Figures
Abstract
Background
Strongyloidiasis is a persistent human parasitic infection caused by the intestinal nematode, Strongyloides stercoralis. The parasite has a world-wide distribution, particularly in tropical and subtropical regions with poor sanitary conditions. Since individuals with strongyloidiasis are typically asymptomatic, the infection can persist for decades without detection. Problems arise when individuals with unrecognized S. stercoralis infection are immunosuppressed, which can lead to hyper-infection syndrome and disseminated disease with an associated high mortality if untreated. Therefore a rapid, sensitive and easy to use method of diagnosing Strongyloides infection may improve the clinical management of this disease.
Methodology/Principal Findings
An immunological assay for diagnosing strongyloidiasis was developed on a novel diffraction-based optical bionsensor technology. The test employs a 31-kDa recombinant antigen called NIE derived from Strongyloides stercoralis L3-stage larvae. Assay performance was tested using retrospectively collected sera from patients with parasitologically confirmed strongyloidiasis and control sera from healthy individuals or those with other parasitoses including schistosomiasis, trichinosis, echinococcosis or amebiasis who were seronegative using the NIE ELISA assay. If we consider the control group as the true negative group, the assay readily differentiated S. stercoralis-infected patients from controls detecting 96.3% of the positive cases, and with no cross reactivity observed in the control group These results were in excellent agreement (κ = 0.98) with results obtained by an NIE-based enzyme-linked immunosorbent assay (ELISA). A further 44 sera from patients with suspected S. stercoralis infection were analyzed and showed 91% agreement with the NIE ELISA.
Conclusions/Significance
In summary, this test provides high sensitivity detection of serum IgG against the NIE Strongyloides antigen. The assay is easy to perform and provides results in less than 30 minutes, making this platform amenable to rapid near-patient screening with minimal technical expertise.
Author Summary
A rapid and sensitive serodiagnostic assay for strongyloidiasis based on a 31-kDa recombinant antigen from Strongyloides stercoralis (NIE) was developed using a novel diffraction-based optical biosensor technology. Assay performance was tested using retrospectively collected sera from patients with parasitologically confirmed strongyloidiasis (n = 54) and control sera from healthy individuals (n = 7) or those with other parasitoses including schistosomiasis, trichinosis, echinococcosis or amebiasis (n = 40). If we consider the control group as the true negative group, the assay readily differentiated S. stercoralis-infected patients from controls detecting 96.3% of the positive cases, and with no cross reactivity observed in the control group. These results were in excellent agreement (κ = 0.98) with results obtained by an NIE-based enzyme-linked immunosorbent assay (ELISA). A further 44 sera from patients with suspected S. stercoralis infection were analyzed and showed 91% agreement with the NIE ELISA. This test provides high sensitivity detection of serum IgG against the NIE Strongyloides antigen. The assay is easy to perform and provides results in less than 30 minutes, making this platform amenable to rapid near-patient screening with minimal technical expertise.
Citation: Pak BJ, Vasquez-Camargo F, Kalinichenko E, Chiodini PL, Nutman TB, Tanowitz HB, et al. (2014) Development of a Rapid Serological Assay for the Diagnosis of Strongyloidiasis Using a Novel Diffraction-Based Biosensor Technology. PLoS Negl Trop Dis 8(8): e3002. https://doi.org/10.1371/journal.pntd.0003002
Editor: Michael Cappello, Yale Child Health Research Center, United States of America
Received: December 16, 2014; Accepted: May 31, 2014; Published: August 7, 2014
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: The National Reference Centre for Parasitology is supported by Public Health Agency of Canada/National Microbiology Laboratory grant HT070-010033. The study was supported by the Foundation of the Montreal General Hospital and the Research Institute of the McGill University Health Centre. PLC was supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre Infection Theme. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: Brian J. Pak, Evgeniya Kalinichenko, and Paul T. Smith are employed by a commercial company, Axela, Inc. This does not alter our adherence to all PLOS NTDs policies on sharing data and materials.
Introduction
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.
(A) Each sensor consists of a contiguous array of 8 assay locations spotted on the bottom of a 10 µL flow channel where reagents and samples are introduced. Each assay location is comprised of a repeating pattern of capture molecules arranged in a defined series of parallel lines creating a diffraction grating. (B) Illumination of an assay spot with a laser generates a predictable diffraction image. The intensity of the diffraction image is monitored in real time by a photodiode detector. (C) Increases in the height (h) of the diffraction grating due to molecular binding events results in a proportionate increase in the diffraction image intensity (ΔDI).
Materials and Methods
Ethics statement
Serum samples were obtained from multiple reference laboratories including the Canadian National Reference Centre for Parasitology (NRCP, Montreal, QC), University College London Hospitals (London, UK), the National Institutes of Allergy and Infectious Diseases (Bethesda, MD), the Centers for Disease Control and Prevention (Atlanta, GA) and the Albert Einstein College of Medicine (Bronx, NY) and were considered exempt. All samples used in this study were anonymized.
