Comparative DNA extraction methods to use for LAMP assay as molecular diagnosis of human schistosomes from urine samples

Abstract

Background

Schistosomiasis in Africa is an ongoing public health problem that is caused by two major human species, Schistosoma mansoni and S. haematobium, which often cause concurrent infections. Due to the global goal of controlling or eliminating schistosomiasis as a public health problem, the issue of diagnostic sensitivity has become more critical in the assessment of program success. In that regard, the World Health Organization (WHO) has drawn attention to the need for field-applicable tests with high specificity and sensitivity.

Methods

To address this, we have evaluated the amplification of S. mansoni and S. haematobium cell-free repeat DNA by loop-mediated isothermal amplification (LAMP) from field-collected filtered urine samples from 30 school children in Zambia. We have used four DNA extraction techniques (Qiagen and LAMP-PURE (LP): column-based DNA extraction technique, Chelex, and heating: rapid DNA extraction technique) to determine their impact on LAMP sensitivity and specificity, along with cost analysis and person-time involvement for each approach.

Results

Both Qiagen and LP extraction detected positive infections, but Qiagen extraction is more cost-effective than LP. DNA extraction by LP is the fastest (average 20 min.) compared to the other three methods, although it is the most expensive, including amplification ($9.35 compared to $4.90 for heating extraction and amplification). Chelex extraction is slower and simpler than LP and detects 20% more positive infections than heating. Heating extraction is very fast, inexpensive, and simple to perform. However, LAMP amplification for heating-extracted samples resulted in false negatives, possibly indicating the presence of inhibitor(s).

Conclusions

We have demonstrated the sensitivity, cost-effectiveness, and time requirement of LAMP with four different DNA extraction approaches for the detection of two schistosome parasite species from a single field-collected urine sample.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12879-025-11370-y.

Introduction

Schistosomiasis is the second most prevalent and socio-economically devastating parasitic disease worldwide after malaria and affects approximately 230 million people ¹. It is a major source of morbidity and mortality in sub-Saharan Africa [1, 2]. Children aged 6–15 years have the highest infection burden [3], which has been associated with growth delays, delayed cognition, attention deficit disorders, poor performance in school, and a negative effect on the overall growth and quality of the child’s life [4, 5]. In Africa, schistosomiasis is an ongoing public health problem, which in recent times has attracted a major campaign to control this parasitic disease via Mass Drug Administration (MDA) by using Praziquantel. The diagnostic challenges are significant because this chronic parasitic disease infects 230 million people globally [5] and causes the estimated worldwide death of over 200,000 people every year [6].

The differential diagnosis of two major concurrent human schistosomes, namely Schistosoma mansoni and S. haematobium [5], is an involved process. It requires both stool (S. mansoni) and urine (S. haematobium), which are standard procedures and low in sensitivity [7, 8]. In addition, lightly infected individuals continually shed schistosome eggs through defecation/urination into the environment. This helps maintain a continual life cycle through the intermediate snail host (Biomphalaria spp. for S. mansoni, and Bulinus spp. for S. haematobium). The eggs may not be detected using the standard diagnostic methods based on stool and urine currently used in the field. The issue of diagnostic sensitivity has recently become more critical due to the assessment of program effectiveness, especially after the MDA. In addition, the success of MDA-based control strategies due to the decreasing infection levels depends on an accurate and sensitive diagnostic test [9]. This could be appropriate if both schistosome species can be detected from one sample type, such as urine. WHO recommended that lightly infected people be identified, especially after MDA, as they shed eggs, which go through snails (intermediate host) and keep the disease transmission going. WHO also has drawn attention to the need for diagnostic tests with high specificity and improved sensitivity [10–12].Evaluation of a molecular test based on field-collected urine samples across age groups will be a huge paradigm shift in schistosome diagnosis, as it would greatly increase the ease with which specimen collections are made as well as significantly increase the accuracy of the diagnosis of both species simultaneously [13–15]. The WHO recommended Kato-Katz (KK) and Circulating Cathodic Antigen (CCA) for S. mansoni, and urine filtration for S. haematobium lacks sensitivity, and hematuria (blood in urine) for S. haematobium lacks specificity. These procedures involve examining both stool and urine for eggs and blood and urine for the presence of parasite antigen. Logistically and technically, such tests are time-consuming and inadequately sensitive [7, 8]. Our unique approach of sample collection, transport, and storage for molecular diagnostic tests is entirely novel, and there is no need to store and process stool and whole blood, or store whole urine to scrutinize it for eggs. We have developed a novel, highly sensitive, and specific molecular approach by capturing parasite DNA on dried filter paper and amplifying species-specific cell-free repeat DNA via loop-mediated isothermal amplification (LAMP) for both parasites from multiple geographic locations [16]. The LAMP can be used for long-term epidemiological studies to evaluate remaining disease prevalence, distribution, and success of control intervention, as well as a surveillance tool. Over the years, we have performed amplification of parasite repeat DNA from urine sediment captured on filter papers by species-specific primers for S. mansoni and S. haematobium [16, 17], Strongyloides stercoralis [18], Plasmodium falciparum [19], and for Trypanosoma cruzi [20] through PCR. Previously, both S. mansoni and S. haematobium have also been detected from the same urine sediment individually by LAMP from the samples collected in Ghana [21].

