A study on intestinal parasitic infections among school children in Karaikal.

INTRODUCTION: Intestinal parasitic infections (IPIs) play a major role in global disease burden with significant morbidity. The most vulnerable age group was school going children and is transmitted through soil. About 90% of infected individuals remain asymptomatic. The present study was designed to screen for IPI among school children in Karaikal, to identify the asymptomatic infections and to assess the type and occurrence of IPIs.
METHODOLOGY: A cross-sectional study was carried out from July to September 2018 among school children in the age group of 6-14 years after getting ethical clearance. A total of 335 single stool samples were collected. The samples were subjected to macroscopic examination, microscopic examination and subjected to concentration techniques such as salt floatation and formal ether sedimentation technique. Two separate fresh stool smears were made on the microscopic slides for trichrome and modified acid-fast staining. The results were calculated as percentage, frequency/proportion, and Chi-square test using IBM SPSS software version 19.
RESULTS: Only 90 (28%) out of 324 stool samples were positive for the presence of intestinal parasites. The sensitivity of formal ether sedimentation technique (58%) was higher than other techniques. None of the ova of helminths detected. Modified acid-fast staining was negative for coccidian parasites.
CONCLUSIONS: The low prevalence of protozoan parasites and total absence of helminths in the study revealed the effective role of nationwide deworming program and Swachh Bharat Abhiyan program. However, anthelminthic does not cover the protozoan parasites and it exists among asymptomatic healthy population. Copyright:

INTRODUCTION

Intestinal parasitic infections (IPIs) are the major contributors of global disease burden among the parasitic infection and include Ascaris, Entamoeba, Toxoplasma, Cyclospora, Giardia, and Cryptosporidium.[1] As per the WHO's 2020, Roadmap on neglected tropical diseases (NTDs), soil transmitted helminth infections (STHI) due to Hookworms, Ascaris lumbricoides, and Trichuris trichiura are grouped one among the 17 NTDs and considered to be a major public health problem.[2] In order to focus on elimination of STHI, the WHO has adopted preventive chemotherapy with single dose of albendazole (400 mg) or mebendazole (500 mg) annually. Although it helps to reduce the worm burden among heavily infected individual and thereby preventing the morbidity, it does not break the cycle of infection and reinfection. As a result, people living in endemic areas continue to be at high risk of infection.

In India, there exist diverse parasite combinations in different region and pose great challenges in the elimination of IPIs. In South India, Hookworm infestations are higher when compared to T. trichiura and A. lumbricoides.[3] Among the intestinal protozoa, asymptomatic cyst passage of Giardiasis was found to be as high as 50%.[4] Children are the most vulnerable age group with highest risk for intestinal parasites transmitted through soil. About 90% of infected individuals remain asymptomatic.[5] This is due to poor personal hygiene, lack of access to potable water, poverty, illiteracy, frequent outdoor exposure, high-risk behavior, and hot and humid tropical climate.[6] The IPIs were not only associated with infection but also produces significant morbidity such as vitamin deficiencies, iron deficiency anemia, poor educational performance with worse cognitive outcomes particularly in polyparasitised children.[6]

A recent study from Karaikal reported 30.64% prevalence of IPI among school children[7] and so far, no studies have been conducted on polyparasitism. Moreover, the nationwide drive on deworming program was launched by Indian Government on February 2015 onward and continued till date. This deworming program consists of biannual treatment with albendazole 400 mg through mass drug administration (MDA).[8] This program targets the school aged children and there is no active surveillance on the occurrence of IPI from this region. The active surveillance helps to identify the intestinal parasites that are required not only for treatment but also help to prevent the transmission of infection. The present study was carried out to screen for IPI among school children in Karaikal, which will help in identifying the children with asymptomatic infection. The findings will help to assess the type and occurrence of IPIs, i.e., polyparasitism.

METHODOLOGY

Study type and design

A cross-sectional descriptive study was conducted among school children in Karaikal district, Puducherry, India.

Study period

The study was carried out from July to September 2018.

