Blastocystis spp. infection in cases of diarrhea: A pilot study from a tertiary care teaching hospital in Rishikesh, Uttarakhand, with a brief review of literature.

CONTEXT: Intestinal parasitic infections (IPI) are among the most common infections throughout the world. Blastocystis spp. is a mysterious parasite which is commonly encountered in tropical countries. Its pathogenic status is unknown and there is a paucity of literature about this organism from the state of Uttarakhand, India. AIMS: The aim was to estimate the prevalence of Blastocystis spp. in diarrheal stools. SETTINGS AND
DESIGN: This was a cross-sectional study conducted from January 2018 to July 2019. SUBJECTS AND METHODS: Nonrepetitive stool samples of 187 consecutive patients of diarrhea attending the inpatient department and outpatient department of a tertiary care teaching hospital located in Rishikesh, Uttarakhand, were collected after obtaining informed written consent. These samples were subjected to wet mount microscopy and permanent staining. STATISTICAL ANALYSIS USED: Fisher's exact test and Kappa coefficient were used in this study.
RESULTS: The mean age ± standard deviation of the patients was 36.04 ± 11.31 years with a male-to-female ratio of 1.49:1. The prevalence of IPI was 36.09%. Giardia intestinalis was the most common parasite. Blastocystis spp. was observed in 6.42% of the stool samples, majority of which were obtained from cases of chronic diarrhea. Moderate agreement (0.48) was observed between wet mount microscopy and permanent staining in the identification of Blastocystis spp.
CONCLUSIONS: This is the first study to assess the burden and role of different epidemiological and clinical profiles of Blastocystis spp. in Uttarakhand. More studies are required to know its pathogenesis and its role as opportunistic pathogen. Copyright:

INTRODUCTION

Intestinal parasitic infections (IPI) are among the most common infections throughout the world. It is globally endemic and has been described as constituting the greatest single worldwide cause of illness and disease. This is related to lack of sanitation, unsafe water supply, and lack of health education and therefore they occur wherever there is poverty. The poor people of underdeveloped nations experience a cycle where undernutrition and repeated infections lead to excess morbidity that can continue from generation to generation.[1]

About 3.5 billion people are infected with some kind of intestinal parasite in the world according to the World Health Organization.[2] People of all ages are affected by this cycle of prevalent parasitic infections; however, children are the worst affected. Asia alone accounts for 70% of this burden and India being one of the largest contributors to the global burden with a national population prevalence of 21%.[3]

Some intestinal parasites are pathogenic, such as Giardia intestinalis and Entamoeba histolytica, and some are nonpathogenic forms, living in the gastrointestinal tract as commensals. Blastocystis species (spp.) is a unicellular, anaerobic protozoan parasite and is one of the most common parasites found in the gastrointestinal tract of humans, animals, and birds spreading widely in the tropical climate areas.[4] The organism was first identified more than 100 years ago by Brittain and Swayne and named by Alexeieff in 1911.[5] It has marked morphologic variability, 5–40 μm lacks cell wall but contains mitochondria, Golgi apparatus, and smooth and rough endoplasmic reticula and has been found in different morphological forms vacuolar, granular, amoeboid and cystic.

Based on the small subunit ribosomal RNA gene (SSUrRNA), Blastocystis has been classified into 17 different subtypes (STs). STs 1–9 and 12 are identified in humans with ST3 being most frequently isolated in various epidemiologic surveys.[6]

The infection is transmitted feco-orally and is associated with poor hygiene practices. Clinical characteristics of the disease are nonspecific; the number of parasites found in stool specimens determines the severity of symptoms and signs of infection. Blastocystis spp. may be associated with asymptomatic carrier state or it may present with diarrhea, nausea, anorexia, abdominal cramps, bloating, flatulence, and fatigue. It is found to be associated with inflammatory bowel syndrome (IBS), urticaria, chronic kidney disease, arthritis, and inflammatory bowel disease (IBD) also.[78]

In India, data on Blastocystis spp. are very less and derived mainly from direct stool microscopy. Various alternative methods of detection are culture and molecular methods.[910] Although these methods are more time-consuming and costly, they are considered to be more sensitive in detecting Blastocystis spp. but are not commonly used in diagnostic laboratory.[11]

Our main objective was to estimate the prevalence of Blastocystis spp. in cases of diarrhea and its epidemiological profile in Uttarakhand as very few studies are available on this.