Patient serum samples
Positive “gold standard” serum samples (confirmed stool positive for S. stercoralis; n = 54) were obtained from multiple reference laboratories. Negative control samples (n = 47) consisted of sera obtained from: 1) healthy individuals residing in Canada with no prior history of travel outside of Canada (n = 7); and 2) individuals with confirmed diagnosis of other parasitic infections including trichinosis (n = 8), filariasis (n = 9), schistosomiasis (n = 9), echinococcosis (n = 6) and amebiasis (n = 8), and were negative for Strongyloides by an ‘in-house’ NIE-based ELISA (NRCP). All of the selected control samples displayed very high ELISA optimal density (OD) in their assays for the respective antibody. A further 44 samples from patients with suspected S. stercoralis (i.e.: serology was requested by the treating physician) were obtained from the NRCP. All serum samples were stored at −80°C until use.
Study design
Two sub-studies were performed: i) serum taken from patients with stool positive for Strongyloides vs negative controls who were healthy or had other parasites found. We used this retrospective study to set a threshold for the assay and derive the sensitivity and specificity estimates; ii) a second study was performed prospectively using serum from the same pool as well as 44 others with possible infection (i.e.: serology ordered by treating physician). This study evaluated the agreement between the NIE ELISA and the new test.
Recombinant NIE antigen
NIE cDNA cloned into pET30b plasmid was generously provided by Dr. Franklin Neva (National Institutes of Health, Bethesda, MD). The plasmid was transformed into E. coli strain BL21 (DE3) and the NIE was isolated from insoluble inclusion bodies at the NRCP as previously described [30]. Briefly, the purified NIE protein contained a plasmid-encoded 52 amino acids including six His tags at its N-terminal. The NIE fusion protein was purified using the His Bind Kit (Novagen, Inc., Billerica, MA), concentrated by ultrafiltration and then run on a size exclusion column (Superdex 75 HiLoad 16/60; Amersham Pharmacia Biotech, Baie d'Urfe, QC) to remove high molecular weight contaminants. Following a series of dialysis reactions in decreasing concentrations of urea (5-0.25 M in 20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 5 mM EDTA, 2 mM DTT) to allow slow renaturation, the eluates were dialysed against PBS for 4 hours.
Oligonucleotide-based addressing system and NIE conjugation
The panelPlus oligonucleotide-based addressing system was used for NIE immobilization onto dotLab Sensors (Axela, Inc., Toronto, ON). This approach is based on oligonucleotide hybridization to target the immobilization of capture molecules to specific locations on dotLab Sensors. The system consists of complementary pairs of 30-bp oligonucleotides, one of which is used to tag capture molecules with the other pre-coated on the dotLab Sensors. Incubation of oligonucleotide-conjugated capture molecules in panelPlus Sensors (Axela, Inc., Toronto, ON) results in their immobilization onto the sensor surface. The panelPlus system allows for either replicate analysis of individual assays or multiplexing capabilities using sensors pre-coated with several different oligonucleotides. Recombinant NIE was conjugated to D oligonucleotides using the panelPlus Labeling Kit (Axela, Inc., Toronto, ON) following the manufacturer's recommended protocol. NIE conjugation was performed at roughly a three oligonucleotide to one NIE molar ratio.
NIE dot assay
All assays were performed on the dotLab mX System using panelPlus D Sensors (Axela, Inc., Toronto, ON). These sensors are provided with a capture surface consisting of D anchor oligonucleotides complementary to the D oligonucleotides conjugated to recombinant NIE. Serological assays were performed using the panelPlus Serology Kit (Axela, Inc., Toronto, ON) with a running buffer of HEPES buffered saline containing 0.1% Tween-20 (HBST). Briefly, sensors were blocked with blocking buffer, followed by a two minute incubation with oligonucleotide D-conjugated recombinant NIE (NIE@D; 231 ng/assay) resulting in NIE immobilization on the sensor surface. The sensors were washed with running buffer, and then incubated for three minutes with a 1∶20 dilution of patient serum (3.5 µL of neat serum). Following a brief wash, antibody binding signal was amplified using a 1∶10 dilution of goat anti-human IgG antibody-coated 40 nm gold colloid (BioAssay Works, LLC., Ijamsville, MD). Assays were performed with three-spot monitoring yielding three separate serum antibody measurements per assay.
