Skip to main content Accessibility help


  • Access
  • Cited by 7


      • Send article to Kindle

        To send this article to your Kindle, first ensure is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about sending to your Kindle. Find out more about sending to your Kindle.

        Note you can select to send to either the or variations. ‘’ emails are free but can only be sent to your device when it is connected to wi-fi. ‘’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

        Find out more about the Kindle Personal Document Service.

        Microsporidial infections due to Encephalitozoon intestinalis in non-HIV-infected patients with chronic diarrhoea
        Available formats

        Send article to Dropbox

        To send this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Dropbox.

        Microsporidial infections due to Encephalitozoon intestinalis in non-HIV-infected patients with chronic diarrhoea
        Available formats

        Send article to Google Drive

        To send this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Google Drive.

        Microsporidial infections due to Encephalitozoon intestinalis in non-HIV-infected patients with chronic diarrhoea
        Available formats
Export citation


We determined the prevalence of microsporidia Enterocytozoon (Ent.) bieneusi and Encephalitozoon (E.) intestinalis infection in patients with chronic diarrhoea and hepatocellular carcinoma (HCC). A total of 330 stool samples were examined from 171 (52%) patients with chronic diarrhoea, 18 (5%) with HCC while 141 (43%) were controls. Stool microscopy, polymerase chain reaction (PCR) with species-specific primers for Ent. bieneusi and E. intestinalis and sequencing were carried out. Microsporidia were found by trichrome staining in 11/330 (3%) and E. intestinalis by PCR in 13/330 (4%) while Ent. bieneusi was not detected. PCR for E. intestinalis was positive in 8/171 (5%) stool samples from patients with chronic diarrhoea, 2/141 (1·4%) samples from healthy controls and in 3/18 (17%) samples from patients with HCC. In the chronic diarrhoea group, E. intestinalis was positive in 4/171 (2·3%) (P=0·69) stool samples compared to 2/18 (11%) (P=0·06) in the HCC group and 2/141 (1·4%) from healthy controls. E. intestinalis infection was significantly associated with chronic diarrhoea and HCC in these patients who were negative for HIV. Stool examination with trichrome or species-specific PCR for microsporidia may help establish the cause of chronic diarrhoea.


Microsporidia are considered to be emerging and opportunistic infections in humans. Enterocytozoon (Ent.) bieneusi and Encephalitozoon (E.) intestinalis are the most common microsporidia infecting humans that are associated with diarrhoea and systemic disease. Persistent or self-limiting diarrhoea are common symptoms associated with microsporidiosis in both immunodeficient or immunocompetent individuals. Microsporidial spores appear to be relatively resistant under environmental conditions. Species of microsporidia infecting humans and animals have been identified in water sources, raising concern about waterborne transmission. In the developing world, parasitic disease contributes heavily to the burden of diarrhoeal diseases. Patients, who are malnourished, transplant recipients and those with acquired immune deficiency syndrome (AIDS), are especially at risk of increased morbidity and mortality from microsporidiosis. Twenty to forty per cent of HIV-positive individuals in the developing world develop microsporidiosis [14].

Microsporidia are small (1–2 μm), single-celled, obligate intracellular parasites characterized by a polar filament that is used for invasion of the host cell [5]. Mature microsporidial spores have thick walls and can pass through water treatment filters and are resistant to chlorine at concentrations used to treat drinking water. Microsporidial spores have been found in drinking water sources, soil and domestic and wild animals, suggesting the possibility of waterborne, foodborne and zoonotic transmission. Microsporidia are on the Contaminant Candidate List because their transmission routes are unknown. Spore identification, removal and inactivation in drinking water are difficult and human infections are difficult to treat [6, 7]. Microsporidial spores detected in a variety of surface waters are implicated as a source of human infection based on epidemiological data [813]. Infections with microsporidia in immunocompetent individuals such as travellers have also been described [5, 14, 15].