LAMP utilizes 4–6 primers that amplify six different regions of the target DNA, which occur at one constant temperature, and positive reactions will yield changes in color or turbidity [22]. It is more resistant to inhibitors than polymerase chain reaction (PCR) because it uses Bst polymerase (DNA strand displacement instead of DNA strand denaturing). The sensitivity of LAMP has been demonstrated to be greater than that of PCR [23]. With LAMP, reactions occur quicker than PCR, and DNA fragments are amplified independently of the standard thermocycler and electrophoresis, thereby removing the need for special reagents, hence saving time and money.

We have detected duo schistosome species by PCR by amplifying cell-free repeat DNA from filtered urine samples collected from Zambia. Detection of cell-free repeat DNA is viable, more sensitive, and a more effective biomarker than a gene-based marker [16, 17]. Mostly non-coding fragments with no protein-coding function, and evolve faster than the rest of the genome, which enables high detection sensitivity and specificity [24]. The concerted evolution allows the sequence homogenization of the repeat fragments within species [10]. The repeat fragments that have been chosen for this project are species-specific. For S. mansoni, PCR will be carried out by amplifying a highly repeated 121 bp Sm1-7 fragment of S. mansoni [1, 25]. For S. haematobium 121 bp Dra, 1 1-repeat fragment will be amplified [26]. For both schistosome species, the repeat fragments comprise 12–16% of each parasite genome (~ 600,000 copies per cell), are species-specific, and occur in different regions of the genomes of these two schistosome parasites, and it is unlikely to observe cross-amplification.

In the current study, urine samples previously collected from school-aged children (ages 9–14) from Zambia have been used along with four different sample preparation procedures and amplified via LAMP to optimize parasite-specific DNA detection for two major human schistosome parasites. The current study also determined the sensitivity and specificity of different extraction techniques and how that impacts LAMP amplification, along with the cost analysis and person-time involvement for each extraction technique. This work is in conjunction with our future endeavor that involves the implementation of a parasite-specific repeat DNA detection (via molecular approach) both in the field and in clinics as a Point of Care (POC) diagnosis, along with a common sample preparation procedure within different endemic settings. To do that, we first have to demonstrate the efficiency and cost-effectiveness of the LAMP method (which can be used as a POC) to detect low-intensity parasite infections in field conditions. This is the first study of its kind to improve diagnosis for both schistosome species, which could be applied in the field in the future.

The objective of the study involved detecting S. mansoni and S. haematobium infection via LAMP amplification from DNA extracted by four DNA extraction techniques from a single urine specimen. In addition, the study also aimed to statistically compare the positive and negative samples by four DNA extraction methods, their cost-effectiveness, and the time requirement for LAMP amplification.

