Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Diagnosis of Schistosoma mansoni Infection in Faecal Samples

Human intestinal schistosomiasis is caused by the blood fluke, Schistosoma mansoni. With intensified efforts to control schistosomiasis by mass drug administration using praziquantel (PZQ), there is an urgent need to have accessible, quality-assured diagnostic tests for case detection and disease surveillance and for monitoring efficacy of treatment and other interventions. Current diagnostic tools are limited by suboptimal sensitivity, slow turn-around-time, affordability, and inability to distinguish current from past infections. We describe a simple and rapid diagnostic assay, based on the loop-mediated isothermal amplification (LAMP) technology for diagnosis of S. mansoni infection in human faecal samples. The LAMP primers used in this assay were previously described and they target a 121-bp DNA repeat sequence in S. mansoni. The LAMP assay was optimized at an isothermal temperature of 63°C for 1 hour. The amplified DNA was either visualized under ultraviolet light after electrophoresis or by directly observing the color change after staining the amplicons with CYBR Green dye. The LAMP assay was evaluated against the microscopy-based procedure and the results were analysed using Cohen's kappa coefficient to determine the degree of agreement between the two techniques. The LAMP assay reliably detected S. mansoni ova DNA in faecal samples and parasite DNA in amounts as low as 32fg. When the assay was tested for specificity against other faecal-based soil-transmitted helminths (STH), no cross-reactivity was observed. The LAMP assay was superior to the Kato-Katz assay with a 97% specificity; a high positivity score reliably detecting S. mansoni and a Kappa Coefficient of 0.9 suggested an exceptional agreement between the two techniques. The LAMP assay developed has great potential for application in field settings to support S. mansoni control and elimination campaigns.


Introduction
Human schistosomiasis is a snail-borne parasitic infection caused by blood flukes in the genus Schistosoma and is among the neglected tropical diseases (NTDs) targeted for elimination [1]. It remains a major public health problem in the tropical and subtropical regions of the world, with an estimated 207 million people infected worldwide, mostly children, and another 779 million people being at risk of becoming infected [2]. S. mansoni, one of the three major elimination efforts must be supported by suitable diagnostic tests for case detection, for reliably evaluating efficacy of chemotherapy, and for disease surveillance in control programs. As schistosomiasis control efforts intensify in the endemic areas (with renewed interest in the control of neglected tropical diseases) and become effective, prevalence and intensities of infection will most likely drastically reduce, and so more sensitive diagnostic tests will become necessary for case detection, especially, in low intensity transmission areas or for evaluating efficacy of chemotherapy and disease surveillance in schistosomiasis control programs [5]. Ideally, the diagnostic test should be simple to perform, inexpensive, rapid, specific, and sensitive enough, to detect low intensity infections, and capable of handling bulk samples at a go. While rapid immunodiagnostic tests based on antibody detection may be useful, they may not distinguish between previous and current infections. Also, the urine-based CCA assay and other antigen detection tests offer greater sensitivity and rapidity and are suitable for analysis of large samples [6,7]. However these have not been evaluated adequately under different field settings. Microscopy is the gold standard for diagnosis of S. mansoni but it is not sensitive enough to detect low intensity infections; it is tedious and cumbersome to perform and requires expertise to make a diagnosis. Molecular diagnostic tools based on the polymerase chain reaction (PCR) technology offer greater sensitivity and robustness [8][9][10] but require costly equipment and are not suitable for field use.
Diagnostic tests based on the loop-mediated isothermal amplification (LAMP) technology on the other hand promise to revolutionize infection diagnosis, in particular, as a tool for point of care diagnosis. The LAMP assay is relatively simple to perform and rapid, does not require sophisticated equipment, can easily be adapted for visual detection of amplified products, and can potentially be performed under field conditions [11]. The LAMP technology has several advantages over the conventional PCR-based procedures, and, in particular, it can be performed at isothermal temperatures in the range 60-65 ∘ C; it is highly specific, and offers efficiency [12]. Besides, the large amounts of a highly insoluble magnesium pyrophosphate salt produced as a byproduct during the LAMP reaction facilitates visual detection of the amplification products, thus further simplifying the procedure, and can potentially be performed under field conditions [11,12].
LAMP assays have been developed for detection of S. mansoni infection in snails [13] and S. japonicum eggs [14], Ascaris lumbricoides [15], and hookworm [16] infections in faecal samples. The LAMP assay for diagnosis of S. japonicum infection was found to be equally or more sensitive than the PCR assay for detection of the parasite infection [14]. LAMP assays have also been developed for detection of African trypanosome parasites [17], Plasmodium parasites [18], S. haematobium [13], and Taenia species [19].
In a previous study, Abbasi et al. [13] described identification of S. mansoni DNA in infected snails from early prepatency. In this paper, we describe a LAMP assay based on the amplification of a 121-bp DNA repeat sequence for the diagnosis of S. mansoni infection in faecal samples.