Study setting

This study was undertaken in Karaikal district (semi-urban area), i.e., 135 Km from Punducherry on the East Coast. It is surrounded by the Nagappattinam and Thiruvarur Districts of Tamil Nadu State, with the area of 161km2 presented with the population of 200,222 inhabitants as per 2011 census.

Study population

School going children in the age group of 6–14 years with no symptoms of abdominal pain, vomiting were included in the study.

Sample size

A total of 335 stool samples were collected as per the sample size calculation[79] among the school children.

Sampling technique

Permission was obtained from school education department to conduct the study. Consent from respective school principal was also taken from eight different schools. One day prior to the study, meeting was arranged with school children in the age group of 6–14 years to explain the purpose of the study. All the interested participants were asked to enroll their names and explained about the collection of stool specimen. The enrolled participants were provided with a sterile container along with spatula. In addition, information through leaflet was given to the parents along with the consent/ascent form and unstructured questionnaire.

Sample collection

A single fresh stool sample was collected without any preservatives from each subject on the following day along with filled in forms. The samples were transported to the Department of Microbiology within 1–2 h for further examination.

Slide preparation and examination

The collected samples were subjected to macroscopic examination to observe for the presence of color, consistency, blood, mucus, or presence of segments. Microscopic examination was done with saline and Lugol's iodine wet mount to observe the parasite eggs, cyst, and trophozoite.[10] All the samples were subjected to concentration technique such as salt floatation and formal ether sedimentation technique. Two separate fresh stool smears were made on the microscopic slides for trichrome and modified acid-fast staining. All the preparations were screened by senior and junior faculties along with the student. Based on the shape, size and number of nuclei in the cyst, Entamoeba histolytica, and Entamoeba coli were identified in the wet mount. The presence of oval shape with central axostyle and a pair of nuclei helps to identify Giardia cysts. The various morphological forms of Blastocystis were identified.[11]

Data collection techniques and data analysis

Sociodemographic data and other variables related to personal hygiene were collected using unstructured questionnaire and all the results were recorded into Microsoft Excel sheet and the results were calculated as percentage, frequency/proportion, and Chi-square test was performed using IBM India Private Ltd, Bengaluru, Karnataka, India for statistical significance.

Ethical approval

The study was conducted after getting ethical clearance (JIP/IEC/2018/258 on Date: July 27, 2018) to ensure consent and confidentiality of the participants.

RESULTS

A total of 335 stool samples were collected. Of this, 11 stool samples were excluded due to ambiguous numbering, insufficient samples, and incomplete consent form. Of the 324 stool sample processed for the identification of ova/cyst, only 90 (28%) stool samples were positive for the presence of intestinal parasites (pathogenic and nonpathogenic). The remaining 234 (72%) stool samples were negative for intestinal parasites (pathogenic and nonpathogenic).

In the present study, varying sensitivity was shown by different techniques that were used to identify the intestinal parasites [Table 1]. Among all these techniques, higher sensitivity for the presence of intestinal parasites (pathogenic and nonpathogenic) was shown with formal ether sedimentation technique (58%) whereas salt floatation technique showed the lower sensitivity (28%). The sensitivity of both iodine wet mount and trichrome staining was found to be similar (46%) whereas saline wet mount showed a slightly lower sensitivity (40%). In this study, modified acid-fast staining to detect coccidian protozoa parasite was found to be negative.

Table 1

Comparison of different techniques for stool samples

Name of the technique	Number positive (n=90), n (%)
Saline wet mount	36 (40)
Iodine wet mount	41 (46)
Salt floatation	25 (28)
Formal - ether sedimentation	52 (58)
Trichrome staining	41 (46)

The intestinal parasite found in either one or all of the technique was considered as positive. Therefore, 90 stool samples were positive for intestinal parasites (pathogenic and nonpathogenic). The most commonly detected intestinal parasites includes E. histolytica/E. dispar/Entamoeba moshkovskii in 53 stool samples (59%), followed by Giardia lamblia in 28 (31%), E. coli in 10 (11%), and Blastocystis spp in 8 (9%) stool samples as either single or dual/multiple parasites, respectively [Figure 1]. None of the ova of helminths were detected using all techniques from stool samples of school children.

