Strongyloidiasis is a persistent human parasitic disease caused by the intestinal nematode, Strongyloides stercoralis. It is endemic in the tropical and subtropical regions of the world where sanitary conditions are poor, and is increasing in prevalence even in resource-rich settings due to widespread travel and migration [1]–[3]. Worldwide, strongyloidiasis is estimated to affect at least 370 million people [4]. The exact prevalence of strongyloidiasis is not known because in many tropical and subtropical countries S. stercoralis can infect up to 60% of the population [5]. The majority of infected individuals are either asymptomatic or display intermittent, subtle, non-specific clinical symptoms that do not come to medical attention. Moreover, due to the unusual ability of S. stercoralis to auto-infect, infection can be life-long with most patients remaining unaware of their infection [5], [6]. Immunosuppression in infected patients, particularly with corticosteroids, can lead to a hyper-infection syndrome with uncontrolled dissemination of larvae and an associated mortality of up to 80% if untreated [7]–[12].

Currently, several imperfect methods exist for diagnosing strongyloidiasis. Stool examination with microscopic identification of larvae is considered the gold standard diagnostic procedure [13], [14]), showing good specificity with experienced staff. However, because of low numbers of adult parasites and irregular larval output during chronic, asymptomatic disease, this method lacks sensitivity, with false negative results in up to 70% of proven infections [13], [15]–[17]. Diagnostic sensitivity can be improved by analyzing serial stool samples [14], [17]–[19], larval enrichment from fecal samples by Baermann or concentration methods, or by agar plate coproculture [13], [14], [18], [20]. However, these approaches are time consuming, require a fresh stool sample and special technical training, and still lack sufficient sensitivity since they rely on the presence of intermittently shed larvae in the stool. Immunological approaches for detecting parasite-specific antibodies in serum by indirect enzyme-linked immuosorbent assays (ELISA) are well-described [6], [13], [21]–[28]. Most employ crude S. stercoralis filariform larvae extract and achieve reasonably high diagnostic sensitivity (∼85%) but lower specificity because of cross-reactivity with other tissue helminth infections [6], [13], [29]. While generally effective and suitable for batch testing, ELISA-based tests require moderately-sophisticated laboratory facilities to perform, limiting their use in many regions where S. stercoralis is endemic.

To circumvent the cross-reactivity associated with crude larval extracts, focus has recently turned to the use of recombinant antigens for Strongyloides serodiagnostics. In 2002, Ravi and colleagues [30] identified a 31 kDa recombinant antigen from an S. stercoralis L3 cDNA library which they named NIE. An NIE-based immunoassay had excellent sensitivity and did not cross-react with samples from individuals with other parasitic infections [13], [28], [31].

In this report, we describe a rapid and high-sensitivity assay for S. stercoralis antibody using recombinant NIE antigen and a novel diffraction-based optical biosensor technology. The dotLab mX System (Axela, Inc., Toronto, ON) utilizes diffractive optics technology (dot) [32], [33] to provide label-free analysis of biomolecular interactions in real-time. Interactions occur in high precision, disposable, plastic biosensors which consist of a linear array of assay spots along the bottom of a 10 µL flow channel (Figure 1A). Each spot is comprised of capture molecules arranged in a defined pattern of parallel lines creating a diffraction grating. When illuminated with a laser, the grating generates a predictable diffraction image (Figure 1B) that increases in intensity as ligands bind. Changes in diffraction image intensity are monitored using a photodiode detector and yield real-time measurement of the binding events (Figure 1C). This approach combines the benefits of improved assay specificity using recombinant NIE with a robust platform that provides rapid, high-sensitivity results.

Due to the subclinical nature of most infections with S. stercoralis and its ability to auto-infect, strongyloidiasis is a persistent disease that can remain undetected for decades following initial exposure. With increasing use of corticosteroids and other immunosuppressive/immunomodulatory therapies for the treatment of a wide variety of disease states [12], [34]–[36], there is considerable cumulative life-time risk of release of S. stercoralis from immune control. Disseminated disease in these individuals can be associated with high mortality [12], [37]–[40]. Serological assays performed by enzyme-linked immunosorbent assay (ELISA) using crude extract of infective larvae have emerged as an alternative method of diagnosing Strongyloides infections [23], [25], [28], [29], [41]. Although relatively simple, these assays still have several limitations in regions where Strongyloides is endemic including availability, turn-around-time and the requirement for moderately-sophisticated laboratory infrastructure. For critically ill patients with hyper-infection syndrome and importantly, screening those at risk of harboring occult Strongyloides infection prior to their starting immunosuppression, a truly rapid test would have real advantages, though antibody may not be detectable in advanced immunosuppression [13], [42], [43].

