Indian Journal of Medical Microbiology IAMM  | About us |  Subscription |  e-Alerts  | Feedback |  Login   
  Print this page Email this page   Small font sizeDefault font sizeIncrease font size
 Home | Ahead of Print | Current Issue | Archives | Search | Instructions  
Users Online: 904 Official Publication of Indian Association of Medical Microbiologists 
  Search
 
  
 ~  Similar in PUBMED
 ~  Search Pubmed for
 ~  Search in Google Scholar for
 ~Related articles
 ~  Article in PDF (307 KB)
 ~  Citation Manager
 ~  Access Statistics
 ~  Reader Comments
 ~  Email Alert *
 ~  Add to My List *
* Registration required (free)  

 
 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Acknowledgments
 ~  References
 ~  Article Tables

 Article Access Statistics
    Viewed2365    
    Printed93    
    Emailed0    
    PDF Downloaded267    
    Comments [Add]    

Recommend this journal

 


 
  Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 32  |  Issue : 1  |  Page : 19-25
 

Molecular characterisation of Giardia intestinalis assemblages from human isolates at a tertiary care centre of India


1 Department of Microbiology, AIIMS, New Delhi, India
2 Department of Gastroenterology and Human Nutrition , AIIMS, New Delhi, India
3 Pediatric Biology Center, Translational Health Science and Technology Institute, Gurgaon, Haryana, India

Date of Submission18-Jun-2013
Date of Acceptance10-Oct-2013
Date of Web Publication4-Jan-2014

Correspondence Address:
B R Mirdha
Department of Microbiology, AIIMS, New Delhi
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.124290

Rights and Permissions

 ~ Abstract 

Purpose: The aim of the study was to determine the genetic heterogeneity of Giardia intestinalis isolates detected in stool samples of the study population using polymerase chain reaction assay and restriction fragment length polymorphism. We also tried to correlate the association/differences between the clinical symptomatology and infection by different assemblages (genotypes) of G. intestinalis. Materials and Methods: This cross-sectional study was conducted from April 2008 to June 2010. A total of 40 adults (n = 40) and 42 children (n = 42) below the age of 12 years with the clinical suspicion of giardiasis and with the onset of one or more of the following five symptoms, i.e., loose stool, nausea, weight loss, fatigue and foul smelling faeces and confirmed laboratory diagnosis of giardiasis at least once during the current episode of diarrhoea were included in this study. Results: Of the 82 patients (males 66) enrolled in the study, 70 (85%) presented with diarrhoea (56 males) and 12 (15%) without diarrhoea (10 males). Out of 70 diarrheic patients, 61 (87%) had chronic diarrhoea, 8 (11.5%) had acute diarrhoea and 1 (1.5%) had persistent diarrhoea. Of the total patients, 63 (77%) were clinically assessed and were apparently immunocompetent, whereas, 19 (23%) immunocompromised patients had different underlying conditions besides giardiasis. Genotyping identified all 82 (100%) isolates as assemblage B. Conclusion: We found that assemblage B of G. intestinalis presents with all kinds of clinical features ranging from asymptomatic carriage to acute, persistent or chronic diarrhoea.


Keywords: Assemblages, diarrhoea, genotyping, Giardia intestinalis, polymerase chain reaction and restriction fragment length polymorphism, triose phosphate isomerase gene


How to cite this article:
Tak V, Mirdha B R, Yadav P, Vyas P, Makharia G K, Bhatnagar S. Molecular characterisation of Giardia intestinalis assemblages from human isolates at a tertiary care centre of India. Indian J Med Microbiol 2014;32:19-25

How to cite this URL:
Tak V, Mirdha B R, Yadav P, Vyas P, Makharia G K, Bhatnagar S. Molecular characterisation of Giardia intestinalis assemblages from human isolates at a tertiary care centre of India. Indian J Med Microbiol [serial online] 2014 [cited 2018 Sep 21];32:19-25. Available from: http://www.ijmm.org/text.asp?2014/32/1/19/124290



 ~ Introduction Top


Giardia intestinalis (synonym: Giardia duodenalis and Giardia lamblia) is an intestinal amitochondriate flagellate protozoan considered as the most common cause of parasitic diarrhoea world-wide. It is also recognised as one of the common causes of traveller's diarrhoea. [1] Giardiasis causes about 2.5 million cases of diarrhoea and nutritional deficiencies in children in developing countries [2] and has been included as a part of World Health Organisation neglected disease initiative since 2004. It is more prevalent in children especially, 1-5 years of age (4-42%) and particularly those from developing countries and those who are malnourished than in adults. [3],[4] G. lamblia has estimated prevalence rates of 20-30% in developing countries and 2-5% in developed countries. [5],[6]

