Comparison of diagnostic performance of single and multiple fecal sampling in the detection of soil-transmitted helminths in school-aged children

Abstract

Soil-transmitted helminths (STHs) are widely distributed globally and India is a significant contributor to the overall global burden of the disease. Microscopic methods like Kato-Katz (K-K) thick smear and direct smear microscopy by wet mount (DSM) are widely used for STH diagnosis due to their ease in performance. Still, low sensitivity proves to be a significant limitation of these methods. This study explores the diagnostic performance of two and three consecutive-day stool samples compared to the common practice of single stool sample examination. We observed that the three consecutive-day stool examination technique increased overall helminth positivity from 12% to 16.3% in K-K and 11.5–15.9% in DSM, indicating that multiple sampling can diagnose intestinal helminthiasis more accurately. A significant increase in the intensities of hookworms (by 37.5%; p value: 0.001) and Trichuris trichiura (by 47.8%; p value: 0.037) (measured in terms of fecal egg count) was also observed. The methods undertaken in the current study are comparable in detecting the helminths as the marginal increase in positivity by K-K (16.32% vs. 15.86%) was statistically insignificant.

Introduction

Roundworm (Ascaris lumbricoides), Whipworm (Trichuris trichiura), and Hookworm (Ancylostoma duodenale, Necator americanus) infections are the most common human parasitic disease in the world. They are together clubbed as 'Soil-Transmitted Helminth' (STH) infections because they are diagnosed using the same laboratory techniques and they respond to the same medicines (WHO 2020). Globally, more than 1.5 billion people, i.e., around one-fifth of the world's population, are estimated to be infected with STH infections, and India has the highest burden of STH infections worldwide (Abraham et al., 2018; WHO 2020). Prevalence and intensity of STH are determined by time-to-time epidemiologic surveillance based on conventional diagnostic methods like microscopy-based stool examination for the presence of helminthic eggs or sometimes larvae or adult worm (Abraham et al., 2018). Diagnostics also provide the information required to assess and implement control programs. However, these methods lack sensitivity and have a high false negativity rate due to factors like intermittent shedding of eggs in the stool, early disintegration of hookworm eggs from slides, need of trained personnel, etc. Molecular methods with higher sensitivity (e.g., Polymerase Chain Reaction) and serological methods can be used for their diagnosis but are expensive and not readily available in developing countries. Moreover, the lack of a reference (Gold standard) test makes it more challenging to assess the diagnostic testing method (Khurana and Sethi 2017; WHO 2002).

Hence, we undertook this study to observe the diagnostic performance of multiple fecal samples over a single sample in detecting STH and estimate its severity by the two commonly employed microscopic methods.

Material and methods

This study was part of a cross-sectional survey carried out at Barpeta district of Assam in June–August 2017 to assess the prevalence and intensity of STH in school-aged children. Institutional ethics clearance was obtained (No.: FAAMC&H/ 128/Pt./2017/3484, Dated June 8, 2017) from our tertiary care teaching hospital. The researchers selected children aged 5–13 years, who were equally distributed over age and gender, from four randomly selected school sand included them in the study. Informed consent was taken from the children’s parents or teachers, and the methods involved in the study were explained to them in detail. Three wide-mouthed, screw-capped, leak-proof, clean containers with the name and age written on the label outside were given to each child. Two of the researchers instructed the participating students to collect three early-morning fresh stool samples over subsequent days and submit them on arrival at school. Samples were collected at the beginning of school timing over the next six days and were immediately transported to the laboratory of FAAMCH in cold boxes. Only those students who submitted properly labeled three-morning samples were enrolled in the study. Children who submitted three viable stool samples over six days with filled questionnaires were enrolled in the study.

On arrival, the specimens were processed for the wet mount (Direct Smear Microscopy, DSM) and Kato-Katz (K-K) thick smear within 1 h of receipt. Macroscopic examination of stool was done to observe its consistency, presence of blood mucous, or any parasitic elements. A portion of stool about the size of a matchstick head was taken, and a thin, smooth smear was made on a slide with saline and iodine. It was covered with a coverslip and examined first under the low-power (10X) and then under high power (40X) microscope. Two researchers examined slides individually to eliminate observer bias, and quality control of each negative slide was done and confirmed by a senior microbiologist. Ova were identified by their characteristic morphology and bile staining properties. A child was considered positive for STH infection if at least one of his/her fecal samples tested positive for one or more species of STHs. To estimate infection intensity, WHO-recommended K-K thick smear technique was employed. A single thick K-K smear was made on a slide for each sample using a standard template with a hole diameter of 6 mm and depth of 1.5 mm, holding 41.7 mg feces. Within 30–60 min, the slides were examined under a light microscope, and the number of helminth eggs, if present, was counted and recorded. Slides were again re-examined after 2 h. The total number of eggs in a slide is multiplied by 24 to quantify helminth eggs per gram of stool. The same processes were repeated for the second and third samples on the subsequent days.

