences obtained from the study were submitted to NCBI GenBank with accession numbers KC244560-KC244638, JQ062932-JQ062960, KC244481-KC244505, JN984745-JN984774, JN984687-JN984715 and KF355975- KF355998. The sequences of all representative repeat patterns from particular loci were aligned using ClustalW multiple alignment program of MEGA Version 4 software [17] and edited manually. The alignments thus obtained were used for constructing phylogenetic tree through “Generalized Time Reversal (GTR) + gamma” substitution model of SeaView Graphical Interface version 4 software [18]. Association of repeat patterns with specific clinical outcome was evaluated by Epi-Info ver 3.5.4 software [19]. 3. Result Amplified PCR products of tRNA linked STR loci were highly variable in size within our study isolates, while PCR products of CHI locus were almost same in size (Fig. 1). This could confer the possibility of recombinational insertion or deletion within the intergenic regions leading to the generation of differential band pattern. Sequence analysis of amplified PCR products revealed considerable amount of polymorphism depending on repeat types, numbers and orientation within the target loci among our 51 study isolates. 3.1. Repeat polymorphism in Chitinase (CHI) locus CHI locus was moderately polymorphic, showing 13 different repeat patterns where 2 of them resemble with previously reported patterns of Eh NIH: 200 and HM-1: IMSS strain [13,16]. While the remaining 11 repeat patterns were newly identified and assigned as Indian Chitinase 1, 2, 3 to 11 (i.e. IND1CHI to IND11CHI) (Fig. 2).

2.2. PCR amplification, DNA sequencing and analysis

3.2. Repeat polymorphism in tRNA linked STR loci

Nested PCR amplification was performed with Eh specific primer pairs, targeting a part of Chitinase (CHI) gene as well as three tRNA linked STR loci i.e., DA-H, NK2-H and R-R [8,13] (Supplementary Table 2). In all cases the reaction was carried out in 50 μl reaction volume containing approximately 0.4 μg and 0.1 μg of template DNA for primary and nested PCR respectively, 10 pM of each primer, 1.5 mM MgCl2, 1 μg of Bovine Serum Albumin (SIGMA, USA), 200 μM dNTP and 2.5 U of Taq DNA polymerase (Bioline, USA) with the reaction parameters as initial denaturation for 15 mins or 4 mins (Primary and Nested respectively) at 95°C. This was followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 50°C (DA-H) and 60°C (CHI, NK2-H and R-R) for 30 s, extension at 72°C for 1 min. This was followed by the final extension for 10 mins at 72°C. The amplified PCR products were separated, according to their size by electrophoresis on 1.5% agarose gels (SIGMA, USA). Amplicons of the expected sizes were extracted from gels and purified (ROCHE, Germany), and their yield was verified again by electrophoresis. Purified PCR products were sequenced directly with specific primers (marked with a in Supplementary Table 2) using the ‘BigDye Terminator V3.1 cycle sequencing kit’ (Applied Biosystems, USA) as per the manufacturer's protocol. The labeled DNA fragments were purified by sodium acetate and ethanol precipitation. The sequencing was performed in an ABI 310 PRISM Automated Genetic Analyzer. The nucleotide sequences were then analyzed with the tandem repeat finder software (http://tandem.bu.edu/trf/trf.html) [14] and each unique repeat unit was coded with a particular color for better understanding (Fig. 2a, b, c and d). The identified repeat patterns that had been reported previously [9,11–13,15,16] were named according to their existing nomenclature. Newly identified repeat patterns were assigned by alphanumerical codes beginning with ‘IND’ (to indicate their Indian origin) followed by ‘repeat pattern number’ (i.e. 1, 2, 3…. etc.) and ‘name of corresponding loci’ (i.e. DA, NK, RR

