Recent advances in nucleic acid-based methods for detection of helminth infections and the perspective of biosensors for future development

Abstract

Pathogenic helminth infections are responsible for severe health problems and economic losses worldwide. Timely and accurate diagnosis of helminth infections is critical for adopting suitable strategies for pathogen control. Here, we review recent advances in nucleic acid-based diagnostic methods, including polymerase chain reaction, quantitative qPCR, loop-mediated isothermal amplification and recombinase polymerase amplification, and discuss their advantages and disadvantages for diagnosing helminth infections. In addition, we highlight recent advances in biosensors for the detection of nucleic acid biomarkers that can potentially be used for the diagnosis of helminth infection.

Introduction

Helminths are invertebrates morphologically characterized by elongated, flat or round bodies, and include trematodes, cestodes and nematodes (Kaliappan et al., 2013). Among them, soil-transmitted helminths (STHs) and schistosomes are responsible for the most prevalent parasitic infection of humans living in resource-limited areas of developing countries. Studies indicate that STHs, such as Ascaris lumbricoides and Trichuris trichiura, as well as hookworms, affect over 1.5 billion people worldwide (WHO, 2019a). Schistosomiasis caused by the genus Schistosoma affects at least 220.8 million people worldwide (WHO, 2019b). Currently, it is still prevalent in 78 countries where 700 million people are at the risk of contracting schistosomiasis. The World Health Organization has already developed a roadmap to control and eliminate the related neglected tropical diseases caused by these helminths. The major control strategy against these helminth infections is mass drug administration (Warren et al., 2001; Minetti et al., 2016). Consequently, it is critical to timely and accurately identify helminth infections to determine whether drug treatment is working efficiently and then deploy appropriate strategies for disease intervention. Currently, several ‘gold standard’ techniques are used for diagnosing human infection including the identification of parasites in blood (lymphatic filariasis (LF)) or skin snip biopsy (onchocerciasis), eggs or larvae in stools or urine (STHs and schistosomiasis) by microscopy and the detection of antibodies or pathogen antigens circulating in blood (onchocerciasis, LF and schistosomiasis) or urine (schistosomiasis) by immunological methods. However, these approaches usually have some limitations such as relatively low sensitivity (particularly for low and mid intensity infections), relatively low-throughput, increased time-consumption, skilled personnel being required for operation and lack of onsite applicability. In addition, serological methods for helminth diagnosis have limited ability to distinguish between ongoing and previous infections (Hinz et al., 2017).

It is essential to develop rapid and sensitive techniques with high-throughput for diagnosing helminth infections. Immunodiagnostic techniques such as the detection of antibodies and antigens, and molecular diagnoses such as polymerase chain reaction (PCR), quantitative qPCR (qPCR), loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) can fulfil these requirements. In addition, nucleic acid-based methods also offer the most rapid and sensitive detection with the potential to distinguish different species and to monitor potential genetic variation in pathogen populations owing to selection pressure caused by drug usage. Although the broad application of current nucleic acid-based detection methods for surveying, monitoring and evaluating helminth control programs have so far been hindered by some obvious flaws such as lack of standards of sample preparation and nucleic acid isolation, lack of suitable internal control for target deoxyribonucleic acid (DNA)/(ribonucleic acid) RNA amplification, potentially high background noise, potential amplification inhibition and lack of accurate data interpretation for intensity of infection, these techniques may best guide decision making for controlling helminth diseases because of their higher sensitivity, rapid and high-throughput (Corrado et al., 2016). Hence, we mainly focused on nucleic acid-based diagnostic methods including PCR, qPCR, LAMP and RPA, reviewed their recent advances and highlighted the potential usage of biosensors, a modern technique, for the diagnosis of helminth infection.

Nucleic acid-based methods for detection of helminth infections

To date, several different nucleic acid-based methods have been available for the detection of helminth infections such as PCR, multiplex PCR, qPCR, LAMP and RPA. These methods offer considerable advantages such as precision, timeliness and sensitivity of parasite detection. We have briefly reviewed recent advances in each method and then discussed their utility in the diagnosis of helminth infections in the following sections.