This study was conducted to determine the incidence of E. intestinalis and Ent. bieneusi infection in our patients presenting with chronic diarrhoea and to compare them with healthy controls and those with hepatocellular carcinoma (HCC) superimposed on chronic liver disease associated with hepatitis C virus (HCV). In patients with HCC, impaired immunological competence and malnourishment are known to cause an increase in their susceptibility to infection [16]. The phagocytic and bactericidal activities of neutrophils and the percentage of natural killer cells are significantly reduced in patients with HCC [16]. Moreover, dysregulation of both type 1-related and type 2-related host immunity has been described in patients with HCV-associated HCC [17].



The stool samples of 330 patients were examined. Of these, 171 patients had chronic diarrhoea with >3 stools a day for at least 4 weeks and <12 weeks. Individual controls (n=141) consisted of healthy members of paramedical staff without diarrhoea and 18 (5%) with HCC associated with chronic liver disease with hepatitis C. There were 222 (67%) males and 108 (33%) females with a mean age of 41±14 years (range 15–83 years). The patients attended the Gastroenterology Outpatient clinic at the Aga Khan University, Karachi between November 2008 and December 2010. The mean age of patients with chronic diarrhoea was 40±15 years (range 16–83) with a male:female ratio of 13:6. The male predominance reflects the tendency of male patients to present more commonly to consulting clinic with this complaint. In healthy controls, the mean age of patients was 42±14 years. All patients underwent history, physical examination, complete blood count, liver function test, serum creatinine, electrolytes, stool microscopy and polymerase chain reaction (PCR) for Ent. bieneusi and E. intestinalis. HIV status was negative in all patients and controls. A note was made of the presence of protozoa such as Blastocystis hominis, Giardia lamblia, Entamoeba, etc. on stool microscopic examination. HCC was diagnosed on the basis of α-fetoprotein level and ultrasound. Serology and PCR were performed for hepatitis B (HBV), HCV and delta (D) virus. All patients with HCC were positive for HCV. The study was approved by the institutional ethics review committee and was performed in accordance with the ethical standards laid down in the Declaration of Helsinki, 1964. All persons gave their informed consent prior to their inclusion in the study. All the stool specimens were processed by microscopy with modified trichrome staining and PCR. The diagnosis of microsporidial infection was made when both stool microscopy and PCR were positive. A microbiological investigation was also performed to detect Salmonella spp., Campylobacter jejuni, Clostridium difficile and Vibrio cholerae. However, a viral screen was not performed on stool specimens due to cost restrictions.

Microscopy of faecal smear

Faecal sample microscopy for demonstrating microsporidia was performed as described previously [6]. Wet smears in saline and iodine were examined under both low-power (×10) and high-power (×40–100) for the presence of parasites. Stool smears were fixed in ethanol and stained with modified trichrome stain in order to detect microsporidia [3].

Extraction of genomic DNA

Stool DNA was extracted by using Stool DNA Extraction kit (Qiagen, USA) according to the manufacturer's protocol. Extracted DNA was stored at −20°C until PCR was performed for microsporidia.


The primers V1 (5′-CACCAGGTTGTTCTGCCTGAC-3′) and EB450 (5′-ACTCAGGTGTTATACTCACGTC-3′) described by Zhu et al. [18] were used to amplify Ent. bieneusi DNA. The primers V1 and SI500 (5′-CTCGCTCCTTTACACTCGAA-3′) described by Weiss et al. [19] were used to amplify E. intestinalis DNA. The PCR reaction volume was 25 μl, comprising of 2·5 μl of 10× PCR buffer (Promega, USA), 2·0 μl of 25 mm MgCl2 (Promega), 0·4 μl dNTP mixture (10 mm of each dNTP, Promega), 0·5 μl (5 IU/μl) of Taq polymerase (Promega), 1 μl (0·25 μm) primers (IDT) and 2·0 μl of template DNA.

For the two sets of primers, one amplification cycle consisted of an initial denaturation of target DNA at 94°C for 10 min, followed by denaturation at 94°C for 1 min, primer annealing at 55°C for 2 min, and elongation at 72°C for 3 min. The last elongation step was extended to 10 min. Samples were amplified through 35 consecutive cycles. The PCR products and molecular markers were electrophoresed in 2% agarose gel with Tris-acetate-EDTA electrophoresis buffer. The size markers were 100-bp ladder (Promega). The PCR amplification was repeated at least three times. Bands were visualized by the imaging system (Gel Doc 2000, Gel Documentation System, Bio-Rad, UK).

