MICROSPORIDIOSIS IN MALIGNANCY AFFECTED PATIENTS IN MOSUL , IRAQ

Over nine months period, the incidence of microsporidiosis in patients receiving treatment for malignancy was investigated. A total of 53 patients (27 males and 26 females) were involved in this study. All patients had stool analysis for microsporidium spp. stools from 53 control groups (30 males and 32 females) have also been investigated. Microsporidium spp. spores were detected in stools of 5 males and 3 females among patients treated with anti-cancer therapy. Three patients particularly affected with leukemia showed positive results. It was concluded that advanced stages of malignancy affected patients who received anti-cancer therapy are at risk from infection with this pathogen, while working physicians showed little awareness concerning this opportunistic infection.


INTRODUCTION
he immunocompromised host has one or more defects in the normal defense mechanisms that protect against infectious agents.These defects predispose the individual to an increased risk of severe lifethreatening infections. [1]Protozoa have come to be the predominant parasitic infection in immunocompnomised patients. [2]Among these parasitic infections, microsporidiosis caused by Enterocytozoon bieneusi is now constituted a major disease problem in the seriously immunocompromised population and HIVinfected patients. [3]Human microsporidial infections have been documented globally.The sources of microsporidia infecting humans and their modes of transmission are uncertain.Ingestion of spores is the most probable source of transmission. [4]Although microsporidiosis appears to occur most frequently in persons infected with HIV, it is emerging as an infection in otherwise immunocompromised hosts as well as immunocompetent individuals. [5,6]Patients with severe cellular immunodeficiency appear to be at the highest risk for developing microsporidial disease. [4]he microsporidium E. bieneusi has been associated with diarrhea in recipients of organ transplants and with self-limited watery diarrhea in immuno-competent adults as well as in children. [7]It has also been reported in up to 33% of HIV infected patients with unexplained chronic diarrhea. [8]iagnosis of microsporidiosis was made by light microscopy [9,10] of stool samples, while definitive species identification of microsporidia is made by electron microscopy, antigenic analysis or molecular analysis. [4,9,11]icrosporidiosis may spread from the gut to the upper respiratory tract and kidneys, but it usually responds well to treatment with albendezole. [12]It can also cause a wide range of illness from diarrhea to involvement of CNS,eyes, viscera and muscles. [13]As far as we know, this is the first study of microsporidiosis in Mosul-Iraq, conducted among malignancy affected patients who were regarded as immuno-suppressed patients due to disease pathogenicity or as a consequence of their anticancer regimens like radiation or chemotherapy.

Subjects:
This study was conducted in Mosul city (IRAQ), over a period of nine months from August 2001 to April 2002 in Ibn-Sina teaching hospital, Al-Zahrawi teaching hospital, Hazem Al-Hafeth hospital for nuclear medicine and Al-Razi general hospital.Fifty three malignancy affected patients (27 males and 26 females) were studied.These subjects who were presumably immunocopromised patients received cytotoxic drugs and/or radiation therapy.Their age varied from 6 to > 55 years.The control group was composed of 53 (apparently healthy) individuals (30 males and 23 females), their age range from 5 to > 57 years.None had any past or present history suggesting malignant diseases, nor had they received any type of anti-cancer therapy.They were either patient's relatives or subjects who were randomly selected.

Specimens:
Stool samples were collected from both patients and controls.The preservatives used for stool samples were sodium acetate, acetic acid formaldehyde and potassium dicromate (K 2 CrO 4 ).Two stool samples were obtained from each subject.The first sample was obtained before receiving the anti-cancer therapy, and the second sample obtained after the patient had received the therapy.Only one stool sample was obtained from each individual of control group.Fecal samples were examined microscopically (wet mounts) and macroscopically by the direct method using normal saline and lugol's iodine.Zinc sulfate flotation technique was also used as a concentration method for processing the stool samples.The Weber technique (Trichrome stain) modified by Deluol and Mahoun for microsporidia [6,14] was used to detect microsporidic spores, which appeared small, oval shaped, stained pink in colour with distinct colourless vacuoles of varying coloured spores appeared very well against the Malachite green background.

