Diagnostic performance evaluation of ParaEgg for identifying intestinal helminthiasis: A comparative study with conventional copromicroscopy

Abstract

Background

Intestinal helminthiasis remains a significant public health concern in Bangladesh, affecting both humans and animals. Conventional copromicroscopic methods, though widely used, often lack sensitivity, particularly in areas with low prevalence and intensity of infection. ParaEgg, a new diagnostic tool, has been developed to improve the efficiency of copromicroscopic detection. This study aimed to evaluate the diagnostic performance of ParaEgg in detecting intestinal helminth infections in humans and dogs compared to commonly used traditional methods.

Methods

A cross-sectional study was conducted from August to November 2024, analyzing 100 human stool samples and 100 dog fecal samples. For human samples, ParaEgg was compared with Formalin-Ether Concentration Technique (FET), Sodium Nitrate Flotation (SNF), Harada Mori Technique (HM), and Kato-Katz Smear (KK). For animal samples, ParaEgg was evaluated against FET, SNF, and HM. Diagnostic performance was assessed using sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), with the composite results of all methods serving as the gold standard. Additionally, ParaEgg's efficacy was evaluated using naturally infected and experimentally seeded (Trichuris and Ascaris eggs) fecal samples.

Results

The overall helminth infection rates were 24 % in humans and 53 % in dogs. In humans, five types of helminth genera (Ascaris, Trichuris, Enterobius, hookworm, and Hymenolepis) were detected, while six genera (Toxocara, Trichuris, Spirometra, hookworm, Alaria, and Hymenolepis) were identified in dogs. In human, ParaEgg detected 24 % of positive cases, closely following Kato-Katz Smear (26 %) and outperforming FET (18 %), SNF (19 %), and HM (9 %). In animal samples, ParaEgg demonstrated superior performance, identifying 53 % of positive cases compared to FET (48 %), SNF (45 %), and HM (29 %). ParaEgg exhibited a sensitivity of 85.7 % and specificity of 95.5 %, closely matching Kato-Katz Smear (sensitivity: 93.7 %, specificity: 95.5 %). Its NPV (80.1 %) and PPV (97.1 %) further confirmed its diagnostic reliability. In experimentally seeded samples, ParaEgg achieved 81.5 % recovery for Trichuris eggs and 89.0 % for Ascaris eggs.

Conclusion

These findings highlight ParaEgg as an effective diagnostic tool, comparable to Kato-Katz Smear and superior to traditional methods. Its ability to detect mixed infections, egg recovery rate and its high sensitivity in both human and animal samples underscore its potential for widespread application in field settings.

1. Background

Intestinal helminthiasis, caused by roundworms, hookworms, and tapeworms, remains a significant public health concern, particularly in low- and middle-income countries (LMICs). These infections are prevalent in both human and animal, contributing to morbidity, malnutrition, and economic losses (Hotez et al., 2008). In humans, intestinal helminthiasis impairing physical and cognitive development, while in animals, it reduces productivity and increases the risk of zoonotic transmission (Bethony et al., 2006; Pullan et al., 2014).

Copromicroscopy techniques, particularly direct smear microscopy and flotation-based methods, remain the cornerstone of helminth diagnosis in field settings across low-income countries, owing to their affordability and simplicity (Knopp et al., 2008). In Bangladesh, the most widely used low-cost diagnostic methods include the Formalin-Ether Concentration Test (FECT), Sodium Nitrate Flotation (SNF), the Harada-Mori Culture Technique (HM), and the Kato-Katz Thick Smear (KK). However, these methods face significant limitations, particularly in areas with low infection intensity. Their sensitivity decreases markedly when egg output is low, leading to underestimation of prevalence and potential false-negative results (Levecke et al., 2011). The performance of these techniques is further compromised by variability in sample quality, uneven egg distribution in stool, and rapid degradation of diagnostic stages, especially under tropical conditions. For instance, KK Smear, while highly specific, has limited sensitivity in low-intensity infections, and the FET and SNF methods may fail to detect certain parasite species like hookworms due to technical limitations (Utzinger et al., 2008). These diagnostic challenges hinder effective surveillance and evaluation of control interventions in endemic low-resource areas. These shortcomings underscore the need for more reliable and efficient diagnostic tools to improve the accuracy of intestinal helminthiasis detection.

