Indian Journal of Medical Microbiology, (2014) 32(4): 378-382
1
Original Article
Refractory giardiasis: A molecular appraisal from a tertiary care centre in India P Yadav, V Tak, *BR Mirdha, GK Makharia
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’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. Key words: Genetic heterogeneity, Giardia lamblia, treatment failure
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. *Corresponding author (email: ) Department of Microbiology (PY, VT, BRM), Department of Gastroenterology and Human Nutrition (GKM), All India Institute of Medical Sciences, New Delhi, India. Received: 3-09-2013 Accepted: 7-02-2014 Access this article online Quick Response Code:
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.142236
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
October - December 2014
Yadav, et al.: Refractory giardiasis
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
379
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 [Figures 1 and 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
1
Original Article
Refractory giardiasis: A molecular appraisal from a tertiary care centre in India P Yadav, V Tak, *BR Mirdha, GK Makharia
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’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. Key words: Genetic heterogeneity, Giardia lamblia, treatment failure
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. *Corresponding author (email: ) Department of Microbiology (PY, VT, BRM), Department of Gastroenterology and Human Nutrition (GKM), All India Institute of Medical Sciences, New Delhi, India. Received: 3-09-2013 Accepted: 7-02-2014 Access this article online Quick Response Code:
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.142236
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
October - December 2014
Yadav, et al.: Refractory giardiasis
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
379
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 [Figures 1 and 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