Sequence analysis of small subunit unit ribosomal RNA and BLAST query

The DNA fragments amplified by E. intestinalis PCRs were purified by Qiagen quick PCR purification kit (Qiagen) and sequenced using both the forward and reverse primers (Table 1) to verify that they represented the E. intestinalis small subunit unit ribosomal RNA gene. Sequence analysis was performed by Macrogen (South Korea). Sequence comparison was carried out using the Blast program and the GenBank database.

Table 1. Characteristics of patients in different groups (N=330)

HCC, Hepatocellular carcinoma; PCR, polymerase chain reaction.

Values given are n (%) unless stated otherwise.

A P value of <0·05 was considered as statistically significant.

* Comparison between groups assessed using the χ2 test.

Comparison between groups assessed using the likelihood ratio test.

Statistical method

Results are expressed as mean±standard deviation for continuous variables (e.g. age) and number (percentage) for categorical data (e.g. gender, stool culture, diarrhoea, etc.). Univariate analysis was performed by using the independent sample t test, Pearson χ2 test and Fisher's exact test where appropriate. Comparison between groups was assessed using the χ2 test, Fisher's exact test or likelihood ratio test, as appropriate. The concordance between stool microscopy and PCR was determined by the kappa (κ) test. A P value of <0·05 was considered statistically significant. All P values were two-sided. Statistical interpretation of data was performed by using the computerized software program SPSS version 19.0 (SPSS Inc., USA).



A total of 193/330 (58%) stool samples were submitted from patients with diarrhoea while 171/330 (52%) had abdominal discomfort or pain.

Stool microscopy

Stool microscopy revealed B. hominis in 110/330 (33%), Entamoeba dispar in 53/330 (16%), G. lamblia in 19/330 (6%), Entamoeba histolytica in 18/330 (6%) and microsporidia in 11/330 (3%), respectively. Microsporidia was also found by microscopy with trichrome staining in 11/330 (3%) and PCR for E. intestinalis in 13/330 (4%) (Table 1). There were no cases of microsporidia that were found by microscopy and not detected by PCR. Eight of 330 (2·4%) had microsporidia detected by both microscopy and PCR (Table 1). Only 4/18 (22%) with HCC had symptoms of diarrhoea.

Diagnostic yield of various methods for microsporidia

Microscopy with staining detected microsporidia in 6/171 (4%) stool specimens from patients with chronic diarrhoea, 2/141 (1·4%) stool specimens from controls and in 3/18 (17%) stool specimens from patients with HCC (Table 1). PCR of E. intestinalis was positive in 8/171 (5%) cases with chronic diarrhoea, 2/141 (1·4%) in controls and in 3/18 (17%) with HCC (Table 1) while Ent. bieneusi was negative. The correlation between microscopy with staining and PCR for E. intestinalis was 8 (73%) (κ=0·654, P<0·001).

Comparison of distribution of E. intestinalis in different groups

Four of 171 (2·3%) stool specimens from patients with chronic diarrhoea, 2/141 (1·4%) from controls and 2/18 (11%) with HCC had E. intestinalis infection (Table 1). In stool specimens from patients with chronic diarrhoea, E. intestinalis was positive in 4/171 (2·3%) compared to 2/141 (1·4%) in stool specimens from controls (P=0·69) (Table 2). In stool specimens from patients with HCC, 2/18 (11%) were positive for E. intestinalis compared to 2/141 (1·4%) in controls (P=0·06) (Table 2).

Table 2. Comparison of different groups with both microscopy and PCR for microsporidia

HCC, Hepatocellular carcinoma; PCR, polymerase chain reaction.

Values given are n (%).

A P value of <0·05 was considered as statistically significant.

* Comparison between groups was assessed using Fisher's exact test.

Distribution of mixed infection in different groups

In stool specimens from patients with chronic diarrhoea 5/171(3%) (P<0·001) and 3/18 (17%) (P=0·03) patients with HCC demonstrated E. intestinalis compared to 2/141(1%) healthy controls, respectively (Table 3). Co-infection of E. intestinalis infection was present with B. hominis in 2/171 (1%) and in 1/171 (1%) with both B. hominis and Entamoeba dispar in chronic diarrhoea (Table 3).

