Microsporidiosis is an emerging infectious
disease caused by Microsporidia, an obligate intracellular spore forming eukaryote protist. Though, discovered about 150 years ago, the first well documented case of human microsporidiosis was reported in 1959.1 The disease remained uncommon till HIV pandemic. The first case of human microsporidial infection causing diarrhoea in HIV infected patient was reported in 1985.1 Thereafter, a number of microsporidial infections in HIV infected individuals have been reported indicating its importance as an opportunistic pathogen causing diarrhoea in patients with AIDS.1-3 It may also, cause diarrhoea in other immunocompromised hosts like transplant recipients. In immuno-competent hosts, it causes acute, self limiting diarrhoea.3 Majority of microsporidial infections in HIV/AIDS are caused by two genera, Enterocytozoon bieneusi and Encephalitozoon, of which 90% cases of human intestinal microsporidiosis are caused by Enterocytozoon bieneusi.3 Both opportunistic and potential parasites cause diarrhoea in immunocompromised individuals. Some of these parasites are amenable to specific treatment. By identifying the agent, it is possible to initiate specific treatment where required. Studying the prevalence of parasites in a specific population can help decide empiric therapy in that population.The frequency at which these parasites are associated with diarrhoea varies in the different studies reported.7-10
The present study was conducted in the Department of Microbiology at Seth G.S. Medical College, Parel, Mumbai with an aim to find the incidence and frequency of parasitic agents in HIV positive individuals presenting with diarrhoea with special reference to the detection of Microsporidia in stool samples and to determine the association, if any between CD4+T cell counts and the presence of parasites.
Material and Methods
64 HIV seropositive adults(males and females) complaining of diarrhoea attending the virology out patient department at Seth G.S.Medical College and K.E.M.Hospital, between October, 2004 to September, 2005 were included. Patients who had received antibiotics, antimalarials, antidiarrhoeals or iron preparations within the previous week were excluded from the present study. CD4+ and CD8+ T cell counts of these patients were recorded. A total of 100 stool samples were collected from these patients, which were processed as follows.11
All stool samples were processed directly and after concentration using Formalin Ethyl Acetate sedimentation technique. For the direct process, the uniform stool suspension was used and for the concentrated process, the sediment was analysed. For each process, saline and iodine wet mounts were prepared and observed for microscopic features. Additionally, for each process, two smears were made and stained by the modified trichrome stain and modified Ziehl Neelsen stain for the detection of Microsporidia and other coccidian parasites respectively. The findings of all the techniques performed were recorded and compared.
In the present study, parasites were detected in 35 out of 64 patients included (54.7%). As shown in Table 1, Isospora was the commonest parasite found in 12 patients (18.75%). Microsporidial spores were detected in 11(17.18%) and Cryptosporidium were seen in 2 (3.12%)
The results of the FEA sedimentation technique employed for concentration (Table 1) revealed that Microsporidia and Isospora, its sensitivity was poorer compared to direct examination. Only 5 of 11 Microsporidia and 4 of 12 Isospora could be detected by this concentration method
A comparison of CD4+T cell counts with presence of parasites revealed (Table 2) that the maximum number of parasites(66.6%) were seen in patients with CD4+T cell counts between 50 and 200. 9 of 12 Isospora, 5 of 11 Microsporidia, 1 of 2 Cryptosporidia were seen in patients with CD4+T cell counts between 50 and 200. When the parasite yield was compared with the consistency of stool samples (Table 3), the maximum yield of parasites was noted in watery stools (66.7%), 9 of 12 Isospora (75%), 6 of 11 (54.5%) Microsporidial spores and both Cryptosporidium oocysts were seen in watery stools.
|Fig. 1 : Oocyst of isospora belli as seen in oil immersion (modified zn staining).
||Fig. 2 Oocyst of cryptosporidium in modified ziehl neelsen stain.
||Fig. 3 : Microsporidial spores as seen in oil immersion (Modified trichrome) (x 1000)
Intestinal infections are a major cause of morbidity and mortality in patients infected with HIV. The present study was carried out to find the incidence of parasitic agents causing diarrhoea in HIV infected individuals.
In the present study, parasites could be detected in the stool samples of 35 out of 64 patients.
Isospora was the commonest parasite found in 12 patients (18.75%).This is comparable with the findings of other workers.8,14 This is higher than the incidence reported in western literature probably because of the widespread use of cotrimoxazole for the prophylaxis of Pneumocystis carinii pneumonia in their patients and it is also effective against Isospora.15
Microsporidia accounted for 17.18%. This is probably the first time in Mumbai that Microsporidia has been detected in a significant proportion of HIV positive patients with diarrhoea, thus highlighting their importance as an emerging pathogen in HIV/AIDS. Kairon et al from Pune10 have reported a prevalence of Microsporidia of 12.9% which is comparable to the findings of our study. The prevalence of microsporidiosis among HIV infected individuals with diarrhoea ranges from 2-50% and this variation may be due to the difference in the distribution of the parasite in each geographical area and the difference in the diagnostic method used for their detection.1 Thus, the results from the present study and that from the neighbouring city, Pune indicate that Microsporidia are emerging as a significant pathogen in HIV seropositive individuals suffering from diarrhoea in this area.