In recent years, efforts have been made to develop innovative diagnostic tools to address the limitations of traditional copromicroscopy. One such tool is the “ParaEgg,” developed by the Korea Disease Prevention and Control Agency (KDCA) (Lee et al., 2024). ParaEgg is designed to enhance the efficiency of copromicroscopy by improving the isolation, and visualization of parasitic eggs in stool samples. Its potential to outperform traditional methods in terms of sensitivity, specificity, and predictive values makes it a promising candidate for routine diagnostic use in both medical and veterinary parasitology. The necessity for new diagnostic tools like ParaEgg is further emphasized by the high prevalence of intestinal helminthiasis in endemic regions. For example, studies in Bangladesh have reported high infection rates among school-aged children and domestic animals, highlighting the urgent need for accurate and accessible diagnostic methods (Mukutmoni et al., 2023; Nath et al., 2022). Improved diagnostics can facilitate early detection, timely treatment, and effective control measures, ultimately reducing the burden of intestinal helminthiasis on public health and animal husbandry.

This study aims to evaluate the diagnostic performance of ParaEgg in comparison to traditional copromicroscopy methods for detecting intestinal helminths. By assessing its sensitivity, specificity, predictive values and filed cost, this research seeks to determine whether ParaEgg can serve as a viable alternative or complement to existing diagnostic techniques in the field settings of Bangladesh. The findings of this study have the potential to inform policy decisions and improve diagnostic protocols, particularly in resource-limited settings where intestinal helminthiasis remains a persistent challenge.

2. Materials and methods

2.1. Study design and area

A cross-sectional study was conducted in the Sylhet district of Bangladesh between August and November 2024. Located in the northeastern region (24°54′23.77″N, 91°50′50.29″E), Sylhet lies within an ecological hilly zone and is characterized by a high prevalence of intestinal parasitic infections. This burden is particularly pronounced in urban slums and tea garden communities, where compromised sanitation, suboptimal hygiene practices, and limited access to clean water increase the risk of parasitic transmission (Nath et al., 2022). These factors make Sylhet an ideal location for evaluating the performance of diagnostic tools.

2.2. Sample collection procedure

A total of 100 human stool samples were collected, with 50 samples obtained from school-aged children (5–15 years) and 50 from adults (above 15 years). Of these, 38 (38 %) were males, and 62 (62 %) were females. Participants were selected using a simple random sampling method from local schools and community. Individuals who had taken antiparasitic drugs within the past month or refused to participate were excluded from the study. Additionally, 100 fecal samples were collected from free-roaming stray dogs in the same area. Stool samples were collected from each participant using sterilized stool cups. Fresh samples were transported to the Parasitology Laboratory, Sylhet Agricultural University, Bangladesh, for analysis. Each sample was processed using five diagnostic methods: ParaEgg, Formalin-Ether Concentration Test, Sodium Nitrate Flotation, Harada Mori Technique, and Kato-Katz Smear. The performance of each method was evaluated against a composite “Gold” standard, which combined the results of all five techniques.

3. Diagnostic techniques

3.1. ParaEgg method

The ParaEgg diagnostic procedure involves several standardized steps to optimize parasite detection. A conical tube containing distilled water is labeled with the specimen name and securely capped. A filter insert is placed into the tube, and approximately 0.5 g of the stool sample is added using a specimen collection spoon. The tube is sealed and mixed in a vortex mixer until the sample is homogenized. After centrifugation at 2000 rpm for 3 min, the filter insert is removed and discarded. Next, 3 ml of ether is added to the tube, which is then covered and mixed again using a vortex mixer. The sample is centrifuged a second time at 3000 rpm for 3 min, and the supernatant is discarded, leaving only the precipitate. The precipitate is stored in a cool place until microscopic examination is performed (Lee et al., 2024).

3.2. Formalin-Ether Concentration Test (FET)

Approximately 0.5 g of faeces was mixed with 10 ml of normal saline in a glass container and stirred thoroughly. The mixture was strained through two layers of gauze into a 15 ml centrifuge tube. Next, 2.5 ml of 10 % formaldehyde and 1 ml of ether were added. The solution was mixed well and centrifuged at 1000 rpm for 3 min. The supernatant was discarded, and slides were prepared from the sediment. Two slides were prepared (one with saline and the other with iodine), covered with a cover slip, and examined under a microscope (Garcia, 2001).

