Detection of Blastocystis species in the stool of immuno-compromised patients by using two different diagnostic techniques

Introduction

Blastocystis is the most frequently isolated protozoan parasite in human faeces in both developing and developed countries with variable prevalence⁽¹⁾.

The life cycle of Blastocystis spp. includes some or all of the following stages: Vacuolar, multivacuolar, amoeboid, granular and cyst stages⁽¹⁾. Transmission of Blastocystis spp. occurs by  faeco- oral route ( via contaminated water or food) where the cyst form is the only form responsible for that transmission ⁽²⁾.

Symptoms of Blastocystis spp. infection in immuno-competent people are ranged from asymptomatic ⁽³⁾ to non specific and non-invasive gastrointestinal  (GIT) symptoms as Nausea, anorexia, abdominal pain, flatulence and acute or chronic diarrhea ⁽⁴⁾. While invasive complications can occur in immuno-compromised patients. In addition, Blastocystis spp. maybe also associated with irritable bowel syndrome and inflammatory bowel disease ⁽⁵⁾.

Impaired or weakened immune system in immuno-compromised patients (e.g., diabetes

mellitus, renal diseases, malignant diseases, hematologic diseases, malnutrition and patients with long duration of corticosteroid therapy) makes the symptoms of blastocystosis more severe ⁽⁶⁾.

Thus, this study aimed to detect Blastocystis spp. in the stool samples of different groups of immuno-compromised patients by two different diagnostic techniques.

Patients and methods

Ethical approval

The study protocol was approved by the scientific ethical committee of the Department of Medical Parasitology, Faculty of Medicine, Minia University at their monthly meeting on January 2021( Date 18 January 2021, Approval No. 2:2021). A written informed consent was obtained from every patient after full explanation of the purpose and the technique of this study.

Study design and setting

This study was a cross sectional study. It was carried out on 200 participants (150 immuno-compromised and 50 healthy control) who enrolled from inpatient and outpatient clinics of Minia Tropical Hospital, Minia National Oncology Institute, Minia University Hospitals, Minia District, Egypt during the period from September 2021 to December 2022.

The participants were classified into 4 groups: Each group was consisted of 50 patients, whereas, the first three groups were immuno-compromised patients. (group I): Patients were having different types of cancer, (group II): Patients were having uncontrolled diabetes mellitus (DM), (group III): Patients were suffering from chronic renal diseases while (group IV): People were healthy used as a control. Their ages were ranged from 15-50 years.

Stool examination

Fresh stool samples were collected from each patient in this study. Samples were collected in sterile labeled plastic containers. The stool samples were transferred to the Laboratory of Medical Parasitology Department, Faculty of Medicine, Minia University, to be examined immediately. 

1- Microscopic examination by direct Wet Mount technique (Saline and Iodine preparation):

A small amount (about 2 mg_s) of feces was mixed in a drop of physiological saline or dilute Lugol’s iodine and then placed on a clean glass slide. The glass slide was covered with cover slide. The slides were microscopically examined initially under the low power (10 x), then the high power (40 x) and oil immersion (100 x) ⁽⁷⁾.

2- Culture on Jones’ medium:

Fifty mg_s from each fresh stool sample were inoculated in Jones’ medium supplemented with 10 % horse serum. The stool portion was mixed with the culture medium with a clean glass rod. Each tube was labeled with the patient's name and incubated at 37º C for 48-72 hours to increase the amount of Blastocystis isolates. Then examination of the culture for Blastocystis spp. was done, one drop of supernatant using a sterile Pasteur pipette was put on a slide and covered by a cover slip and then examined with x100 objective (with oil immersion) ⁽⁸⁾.

Statistical analysis

Data were entered and analysed using SPSS software (version 20 USA Chicago). Quantitative data displayed as mean and standard deviation, qualitative data as frequency and percent. P value is considered significant at <0.05. Chi squared test and z test for comparison of categorical data.

Results

By direct wet mount technique (saline and iodine preparations); the percentage of Blastocystis spp. positive samples was higher in immuno-compromised group than in control group (83.3% and 40% respectively). These data were statistically significant (P value <0.001). After culture on Jones’ medium, the percentage of positive samples was increased in both groups (96% and 60%). These data were statistically significant p value <0.001 as shown in table (1).

Furthermore, the sensitivity of direct wet mount technique was less than that in the culture on Jones’media (83.3% and 96% respectively). However, the specificity of direct wet mount technique was high when compared to that of the culture on Jones’ media (60% and 40%) as shown in table (2).

Blastocystis spp. positive samples were higher in different immuno-compromised groups, I, II and III compared to that in the healthy control (86%, 90%, 74% and 40% respectively). These data were statistically significant p value <0.001 by direct wet mount technique. However, there was no statistically significant difference in percentage of Blastocystis spp. detected among different immuno-compro-mised groups (I, II and III). The Percentage of positive Blastocystis spp. samples was increased up to 96%, 98%, 94% and 60% after culture on Jones’ media in the four different studied groups respectively. There was statistical significant difference in percentage of Blastocystis spp. in different immuno-compromised groups with control group (P value <0.001). However, there was no statistical significant difference among the different immuno-compromised groups.

There was a significant increase in the percentage of positive Blastocystis spp. detected in each immuno-compromised group   after culture on Jones media when compared to the percentage of positive Blastocystis spp. detected by direct wet mount technique (Saline and iodine preparations), in (group I) cancer patients (96% and 86%), (group II) uncontrolled DM patients (98% and 90%), (group III) chronic renal patients (94% and 74%) as shown in table (3).