SUBJECTS AND METHODS

The cross-sectional study was conducted in the Parasitology Laboratory of Microbiology at Tertiary Care Institute of Rishikesh. It included 187 consecutive patients of diarrhea attending inpatient department and outpatient department, after taking voluntary, informed written consent. The study was done after taking approval of the Institutional Ethics Committee vide letter number AIIMS/IEC/18/112.

Inclusion criteria

Patients of diarrhea

Age – 1 year and above

Both sexes.

Exclusion criteria

Pregnant females

Children <1 year

Patients on any antibiotics/probiotics

Patients who have taken any antiparasitic drug in the previous 2 weeks.

The overall methodology of the study is depicted in Figure 1.

Figure 1

Overall methodology of the study

Categorical variables were presented as proportions, whereas continuous variables were presented as mean with standard deviation (SD). Comparison of categorical variables was done by Fisher's exact test. Kappa coefficient was calculated for agreement. All statistical tools were two-tailed and a significant level P < 0.05 was used. All statistical tests were performed using the Statistical Package for the Social Sciences version 23 (SPSS Inc., Chicago, USA).

RESULTS

General characteristics

A total of 187 cases of diarrhea from Uttarakhand and neighboring states were included in the study. The mean age of patients was 36.04 ± 11.31 years (±SD); the most common age group was 31–60 years (48.13%) with a male-to-female ratio of 1.49:1. Most of the diarrheal cases were reported in summers (41.18%) and commonly affected those who used untreated drinking water (71.12%). The cases of diarrhea were classified into acute (92.51%), persistent (2.14%), and chronic (5.35%), out of which majority was acute watery type of diarrhea (86.10%). Pain abdomen was other presenting symptom commonly associated with diarrhea (79.14%). Only 5.35% of cases had comorbidities such as diabetes mellitus, hypertension, IBDs, and carcinoma [Table 1]. No pathogenic bacteria causing diarrhea were isolated on culture.

Table 1

Distribution of Blastocystis spp. prevalence according to clinico- demographic and epidemiological factors

	Number (Percentage %)	Blastocystis (%)	P (Fisher’s exact test)
Gender
Male	112 (59.89%)	8 (7.14%)	0.78
Female	75 (40.11%)	4 (5.33%)
Age
1-14 years	17 (9.09%)	0	0.58
15-30 yrs	64 (34.22%)	5 (7.81%)	1.00
31-60 yrs	90 (48.13%)	6 (6.67%)
>60 yrs	16 (8.56%)	1 (6.25%)	1.00
Season wise
Winter (November, December, January)	24 (12.83%)	1 (4.17%)	1.00
Spring (February, March, April)	69 (36.90%)	4 (5.80%)	1.00
Summer (May, June, July)	77 (41.18%)	5 (6.49%)
Autumn (August, September, October)	17 (9.09%)	2 (11.76%)	0.60
Food type
Vegetarian	91 (48.66%)	6 (6.59%)	1.00
Veg & non veg both	96 (51.34%)	6 (6.25%)
Travel history
Present	17 (9.09%)	0	0.060
Absent	170 (90.91%)	12 (7.06%)
Type of drinking water
Treated	54 (28.88%)	2 (3.70%)	0.51
Untreated	133 (71.12%)	10 (7.52%)
Duration of diarrhea
Acute	173 (92.51%)	8 (4.62%)
Persistent	4 (2.14%)	1 (25%)	0.18
Chronic	10 (5.35%)	3 (30%)	0.01
Type of diarrhea
Acute Watery	161 (86.10%)	4 (2.48%)
Acute Bloody	12 (6.42%)	4 (33.33%)	0.0008
Persistent	14 (7.48%)	4 (28.57%)	0.001
Pain abdomen
Present	148 (79.14%)	12 (8.11%)	0.07
Absent	39 (20.86%)	0
Fever
Present	32 (17.11%)	2 (6.25%)	1.00
Absent	155 (82.89%)	10 (6.45%)
Vomiting
Present	42 (22.46%)	1 (2.38%)	0.30
Absent	145 (77.54%)	11 (7.59%)
Bloating
Present	70 (37.43%)	4 (5.71%)	1.00
Absent	117 (62.57%)	8 (6.84%)
Comorbidities
Present	37 (5.35%)	10 (27.03%)	0.001
Absent	150 (72.75%)	2 (1.33%)