PCR

The PCR is based on using the ability of DNA polymerase to synthesize a new strand of DNA complementary to the template strand. The technique has been successfully used for detecting schistosome DNA from host serum, urine and faeces as well as water containing cercaria and infected snails (Enk et al., 2012; Sarhan et al., 2015; Sengupta et al., 2019). A new PCR-based approach has been shown to identify different Schistosoma species (S. mansoni, S. japonicum, S. haematobium, S. intercalatum and S. bovis) and is applicable to human urine samples with high specificity and sensitivity (Sandoval et al., 2006). Recently, a study documented that PCR assay targeting the 121-bp tandem repeat sequence sensitive for S. mansoni and the Dra1 tandem repeat sequence specific for S. haematobium can distinguish Schistosoma species for diagnosis of early schistosomiasis (Bonnefond et al., 2019). For filarial pathogens, the amplification of genomic DNA repeat sequences (188 bp DNA repeat sequence) SspI from Wuchereria bancrofti (Zhong et al., 1996), Hha I repeat region from Brugia malayi (Fischer et al., 2000), LLMF72 or a repeat region (15r3) from Loa loa (Fink et al., 2011) and the O-150 repeat sequence from Onchocerca volvulus (Toé et al., 1998) by the PCR has been used to detect pathogen infection. In addition, the PCR can also amplify W. bancrofti DNA from host sputum, serum and urine with high diagnostic accuracy (Kagai et al., 2008; Ximenes et al., 2014). Moreover, the detection of Taenia solium DNA from cerebrospinal fluid (CSF) samples by the PCR has diagnostic potential for neurocysticercosis, which cannot be detected using radiology or serology (Michelet et al., 2011; Chaya and Parija, 2014). Furthermore, the PCR was used to amplify pathogen-specific DNA in host saliva and urine, which may become a new type of non-invasive biomarker for the diagnosis of schistosomiasis japonica (Weerakoon and McManus, 2016). In addition, this has been shown to have good potential for detecting early stage of schistosome infection by amplifying schistosome specific DNA from host sera using the PCR (Xia et al., 2009; Kato-Hayashi et al., 2010).

The traditional PCR can be modified by incorporating additional primers and amplification to increase the sensitivity for detecting helminth infection. For example, a two-step semi-nested PCR for amplifying Oesophagostomum bifurcum egg DNA in human faecal samples was shown to achieve a specificity of 100% (Verweij et al., 2000). In addition, the semi-nested PCR technique showed a 1 000 000-fold increase to the detection limit for W. bancrofti, compared with that of the conventional PCR (Ximenes et al., 2014).

By incorporating fluorescent dyes or fluorescently labelled oligonucleotide probes, the amplified DNA can be quantified. This is called a real-time qPCR, which is now one of the most widely used tools in research and clinical diagnosis. The qPCR has been used to detect schistosomes, STHs (e.g. S. stercoralis, T. trichiura, A. duodenale, N. americanus and A. lumbricoides) and O. volvulus infection (Morales-Hojas et al., 2001; Lier et al., 2009; O'Connell and Nutman, 2016a; Prince-Guerra et al., 2018). In schistosomes, the qPCR was shown to identify considerably more positive cases than the Kato–Katz method and could be adopted as a suitable strategy for schistosomiasis elimination (He et al., 2018).

Recently, microRNAs (miRNAs) have been considered as a new class of biomarkers for diagnosing many diseases, particularly for circulating miRNAs (Cheng, 2015). For example, Cheng and coworkers (2013) identified five schistosome-specific miRNAs (Bantam, miR-3479, sja-miR-8185, miR-10 and miR-3096) in the plasma of rabbits infected with S. japonicum. Four out of these five miRNAs were shown to be significantly abundant in plasma of S. japonicum-infected mice, suggesting these miRNAs can serve as a potential biomarker for schistosomiasis diagnosis. More interestingly, several further studies demonstrated that parasite-derived miRNAs (miR-277, miR-3479-3p and bantam) could detect individuals infected with S. mansoni or S. haematobium from low and high-infection intensity sites by the real time qPCR (Hoy et al., 2014; Meningher et al., 2017).

Apart from diagnosis, the PCR and qPCR can also be used to distinguish between species of parasites. For example, a copro-PCR method targeting a segment of the internal transcribed spacer (ITS) region and COX1 gene was shown to distinguish between O. viverrini and C. sinensis; results suggested that C. sinensis was present in an area in central Thailand where it had not been previously detected (Traub et al., 2009; Cai et al., 2014). In addition, the PCR and qPCR for diagnosis of STH infections and for monitoring genetic variation of helminths under mass drug administration have been elegantly reviewed (Corrado et al., 2016; O'Connell and Nutman, 2016b; Papaiakovou et al., 2019). The qPCR was able to detect and distinguish filarial infection in domestic cats among B. malayi, D. immitis and D. repens as well as A. (Dipetalonema) reconditum, showing more sensitivity than microfilariae detection (Wongkamchai et al., 2014).

