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 ~  Abstract
 ~  Materials and Me...
 ~  Results
 ~  Discussion
 ~  Acknowledgement
 ~  References

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Year : 2004  |  Volume : 22  |  Issue : 2  |  Page : 119-122
 

Electron microscopy identification of microsporidia (enterocytozoon bieneusi) and cyclospora cayetanensis from stool samples of HIV infected patients


Dept. of Microbiology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai - 600 113, Tamil Nadu, India

Correspondence Address:
Dept. of Microbiology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai - 600 113, Tamil Nadu, India

 ~ Abstract 

Microsporidia (Enterocytozoon bieneusi) and Cyclospora cayetanensis have been reported worldwide causing diarrhoea in AIDS patients. Stool samples from HIV infected patients were subjected to routine examination for parasites, followed by special staining techniques to detect microsporidia and Cyclospora cayetanensis. Confirmed positive cases of these parasites were further processed for electron microscopy identity of the parasites and characteristic details. Scanning and transmission electron microscopy showed better morphological and structural details of the parasites.

How to cite this article:
Satheeshkumar S, Ananthan S. Electron microscopy identification of microsporidia (enterocytozoon bieneusi) and cyclospora cayetanensis from stool samples of HIV infected patients. Indian J Med Microbiol 2004;22:119-22


How to cite this URL:
Satheeshkumar S, Ananthan S. Electron microscopy identification of microsporidia (enterocytozoon bieneusi) and cyclospora cayetanensis from stool samples of HIV infected patients. Indian J Med Microbiol [serial online] 2004 [cited 2019 Nov 17];22:119-22. Available from: http://www.ijmm.org/text.asp?2004/22/2/119/8085


Numerous gastrointestinal pathogens have emerged in recent years including two important types of protozoa: Microsporidia and Cyclospora cayetanensis.[1] Microsporidia are obligate, intracellular spore forming parasites that are emerging as opportunistic pathogens in the setting of AIDS. Microsporidia are one of the leading causes of diarrhoea in patients infected with HIV.[2],[3] In the severely immunocompromised patients almost all cases of intestinal microsporidiosis result from infection with Enterocytozoon bieneusi and a much smaller number are caused by Encephalitozoon (Septata) intestinalis.[4]
The parasite Cyclospora is widely distributed throughout the world and infection with it is associated with prolonged watery diarrhoea. Cyclospora was first noted in the intestine of moles in 1881 and was identified as a cause of human infection in 1977 in Papua New Guinea. Before 1996 only three outbreaks of cyclosporiasis had been reported in the Unites States.[5] Sporadic cases were limited to international travellers and usually present with watery diarrhoea in immunocompromised patients with duration of four months.[6]
In recent years, many techniques have been evolved to diagnose intestinal parasitic infection but still electron microscopy (SEM and TEM) remains a gold standard in terms of detection, speciation, morphological and structural details of the organisms. In view of the above, the present study was undertaken to identify Microsporidia (Enterocytozoon bieneusi) and Cyclospora cayetanensis by scanning and transmission electron microscopy from confirmed positive stool samples obtained from HIV infected patients.

 ~ Materials and Methods Top

Stool specimens obtained from HIV infected patients were routinely processed for the detection of enteric parasites and bacterial pathogens. Parasitic investigations included direct microscopic examination of saline and wet mounts of fresh faeces and further concentrated by formol-ether sedimentation method.[7] Direct smears of stool samples and smears made from the deposits of sedimentation were stained with modified trichrome stain for the detection of microsporidial spores.[8] For the detection of Cyclospora cayetanensis, modified Kinyoun acid fast stain was used.[9]
Stool smears of Microsporidia (Enterocytozoon bieneusi) and Cyclospora cayetanensis positive cases were sent to division of parasitic diseases, CDC, Atlanta to confirm the diagnosis. Confirmed positive cases of Microsporidia and Cyclospora cayetanensis were subjected to scanning and transmission electron microscopy.
Scanning Electron Microscopy[10]
Stool samples were fixed in 5% (v/v) gluteraldehyde in 0.1M phosphate buffer pH 7.3 for 24 hours. Samples were washed in phosphate buffer prior to post fixation in 1% (w/v) osmium tetraoxide in 0.1M-phosphate buffer for 24 hours followed by washing in the same buffer, then dehydrated using an ethanol series (10-100% v/v) and critical point dried. The dried samples were mounted on specimen stubs using scotch 3M double side adhesive tape and coated with gold to a thickness of 100 A0. Coated samples were viewed in a Hitachi scanning microscope at 20 KV and photographed.
Transmission Electron Microscopy[11]
Negative staining was performed due to low quantity of parasites in the stool samples. Formvar layer was made using 1-2mL of chloroform and 0.2% formvar. After layer was formed, copper grids (300m mesh, sigma) were subjected to coating by placing over the layer. After few minutes, coated grids were kept for drying to two hours and positive samples (10L) were placed over the coated grid for 10-20 minutes. Samples loaded grids were stained with 2% uranyl acetate for 15 seconds and kept for drying. Samples were screened in Philips EM. Appropriate pictures were taken at 60 KV acceleration voltages.