3.3. Kato-Katz technique

The Kato-Katz quantitative cellophane thick smear method was used. Sieved stool was transferred to a template that delivered 41.7 mg of stool. The stool was covered with cellophane previously soaked in malachite green. The slides were examined microscopically for the identification of parasite eggs or cysts (World Health Organization (WHO), 2004).

3.4. Sodium Nitrate Flotation (SNF)

Sodium Nitrate Flotation is a technique used to concentrate parasite eggs based on their buoyancy. Approximately 1 g of stool was mixed with 10 ml of saturated sodium nitrate solution in a centrifuge tube. The mixture was strained through a sieve to remove debris and then centrifuged at 1500 rpm for 5 min. After centrifugation, the tube was filled with sodium nitrate solution until a meniscus formed at the top. A coverslip was placed on the tube and left for 10 min to allow eggs to float to the surface. The coverslip was then transferred to a slide and examined under a microscope (World Health Organization (WHO), 2004).

3.5. Harada Mori Technique

The Harada Mori Technique is a culture-based method used to detect larvae of hookworms and Strongyloides species. Approximately 0.5 g of stool was placed on a filter paper strip in a test tube containing 2 ml of distilled water. The tube was incubated at 28 °C for 7–10 days. After incubation, the water was examined microscopically for the presence of larvae (Harada and Mori, 1955).

3.6. Efficacy assessment

Experimentally seeded fecal samples from unifected human were used to evaluate the ParaEgg method with two reference methods (FET, and SNF). Ten samples from uninfected individuals were selected randomly and homogenized. Each sample was then divided into 3 separate parts and analyzed separately by ParaEgg and reference methods. The sample was divided into 3 parts of 3 g each and then further subdivided into 3 groups (I, II & III). Group ‘I' soil samples were seeded with Trichuris eggs, group ‘II' with Ascaris eggs, and group ‘III' as the negative control. Ten (10) ml of concentrated egg solution containing (200 ± 10) eggs per 100 μl of aliquot was added to each sample and thoroughly homogenized. Parasitic eggs isolated from fecal samples were used for experimental seeding. The stock solution concentration was estimated by counting number of eggs per 100 μl of aliquot. Five trials were conducted under each technique and the experiment was repeated for 10 separate samples.

3.7. Data analysis

Data were entered and analyzed using STATA version 17. Since no single method is considered the “Gold” standard for detecting intestinal parasites, the combined results of all five diagnostic methods were used as the composite “Gold” standard. The operational characteristics (sensitivity, specificity, positive predictive value, negative predictive value, and kappa value) of each diagnostic test were calculated against this composite standard. Sensitivity and negative predictive value (NPV) were particularly emphasized to evaluate the diagnostic accuracy of ParaEgg and other methods.

4. Results

The study found that 26 % of the 100 human samples and 53 % of the 100 dog samples tested positive for at least one helminth species, indicating a higher prevalence of helminth infections in the study area.

4.1. Detection rates of diagnostic methods in humans

Among the human participants, infection prevalence varied by sex and age group (Table 1). Helminth infections were detected in 11 of 38 males (28.9 %) and 15 of 62 females (24.2 %). Age-stratified analysis showed a higher prevalence among individuals aged 5–15 years, with 19 of 50 (38 %) testing positive, compared to 7 of 50 (14 %) individuals aged over 15 years. These findings indicate a higher susceptibility to helminth infections among younger individuals.

Table 1.

Helminth species by sex and age group in human.

Population	Number (and %) infected
	n	Any helminth species
Sex
Male	38	11 (28.9)
Female	62	15 (24.2)
Age
5-15	50	19 (38.0)
>15 years	50	7 (14.0)

Among the samples analyzed, ParaEgg demonstrated a high detection rate, identifying 24 positive cases (24 %). This was higher than FET (18 %), SNF (19 %), and HM (9 %, larvae only). The KK Smear method showed the highest detection rate (26 %), closely followed by ParaEgg. HM had the lowest detection rate, indicating its limited effectiveness in identifying positive cases (Table 2). (See Fig. 1, Fig. 2.).

Table 2.

Intestinal parasites identified in each diagnostic test in humans.