Unlike the conventional qPCR, the Droplet Digital™ PCR (ddPCR) can perform absolute quantification of PCR products without the need for generating standard curves (Elmahalawy et al., 2018). In principle, the ddPCR can fractionate a loading sample into thousands to millions of droplets, and PCR amplification of the template molecules occurs in each individual droplet, followed by a real time or endpoint detection of the amplification. The distribution of target sequences into droplets is described by the Poisson distribution, thus allowing accurate and absolute quantification of the target without using reference materials with known target concentrations (Gutierrez-Aguirre et al., 2015). The ddPCR has been successfully used to detect W. bancrofti DNA (targeting the long DNA repeat (LDR) element) and B. malayi DNA (targeting the Hha I element) in host blood samples and mosquito vectors with high sensitivity and specificity (Jongthawin et al., 2016). In gastrointestinal (GI) nematode parasitic infection, the ddPCR can detect and distinguish major GI nematodes by targeting the internal transcribed spacer region 2 (ITS2) of the ribosomal RNA gene in ovine parasites such as Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus species (Elmahalawy et al., 2018). In addition, the ddPCR assay exhibited a high level of diagnostic performance compared with Kato–Katz and antibody-based ELISA for diagnosis of Schistosoma infection although it is expensive and requires specialized equipment (Weerakoon et al., 2017a,b; Cai et al., 2019).

Multiplex PCR

The multiplex PCR can amplify several different DNA sequences simultaneously using multiple primers (Touron et al., 2005; Hawkins and Guest, 2017). Using selected target sequences for amplification, this PCR approach can not only detect the pathogen's genotype, but also distinguish among species. For example, S. mansoni and S. haematobium from a set of duplicate stool samples can be distinguished using primers for species-specific amplification of cytochrome C oxidase genes by the multiplex PCR, and the results showed a significant correlation with microscopic egg counts for S. mansoni in stool and S. haematobium in urine (ten Hove et al., 2008). In addition, the multiplex PCR was also shown to distinguish between differential infections by T. solium and T. asiatica (Wicht et al., 2010; Jeon and Eom, 2013). Moreover, the multiplex PCR has been shown to discriminate N. americanus, A lumbricoides and T. trichiura infections from faecal samples (Phuphisut et al., 2014). In the Philippines, a study indicated that the multiplex PCR could simultaneously detect two cestode parasites T. saginata and T. solium alongside A. lumbricoides and hookworms in stool samples (Gordon et al., 2015). Moreover, a recent study indicated that a multiplex single-tube PCR assay can specifically discriminate trematode, cestode and nematode species by targeting the 28S rRNA gene (trematode), COX1 gene (cestode) and COX1 gene (nematode) (Wong et al., 2019).

By incorporating Luminex beads or fluorescent dye, the multiplex PCR can be modified as qPCR to improve its sensitivity and specificity. For example, several helminths including A. lumbricoides, A. duodenale, N. americanus and S. stercoralis can be quantitatively co-detected using a Luminex bead-based multiplex qPCR, showing high sensitivity (90–100%) and specificity (95–100%) from 319 stool samples (Taniuchi et al., 2011). In addition, a multiplex qPCR for detecting N. americanus and A. duodenale also showed 100% specificity and 98.5 to 100% sensitivity (Verweij et al., 2007). Moreover, a pentaplex qPCR was not only able to simultaneously detect A. duodenale, N. americanus, A. lumbricoides and S. stercoralis but also to show significantly increased sensitivity of detection (up to 62.3% compared with the detection of microscopic examination of 7.8%) for one or more helminth species (Basuni et al., 2011). Recent studies also indicated that a multiplex TaqMan qPCR assay can specifically identify encapsulated Trichinella species in North America (Almeida et al., 2018), and a multiplex qPCR can detect and quantify DNA from F. hepatica and the intermediate snail host, A. tomentosa, in water samples (Rathinasamy et al., 2018).