Methods

Study design

111 urine specimens were collected on filter paper upon filtration of approximately 20mL of collected urine from school children (aged 9–14 years) from two schistosome-endemic regions of Zambia [27]. Our unique approach of sample collection, transport, and storage for molecular diagnostic tests is entirely novel, and there is no need to collect and process stool, whole urine, or whole blood to scrutinize them for eggs. Four different DNA extraction techniques were employed, and species-specific cell-free repeat DNA was amplified via LAMP to determine the presence of S. mansoni and S. haematobium from a single urine sample (Fig. 1). QIAamp had been used as a reference technique due to its filter-based extraction technique and usage in our previous studies. Gel electrophoresis was used to confirm positive vs. negative amplification. A detailed cost analysis was done for each DNA extraction technique and LAMP amplification combined. A similar analysis was done for the time requirement for each type of DNA extraction (Fig. 1). A statistical test was done to compare the sensitivity and specificity between different DNA extraction techniques and their impact on LAMP amplification. The required cost for each extraction and person-time involvement was also statistically evaluated for each approach (detailed description is below).

Fig. 1.

The schematic diagram of the conducted study highlights urine sample collection, DNA extraction, LAMP amplification, and cost analysis

30 samples were chosen for this study based on their positive and negative infection status and for four possible combinations (Table 1): (1) S. mansoni + and S. haematobium +, (2) S. mansoni - and S. haematobium -, (3) S. mansoni + and S. haematobium -, (4) S. mansoni - and S. haematobium + (Fig. 1).

Table 1.

Information about the filtered urine samples used in this study. The infection status of samples was determined by PCR amplification. i.e., + = positive infection and– = negative infection

Combination	# of samples
S. mansoni +/S. haematobium +	8
S. mansoni -/S. haematobium -	7
S. mansoni +/S. haematobium -	8
S. mansoni -/S. haematobium +	7
Total	30

Study sites and population

The study was approved by the institutional review board (IRB) of Marquette University, USA (IRB # HR-3116) and by ERES Converge, Zambia (IRB # 2016-Apr-002). The study adhered to the Declaration of Helsinki. The study was voluntary, and informed consent to participate was obtained from the parents or legal guardians of the children participant.

Filtered urine samples were collected as part of a previous study from the school children from the Chongwe district of Lusaka Province and the Siavonga district of Southern Province in Zambia. Chongwe River runs through the Chongwe district, while the Siavonga district is on the lakeshores of Lake Kariba, the World’s largest artificial lake by volume and the second-largest man-made lake in the world [27]. Both districts were known to be endemic for both S. mansoni and S. haematobium based on previous mapping studies. In addition, these districts are part of an ongoing Schistosomiasis MDA program. Sample collection was done approximately one month after the first round of MDA. Therefore, infection intensity was expected to be low. All samples were filtered through Whatman #3 filter paper, left to air dry on a clean bench surface with a net covering to prevent any contamination, and then individually packed in Ziploc bags with desiccant to prevent degradation of the genetic material. All dried filtered urine samples were shipped at room temperature and kept at 4 °C in the laboratory in the USA.

DNA extraction

Each filter paper containing dried urine sediment was split into four quadrants, which essentially produced four replicates, and each replicate was used for one type of DNA extraction. In total, there were 120 DNA samples (30 samples x 4 types of extraction). The four DNA extraction protocols used were heating, Chelex, QIAamp (Qiagen, MD) blood and tissue mini kit, and loop-mediated isothermal amplification– procedure for ultra-rapid extraction (LAMP-PURE) (Fig. 1). A standard paper punch was used to punch 12–15 holes (~ 1 mm in diameter) from one sample’s first filter paper quadrant. The paper puncher was washed with 10% bleach and water after use on each sample to minimize the chance of contamination.