Characteristics of infection with Blastocystis spp.

The prevalence of IPI was 36.90%; G. intestinalis was the predominant parasite, followed by Blastocystis spp. [Table 2].

Table 2

Summary of prevalence of intestinal parasites observed in our study

Parasites Single infections	Number (n=69)	Percentage
Protozoan
Giardia intestinalis	25	36.23
Blastocystis	4	5.79
Cryptosporidium	12	17.39
Cystoisospora belli	3	4.35
Entamoeba	2	2.90
Entamoeba coli	1	1.45
Iodamoeba butschlii	1	1.45
Helminth
Hookworm	3	4.35
Hymenolepis nana	2	2.90
Ascaris lumbricoides	1	1.45
Strongyloides stercoralis	5	7.25
Dual infections
Hookworm + Trichuris trichiura	1	1.45
G. intestinalis + H. nana	1	1.45
Entamoeba + Blastocystis	7	10.14
A. lumbricoides + Blastocystis	1	1.45

Blastocystis spp. was found in 6.42% of cases of diarrhea. The mean age of patients was 38.08 ± 15.67(±SD)’; the most common age group was 15–30 years with males being more affected (7.14%). Coinfection was observed in 66.67% (58.33% coinfection with Entamoeba spp. and 8.33% with Ascaris lumbricoides).

Majority of cases of Blastocystis spp. infection were reported in the month of August to October (11.76%) and more in persons using untreated drinking water (7.52%).

Blastocystis spp. was mainly found in cases of chronic diarrhea (30%) and with history of pain abdomen. Comorbidity was found in 27.03% and IBD was the most common (58.33%) [Table 1].

Wet mount microscopy helped us identify only 33.33%; 66.67% were additionally identified by permanent staining methods (trichrome and iron hematoxylin stain). The coefficient of agreement (Kappa) between wet mount and permanent staining was calculated and the value was interpreted as moderate agreement (Kappa coefficient = 0.48) as shown in Table 3. The findings of wet mount microscopy and permanent staining are depicted in Figures 2 and 3, respectively.

Table 3

Agreement analysis between Wet mount microscopy & Permanent staining w.r.t. Blastocystis spp.

	Permanent staining Blastocystis spp. present	Permanent staining Blastocystis spp. absent	Total	Kappa coefficient
Wet mount Blastocystis spp. present	4	0	4	0.48
Wet mount Blastocystis spp. absent	8	175	183
Total	12	175	187

*Kappa values <0: no agreement, 0.00-0.20: slight agreement, 0.21-0.40: Fair agreement, 0.41-0.60: Moderate agreement, 0.61-0.80: Substantial agreement, 0.81-1.00: Almost perfect agreement.[43]

Figure 2

Wet mount microscopic findings of Blastocystis spp. (400×)

Figure 3

Trichrome stained preparation showing Blastocystis spp. (1000×)

DISCUSSION

IPI are one of the most important social health problems commonly encountered in developing countries, especially India. It is an important public health problem worldwide and is predominantly found in tropics, subtropics, and resource-poor settings, mainly due to poor sanitation, nonavailability of clean drinking water, and inadequate personal hygiene.