LAMP Primers Used.
The LAMP primers used to perform the present assay were those previously described by Abbasi et al. [13] and target a 121-bp DNA repeat sequence in S. mansoni, and whose sequences are shown in Table 1.

Test Parasite for Assay Development.
A laboratory maintained isolate of S. mansoni originally obtained from a naturally infected patient from Kisumu, western Kenya, and which is routinely maintained in the laboratory at the Kenya Medical Research Institute (KEMRI) in Nairobi through Swiss albino mice and Biomphalaria sudanica snails was used as a test parasite in the development and optimization of the LAMP assay for detection of S. mansoni infection in faecal samples. S. mansoni cercariae shed by lab infected snails and approximately 100 cercariae were used to infect individual mice through the percutaneous route essentially, as previously described by Smithers and Terry [20]. Four weeks after infection, the mice faecal samples were collected by placing mice in about 50 ml of a normal saline solution in a 500-ml glass beaker at room temperature to induce defecation [21]. The presence of S. mansoni eggs in mice faecal samples was confirmed by examining faecal smears prepared according to the Kato-Katz procedure [22], under a compound microscope. The parasite eggs on the faecal smear were then counted, and the number of parasite eggs per gram (epg) of faecal sample was calculated. The S. mansoni adult worms were then harvested from the infected mice by perfusion as previously described by Smithers and Terry [20], and the recovered worms were preserved in 95% ethanol. These parasite specimens were then used in the development and optimization of the LAMP assay. Similarly, faecal samples were obtained from school children in Mwea, who had tested positive for S. mansoni by the Kato-Katz procedure and whose parents or guardians had given consent for their children to participate in this study. Mwea division is an irrigation area in central Kenya, where schistosomiasis is endemic. The human faecal samples were used in the evaluation of the LAMP assay.

Parasite DNA Extraction.
Genomic DNA was extracted from freshly collected mice or human faecal samples using the QIAMP fast DNA stool mini kit from Qiagen (Catalogue No. 51604) following the manufacturer's instructions. Briefly, 0.2 g of stool sample was weighed out and placed in 2 ml tubes. Approximately 1 ml of inhibit EX buffer was added to the tube containing faecal sample followed by 1 min vortexing. The suspension was then heated at 95 ∘ C for 5 min. Proteinase K (15 l) was added to a new tube followed by addition of the supernatant then centrifuged at 14,000rpm. Buffer AL (200 l) was added and mixed by vortexing for 15s followed by incubation in a water bath for 10mins at 70 ∘ C. 200 l of absolute ethanol was then added followed by thorough mixing. From this mixture, 600 l of the lysate was pipetted into a spin column and centrifuged at maximum speed (14000rpm) for 1 min, and the supernatant was discarded. Buffer (AW1) was added followed by centrifugation at max speed for 1 min then supernatant was discarded. 500 l buffer 2 (AW2) was then added and centrifuged for 3 mins at max speed. The supernatant was then discarded and elution performed using 200 l ATE buffer. DNA extraction for adult worms was done using a modified method previously described by Truett et al. [22]. Extracted DNA was stored at −20 ∘ C until being used for subsequent assay.

Amplicon
Detection. The amplified DNA was visualized under ultraviolet light at 320nm after electophoresis on 2% standard agarose gel for 1 hour and then photographed in black and white. Direct detection of amplicons in a reaction tube was also done by direct observation of the reaction with unaided eye after addition of 1:10 dilution 1:10 SYBR I Green dye (Invitrogen, Carlsbad, CA) to the amplicon. Under these conditions, the color in the reaction tube changed from orange to green in the presence of positive LAMP amplicons.

