|Year : 2015 | Volume
| Issue : 3 | Page : 357-363
Intestinal microsporidiosis in renal transplant recipients: Prevalence, predictors of occurrence and genetic characterization
U Ghoshal1, S Khanduja1, P Pant1, KN Prasad1, TN Dhole1, RK Sharma2, UC Ghoshal3
1 Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
2 Department of Nephrology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
3 Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
|Date of Submission||29-Apr-2014|
|Date of Acceptance||16-Dec-2014|
|Date of Web Publication||12-Jun-2015|
Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh
Source of Support: Indian Council of Medical Research, New Delhi., Conflict of Interest: None
Purpose: Intestinal microsporidiosis, which occurs in immunocompromised states such as acquired immunodeficiency syndrome, has rarely been studied in patients with renal transplantation (RT) on immunosuppressive therapy. Materials and Methods: Three hundred and twenty-four consecutive RT recipients on immunosuppressive treatment and 170 healthy subjects were evaluated for intestinal microsporidiosis and other parasites by modified trichrome staining, wet mount using normal saline, iodine and polymerase chain reaction (PCR). Clinical, demographic and laboratory parameters associated with occurrence of intestinal microsporidiosis were studied using univariate and multivariate analysis. The species of microsporidia were studied using PCR-restriction fragment length polymorphism (RFLP). Patients were treated with albendazole (400 mg twice daily for 2 weeks). Results: Of 324 RT recipients initially screened, 52 were excluded from final analysis due to incomplete data. Patients with RT [n = 272, age 42 ± 12.54 years, 222 (81.6%) male] more often had microsporidiosis than healthy subjects by modified trichrome stain and PCR [n = 170, age 33.8 ± 6.7 years, 123 (72.3%) male] [16/272 (5.8%) vs. 0/170 (0%), P < 0.001]. Patients with intestinal microsporidiosis were younger (33.9 ± 8.3 years vs. 42.3 ± 12.6 years; P = 0.009), had diarrhoea more often (13/16, 81% vs. 123/256, 48%; P = 0.02), which was longer in duration (60, 32.5-105 days vs. 12, 6.2-18 days; P < 0.001) and had associated giardiasis (2/16, 12.5% vs. 2/256, 0.8%; P = 0.018). Younger age, presence of diarrhoea and associated giardiasis were significant on multivariate analysis. Enterocytozoon bieneusi was detected in 15/16 (93%) patients with intestinal microsporidiosis. Conclusion: Intestinal microsporidiosis occurs frequently in patients with RT on immunosuppressive treatment, particularly among younger patients with longer diarrhoea duration and associated giardiasis. E. bieneusi is the major species identified among these patients.
Keywords: Diarrhoea, microsporidia, renal transplant
|How to cite this article:|
Ghoshal U, Khanduja S, Pant P, Prasad K N, Dhole T N, Sharma R K, Ghoshal U C. Intestinal microsporidiosis in renal transplant recipients: Prevalence, predictors of occurrence and genetic characterization. Indian J Med Microbiol 2015;33:357-63
|How to cite this URL:|
Ghoshal U, Khanduja S, Pant P, Prasad K N, Dhole T N, Sharma R K, Ghoshal U C. Intestinal microsporidiosis in renal transplant recipients: Prevalence, predictors of occurrence and genetic characterization. Indian J Med Microbiol [serial online] 2015 [cited 2019 Nov 12];33:357-63. Available from: http://www.ijmm.org/text.asp?2015/33/3/357/158551
| ~ Introduction|| |
Microsporidia are emerging opportunistic pathogens in immunocompromised patients.  Intestinal microsporidiosis is known to cause diarrhoea with significant morbidity and mortality in them.  Microsporidiosis commonly occurs in association with human immunodeficiency virus (HIV) infection, with a prevalence ranging from 0.1% to 50%. 