Method	No. of Examined (N)	Positive [N (%)]	Negative [N (%)]
ParaEgg	100	24 (24 %)	76 (76 %)
FET	100	18 (18 %)	82 (82 %)
SNF	100	19 (19 %)	81 (81 %)
HM	100	9 (9 %)	91 (91 %)
Kato-Katz Smear	100	26 (26 %)	74 (74 %)

Fig. 1.

ParaEgg tools and its parts.

Fig. 2.

Helminths ova isolated using ParaEgg. A. Ascaris egg; B. Trichuris egg; C. Hookworm egg; D. Sprirometra egg; E. Hymenolepis egg; F. Toxocara egg.

4.2. Detection of specific helminths in humans

ParaEgg demonstrated strong performance in detecting specific helminth species in human stool samples. It identified 15 cases of Ascaris, matching the KK Smear and outperforming other methods. For Trichuris, ParaEgg detected 3 cases, consistent with FET and SNF but superior to HM, which failed to detect any cases. ParaEgg also detected 1 case of Enterobius, similar to SNF but lower than Kato-Katz Smear (2 cases). In detecting Hookworm, ParaEgg identified 6 cases, fewer than HM and Kato-Katz Smear. For Hymenolepis, only ParaEgg and FET detected 1 case each. Additionally, two mixed infections were found during this study (Table 3).

Table 3.

The ability of diagnostic techniques to detect helminths in humans.

Method	Ascaris	Trichuris	Enterobius	Hookworm (egg/larvae)	Hymenolepis
ParaEgg	15	3	1	6	1
FET	11	3	–	5	1
SNF	14	3	1	3	–
HM	–	–	–	9	–
Kato-Katz	15	3	2	8	–

* Two mixed infections were detected.

4.3. Detection rates of diagnostic methods in animals

In animal fecal samples, ParaEgg outperformed traditional methods, identifying 53 positive cases out of 100 (53 %). FET detected 48 cases, SNF detected 45 cases, and HM detected only 29 cases. The negative detection rates were inversely proportional, with ParaEgg having the lowest (47 cases) and HM the highest (71 cases). These results highlight ParaEgg's superior sensitivity in detecting intestinal parasites in animals (Table 4).

Table 4.

Intestinal parasites identified in each diagnostic test in animals.

Method	No. of Examined (N)	Positive [N (%)]	Negative [N (%)]
ParaEgg	100	53 (53 %)	47 (47 %)
FET	100	48 (48 %)	52 (52 %)
SNF	100	45 (45 %)	55 (55 %)
HM	100	29 (29 %)	71 (71 %)

4.4. Detection of specific helminths in animals

ParaEgg demonstrated the highest detection rate for Toxocara (19 cases), outperforming FET and SNF (18 cases each). For Trichuris, all methods except HM detected 12 cases. ParaEgg and FET identified 9 cases of Spirometra, while SNF detected 5 cases and HM failed to detect any. In Hookworm detection, ParaEgg and HM identified 14 cases, outperforming FET (9 cases) and SNF (12 cases). ParaEgg demonstrated high efficiency in identifying hookworm larvae. Alaria was detected only by FET and SNF (1 case each), while Hymenolepis was detected by all methods except HM. Seven mixed infections were noted, underscoring the complexity of parasite infestations in animals (Table 5).

Table 5.

The ability of diagnostic techniques to detect helminths in dogs.

Method	Toxocara	Trichuris	Spirometra	Hookworm (egg/larvae)	Alaria	Hymenolepis
ParaEgg	19	12	9	14	–	2
FET	18	12	9	9	1	2
SNF	18	12	5	12	1	2
HM	–	–	–	14	–	–

Note: Seven mixed infections were detected.

4.5. Performance of diagnostic techniques

The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and kappa values of the diagnostic methods were calculated against the composite “Gold” standard. Kato-Katz Smear exhibited the highest sensitivity (93.7 %), followed closely by ParaEgg (85.7 %). FET (61.7 %), SNF (63.1 %), and HM (48.9 %) showed significantly lower sensitivity. Specificity was highest for FET and HM (98.0 %), while ParaEgg and Kato-Katz Smear had a specificity of 95.5 %. ParaEgg and Kato-Katz Smear also demonstrated the highest PPV (97.1 % and 98.4 %, respectively) and NPV (80.1 % and 81.5 %, respectively). The kappa value, indicating agreement with the Gold Standard, was highest for Kato-Katz Smear (0.83) and ParaEgg (0.81), while HM had the lowest (0.26). These results confirm that ParaEgg is a highly effective diagnostic tool, closely matching the performance of Kato-Katz Smear and outperforming traditional methods (Table 6).