IPI is an important cause of morbidity worldwide and mainly affects children. It is usually associated with diarrhea and other intestinal manifestations such as nausea, anorexia, flatulence, and abdominal cramps. It affects nutrition and as a result of morbidity and leads to detrimental effects such as poor cognitive performance and physical growth.[12] Cumulative records of IPI since 2010 from various Indian studies show a prevalence ranging from 6.63% to 62%. G. intestinalis was found to be the most common. Incidentally, Blastocystis spp. is usually not considered pathogenic and is thus not routinely reported by medical parasitologists, which may have led to it being underreported [Table 4].

Table 4

Comparative study of prevalence of IPI in India

Study	Study population	Sample size	Prevalence	G. intestinalis	Entamoeba	A. lumbricoides	H.nana	Strongyloides	Blastocystis spp.	A. duodenale
North India
Bansal et al.[44] 2018	Children age <10 years recruited from four schools of Rishikesh	461	24.3%	15.2%	2.17%	0.86%	2.38%	-	-	2.17%
Yadav et al.[13] 2016	Transplant patients	88	-	11%	-	-	11%	-	3%	-
Kotian et al.[45] 2014	Patientsattending OPD & IPD of Tertiary care centre	327	11.62%	3.06%	0.92%	-	2.14%	-	-	2.75%
Das et al [14]	IBS cases & control	150 cases 100 controls	-	-	-	-	-	-	Cases 33.3% Controls 15%	-
Kumar et al.[46] 2014	All Patients attending OPD & IPD except cases of diarrhoea or dysentery	2732	49.38%	-	-	46.88%	0.21%	-	-	-
Singh et al.[47] 2013	Symptomatic opd patients	20751	6.68%	58.5%	32%	5.8%	-	-	-	12.8%
South India
Langbang et al.[48] 2019	symptomatic and asymptomatic Rural & urban population	1006	30.4%	15.8%	4.9%	0.4%	0.4%	-	6.2%	2.2%
Tenali et al.[49] 2018	5-18 years children suffering from diarrhoea	1246	22.95%	12.07%	31.76%	22.57%	-	-	-	19.95%
Mareeswaran et al.[50] 2018	Patients attending the OPD of field practice areas of medical college	390	36.4%	0.5%	28.5%	2.6%	-	-	-	2.17%
Praharaj et al.[51] 2017	all stool samples sent to the parasitology laboratory for routine screening except HIV positive patients	257,588	8.9%	3.5%	8.2%	11.2%	3.3%	10%	0.22%	1.6%
Kumar et al.[52] 2016	Both symptomatic & asymptomaticOPD & IPD patients	1508	22.21%	7.8%	39.7%	11.34%	-	5%	13.4%	8.7%
Padukone et al.[11]	symptomatic & asymptomatic patientscoming to Tertiary care centre	279	-	-	-	-	-	-	37.63%	-
Rayan et al.[53] 2010	School going children of 5-11 years age	100 children were from the urban setting	54.7%	14%	14%	4%	1%	-	18%
	95 children from the rural community	62%	17.9%	4.2%	1.1%	1.1%	-	14.7%	-
West India
Saurabh et al.[54] 2017	Symptomatic opd & IPD patients	968	16.3%	23.12%	1%	1.15%	2.9%	-	6.94%	0.57%
Shobha et al.[55]	Symptomatic & asymptomatic Slum dwellers	880	15.19%	5.45%	-	2.04%	1.13%	-	-	25.7%

