The cross-sectional study was carried out in Department of Microbiology at a tertiary care hospital. The research study was approved by Sumandeep Vidyapeeth Institutional Ethics Committee. [SVIEC/ON/PhD/17018]. Patients on immunosuppressant; fulfilling inclusion criteria, during three months between June 2019 to September 2019 were studied. Patients were informed and appropriate consent was obtained before being recruited for the study.

Those patients who visited the hospital with illness like HIV, diabetes mellitus, tuberculosis, liver cirrhosis as well as positive Hepatitis B, taking steroid, on cytotoxic chemotherapy and immunosuppressant drugs etc., were considered as immunosuppressive patients and were included in the study. Inappropriate quality of stool sample was the exclusion criteria.

Total 120 immunosuppressive patients were included in this study. Each stool sample was collected in a clean, dry, leak proof, well-labeled wide mouth stool container with name and identification number and processed in the Parasitology section of the Microbiology department. Necessary data of patients were obtained from the Medical Record Department (MRD).

Macroscopic examination: Macroscopic analysis was done in terms of color and consistency and presence of adult worms as well as segments of tapeworm and larvae.

Microscopic examination: After macroscopic examination all samples were examined by direct microscopic method. Wet mount of the samples were performed as saline and iodine mounts to identify trophozoite and cysts of protozoan parasites as well as ova and larva. For the detection of intestinal coccidians Cryptosporidium parvum, Cyclospora and Isospora belli Modified Ziehl-Neelsen (MZN) stain was performed. Formal ether concentration technique was also done to detect eggs and larva of intestinal parasites [15].

For the statistical analysis, Graph Pad Prism version 8 was used. Chi-square (χ²) test was performed and a p-value ≤0.05 was considered as statistically significant.

Out of total 120 patients, 20 (16.7%) were found to be infected with parasite. Among all 120 immunosuppressive patients, 80 (66.7%) were males and 40 (33.3%) were females. Among the male population, 11 (9.2%) and among females, 9 (7.5%) were found to be positive for the infection. The age range of the population was 21-60 years, the mean age being 43.1±6.5 years. The age group of 41-50 years showed maximum parasitic infections. When two groups having highest (7 out of 46; 5.8% in age group of 41-50) and lowest prevalence (3 out of 27, 2.5% in age group 31-40) of parasitic infestation were compared; there was no statistical significant difference (p-value=0.73). No statistical significant difference was observed for socio-demographic variables like gender, age, education, employment, hygiene methods, (washing of hands before taking meals and after defecation), as well as with level of sanitation in form of defecation in open or having facility of toilet [Table/Fig-1].

Prevalence of parasitic infection was found to be high in HIV (24%), diabetes (14.75%), tuberculosis (17.65%) and patients having Hepatitis B infection (18.18%) while no parasitic infection was present in miscellaneous group [Table/Fig-2].

As shown in [Table/Fig-3], among the total 20 positive samples; 15 samples (75%) showed the presence of Protozoa in which 10 samples (50%) showed presence of Entamoeba histolytica [Table/Fig-4], followed by Cryptosporidium parvum [Table/Fig-5] in four samples (20%) and Giardia lamblia in one sample (5%). The presence of Helminths was found in five samples (25%), of which three samples (15%) showed the presence of Ascaris lumbricoides [Table/Fig-6] and Taenia species eggs [Table/Fig-7] were found in two samples (10%) [Table/Fig-3]. Prevalence ratio of protozoan to helminths parasites was 3:1.

[Table/Fig-4]:

Stool microscopy showing cyst of Entamoeba histolytica in saline wet mount (40X).

[Table/Fig-5]:

Modified ZN stain showing Cryptosporidium parvum in stool sample under oil immersion field (100X power).

[Table/Fig-6]:

Stool microscopy showing ova of Ascaris lumbricoides in saline wet mount (40X).

[Table/Fig-7]:

Stool microscopy showing Taenia species ovum in saline wet mount (40X).