Table 3. Distribution of protozoa parasite in different groups

Values given are n (%).

* Comparison between groups was assessed using the likelihood ratio.

A P value of <0·05 was considered as statistically significant.

Sequence analysis of small subunit unit ribosomal RNA and BLAST query

The DNA fragments amplified by PCR were purified by Qiagen quick PCR purification kit (Qiagen) and sequenced using both the forward and reverse primers V1 and SI520 to verify that they represented E. intestinalis small subunit unit ribosomal RNA gene with GenBank accession numbers JF932504, JF932505, JF932506 and JF932507. Homology of the DNA sequences to published sequences was determined using the BLAST window on the National Centre for Biotechnology Information (NCBI) site at PCR product sequences aligned well with the sequences of E. intestinalis CP001951.1, CP001946.1, CP001945.1, CP001942.1 and DQ453122.1.


In non-immunocompromised patients, both asymptomatic and symptomatic microsporidial infections have been described, which are usually seen in travellers or residents of tropical areas. The majority of symptomatic infections in humans occur in patients with HIV infection who are significantly immunocompromised. The elderly, with a mean age 75 years were identified as at risk of Ent. bieneusi infection when presenting with chronic diarrhoea [20]. Case reports have also documented travellers' diarrhoea caused by Ent. bieneusi in normal hosts [12, 21, 22]. E. intestinalis has been found in stool samples of travellers' with chronic diarrhea [23].

This study highlights E. intestinalis as the cause of chronic diarrhoea among other more common parasites such as G. lamblia, Entamoeba histolytica, etc. The point prevalence of microsporidial infection in patients with chronic diarrhoea was rather low (4% for microscopy, 5% for PCR, and 2·3% for both microscopy and PCR) and not much different from controls (i.e. 1·4% for both microscopy and PCR). E. intestinalis was shown in patients with HCC, chronic diarrhoea and in controls. There was a relatively high percentage of microsporidial infection in HCC patients. However, the number of patients was too small in this group to make a meaningful comparison with the other groups of chronic diarrhoea and controls. Moreover, the lack of follow-up does not allow an assessment of the significance and duration of microsporidial infection in this group. Co-infection of E. intestinalis with B. hominis was demonstrated in two cases and with B. hominis and Entamoeba dispar in one case with chronic diarrhoea. However, none of the cases with HCC described here had E. intestinalis infection combined with any of the other parasites. There were only isolated infections with B. hominis, Entamoeba dispar or G. lamblia (Table 3). Testing with modified trichrome staining and PCR for E. intestinalis demonstrated a high degree of concordance. These parasites are easily detected by light microscopy when infections are heavy. However, early infections without spores, or light infections with low numbers of spores, can easily be missed. E. intestinalis was detected in 13 cases by PCR and in only 11 by modified trichrome staining. It was probably missed by staining in cases where spores were low in numbers. It is surprising that E. intestinalis was identified while Ent. bieneusi was not detected in any group in this study. This could be explained by the lack of exposure to the environmental sources of Ent. bieneusi such as pigs, cats and cattle, while E. intestinalis occur in goats and donkeys which are common in urban areas of Pakistan, being kept as pets or for pulling carts for transportation of goods [24, 25]. The presence of E. intestinalis has also been confirmed in tertiary sewage effluent, surface water and groundwater [26]. Locally, water used for domestic purposes is usually supplied by the underground city municipal pipeline which is in close proximity to the sewage pipeline. Over the years with wear and tear they have been known to break down causing undetected contamination of domestic water supply by sewage. Moreover, in studies from neighbouring India, the prevalence of intestinal parasitic infections in HIV-positive patients with diarrhoea reported Ent. bieneusi in only 2·5% [27] while Cryptosporidium parvum and G. lamblia were the most common infecting parasites in these patients [27, 28].