 ~ Results Top

Under Scanning electron microscopy (SEM), revealed an ovoid shaped Microsporidia (Enterocytozoon bieneusi) spore measuring approximately 1.5 by 0.9m [Figure - 1], bar 3m) Anterior side of the spore showed a small indentation and other structural details were limited. Cyclospora cayetanensis revealed as spherical ball approximately measuring 8-10m [Figure - 2], bar 1m.
Under transmission electron microscopy (TEM), Microsporidia (Enterocytozoon bieneusi) showed a diagnostic feature of electron dense outer layer of the wall (exospores), which is known to be chitin rich in nature, and the electron lucent inner layer (endospore). The nucleus was not recognized. Since negative staining was performed the characteristic polar tubules of microsporidia were not visible [Figure - 3].
Cyclospora cayetanensis revealed a roughly spherical organism 6-8m in diameter. The organism comprised of an outer fibrillar coat, a thinner cell wall and a cell membrane enclosing the cytoplasm. The nuclei, endoplasmic reticulum or mitochondria were not identified [Figure:4].

 ~ Discussion Top

The present study was planned to highlight the importance of electron microscopy identification and documentation of Microsporidia and Cyclospora cayetanensis from stool samples. These coccidian parasites are increasingly recognized as a group of emerging protozoal agents that infect immunocompromised patients with AIDS.[2]
Increased incidence and various reports of these parasites warrant further characterization of the parasites. Scanning and transmission electron microscopy were performed to identify and observe the morphological and structural details of the parasites. Our finding in the study related to morphology and other structural details were found to be similar as reported from different parts of the world.[12],[13] The present study, to the best of our knowledge, is the first documentation of electron microscopic details of Microsporidia (Enterocytozoon bieneusi) and Cyclospora cayetanensis from HIV infected patients in Chennai.
Although TEM and SEM remain as gold standards for the diagnosis of this organism, its value as a confirmatory tool or stand - alone test is questionable in cases of very low number of spores and oocysts from specimens of HIV negative immunocompromised patients. HIV positive patients infected with Microsporidia or Cyclospora may benefit from treatment with albendazole or trimethoprim methoxazole respectively, which may result in a shortened duration of symptoms and the prevention and delay of microsporidian or cyclosporian dissemination.

 ~ Acknowledgement Top

The authors gratefully acknowledge the help of Dr.Stephanie P.Johnston, division of parasitic diseases, CDC, Atlanta for confirming the diagnosis. We are thankful to Dr. Andrew Chandramohan, Central Instrumentation Laboratory, Madras Veterinary College, Chennai and to Dr. Kathiravan, Centre for advanced studies, School of Life sciences, University of Madras, Chennai, for technical help and processing of the specimens to view under TEM and SEM respectively. 

 ~ References Top

1.Cegieslki JP, Ortega YR, Mc Kee S, Madden JF, Gaido L, Schwartz DA, et al. Cryptosporidium, Enterocytozoon and Cyclospora infection to pediatric and adult patients with diarrhoea in Tanzania. Clin Infect Dis 1994;28:314-321.  Back to cited text no. 1    
2.Weber R, Bryan RT, Schwartz DA, Owen RL. Human microsporidial infections. Clin Microbiol Rev 1994;7:426-461.  Back to cited text no. 2    
3.Schwartz DA, Sobottka I, Leitch GJ, Cali A, Visvesvara GS. Pathology of microsporidiosis, emerging parasitic infections in patients with acquired immunodeficiency syndrome. Arch Pathol Lab Med 1996;120:173-188.  Back to cited text no. 3    
4.Franzen C, Schwartz DA, Visveswara GS, Muller A, Schwenk A, Salzberger B. Immunologically confirmed disseminated asymptomatic Encephalitozoon cuniculi infection of the gastrointestinal tract in a patient with AIDS. Clin Infect Dis 1995;21:1480-1484.  Back to cited text no. 4    
5.Brow GH, Rotschafer JC. Cyclospora: Review of an emerging parasite. Pharmocotherapy 1999; 19(1): 70 - 75.  Back to cited text no. 5    
6.Satheeshkumar S, Ananthan S, Lakshmi P. Intestinal infection in HIV infected patients with diarrhoea in Chennai. Ind J Med Micro 2002:20(2):88-91.  Back to cited text no. 6    
7.Ash LR, Orihel TC: Parasites: A guide to laboratory procedures and identification - 1st Edition : (American Society of Clinical pathologist, Chicago) 1987:5-53.  Back to cited text no. 7    
8.Weber R, Bryan RT, Owen RL, Wilcox CM, Gorelkin L, Visvesvara GS. Improved light microscopical detection of microsporidia spores in stool and duodenal aspirates. The New Eng J Med 1992;326:161-166.  Back to cited text no. 8    
9.Ma P, Soave R. Three step stool examination for cryptosporidiosis in 10 homosexual men with protracted watery diarrhoea. J Infect Dis 1983;147:824-828.  Back to cited text no. 9    
10.Milloing G. Advantages of phosphate buffer for osmium tetraoxide solution in fixation. J Appl Phys 1961;32:1637.  Back to cited text no. 10    
11.Dennehy PH, Gaunlett BR, Spangenber SE. Choice of reference assay for the detection in fecal specimens, electron microscopy versus enzyme immunoassay. J Clin Micro 1990;28:1280-1283.  Back to cited text no. 11    
12.Ortega YR, Sterling CR, Gilman RH, Cama VA, Diaz F. Cyclospora species - A new protozoan pathogen of humans. The New Eng J Med 1993;6:1308-1312.  Back to cited text no. 12    
13.Gool VT, Hollister WS, Schattenkerk JE, et al. Diagnosis of Enterocytozoon bieneusi microsporidiosis in AIDS patients by recovery of spores from faeces Lancet 1990;336:697-668.  Back to cited text no. 13    
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2004 - Indian Journal of Medical Microbiology
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