Evaluation of different concentration techniques for microscopic diagnosis of protozoa and helminths in stool samples of children

ABSTRACT

Introduction:

Intestinal parasitic infections (IPIs) are prevalent among children in developing countries, particularly in tropical and subtropical regions. These infections significantly impact physical and intellectual development and exacerbate nutritional deficiencies in early childhood.

Materials and Methods:

This hospital-based cross-sectional study, conducted at AIIMS, Gorakhpur, from July to December 2023, involved 110 children aged six months to five years with diarrhea. The study compared the diagnostic performance of routine wet mount examination and stool concentration techniques (Formol-Ether Acetate Concentration [FAC] and Formal-Ether Concentration [FEC]) for identifying intestinal parasites.

Result:

FAC detected parasites in 75% of cases, FEC in 62%, and wet mount in 41%. Protozoan infections were predominant, with Blastocystis hominis, Entamoeba coli, Entamoeba histolytica, and Giardia lamblia being the most common. Dual infections were better detected by concentration methods.

Conclusion:

The study highlighted the higher sensitivity of FAC over FEC and wet mount techniques. Environmental, socioeconomic, and geographical factors influenced the prevalence and distribution of IPIs. The study emphasizes the need for improved public health measures, including health education, safe water, and sanitation facilities. The formol–ether acetate concentration technique is recommended for its higher recovery rate, safety, and feasibility in rural settings, requiring minimal infrastructure.

Introduction

Intestinal parasitic infections (IPIs) are the most common infections among children in developing countries. In tropical and subtropical regions, infectious parasitic diseases, such as soil-transmitted helminths and parasitic intestinal protozoa, are known to be the leading causes of illness and disease worldwide.[1,2] IPIs not only result in mortality and morbidity but also have a detrimental impact on physical and intellectual development and exacerbate nutritional deficiencies in early childhood.[3,4,5]

Ascaris lumbricoides, Giardia duodenalis, Cryptosporidium parvum, Entamoeba histolytica, Entamoeba coli, Hookworm, Hymenolepis nana, Tapeworm, are the most common parasites that cause acute diarrhoeal illnesses in children under the age of five.[6,7]

The distribution and prevalence of intestinal parasitic infections are influenced by environmental, socioeconomic, and geographical factors, leading to variations in their occurrence from region to region.[8,9]

Intestinal parasitic infections lead to many clinical manifestations among which diarrhea with abdominal cramping, weight loss, vomiting, and flatulence are the most common symptoms.[10,11,12] Globally, with more than 1.7 million cases of diarrhoeal disease and more than 0.44 million deaths among children under the age of five, diarrhea was among the leading causes of death worldwide.[13,14] A study by Kamath et al.[15] (2018), specified that there is more prevalence of childhood diarrhea deaths among the states of Uttar Pradesh and Assam than in other states of India. Therefore, various techniques have been employed for the examination of intestinal parasitic diseases.[16]

Material and Methods

The study is a hospital-based cross-sectional study conducted in the Department of Paediatrics and the Department of Microbiology at All India Institute of Medical Sciences, Gorakhpur, over six months from July 2023 to December 2023. The study participants include children aged six months to five years. The sample size of the study is 110. Inclusion criteria for the study are children aged six months to five years who have diarrhea and whose parents or guardians are compliant with the study. Exclusion criteria include children who have already received antibiotics or antiparasitic drugs before coming to the hospital, children in an immunocompromised state or condition, those on corticosteroids or similar immunosuppressive medications, and children whose parents or guardians are non-compliant with the study.

The study aims to compare the diagnostic performance of routine wet mount examination and stool concentration techniques for intestinal parasite identification and to assess the hospital-based prevalence of intestinal parasitic infections among children having diarrhea.

Methods: Stool samples were collected from the Department of Paediatrics of children aged six months to five years having diarrhea in sterile wide-mouth plastic containers and were carefully labeled and transported to the Parasitology laboratory, Department of Microbiology, and examined on the same day. The analysis of each stool sample was performed by the following methods:

1. Macroscopic Examination: Each stool sample was visually inspected without magnification to assess characteristics such as color, consistency, presence of blood or mucus, as well as the appearance of any adult worms.

