Indian Journal of Medical Microbiology Home 

ORIGINAL ARTICLE
[Download PDF]
Year : 2014  |  Volume : 32  |  Issue : 4  |  Page : 378--382

Refractory giardiasis: A molecular appraisal from a tertiary care centre in India

P Yadav1, V Tak1, BR Mirdha1, GK Makharia2,  
1 Department of Microbiology, Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi, India
2 Department of Human Nutrition, All India Institute of Medical Sciences, New Delhi, India

Correspondence Address:
V Tak
Department of Microbiology, Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi
India

Abstract

Purpose: The intestinal flagellate Giardia lamblia includes many genetically distinct assemblages, of which assemblage A and B, predominantly infect humans. Nitroimidazoles derivatives (metronidazole and tinidazole) and nitazoxanide are some of the therapeutic agents for treatment of giardiasis. Nevertheless, some individuals with giardiasis are non-responsive to standard therapy. The present study highlights cases of refractory giardiasis and attempts to elucidate if genetic heterogeneity in the parasite is associated with treatment failure. Materials and Methods: Three stool samples were obtained on three consecutive days from 4000 patients with diarrhoea and were microscopically examined for the detection of trophozoites, and/or cysts, using both normal saline and Lugol«SQ»s iodine. A hemi-nested polymerase chain reaction (PCR) assay using triose phosphate isomerase (tpi) as the target gene was performed to determine the assemblages. Sequencing of the PCR products of the patients showing failure to treatment of giardiasis was also performed. Results: Two per cent (82/4000) of the total patients were microscopically positive for Giardia lamblia in the stool samples. All these patients were treated with metronidazole/tinidazole as per the standard regimens. However, eight patients showed treatment failure to giardiasis as stool examinations were repeatedly positive even after treatment with multiple courses of anti-giardial therapy. Genetic characterisation of all eight Giardia isolates showed that they belonged to Assemblage B and had homogeneous sequences. These patients were either treated with extended regimens or with combination therapy of anti-giardials. Conclusion: In our experience, combination of two or more drugs for a longer duration is the treatment modality to treat refractory giardiasis.

How to cite this article:
Yadav P, Tak V, Mirdha B R, Makharia G K. Refractory giardiasis: A molecular appraisal from a tertiary care centre in India.Indian J Med Microbiol 2014;32:378-382

How to cite this URL:
Yadav P, Tak V, Mirdha B R, Makharia G K. Refractory giardiasis: A molecular appraisal from a tertiary care centre in India. Indian J Med Microbiol [serial online] 2014 [cited 2020 Nov 27 ];32:378-382
Available from: https://www.ijmm.org/text.asp?2014/32/4/378/142236

Full Text

 Introduction



Giardia lamblia is the most common pathogenic flagellate worldwide responsible for both acute and chronic parasitic diarrhoea. [1] Giardia species have so far been divided into eight different groups (assemblages A to H) or genotypes based on broad range of host specificity. While six genotypes (C to H) are host specific and infect non-human species, the genotypes that exclusively cause human infections are assemblage A and B. [2] Further, intra-species variations exist within these assemblages and are referred to as AI and AII and BIII and BIV in both assemblage A and B, respectively.

Giardiasis is a treatable disease and a number of therapeutic drugs are available for effective treatment. Nitroimidazoles derivatives such as metronidazole and tinidazole and nitazoxanide are the drugs of choice with efficacy rates ranging from 60% to 100%. [3],[4] Paromomycin, furazolidone, quinacrine and albendazole are the other alternatives for giardiasis. [5] Nevertheless, 20% of the individuals experience treatment failure despite standard therapy for desired duration. [6]

Treatment failure to giardiasis has been defined as the presence of the parasitic protozoa in at least one of the three consecutive stool samples of the infected patient along with persistence of symptoms such as diarrhoea, bloating, abdominal pain, weight loss after completion of one or more courses of standard treatment. [3] The probable potential causes of treatment failures that have been reported in the literature are (i) re-infection, (ii) inadequate drug levels in the tissue, (iii) concomitant immunosuppression, (iv) resistance to the drug, (v) sequestration in the gall bladder or pancreatic ducts and (vi) due to unknown reasons. [7]