Determination of the Sensitivity and Specificity of the LAMP Assay.
Specificity of the LAMP assay was carried out using S. mansoni specific primers to amplify DNA of other co-occurring soil-transmitted helminths including Trichuris trichiura, hookworm, and Ascaris lumbricoides.
To determine the sensitivity of the LAMP assay by establishing the lower detection limit, the genomic DNA concentration was determined using a Qubit5 followed by a 10fold serial dilution of the DNA. The successive serially diluted DNA samples were then amplified with LAMP primers to determine assay sensitivity.

Parasite Infection Intensity and Sensitivity Determination.
Faecal samples obtained from infected children with varying infection intensities of S. mansoni and categorized as low (1-99 epg), moderate (100-399 epg), and high (>400 epg) infection based on schistosome egg counts on the Kato-Katz faecal smears, and according to the WHO criteria [23], were used in this experiment. The ability of the LAMP assay to detect and differentiate "low", "moderate", and "high" infection intensities was determined, and the results were compared with those obtained using the Kato-Katz procedure.

Specificity
Test. The ability of the LAMP technique to specifically detect S. mansoni in faecal samples, and not other helminths such as the soil-transmitted helminths (STH), which often co-occur with S. mansoni in the same individual or within the same locality (and whose eggs and/or larval stages also occur in the host faecal samples), was determined, given that other helminths can potentially interfere with the specificity of the LAMP assay for detection of S. mansoni in faecal samples. DNA was extracted from faecal samples of individuals infected with Ascaris lumbricoides, hookworm, or Trichuris trichiura (common parasites that often co-occur with S. mansoni in endemic areas), and amplified in the assay using the S. mansoni LAMP specific primers.

Comparison of the Results of the LAMP Assay with Those of the Kato-Katz Procedure.
Comparisons were made between the results of the LAMP assay and those of the Kato-Katz procedure (which is the standard test for diagnosis of S. mansoni infection) using Cohen's kappa coefficient procedure described by Viera [24] to establish the degree of agreement between the two techniques. This was calculated by the formula K= (OA-AC / (1-AC), where K is Kappa coefficient, OA is observed agreement, and AC is agreement by chance.

Study Approvals. This study was approved by the Scientific and Ethics Review Unit (SERU) of the Kenya Medical
Research Institute.

Amplification of S. mansoni DNA Using LAMP Primers
Targeting the 121-bp Repeat Sequence. The LAMP primers used in this study consistently amplified the 121-bp repeat sequence (ladder like sequence) in the different life cycle stages of S. mansoni under the isothermal amplification conditions of 63 ∘ C for 60 min as anticipated. These results showed that the developed LAMP assay could successfully and reliably amplify the target 121 pb repeat sequence in S. mansoni across all the larval and adult stage of the parasite (Figures 1(a) and 1(b)).  Figure 2(a) shows separation of amplification products on agarose gel after electrophoresis, and Figure 2(b) shows LAMP products in reaction tubes after staining with SYBR Green I dye.

Sensitivity of the LAMP Assay.
The ability of LAMP technique to detect the lowest amount of S. mansoni egg DNA present in a faecal sample was determined after serial dilutions of the sample. Tenfold serial dilutions of total genomic DNA derived from faecal sample obtained from individuals infected with S. mansoni was used to test the sensitivity of the LAMP assay. The results of this experiment are presented in Figure 3. It was observed that the lowest detection limit of the LAMP reaction was as low as 32fg of S. mansoni egg DNA in a faecal sample. Direct observation of the amplicons in reaction tubes after staining with SYBR Green I dye indicated that all the serially diluted samples were positive for S. mansoni DNA because the samples color turned from orange to green, except for those that had less than 32fg of DNA and the negative control sample, as shown on the agarose gel picture in Figure 3.

Specificity of the LAMP Assay.
When faecal samples containing DNA of Ascaris lumbricoides, hookworms, or Trichuris trichiura eggs (common parasites that often cooccur with S. mansoni in endemic areas) were amplified in the LAMP assay using the S. mansoni LAMP specific primers, no amplicons were detected for A. lumbricoides, hookworm, or T. trichiura, and only samples with S. mansoni eggs were amplified. Figure   individuals yielded positive results in the LAMP assay while samples from individuals infected with the soil-transmitted helminths (STH) tested gave negative results in the assay. These results suggest that the assay is specific for S. mansoni and will not detect the STH, whose eggs and/or larval stages also commonly occur in human faecal samples.