Heating extraction

This was non-filter-based, crude DNA extraction. The 12 paper discs were immersed in 800 µl nuclease-free water in 2 mL Eppendorf tubes, then incubated at 95 °C for 20 min. The tubes were then centrifuged at 7000xg for 1 min, and the supernatant (approximately 700 µl) was transferred to a clean 1.5 mL Eppendorf tube. DNA concentration was quantified using NanoDrop (Thermo Scientific, DE), and the initial concentration ranged between 100 and 600 ng/µl. The 260/280 ratio ranged for Qiagen 0.2–1.6, LP 0.5–1.5, Heating 0.6–1.7, and Chelex 0.5–1.7. On the other hand, the 260/230 ratio ranged for Qiagen 0.1–1.5, LP 0.2–0.5, Heating 0.2–0.5, and Chelex 0.2–0.3. So, each sample was diluted to approximately 3–5 ng/µl (+/− 0.5 ng/µl) using nuclease-free water of a 50 µl aliquot, as the LAMP reaction used 3–5 ng/µl of extracted DNA. The stock and aliquot of heating extracted DNA samples were stored at −20 °C freezer until LAMP amplification was conducted (probably a month).

Chelex extraction

Chelex was a fast and non-filter-based crude DNA extraction. 12–15 discs were immersed in 10% Chelex suspension (450 µl nuclease-free water and 45 mg Chelex). The tubes were vortexed for 10–15 s, spun for 10–15 s at 5000xg in a microcentrifuge, then incubated at 95 °C for 20 min. After incubation, the tubes were centrifuged at 12,000 rpm for 1 min, and 200 µl of supernatant was transferred to a clean 1.5 mL tube. The quantification, dilution, and storage were the same as heating extraction.

QIAamp blood and tissue mini kit extraction

QIAamp DNA Blood and Tissue Mini Kit (Qiagen, MD) was a nitrocellulose filter-based pure DNA extraction kit. The extraction, quantification, and storage are the same as described in our previous study [27]. These samples were not diluted.

LAMP-PURE extraction

LAMP-PURE extraction kit (Eiken Chemical, Tokyo, Japan) was a fast and non-filter-based semi-pure DNA extraction. The tubes with paper punch and water were incubated for 10 min at 95 °C. DNA was extracted from the reconstituted urine following the manufacturer’s protocol, which consisted of a series of extraction and purification steps. Following heat shock, the urine was added to a DNA extraction solution and mixed by inversion. Then, the mixture was treated with an adsorbent material to neutralize the solution and remove possible LAMP inhibitors present in the sample. A syringe was then attached, and the reaction tube was squeezed to extract DNA into a clean Eppendorf tube. As LAMP-PURE was a crude process and resulted in a very high DNA concentration (40–760 ng/µl) for all samples, all samples were diluted to 50 µl of approximately 5 ng/µl concentration. The aliquot was then stored in a −20 °C freezer until LAMP amplification.

LAMP amplification and detection

Cell-free repeat DNA fragments from all 120 extracted DNA samples were amplified via LAMP using two sets of species-specific primers [28]. to determine the presence of either S. mansoni or S. haematobium, or both species. The 10µL reaction mixture consisted of 4 µl of 2X buffer mix (composed of 10 LAMP buffer, dNTPs, and betaine), 0.5 µl each of 40 pmol FIP and BIP primers, 0.5 µl each of 5 pmol F3 and B3 primers, 0.5 µl MgCl2, 1 µl Bst polymerase (Lucigen, WI), 2 µl DNA (3–5 ng/µl in concentration), and the rest nuclease-free water. Four different controls were used for every LAMP reaction: S. mansoni and S. haematobium genomic DNA (BEI Resources, VA) as positive control or negative control, nuclease-free water as water control, and DNA extracted from urine collected in the USA, who were not exposed to schistosomiasis as a no-template control. LAMP amplification occurred in an automated thermocycler at 63 °C for 2 h and then at 80 °C for 5 min for enzyme inactivation. Each sample extracted by four different extraction methods was tested two times before making a conclusive decision about positive or negative. The amplification was repeated if there was any issue of contamination.

Gel electrophoresis was done on all LAMP amplified products to confirm amplification. 5 µl of LAMP product was run on a 2% agarose gel stained with SYBR Green I (Life Technologies, NY) and a 50 bp DNA ladder (New England BioLabs, MA). A gel picture was taken using the Azure C200 gel documentation system (Azure Biosystems, CA) and was stored for future reference. LAMP amplification was observed for both S. mansoni and S. haematobium for DNA extracted by four different methods. The detection of LAMP amplified product via SYBR Green I was confirmed by running all amplified samples on 2% agarose gel electrophoresis (Fig. 2). A sample that changed color from orange to yellow-green in the presence of LAMP amplicon was regarded as positive.