 Materials and Methods



During the study period of 26 months (April 2008 to June 2010), three consecutive stool samples from patients with complaints of diarrhoea and other gastro-intestinal disorders were collected from 4000 patients to determine parasitic causes of diarrhoea. Three stool samples obtained on three consecutive days from each of these patients were examined for detection of trophozoites, cysts, oocysts and ova by light microscopy using both normal saline and Lugol's iodine wet-mount preparations. For formed stool specimens, formol-ether sedimentation concentration technique was performed. [8] Modified Ziehl-Neelsen staining was also performed for the detection of oocysts of intestinal coccidia. [8]

Stool samples were also obtained after the completion of treatment. Response to the treatment was assessed by both the parasitological clearance and improvement in clinical symptomatology.

DNA was isolated from the clinical specimens that were positive for Giardia lamblia using a QIAamp DNA Stool Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions. The extracted DNA was stored at -70°C until further use. A two-step or hemi-nested polymerase chain reaction (PCR) assay was performed using triose phosphate isomerase (tpi) as the target gene to carry out the assemblage study. [9] PCR products were visualised by electrophoresis after ethidium bromide staining. PCR products of the patients showing failure to treatment of giardiasis were further purified (MinElute Gel Extraction Kit, Qiagen) and sequenced on both strands (by Chromous Biotech, Germany). Electrophenograms and sequences were analysed with that of reference sequence (GenBank IDAF069561) from GenBank using Clustal W (http://www.clustalw.genome.jp) software. Phylogenetic trees were constructed for the tpi locus with additional isolates from GenBank. The evolutionary history was inferred using the neighbor-joining method. [10] The bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the taxa analysed. [11] The evolutionary distances were computed using the Maximum Composite Likelihood method [12] and were in the units of the number of base substitutions per site. All ambiguous positions were removed for each sequence pair. Evolutionary analyses were conducted using MEGA5. [13]

Ethical approval

The necessary ethical clearance was also obtained from institutional ethics committee to conduct the study.

 Results



A total of 82 (2%, 82/4000) of the total patients were positive for Giardia cysts and/or trophozoites in the stool samples. This group comprised of 40 (49%, 40/82) adults and 42 (51%, 42/82) children. Among adults and children, the male to female ratio was 4:1 (32:8) and 17:4 (34:8), respectively. All these patients were treated with metronidazole/tinidazole as per the standard regimen. [14] However, eight (9.8%, 8/82) patients had treatment failure to giardiasis as the examinations of stool samples were repeatedly positive even after treatment with multiple courses of anti-giardial therapy. These eight patients included six immunocompetent and two immunocompromised (five adults and three children) patients. The six immunocompetent patients were investigated for their antibody profile and human immunodeficiency virus (HIV) status. All of them were HIV sero-negative and had antibody profile within normal limits. The two immunocompromised patients comprised of an adult patient infected with HIV and a child who had acute lymphoid leukaemia (ALL). Both the immunocompromised patients had concomitant cryptosporidiosis.

Genetic characterisation of all eight Giardia isolates was performed at the tpi locus. All of them belonged to Assemblage B. PCR products from representative six isolates with refractory giardiasis were sequenced, and all were found to be of homogeneous sequences [Figure 1] and [Figure 2].

Both the immunocompromised patients (n = 2) received specific treatment for both giardiasis (metronidazole) and cryptosporidiosis (nitazoxanide) as per the standard regimen [Table 1]. The HIV patient had CD4 count of <200 cells/µl and was also receiving highly active anti-retroviral treatment (HAART). In spite of this, he failed to respond to the treatment and the stool samples were positive for both the protozoa till the 10th day. The treatment was further continued with combination therapy of metronidazole and nitazoxanide for another 10 and 20 days, respectively. This extended regimen led to improvement of symptoms and subsequent stool samples examination was negative. However, both these two patients had recurrence of infections with the same parasites after a month. Both the patients were followed up for nearly 2 months. The ALL patient was treated according to the same protocol, however, he had a fatal outcome.{Figure 1}{Figure 2}{Table 1}

The six immunocompetent patients with giardiasis were treated with different treatment regimens as summarised in [Table 1]. Partial or complete clinical and parasitological improvement was achieved in all the patients.