This is the first report of microsporidial infection from Pakistan. It supports the need to include microsporidia in the differential diagnoses for causes of chronic diarrhoea in both HIV-infected [2] and non-HIV-infected groups of patients. Specific stains for microsporidia should be requested in particular cases since routine examination for ova and parasites does not usually detect microsporidia spores. Faecal leukocytes and blood are commonly absent since microsporidial infection is not associated with a significant inflammatory reaction. PCR of microsporidia can be used for quick detection of microsporidial infection [29]. Modified trichrome staining has a reported sensitivity and specificity of 100% and 83%, respectively [30]. In our study there was a significant concordance between PCR and modified Trichrome staining of the specimen. Available fluorescent techniques including Uvitex 2B (Ciba Geigy, UK), Calcofluor White M2R (Sigma, USA), the FungiFluor kit (Polysciences, USA) and Fungiqual A have similar sensitivity and specificity to modified trichrome staining [13, 31, 32]. However, microscopy is known to be highly dependent on the expertise of the examiner. Monoclonal antibodies to Encephalitozoon spp. have been described as improving detection of microsporidia in clinical specimens [33, 34]. Other techniques include serological assays (which detect IgM and IgG anti-microsporidial antibodies), tissue culture, and indirect immunofluorescence and PCR [13, 18, 35, 36]. Multiplex FISH assay has been described as more sensitive than both Chromotrope-2R and Calcofluor White M2R stains and it has been used for assessing spore-shedding intensity in intestinal microsporidiosis and identification of microsporidial spores [36]. In conclusion, microsporidial infection may present with chronic diarrhoea. An examination of stool specimen with modified trichrome staining or PCR for E. intestinalis should be performed for selected patients with intractable symptoms of diarrhoea.


This study was supported by the Higher Educational Commission Grant (ref. 20-774/R&D/06/267). We are grateful to the staff of the Juma Building Research Laboratory for their help during this study.