Alternative staining methods are used for the demonstration of opportunistic protozoan parasites in stool. Of them, the most common is the modified Ziehl Neelsen staining technique which does not stain Microsporidia. Hence, unless special staining technique like modified trichrome, acid fast trichrome or the use of optical brighteners with fluorescence microscopy are employed, the parasite may go undetected.2,11,12,13 In the past, diagnosis of microsporidiosis depended upon Transmission Electron Microscopy, which is time consuming, expensive, requires much expertise and is an invasive technique involving small intestinal biopsy. In the present study, we have used the Weber green modified trichrome technique to stain Microsporidia.11 Positive control slides were provided by NICED, Kolkata.
The distribution of parasites in our patients showed some interesting findings. Cryptosporidiosis which is reported as a predominant pathogen in other Indian studies,8,9,16 accounted for only 3.12% of the total parasites detected. A similar low prevalence has been reported by Pune workers.10
In the present study, similarly, Cyclospora was not detected from any stool samples though other studies from India have reported a low prevalence of cyclosporidiasis.8-10
Parasite yield can be significantly improved by employing specific concentration methods on stool samples.Concentration by FEA sedimentation is a recommended technique for improving yield of parasites. We found it useful for the recovery of oocysts of Cryptosporidium. However, the technique distorted the Isospora oocysts and altered their staining properties as well. It also led to substantial loss of microsporidial spores. Based on these findings, FEA sedimentation can not be recommended as a preferred concentration technique.
In the present study, we found that watery stools yielded the maximum number of parasites (66.6%). These findings are consistent with well established studies which state that more number of oocysts of these parasites are shed and consequently detected as the stool frequency becomes more liquid. Of the 11 Microsporidia detected, 6 (54.55%) were detected in watery and liquid stool specimens. However, an almost equal number (5 of 11) were found in semiformed faeces. The present study has been conducted in symptomatic patients with diarrhoea, however asymptomatic carriage of microsporidial spores has been reported.23
When the parasite yield was correlated with CD4+T cell counts, it was seen that maximum parasite yield was in those patients with CD4+T cell counts between 50 and 200 (66.6%). It was also interesting to note that parasite yield was lower (30.8%) in patients with CD4+T cell counts less than 50. This is probably because infection due to CMV and HIV enteropathy is a more common cause of diarrhoea with severe reduction in CD4+T cell counts.1
Thus, Microsporidia are emerging as significant parasites in HIV infected patients with diarrhoea in Mumbai. Concentration of stool samples by Formalin Ethyl Acetate sedimentation technique has been found to be unsatisfactory for Microsporidia.
- Weiss CM. Microsporidiosis, In Mandell GL, Bennett JE, Dolin R. (ed) Principles and Practice of Infectious Diseases, 6th edition, Elsevier, Philadelphia, PA. 2005; p 3237-54.
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- Sobottka I, Schmetz C, Schottelius J. Microsporidia. In Dionisio D (ed). Textbook-Atlas of Intestinal Infections in AIDS. Springer-Verlag Italia, Milano. 2003: p 305-319.
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- Satheesh KS, Ananthan S, Lakshmi P. Intestinal parasitic infections in HIV-infected patients with diarrhoea in Chennai. Indian J Med Microbiol 2002; 20 (2) : 88-91.
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- Kairon R, et al. Study on opportunistic enteric parasites in HIV-seropositive adult patients hospitalized for diarrhoea. National AIDS Research Institute. Annual Report 2003; 2003-4.
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- Fisk TL, Keystone JS, Kozarsky P. Cyclospora cayetanensis, Isospora belli, Sarcocystis species, Balantidium coli and Blastocystis hominis, In Mandell GL, Bennett JE, Dolin R (ed) Principles and Practice of Infectious Diseases, 6th edition, Elsevier, Philadelphia, PA. 2005: p 3228-34.
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- Germani Y, Minssart P, Vohito M, et al. Etiologies of acute, persistent and dysenteric diarrhoeas in adults in Bangui, Central African Republic, in relation to human immunodeficiency virus serostatus. Am J Trop Med Hyg 1998; 59 (6) : 1008-14.
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- Rabeneck L, Gyorkey F, Genta RM, et al. The role of microsporidia in the pathogenesis of HIV-related chronic diarrhoea. Annals Int Med 1993; 119 : 895-99.