Loop-mediated isothermal amplification (LAMP)

Although the PCR and multiplex PCR offer significant advantages for detecting helminth infections such as more sensitivity and specificity, high-throughput and cost-effectiveness, both methods are heavily dependent on a particular instrument or elaborate detection methods. Alternatively, LAMP can amplify target DNA by DNA polymerase based on a set of specific primers under a constant temperature of 60–65°C without a thermocycler and accumulated 10⁹ copies of target DNA in less than an hour, with a detection limit of a few copies. More importantly, the amplification endpoint could be observed using agarose gel electrophoresis for visual observation of DNA under UV light and using turbidity determination that is more suitable for field research. As a pyrophosphate ion is released once a nucleotide is added to the DNA strands, a visible white precipitate of magnesium pyrophosphate is formed and used to determine whether the target nucleic acid was amplified or not.

The use of LAMP for helminth detection has been recently summarized and reviewed (Amoah et al., 2017; Deng et al., 2019). Here, we highlight only the studies related to schistosomes. LAMP has been successfully used to amplify the SjR2 gene of S. japonicum from the blood samples of infected rabbits at 3^rd day post-infection, indicating the potential of early detection of S. japonicum infection (Xu et al., 2015). In addition, by targeting S. haematobium ribosomal intergenic spacer DNA, LAMP was shown to detect S. haematobium DNA in heated pellets from patients’ urine samples without requiring a complicated procedure for DNA extraction with 100% sensitivity and 86.67% specificity (Gandasegui et al., 2015). Moreover, LAMP can detect miracidium DNA at the first day of snail exposure (intermediate hosts) to Schistosoma (Abbasi et al., 2010) and also detect infection in pooled samples of field-collected snails (Tong et al., 2015). Furthermore, LAMP was shown to be an effective means to detect low-intensity infection of schistosomiasis (Lodh et al., 2017). Overall, these studies suggested that LAMP might become a new surveillance and response tool for schistosomiasis control.

Compared to the PCR, LAMP has better sensitivity that could enhance the effectiveness of surveillance and mass drug administration of control programs for helminth diseases. However, primer design is a critical part of LAMP assay, because the use of multiple primers for LAMP assay may increase the risk of primer–primer hybridization, resulting in template-free amplification that needs to further evaluate false-positive outcomes (Hsieh et al., 2014). Similar to the PCR, LAMP cannot be used for detecting unknown or unsequenced targets of pathogens since specific DNA sequences are required for primer design (Mori and Notomi, 2020).

Recombinase polymerase amplification (RPA)

RPA is another fast and sensitive isothermal amplification-based method for amplifying target DNA or RNA using a bacteriophage derived recombinase in combination with a polymerase (Piepenburg et al., 2006; Euler et al., 2012). RPA comprises three core enzymes including a recombinase, a single-stranded DNA-binding protein (SSB) and a strand-displacing polymerase. Recombinases pair oligonucleotide primers with a homologous sequence, and the SSB binds to displaced strands to prevent primer displacement (Piepenburg et al., 2006). Finally, the strand displacing polymerase begins DNA synthesis where the primer has bound to the target DNA (Euler et al., 2012).

Studies have demonstrated that RPA can detect small amounts of viral, bacterial and parasitic DNA with high sensitivity and specificity (Shahin et al., 2018). In helminths, most of these studies focused on schistosomes. For example, RPA was used to amplify the SjR2 gene of S. japonicum, and the result indicated that this assay could detect 0.9 fg S. japonicum DNA within 15 min and distinguish S. japonicum from other worm species (Xing et al., 2017). In addition, RPA combined with oligochromatographic lateral flow strips was shown to detect low levels of S. haematobium DNA (Dra1) from crude urine (Rosser et al., 2015). Similarly, an RPA-based lateral flow was also developed in S. mansoni by amplifying the 28S rDNA region and the result indicated that the lower limit of detection for this assay was 10 pg DNA with the lower test parameters permitting sufficient amplification being 6 min and 25°C (Poulton and Webster, 2018).

RPA can amplify as low as 1–10 DNA/RNA target copies with a rapid turnaround time (less than 20 min) and simple set-up at a single low temperature (37–42°C). Consequently, it provides significant advantages compared with other isothermal methods such as LAMP. In addition, RPA has been shown to amplify diverse targets, including RNA, miRNA, ssDNA and dsDNA from a wide variety of organisms and samples (Euler et al., 2012). It may be necessary to develop an RPA assay to detect pathogen-specific small RNAs such as miRNAs in future for diagnosing helminth infections. Moreover, RPA has good potential for field application and the use of this method can reduce diagnostic expenses in resource-limited countries (Ajibola et al., 2018). Although PCR, LAMP and RPA methods can also be ‘sensed’ by incorporating a fluorescent dye or probe and the amplified signal can be detected and presented by an electrical device, these methods usually carry disadvantages such as requiring a multi-step set up, being time-consuming, requiring a trained operator for use and being difficult to apply in the field.