Prevalence

In our study, all 187 samples were screened by direct wet mount microscopy, trichrome staining, iron hematoxylin staining, and modified Ziehl–Neelsen staining. The prevalence of IPI was 36.90%; G. intestinalis was the predominant parasite, followed by Blastocystis spp (6.42%). The prevalence of Blastocystis spp. in other Indian studies ranges from 3.00 to 37.63.[111314] Padukone et al. from Puducherry screened a total of 279 stool samples and reported Blastocystis spp. in 37.63%.[11] In North India, two studies were conducted on different study population; Yadav et al. in 2014 conducted a study on IPI in transplant patients and found a prevalence of 3%.[13] Das et al. did a case–control study for molecular characterization and subtyping of Blastocystis spp. in IBS patients and found a prevalence of 33.3%.[14] In our study, the prevalence was found to be 6.42%. Although PCR is the most sensitive method, it could not be used in our study due to financial constraints. The diagnostic method used was microscopy only and the sensitivity of microscopy is very low as compared to the culture and molecular method and we could obtain only one sample per patient which can also be a reason for the lower positivity rate.[91114]

Differences in the study population, geographical location, methods of detection, and expertise can lead to varied results. Indian studies on IPI including Blastocystis spp. have found it to be one among the top 3 IPI [Table 4], similarly, in our study, we found it to be the second most common IPI in cases of diarrhea.

Internationally, the existing information suggests that this infection is more prevalent in developing countries with prevalence ranging from 6.52% to 100%.[8151617181920212223]

The study found Blastocystis infection to be more prevalent in males and 15–30 years with an overall male-to-female ratio of 2:1. This was in concordance with studies done by Zanetti et al.,[18] Sedhighi et al.,[16] Pipatsatitpong et al.,[24] and Osman et al.[22] Infections in children and adolescents have also been reported.[822]

Mode of transmission

Due to lack of proper animal mode, the life cycle of Blastocystis spp. is not yet fully elucidated. Studies have proved that cysts are the transmissible forms of Blastocystis and are transmitted by the fecal–oral route.[2526] Ingestion of contaminated drinking water is a potential risk factor for Blastocystis spp. infection. And our study also showed a higher prevalence of Blastocystis spp. infection in cases using untreated drinking water (83.33%). This finding was similar to studies done by Abdulsalam et al. and Osman et al.[822]

Clinical features

Clinical manifestations of the disease are nonspecific and range from mild, moderate to severe acute, and chronic events. Blastocystis is generally considered noninvasive, but studies on mice inoculated with high doses of Blastocystis have shown a loss of weight in mice and onset of diarrhea.[262728] Studies have also demonstrated that Blastocystis can invade the epithelium of rat colon and attack lamina propria, submucosa, and muscle layers, in view of the increased levels of hyaluronidase in the urine of rats infected with Blastocystis[2930] Blastocystis spp. can be detected in both symptomatic and asymptomatic patients.[31] Abdominal pain and diarrhea are the most common symptoms. Other symptoms include nausea, anorexia, bloating, and perianal itching. Blastocystosis is associated with IBS and nonspecific colitis along with chronic IBD.[3233]

Several studies suggested that the protist could be a potential pathogen in both immunocompetent and immunocompromised patients.[20] A recent study indicated that Blastocystis was a common member of the intestinal flora in healthy people, and various STs of the protist could also colonize the gastrointestinal tract resulting in the asymptomatic carriage.[34]

In our study, the prevalence was higher in cases of chronic diarrhea (30%). All cases had a history of pain abdomen. Other presenting symptoms were vomiting, bloating, and fever. Most cases of Blastocystis infection were associated with comorbidities (83.33%), of which IBD was most common (58.33%).

Most of the cases of diarrhea were associated with coinfection of Blastocystis spp. with other IPI, e.g., Entamoeba spp. and we had taken single fecal sample testing which does not make it pathogen. We should have tested two or more samples, which was not feasible due to the cross-sectional type of study and lack of follow-up in patients. Hence, there is absence of evidence of Blastocystis spp. as a definite pathogen. In order to understand the pathogenic potential and association with comorbidities and immune status, further research based on molecular assays is needed.