G. intestinalis has been described as a species complex comprising of seven assemblages. [7] The assemblages A and B are potentially zoonotic and C, D, E, F and G appears to infect only specific hosts. [8] "Slow" evolving markers such as small-subunit ribosome ribonucleic acid can be used to distinguish major assemblages, whereas "fast" evolving markers, such as the triose phosphate isomerase (tpi) gene, β-giardin gene and the glutamate dehydrogenase gene, allow identification of different subgenotypes within each assemblage. [9],[10],[11],[12],[13],[14],[15] Assemblage A Group I has been detected in livestock, cats, dogs, whereas, assemblage A Group II is being confined to humans. [16]

The aim of the study was to determine the genetic heterogeneity of G. intestinalis isolates detected in stool samples of the study population using polymerase chain reaction (PCR) assay and restriction fragment length polymorphism (RFLP). The study also attempted to correlate the association between the clinical symptomatology and infection by different assemblages (genotypes) of G. intestinalis.


 ~ Materials and Methods Top


Study population and specimen collection

This cross-sectional study was conducted from April 2008 to June 2010. Three consecutive stool samples on three consecutive days were obtained from each patient enrolled for the study. A total of 246 samples were collected from patients. A total of 40 adults (n = 40) and 42 children (n = 42) below the age of 12 years with clinical suspicion of giardiasis and with the onset of one or more of the following five symptoms, i.e. loose stool, nausea, weight loss, fatigue and foul smelling faeces and confirmed laboratory diagnosis of giardiasis at least once during the current episode of diarrhoea were included in the study. A confirmed case of giardiasis was defined as the detection of either cysts or trophozoites or both stages of Giardia, by light microscopy.

Examination of clinical samples

Stool specimens were examined for intestinal parasites immediately after the collection of the sample in normal saline and Lugol's iodine wet mount preparation. Formed stool specimens could be processed for the formol-ether sedimentation concentration technique. [17] Staining by modified Kinyoun's acid-fast staining for coccidian oocysts using the standard methods was performed in all the clinical samples. [18]

Demographic information collection

Information pertaining to age, gender, weight, detailed history of the patient's illnesses, type of dwelling, source and treatment of drinking water, food hygiene, number of pets owned by the household etc., was obtained from each patient using a structured questionnaire. Informed/written consent was obtained from all the patients including parents/guardian of the paediatric patients.

Deoxyribonucleic acid extraction and PCR assay

DNA was extracted directly from 200 mg of the fresh non-preserved stool samples that were positive for Giardia species using a QIAamp DNA Stool Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions, except that the mixture was lysed in the lysis buffer at 95°C for 1 h including an initial step of cyst disruption using 8-10 glass beads of 0.5 mm diameter. Samples for which the PCR amplification was unsuccessful, a further DNA purification using polyvinylpyrrolidone was performed as described by Lawson et al. [19] The extracted DNA was stored at −70°C until further use.

A two-step or heminested PCR amplification assay, with Phase I comprising of a single duplex reaction and Phase II comprising of two individual reactions were performed using tpi as the target gene. Previously published primers and protocol was followed at the tpi locus for the detection of assemblage of Giardia species, particularly assemblage A and B. [9] Positive control DNA for both assemblage A and B of G. intestinalis were obtained from CMC Vellore, India (Courtesy: Dr. Sitara Swarna Rao). Autoclaved double distilled water was used as a negative control.

RFLP analysis

Rsa
I restriction sites were identified and RFLP was performed for genotyping assemblage A into the subgroups A-I or A-II based on the predicted restriction digestion products of 437 and 39 bp for assemblage A Group I and 235, 202 and 39 bp for assemblage A Group II. [9] RFLP analysis was performed by digesting 5 ∝l of the tpiA-PCR or tpiB-PCR product with 5U of RsaI restriction enzyme in 1X enzyme buffer (New England Biolabs) in a final volume of 30 ∝l for 3 h at 37°C.

PCR amplified product and restriction fragment detection

PCR products and restriction fragments were separated by horizontal electrophoresis in 1.5 and 2% agarose gels, respectively, with ethidium bromide staining and were recorded by ultraviolet transillumination with type 667 film (Polaroid Ltd., St. Albans, United Kingdom). Secondary PCR products corresponding to 140 bp for assemblage B and 476 bp for assemblage A were visualised in a gel documentation system (Chemi Imager 5.5, Alpha Innotech Inc. USA).