tRNA linked DA-H locus was seemed to be least polymorphic among all target loci. A total of 11 different repeat patterns were observed where three of them (i.e. 5DA, 6DA and 14DA) were previously reported (Fig. 2) [9,15]. The other eight were newly identified and assigned as- IND2DA to IND9DA (nomenclature followed the previous report from India by Zermeno et al.) [11]. Comparatively high degree of polymorphism was observed in case of R-R locus. Depending upon the size, numbers and orientation of the repeats, 17 different STR patterns were observed and all of them were newly identified and assigned as IND1RR to IND17RR (Fig. 2). tRNA linked N-K2 loci was found to be most polymorphic and exhibit as much as 22 different repeat patterns among 51 study isolates. 18 of them were newly identified and assigned as - IND6NK to IND23NK (nomenclature followed the previous report from India by Zermeno et al.) [11]. The other 4 (i.e. 18NK, 3NK, 8NK and 16NK) resembles with previously reported patterns [9] (Fig. 2). All repeat information of study isolates has been provided in Supplementary Table 1. 3.3. Distribution of Repeat patterns among three different outcome groups Outcome specific repeat pattern in both CHI and tRNA linked STR locus were observed in our study. For example, patterns like IND3CHI – IND6CHI, IND8DA, 3NK, 8NK, 16NK and IND12RR were found only in liver abscess (LA) group whereas, IND1CHI, IND2CHI, 5DA, IND12NK, IND14NK, IND1RR and IND4RR were only present in diarrheal (D) group. Similarly, IND7CHI, IND7DA, IND22NK, IND23NK and IND17RR were exclusive for asymptomatic (AS) group. The appearance of 5DA as sole diarrheal pattern was somehow incongruous with previous report by Escueta-de Cadiz et al., where 5DA was associated with all three outcomes [15]. However, similar type of observation was also previously reported in another tRNA linked STR loci R-R. For example, Ali et al. have reported that repeat pattern 5RR was solely associated

310

K. Das et al. / Parasitology International 63 (2014) 308–314

Fig. 1. Agarose gel electrophoresis of amplified PCR products of target loci: Fragment length polymorphisms of 14 representative isolates of one protein coding i.e. (a) Chitinase (CHI) of band size around 550 bp and 3 tRNA-linked STR loci i.e. (b) DA-H of band size ranging from 300–400 bp, (c) NK-2 ranging from 400–600 bp, (d) R-R ranging from 400–600 bp. Lane1 and Lane 17 corresponds to the 50 bp DNA marker, Lanes 2–15 correspond to PCR amplified products of corresponding loci of 14 representative isolates, Lane 16 corresponding to negative control.

with asymptomatic (AS) outcome within their study population from Bangladesh [20]. In contrast, Feng et al. have reported that 5RR was associated with both liver abscess (LA) and colitis in China [21], and interestingly study by Escueta-de Cadiz et al. revealed that 5RR was found in all three disease outcomes in Japan (i.e. LA, AS and D group) [15]. Repeat patterns of CHI locus that resembles previously reported repeat pattern of Eh NIH: 200 and HM-1: IMSS strains were found in all three disease outcome group. But in contrast, no such repeat patterns were observed in tRNA linked loci which are found in all three disease group, suggesting more specific distribution and resolution of tRNA linked loci which could highlight any possible relation of repeat patterns with the disease outcome. For example, 6DA, 18NK and IND2RR were found both in LA and D group. Similarly, 14DA, IND13NK and IND16NK were common among LA and AS group (Table 1). The association between the repeat pattern and disease outcome were further analyzed to justify these observations. 3.3.1. Association between repeat patterns and outcome of infection Repeat pattern of CHI locus that resembles repeat pattern of Eh NIH: 200 strains showed strong positive association with D group (p = 0.00001) and a strong negative association with LA group (p = 0.0014). In contrast the HM-1: IMSS like repeat pattern of the same loci showed a negative association with D group (p = 0.0173). Moreover, 6DA of DA-H loci showed strong positive association with LA (p = 0.0026), and a negative association with D group (p = 0.0290). Similarly, 14DA exhibit a strong positive association with both LA (p = 0.0096) and AS group (p = 0.0026), but a negative association with D group (p = 0.000004). In N-K2 locus, 18NK showed strong positive association with D (p = 0.0008), but a negative association with LA (p = 0.0116). Whereas, IND11RR of RR locus was found to be positively associated with AS group (p = 0.0082), but negatively associated with D group (p = 0.0004) (Table 2). This result corroborates with a previous report by Ali et al., where 5RR was associated with asymptomatic outcome and 10RR was associated with symptomatic outcome [20]. Detailed analysis by Epi-Info software has been provided as supplementary data (Supplementary Data 3, 4, 5, 6). 3.4. Phylogenetic analysis between representative repeat patterns of STR loci STR patterns found exclusively in a particular disease outcome group (Table 1) showed a distinct cluster formation in phylogenetic trees (Figs. 3, 4, 5). Especially the STR patterns found only in the diarrheal isolates (exclusive ‘D’ patterns) showed a distinct position in all trees (i.e. ‘D’ group). A separate cluster with STR patterns exclusive