Biosensors for nucleic acid biomarker detection

With the development of modern electrical and chemical techniques, biosensors, a powerful and innovative analytical device shows excellent characterizations, being highly sensitive, rapid, simple, low-cost and field-applicable that can be widely used for drug discovery, diagnosis, biomedicine, food safety, processing and environmental monitoring.

The first biosensor invented by Clark and Lyons (1962); Cheng and coworkers (2013) for measuring glucose levels in biological samples was based on electrochemical detection of oxygen or hydrogen peroxide (Fracchiolla et al., 2013). Since then, incredible progress has been made in terms of both the technology and applications of biosensors. Generally, biosensors consist of at least three elements, namely, a biological capture molecule (bioreceptor), a method of converting capture molecule-target interactions into a signal (transducer) and a data output system. A bioreceptor is an immobilized sensitive biological element (antibody, DNA probe or enzyme) recognizing the analytic (antigen, complementary DNA or enzyme substrate). A transducer is used to convert the (bio) chemical signal resulting from the interaction of the analytic with the bioreceptor into an electronic signal. The intensity of signals generated corresponds to the analyte concentration.

Currently, most reported biosensors have been designed to detect nucleic acid biomarkers and protein biomarkers related to pathogen infection. Nucleic acid biomarker-based biosensors show good potential for the detection and diagnosis of various diseases such as cancer, diabetes, cardiovascular diseases, tuberculosis, hepatitis, dengue and food-borne diseases such as diarrhea, cholera and salmonellosis (Bora et al., 2013). Based on transducer methods, these biosensors can be divided into electrochemical biosensors and optical biosensors.

Electrochemical detection techniques

Various electrochemical biosensors have been reported to detect nucleic acid biomarkers during parasite infection, particularly protozoan infections. For example, Mohan and coworkers used a kind of sol–gel synthesized nickel oxide (NiO) film deposited onto an indium tin oxide (ITO) coated glass plate to immobilize a 23-mer DNA sequence (oligonucleotide) from 18S rRNA gene sequences from L. donovani for the development of a DNA biosensor. Response studies of the ss-DNA/NiO/ITO bioelectrode in the presence of methylene blue redox dye as a redox mediator indicated a linear response in the wide concentration range of 2 pg/mL to 2 μg/mL of complementary target genomic DNA (parasite DNA), suggesting the potential of this biosensor for diagnosis of Visceral leishmaniasis (Mohan et al., 2011). In addition, a gold nanoleaf-based biosensor was constructed by immobilizing Leishmania major-specific DNA for detecting parasite infection. Further analysis indicated that the biosensor could detect a synthetic DNA target in the range of 1.0 × 10⁻¹⁰ to 1.0 × 10⁻¹⁹ mol L⁻¹ with a detection limit of 1.8 × 10⁻²⁰ mol L⁻¹, and genomic DNA in the range of 0.5–20 ng μL⁻¹ with a detection limit of 0.07 ng μL⁻¹. More importantly, the biosensor could not only detect L. major in patient samples but also distinguish L. major from a non-complementary-sequence oligonucleotide and L. tropica with high selectivity (Moradi et al., 2016).

Recently, several electrochemical biosensors were also developed for detecting nucleic acid biomarkers for helminth infection. For example, Santos et al. used AuNPs that are the most commonly used nanomaterial in biosensor development through biomolecule conjugation and then obtained compelling sol–gel derivative films to immobilize AuNPs using the organosilane, 3-mercaptopropyltrimethoxysilane. Further analyses indicated that the biosensor could recognize the S. mansoni genome sequence at different concentrations in samples of urine, CSF and serum with the limit detection of 0.6 pg μL⁻¹ (Santos et al., 2019). The authors also reported the development of another biosensor by using a self-assembled monolayer of mercaptobenzoic acid immobilizing nanostructures composed of gold nanoparticles (AuNPs) and magnetite nanoparticles (Fe₃O₄_NPs) to recognize specific nucleotide sequence of S. mansoni present in CSF and serum samples, and this biosensor showed a DNA detection limit of 0.781 and 0.685 pg μL⁻¹ (Santos et al., 2017).

