|Year : 2014 | Volume
| 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
|Date of Submission||03-Sep-2014|
|Date of Acceptance||07-Feb-2014|
|Date of Web Publication||4-Oct-2014|
Department of Microbiology, Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi
Source of Support: None, Conflict of Interest: None
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'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.
Keywords: Genetic heterogeneity, Giardia lamblia, treatment failure
|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;32:378-82
|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 2019 Nov 15];32:378-82. Available from: http://www.ijmm.org/text.asp?2014/32/4/378/142236
| ~ Introduction|| |
Giardia lamblia is the most common pathogenic flagellate worldwide responsible for both acute and chronic parasitic diarrhoea.  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.  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%. , Paromomycin, furazolidone, quinacrine and albendazole are the other alternatives for giardiasis.  Nevertheless, 20% of the individuals experience treatment failure despite standard therapy for desired duration. 
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.  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. 
| ~ 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.  Modified Ziehl-Neelsen staining was also performed for the detection of oocysts of intestinal coccidia. 
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.  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.  The bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the taxa analysed.  The evolutionary distances were computed using the Maximum Composite Likelihood method  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. 
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.  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: Sequence alignment of the (GI_3.......GI_2) with reference sequences retrieved from the (JN579668.1.........JF918520.1). The dots represent homogeneity in the sequences with the reference sequence|
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|Figure 2: The evolutionary history was inferred using the neighbor-joining method. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analysed. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. Evolutionary analyses were conducted in MEGA5 Evolutionary relationship of taxa with reference sequences|
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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. , 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.  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)  and Spain 5.8% (10/170). 
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. ,, 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,  whereas assemblage B was the predominant genotype in children (40/50).  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.  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.  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. , Among all these loci, tpi gene has the highest degree of polymorphisms. 
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.  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. , 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.  Nitazoxanide has also been used in successful treatment of metronidazole-resistant giardiasis in a patient with acquired immunodeficiency syndrome.  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.
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[Figure 1], [Figure 2]