Ethical clearance

The study was approved by the Institutional Ethics Committee. To adhere to ethical norms for using human subjects for medical research, all patients and/or their guardians/parents were informed about the objectives and goals of the present study. Physicians and laboratory personnel explained the results of the tests and in case of positive results; the study population received an appropriate treatment. Necessary information regarding treatment and prevention was also provided.


 ~ Results Top


Out of the 82 patients, 66 males were positive for giardiasis, 70 (85%) were presented with the complaints of diarrhoea of which 56 males and 12 (15%) had infrequent episodes of diarrhoea (10 males). Out of 70 diarrheic patients, 61 (87%) had chronic diarrhoea, 8 (11.5%) had acute diarrhoea and 1 (1.5%) had persistent diarrhoea. Of the total patients, 63 (77%) were clinically assessed and were apparently immunocompetent, whereas, 19 (23%) patients had different immunocompromised underlying conditions and 4 (21%) had human immunodeficiency virus, 3 (16%) renal transplant recipients, 3 (16%) common variable immunodeficiency, 6 (31%) patients with malignancies and 4 (21%) had acute lymphoid leukaemia, 1 (5%) acute myeloid leukaemia, 1 (5%) neuroblastoma, 2 (11%) patients on prolonged steroid therapy for Nephrotic syndrome and 1 (5%) had Cushing's syndrome.

Adults

Of the 40 (48.5%) adult patients 85% had diarrhoea of which 33 (97%) had chronic diarrhoea and only 1 (3%) had acute diarrhoea [Table 1]. The total patients enrolled included 31 (77.5%) clinically apparent immunocompetent whereas 9 (22.5%) had some underlying conditions leading to immunocompromised state.
Table 1: Distribution of age and gender among study subjects (n=82)

Click here to view


Children

Of the 42 (51.5%) paediatric patients, 36 (86%) presented with diarrhoea of which, 28 (78%) had chronic diarrhoea followed by acute diarrhoea in 7 (19%) and persistent diarrhoea in 1 (3%) [Table 1].

PCR and genotyping

All the 82 stool specimens that were microscopically positive for G. intestinalis cyst and/or trophozoites were subjected to PCR assay using tpi gene. A hemi-nested product of 140 bp in size of G. intestinalis indicated positive amplification. Genotyping of all 82 (100%) isolates were determined as assemblage B.

Distribution of co-infecting parasites

Nearly 32% (26/82) patients with Giardia infection harboured some pathogenic or non-pathogenic parasites. Of these, 14 (54%) patients were infected with pathogenic and 12 (46%) with non-pathogenic parasites. The prevalence of multiple parasitic infections was slightly higher in children (11/21, 52%) than in adults (10/21, 48%) with diarrhoea. Of all the co-infecting parasites, Cryptosporidium spp. (7/16, 27%) was the most common pathogenic parasite, whereas Blastocystis hominis (7/23, 27%) and Endolimax nana (7/23, 27%) were the most common non-pathogenic parasites.


 ~ Discussion Top


The present study provides, for the 1 st time, information on the distribution of the genotypes of G. intestinalis isolates by PCR amplification using tpi gene as the target gene from patients with giardiasis in and around Delhi. In our observation, all 82 (100%) isolates of G. intestinalis were genotypically characterised as assemblage B type. Similar findings have also been reported from previous studies conducted in India (Hyderabad and Kolkata) (100%, n = 10), [20] (Vellore) (87%, n = 101), [21] (Chandigarh) (58%, n = 12) [22] and from other parts of the world Malaysia (97.62%, n = 42), [23] Peru (76%, n = 25) [24] and United Kingdom (64%, n = 33). [9] The comparative prevalence and distribution of Giardia assemblages as observed in various studies conducted in different parts of the world is tabulated in [Table 2]. Our findings are in agreement with the increased overall prevalence of assemblage B (60-69%) in the world compared with assemblage A (26-35%) and mixed assemblage A + B (5-6%). [7],[26]
Table 2: Global prevalence of Giardia intestinalis assemblages A and B and correlation with presenting clinical symptoms

Click here to view


In some of the previous studies, assemblage B has been correlated with severe giardiasis or symptomatic diarrhoea Homan and Mank [16] Gelanew et al., [27] while other studies reported a significant association between assemblage A and the presence of severe symptoms and disease Read et al., [28] and Haque et al. [25] The results of previous global studies attempting correlation of clinical symptoms with prevalent Giardia assemblages are tabulated in [Table 2]. As all our cases were found to be assemblage B, the correlation of different assemblages in symptomatic and asymptomatic giardiasis could not be ascertained. At the same, we found that assemblage B of G. intestinalis presents with all kinds of clinical features ranging from asymptomatic carriage to acute, persistent or chronic diarrhoea.