Laboratory diagnosis

This involves the demonstration of different morphological forms such as vacuolar, granular, ameboid, and cystic using the wet mount method. Studies have shown that each of the vegetative forms can freely transform into other vegetative forms as a random, continuous process occurring in vivo. As a result of this transformation process, a plethora of intermediate forms is usually present in fresh fecal samples. Owing to various undefined appearances, the morphology of these intermediate forms cannot be well characterized and is hence often overlooked and not reported.[35]

For diagnosis of Blastocystis infection to be made, several stool samples (at least 3) are required, more than 5 cysts in the visual field without other parasites.[36]

Stained preparations

Giemsa, Gram, Wright, and Iron hematoxylin can be used. The trichrome stain is a routinely employed stain and studies have shown that it is more sensitive for the detection of intestinal protozoa and Blastocystis spp. than the wet mount.[31] It typically appears 6–40 μ in diameter with a large central body surrounded by up to 6 small nuclei. The central body stains are characteristic red, green, or blue.[37]

Other methods

Different Xenic and Axenic medias are used for culture, serodiagnosis can be done by ELISA & IFA & molecular methods can also be used for diagnosis.[31343839] Cultures can be done on various media such as Löwenstein–Jensen medium, Jones' or Boeck-Drbohlav condensed medium under anaerobic conditions. Jones' medium is the medium of choice for studies involving cultures.[38] Axenic cultures demonstrate a rich growth in different media. Axenic cultures of Blastocystis isolates are very important for molecular and biochemical research.[3940] In vitro culture increases the sensitivity of detection compared to that of direct microscopy.[113141]

Blastocystis infection leads to IgG and IgA responses, as detected by IFA and ELISA tests.[4243444546474849505152535455] There is limited knowledge of host immune response to Blastocystis spp. & apparent antigenic diversity of parasite. Hence, serology is not included in routine laboratory diagnosis and limited to epidemiological and serological studies.[31]

Molecular methods

Amplification of Blastocystis DNA obtained from fresh stool samples or culture is convenient for the purpose of epidemiological and screening studies. Genotyping should be included in the analysis as well. The development of a real-time polymerase chain reaction (PCR) method for sufficiently sensitive and rapid detection of Blastocystis and the ability to differentiate between the genotypes present in the specimen would be equally useful in screening and epidemiological studies.[5]

In our study, 4/12 (33.33%) were identified by direct wet mount microscopy and 8/12 (66.67%) were additionally identified by permanent staining methods (trichrome and iron hematoxylin stain). This was in agreement with studies done by Karaman et al. and Elghareeb et al. who concluded that trichrome stain is better method for detection of Blastocystis than the direct wet mount.[1941]

The limitations of the current study were as follows:

Since this was a cross-sectional study, the etiological role of Blastocystis in causing diarrhea could not be clearly established

Single stool examination may be insufficient to detect IP due to intermittent shedding in feces, hence the prevalence of IPI and Blastocystis spp. may be underestimated

Single observer reporting for different microscopic methods might have led to bias

Lower sensitivity of microscopy compounded by lack of technical expertise might have led to underreporting or overreporting of this protozoan

Due to financial constraints, molecular-based assays could not be used and different STs of Blastocysts could not be identified.

CONCLUSION

To conclude, this is the first study to assess the burden and role of different epidemiological and clinical profiles of Blastocystis spp. in Uttarakhand. Our study showed Blastocystis spp. as the second most common intestinal parasite causing diarrhea. Blastocystis spp. infection was associated with the use of untreated drinking water. Hence, necessary interventions such as improving the quality of drinking water and awareness program on personal and environmental hygiene are required. Although no conclusions can be derived from this study regarding the causal association of diarrhea and Blastocystis spp., it still helps to understand the prevalence of Blastocystis spp. and other IPIs in Uttarakhand and nearby states, where information on the subject is limited.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.