2. Direct Microscopic Examination using Saline and Iodine Preparations: A small portion of the stool sample was mixed with saline (0.9% NaCl) and iodine on a glass slide. Subsequently, a cover slip was placed over the mixture, and it was examined under a microscope.

3. Concentration Microscopic Examination:

Formalin-Ethyl Acetate Concentration Method: Fecal specimens underwent emulsification by combining approximately 1 g of stool with 7 mL of 10% formol saline, followed by a 10-minute fixation period. The mixture was then strained through three folds of gauze. The resulting filtrate was combined with 3 mL of ethyl acetate and centrifuged at 1500 rpm for 5 minutes. After settling, the supernatant was removed, and two drops of the sediment were placed on a slide, covered with a cover slip, and examined under a microscope first at 10× and then at 40× magnification for parasite detection.

Formalin–Ether Concentration Method: Stool specimens were processed according to a standardized formalin–ether procedure. Specifically, one gram of stool was added to a clean conical centrifuge tube containing 7 mL of 10% formol water. The suspension was then filtered through a sieve into a 15 mL conical centrifuge tube. Next, 4 mL of diethyl ether was added to the formalin solution, followed by centrifugation at 300 rpm for 1 minute. The supernatant was discarded, and a smear was prepared from the sediment on a slide [Table 1]. Finally, the slide was examined under a microscope, initially at 10 × magnification and subsequently at 40 × magnification.[17]

Table 1.

Characteristics of different procedures[17]

Procedure	Method	Solvent	Specimen Volume
Direct mount	Direct Stool Mount	0.9%NaCl/Iodine	1-2 drops
Ethyl acetate	Filteration	Ethyl acetate	1 gm of feces
Formol Ether concentration	Filteration/Sedimentation	Formol ether	1gm

Data entry and statistical analysis: The data was entered in a computer-generated Excel sheet. Data cleaning, duplication of entry, or cross-checking of data was done to ensure quality. Descriptive and statistical analysis was done as per the study requirement.

Ethical permission: Ethical permission was taken from the Institutional Ethical Board (IHEC Ref no-IHEC/AIIMSGKP/BMR/169/2023 dated-20/05/2023) AIIMS, Gorakhpur. Participation consent was taken from all participants. A participation information sheet was provided to all participants.

Observation and Results

A total of 110 stool samples were collected from children under 5 years of age from 44 (40%) males and 66 (60%) females. The results of three different diagnostic tests for parasitological examinations indicated that the FAC (Formalin Ethyl Acetate Concentration) technique could detect parasites in 82 (75%) followed by the Formal Ether Concentration (FEC) method in which 68 (62%) and direct wet mount technique 45 (41%), as illustrated in Table 2. Qualitatively, the parasites (9 species) were detected by all the methods but with a different recovery rate. Protozoan infections were predominant, with B. hominis, Entamoeba coli, Entamoeba histolytica, and G. lamblia being the most commonly identified species.

Table 2.

Concentration factors and extraction yield of stool procedures for helminth and protozoa species

Parasites observed	Wet mount n (%)	Formol ether concentration (FEC) n (%)	Formol ethyl acetate concentration (FAC) n (%)
1. Protozoal cysts
• Blastocytis hominis	4 (9%)	10 (15%)	12 (15%)
• Entamoeba coli	6 (14%)	8 (12%)	8 (10%)
• Entamoeba histolytica	13 (31%)	18 (26%)	20 (24%)
• Giardia lamblia	9 (20%)	12 (18%)	13 (16%)
2. Helminth eggs and larvae
• Hymenolepis nana	2 (1%)	4 (6%)	5 (6%)
• Ascaris lumbricoides	4 (10%)	4 (6%)	7 (8%)
• Strongyloides stercoralis	1 (2%)	2 (3%)	4 (5%)
3. Hookworm eggs
• Trichuria trichuris	1 (2%)	3 (4%)	3 (4%)
• Taenia sp	5 (11%)	7 (10%)	10 (12%)
Total (n/110)	45 (41%)	68 (62%)	82 (75%)

Dual infection was seen in 2 cases (6.25%). Both concentration techniques could detect E. histolytica cyst with Ascaris lumbricoides eggs infection in one sample while the other having Ascaris lumbricoides eggs (fertilized and unfertilized both) with Strongyloides stercoralis larva was detected only by FAC. Thus, proving the increased sensitivity of FAC over FEC even in multiple infections. The various clinical features have been demonstrated in Table 3.