Discussion

Blastocystis was the most frequently isolated protozoan parasite from stool samples with high prevalence in immuno-compromised patients⁽⁹⁾.

This study aimed to detect Blastocystis spp. in immuno-compromised groups by two diagnostic techniques (direct wet mount technique and culture on Jones’ media) which are generally considered the gold standard in the diagnosis of Blastocystis  spp. infection ⁽¹⁰⁾. 

The percentage of Blastocystis spp. detected by direct wet mount technique was higher in immuno-compromised group than that detected in the control group (83.3% and 40% respectively). These results were approved after culturing on Jones media where the percentage was increased in both groups (96% and 60% respectively). These data were statistically significant p value <0.001 in immuno-compromised groups. The direct wet mount technique showed low sensitivity 83.3% and high specificity 60%, while culture on Jones media showed high sensitivity 96% and low specificity 40%.

This result was agreed  with  Gabrielli and others who found that  Blastocystis spp. were frequently detected in the immuno-compromised patients compared to that of the control group⁽¹¹⁾. While Eassa and others recorded that the prevalence of Blastocystis spp. were higher in both immuno-compromised patients and control group⁽¹²⁾.

On the other hand, this result disagreed by several researchers  who  documented  that Blastocystis spp. infections were higher in the control group due to poor hygienic conditioned environment^{(13, 14)}. The lower percentages among immuno-compromised group could be explained by Eassa and others who stated that immuno- compromised patients might  be less active, losing appetite and having more care from their relatives thus,  they were less exposed to infection than that of the normally active ones ⁽¹²⁾.  

Many researchers stated that the traditional diagnostic technique performed for detection of Blastocystis   spp. as direct smear with or without staining had a lower detection rate than that with fecal cultures^(15-17),  while Sarzhanov and others stated that the culture method was the best in the diagnosis of Blastocystis spp. ⁽¹³⁾

Some studies found that the number of Blastocystis spp. positive cases detected were increased by the use of Jones medium compared to that detected by the direct method due to its high sensitivity, convenience and simplicity^{(17, 18)}.

In the present study, there was a high significant difference between Blastocystis spp. infection in cancer patients compared to Blastocystis spp. infection in the healthy control (P value <0.001). This was agreed by Mohamed and others who reported a significant higher prevalence of Blastocystis spp. infection in

cancer patients when compared to that in the control group⁽¹⁹⁾. Asghari and others stated that Blastocystis spp. infections were frequently detected among cancer patients who received radio or chemotherapy without statistical significance⁽²⁰⁾. Yersal stated that Blastocystis   spp. infections were more frequent in the patients who had received eight or more cycles of chemotherapy compared to those who had received less or none cycles of chemotherapy⁽¹⁸⁾. These findings confirmed the association between Blastocystis spp. and cancer patients especially colorectal cancer⁽²¹⁾.

Although, this was disagreed by Ali and others who found that there was no significant statistical difference between the prevalence of Blastocystis spp. infections  in cancer patients and that in the control group⁽²²⁾. Also, Allemani and others found that the prevalence of Blastocystis spp. infections detected in the cancer patients were low⁽²³⁾.

The lower percentage of Blastocystis spp. infection among immuno-compromised cancer group could be explained by the pharmacologic effect of cytotoxic drugs upon the parasite⁽²⁴⁾.

In the present study, there was high association between Blastocystis spp. infections and uncontrolled diabetic patients compared to Blastocystis spp. infection in the healthy control (P value <0.001). This  was agreed by Popruk and others who stated that Blastocystis spp. infections were significantly more prevalent in patients with DM compared to that in the control group⁽²⁵⁾. This might be  due to impaired cellular immunity in immuno-compromised diabetic patients and metabolic imbalance^{(26, 27)}.

Laodim and others found that Blastocystis spp. were common in 25.8% of diabetic patients⁽²⁸⁾. Moreover, Mohtashamipour and others stated that the risk of intestinal parasites was 3.6 times more  in the diabetic patients than that in the healthy control, especially with Blastocystis spp., which flourished in the damaged intestinal mucosal integrity caused by uncontrolled DM resulting in sever GIT complaints ⁽²⁹⁾. Also, Drawany and others reported that Blastocystis was considered the second major intestinal parasite among the diabetic patients ⁽³⁰⁾.

In this study, there was a high significant difference between Blastocystis spp. infection in the renal patients compared to Blastocystis spp. infection in the healthy control (P value <0.001). These data were matched with many researchers who stated that renal patients were considered at a high risk for Blastocystis spp. infection and these researchers recommended that Blastocystis spp. might not be ignored in routine examination of immuno-compromised renal patients^(31-33). This might be explained by the effects of uremic toxins, alterations in the gut microbiota and the colonic flora in the patients with chronic renal disease⁽³⁴⁾.

Among different immuno-compromised groups, the percentage of Blastocystis spp. positive samples detected by direct wet mount technique was high and increased after culture on Jones’media with no statistical significant difference between different immuno-compromised groups. This was agreed by Asghari and others who reported that among different immuno-compromised groups examined by direct microscopy, Blastocystis spp. infection was high in cancer and renal patients⁽³⁵⁾. Also, Zanetti documented that among different studied immuno-compromised groups, renal patients were the most susceptible to the Blastocystis spp.  infection⁽³⁶⁾.  Other  studies stated that Blastocystis spp. might cause opportunistic infection in the immuno-compromised hosts as cancer and renal patients^(31,37).