 Discussion



The present study represents a group of patients who had treatment failure to giardiasis. Several factors that are responsible for chronic infections caused by Giardia reverting to refractory giardiasis have been postulated. Host factors such as age, immune status, history of any previous ailments and altered microbiota along with various parasitic factors (association with a particular genotype, rate of multiplication) have also been incriminated. [15],[16] At biochemical level, pyruvate: Ferredoxin oxidoreductase (PFO) along with ferredoxin I gets down-regulated resulting in decrease in activation of the drug and subsequent resistance. [17] In the present study, 9.8% of patients had refractory giardiasis. Similar rates of refractory giardiasis have also been observed from Norway 3.2% (38/1200) [18] and Spain 5.8% (10/170). [3]

Genotyping using tpi as the target gene was performed in all the 82 isolates (data not shown) to investigate whether different genotypes and/or intra-species variations within the genotypes have variable effect on clinical symptomatology and response to treatment. Prevalence of Assemblages A and B in humans varies considerably from country to country. [19],[20],[21] In India, only two earlier studies based on restriction fragment length polymorphism (RFLP) analysis of the triose phosphate isomerase (tpi) locus have identified Giardia assemblages. Assemblage A as the frequently associated genotype with giardiasis was reported by Paintlia, [22] whereas assemblage B was the predominant genotype in children (40/50). [23] In the present study, all the Giardia isolates belonged to assemblage B.

In a study from Sweden, overlapping nucleotides or nucleotide substitutions have been observed in assemblage B sequences. These changes occurred at the ί-giardin gene (52%), at the tpi gene (59%) and at the gdh gene (64%) of the isolates. [24] In another study from Norway, genetic characterisation of parasites with refractory giardiasis, however, showed more heterogeneity with 10 different sequence profiles using gdh and β-giardin as the target genes. [25] In the present study, sequence profile of six of the eight refractory cases showed complete genetic homogeneity at the tpi locus with no differences both at the nucleotide and amino acid levels in comparison to the reference sequence. Phylogenetic characterisation based on the nucleotide sequences of gdh, tpi, β-giardin, elongation factor 1 (EF1) and SSU rRNA genes indicate a high degree of genetic variability within both assemblages A and B. [26],[27] Among all these loci, tpi gene has the highest degree of polymorphisms. [27]

Refractory giardiasis is difficult to treat especially in immunosuppressed individuals. Patients with HIV/AIDS with low CD4 + cell counts are even more difficult to treat and cure. [28] Treatment of giardiasis with longer duration or repeated courses and/or higher doses of the standard drug or a combination of drugs is recommended in case of treatment failure. [29],[30] Nitazoxanide besides being the potential drug in treating Cryptosporidium is generally well tolerated in patients with intestinal giardiasis and can be used in cases of failure to metronidazole treatment. [3] Nitazoxanide has also been used in successful treatment of metronidazole-resistant giardiasis in a patient with acquired immunodeficiency syndrome. [31] In the present study, nitazoxanide was used in combination with metronidazole in treatment of HIV-infected adult and the child with ALL.

In conclusion, present study reinforces the role of combination therapy in treating patients with giardiasis refractory to metronidazole treatment. The newer drug nitazoxanide can be used as an adjunct therapy for the treatment of giardiasis besides cryptosporidiosis in patients with poor response to metronidazole and albendazole.

 Acknowledgement



The study was supported by a financial grant to the corresponding author, by Indian Council of Medical Research (ICMR), Department of Health Research, Government of India, New Delhi, India.