Multiple parasitism was observed in 32% of our study subjects with Cryptosporidium spp. (27%) being the most common co-infecting pathogenic parasite. Mehraj et al., [29] have reported 10% cases and Peréz Cordón et al., [24] have reported 45.6% cases of multiple parasitism in their respective studies. Kohli et al., [30] in Brazil also reported co-infection of Giardia and Cryptosporidium spp. in 17.54% of their study, which was also the commonest co-infection. The dynamics of such co-infection has not been delineated so far, whether it is a coincidental finding or synergistic symbiosis needs to be explained. Non-pathogenic parasites causing co-infections in our study were B. hominis (27%) and E. nana (27%). Peréz Cordón et al., [24] have also reported B. hominis and G. intestinalis as the most common co-infection in their study. The plausible reason for these co-infections may be due to common vehicles of transmission by contaminated water or food by faeco-oral route.

Limitations of the study

As the study was conducted in a tertiary health-care centre, the results obtained in this study may be a tiny glimpse of the broader picture in the community. Therefore, to get a more comprehensive view of the various issues associated with giardiasis and the prevalent genotypes leading to disease manifestations may be obtained by a larger community-based prospective case-control study with adequate follow-up.


 ~ Acknowledgments Top


We would like to acknowledge the kind support of Dr. Sitara Swarna Rao of CMC Vellore who provided us with the positive controls of both assemblage A and B of G. intestinalis.

 
 ~ References Top

1.Ekdahl K, Andersson Y. Imported giardiasis: Impact of international travel, immigration, and adoption. Am J Trop Med Hyg 2005;72:825-30.  Back to cited text no. 1
[PUBMED]    
2.WHO. Intestinal parasites control: Burden and trends. WHO Division of Control of Tropical Diseases. Geneva, Switzerland: World Health Organization; 1998.  Back to cited text no. 2
    
3.Miotti PG, Gilman RH, Santosham M, Ryder RW, Yolken RH. Age-related rate of seropositivity of antibody to Giardia lamblia in four diverse populations. J Clin Microbiol 1986;24:972-5.  Back to cited text no. 3
[PUBMED]    
4.Nygård K, Schimmer B, Søbstad Ø, Walde A, Tveit I, Langeland N, et al. A large community outbreak of waterborne giardiasis-delayed detection in a non-endemic urban area. BMC Public Health 2006;6:141.  Back to cited text no. 4
    
5.WHO. Control of Tropical Diseases. Geneva, Switzerland: World Health Organization; 1998.  Back to cited text no. 5
    
6.Thompson RC. Giardiasis as a reemerging infectious disease and its zoonotic potential. Int J Parasitol 2000;30:1259-67.  Back to cited text no. 6
[PUBMED]    
7.Cacciò SM, Ryan U. Molecular epidemiology of giardiasis. Mol Biochem Parasitol 2008;160:75-80.  Back to cited text no. 7
    
8.Monis PT, Thompson RC. Cryptosporidium and Giardia zoonosis: Fact or fiction? Infect Genet Evol 2003;3:233-44.  Back to cited text no. 8
[PUBMED]    
9.Amar CF, Dear PH, Pedraza-Díaz S, Looker N, Linnane E, McLauchlin J. Sensitive PCR-restriction fragment length polymorphism assay for detection and genotyping of Giardia duodenalis in human feces. J Clin Microbiol 2002;40:446-52.  Back to cited text no. 9
    
10.Mahbubani MH, Schaefer FW 3 rd , Jones DD, Bej AK. Detection of Giardia in environmental waters by immuno-PCR amplification methods. Curr Microbiol 1998;36:107-13.  Back to cited text no. 10
    
11.Monis PT, Andrews RH, Mayrhofer G, Ey PL. Molecular systematics of the parasitic protozoan Giardia intestinalis. Mol Biol Evol 1999;16:1135-44.  Back to cited text no. 11
[PUBMED]    
12.Adam RD. The Giardia lamblia genome. Int J Parasitol 2000;30:475-84.  Back to cited text no. 12
[PUBMED]    
13.Cacciò SM, De Giacomo M, Pozio E. Sequence analysis of the beta-giardin gene and development of a polymerase chain reaction-restriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples. Int J Parasitol 2002;32:1023-30.  Back to cited text no. 13
    