Patients undergoing renal transplantation (RT) are immunosuppressed as they receive multiple drugs to prevent rejection of the transplanted kidney. Though these patients are markedly immunosuppressed as evidenced by frequent occurrence of other opportunistic infections such as cytomegalovirus,  data on intestinal microsporidiosis are scanty in them. Only 31 cases of microsporidiosis in RT recipients have been reported worldwide till now. ,,,,,,, Therefore, there is a need to evaluate the risk factors and clinical features of intestinal microsporidiosis in RT recipients, as clinicians need to suspect this condition early for undertaking appropriate diagnostic evaluation and treatment.
The species of microsporidia, namely, Enterocytozoon bieneusi (E. bieneusi) and Encephalitozoon intestinalis (E. intestinalis) are commonly known to cause intestinal microsporidiosis in humans.  However, one study reported Encephalitozoon hellem (E. hellem) as cause of intestinal microsporidiosis in cancer patients.  Some of these species respond to currently available treatment, others do not.  Knowledge about prevalent species of the organism is important while deciding on treatment and prognosis. There are a few case reports ,,,,,,, but to the best of our knowledge, no prospective case control study has yet been reported; moreover, no study evaluated species of microsporidia infecting patients with RT.
Accordingly, we undertook a study with following aims, (a) to evaluate the prevalence of microsporidiosis among RT recipients as compared with healthy subjects, (b) to analyze the predictors of occurrence of this condition among them and (c) to study the species of microsporidia by genetic characterization among patients with RT.
| ~ Materials and Methods|| |
Study subjects and protocol
Three hundred and twenty-four consecutive patients with RT (HIV negative) attending Nephrology service of a tertiary care institution were evaluated for intestinal microsporidiosis during January 2006 to June 2013. One hundred and seventy healthy subjects were similarly evaluated as controls. Data on demographic, clinical and laboratory parameters were recorded in a standard questionnaire in each patient. No patient took anti-microsporidial drugs in recent past. The study was approved by Institutional Ethics Committee (PGI/DIR/RC/1085/2007).
Three consecutive fresh stool samples from each patient and control were collected. Samples were subjected to microscopic examination and a part was stored at −40°C in normal saline for deoxyribonucleic acid (DNA) extraction. Blood samples were also collected in ethylenediaminetetraacetic acid (EDTA) vial for CD4 count.
Three consecutive stool samples were subjected to routine microscopy by direct wet mount technique using both normal saline and iodine and observed under light microscope to look for ova and cysts of parasites. Non-pathogenic amoeba and Blastocystis were not included as their role in diarrhoea is yet not well-established. Thereafter, formol ether concentration was performed and the smears were stained with Weber's modified trichrome stain  with certain modifications and Kinyoun's stain. Briefly, fast green was used as counter stain and the smear was incubated in trichrome stain at 50°C for 10-12 minutes. Each stained smear was observed under total magnification of 1000X. A plus grading system was opted for counting spores in different fields of the smear [Table 1]. Stool samples were subjected to culture on Mac Conkey, deoxycholate citrate agar (DCA) and dextrose sorbitolrhamnose agar (DSRA) media for common bacterial agents like Salmonella More Details, Shigella and enterohaemorrhagic E. coli. CD4 + cells were counted using Becton Dickinson (BD) fluorescence-activated cell sorting (FACS) flow CD4/CD3 reagent kit (BD Biosciences, CA, USA).