Fig. 2.

LAMP results obtained by adding SYBR Green immediately after amplification were compared against gel electrophoresis for amplification of cell-free repeat DNA for Schistosoma mansoni. Four different DNA extractions were compared for six samples. Gel electrophoresis-positive band appeared as smears due to the production of considerably more amplified fragments than PCR and for negative samples no smear was visible. Positive (+) (Yellow-green color) and negative (-) (Orange color) amplifications are highlighted

A second detection method was performed using the remaining 5 µl of LAMP product. 1 µl (1:20 dilution) of SYBR Green I (Life Technologies, NY) was added to each tube. The tubes were vortexed, spun in a microcentrifuge, and observed for color change under visible light (orange = negative amplification, yellow = positive amplification). A picture was taken of each set of tubes and stored for future reference.

Cost estimation and recording of person-time requirements

DNA extraction was done in a batch of 15 samples per DNA extraction method, and the time needed for each type of extraction procedure (for 15 samples) was recorded. Each step of extraction was timed separately: preparation time (labeling tubes, etc.), extraction time (hands-on work), heat shock time, and quantification time. The timing of these steps was added. Time spent on the shaker (overnight for Qiagen extraction) was not included in the total time. For each extraction method, the total time for the two batches (a total of 30 samples) was averaged to calculate the total average time for a batch. The total average time was divided by the total samples (30) to determine the individual average time.

For each of the techniques applied, the cost estimation was done as follows: (a) DNA extraction material price in bulk divided by the bulk number to get the value for one sample for each ingredient. Then all the individual ingredient values are used to estimate the cost of one sample extraction. (b) The ingredients required for LAMP were purchased in bulk from commercial vendors and divided that price by the bulk number to get the value for one sample amplification for each ingredient. Then, all the costs of individual ingredients were determined to estimate the cost of one sample amplification. (c) The figure or value for DNA extraction and LAMP amplification for one sample was added to estimate the total cost per sample.

Statistical analyses

Data was collected for positive and negative amplification by S. mansoni and S. haematobium-specific LAMP for all four types of DNA extraction. The statistical analyses were done by converting the results of LAMP amplification to numerical values, such as 1 = positive and 0 = negative. The total positive and negative infections for each schistosome species for each type of DNA extraction were calculated by JMP 12 (JMP^® v12, SAS Institute Inc., Cary, North Carolina, USA). Data was recorded for person-time (including gap time) involvement for each type of DNA extraction for individual samples, and then for a total (30) number of samples, and LAMP amplification separately. The sensitivity and specificity of LAMP amplification for four different DNA extractions were calculated using MedCalc 12.4.0 (MedCalc Software, Ostend, Belgium). These were compared against PCR amplification that was recorded in the previous study.

Results

LAMP amplification for four different DNA extraction

In gel electrophoresis, the positive amplification had a smeared band appearance due to the presence of the LAMP amplicon. For the negative sample, there was no change of color (remained orange) and no band appearance on the agarose gel, signifying the absence of LAMP amplicon (Chelex negatives: S2, S3, and S4; Heating negatives: S1, S3, and S4). This was evident for a few chelex, and heating extracted DNA samples (Fig. 2). It should be noted that LAMP bands for chelex (S1 and S5) and heating (S5 and S6, Fig. 2) were weak and not well reproduced in the photograph. The results for the control samples were consistent throughout the experiment. Positive controls reliably showed amplification, confirming the successful detection of the target species through the use of species-specific primers. In contrast, both the no-template control and the water control consistently yielded negative results, indicating the absence of contamination or non-specific amplification. If any amplification was observed in either the no-template or water controls, the experiment was repeated to ensure the validity and accuracy of the findings.