Figure 1

(a) Saline wet mount – Cyst of Entamoeba spp, (b) Iodine wet mount – Cyst of Blastocystis spp, (c) Trichrome stain- Cyst of Entamoeba spp, (d) Trichrome stain – Cyst of Giardia lamblia, (e) Iodine wet mount – Cyst of Entamoeba spp

Single parasite was detected in significant proportion (73%) when compared to double (26%) and multiple parasites (1%) [Table 2]. Among the double parasites, 17 of the stool samples showed E. histolytica/E. dispar/E. moshkovskii with E. coli, followed by 4 samples with E. histolytica/E. dispar/E. moshkovskii with G. lamblia and one stool sample each for G. lamblia with E. coli and E. histolytica/E. dispar/E. moshkovskii with Blastocystis spp. separately.

Table 2

Presence of single and multiple parasites (pathogenic and nonpathogenic) in stool samples

Number of parasites	Name of the parasites	Number of stool sample positive
Single parasite	G. lamblia	22
	E. histolytica, E. dispar/E. moshkovskii	30
	E. coli	8
	Blastocystis spp.	6
Total (%)		66 (73)
Double parasites	G. lamblia + E. histolytica/E. dispar/E. moshkovskii	4
	G. lamblia + E. coli	1
	E. histolytica/E. dispar/E. moshkovskii + E. coli	17
	E. histolytica/E. dispar/E. moshkovskii + Blastocystis spp.	1
Total (%)		23 (26)
Multiple parasites	G. lamblia + E. histolytica/E. dispar/E. moshkovskii + Blastocystis spp.	1
Total (%)		1 (1)

G. lamblia: Giardia lamblia, E. histolytica: Entamoeba histolytica, E. dispar: Entamoeba dispar, E. moshkovskii: Entamoeba moshkovskii, E. coli: Escherichia coli

The stool samples collected from school children of 6–14 years of age showed that there was a statistically significant difference (P < 0.05) in the number of intestinal parasites (pathogenic and nonpathogenic) from high school children (>10–14 years) to that of primary school children (6–10 years) [Table 3]. In the present study, there was no statistically significant difference (P > 0.05) in the percentage of positivity of intestinal parasites from stool sample of male and female school children [Figure 2].

Table 3

Age-wise occurrence of intestinal (pathogenic and nonpathogenic) in stool samples

Age group (n)	Total number positive for stool (n=90)	List of parasites
Primary school (6-10 years) (n=119)	33	Single parasite
		Giardia (8), E. histolytica/E. dispar/E. moshkovskii (12), E. coli (5), Blastocystis (2)
		Double parasites
		E. histolytica/E. dispar/E. moshkovskii + E. coli (5)
		Giardia + E. histolytica/E. dispar/E. moshkovskii (1)
High school (10-14 years) (n=205)	57	Single parasite
		Giardia (14), E. histolytica/E. dispar/E. moshkovskii (18), E. coli (3), Blastocystis (4)
		Double parasites
		E. histolytica/E. dispar/E. moshkovskii + E. coli (12)
		Giardia + E. histolytica/E. dispar/E. moshkovskii (3)
		Giardia + E. coli (1)
		E. histolytica/E. dispar/E. moshkovskii + Blastocystis (1)
		Multiple parasites
		E. histolytica/E. dispar/E. moshkovskii + Blastocystis + Giardia (1)

G. lamblia: Giardia lamblia, E. histolytica: Entamoeba histolytica, E. dispar: Entamoeba dispar, E. moshkovskii: Entamoeba moshkovskii, E. coli: Entamoeba coli

Figure 2

Percentage of intestinal parasites from stool sample of male and female school children

DISCUSSION

In developing country like India, IPI varied from region to region and any updates on the distribution and extent of IPI in a given area form the basis for planning and evaluation of any intervention program of that area. Therefore, in the present study, the aim was to identify the occurrence of polyparasitism and asymptomatic infection among school children.