for asymptomatic isolates (exclusive ‘AS’ patterns) was observed in the R-R tree (i.e. ‘AS’ group). N-K2 being the most polymorphic loci also showed much higher resolution and specific distribution with two distinct clusters (i.e. ‘D’ group and ‘LA’ group) of exclusive ‘D’ and exclusive ‘LA’ patterns (STR patterns found only in the liver abscess causing isolates) and another mixed cluster were having both exclusive ‘LA’ and exclusive ‘AS’ patterns. Similar kind of mixed cluster with exclusive ‘LA’ and ‘AS’ patterns was also observed in DA-H tree. As per the cluster analysis the ‘D’ group always showed a distinct position unlike that of ‘AS’ and ‘LA’. The ‘AS’ and ‘LA’ STR patterns were found to be existing in close proximity as well as in same clusters which supports the finding of the association analysis i.e. isolates causing Liver Abscess and Asymptomatic outcome may be closely related or belongs to similar lineage. Since, the location of repetitive tRNA arrays can't be mapped to the E. histolytica genome [22], duplication and divergence of STR arrays occurred independently for each STR locus [9]; concatenated phylogenetic trees based on the complete data set (i.e., multiple genetic markers) for each sample do not provide useful information on genetic markers that have association with disease outcomes. For independently segregating unlinked markers such as tRNA-linked STR loci of Entamoeba, phylogenetic analysis of independent loci should be able to give insights into strain diversification. 4. Discussion Genome information of infecting strains from the endemic areas throughout the world is needed to interpret the role of parasite genome in outcome of amoebic infection [23]. Selection of appropriate genetic markers is required for an optimum genotyping system. tRNA linked STR loci has been preferred as high resolution genotyping tool for its unique features over conventional genetic markers. An estimated 4500 copies of tandemly arrayed tRNA genes (10% of whole genome) are found in E. histolytica [9,24]. The intergenic regions of these tRNA genes comprised of short tandemly repeated sequences (STRs) which resembles the micro/mini satellites of eukaryotic genomes. The only difference is that unlike randomly dispersed micro/mini satellites, STRs form a part of a larger unit which is itself tandemly arrayed [9]. tRNA genes are thought to be “hotspots” for recombination probably due to the formation of replication fork barriers by RNA Polymerase III transcription complex leading to the pause of replication [25,26]. The intergenic tRNA regions are A + T rich resulting in possibility of frequent tandem duplication due to chance mutation [27,28]. Moreover, the average numbers of STRs in the intergenic tRNA regions remained stable in E. histolytica for several years for a particular strain in a continuous culture [8]. These particular features make it a very useful

K. Das et al. / Parasitology International 63 (2014) 308–314

311

Fig. 2. Schematic representation of repeat patterns of each loci based on the nucleotide sequences of our study isolates: tRNA genes are depicted in arrows, repeat unit in rectangles (with specific color coding) and non-repeat regions are shown in lines. Newly identified repeat patterns were assigned by alphanumerical codes beginning with ‘IND’ (to indicate their Indian origin) followed by ‘repeat pattern number’ (i.e. 1, 2, 3…. etc.) and ‘name of corresponding loci’ (i.e. DA, NK, RR and CHI). (a) Chitinase (CHI) loci: 11 new repeat patterns were assigned (i.e. IND1CHI to IND11CHI), (b) DA-H loci: 8 new repeat patterns were assigned (i.e. IND2DA to IND9DA), (c) R-R loci: 17 new repeat patterns were assigned (i.e. IND1RR- IND17RR), (d) N-K2 loci: 18 new repeat patterns were named as IND6NK to IND23NK.