|Table 1: Plus grading system for counting spores in different fields of smear |
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Three consecutive stool samples collected from each patient and control were pooled in one aliquot each. DNA was extracted from pooled stool samples using QIAamp Qiagen mini stool kit (Qiagen Inc., Valencia, CA, USA) following manufacturer's instruction with some modifications. Briefly, the sample was suspended in phosphate buffered saline (PBS) and subjected to centrifugation. The suspension was heated at 80°C for 10 minutes. The extracted fecal DNA was subjected to amplify the conserved region of small subunit ribosomal ribonucleic acid (SSU rRNA) gene of Enterocytozoon bieneusi and Encephalitozoon species using previously published forward primer C1 (5'CACCAGGTTGATTCTGCC-3') and reverse primer C2 (5'GTGACGGGCGGTGTGTAC-3'). The DNA was amplified in a 50-μl reaction mixture including 12.5 pmol of each primer, 200 mM each de-oxynucleosidetriphosphate, 2 mM MgCl2 and 1U of Taq DNA polymerase. After initial denaturation of the DNA at 94°C for 5 minutes, 30 cycles were run as follows: Denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, and elongation at 72°C for 1 minute, with 5 minutes of final extension at 72°C after 30 cycles. Amplified products were electrophoretically analyzed on agarose gel and stained with ethidium bromide.
The amplified fragments were subjected to digestion using restriction endonucleases HinfI and HindIII to differentiate between four human Microsporidia species, E. bieneusi, E. intestinalis, E. cuniculi and E. hellem. Ten microlitres of amplified DNA was digested with 5 unit of HinfI and HindIII in a final volume of 15 μl. Digested fragments were analyzed on 2.5% agarose gel stained with ethidium bromide. Amplified fragments were analyzed electrophoretically on agarose gel.
To know nucleotide sequences, purified PCR product were sequenced using Microsporidia specific SSU rRNA's forward primer by fluorescent label ABI's AmpliTaq FS dye terminators on ABI model 3100. Electropherograms were analyzed and aligned with previously reported sequences of Microsporidia using standard software (Chromas program, Technelysium Pty Ltd, Sydney, Australia and BioEdit v7.0.5, Ibis Biosciences, Carlsbad, California, respectively). Identified sequences were submitted to National centre for biotechnology information (NCBI).
Detection of microsporidia either on microscopy in one or more stool sample or its DNA on molecular analysis or both was considered diagnostic of intestinal microsporidiosis.
Treatment and follow-up
Patients were followed up after treatment with albendazole (400 mg twice daily for 2 weeks). Stool microscopy and PCR were repeated in patients agreeing for it and symptoms including diarrhoea, duration of diarrhoea, number of stools/day, character of stool and abdominal pain were re-evaluated on follow-up. On repeat stool microscopy, plus grading system was applied to count the number of spores in the smear.
Data were checked for normal distribution using Shapiro-Wilk test. Categorical, parametric and non-parametric continuous data were presented as proportion, mean, standard deviation, median and inter-quartile range, respectively. Chi-square, Fisher's exact, unpaired t and Wilcoxon rank sum tests were used to compare between categorical, continuous parametric and non-parametric data, respectively. P values below 0.05 were considered significant for all statistical analysis. Statistical analysis was done using R, Epicalc and R-studio software (R development core team, Vienna, Austria) and statistical package of social sciences (SPSS) version 15 (SPSS, Inc., Chicago, IL, USA).
| ~ Results|| |
Of 324 RT recipients initially screened, 52 were excluded from the final analysis due to incomplete data (lesser than three stool samples or inadequate clinical records). A total of 816 stool samples were collected from 272 RT recipients. The demographic (age and gender) and clinical parameters of 272 patients are shown in [Table 2]. The controls (n = 170) were comparable to RT patients in age and gender.
|Table 2: Demographic, clinical and laboratory parameters of patients with and without microsporidiosis with renal transplantation |
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Detection of microsporidia and other parasites among RT patients and controls
A total of 16/272 (5.8%) patients and none of the healthy controls had intestinal microsporidiosis (P < 0.001). [Figure 1] shows the spores of Microsporidia observed in 1000X magnification. [Table 3] shows the spectrum of parasites detected in RT recipients by microscopy. Of 16 patients with microsporidiosis, 12 were positive by both microscopy and PCR, 1 by microscopy alone and 3 by PCR alone. Microsporidiosis was diagnosed within mean duration of 4.3 ± 2.5 years (range, 4 months-9 years) after transplantation.
