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CORRESPONDENCE
Year : 2011  |  Volume : 134  |  Issue : 6  |  Page : 982-984

Performance of microscopy for detection of microsporidial spores from stool samples of HIV infected individuals with diarrhoea


1 National AIDS Research Institute , Pune, India
2 Naidu Municipal Corporation Hospital, Pune, India

Date of Web Publication6-Feb-2012

Correspondence Address:
Sangeeta Kulkarni
Department of Microbiology & Clinical Pathology, National AIDS Research Institute (ICMR), Pune 411 026
India
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DOI: 10.4103/0971-5916.92646

PMID: 22310832

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How to cite this article:
Kulkarni S, Patsute S, Chandane M, Risbud A. Performance of microscopy for detection of microsporidial spores from stool samples of HIV infected individuals with diarrhoea. Indian J Med Res 2011;134:982-4

How to cite this URL:
Kulkarni S, Patsute S, Chandane M, Risbud A. Performance of microscopy for detection of microsporidial spores from stool samples of HIV infected individuals with diarrhoea. Indian J Med Res [serial online] 2011 [cited 2014 Oct 1];134:982-4. Available from: http://www.ijmr.org.in/text.asp?2011/134/6/982/92646

Sir,

Intestinal microsporidiosis is the most common cause of chronic diarrhoea in treatment-naïve HIV infected patients [1] . Intestinal microsporidiosis due to Encephalitozoon (Septata) intestinalis and Enterocytozoon bieneusi are also most frequently reported among immunocompromised patients such as transplant recipients [2],[3],[4] . Laboratory diagnosis of microsporidiosis was initially based on the detection of microsporidial spores in stool specimens by light microscopy using special stains [5] . Electron microscopy, wherever possible, was also used for detection of microsporidia [6],[7],[8] .

Diagnosis of intestinal microsporidiosis has been markedly improved by the use of Weber's chromotrope stain and its modified procedure [1],[5],[9] , and fluorescence staining with fluorochromes such as Uvitex 2B or calcoflour white which binds to the chitin in the endospore layer of the microsporidial spore [1],[8],[10],[11] . Molecular technique such as polymerase chain reaction (PCR) became available later for confirmation of micropsoridia. Additionally, species specific PCR can be used to identify different species of microsporidia [12],[13],[14],[15],[16] . However, due to resource constraints, most laboratories rely on the use of microscopy of stool samples for diagnosis of intestinal microsporidiosis. Limited studies have been carried out in India on detection of microsporidia [8],[17],[18] . We compared light microscopy with modified trichome stain, fluorescence microscopy using Uvitex 2B and PCR to detect microsporidia in HIV infected individuals with diarrhoea during January 2009 to May 2010 on consecutive HIV infected patients with diarrhoea admitted at Naidu Municipal Corporation Hospital, Pune. The study protocol was approved by the institutional ethics committee & Naidu Municipal Corporation Hospital.

The study included 331 patients [65 (51 males, 14 females)] HIV infected adults >18 yr and 266 HIV (uninfected individuals) with diarrhoea. This study was carried out only on HIV infected individuals with diarrhoea. Study patients were interviewed using the structured questionnaire. Diarrhoea was defined as two or more liquid or three or more soft stools per day All the patients had history of diarrhoea of <14 days and were treatment-naïve HIV-infected individuals. Stool samples were collected in wide mouth, leak proof, clean sterile containers and then transported to National AIDS Research Institute (NARI) within 4 h of collection. If there was a delay in the transportation, the samples were preserved at 4 o C. The samples were immediately processed after receiving at NARI for microscopy by the conventional method [8] . Stool samples were stained by modified trichome and Uvitex 2B by the method described earlier [4],[6],[8] , and were subjected to light microscopy and fluorescence microscopy, respectively. A portion of the stool was stored at -70 o C for further molecular analysis. DNA was extracted from frozen samples using the QIA amplication DNA tissue kit (Qiagen, Inc, Germany). PCR was performed using specific primers as described by Najla et al[19] .