NIE ELISA
All serum samples were also tested by an NIE ELISA that was developed and validated at the NRCP. In brief, 96-well microtiter plates (Immulon 2; Thermo Labsystems, Franklin, MA) were coated overnight at 4°C with recombinant NIE diluted in 18 mM Na2CO3, 45 mM NaHCO3, pH 9.6. Wells were washed four times with phosphate-buffered saline containing 0.05% Tween 20 (PBST) and then blocked using 100 µL of 2% BSA in PBST. One hundred microliters of diluted test sera (1∶200) was added to each of the wells and incubated for one hour at 37°C. Following four washes with PBST, 100 µL of horseradish peroxidase (HRP)-conjugated goat anti-human antibodies (1∶16,000 dilution; PerkinElmer, Waltham, MA) was added to the wells for 30 minutes. The wells were washed three times with PBST and then 100 µL of 3,3′,5,5′-tetramethyl-benzidine (TMB) substrate (Millipore Corp., Billerica, MA) was dispensed into each well. After 10 minutes, the reaction was stopped by the addition of H2SO4. The plates were read in a spectrophotometer at 450 nm. Samples with optical densities (OD)<0.2 were considered negative, OD≥0.2 but <0.3 were considered equivocal and OD≥0.3 were considered positive for Strongyloides infection.
Data analysis
The amplitude of the 40 nm gold colloid binding signal (ΔGNP) was normalized to the amplitude of the NIE@D binding curve (ΔNIE) to account for slight variations in sensor surface binding capacity yielding a normalized diffractional intensity (nDI) value that is proportional to antibody titer. Binding curve amplitudes and nDI calculations were performed using the Quantitation Editor module in version 1.1.3.4 of the dotLab Software (rev 8170; Axela, Inc., Toronto, ON). Assays were performed with three-spot monitoring, generating three independent titer measurements. An exclusion criterion of ΔNIE<0.075 DI or ΔNIE>0.310 was used to omit spots within a sensor. Each serum sample was analyzed twice and the average of the duplicate assays was taken as the measurement of antibody titer. For sample classification, a cutoff of five standard deviations above the mean nDI of the control samples was taken as the diagnostic threshold.
Statistical analysis
Assay variability was evaluated based on the replicate analysis (n = 5) of a Strongyloides positive serum sample performed with three spot monitoring. Intra-assay reproducibility was calculated as the average coefficient of variance (CV) of the three spots monitored per assay while inter-assay variability was as determined by the CV of the five assays. Comparisons between parasitologically proven Strongyloides and control groups were performed using the Mann-Whitney U test. A p value of ≤0.05 was considered statistically significant. Concordance between the NIE dot assay and NIE ELISA was determined using Cohen's kappa coefficient (κ).
Results
NIE dot-based serological assay
To determine the optimal serum concentration for antibody detection sensitivity, a series of serum dilutions were analyzed. A Strongyloides positive serum sample was tested at dilutions of 1∶10, 1∶20, 1∶50 and 1∶100. As shown in Figure 2, the highest antibody signal was obtained at serum dilutions of 1∶10 and 1∶20 with no difference in signal between the two dilutions (p = 0.17). However, a significant reduction in signal was observed between dilutions of 1∶20 and 1∶50 (p<0.001). Therefore, in order to obtain maximal signal at the highest possible dilution, a serum dilution of 1∶20 was used for all assays.
A series of different dilutions of Strongyloides positive serum were analyzed to determine the optimal serum concentration for use in dot-serology assays. Dilutions of 1∶10 and 1∶20 generated the highest antibody signal with no differences between the two dilutions (p = 0.17), while a significant decrease in signal intensity was observed between 1∶20 and 1∶50 dilutions (p<0.001). A serum dilution of 1∶20 was determined to be optimal for the dot-based Strongyloides assay. Data represent mean ± SD.
Figure 3 represents a typical trace obtained from the analysis of a Strongyloides positive serum sample on the dotLab mX System. The regions of the trace highlighted in pink correspond to the incubation of each of the reagents used in the assay while the non-highlighted regions represent washes with running buffer. Sample delivery to the sensors, incubations and washes were fully automated on the dotLab mX System and total assay time was less than 30 minutes. Assay reproducibility was determined based on five replicate analysis of a pooled Strongyloides positive serum sample performed with three-spot monitoring. These results displayed good reproducibility with an intra-assay and inter-assay CV of 9.9% and 14.0% respectively.
The dotLab mX System outputs a real time trace displaying each reagent incubation and wash step in the assay. Note the binding curves representing the immobilization of NIE@D conjugate and serum anti-NIE antibodies. Significant signal amplification is achieved using anti-human IgG antibody conjugated gold nanoparticles (GNP). The three superimposed traces represent the results of a single assay performed with three spot monitoring.