LAMP amplification for DNA extracted by four different extraction methods was compared against the PCR amplification results for the same samples (Table 2). For both S. mansoni and S. haematobium, LAMP detected all the positive samples previously detected by PCR for DNA extracted by Qiagen and LP. This was expected as LAMP is equally sensitive as PCR (Abbasi et al., 2010). In the case of Chelex detection of PCR-positive samples, it was higher for S. haematobium (90%) compared to S. mansoni (73.3%). Also, LAMP missed some of the PCR-positive samples for chelex (Table 2). For heating extraction, the LAMP amplification result was almost the same as chelex with some missed PCR-positive samples.

Table 2.

LAMP amplification frequency for DNA extracted by four different DNA extraction methods for Schistosoma mansoni and S. haematobium

Schistosome species		LAMP amplification
		Qiagen QIAmp kit	LAMP-PURE	Chelex	Heating
S. mansoni	PCR Positive	28 (93.3%)	28 (93.3%)	22 (73.3%)	21 (70%)
	PCR Negative	2 (6.7%)	2 (6.7%)	8 (26.7%)	9 (30%)
S. haematobium	PCR Positive	17 (56.7%)	21 (70%)	27 (90%)	27 (90%)
	PCR Negative	13 (43.3%)	9 (30%)	3 (10%)	3 (10%)

Time and cost analysis

DNA extraction by LAMP-PURE (LP) was the fastest (21 min) compared to the other three methods and yielded a substantial volume of DNA (40 ng/µl– 754 ng/µl). Chelex extraction was a simpler, non-filter-based technique (100 ng/µl– 950 ng/µl) and required 28 min (second to LP) to perform. Extraction by heating was also very fast (30 min.), fastest for a cluster (30 samples) of samples, and arguably the simplest to perform (produced approximately 700 ng/µl). Both Chelex and heating extraction yielded a considerable volume of DNA. Qiagen extraction took the longest (43 min) due to a filter-based technique (0.35 ng/µl– 5 ng/µl) and an overnight wait period (Table 3).

Table 3.

Time required for DNA extraction for individual samples and clusters of samples by four different DNA extraction methods

DNA extraction type	Filter-based/non-filter-based	Overall DNA yield (concentration)	Individual sample extraction time	The cumulative time requirement for extraction (30 samples)	LAMP Amplification time
Qiagen QIAmp kit	Filter based	0.39ng/µl– 282ng/µl	43 min.	1 h 25 min. + 12 h	1 h 35 min.
LAMP-PURE*	Non-filter based	40ng/µl– 754ng/µl	21 min.	2 h 56 min.	1 h 35 min.
Heating	Non-filter based	124ng/µl– 939ng/µl	30 min.	2 h 9 min.	1 h 35 min.
Chelex	Non-filter based	106ng/µl– 559ng/µl	28 min.	2 h 15 min.	1 h 35 min.

$ = Includes preparation, extraction, wait, and quantification time

* = Loop-mediated isothermal amplification– purified ultra-rapid extraction (LAMP-PURE)

Heating was the least expensive extraction method (extraction: $1.00/sample and extraction + amplification: $4.90/sample). The Chelex per-sample extraction cost ($2.60) and amplification cost ($6.50) were lower than LAMP-PURE and Qiagen. LAMP-PURE extraction and amplification were the most expensive ($5.45 and $9.35). However, per sample extraction cost and amplification cost for Qiagen were lower than LAMP-PURE ($4.00 and $7.90; Table 4).

Table 4.

Cost analysis for four different DNA extraction methods and LAMP amplification. I.e., calculations are based on single and multiple samples and include the cost of plastic supplies

DNA extraction type	Extraction cost/sample	LAMP test cost/sample	Total for one sample (extraction + amplification)	Total for 30 samples (extraction + amplification)
Qiagen QIAmp kit	$4.00	$3.90	$7.90	$237.00
LAMP-PURE	$5.45	$3.90	$9.35	$280.50
Chelex	$2.60	$3.90	$6.50	$195.00
Heating	$1.00	$3.90	$4.90	$147.00

Sensitivity and specificity analysis

For S. mansoni, Qiagen and LAMP-PURE both had 100% sensitivity and specificity for all the amplified samples. Whereas Chelex and heating both had 100% specificity, but sensitivity was 72.73% and 76.19% respectively (Table 5). For S. haematobium, LAMP amplification for all four reactions had 100% specificity, but sensitivity was variable (94.44% − 100%, Table 5).