The median duration of diarrhoea was found to be 3 days (IQR 2.75 - 5) and median number of episodes of diarrhoea was noted as 6 in HIV-infected individuals (n=65). The mean age was found to be 35.1 yr ranging from 18-60 yr. Male (n=51) outnumbered the females (n=14). The CD4 counts were available only for 35 patients [CD4 count 200-499 cells/μl (n= 29) and CD4 count ≥500 cells /μl (n=6)] Examination of 65 stool samples from 331 patients by light microscopy with modified trichome stain showed microsporidial spores (oval shaped, measuring 1-2 μm in diameter under oil immersion) in four samples. Fluorescence microscopy using Uvitex 2B also showed microsporidial spores in the same four (6.2%) HIV-infected individuals. PCR detected microsporidial spores in stool samples of five (7.7%) individuals including four found positive by light and fluorescent microscopy [Figure 1]. Of these five samples positive by PCR, three were identified as Enterocytozoon bieneusi and two were identified as Encephalitozoon intestinalis. A repeat light microscopy and fluorescent microscopy on one sample positive only by PCR did not yield positive results. Thus, the results, though limited by a small number of positives, indicate that trichome and Uvitex 2B stains work well for diagnosis of intestinal microsporidiosis. PCR as expected was more sensitive and yielded one additional positive sample.
Figure 1: (a): Stool smear stained with modified trichome stain showing microsporidial spores (arrow); (b): Stool smear stained by Uvitex 2B & examined with UV light. spores of microsporidia show typical elongated shape (arrow); (c): Agaraose gel electrophoresis of PCR product agarose gel electrophoresis of polymerase chain reaction (PCR product. Lane1: Marker 100 bp, Lane 2: positive sample, Lane 3: negative control and Lane 4: positive control).

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In conclusion, with well trained laboratory staff both the Uvitex 2B and modified trichome stain can be used for microsporidia detection. PCR can increase sensitivity and species identification. The study was limited by small sample size and small number of positives and hence warrants additional testing on a large number of samples.


   Acknowledgment Top


Authors thank Naidu hospital staff for providing support for recruitment of patients and collection of samples. Authors also acknowledge the Director, NARI, Pune, for extending support for the study.

 
   References Top

1.Mohammed H, Endeshaw T, Kebede A, Defera M. Comparison of Chromotrope 2R and Uvitex 2B for the detection of intestinal microsporidial spores in stool specimens of HIV patients attending Nekempte Hospital, West Ethiopia. Ethiop Med J 2009; 47 : 233-7.  Back to cited text no. 1
    
2.Chabchoub N, Abdelmalek R, Mellouli F, Kanoun F, Thellier M, Bouratbine A, et al. Genetic identification of intestinal microsporidia species in immunocompromised patients in Tunisia. Am J Trop Med Hyg 2009; 80 : 24-7.  Back to cited text no. 2
    
3.Galván AL, Sánchez AM, Valentín MA, Henriques-Gil N, Izquierdo F, Fenoy S, et al. First cases of microsporidiosis in transplant recipients in Spain and review of the literature. J Clin Microbiol 2011; 49 : 1301-6.   Back to cited text no. 3
    
4.Talabani H, Sarfati C, Pillebout E, van Gool T, Derouin F, Menotti J. Disseminated infection with a new genovar of Encephalitozoon cuniculi in a renal transplant recipient. J Clin Microbiol 2010; 48 : 2651-3.  Back to cited text no. 4
    
5.Endeshaw TA, Kebede JJ, Verweij A, Zewide K, Tsige Y, Abraham D, et al. Intestinal microsporidiosis in diarrheal patients infected with human immunodeficiency virus-1 in Addis Ababa Ethiopia. Jpn J Infect Dis 2006; 59 : 306-10.  Back to cited text no. 5
    
6.Polley SD, Boadi S, Watson J, Curry A, Chiodini PL. Detection and species identification of microsporidial infections using SYBR Green real-time PCR. J Med Microbiol 2011; 60 : 459-66.   Back to cited text no. 6
    