References

1Nash TE, Herrington DA, Losonsky GA, Levine MM. Experimental human infections with Giardia lamblia. J Infect Dis 1987;156:974-84.
2Thompson RC, Hopkins RM, Homan WL. Nomenclature and genetic groupings of Giardia infecting mammals. Parasitol Today 2000;16:210-3.
3Lopez-Velez R, Batlle C, Jimenez C, Navarro M, Norman F, Perez-Molina J. Short course combination therapy for giardiasis after nitroimidazole failure. Am J Trop Med Hyg 2010;83:171-3.
4The treatment of diarrhea-A manual for physicians and other senior health workers. Available from: www.who.int/chd/publications/cdd/textrev4.htm [Last accessed on 2013 Nov 12].
5Petri WA. Treatment of giardiasis. Curr Treat Options Gastroenterol 2005;8:13-7.
6Upcroft P. Drug resistance in Giardia: Clinical versus laboratory isolates. Drug Resist Updat 1998;1:166-8.
7Nash TE, Ohl CA, Thomas E, Subramanian G, Keiser P, Moore TA. Treatment of patients with refractory giardiasis. Clin Infect Dis 2001;33:22-8.
8Casemore DP, Armstrong M, Sands RL. Laboratory diagnosis of cryptosporidiosis. J Clin Pathol 1985;38:1337-41.
9Amar CF, Dear PH, Pedraza-Diaz 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.
10Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406-25.
11Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 1985;39:783-91.
12Tamura K, Nei M, Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci U S A. 2004;101:11030-5.
13Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011;28:2731-9.
14Gardner TB, Hill DR. Treatment of giardiasis. Clin Microbiol Rev 2001;14:114-28.
15Röxstrom-Lindquist K, Palm D, Reiner D, Ringqvist E, Svärd SG. Giardia immunity--an update. Trends Parasitol 2006;22:26-31.
16Robertson LJ, Hanevik K, Escobedo AA, Morch K, Langeland N. Giardiasis--why do the symptoms sometimes never stop? Trends Parasitol 2010;26:75-82.
17Land KM, Johnson PJ. Molecular basis of metronidazole resistance in pathogenic bacteria and protozoa. Drug Resist Updat 1999;2:289-94.
18Mørch K, Hanevik K, Robertson LJ, Strand EA, Langeland N. Treatment-ladder and genetic characterisation of parasites in refractory giardiasis after an outbreak in Norway. J Infect 2008;56:268-73.
19Gelanew 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.
20Caccio SM, Ryan U. Molecular epidemiology of giardiasis. Mol Biochem Parasitol 2008;160:75-80.
21Lalle M, Bruschi F, Castagna B, Campa M, Pozio E, Cacciò SM. High genetic polymorphism among Giardia duodenalis isolates from Sahrawi children. Trans R Soc Trop Med Hyg 2009;103:834-8.
22Paintlia AS, Descoteaux S, Spencer B, Chakraborti A, Ganguly NK, Mahajan RC, et al. Giardia lamblia groups A and B among young adults in India. Clin Infect Dis 1998;26:190-1.
23Ajjampur 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.
24Lebbad M, Petersson I, Karlsson L, Botero-Kleiven S, Andersson JO, Svenungsson B, et al. Multilocus genotyping of human Giardia isolates suggests limited zoonotic transmission and association between assemblage B and flatulence in children. PLoS Negl Trop Dis 2011;5:e1262.
25Robertson L, Hermansen L, Gjerde BK, Strand E, Alvsvåg JO, Langeland N. Application of genotyping during an extensive outbreak of waterborne giardiasis in Bergen, Norway, during autumn and winter 2004. Appl Environ Microbiol 2006;72:2212-7.
26van Keulen H, Campbell SR, Erlandsen SL, Jarrol L. Cloning and restriction enzyme mapping of ribosomal DNA of Giardia duodenalis, Giardia ardeae and Giardia muris. Mol Biochem Parasitol 1991;46:275-84.
27Sulaiman 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.
28Aronson NE, Cheney C, Rholl V, Burris D, Hadro N. Biliary giardiasis in a human immunodeficiency virus. J Clin Microbiol 2001;33:167-70.
29Lemée V, Zaharia I, Nevez G, Rabodonirina M, Brasseur P, Ballet JJ, et al. Metronidazole and albendazole susceptibility of 11 clinical isolates of Giardia duodenalis from France. J Antimicrob Chemother 2000;46:819-21.
30Hill DR, Nash TE. Giardia lamblia. In: Mandell GL, Bennett JE, Dolin R, editor. Mandell, Douglas and Bennett's Principles and Practice of Infectious Diseases. 7th ed. Elsevier Churchill Livingstone; 2009. p. 3527-534.
31Abboud P, Lemée V, Gargala G, Brasseur P, Ballet JJ, Borsa-Lebas F, et al. Successful treatment of metronidazole- and albendazole-resistant giardiasis with nitazoxanide in a patient with acquired immunodeficiency syndrome. Clin Infect Dis 2001;32:1792-4.