Statistical tests were performed using SPSS (v17.0). Fisher's exact test was applied for comparisons among proportions, and test function was used to compare arithmetic means of fecal egg counts (FEC) between single and multiple day stool examinations. The significance limit was set at p values ≤ 0.05.

Results

Five hundred sixty students of age group 5–13 years were selected. Among them, 435 children with complete data record and three viable stool samples over six days were included in the analysis (compliance rate 77.68%). Two hundred thirty-seven (54.5%) of the included subjects were males, and 198 (45.5%) were females.

Tables 1 & 2 present findings on the increase in calculated per-species prevalence and infection intensities from the examination of two and three consecutive day stool samples compared with a single sample. The observed overall prevalence of infection with any of the three main species of STH was found to be 16.32% by K-K method 95% confidence interval(CI) = 12.85–19.79) and 15.63%by DSM[95%CI = 12.35–19.39] with three-day samples. In contrast, a single sample examination yielded a prevalence of 11.95%[95%CI = 9.06–15.38] and 11.49% [95%CI = 8.65–14.87] by the two methods, respectively (Fig. 1). Thus, a significant increase of 26.76% (p value = 0.040)and 26.47% (p value = 0.046) was observed by triplicate K-K and DSM, respectively.

Table 1.

Prevalence and Intensity of STH infection after examining single, 2-day, and 3-day stool samples

	Day 1 sample	Day 1 + 2* n, (%)	Day 1 + 2 + 3 † n, (%)	Methods
Overall prevalence ‡	52 (12)	62 (14.3)	71 (16.3)	KK
	50 (11.5)	60 (13.8)	68 (15.9)	DSM
Prevalence of individual helminth n, (%)
A. lumbricoides	36 (8.3)	39 (9)	41 (9.5)	KK
	35(8.04)	38 (8.7)	40 (9.2)	DSM
Hookworm	20 (4.6)	27 (6.2)	32 (7.4)	KK
	19 (4.4)	26(5.98)	32 (7.4)	DSM
T. trichiura	16(3.7)	18 (4.1)	23 (5.3)	KK
	15 (3.4)	17 (3.9)	21 (4.8)	DSM
Mean Fecal Egg Count among infected, (FEC) in EPG
A. lumbricoides	836.68	895.39	922.73	KK
Hookworm	42.88	48.5	62.71	KK
T. trichiura	103.17	140.96	197.69	KK

KK Kato-Katz thick smear, DSM direct smear microscopy (in normal saline), FEC Fecal egg count, EPG Eggs per gram

^*Cumulative coproscopic findings of 1st day and a subsequent day (2-samples)

^†Cumulative coproscopic findings of 1st day and 2 subsequent days (3-samples)

^‡Presence of at least one of the three species of STH

Table 2.

Diagnostic performance of examining multiple (2-day and 3-day) stool sample over single stool sample examination

	Performance of 3-day sample examination		Performance of 2-day sample examination		Method
Prevalence	% increase in prevalence [95%CI]	p-value	% increase in prevalence [95%CI]	p-value
Overall	26.76% [16.9–38.6]	0.040	16.13% [6.9–27.7]	0.183	KK
	26.47% [16.0–38.6]	0.046	16.67% [7.2–26.1]	0.179	DSM
A. lumbricoides	12.20% [4.1–25.7]	0.317	7.69% [1.6–26.8]	0.405	KK
	12.5% [4.2–26.8]	0.315	7.89% [1.7–21.4]	0.403	DSM
Hookworm	37.5% [21.1–56.3]	0.050	25.9% [11.1–46.3]	0.184	KK
	40.6% [11.1–46.3]	0.041	26.9% [11.6–47.8]	0.179	DSM
T. trichiura	30.43% [13.2–52.9]	0.163	11.1% [1.4–34.7]	0.431	KK
	28.57% [9.3–47.9]	0.198	11.76% [1.5–36.4]	0.429	DSM

Intensity	% difference in mean FEC (EPG)	p-value	% difference in mean FEC (EPG)	p-value
A. lumbricoides	9.33%	0.810	6.56%	0.869	KK
Hookworm	31.62%	0.001	11.54%	0.346	KK
T. trichiura	47.81%	0.037	26.81%	0.412	KK

Bold values indicate sub-heading within the table which is essential in the context of result

CI Confidence interval, FEC Fecal egg count, EPG Eggs per gram, KK Kato-Katz thick smear; DSM direct smear microscopy (in normal saline)

Fig. 1.

