Abstract
Once thought to be a rare entity, Cyclospora cayetanensis is now an established cause of chronic diarrhoea in immunosuppressed individuals. This paper reports two cases of cyclosporiais in HIV infected individuals seeking medical attention in K.E.M. Hospital, Parel, Mumbai.

Introduction

Opportunistic parasitic infections have played a major role in the clinical manifestation of AIDS. It has been observed in the last few years in developed countries that there is a remarkable decrease in morbidity and mortality related to AIDS. This is due to the introduction of potent antiretroviral treatment, However, in the developing world, intestinal parasitosis still remains a potential threat to patients and diarrhoea remains an emerging symptom in HIV infected individuals
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Cyclospora cayetanensis is one of these opportunistic parasites. It is a coccidian parasite and the only species known to infect man and has been the cause of cyclosporiasis leading to prolonged, severe and recurrent diarrhoea in HIV infected and other immunosuppressed individuals.¹

Cyclospora cayetanensis was first described in humans in Papua New Guinea in 1977.¹ It eluded taxonomic classification for a long time. In 1993, Ortega⁷ confirmed its genus as Cyclospora by successfully inducing sporulation. It was then named Cyclospora cayetanensis after the Universidad Peruana Cayetano Heredia in Lima, Peru, which was a major site of research on this infection. The prevalence of cyclosporiasis varies with the geographical location. A large study by Pape et al from Haiti² has reported a prevalence of cyclosporiasis of 11% in HIV positive individuals. However, Indian studies from Chennai by Satheesh et al,³ from Chandigarh by Mohandas et al4 and from Pune Kairon et al⁵ have reported a prevalence of cyclocporiasis to be 1%, 1.6% and 3.33% respectively.

We present two cases of cyclosporiasis in HIV infected individuals with chronic diarrhea who sought medical advice at K.E.M. Hospital, Parel, Mumbai.

Case 1
A 35 year old HIV infected male presented with watery diarrhoea of two months duration (6-8 passages/day). He had no other major complaints.His CD4+T cell count was 103 cells/µl .His systemic examination was found to be normal.

Findings
Motile trophozoites of Giardia lamblia and oocysts of Cyclospora were seen in saline mount. The oocysts appeared as non-refractile structures of about 8-10 µm with inclusions. Wet mounts of concentrated deposits did not show the presence of oocysts of Cyclospora. The modified ZN stained smears made of direct as well as deposit after concentration did not reveal any Cyclospora.

This patient was treated with Metronidazole and trimethoprim-suphamethoxazole combination.

Fig. 1 : Photomicrograph 1 showing acid-fast, bright pink oocysts of cyclospona. (Modified ZN stain).	Fig. 2 : Photomicrograph 2 showing ill-defined internal structure. (Modified ZN stain)	Fig. 3 : Photomicrograph 3 showing glassy, wrinkled appearance of cyclospora oocysts. (Modified ZN strain).

Case 2
A 35 year old HIV infected male presented with watery diarrhoea with severe abdominal colicky pain and low grade fever for three days (10-12 passages/day) and was admitted to our hospital as a case of acute gastroenteritis. His CD4+T cell count was 80 cells/µl. His systemic examination was found to be normal.

Findings
Oocysts of Cyclospora were seen in saline mount. The appearance of oocysts was same as that seen in case 1. Wet mounts of concentrated deposits did not show the presence of oocysts of Cyclospora. The modified ZN stained smears made of direct stool sample revealed acid-fast, bright pink, about 8-10 µm spherical oocysts of cyclospora. (as shown in photomicrograph 1) They also showed ill-defined internal structures (as shown in the photomicrograph 2) Whereas some had glassy, wrinkled appearance (as shown in the photomicrograph 3) The smear of the deposit after concentration did not reveal any Cyclospora.

Microsporidial spores were not seen on modified trichrome stain in smears prepared from both the cases.

This patient was treated with trimethoprim-sulphamethoxazole combination.

Both these patients were not on any anti-retroviral therapy or any other anti-parasitic agents.

Stool samples were examined for the detection of opportunistic parasites.⁶

Gross examination of the stool samples was done, which showed watery consistency and did not reveal any visible blood, mucus or adult parasites. Microscopic examination using saline and iodine was performed. Two smears were prepared directly from each sample for staining with a) Modified Ziehl Neelsen stain and b) Modified trichrome stain. Formalin Ethyl Acetate sedimentation technique was also performed for concentrating parasites. A wet mount and two smears were also made from the sediment. The smears were stained by the same stains as above.⁶

In both these cases, second sample of stool could not be obtained to check parasitological cure due to patient's non-compliance. (Case 2 who was admitted took discharge against medical advise on the same day). Stool of both these patients also could not be processed for bacteriological culture for the same reason.

It is not possible to differentiate between cyclosporiasis and cryptosporidiosis clinically as the clinical presentation of both these diseases is similar. Both these present with protracted, debilitating diarrhea and are AIDS defining illnesses. Also, it is very commonly seen that very few oocysts are shed in stool. Hence, oocysts could be missed if the load is low.

However, the treatment for both these is different. Cyclosporiasis is easily treated with oral trimethoprim-sulphamethoxazole combination, making early diagnosis of cyclosporiasis essential.

Laboratory diagnosis of cyclosporiasis is based on the demonstration of oocysts of cyclospora in faeces by wet mount examination and modified Ziehl Neelsen stain. The oocysts of Cyclospora may show variable staining characteristics, which was also seen in the present study. All may not appear as acid fast structures. However, if they pick up the modified Ziehl Neelsen stain, they appear as bright pink, acid fast, 8-10 µm in diameter. Some oocysts may take on a glassy wrinkled appearance. It may also have ill-defined internal structure that take on a dark filamentous stain.

The parasite can also be demonstrated by safranine staining and microwave heating, a technique requiring standardization. Ultraviolet epifluorescence microscopy lacking specificity or PCR has not been yet been evaluated in diagnostic clinical settings.¹

Though Formalin Ethyl Acetate sedimentation is a recommended technique for optimal detection of parasites, it could not detect oocysts of cyclospora in the present study. However, it is difficult to comment on the usefulness of this procedure since only two stool samples yielded Cyclospora.

Hence, for optimal diagnosis of cyclosporiasis, it is essential to perform a complete stool examination, including a wet mount of direct and concentrated stool specimen using the sediment and staining of smears by modified ZN technique in all clinical laboratories.

References

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