The overall occurrence of intestinal parasites (pathogenic and nonpathogenic) was found to be 28% in the stool sample of school children from this area using different techniques. In addition, the present study also showed a difference in the occurrence rate between individual parasites. Earlier report from this area had mentioned a prevalence of 30.64% of pathogenic parasites. In India, the overall prevalence of pathogenic parasites ranges from 12.5% to 91%.[7] It was suggested that this variation in the prevalence of infection in different studies could be attributed to the time of the study and geographical difference in the area. Such low prevalence of IPI had also been reported in some other studies.[1213] It was suggested that better awareness of personal hygiene, sanitation, and drinking water facility in those area was the main reason for reduction in the occurrence rate. Moreover, the use of single stool sample results in underreporting of prevalence whereas multiple stool samples will help to identify the actual prevalence of infection.[11] Although the use of multiple sample increases sensitivity of detection, in the present study, the collection of more than one sample was inconvenient.

The accurate diagnosis of the stool sample is a challenging task and depends on the skilled microscopist and quality assurance of stool sample from collection to processing. Thus, diagnosis plays an important role in prevention of morbidity, complication and in the active surveillance for control program. In the present study, the sedimentation method (58%) showed higher sensitivity followed by iodine wet mount and trichrome staining (46%). Lowest sensitivity was shown with salt flotation (28%) and this could be due to poor identification as there was a chance of distortion of some parasite morphology with high specific gravity of the salt solution.[11]

Direct microscopy using fresh stool specimen is the best technique for identification of IPI in resource poor settings. Although microscopy is the gold standard, a study from a Hyderabad suggested that direct microscopy methods used for detection of parasites had lower sensitivity whereas formal ether concentration had increased the prevalence rate.[14] It also suggested that when parasite load was very low and/or missed out, the concentration methods allowed the detection of intestinal parasite. Moreover, the use of different technique could able to detect more than one parasite and or co-infections.[15]

In the present study, the modified acid-fast staining used to identify coccidian parasite. Like oocyst of Cryptosporidium spp, Cyclospora spp, and Cystoisospora spp showed negative results. It was reported that for 100% detection of coccidian parasites from formed stool, a concentration of 50,000–500,000 oocysts/gram stool is required.[15] This might be reason for negative results of coccidian parasites from our study. Moreover, the study population was healthy school children whereas coccidian parasites were more common in immunocompromised individual.

In the present study, only intestinal protozoan parasites were detected. Similarly, other studies reported higher prevalence of protozoan parasites and low prevalence of helminths[1617] and suggested the transmission was through contaminated food and water.

Among the protozoan parasites, E. histolytica showed higher occurrence (59%) in our study. The global prevalence of Entamoeba spp., ranges from 2% to 60% whereas in the Indian scenario, it was 3.6%–7.4%/.[18] Our results were consistent with other studies from South India.[1920] The difference between the cysts of E. histolytica, E. dispar, E. moshkovskii was difficult with the microscopy due to its morphological similarity. Moreover, E. dispar is nonpathogenic that occurs 10 times more common than E. histolytica. Hence, the cyst was reported as E. histolytica/E. dispar/E. moshkovskii and the correct etiology is identified with immunoassays or by molecular methods.[1117]

In the present study, Giardia was the next most common intestinal parasite causing infection with the prevalence of 31%. Globally, the prevalence of Giardia spp., peaks up to 30%[4] and in developing countries, it ranges between 10% and 50%.[21] The recent studies also reported Giardia as the most common protozoa.[1622] The most important risk factor for transmission was through environmental contamination of water supply with human waste. This has been proved by isolation from water supplies in different parts of the world.[16] In the present study, Giardia was common in school children and they were found to be asymptomatic, as stool sample is collected among healthy population. The prevalence of Giardia as asymptomatic infection has been reported in other studies.[423]

The present study showed the occurrence of 9% for Blastocystis spp. The worldwide prevalence of Blastocystis ranges from 0.5% to 62%.[24] Based on the molecular study from India, the prevalence of Blastocystis spp was 27% among healthy population.[25] However, the studies based on microscopic techniques have reported lesser prevalence rate ranging from 3% to 8%.[26] Another reason suggested for the occurrence of Blastocystis spp among healthy population was due to its more common existence in the coastal regions.[17]

The lack of differentiation of E. histolytica/E. dispar/E. moshkovskii by conventional method has made to considered as nonpathogenic. Similarly, Blastocystis spp occur in healthy individual and its true prevalence as pathogen was not known. Hence, the true pathogen in the present study was G. lamblia. Therefore, the asymptomatic occurrence of IPI among school children was 31%.