Infection prevalence (%) of overall STH infection in relation to number of fecal samples examined. S1: Results of stool examination of day 1; S2: Sum of all positive results obtained by examining 2 consecutive day samples; S3: Sum of all positive results obtained by examining 3 consecutive day samples

Individually, at the species level, an examination of three K-K thick smear increased the positivity rate (Fig. 2) from 8.3%[95%CI = 5.7–10.9], 4.6%[95%CI = 2.6–6.6], and 3.7%[95%CI = 1.9–5.5], to 9.43%[95%CI = 6.68–12.17], 7.36%[95%CI = 4.90–9.81] and 5.29% [95%CI = 3.18–7.39] for Ascaris, Hookworm and Trichuris, respectively. A striking increase of 37.5% (K-K) and 40.6% (DSM) in Hookworm positivity was observed (p value = 0.050 in K-K and 0.046 in DSM).

Fig. 2.

Infection prevalence (%) of individual soil-transmitted helminths in relation to number of fecal samples examined. S1: Results of stool examination of day 1; S2: sum of all positive results obtained by examining 2 consecutive day samples; S3: Sum of all positive results obtained by examining 3 consecutive day samples

A linear increase in the cumulative mean fecal egg count with increased sample numbers was also noticed (Table1). The observed increase in infection intensity is statistically significant for Hookworm (p value = 0.001) and T. trichiura (p value = 0.037) (Table2).

The K-K thick smear method detected more STH infections than DSM (16.32% vs. 15.86%); however, the difference was not statistically significant. The two methods identified Hookworm equally (7.4%), and marginally higher A. lumbricoides (9.5 vs. 9.2) and T. trichiura (5.3% vs. 4.8%) were detected by the K-K method (Table1).

Discussion

Estimating STH prevalence in a given region from time to time is pivotal in understanding the disease load and monitoring the effectiveness of mass deworming strategies (WHO 2002). WHO recommends coproscopic methods like K-K thick smear method to determine the prevalence and intensity of STH infection (Cools et al., 2017; WHO 2002).Moreover, in most diagnostic settings, DSM (in normal saline) of a single stool sample is still widely used to diagnose intestinal parasitosis. Although these methods are effortless to perform and are relatively cheap, they may fail to detect helminth infections of lower intensity (Khurana and Sethi. 2017; Lim et al., 2018).

In this study, we tried to evaluate the diagnostic performance of multiple stool samples versus a single stool sample in detecting STH by K-K and DSM. We observed that an increase in the number of sampling efforts results in higher observed helminth positivity. The observed STH infection prevalence increased by around 26% in both methods. At the species level, too, a significant increase was seen in the detection of hookworms (by 37–40%) and T. trichiura (by 28–30%).Our findings are in accordance with the study by Liu et al. (Liu et al. 2017). Knopp et al. observed that helminth prevalence obtained by examining three stool samples is close to 'true' prevalence predicted by the mathematical model (Knopp et al. 2008).

Our study further points out that a single K-K examination can miss low-intensity Hookworm (p value = 0.001) and T. trichiura infections (p value = 0.037) and, to some extent, A. lumbricoides. Other methods like FLOTAC/Mini-FLOTAC, qPCR, TF-test,etc., are more sensitive microscopic methods (Knopp et al., 2009; Cools et al. 2019; Xavier et al. 2012) but are relatively expensive and labor-intensive for resource-scant laboratory settings. Increasing sampling efforts to a 3-consecutive-day sample can equate the outcome to these more sensitive methods (Nikolay et al., 2014).

Conclusion

Multiple stool samples (3-consecutive day samples), compared to commonly employed single stool samples, can enhance the diagnostic yield in STH infection by stool microscopy. This will help achieve a better estimation of the disease load in the community and thereby help epidemiologists access the progress of mass-drug administration in school-aged children.

Author's contribution

SD: Conceptualization; Data curation; formal analysis; Investigation; Methodology; Resources; Formal analysis Software; Validation; Visualization; Writing—original draft. DB: Resources; Supervision; Investigation; Visualization; Data curation; Methodology. HS: Methodology; Review & editing; Software; Validation; Formal analysis. DK: Conceptualization; Project administration; Investigation; Data curation; Validation; Visualization; Review & editing; Supervision

Funding

None.

Availability of data and materials

The data sets in this study are available from the corresponding author on reasonable request.

Compliance with ethical standards

Conflict of interest

The author(s) declare that they have no competing interests.

Ethics approval and consent to participate

This study was reviewed and ethically approved by Institutional Ethics committee of Fakhruddin Ali Ahmed Medical College, Barpeta, Assam. Permission to conduct the study was also obtained from the School Principals. The objective of the study was explained to parents, school teachers and students before data collection. Data collection and stool samples were collected after obtaining written consent from parents.