Apart from the detection of nuclear acid biomarkers, some biosensors were also developed to detect specific antibodies/antigens related to helminth infection. For example, a piezoelectric immunosensor assay was developed with immobilizing immunoglobulin G (IgG) as a probe to detect S. japonicum circulating antigens. By immobilizing purified IgG from infected rabbit's sera with S. japonicum, the developed biosensor was shown to specifically detect S. japonicum circulating antigens (Cheng et al., 2008). In addition, a superhydrophobic surface-based magnetic electrochemical immunoassay for the detection of S. japonicum antibodies was also developed and the results indicated that the biosensor is quantitatively sensitive to SjAb concentrations ranging from 2 ng mL⁻¹ to 15 μg mL⁻¹, with a detection limit of ~1.3 ng mL⁻¹ (Nie et al., 2012). Moreover, an electrochemical immunosensor was also developed by immobilizing W. bancrofti epitope antigens (Wb/ALT2-A5) on a screen-printed carbon electrode by their amine groups via chitosan film by coupling with glutaraldehyde as the crosslinker. The detection limitation of this biosensor was 0.002 μg mL⁻¹ for human serum with a sensitivity of 1.86 μA (Prado et al., 2018).

Optical detection techniques

Optical detection techniques are those that can sense phenomena related to the interaction of target compounds with the analytes. Usually, these biosensors can measure luminescence, fluorescence or colour changes. Several biosensors have used optical detection techniques to detect nucleic acid biomarkers for parasite infection, particularly for malaria. For example, Ittarat and coworkers described a genosensor based on quartz crystal microbalance (QCM) to detect malaria infection. In this method, the QCM surface was immobilized with a malaria biotinylated probe. Specific DNA fragments of malaria-infected blood were amplified. Hybridization between the amplified products and the immobilized probe resulted in quartz frequency shifts that were measured by an in-house frequency counter. Further analysis indicated that this biosensor can differentially diagnose blood infected with P. falciparum from that infected with P. vivax and no cross reaction with human DNA (Ittarat et al., 2013). In helminths, a diffraction-based optical biosensor was developed although it detects a protein biomarker (a 31-kDa recombinant antigen called NIE derived from S. stercoralis) to diagnose strongyloidiasis. The biosensor showed excellent agreement (κ = 0.98) with results obtained by an NIE-based ELISA method (Pak et al., 2014).

In addition, as stated above, miRNAs are recognized a novel kind of biomarker for disease diagnosis, particularly for cancer, for example in early diagnostics, predicting responses to therapy, monitoring relapse and molecular classification of tumours. A recent study documented that circulating miRNAs associated with cancer derived from serum can be detected with a biosensor using DNA concatemer amplification (Hong et al., 2013). If sensitive and effective nucleic acid biomarkers can be identified during helminth infection, and suitable and effective probe layers can be immobilized on a transducer surface, we believe that electrochemical biosensors can become a next generation technique for diagnosis of helminth infections.

All the nucleic acid-based methods discussed and the related targets for detecting helminth infections are summarized and listed in Table 1.

Table 1.