tool for the quantification of evolutionary divergence of this fascinating parasite. The goal of our study was to generate an idea about the genetic pattern and the diversity of E. histolytica isolates obtained from different disease outcome based on specific genetic markers. To perform an optimal genotyping, we have included the protein coding regions such as CHI along with tRNA linked DA-H, N-K2 and R-R loci. Depending on the size of PCR products obtained from local E. histolytica isolates, CHI locus represents lesser polymorphism compared to other loci. Moreover, detailed sequence analysis and repeat assignments revealed that mostly dominant repeat patterns resembles with previously reported repeat pattern of E. histolytica NIH: 200 and HM-1: IMSS strain and were found in all three disease groups (i.e. common repeat patterns). But detailed analysis of STR loci revealed some interesting scenario. Most of repeat patterns were newly assigned and possess specific relation with diseases outcome groups. No repeat patterns were found which were present in all

three disease groups. So, it could be assumed that the STR loci may have comparatively high degree of resolution for determining the relationship between parasite genotypes and outcome of amoebic infection. To justify this finding, attempts were made to determine whether any statistically significant association exists between repeat patterns and disease outcome. It revealed that repeat patterns which showed strong positive association with LA (liver abscess), also showed strong positive association with AS (asymptomatic) or vice versa (in case of 14DA of DA-H locus). Whereas repeat patterns which showed positive association with D (diarrhea), showed strong negative association with LA (in case of 6DA, 14DA, 18NK and NIH: 200) and AS group (in case of 14DA and IND11RR). So, isolates derived from liver abscess and asymptomatic cases may be genetically close or may have originated from similar lineage rather than, isolates from diarrheal outcome. The finding was well supported by the clustal analysis where STR patterns exclusive for asymptomatic (AS) and liver abscess (LA) outcome forms a single

312

K. Das et al. / Parasitology International 63 (2014) 308–314 Table 1 Distribution of repeat patterns among disease groups.

K. Das et al. / Parasitology International 63 (2014) 308–314

313

Table 2 Repeat patterns of target loci showing significant association with disease outcomes. Loci

Repeat pattern

Liver abscess (LA)

Diarrheal (D)

Asymptomatic (AS)

D-AH

6DA

Co-eff: 0.507a p = 0.0026b Co-eff: 0.403a p = 0.0096b Co-eff: −0.404a p = 0.0116b Xc

Co-eff: −0.385a p = 0.029b Co-eff: −0.684a p = 0.000004b Co-effa: 0.532 p = 0.0008b Co-eff: −0.619a p = 0.0004b Co-eff: 0.577a p = 0.00001b Co-eff: −0.456a p = 0.0173b

Xc

14DA N-K2

18NK

R-R

IND11RR

Chitinase (CHI)

NIH:200 HM1:IMSS

Co-eff: −0.429a p = 0.0014b Xc

Co-eff: 0.281a p = 0.0026b Xc Co-eff: 0.286a p = 0.0082b Xc Xc

a Correlation co-efficient value of the particular association indicate whether the association is positive or negative. b Probability value of the particular association. c Does not have any significant association with disease outcomes.

cluster in DA-H and NK-2 tree, whereas STR patterns exclusive for diarrheal (D) outcome always forms a separate cluster in all three trees. It is obvious from the above observations that the LA and AS isolates are genetically or evolutionary more closer in compare to the D isolates. Fig. 4. Phylogenetic relation among STR patterns of N-K2-H loci: The sequences of repeat patterns from N-K2-H loci obtained from the genetic analysis of study isolates were aligned using ClustalW multiple alignment program of MEGA Ver 4 software. The alignment thus obtained was used for constructing phylogenetic tree through “Generalized Time Reversal (GTR) + gamma” substitution model of SeaView Graphical Interface Ver.4 software using a maximum likelihood matrix algorithm. One mixed “AS + LA” group and two distinct “D” group and “LA” group can be assigned. Mixed “AS + LA” group contains STR patterns found in both asymptomatic and liver abscess outcomes. “D” group contain STR patterns found exclusively in diarrheal outcome. “LA” group contain STR patterns found exclusively in liver abscess outcome.