7.Chen YP, Evans JD, Murphy C, Gutell R, Zuker M, Gundensen-Rindal D, et al. Morphological, molecular, and phylogenetic characterization of Nosema ceranae, a microsporidian parasite isolated from the European honey bee, Apis mellifera. J Eukaryot Microbiol 2009; 56 : 142-7.  Back to cited text no. 7
    
8.Satheeshkumar S, Ananthan S. Electron microscopy identification of microdporidia (Enterocytozoon bieneusi) and Cyclospora cayentanesis from stool samples of HIV infected patients. Indian J Med Microbiol 2004; 22 : 119-22.  Back to cited text no. 8
    
9.Anane S, Kaouech E, Belhadj S, Abdelmalek R, Ammari L, Ben Othman T, et al. Identification of Enterocytozoon bieneusi by PCR in stools of Tunisian immunocompromised patients. Pathol Biol (Paris) 2011; 59 : 234-9.  Back to cited text no. 9
    
10.Endeshaw T, Kebede A, Verweij JJ, Wolday D, Zewide A, Tsige K, et al. Detection of intestinal microsporidiosis in diarrhoeal patients infected with the human immunodeficiency virus (HIV-1) using PCR and Uvitex-2B stain. Ethiop Med J 2005; 43 : 97-101.   Back to cited text no. 10
    
11.Diagnostic procedures for stool specimens. Available from: http//www.dpd.cdc.gov, accessed on January 3, 2009.  Back to cited text no. 11
    
12.Sokolova OI, Demyanov AV, Bowers LC, Didier ES, Yakovlev AV, Skarlato SO, et al. Emerging microsporidian infections in Russian HIV-infected patients. J Clin Microbiol 2011; 49 : 2102-8.  Back to cited text no. 12
    
13.Espern A, Morio F, Miegeville M, Illa H, Abdoulaye M, Meyssonnier V, et al. Molecular study of microsporidiosis due to Enterocytozoon bieneusi and Encephalitozoon intestinalis among human immunodeficiency virus-infected patients from two geographical areas : Niamey, Niger and Hanoi, Vietnam. J Clin Microbiol 2007; 45 : 2999-3002.  Back to cited text no. 13
    
14.Lono A, Kumar S, Chye TT. Detection of microsporidia in local HIV-positive population in Malaysia. Trans R Soc Trop Med Hyg 2011; 105 : 409-13.  Back to cited text no. 14
    
15.Samie A, Obi CL, Tzipori S, Weiss LM, Guerrant RL. Microsporidiosis in south Africa: PCR detection in stool samples of HIV positive and HIV-negative individuals and school children in Vhembe district, Limpopo Province. Trans R Soc Trop Med Hyg 2007; 101 : 547-54.  Back to cited text no. 15
    
16.Raccurt CP, Fouché B, Agnamey P, Menotti J, Chouaki T, Totet A, et al. Presence of Enterocytozoon bieneusi associated with intestinal coccidia in patients with chronic diarrhea visiting an HIV center in Haiti. Am J Trop Med Hyg 2008; 79 : 579-80.  Back to cited text no. 16
    
17.Reddy AK, Balne PK, Gaje K, Garg P. PCR for the diagnosis and species identification of microsporidia in patients with keratitis. Clin Microbiol Infect 2011; 17 : 476-8.  Back to cited text no. 17
    
18.Dwivedi KK, Prasad G, Saini S, Mahajan S, Lal S, Baveja UK. Enteric opportunistic parasites among HIV infected individuals: associated risk factors and immune status. Jpn J Infect Dis 2007; 60 : 76-81.  Back to cited text no. 18
    
19.Najla C, Rim A, Fethi M, Fakher K, Marc T, Aida B, et al. Genetic identification of intestinal microsporidia species in immunocompromised patients in Tunisia. Am J Med Hyg 2009; 80 : 24-7.  Back to cited text no. 19
    


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