In this study the overall prevalence of parasitic infection in immunosuppressive patients was 16.7%. This study was primarily done to find out the risk factors involved in developing parasitic infection in immunosuppressive patients. Study done by Hailegebriel T showed that various socio-demographic factors like size of the family, hygienic practices like hand washing, nail cutting and wearing shoes had relation with intestinal parasitic infection [16]. In immunosuppressed patients, if same holds true, then there will be the need for additional interventional strategy. Moudgil V et al., in their study found co-prevalence of diabetes with parasitic infection of 8.6% [17]. In a study by Sabah AA and Temsah AG they showed parasitic infection amongst diabetic patients had predominance of E. histolytica, however their was no significant difference between patients having diabetes and controls [18]. They assumed that E. histolytica can be a factor for poor sanitation and environment pollution. Predominance of E. histolytica, was found in present study also however no significant relation was found with socio-demographic risk factors. It can therefore be presumed that immunosuppression was responsible for parasitic infections than socio-demographic factors; however a larger population based study may be needed for significant conclusion. Immunosuppression due to HIV may also have opportunistic infection by enteric parasite. In this study of 120 patients having immunosuppression, 24 were HIV positive of which 6 had parasitic infection (25%). Swathirajan CR et al., studied 829 HIV positive individuals of which 23.4% had enteric parasitic infections [19]. Study done by Ghoshal U et al., on detection of opportunistic parasitic pathogens in immunocompromised patients, of 10,233 stool samples received in 11 years by Institute parasitology laboratory, 64 were found to excrete oocysts of Cystoisospora, of which 37/64 (57.81%) were HIV positive [20]. The prevalence of parasitic infection varies between different countries, places or even regions. Hence, present study shows relatively lower prevalence than other studies. This could be because of different method adopted for finding out this data.

In this study, Entamoeba histolytica was the most common parasite observed followed by Cryptosporidium parvum, Ascaris lumbricoides, Taenia species and Giardia lamblia was least observed parasite. Study carried out in immunocompetent and immunocopromised patients have different parasitic infestations; like opportunistic enteric parasitic infections like cystoisosporiasis is greater in HIV positive than in negative individuals [19]. In another study by Kaniyarakkal V et al., [21] on 200 seropositive HIV patients, Cystoisospora and Cryptosporidium spp were most commonly observed parasite followed by Microsporidium spores and Chilomastix mesnili. Cystoisospora spp, common opportunistic parasitic infestation in HIV positive individuals can cause diarrhoea and may be related to poor sanitation and lack of hygiene. Cryptosporidium and Microsporidium, are associated with severe immunosuppression that may require high index of suspicion and need different staining methods for diagnosis.

Study carried out by Saraswathi R et al., from patients of different speciality units of rural teaching hospital, 55 of 726 stool samples (7.6%) were positive for intestinal parasites [22]. In present study, of 120 stool samples of immunocompromised patients, 20 (16.7%) had parasitic infection. Present study and other studies have reported higher prevalence of intestinal parasites in immunocompromised patients than reported by studies in immunocompetent and general population [18,19,22,23]. Gender related distribution of parasitic infection is reported higher in males in comparison with females in general population, for the reason that males are more exposed to environment [14,22]. There is lack of specific gender related intestinal parasite prevalence data and also of their relation to socio-demograhic risk factors in immunocopromised individuals. Present study did not show significant gender wise difference and it also did not show any significant connection between the prevalence of parasite infections and socio-demographic variables of immunocopromised patients. Study by Shahdoust S et al., showed no significant association between the prevalence of parasitic infestation and the variables of socio-demographic data in patients who were referred to hospital of Tonekabon Mazandaran province, Iran [14]. Studies done in general population as well as in immunocompromised patients recommend for improvement on local risk factors like poor sanitation, contact with soil, contact with domestic animal and others, which requires community education in health and hygiene [5,23,24].

In present study of 120 immunosuppressed patients, 61 had DM of which 9 (14.75%) had intestinal parasitic infection. In a case control study by Mohtashamipour M et al., on 118 DM patients and same number of control group healthy individuals, parasitic infection prevalence was 26.3% in diabetic patients which was significantly higher than in control group [5].

Immunosuppression is an important risk factor for development of parasitic infections. Though some of the evidences generated in general population can be broadly applied to immunosuppressed patients, there seems to be a distinctive and separate pathophysiological mechanism, prevalence, profile and type of parasite involvement in immunosuppressed patients [16,17,19,20]. For better management of such patients, diseases causing immunosuppression and parasitic involvement, like diabetes should be linked with blood sugar and HbA1c control; HIV/AIDS to be related to CD4 count and ART treatment and post transplant as well as patients having malignancy to type and duration of immunomodulators/chemotherapeutic/drugs [4,5,10,11,18].

One of the limitations of the study was a small sample size. Larger sample with more representation of different groups of immunosuppressed patients like Diabetes/HIV/AIDS/Post transplant group can give better insight.

The prevalence of parasite infection among immunosuppressive patients was 16.7% in this study. Entamoeba histolytica was the most common observed parasite followed by Cryptosporidium parvum. There was no significant relation between socio-demographic risk factor and parasitic infections in immunosuppressed patients. Mostly of the time parasitic infection can be diagnosed by stool microscopy. Risk factors and profile of parasitic infections can vary in immunosuppressed patients, which require further research so that treatment and prevention can help to decrease the morbidity in immunosuppressed patients.