|Figure 1: Spores of microsporidia in the stool sample of a renal transplantation (RT) recipient under x1000 magnification in light microscope|
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|Table 3: Parasites detected in renal transplant recipients by microscopy |
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Factors associated with occurrence of microsporidiosis among RT recipients
[Table 2] shows the univariate analysis of factors associated with occurrence of intestinal microsporidiosis among RT recipients. Patients with intestinal microsporidiosis were younger in age, more often had diarrhoea and associated giardiasis. Diarrhoea was longer in duration with higher frequency. Other clinical manifestations including fever, cough, nausea and vomiting, abdominal pain were comparable among patients. History of keeping pets at home was comparable among patients with and without intestinal microsporidiosis (5/12, 42% vs. 37/111, 33%, P = 0.5). Similarly, history of drinking filtered or unfiltered water was comparable among patients with and without intestinal microsporidiosis (8/12, 66.7% vs. 70/111, 63%, P = 1.00). On multivariate analysis, intestinal microsporidiosis was more often associated with younger age of patients, presence of diarrhoea and associated giardiasis [Table 4]. No other parasite except Giardia (2/16, 12.5%) was detected in patients with microsporidia.
|Table 4: Result of multivariate analysis of factors associated with microsporidia infection |
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Of 272 patients, 15 (5.5%) were positive by PCR. A 1200 bp amplified fragment of microsporidia was observed using the specified primers [Figure 2]. The amplified product subjected to enzymatic digestion using restriction enzyme HinfI and HindIII displayed one restriction site with two bands of 230bp, 940bp and 386bp, 784bp, respectively [Figure 3]. This pattern of bands suggests infection with E. bieneusi which was confirmed by sequencing. E. intestinalis, E. hellem and E. cuniculi were not detected in any patient.
|Figure 2: An agarose gel showing polymerase chain reaction (PCR) products of human fecal samples with microsporidia. Lane M, 50 bp ladder; Lane PC, positive control for Microsporidia; Lane 1-3, Microsporidia positive samples; Lane NC, negative control|
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|Figure 3: Digestion pattern of polymerase chain reaction (PCR) products using restriction enzyme HinfI and Hind III. Lane M, 100 bp ladder; Lane PC, positive control for E. bieneusi using HinfI; Lane 2 and 4, samples positive for E. bieneusi using HindIII; Lane 3, samples positive for E. bieneusi using HinfI|
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Nucleotide accession number
The gene bank accession number for sequences submitted are KF148056 and KF148057.
Treatment and follow-up
Three patients died and eight were lost to follow-up before administering anti-microsporidial treatment. Five patients was evaluated after two week treatment. Of them, all the patients became asymptomatic after treatment. Spores of microsporidia were detected in three patients while two patients became negative for microsporidia on follow up by microscopy and PCR [Table 5].
|Table 5: Effect of treatment with albendazole on frequency of spores of microsporidia by microscopy in five follow-up patients after 2 weeks of treatment |
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| ~ Discussion|| |
In the present study, 5.8% RT recipients had intestinal microsporidiosis. On univariate analysis, patients with microsporidiosis were younger in age, had diarrhoea, which was longer in duration and had associated giardiasis. However, on multivariate analysis, all factors except duration of diarrhoea were associated with microsporidiosis. E. bieneusi was the commonest species identified to cause intestinal microsporidiosis in RT recipients.