Table 5.

The sensitivity and specificity analysis of LAMP amplification for DNA extracted by four different DNA extraction methods and via PCR was conducted as part of the previous study for both Schistosoma mansoni and S. haematobium

Schistosome species		PCR	LAMP amplification
			Qiagen QIAmp kit	LAMP-PURE	Chelex	Heating
S. mansoni	Sensitivity	57.14%	100%	100%	72.73%	76.19%
	Specificity	100%	100%	100%	100%	100%
S. haematobium	Sensitivity	83.33%	94.44%	95.45%	100%	96.43%
	Specificity	100%	100%	100%	100%	100%

Discussion

The performance of the LAMP assay in this study was influenced significantly by the DNA extraction method based on urine samples collected on filter paper. The four extraction techniques - Qiagen, LP, Chelex, and heating differed in cost, speed, complexity, and DNA purity, which in turn impacted LAMP sensitivity and specificity.

All four methods were adapted to extract DNA from filter papers, which allows easy transportation and storage of urine samples, and our findings reaffirm their effectiveness as a matrix for recovering Schistosoma DNA. Among the methods, LP extraction offered the highest sensitivity and simplicity, though at a higher cost. It was also the fastest and provided DNA of high purity with minimal inhibitor presence, a crucial factor given LAMP’s vulnerability to chemical inhibitors, despite its generally higher inhibitor tolerance compared to PCR.

Chelex extraction was slower but still outperformed the heating method in terms of LAMP detection sensitivity. Heating was the most rapid and affordable but frequently led to false negatives, especially for S. mansoni, likely due to residual inhibitors or lower DNA yield. This highlights that while LAMP can be robust against some inhibitors due to Bst polymerase, extraction chemistry and purity still critically impact assay performance.

The Qiagen kit, although not originally designed for paper-filter DNA extraction, was successfully adapted and performed comparably to LP, offering a more cost-effective yet still sensitive solution. Its silica column-based chemistry ensures good DNA purity, minimizing the risk of LAMP inhibition.

Other studies have similarly evaluated extraction methods for LAMP, particularly in the context of other pathogens. For example, studies on Plasmodium spp. and Trypanosoma have shown that DNA extracted from dried blood spots using Chelex or heat methods can perform well in LAMP, though with some trade-offs in sensitivity [29, 30]. These findings correlate with ours, confirming that while LAMP is adaptable, the extraction method must match the sample matrix and desired diagnostic sensitivity.

LAMP can be used for long-term epidemiological studies to evaluate remaining disease prevalence, distribution, and success of MDA control intervention, as well as used as an overall surveillance tool. Our findings demonstrated that the urine collection via the filter paper method is cost-effective and efficient for collecting parasite DNA coming from urine for easy storage, transportation, and processing for extraction. We have demonstrated this in our previous studies in different geographic locations [2, 25]. The single urine filter paper can be used for the detection of both Schistosome species multiple times, like in this study (four different DNA extractions). LAMP is used to amplify cell-free repeat DNA, which is a viable, more sensitive, and more effective biomarker than a gene-based marker [16, 31]. Our approach to sample collection, transport, and storage for molecular diagnostic tests is entirely novel, and there is no need to collect and process stool, whole urine, or whole blood to scrutinize eggs. Now the efficacy of the LAMP assay has been demonstrated in the current study by using different DNA extraction as it has been regarded as a stumbling block for molecular assays for control and surveillance purposes for Schistosomiasis. Out of four DNA extractions, LP can be used effectively as it requires minimal steps, is easy to use, and has less chance of contamination (confined environment) with very high sensitivity and specificity for both Schistosoma species. The cost of LP can be an issue, although it can be minimized with greater use and more manufacturers producing the extraction kit. The sample size per extraction method could be a limiting factor, although the general findings were similar to our previous findings [28].