Repeat patterns exclusive for AS and LA outcomes belong to same clusters and showed a similar association pattern with disease outcome which supports the previous findings of high genetic similarity or relatedness between E. histolytica isolates from AS and LA outcomes [4,21,22,29]. Since, amoeba trophozoites migrate from intestine to liver via bloodstream to cause amoebic liver abscess [12], individuals with persistent asymptomatic E. histolytica infection may be under high risk of developing amoebic liver abscess formation in future. So, decreasing trend of E. histolytica infection among diarrheal patients in a tropical country [30,10] may not at all be a signs of relief but may turn into an epidemic of ALA in future and further detailed investigation of the individuals with asymptomatic E. histolytica infection is always required. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.parint.2013.11.014. Acknowledgments

Fig. 3. Phylogenetic relation among STR patterns of R-R loci: The sequences of repeat patterns from R-R loci obtained from the genetic analysis of study isolates were aligned using ClustalW multiple alignment program of MEGA Ver 4 software. The alignment thus obtained was used for constructing phylogenetic tree through “Generalized Time Reversal (GTR) + gamma” substitution model of SeaView Graphical Interface Ver.4 software using a maximum likelihood matrix algorithm. Two distinct (named as “D” group and “AS” group) as well as a mixed cluster can be assigned. “D” group contain STR patterns found exclusively in diarrheal outcome, whereas “AS” group contain STR patterns exclusive for asymptomatic outcome.

This study was jointly supported by a grant from National Institute of Infectious Diseases, Japan, Okayama University Program of Founding Research Centre for Emerging and Re-emerging Infectious Disease, Ministry of Education, Culture, Sports, Science and Technology of Japan and Japan Health and Science Foundation, Govt. of Japan. The study was also aided by Indian Council of Medical Research, Govt. of India for providing Senior Research Fellowship to K. D. The authors acknowledge Prof. Seiki Kobayashi, Keio University Japan for his constructive suggestions, comments, support and immense help throughout the entire study; Dr. Escuta-de Cadiz Aleyla NIID Japan for her excellent support regarding the analysis of STR. We also thank Dr. Mrinmoy Ghosh, Principal, Infectious Diseases and Beliaghata General Hospital, Kolkata, for the arrangement of the whole stool collection procedure. Authors would like to thank Mrs. Debarati Ganguly for her immense help regarding the English language correction.

314

K. Das et al. / Parasitology International 63 (2014) 308–314

Fig. 5. Phylogenetic relation among STR patterns of DA-H loci: The sequences of repeat patterns from DA-H loci obtained from the genetic analysis of study isolates were aligned using ClustalW multiple alignment program of MEGA Ver 4 software. The alignment thus obtained was used for constructing phylogenetic tree through “Generalized Time Reversal (GTR) + gamma” substitution model of SeaView Graphical Interface Ver.4 software using a maximum likelihood matrix algorithm. One mixed “AS + LA” group and a distinct “D” group can be assigned. Mixed “AS + LA” group contains STR patterns found in both asymptomatic and liver abscess outcomes.

References [1] W.H.O., PAHO, UNESCO. A consultation with experts on amebiasis. Epidemiol Bull 1997;18:13–4. [2] Stanley Jr SL. Amoebiasis. Lancet 2003;361:1025–34. [3] Mehmet T, Petri Jr WA. Laboratory diagnosis of amebiasis. Clin Microbiol Rev 2003;16(4):713–29. [4] Ali IKM, Mondal U, Roy S, Haque R, Petri Jr WA, Clark CG. Evidence for a link between parasite genotype and outcome of infection with Entamoeba histolytica. J Clin Microbiol 2007;45:285–9. [5] Duggal PR, Haque R, Roy S, Mondal D, Sack RB, Farr BM. Influence of human leukocyte antigen class II alleles on susceptibility to Entamoeba histolytica infection in Bangladeshi children. J Infect Dis 2004;189:520–6.