14.Lu S, Wen J, Li J, Wang F. DNA sequence analysis of the triose phosphate isomerase gene from isolates of Giardia lamblia. Chin Med J (Engl) 2002;115:99-102.  Back to cited text no. 14
[PUBMED]    
15.Rimhanen-Finne R, Hörman A, Ronkainen P, Hänninen ML. An IC-PCR method for detection of Cryptosporidium and Giardia in natural surface waters in Finland. J Microbiol Methods 2002;50:299-303.  Back to cited text no. 15
    
16.Homan WL, Mank TG. Human giardiasis: Genotype linked differences in clinical symptomatology. Int J Parasitol 2001;31:822-6.  Back to cited text no. 16
[PUBMED]    
17.Basic Laboratory Methods in Medical Parasitology. Geneva, Switzerland: World Health Organization; 1993. p. 9-33.  Back to cited text no. 17
    
18.Garcia LS, Bruckner DA, Brewer TC, Shimizu RY. Techniques for the recovery and identification of Cryptosporidium oocysts from stool specimens. J Clin Microbiol 1983;18:185-90.  Back to cited text no. 18
[PUBMED]    
19.Lawson AJ, Linton D, Stanley J, Owen RJ. Polymerase chain reaction detection and speciation of Campylobacter upsaliensis and C. helveticus in human faeces and comparison with culture techniques. J Appl Microbiol 1997;83:375-80.  Back to cited text no. 19
[PUBMED]    
20.Sulaiman IM, Fayer R, Bern C, Gilman RH, Trout JM, Schantz PM, et al. Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis. Emerg Infect Dis 2003;9:1444-52.  Back to cited text no. 20
[PUBMED]    
21.Ajjampur SS, Sankaran P, Kannan A, Sathyakumar K, Sarkar R, Gladstone BP, et al. Giardia duodenalis assemblages associated with diarrhea in children in South India identified by PCR-RFLP. Am J Trop Med Hyg 2009;80:16-9.  Back to cited text no. 21
[PUBMED]    
22.Paintlia AS, Descoteaux S, Spencer B, Chakraborty A, Ganguly NK, Mahajan RC, et al. Giardia lamblia groups A and B among young adults in India. Clin Infect Dis 1998;26:190-91.  Back to cited text no. 22
    
23.Mohammed Mahdy AK, Surin J, Wan KL, Mohd-Adnan A, Al-Mekhlafi MS, Lim YA. Giardia intestinalis genotypes: Risk factors and correlation with clinical symptoms. Acta Trop 2009;112:67-70.  Back to cited text no. 23
[PUBMED]    
24.Peréz Cordón G, Cordova Paz Soldan O, Vargas Vásquez F, Velasco Soto JR, Sempere Bordes L, Sánchez Moreno M, et al. Prevalence of enteroparasites and genotyping of Giardia lamblia in Peruvian children. Parasitol Res 2008;103:459-65.  Back to cited text no. 24
    
25.Haque R, Roy S, Kabir M, Stroup SE, Mondal D, Houpt ER. Giardia assemblage A infection and diarrhea in Bangladesh. J Infect Dis 2005;192:2171-3.  Back to cited text no. 25
[PUBMED]    
26.Cacciò SM, Thompson RC, McLauchlin J, Smith HV. Unravelling cryptosporidium and Giardia epidemiology. Trends Parasitol 2005;21:430-7.  Back to cited text no. 26
    
27.Gelanew T, Lalle M, Hailu A, Pozio E, Cacciò SM. Molecular characterization of human isolates of Giardia duodenalis from Ethiopia. Acta Trop 2007;102:92-9.  Back to cited text no. 27
    
28.Read C, Walters J, Robertson ID, Thompson RC. Correlation between genotype of Giardia duodenalis and diarrhoea. Int J Parasitol 2002;32:229-31.  Back to cited text no. 28
    
29.Mehraj V, Hatcher J, Akhtar S, Rafique G, Beg MA. Prevalence and factors associated with intestinal parasitic infection among children in an urban slum of Karachi. PLoS One 2008;3:e3680.  Back to cited text no. 29
[PUBMED]    
30.Kohli A, Bushen OY, Pinkerton RC, Houpt E, Newman RD, Sears CL, et al. Giardia duodenalis assemblage, clinical presentation and markers of intestinal inflammation in Brazilian children. Trans R Soc Trop Med Hyg 2008;102:718-25.  Back to cited text no. 30
[PUBMED]    



 
 
    Tables

  [Table 1], [Table 2]



 

Top
Print this article  Email this article
 

    

2004 - Indian Journal of Medical Microbiology
Published by Wolters Kluwer - Medknow

Online since April 2001, new site since 1st August '04