The two schistosome species usually co-occur with other nematode parasites, and mixed infections are common. Therefore, it was important to exclude any cross-amplification. We eliminated any cross-amplification by using positive genomic DNA controls, DNA from closely related sister species used as controls, negative DNA controls, and water control of the amplified product. Regular decontamination efforts were undertaken by cleaning all the working bench spaces occupied by the machine and apparatus with 10% bleach and diluted 100% ethanol. It is demonstrated by the specificity analysis, as all four DNA extraction amplifications have 100% specificity.

According to the WHO Roadmap for NTDs 2030, new highly sensitive and easily operable diagnostic tests for different prevalence settings and all schistosome species are needed for surveillance [32]. In recent times, many African countries are moving towards the goal of local elimination of Schistosomiasis as a public health problem via MDA, which lowers infection prevalence. As elimination campaigns progress, infections become less severe, and traditional tests cannot detect light infections due to poor sensitivity and specificity. Genetic characterization of schistosome species and molecular diagnosis based on such characteristics are key to controlling the disease [33]. WHO recommends using the Kato-Katz (KK) technique and the Circulating Cathodic Antigen (CCA) test for diagnosing Schistosoma mansoni. In contrast, for Schistosoma haematobium, urine filtration shows low sensitivity, and detecting hematuria (blood in urine) lacks specificity. Whereas LAMP can be used for long-term epidemiological studies to determine the remaining disease prevalence, distribution, and success of control interventions. In the current study, LAMP utilized four primers (2 sets) to amplify six different regions of the target cell-free species-specific DNA, which occurred at one constant temperature, and positive reactions yielded changes in color or turbidity [34]. The amplification is more resistant to inhibitors than polymerase chain reaction (PCR) because it uses Bst polymerase. This is evident because LP, Chelex, and heating extraction are non-filter-based techniques and yield DNA with potential inhibitors. The LAMP amplification was able to detect all PCR-positive [28] samples with higher sensitivity compared to PCR and greater specificity (Tables 2 and 5). It was also demonstrated on prior occasions [23]. Overall, LAMP can be used to detect low-level infection for both Schistosome species with accurate sensitivity and greater specificity.

Conclusions

We have demonstrated the greater sensitivity and specificity, cost-effectiveness, and time requirement of LAMP amplification for four different DNA extraction techniques (filter and non-filter-based) to detect two Schistosome parasite species from a single field-collected urine sample. LAMP using Qiagen-extracted DNA demonstrated the highest overall performance, offering superior sensitivity, specificity, and cost-effectiveness. Highly sensitive and specific tests, such as LAMP, can detect schistosomiasis at the species level at an earlier stage and with a lower level of infection, especially after MDA, than traditional methods, enabling prompt treatment and preventing the progression of the disease to more severe forms. Early detection is crucial for controlling schistosomiasis as a Public Health problem by 2030.

Abbreviations

WHO

World Health Organization

MDA

Mass Drug Administration

POC

Point of Care

KK

Kato-Katz

CCA

Circulating Cathodic Antigen

PCR

Polymerase chain reaction

LAMP

Loop-mediated isothermal amplification

LAMP-PURE

Loop-mediated isothermal amplification– procedure for ultra-rapid extraction

LP

LAMP-PURE

Author contributions

Conceptualization: NL; sample collection, data management, extraction, and analysis: JM, CS, BP, and NL; original draft: NL, and BP; supervision and funding acquisition: NL and BP; revision of the manuscript: JM, and CS. All authors contributed to the article and approved the submitted version.

Funding

Brittany Pulkkila received an ASM Undergraduate Research Fellowship from the American Society for Microbiology. Nilanjan Lodh received an Early Career Research award from the Thrasher Research Fund. The study was possible because of this.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The study was approved by the institutional review board (IRB) of Marquette University, USA (IRB # HR-3116) and by ERES Converge, Zambia (IRB # 2016-Apr-002). The study adhered to the Declaration of Helsinki. The study was voluntary, and informed consent to participate was obtained from the parents or legal guardians of the child participants.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.