Table 6.

Performance of diagnostic techniques against the Gold standard method.

Technique	Sensitivity (95 % CI)	Specificity (95 % CI)	PPV (95 % CI)	NPV (95 % CI)	P, χ2	Kappa Value
ParaEgg	85.7	95.5	97.1	80.1	0.00, 2.9	0.81
		(94.4–94.7)	(96.9–97.7)	(79.6–80.9)
	(84.6–86.9)
FET	61.7	98.0	96.9	43.6	0.00, 94.9	0.43
		(97.6–99.1)	(96.6–97.1)	(43.5–43.9)
	(62.1–62.5)
SNF	63.1	94.4	94.7	51.4	0.00, 94.9	0.39
		(93.5–95.1)	(91.4–91.9)	(41.1–51.9)
	(62.9–63.3)
HM	48.9	98.0	91.3	36.1	0.00, 55.35	0.26
		(97.6–99.1)	(91.1–91.5)	(35.5–36.6)
	(48.8–49.1)
Kato-Katz	93.7	95.5	98.4	81.5	0.00, 2.9	0.83
		(94.4–94.7)	(98.4–98.5)	(80.1–82.3)
	(93.6–93.7)

We also evaluated the sensitivity and specificity of two diagnostic methods, Kato-Katz and ParaEgg, for detecting soil-transmitted helminths in human samples. The study analyzed 100 samples for each of the three STHs: Hookworm, Ascaris, and Trichuris. Both methods demonstrated high specificity (>95 % for all helminths), indicating strong accuracy in identifying true negative cases. However, their sensitivity was moderate to low, ranging from 48.1 % to 54.7 %, suggesting a significant proportion of true infections were missed (Table 7). The performance of Kato-Katz and ParaEgg was comparable, with no clear superiority of one method over the other.

Table 7.

Sensitivity and specificity of Kato-Katz and ParaEgg for soil-transmitted helminths.

Test	Samples	Diagnostic error of Kato Katz (95 % CI)		Diagnostic error of ParaEgg (95 % CI)
		Sensitivity	Specificity	Sensitivity	Specificity
Hookworm	100/9	53.1	96.4	48.1	98.1
		(36.9–67.2)	(91.9–98.8)	(34.6–60.1)	(94.9–99.1)
Ascaris	100/15	54.1	98.9	51.4	98.9
			(97.1–99.2)	(32.2–72.8)	(98.1–99.8)
		(34.9–74.1)
Trichuris	100/3	53.1	95.9	54.7	97.1
			(90.5–98.8)	(37.3–69.7)	(93.1–99.4)
		(39.5–66.9)

In this study, a total of 10 experimentally seeded fecal samples were analyzed to evaluate the efficacy of egg recovery using the ParaEgg method alongside two reference methods. The results, as detailed in Table 8, highlight the performance of each method in isolating eggs from experimentally seeded feces with known quantities of parasite eggs. From a practical perspective, it is evident that none of the methods achieved 100 % recovery efficiency. For Trichuris species, with an experimental parasite load of 200 ± 10 eggs, the ParaEgg method demonstrated superior recovery, isolating 163 eggs (81.5 %), compared to the FET method, which recovered 121 eggs (61.5 %), and the SNF method, which recovered 115 eggs (55.5 %). Similarly, for Ascaris species, also seeded at 200 ± 10 eggs, the ParaEgg method outperformed the reference methods, recovering 178 eggs (89.0 %), while the FET and SNF methods recovered 98 (49.0 %) and 130 (65.0 %) eggs, respectively.

Table 8.

Efficacy of methods for isolating eggs from experimentally seeded faeces.