Earlier study from this area showed the prevalence of helminths such as A. lumbricoides (33.33%), Strongyloides stercoralis (22.80%), Hymenolepis nana (7.71%), Taenia spp (12.28%), Enterobius vermicularis (7%), and T. trichiura (1.75%).[7] However, in the present study, there was total absence of helminths. The reason might be due to deworming with single dose of albendazole given regularly every 6 months once and finally given on February 2018 to the school children by the education department of this region through the nationwide biannual integrated deworming program for MDA. Apart from this massive deworming program of Indian Government from February 2015, the introduction of the “Swachh Bharat Abhiyan” program around this time was also one of the reasons for the absence of soil-transmitted helminth from this area.[8] It was evident from the present study that the single dose was sufficient for the control of STHI, whereas it was not same for protozoal infection. Our findings suggest that there is a need for regular screening of IPI not only among school children but also among adults as protozoa infections were mostly asymptomatic and form barrier for effective management of these infections.

In the present study, polyparasitism was seen with dual parasites (28%) when compared to multiple parasites (1%). The most common double parasites includes E. histolytica/E. dispar/E. moshkovskii with E. coli from 17 stool samples followed by E. histolytica/E. dispar/E. moshkovskii with G. lamblia from 4 stool samples whereas combination of nonpathogenic parasite for 2 stool samples separately. Such combination of dual parasites had also been reported from other studies.[172227] The present study showed the low occurrence of polyparasitism and this correlates with other study from Puducherry.[17] It was reported that nearly 47.4% of them were found to be infected with one or more parasites with no relevant clinical symptoms and were said of to be asymptomatic.

In the present study, the presence of intestinal parasite (pathogenic and nonpathogenic) was found to be significantly higher among high school children (10–14 years) than that of primary school children (6–10 years). Similar findings were reported from study in Nepal.[23] The increase in prevalence of IPI was due to lack of parental control regarding dietary habits and increased outdoor activities.[28] In contrary, the decrease in prevalence of IPI with increase in age of children has been reported in a study in which they have attributed this finding to rise in awareness regarding hygienic practices and environmental sanitation.

In the present study, there was no gender difference in the occurrence of IPI among school children. It was reported from Nepal, the prevalence of IPI was slightly higher among boys.[23] In contrary, Puducherry study showed that females (56%) had higher prevalence of IPI than male 43.92%.[17]

In the present study, behavioral habits were also studied among school children. It was found that only 39% of them used boiled water and 7% used purified water. It was proved that untreated water carries eggs and cyst and such contaminated water acts as a major source of transmission.[16]

Surprisingly, it was found that hand washing practices were high (76%) among school children before and after taking food. However, in the present study, 53.33% had taken food frequently outside. It was reported that food vendors were another cause of direct transmission. The different parasites reported includes E. histolytica (72%), A. lumbricoides (54%), E. vermicularis (27%), and Giardia duodenalis (13%) were transmitted through food vendors.[29]

In the present study, 36% of school children had nail biting habits and 17% of them played with dogs and 26% with cats. This might also be a possible source for the transmission of Giardia cyst in them as it was reported in earlier study.[30]

CONCLUSIONS

The low occurrence of protozoan parasites and total absence of helminths revealed the effective role played by Indian Government through the nationwide deworming program and Swachh Bharat Abhiyan program. However, anthelminthic does not cover the protozoan parasites and it exists among asymptomatic healthy population. The deworming program takes care of children in the age group of 1–19 years and rest of the age group was not treated. Thus, untreated asymptomatic population serves as source of transmission. Therefore, periodic screening of stool sample is needed not only among school children but also among adults to identify, treat, and prevent the transmission of IPI.

Financial support and sponsorship

This study was supported by ICMR-STS 2018, Rs. 10,000/-.

Conflicts of interest

There are no conflicts of interest.