In this study, we describe a rapid and simple serological assay based on real-time optical diffraction (NIE dot) that can accurately differentiate patients infected with S. stercoralis from both healthy individuals and those infected with other tissue parasitic infections. This assay clearly resolved infected individuals from controls with a 133-fold difference in average signal intensity. In this study, we defined the healthy subjects and other parasite controls group as the ‘true negative’ group and the subjects with stool positive Strongyloides group as a ‘true positive’ group. So, using a diagnostic cutoff of five standard deviations above the average signal obtained from all control samples, the NIE dot assay would have a sensitivity of 96.3% and specificity of 100% in this selected population. Although these results were based on a relatively small sample set, these findings represent a significant improvement over ELISAs based on crude S. stercoralis antigen which have reported sensitivity and specificity ranges of 83%–97% and 78%–98% respectively [6], [13], [23]–[26], [28], [41], [44]. Typically these studies showed a strong reciprocal relationship between better specificity at the expense of sensitivity or vice versa. The excellent performance of the NIE dot assay can be partially attributed to the use of recombinant NIE rather than crude antigen. Cross-reactivity of Strongyloides ELISAs based on crude larval antigens is common for subjects with other tissue helminth infections, particularly filariasis and schistosomiasis [23]–[26], [45], [46]. Consistent with our findings, other recent studies using immunoassays based on recombinant NIE had little cross-reactivity [28], [30], [31]. NIE-based assay formats such as ELISA, luciferase immunoprecipitation system (LIPS) and NIE dot have all reported >95% specificity while achieving >97% sensitivity, with the understanding that true negatives are difficult to define. In addition to reduced cross-reactivity, the use of recombinant proteins significantly simplifies the antigen preparation process. Crude Strongyloides antigen is produced from filariform larvae obtained from fecal cultures from heavily infected patients or experimental animals [47], [48]. This process is dangerous (L3 larvae are infective to humans), time-consuming and labor intensive as fecal samples need to be cultured for almost a week, then concentrated and purified to obtain suitable larvae for antigen preparation. Crude larval antigen is therefore difficult to produce reliably and in large quantities, leading to variation between antigen lots. The use of the recombinant NIE antigen therefore represents a significant advance in Strongyloides serodiagnosis.

This study is limited by the lack of control samples from patients infected with other intestinal parasites (e.g.: hookworms, Ascaris lumbricoides). This could theoretically lead to an overestimation of the specificity of dotLab NIE. However, in other hands, the NIE antigen has shown good specificity in this type of specimen [28]. As described by several authors and reviewed by Requena-Mendez [13], in immunosuppressed patients, the sensitivity of the ELISA might be lower. At the time this work was performed, we had access to only one sample from a patient with disseminated Strongyloides on which NIE ELISA was negative. Unfortunately the remaining sample was insufficient to be tested with the dotLab NIE. Further prospective study will be required to validate the performance of the NIE antigen in general and these immunocompromised subjects in particular.

The dotLab mX diffractive optics system used in this study offers a number of distinct advantages over conventional immunoassay platforms. The system is simple to operate and generates results in less than 30 minutes, making it amenable for more general distribution and near-patient settings. The panelPlus oligonucleotide-based addressing system used to immobilize recombinant NIE to the dotLab Sensors facilitates customization of multiplex assays. This system utilizes a library of unique 30-bp oligonucleotides, each of which can be conjugated to a different protein target. Using panelPlus sensors bearing a linear array of spots, each coated with different oligonucleotides complementary to those used for target conjugation, multiple antigens can be immobilized on a single sensor at user designated locations. The dotLab mX System interrogates each spot independently during an assay and yields multiple real-time traces representing molecular interactions that occur on each spot. Therefore, the dotLab mX System using panelPlus has the potential to perform multiplex assays in near-patient settings.

For Strongyloides, a number of different recombinant proteins in addition to NIE have previously been described as potential antigens for serodiagnostic use. These include 5a [49], 12A [49] and SsIR [31]. Multiplex serological assays using a combination or all of these antigens may provide improved assay performance over single antigen tests. Indeed, Ramanathan and colleagues recently showed that an LIPS assay using both NIE and SsIR improved overall assay performance compared to NIE alone [31]. Based on our work, multiplexing one or more Strongyloides antigens with antigens from other pathogens having similar clinical presentations on the dotLab mX System could serve as a rapid screening test in some settings. Lastly, S. stercoralis co-infection with human T cell lymphotropic virus type 1 (HTLV-1) is known to have important clinical implications. HTLV-1infection results in T cell proliferation leading to a shift from a Th2 to Th1 immune response and concomitant increase in interferon gamma (IFN-γ) and interleukin 10 (IL-10), and lower levels of interleukin 4 (IL-4), 5 (IL-5), 13 (IL-13) and parasite-specific IgE [50]–[52]. Co-infected patients are at much greater risk of developing disseminated strongyloidiasis [51], [52]. A multiplex Strongyloides assay that included HTLV-1 screening might improve the management of these patients.

In summary, we have developed a rapid, simple serodiagnostic assay for detecting S. stercoralis IgG using recombinant NIE antigen and a novel, high-sensitivity diffractive optics technology (dot). This assay performed as well as an NIE-based ELISA and LIPS. This platform generates results in less than 30 minutes and is fully automated requiring minimal user intervention, making it potentially attractive for near-patient testing and for use in regions where technical expertise or adequate laboratory facilities may not be available. With the ability to create custom multiplex assays using an oligonucleotide-based addressing system (panelPlus), the dotLab mX System could also be used further to improve Strongyloides serodiagnostics by incorporating multiple recombinant antigens in a multiplex format or by simultaneously screening for clinically relevant co-infections such as HTLV-1.