Table 3.

Clinico-epidemiological characteristics of the patients

Parameter	Number	Percentage
1. Gender
• Male	44	40
• Female	66	60
2. Age
• <1 year (infants)	20	18.01
• 1-3 years (Toddlers)	42	39.40
• 3-5 years (Pre schoolers)	48	43.60
3. Education of the parents
• Literate	63	57
• Illiterate	47	43
4. Occupation of the parents
• Government job	53	48
• Private job	33	30
• Others (Home makers)	24	22
5. Type of residence
• Rural	25	23
• Urban	85	77
6. Housing condition
• Pakka house	46	42
• Kutcha house	64	58
7. Nail biting/thumb sucking
• Yes	17	15
• No	93	85
8. Hand washing source of water
• Running tap water	76	69
• Bucket	32	29
• Others	02	2
9. Hand washing practices
• Yes	67	62
• No	32	29
10. Toilet facilities
• Open field	16	15
• Private/Sharing	94	85
11. Breast feeding for 1st year of life
• Exclusive BF	85	76.4
• BF with complimentary foods	15	14
• No BF	10	8
12. Clinical condition of the patient at OPD
• Fever	75	68
• Vomiting	0	0
• Abdominal distension	36	33
• Loose stools	110	100
13. Drinking water source
• Tap	75	68
• Handpump	33	30
• Tubewell	02	2

Discussion

Parasitic infestations are the major causes of morbidity and mortality in developing countries like India. The data on their prevalence and the sensitivity of various diagnostic methods help clinicians and microbiologists in the diagnosis and management of patients. Diagnosing parasitic infections in humans poses a significant challenge, requiring skilled differentiation among various parasites. Routine diagnostic procedures often lack the necessary sensitivity. Concentration methods are essential for accurately detecting parasites present in small numbers, which may be missed by direct wet mounts alone.

Overall recovery rate: Here, in our study, we proceeded to evaluate the effectiveness of two sedimentation techniques FEC and FAC with routine wet mounts for diagnosing intestinal parasites in specimens that had been identified as positive or negative for the first time. Differences were observed in the recovery of positive samples by each technique, with considerable variability noted from one species to another. Throughout the study, all techniques successfully identified the same intestinal parasite species. In our study, it was found that the FAC technique had a maximum recovery of 75%, which is comparable to the study conducted by Pakdad et al.[18] in Iran, which had a sensitivity of 70% while the sensitivity of FEC was 55.83% comparable to our study (62%). Another study conducted by Moges F et al.[19] detected parasites by FEC and FAC at 79.1% and 73%, respectively, and that of iodine mount was 50%. A study by Parija et al.[20] demonstrated the intestinal parasite recovery rate of 65.26% for formol-ether and 34.74% for direct smear methods, which indicated the superiority of the formol–ether technique over the direct iodine preparation method, which is also similar to our study. In another study by Demeke G et al.,[21] the prevalence of IP was 13.1% and 25.7%, respectively, by WM and FEC. In a study by Mulat Yimer et al.[22] showed that direct wet mount exhibited very low sensitivity for the detection of S. mansoni and hookworm species as compared to the FEC and Kato-Katz techniques. In a study by Young, et al.[23] Ethyl acetate compares favorably with diethyl ether as a solvent in the F-E sedimentation procedure. In our study, no distortion or alteration of parasite morphology occurred with ethyl acetate, and the concentration of organisms was equal to or greater than that with diethyl ether. In general, ethyl acetate seemed to increase the efficiency of the procedure both in the numbers of organisms and in the range of species recovered. It was more effective than diethyl ether in concentrating Giardia cysts and H. nana eggs, which are often not recovered by the original F-E method.

Similarity for both techniques- In our study, the two concentration techniques gave similar results for H. nana and T. trichuris, i.e. 6% and 4% each for both which is comparable to a study by Moges F et al.[19] where similar results were seen for A. lumbricoids, H. nana, T. trichuira, and S. stercoralis.