[6] Ali IKM, Clark CG, Petri Jr WA. Molecular epidemiology of amebiasis. Infect Genet Evol 2008;8:698–707. [7] Mehmet T, Mustafa U, Hasan Y, Zeynep G, Engin A, Gurkan M, et al. Genetic variability of the serine-rich gene of Entamoeba histolytica in clinical isolates from Turkey. Turk J Med Sci 2008;38(3):239–44. [8] Ali IKM, Zaki M, Clark CG. Use of PCR amplification of tRNA gene-linked short tandem repeats for genotyping Entamoeba histolytica. J Clin Microbiol 2005;43:5842–7. [9] Tawari B, Ali IKM, Scott C, Quail MA, Berriman M, Hall N, et al. Patterns of evolution in the unique tRNA gene arrays of the genus Entamoeba. Mol Biol Evol 2008;25:187–98. [10] Mukherjee AK, Chowdhury P, Bhattacharya MK, Ghosh M, Rajendran K, Ganguly S. Hospital-based surveillance of enteric parasites in Kolkata. BMC Res Notes 2009;2:110. [11] Zermeno V, Ximenez C, Moran P, Valadez A, Valenzuela O, Rascon E. Worldwide genealogy of Entamoeba histolytica: An overview to understand haplotype distribution and infection outcome. Infect Genet Evol 2013;17:243–52. [12] Ali IKM, Solaymani-Mohammadi S, Akhter J, Roy S, Gorrini C, Calderaro A, et al. Tissue invasion by Entamoeba histolytica: evidence of genetic selection and/or DNA reorganization events in organ tropism. PLoS Negl Trop Dis 2008;2:e219. [13] 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;40:4081–90. [14] Benson G. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res 1999;27:573–80. [15] Escuta-de Cadiz A, Kobayashi S, Takeuchi T, Tachibana H, Nozaki T. Identification of an avirulent Entamoeba histolytica strain with unique tRNA-linked short tandem repeat markers. Parasitol Int 2010;59:75–81. [16] Ghosh S, Frisardi M, Ramirez-Avila L, Descoteaux S, Sturm-Ramirez K, NewtonSanchez OA, et al. Molecular epidemiology of Entamoeba spp. evidence of a bottleneck (demographic sweep) and transcontinental spread of diploid parasites. J Clin Microbiol 2000;38:3815–21. [17] Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24(8):1596–9. [18] Gouy M, Guindon S, Gascuel O. SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol 2010;27(2):221–4. [19] Dean AG, Arner TG, Sunki GG, Friedman R, Lantiga M, Sangam S, et al. Epi Info™, a database and statistic program for public health professional. Atlanta, Georgia, USA: Centers for Disease Control and Prevention; 2007. [20] Ali IKM, Haque R, Alam F, Kabir M, Siddique A, Petri WA. Evidence for a link between locus R-R sequence type and outcome of infection with Entamoeba histolytica. Clin Microbiol Infect 2012;18:235–7. [21] Feng M, Cai J, Yang B, Fu Y, Min X, Tachibana H, et al. Unique short tandem repeat nucleotide sequences in Entamoeba histolytica isolates from China. Parasitol Res 2012;111:1137–42. [22] Gilchrist CA, Ali IKM, Kabir M, Alam F, Scherbakova S, Ferlanti E, et al. A Multilocus Sequence Typing System (MLST) reveals a high level of diversity and a genetic component to Entamoeba histolytica virulence. BMC Microbiol 2012;12:151. [23] Weedall GD, Hall N. Evolutionary genomics of Entamoeba. Res Microbiol 2011;162:637–45. [24] Clark CG, Ali IKM, Zaki MB, Loftus J, Hall N. Unique organization of tRNA genes in Entamoeba histolytica. Mol Biochem Parasitol 2006;146:24–9. [25] Deshpande AM, Newlon CS. DNA replication fork pause sites dependent on transcription. Science 1996;272:1030–3. [26] Labib K, Hodgson B. Replication fork barriers: pausing for a break or stalling for time? EMBO Rep 2007;8:346–53. [27] Dunham MJ, Badrane H, Ferea T, Adams J, Brown PO, Rosenzweig F, et al. Characteristic genome rearrangements in experimental evolution of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 2002;99:16144–9. [28] Jm Kim, Vanguri S, Boeke JD, Gabriel A, Voytas DF. Transposable element and genome organization: a comprehensive survey of retrotransposons revealed by the complete Saccharomyces cerevisiae genome sequence. Genome Res 1998;8:464–78. [29] P.F. Ayeh-Kumi, I.M. Ali, L.A. Lockhart, C.A. Gilchrist, W.A. Petri, Jr., and R. Haque, Entamoeba histolytica: genetic diversity of clinical isolates from Bangladesh as demonstrated by polymorphisms in the serine-rich gene. Exp Parasitol 2001;99:80–8. [30] Mukherjee AK, Das K, Bhattacharya MK, Nozaki T, Ganguly S. Trend of Entamoeba histolytica infestation in Kolkata. Gut Pathog 2010;2:12.