1. Petry, F. Cryptosporidiosis and Microsporidiosis. Switzerland: Karger, 2006, pp. 1268.
2. Eeftinck Schattenkerk, JK, et al. Clinical significance of small-intestinal microsporidiosis in HIV-1-infected individuals. Lancet 1991; 337: 895898.
3. Gamboa-Dominguez, AJ, et al. Disseminated Encephalitozoon cuniculi infection in a Mexican kidney transplant recipient. Transplantation 2003; 75: 18981900.
4. Sax, PE, et al. Intestinal microsporidiosis occurring in a liver transplant recipient. Transplantation 1995; 60: 617618.
5. Muller, A, et al. Detection of microsporidia in travelers with diarrhoea. Journal of Clinical Microbiology 2001; 39: 16301632.
6. EPA. United States Environmental Protection Agency ( Accessed 5 February 2011.
7. Didier, ES, et al. Therapeutic strategies for human microsporidia infections. Expert Review Anti-Infective Therapy 2005; 3: 419434.
8. Fournier, S, et al. Detection of microsporidia in surface water: a one-year follow-up study. FEMS Immunology Medical Microbiology 2000; 29: 95–100.
9. Graczyk, TK, et al. Human waterborne parasites in zebra mussels (Dreissena polymorpha) from the Shannon River drainage, Ireland. Parasitology Research 2004; 93: 389391.
10. Cotte, L, et al. Waterborne outbreak of intestinal microsporidiosis in persons with and without human immunodeficiency virus infection. Journal of Infectious Diseases 1999; 180: 20032008.
11. Franzen, C, Muller, A. Cryptosporidia and microsporidia: waterborne diseases in the immunocompromised host. Diagnostic Microbiology Infectious Diseases 1999; 34: 245262.
12. Lopez-Velez, R, et al. Microsporidiosis in travelers with diarrhoea from the tropics. Journal of Travel Medicine 1999; 6: 223227.
13. DeGirolami, PC, et al. Diagnosis of intestinal microsporidiosis by examination of stool and duodenal aspirate with Weber's modified trichrome and Uvitex 2B strains. Journal of Clinical Microbiology 1995; 33: 805810.
14. Weber, R, et al. Human microsporidial infections. Clinical Microbiology Review 1994; 7: 426461.
15. Weiss, LM. Microsporidia: emerging pathogenic protists. Acta Tropica 2001; 78: 89–102.
16. Iida, K, et al. Immunological function and nutritional status in patients with hepatocellular carcinoma. Hepatogastroenterology 1999; 6: 24762482.
17. Suruki, RY, et al. Host immune status and incidence of hepatocellular carcinoma among subjects infected with hepatitis C virus: a nested case-control study in Japan. Cancer Epidemiology, Biomarkers and Prevention 2006; 15: 25212525.
18. Zhu, X, et al. Small subunit rRNA sequence of Enterocytozoon bieneusi and its potential diagnostic role with use of the polymerase chain reaction. Journal of Infectious Diseases 1993; 168: 15701575.
19. Weiss, LM, et al. Utility of microsporidian rRNA in diagnosis and phylogeny: a review. Folia Parasitologica 1994; 41: 8190.
20. van Gool, T, et al. High seroprevalence of Encepha-litozoon species in immunocompetent subjects. Journal of Infectious Diseases 1997; 175: 10201024.
21. Lores, B, et al. Intestinal microsporidiosis due to Enterocytozoon bieneusi in elderly human Immunodeficiency virus-negative patients from Vigo, Spain. Clinical Infectious Diseases 2002; 34: 918921.
22. Fournier, S, et al. Microsporidiosis due to Enterocytozoon bieneusi infection as a possible cause of traveller's diarrhea. European Journal Clinical Microbiology Infectious Diseases 1998; 17: 743744.
23. Bryan, RT, Weber, R, Schwartz, DA. Microsporidiosis in patients who are not infected with human immunodeficiency virus. Clinical Infectious Diseases 1997; 24: 534535.
24. Dengjel, B, et al. Zoonotic Potential of Enterocytozoon bieneusi . Journal of Clinical Microbiology 2001; 39: 44954499.
25. Bornay-Llinares, FJ, et al. Immunologic, microscopic, and molecular evidence Of Encephalitozoon intestinalis (Septata intestinalis) in mammals other than humans. Journal of Infectious Diseases 1998; 178: 820826.
26. Dowd, SC, Gerba, CP, Pepper, IL. Confirmation of the human-pathogenic microsporidia Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Vittaforma corneae in water. Applied Environmental Microbiology 1998; 64: 33323335.
27. Mohandas, SR, Sud, A, Malla, N. Prevalence of intestinal parasitic pathogens in HIV-seropositive individuals in Northern India. Japanese Journal of Infectious Diseases 2002; 55: 8384.
28. Becker, ML, et al. Diarrhoeal disease among HIV-infected adults in Karnataka, India: evaluation of risk factors and etiology. American Journal of Tropical Medicine Hygiene 2007; 76: 718722.
29. Weber, R, et al. Improved light-microscopical detection of microsporidia spores in stool and duodenal aspirates. The Enteric Opportunistic Infections Working Group. New England Journal of Medicine 1992; 326: 161166.
30. Ignatius, R, et al. Comparative evaluation of modified trichrome and Uvitex 2B stains for detection of low numbers of microsporidial spores in stool specimens. Journal of Clinical Microbiology 1997; 35: 22662269.
31. Mo, L, Drancourt, M. Monoclonal antibodies for specific detection of Encephalitozoon cuniculi . Clinical Diagnostic Laboratory Immunology 2004; 11: 10601063.
32. Sheoran, AS, et al. Monoclonal antibodies against Enterocytozoon bieneusi of human origin. Clinical Diagnostic Laboratory Immunology 2005; 12: 11091113.
33. Fedorko, DP, Hijazi, YM. Application of molecular techniques to the diagnosis of microsporidial infection. Emerging Infectious Diseases 1996; 2: 183191.
34. Müller, A, et al. A powerful DNA extraction method and PCR for detection of microsporidia in clinical stool specimens. Clinical Diagnostic Laboratory Immunology 1999; 6: 243246.
35. Franzen, C, Muller, A. Molecular techniques for detection, species differentiation and phylogenetic analysis of microsporidia. Clinical Microbiology Review 1999; 12: 243285.
36. Graczyk, TK, et al. Retrospective species identification of microsporidian spores in diarrheic fecal samples from human immunodeficiency virus/AIDS patients by multiplexed fluorescence in situ hybridization. Journal of Clinical Microbiology 2007; 45: 12551260.