Parasite species	Experimental Parasite load	FET		SNF		ParaEgg
		n	%	n	%	n	%
Trichuris	(200 ± 10)	121	61.5	115	55.5	163	81.5
Ascaris	(200 ± 10)	98	49.0	130	65.0	178	89.0

The approximate cost per test varied across the five helminth detection methods evaluated (Table 9). The SNF had the lowest cost at 105 BDT per test, closely followed by the FET at 110 BDT per test. Moderately priced methods included ParaEgg (120 BDT per test) and the KK technique (140 BDT per test), primarily due to their low reagent costs. In contrast, the HM method was the most expensive at 170 BDT per test, attributable to the requirement for specialized culture media and prolonged incubation.

Table 9.

Cost analysis per test (estimated).

Component	FET	SNF	HM	KK	ParaEgg
Reagents & Consumables	50	50	50	15	15
Equipment Usage	20	20	60	95	70
Laboratory Staff Time	20	20	40	20	20
Sample Processing Time	20	25	20	10	15
Total Cost per Test	110	105	170	140	120

Note: Approximate value in Bangladeshi Taka (1 USD is equivalent to 115 BDT).

5. Discussion

The present study evaluated the diagnostic performance of the ParaEgg for detecting intestinal helminth infections in humans and dogs. Lee et al. (Lee et al., 2024) demonstrated that ParaEgg is highly effective in detecting eggs of Clonorchis sinensis, showcasing its potential as a reliable diagnostic tool for this specific helminthic infection. However, the study did not extend its evaluation to the detection of eggs from other helminth species, leaving its broader applicability yet to be explored. Importantly, ParaEgg was developed as an innovative alternative to the pre-treatment step of the FET method, aiming to streamline the diagnostic process while enhancing detection efficiency. Our study demonstrated that ParaEgg exhibited a detection rate comparable to KK Smear and superior to commonly used traditional copromicroscopy methods. These results align with previous studies highlighting the limitations of conventional copromicroscopy in accurately diagnosing helminth infections (Assefa et al., 2014; Nikolay et al., 2014). The high sensitivity and specificity of ParaEgg, in comparison to KK Smear, indicate that ParaEgg can serve as a reliable diagnostic alternative, particularly in low-resource settings where efficient diagnostic tools are needed.

ParaEgg also performed well in detecting specific helminths, particularly Ascaris and Trichuris. However, its detection of hookworm was slightly lower, which could be attributed to differences in the egg recovery efficiency of the methods. Similar findings have been reported in previous studies, where KK Smear was found to be more effective in detecting hookworm due to its ability to recover fresh eggs from stool samples (Glinz et al., 2010). Despite this, ParaEgg outperformed FET and SNF in detecting hookworm, reinforcing its diagnostic efficiency. The KK technique is widely recognized for its simplicity and sensitivity in diagnosing STHs. However, its specificity can be limited, particularly in distinguishing between closely related species. By enhancing resolution during preparation, ParaEgg could enable more precise identification and differentiation of helminth eggs, potentially improving diagnostic accuracy where STHs pose a persistent public health challenge. One notable limitation of the ParaEgg technique lies in its mesh size, which is standardized at 100 μm (Lee et al., 2024). This relatively small pore size may hinder the isolation and detection of larger helminth eggs.

In dogs, ParaEgg outperforms traditional techniques such as FET, SNF, and HM. This superior detection rate suggests that ParaEgg is particularly effective in veterinary parasitology, where reliable diagnostic techniques are crucial for controlling zoonotic helminths. The ability of ParaEgg to detect Toxocara, Trichuris, and Spirometra with greater accuracy than other methods underscore its potential for broader applications in both human and veterinary medicine. Similar studies have emphasized the need for improved diagnostic techniques in animals to prevent the transmission of zoonotic parasites to humans (Traub et al., 2005).

The kappa value analysis further confirmed the strong agreement between ParaEgg and the gold standard method. One of the key advantages of ParaEgg is its high positive predictive value and negative predictive value, which indicate its robustness in confirming positive cases while minimising false negatives. The detection rates of ParaEgg for specific helminth species further validate its efficiency. For Ascaris and Trichuris, ParaEgg matched or outperformed traditional methods, highlighting its suitability for routine diagnostic use. While KK Smear showed slightly higher detection rates for Enterobius and hookworm, ParaEgg still demonstrated competitive performance. In dogs, ParaEgg outperformed FET, SNF, and HM in detecting common helminths such as Toxocara, Spirometra, and Hookworm,. These results align with previous studies that have highlighted the limitations of conventional diagnostic methods, particularly in detecting low-intensity infections (Bergquist et al., 2009; Zhang et al., 2009). The evaluation of ParaEgg for diagnosing STHs revealed that the method exhibits high specificity, indicating its reliability in correctly identifying true negative cases in low-intensity infections (Knopp et al., 2008; Enoe et al., 2000).