RESULTS
The types of malignant diseases among tested patients (immunocopromised) are presented in (Table -1).The results showed that breast carcinoma, leukemia and lymphoma were the most prevalent malignant diseases among our patients (35.85%, 26.42% and 13.21% respectively).The clinical symptoms among tested patients before treatment and after treatment showed that all patients suffered from weight loss, while fever and diarrhea were noticed in 79.61% and 45.28% of treated patients respectively.According to the type of anti-cancer regimen, the spores of microsporidia spp.were detected in stools of 4 patients who had received both type of anti-cancer therapy (cytotoxic and radiation), and in 3 patients who received cytotoxic drugs only.Leukemia affected patients showed the highest rate of infection with microsporidia where 3 patients who had suffered from this type of malignancy were found positive from the total number.Among treated patients, those with advanced stages of malignancy were found with a high percent of infection with microsporidia (9.43%) than those with early stage of malignancy (5.67%).This pattern was also true among untreated patients.

DISCUSSION
In the present study Microsporidia spp. was found among patients who received anti-cancer therapy.It has been reported that fifty eight percent of patients receiving anti-cancer therapy (cytotoxic drugs) were found to have enteric parasites with 8% of Cryptosporidium infection. [15]Both cytotoxic drugs and therapeutic ionizing radiation are considered the chief immunosuppressive agents used by the malignant patients. [16]Parasites were also detected among 7.6% of 105 immunocompromised patients.The immunosuppression was due to cytotoxic drugs, leukemia and malnutrition. [17]In our study the light microscopic diagnosis of faeces from cancer patients for microsporidiosis was determined.Light microscopy (LM) has been used successfully for diagnosis of biopsies from AIDS patients. [18]Those authors concluded that it should be possible to render the diagnosis of intestinal microsporidiosis by (LM) in most cases, while Transmission Electron Microscopy may be needed for minority of cases with low parasite burden.Stool samples were also analyzed by both (LM) and (PCR) techniques, to detect microsporidiosis in patients with AIDS. [19]owever, new simple staining method using modified trichrome stain [6] for faeces and other body fluids have facilitated clinical diagnosis as well as drug evaluation and epidemiological studies.The isolation of microsporidial spores from body fluids, including stools, urine, respiratory secretions and duodenal aspirates, [20] suggesting that these may serve as potentially infective sources for person to person transmission.A study of potentially human pathogenic protozoan parasites in water contaminated with human and animal faeces, showed that 23% of the samples were positive for human pathogenic microsporidia species. [21]icrosporidia are present worldwide, but accurate assessment of their prevalence in general population has not been established. [22]o the best of our knowledge among data available from literatures in Iraq.This is the first report which shows malignancy, affected patients at risk of acquisition of microsporidal infection.Although microsporidium species were initially believed to be a cause of AIDS related diarrhoea, they were subsequently shown to be pathogenic in the conjunctiva, liver, peritoneal cavity and lungs. [23]Our patients suffered from various clinical symptoms including weight loss and diarrhea.The result showed that persistent diarrhea was an important problem in our patients.Some experienced a significantly higher diarrhoeal burden during the course of anti-cancer therapy.A clear association between the presence of microsporidia and diarrhea was established by Coyle et al., [7] who found that 44% of HIV infected patients with diarrhea were infected with microsporidia whereas only 2.3% of the patients without diarrhea were infected.Another study of 73 HIV-infected patients with microsporidiosis indicated that 55% of patients had persistent diarrhea after 6 months and 51% showed weight loss. [21]icrosporidiosis was also found to be a common cause of chronic diarrhea, malabsorption and weight loss in AIDs patients and in cases unexplained diarrhea. [8,18,22]The epidemiology of human microsporidiosis is poorly understood and environmental factors affecting transmission of the organism have not been fully elucidated, which need further investigation.

Table 3 . Types of anti-cancer therapy among tested patients.
43%).No microsporidia spores have been observed in stools of control group.