Nucleic acid-based methods for the detection of helminth infections

Pathogens	Target genes	Detection methods	Types of sample	References
S. japonicum	SjR2	PCR	Faeces	(Fung et al., 2012)
S. mansoni	121-bp tandem repeat sequence	PCR	Sera	(Hussein et al., 2012)
S. mansoni S. haematobium	28S rDNA	PCR	Urine	(Sandoval et al., 2006)
S. haematobium	Dra 1	PCR	Urine	(Ibironke et al., 2011; Lodh et al., 2014)
S. japonicum	COX1	PCR	Sera and urine	(Kato-Hayashi et al., 2015)
S. japonicum	Repetitive retrotransposon SjCHGCS19	PCR	Serum	(Xu et al., 2017)
T. saginata T. solium	HDP2	qPCR	Faeces	(Flores et al., 2018)
W. bancrofti	LDR	qPCR	Blood	(Plichart and Lemoine, 2013)
A. lumbricoides A. duodenale N. americanus S. stercoralis T. trichiur	ITS1, ITS2	qPCR	Faeces	(Mejia et al., 2013)
S. japonicum	NAD6 and NAD1	PCR and qPCR	Faeces	(Gordon et al., 2012)
S. mansoni S. haematobium	ITS2	qPCR	Faeces	(Meurs et al., 2015)
N. americanus A. duodenale A. ceylanicum A. caninum A. braziliense	ITS2	qPCR	Faeces	(Ngui et al., 2012)
T. trichiura A. lumbricoides	18S rDNA and ITS1	qPCR	Faeces	(Liu et al., 2013)
A. lumbricoides A. duodenale N. americanus S. stercoralis	ITS1, ITS2 and 18S rDNA	Multiplex qPCR	Faeces	(Basuni et al., 2011)
T. solium T. saginata	COX1	Multiplex PCR	Faeces	(Jeon et al., 2011)
S. japonicum S. mansoni	NAD1, 2, 6 and COX2 NAD5, 6 and COX2	Multiplex PCR	Faeces	(Gobert et al., 2005)
F. hepatica A. tomentosa	ITS-2	Multiplex PCR	Water	(Rathinasamy et al., 2018)
Trichinella Spp.	ITS1 and ITS2	Multiplex qPCR	Meat	(Almeida et al., 2018)
W. bancrofti	Repeated DNA sequence	Semi nested PCR	Urine and sera	(Ximenes et al., 2014)
W. bancrofti	Long direct repeat element (LDR)	ddPCR	Blood, vectors	(Jongthawin et al., 2016)
B. malayi	HhaI element	ddPCR	Blood, vectors	(Jongthawin et al., 2016)
S. japonicum	NAD 1 and SjR2	ddPCR	Urine, faeces, salivary gland and serum	(Weerakoon et al., 2017a; Weerakoon et al., 2017b; Cai et al., 2019)
H. contortus, T. circumcincta and Trichostrongylus spp.	ITS2	ddPCR	Parasite DNA	(Elmahalawy et al., 2018)
T. solium	HDP2	Semi nested PCR	Cerebrospinal fluid	(Hernández et al., 2008)
S. japonicum	SjR2	LAMP	Serum	(Xu et al., 2010)
S. mansoni	Mitochondrial minisatellite DNA	LAMP	Faeces	(Fernández-Soto et al., 2014)
S. stercoralis	28S rDNA	LAMP	Faeces	(Watts et al., 2014)
C. sinensis	COX1	LAMP	Faeces	(Rahman et al., 2017)
T. trichiura N. americanus A. lumbricoides	β-Tubulin	LAMP	Faeces	(Rashwan et al., 2017)
T. canis	ITS2	LAMP	Faeces	(Khoshakhlagh et al., 2017)
E. multilocularis	COX1	LAMP	Faeces	(Feng et al., 2017)
N. americanus	ITS2	LAMP	Faeces	(Mugambi et al., 2015)
B. malayi B. timori	Hha I	LAMP	Blood	(Poole et al., 2012)
P. westermani	ITS2	LAMP	Sputum and pleural fluid	(Chen et al., 2011)
S. haematobium	Dra 1	RPA	Urine	(Rosser et al., 2015)
S. mansoni	28S rDNA and ITS	RPA	Genomic DNA	(Poulton and Webster, 2018)
S. japonicum	SjAb	Immunosensor	Sera	(Nie et al., 2012)
S. mansoni	Genomic DNA	Genosensor	Urine, cerebrospinal fluid and serum	(Santos et al., 2019)
S. mansoni	Genomic DNA	Genosensor	Cerebrospinal fluid and sera	(Santos et al., 2017)
S. japonicum	S. japonicum circulating antigens	Immunosensor	Sera	(Cheng et al., 2008)
W. bancrofti	W. bancrofti epitope antigens	Immunosensor	Sera	(Prado et al., 2018)

Advantages and limitations of nucleic acid-based methods for detection of helminth infections