Cost analysis revealed significant variation between methods. While marginally more expensive than conventional sedimentation techniques (SNF/FET), ParaEgg represents an optimal solution for resource-limited settings, combining moderate cost with standardized protocols and demonstrated diagnostic reliability (high sensitivity/specificity). This balance of economic feasibility and diagnostic performance makes it particularly suitable for settings requiring reproducible accuracy without substantial cost burden.

The necessity of developing new diagnostic tools such as ParaEgg is underscored by the persistent challenges associated with traditional copromicroscopy (Nath et al., 2021; Mbong Ngwese et al., 2020). The use of chemicals in some methods, such as FET, poses safety concerns. ParaEgg addresses these issues by offering a simple, efficient, and reliable alternative that can enhance diagnostic capacity, particularly in low-resource settings where access to advanced laboratory facilities is limited. In terms of convenience, ParaEgg stands out for its simplicity and ease of use. The procedure is relatively quick, reducing the risk of contamination and human error, which is particularly important in busy clinical environments. The sediment produced by ParaEgg contained fewer dust particles, resulting in clearer images. This improves the ease of interpretation, allowing technicians to more effectively identify parasitic forms without interference from extraneous particles. These attributes make ParaEgg highly suitable for routine use in diagnostic laboratories, where efficiency and reliability are paramount.

Despite the promising results, this study has several limitations. First, due to resource constraints, our experiments were confined to human and canine samples, and the sample size was relatively small, potentially limiting the generalizability of our findings. Future large-scale studies across diverse geographical regions would further validate the efficacy of ParaEgg. Second, the study primarily focused on the detection of helminth eggs rather than larvae. Although ParaEgg demonstrated strong performance in detecting both eggs and larvae for most helminths, its sensitivity for larval detection requires additional investigation. Finally, the evaluation was restricted to a comparison between ParaEgg and conventional copromicroscopy methods, without assessment against molecular diagnostic techniques, which are recognized for superior sensitivity and specificity (Knopp et al., 2008).

6. Conclusion

This study demonstrated that ParaEgg is a highly effective diagnostic tool for detecting intestinal helminths in both humans and animals, showing sensitivity and specificity comparable to KK Smear and superior performance over traditional copromicroscopy techniques. Given its high detection rate, strong agreement with the gold standard method, and applicability in both medical and veterinary parasitology, ParaEgg presents a promising alternative for routine parasitological diagnostics, particularly in resource-limited settings. Further studies with larger sample sizes and additional validation against molecular methods are recommended to enhance the accuracy and applicability of ParaEgg in global helminth control programs.

Availability of data and materials

The datasets and resources used in this current study are available from the corresponding author upon reasonable request.

CRediT authorship contribution statement

Tilak Chandra Nath: Writing – original draft, Methodology, Conceptualization, Validation, Formal analysis, Writing – review & editing, Supervision, Data curation. Heeil Lee: Writing – review & editing, Validation. Md. Mahamudul Hasan: Resources, Methodology, Investigation. Tanmoy Roy Rudro: Methodology, Investigation, Formal analysis. Dipta Das: Methodology, Investigation, Formal analysis. Md. Taufiqur Rahman: Resources, Methodology, Formal analysis. Nandiny Saha Roy: Methodology, Investigation. Pritha Parial: Investigation, Methodology. Proloy Chakraborty Tusher: Methodology, Investigation. Tarek Siddiki: Resources, Methodology, Software, Investigation.

Ethical approval and consent to participate

This study protocol was reviewed and approved by the Sylhet Agricultural University Research System and the Department of Parasitology, Sylhet Agricultural University, Bangladesh (SAURES-UGC-2023-2024-02). Written informed consent was obtained from all adult participants and the parents or guardians of children participating in the study. The corresponding author personally obtained written consent for publication from the participants. This study obtained permission from the Korea Disease Control and Prevention Agency (KDCA) to use ParaEgg, and there is no conflict-of-interest issue.

Declaration of competing interest

There is no conflict of interest declared by any of the authors.