Discussion
The LAMP assay described in this paper provides a simple, rapid, accurate, and reliable means for detecting S. mansoni infection in human faecal samples. While the assay was initially optimized using faecal samples from mice infected with S. mansoni under lab conditions, subsequent evaluation of the assay using faecal samples from S. mansoni infected school children from an endemic locality in Kenya indicated that it could reliably and consistently detect S. mansoni infections in human faecal samples. The assay relies on the amplification of a 121bp repeat sequence present in S. mansoni using primers initially developed for detection of S. mansoni infections in the snail intermediate host [13]. The primers used in this study were, apparently, capable of amplifying the repeat sequence in all the life cycle stages of the parasite. A LAMP assay was recently developed for early detection of S. mansoni infection in rodent faecal samples, and it targeted a sequence corresponding to a mitochondrial minisatellite DNA region [25]. While this assay was found to reliably detect S. mansoni DNA in mouse faecal samples, up to levels within 1fg DNA, it would be interesting to see how this assay will perform using human faecal samples. In the assay developed in the present study, the detection limit was found to be 32 fg DNA. Further evaluation of this assay will certainly be necessary.
Although it remains to be established how the present assay will perform using faecal samples collected in areas where schistosomiasis control and elimination are being implemented using chemotherapy; we have found the assay to perform satisfactorily using human faecal samples with infection intensities in all the 3 categories of the WHO criteria of low (1-99 eggs per g (epg) of faeces), moderate (100-399 epg), and high (>400 epg) infection intensities [23]. However, the assay could not differentiate between the different categories of infection intensities.
The LAMP assay results were generally similar to those of the microscopy-based Kato-Katz procedure, with sensitivity and specificity of the two assays being within the same range.
While the microscopy-based Kato-Katz procedure for detection of S. mansoni infection is considered cumbersome to use and requires expertise to perform, the LAMP assay is relatively simple to perform and it requires no expertise to perform. It can employ a simple process for detection of amplified products which relies on use of SYBR Green I dye, and the products can then be visualized with a naked eye, making it possible to use the assay under field conditions. Furthermore, the assay can potentially be adapted for analysis of several samples at the same time and is therefore ideal for use in resource-limited settings.

Conclusion
In spite of the advantages of the LAMP assay over the microscopy-based Kato-Katz procedure, the LAMP assay developed will require further optimization and refinement, before it can be applied routinely for diagnosis of S. mansoni. Specifically, the DNA extraction procedure needs to be simplified yet remain efficient and inexpensive. Also, availability of a premix version of the reagents will further increase efficiency and turnaround time. Follow-up studies have been carried out for validation and optimization of LAMP assays for practical use in field laboratories for diagnosis of schistosomiasis [26], but the initial study had shortcomings on the field applicability and practical use scale-up [14].
Although this assay was tested in a closed single-tube, there is concern that contamination of the working area could occur if the reaction tube is opened after amplification. As such, prevention of cross-contamination in the test samples is a priority when performing this assay, and this can be realized by use of highly sterilized apparatus, frequent change of gloves between every assay, and strict separation of reagents used in the assay.
In conclusion, good progress has been made in the development of a LAMP test for detection of S. mansoni infection in faecal samples which can potentially also be used to support schistosomiasis control and elimination efforts in the endemic areas. A sensitive and specific diagnostic system for S. mansoni infection is critical for precise detection of the infection as well as for evaluation of mass drug administration (MDA) efforts (the WHO recommended schistosomiasis control and elimination strategy) and as a potential tool for disease epidemiological surveillance and in operational research.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval
This study was approved by the Scientific and Ethics Review Unit (SERU) of the Kenya Medical Research Institute (KEMRI). All study participants found to be infected with S. mansoni and other soil-transmitted helminthes received the WHO recommended standard treatment for STHs of 400mg albendazole [25] in a single dose under the supervision of a qualified clinician. Collection of faecal samples used in the study did not pose any risks or cause any harm to study participants.