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Indian Journal of Medical Microbiology, (2014) 32(1): 19-25

Original Article

Molecular characterisation of Giardia intestinalis assemblages from human isolates at a tertiary care centre of India V Tak, *BR Mirdha, P Yadav, P Vyas, GK Makharia, S Bhatnagar

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

Introduction 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, *Corresponding author (email: ) Departments of Microbiology (VK, BRM, PY, PV), and Gastroenterology and Human Nutrition (GKM), AIIMS, New Delhi, Pediatric Biology Center, (SB), Translational Health Science and Technology Institute, Gurgaon, Haryana, India Received: 18-06-2013 Accepted: 10-10-2013 Access this article online Quick Response Code:

Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.124290

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, -giardin gene and the glutamate dehydrogenase gene, allow identification of different subgenotypes within each assemblage.[9-15] 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.

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Materials and Methods 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.

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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 RsaI 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.

Examination of clinical samples

PCR amplified product and restriction fragment detection

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]

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).

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

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 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,

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Tak, et al.: Genotyping of Giardia intestinalis in human stool samples

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. 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 The present study provides, for the 1st time, information on the distribution of the genotypes of G. intestinalis Table 1: Distribution of age and gender among study subjects (n=82) Children Clinical presentation Adults (n=40) (n=42) Male Female Male Female (n=32) (n=8) (n=34) (n=8) 1 5 2 Acute diarrhoea (n=8) 1 Persistent diarrhoea (n=1) 26 7 23 5 Chronic diarrhoea (n=61) 5 1 5 1 No diarrhoea (n=12)

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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] 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,

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Table 2: Global prevalence of Giardia intestinalis assemblages A and B and correlation with presenting clinical symptoms Country of Sample Study outline Genetic Assemblage Assemblage Mixed Genotypic Reference origin type (numbers) target A (%) B (%) assemblage associations number A+B (%) with symptoms (%) UK Nursery 21 Giardia 21 (100) [9] tpi outbreak (21) isolates from a nursery outbreak India Sporadic (10) Giardia from 10 (100) [20] tpi individuals 1 from Hyderabad and 9 from Kolkata, India 6 (24) 19 (76) [20] Peru Sporadic (25) Giardia from 25 tpi individuals from Lima, Peru 1 (2) 41 (98) Most of the [23] Malaysia Sporadic (42) Giardia from 42 SSU infections with aboriginal tribals rRNA assemblage with or without B were symptoms symptomatic 9 (36) 16 (64) [7] France Sporadic (25) Giardia from 25 tpi fecal isolates Spain Case Giardia from 43 (39.8) 61 (56.5) 4 (3.7) Abdominal [7] tpi control (108) 57 individuals symptoms: 29 with abdominal Assemblage symptoms and AII, 26 51 asymptomatic assemblage B individuals Asymptomatic: 14 Assemblage AII, 35 assemblage B Ivory Coast Soldiers (14) Giardia isolates tpi 14 (100) [7] from 14 French soldiers returning from Ivory Coast USA Sporadic (2) Foodborne 2 (100) [7] tpi infection in 2 individuals from San Francisco, California Turkey Sporadic (44) Giardia in 20 19 (43) 25 (57) Diarrhoea: 17 [7] tpi individuals with assemblage A, diarrhea and 3 assemblage B 24 individuals Other having no diagnosis, no diarrhea but diarrhea: 2 presenting with assemblage A, gastric ulcer 7, 22 assemblage duodenal ulcer B 4, ulcerative colitis 3 and hypertension 3 Contd... www.ijmm.org

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Country of origin

Sample Study outline type (numbers)

Bangladesh Case control (267)

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Tak, et al.: Genotyping of Giardia intestinalis in human stool samples

Giardia mono-infections from 211 individuals with or without diarrhoea

UK Sporadic (1185) The Sporadic (18) Giardia from Netherland 9 individuals with persistent diarrhoea, diarrhoea at presentation and more severe symptoms and 9 with intermittent diarrhoea (alternating episodes) and moderate symptoms Ethiopia Sporadic (59) Giardia from 43 individuals with abdominal symptoms and 14 asymptomatic. No information was available regarding symptomatology of two patients Australia Population Giardia from survey (23) 23 children. faecal samples categorised as diarrhoeal or normal. No other symptom data collected Vellore, Case Giardia from 50 India control (101) children with diarrhoea, and 51 asymptomatic

Table 2: Contd... Genetic Assemblage Assemblage Mixed Genotypic Reference target A (%) B (%) assemblage associations number A+B (%) with symptoms (%) 20 (7.5) 231 (86.5) 16 (6) Diarrhoea: 8 [25] tpi assemblage A, 32 assemblage B Without diarrhoea: 6 assemblage A, 165 assemblage B 265 (22) 843 (71) 77 (7) [26] tpi [16] 9 (50) 9 (50) Severe gdh symptoms, persistent/ actual diarrhoea: 0 assemblage A, 9 assemblage B Milder symptoms, intermittent diarrhoea: 9 assemblage A, 0 assemblage B β-giardin

31 (52)

13 (22)

15 (25)

SSU rRNA

7 (30)

16 (70)

-

tpi

7 (7)

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88 (87)

6 (6)

Abdominal symptoms were significantly more associated with assemblage B (92) as compared to assemblage A (58). (P=0.007) Diarrhoea: 6 assemblage A, 3 assemblage B Normal faeces: 1 assemblage A, 13 assemblage B

[27]

Diarrhoea: 5 assemblage A, 40 assemblage B, 5 mixed infections Asymptomatic: 2 assemblage

[21]

[28]

Contd...

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Country of origin

Sample Study outline type (numbers)

United Kingdom

Sporadic (33)

Chandigarh, Sporadic (12) India

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Table 2: Contd... Genetic Assemblage Assemblage Mixed Genotypic Reference target A (%) B (%) assemblage associations number A+B (%) with symptoms (%) A, 48 assemblage B, 1 mixed infection 9 (27) 21 (64) 3 (9) [9] tpi

Giardia isolates from 33 individuals Giardia from tpi 6 adults with abdominal symptoms (diarrhoea, abdominal pain, loose stools) and 6 with dermatologic problems but no diarrhoea or gastrointestinal complaints

5 (42)

7 (58)

-

Gastrointestinal symptoms: 4 assemblage A, 2 assemblage B Dermatological symptoms: 1 assemblage A, 5 assemblage B

[22]

tpi: Triose phosphate isomerase, β-giardin: Beta-giardin, gdh: Glutamate dehydrogenase, SSU: Small-subunit, rRNA: Ribosome ribonucleic acid

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.

7. 8. 9.

Acknowledgments 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.

10.

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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. 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. Cacciò SM, Thompson RC, McLauchlin J, Smith HV. Unravelling cryptosporidium and Giardia epidemiology. Trends Parasitol 2005;21:430-7. 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. Read C, Walters J, Robertson ID, Thompson RC. Correlation between genotype of Giardia duodenalis and diarrhoea. Int J Parasitol 2002;32:229-31. 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. 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.

How to cite this article: Tak V, Mirdha BR, Yadav P, Vyas P, Makharia GK, 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. Source of Support: Nil, Conflict of Interest: None declared.

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