The present report is the first largest case-control study on microsporidia in renal transplant recipient. To the best of our knowledge, only about 31 cases and a retrospective study on intestinal microsporidiosis in RT recipients have been reported till date. ,,,,,,,, This infection is known to be more prevalent in HIV infected patients with prevalence ranging from 1%−50% depending on the geographic region and diagnostic methods.  Comparatively, a low frequency of intestinal microsporidiosis in RT recipients (6%) is reported in the current study as compared to that reported in HIV-infected patients. Probably this could be due to administration of controlled state of immunosuppressants in renal transplantation. Since, acquired immune deficiency syndrome (AIDS) is a highly immunosuppressed state, it has higher susceptibility to other infections like Entamoeba histolytica, Leishmania.  However, these infections have rarely been reported in RT recipients. ,, Though, the frequency of microsporidia is less in RT recipients, this cannot be overlooked as microsporidia causes high morbidity and mortality.  Also, in the current study, microsporidiosis was diagnosed within mean duration of 4.3 ± 2.5 years (4 months to 9 years) after transplantation which is in concordance with earlier published reports. , This emphasizes the need for evaluating this opportunistic infection in RT recipients within few years after transplantation.
In the present study, patients with microsporidiosis were younger in age (mean, 33.9 ± 8.3 years). Similarly, mean age of microsporidia infected RT recipients as reported in different cases and retrospective study was 45.8 ± 11 and 44 years, respectively. ,,,,,,,, Since transplant recipients with younger age are at an increased risk of infection with microsporidia, such patients should be thoroughly investigated for microsporidiosis. Chronic diarrhoea is a common clinical presentation of immunocompromised patients infected with microsporidia. , In the present study, 12/13 (92.3%) patients with intestinal microsporidiosis had chronic diarrhoea. This is in accordance with earlier published reports. ,,,, A study on HIV patients reported presence of microsporidia in 60% with chronic diarrhoea.  Hence, RT recipients with chronic diarrhoea are at an increased risk of microsporidiosis. There was no association between pets, type of drinking water and presence of microsporidia in the current study. However, giardiasis, a surrogate marker for bad hygiene, was found associated with intestinal microsporidiosis. This is in agreement with previously published study on microsporidiosis showing co-infection with giardia.  Therefore, immunosuppression along with bad hygiene could be a probable reason for microsporidiosis in immunocompromised patients.
E. bieneusi was the commonest species causing intestinal microsporidiosis in HIV infected patients.  However, in other study E. intestinalis was reported as common causative species.  In the current study, E. bieneusi was found to be the predominant species causing intestinal microsporidiosis in RT recipients. The finding of infection of E. bieneusi is in accordance with the previously reported series of patients with RT. ,,,,,, Therefore, E. bieneusi is the common species causing intestinal microsporidiosis in RT recipients. Albendazole, a benzimidazole carbamate with broad antihelminthic and antifungal properties is the drug of choice against microsporidiosis.  This drug is highly effective against Encephalitozoon spp. but not against E. bieneusi.  In the present study, three patients became asymptomatic though spores were seen on follow-up stool samples. This is in accordance with an earlier study on treatment of intestinal microsporidiosis due to E. bieneusi, administration of albendazole (400 mg orally twice a day) resulted in decrease of organism burden without clearing it completely.  Similarly, other study reported an apparent decrease in organism load and resolution of diarrhoea after treatment with Albendazole.  Albendazole attacks the colchicine sensitive sites of tubulin causing an incomplete inhibition of E. bieneusi reproduction. In the current study, two patients became asymptomatic and their stool sample became negative for spores. Therefore, more research and controlled clinical trials are required to assess the potential of albendazole against E. bieneusi.
In conclusion, the present study shows that microsporidia is prevalent cause of diarrhoea in renal transplant recipients. Patients with younger age, chronic diarrhoea and those co-infected with giardia are at increased risk of microsporidiosis. E. bieneusi is the commonest species of microsporidia among RT recipients. Hence, clinicians should look for microsporidia in RT recipients presenting with chronic diarrhoea. Strong clinical suspicion and special methods are required for its diagnosis.
| ~ Acknowledgement|| |
This study was supported by a grant from Indian Council of Medical Research, New Delhi, India (reference no. 5/3/3/3/2008-ECD-I). Sonali Khanduja would also like to acknowledge ICMR fellowship (80/829/2013-ECD-I).
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]