Possibility of infections- In our study, E. histolytica was the commonest species identified followed by Giardia cyst whereas in a study, By Ganguly et al. and Bisht et al. A. lumbricoides was the most common STH infection with a weighted prevalence of 69.6%. The prevalence of hookworm and T. trichiura infections was 22.6% and 4.6%, respectively. The majority (4206, 91.9%) of the children were infected with one STH, while 8.1% (n = 369) and 3 (0.07%) were infected with two and three STH[24,25] similar to our study where dual infections were seen in 2 (6%) cases by FAC technique while in 1 (0.9%) case by FEC technique. Kang G et al.,[6] in their study, showed that the commonest parasitic infection was Hookworm (61.5%), followed by Giardia (53.8%) and Cryptosporidium (39.7%). However, the present study did not show any Cryptosporidium spp in any sample. In the study by Marothi Y et al.[26]’ study et al., the dual infection rate was as low as (1%).

Thus, dual parasite infections were better detected in the concentration methods than direct iodine preparation method. This may be because; ether and acetone can help dissolve debris and fats from stool samples to increase the detection of formol-ether and formol-acetone concentration techniques, respectively.

Clinicoepidemiological features: The prevalence of parasitic infestations was more common in females (60%) as compared to that in males (40%) [Table 3]. Marothi Y et al.[26] showed that the infestations had a female preponderance. Various studies have shown the varying sex prevalence of the parasitic infestations. However, the sex predominance for the parasite infections has still not been confirmed.

Drinking water source: In our study, the predominant source of drinking water was tap water, accounting for 68% of cases. Protozoan parasites can be orally transmitted through the consumption of contaminated water, highlighting the significance of water sources in infection transmission. Municipal water supplies contaminated with human waste have been implicated in numerous large-scale outbreaks of Giardiasis. This risk is particularly pronounced in regions like India, where issues such as poor water quality and faulty sewage infrastructure contribute to widespread water contamination. Rural areas, in particular, face heightened challenges due to the absence of adequate municipal water networks and sewage systems.[25,27]

Rural–urban housing: The majority of children (77%) were urban residents and the remaining 23% were residents of the surrounding rural areas of Eastern Uttar Pradesh. Whereas in another study the prevalence of intestinal parasitic infection was higher in the rural areas.[19]

Sewage disposal-In our study it was found that Open sewage disposal was present in 48% of patients. About 45% of the children had poor hand-washing practices, 69% washed their hands by running tap water and 29% were from stored bucket water. However in another study open defecation was found in 55%, 52.5% used to wash their hand in water stored in an open bucket, and 45% had waste disposal in the vicinity of the house. Thus, emphasizing the need to strengthen the public health measures also there is a need to improve and provide health education, safe water, and sanitation facilities in settings where parasitic infection load is high.[28]

Literacy rate of parents/guardians: Children from parents with illiterate and literate accounted for 47 (43%), and 63 (57%), respectively. Most of the subjects (58%) had kutcha houses. Children with nail-biting and thumb-sucking habits were 15%. Children who were exclusively breastfed for the first year of life 76.4% and those who were breastfed but also given complementary food (14%) and who were no longer being breastfed 8%, respectively. According to the information given by the parents/guardians, 100% had watery diarrhea followed by fever (68%) and abdominal distension (33%), respectively.

Many studies suggest that the parents’/guardians increasing level of education has been considered a protective factor against diarrhea.[29,30,31]

Conclusion

The prevalence of intestinal parasites was under-reported by the wet mount technique In conclusion, we found that the formol–acetone method had a higher recovery rate of parasites than the direct iodine method. Thus, for safety and hazard-free laboratory setup, we recommend the formol–actone concentration technique as an alternative method. Furthermore, concentration techniques offer cost-effectiveness and feasibility in rural settings, requiring minimal infrastructure.

Ethical permission

Ethical permission was taken from Institutional ethical board (IHEC Ref no-IHEC/AIIMSGKP/BMR/169/2023 dated-20/05/2023) AIIMS, Gorakhpur. Participation consent was taken from all participants. Participation information sheet was provided to all participants.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.