Each nucleic acid-based method has its own advantages and limitations. We have summarized them as follows. The PCR is one of the most promising approaches to detect parasite DNA or RNA in human, animal or vector samples owing to its higher diagnostic sensitivity and specificity compared to microscopic examination or immunodiagnostics. However, it has limitations, such as the high cost of the instrumentation required for thermal cycling and signal detection, and the need for well-equipped laboratories (Verweij and Stensvold, 2014). Both qPCR and ddPCR methods can quantitatively determine helminth infection as they are amenable for automation and have relatively high-throughput, whereas the ddPCR can increase the accuracy of quantification at low-target concentrations. Unlike the PCR, LAMP offers sensitivity, high efficiency, rapidity and simplicity for identifying certain infectious diseases, although the specificity is yet to be validated. The main shortcoming of LAMP is the false-positive production caused by non-specific amplification resulting from primers, which could be improved by optimization of the reaction conditions, primer design, detection approaches, etc. RPA offers several advantages such as operation at a low and constant temperature, without the need for an initial denaturation step or the use of multiple primers. Biosensor-based methods have the potential to deliver rapid, accurate and affordable diagnosis of parasite infection. In addition, biosensors can improve diagnosis and follow-up treatment in terms of rapidity, real-time performance and expense compared to current detection technologies such as PCR and ELISA. Furthermore, the use of biosensors offers significant advantages such as small fluid volume manipulation and high-integration capability that facilitates the development of portable devices and ease of use, allowing use by non-specialized personnel in non-centralized laboratories (Sin et al., 2014). The time-consuming preparative steps of gene probe evaluation make it hard to consider these as the basis of biosensors for in situ detection of pathogenic microorganisms. Although extensive efforts have been expended to develop biosensors, a relatively small number of analytics, particularly toxic materials, can be detected using commercially available devices (Alhamoud et al., 2019). Although recent advancements in nanotechnology are facilitating the development of smaller, less-expensive, multiplex, easy-to-use and fully integrated biosensors for diagnostics and food and water safety (Bouguelia et al., 2013; Verbarg et al., 2013; Chung et al., 2015), future research must also focus on simplifying their fabrication techniques, miniaturizing devices and developing the biocompatibility and environmental friendliness of the nanomaterials. Unfortunately, the preparation and manipulation of nanomaterials in a reproducible and economic fashion is still premature. More importantly, the development of point of care devices is the ultimate goal, particularly for human patients, which still faces a significant challenge. Consequently, techniques that employ and combine with chemistry, electrics, optics, material science and mechanical passivation approaches could speed up the development of biosensors for commercial requirements. The advantages and limitations of each nucleic acid-based method are summarized in Table 2.

Table 2.

Summary of advantages and limitations of each nucleic acid-based method

Techniques	Advantages	Limitations	References
PCR	Relatively high specificity and sensitivity; can detect small amounts of target genes; lower turnaround time; relatively high throughput; high dynamic range of detection.	Relatively expensive instruments are required, risk of false negatives in pathogen detection.	(Diguta et al., 2010; Agarwal et al., 2014)
Multiplex PCR	High sensitivity and specificity; can detect multiple genes simultaneously with relatively high-throughput.	Primer design is critical; the primers may interfere with each other during amplification.	(Hajia et al., 2014; Vică et al., 2016)
ddPCR	High sensitivity and specificity; accurate and absolute quantification of the target without using reference materials with known target concentrations; low copy detection for target	Expensive instruments are required.	(Gutierrez-Aguirre et al., 2015)
LAMP	Rapid, highly efficient and cost-effective; denaturation step not required; more rapid than other conventional PCR-based methods; can directly visualize results; less equipment.	Primer design is critical for LAMP assay; risk of false-positive results.	(Tomita et al., 2008; Hsieh et al., 2014; Mori and Notomi, 2020)
RPA	Cost-effective, highly sensitive and specific, short amplification time. Takes place at the ambient temperature without the need for an initial denaturation step or the use of multiple primers.	Risk of false-positive outcomes.	(Daher et al., 2016; Shahin et al., 2018)
Biosensors	High sensitivity and reliability; fast detection; reduction in reagent consumption; detection of small molecules, proteins and nucleic acid.	Not commercially available for most pathogens, further research required for developing the system.	(Sin et al., 2014; Luz et al., 2016; Janissen et al., 2017)

Conclusions

Traditional pathogen detection methods are usually time consuming, and associated with low sensitivity, selectivity or accuracy. Therefore, sensitive and specific methods for diagnosing parasitic diseases are urgently required. Recent advances in PCR, qPCR, ddPCR, LAMP and RPA are proving to be highly sensitive and specific diagnostic approaches. By identifying effective biomarkers during helminth infection, these approaches could be further improved by increasing the sensitivity of detection and diagnosis. Current biosensors have the potential to provide rapid, accurate and affordable diagnoses of parasite infection by offering significant advantages such as small fluid volume manipulation, high integration capability that facilitates the development of portable devices and ease of use. Therefore, the combination of these outcomes with advanced technologies such as nucleic acid-based biosensors may change the current scenario of clinical diagnosis.

Financial support

This study was, in part or in whole, supported by the National Key Research and Development Program of China (2017YFD0501306-3), the National Natural Science Foundation of China (Grants No. 31472187 and 31672550) and the Science & Technology Innovation Program of the Chinese Academy of Agricultural Sciences.

Ethical standards

Not applicable.

Conflict of interest

The authors declare that there are no conflicts of interest