Diagnostic performance of NIE dot assay
The dot-based Strongyloides serological assay using recombinant NIE was effective in distinguishing parasitologically proven S. stercoralis patients from controls. All 54 gold standard Strongyloides serum samples displayed a detectable antibody signal with an average normalized diffractional intensity (nDI) of 17.41 (range 0.36 to 41.22) compared with an average nDI of 0.14 (range −0.10 to 0.83) for the 47 control samples (p<0.001). As shown in Figure 4, a significant difference was found between the nDIs of the gold standard Strongyloides sera and each of the control groups: 1) healthy uninfected (n = 7; p<0.001); 2) trichinosis (n = 8; p<0.001); 3) filariasis (n = 9; p<0.001); 4) schistosomiasis (n = 9; p<0.0001); echinococcosis (n = 6; p<0.001); and amebiasis (n = 8; p<0.001).
The plots summarize the results of gold standard Strongyloides samples (n = 54) and six control groups comprised of healthy individuals (n = 7) as well as those with trichinosis (n = 8), filariasis (n = 9), schistosomiasis (n = 9), echinococcosis (n = 6) and amebiasis (n = 8). The lower and upper boxes represent the samples in the second and third quartile respectively while the error bars above and below the box correspond to the 95th and 5th percentiles. The horizontal lines separating the boxes represent the median and the diamond denotes the mean. X represents the minimum and maximum values. A significant difference was observed between the gold standard Strongyloides and all control groups (p<0.001).
To estimate assay sensitivity and specificity in this particular selection of specimens, a cutoff of 0.93 nDI representing the average nDI of the control samples plus five standard deviations, was used for sample classification. Based on this criterion, 52 of 54 gold standard sera were classified as positive, corresponding to a sensitivity of 96.3%. Assay specificity was 100% with all 47 control sera yielding signal below the cutoff.
Comparison with NIE ELISA
The qualitative agreement between the NIE dot assay and NIE ELISA was determined by calculating the kappa coefficient (κ). Results for all gold standard and control samples showed almost perfect concordance between these two methods with κ = 0.98. The analysis of a further 44 samples from patients with suspected S. stercoralis infection also showed excellent agreement between NIE dot and ELISA with agreement in 40 of 44 (91%) samples. The four discordant samples were classified as Strongyloides positive by NIE ELISA but had OD values just above the defined equivocal threshold. There was no evidence of a pro-zone effect noted in samples with high antibody titers at the 1∶20 serum dilution used.
Discussion
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.
Author Contributions
Conceived and designed the experiments: BJP PTS MN. Performed the experiments: BJP FVC EK MN. Analyzed the data: BJP FVC EK PLC TBN HBT IM PW PTS BJW MDL MN. Contributed reagents/materials/analysis tools: PLC TBN HBT IM PW PTS BJW MDL MN. Wrote the paper: BJP MN.
References
- 1. Hall A, Conway DJ, Anwar KS, Rahman ML (1994) Strongyloides stercoralis in an urban slum community in Bangladesh: factors independently associated with infection. Trans R Soc Trop Med Hyg 88: 527–530.
- 2. Gyorkos TW, Genta RM, Viens P, MacLean JD (1990) Seroepidemiology of Strongyloides infection in the Southeast Asian refugee population in Canada. Am J Epidemiol 132: 257–264.
- 3. Checkley AM, Chiodini PL, Dockrell DH, Bates I, Thwaites GE, et al. (2010) Eosinophilia in returning travellers and migrants from the tropics: UK recommendations for investigation and initial management. J Infect 60: 1–20.
- 4. Bisoffi Z, Buonfrate D, Montresor A, Requena-Mendez A, Munoz J, et al. (2013) Strongyloides stercoralis: a plea for action. PLoS Negl Trop Dis 7: e2214.
- 5. Schar F, Trostdorf U, Giardina F, Khieu V, Muth S, et al. (2013) Strongyloides stercoralis: Global Distribution and Risk Factors. PLoS Negl Trop Dis 7: e2288.
- 6. Bisoffi Z, Buonfrate D, Sequi M, Mejia R, Cimino RO, et al. (2014) Diagnostic accuracy of five serologic tests for Strongyloides stercoralis infection. PLoS Negl Trop Dis 8: e2640.
- 7. Igra-Siegman Y, Kapila R, Sen P, Kaminski ZC, Louria DB (1981) Syndrome of hyperinfection with Strongyloides stercoralis. Rev Infect Dis 3: 397–407.
- 8. Keiser PB, Nutman TB (2004) Strongyloides stercoralis in the Immunocompromised Population. Clin Microbiol Rev 17: 208–217.
- 9. Kaslow JE, Novey HS, Zuch RH, Spear GS (1990) Disseminated strongyloidiasis: an unheralded risk of corticosteroid therapy. J Allergy Clin Immunol 86: 138.
- 10. Basile A, Simzar S, Bentow J, Antelo F, Shitabata P, et al. (2010) Disseminated Strongyloides stercoralis: hyperinfection during medical immunosuppression. J Am Acad Dermatol 63: 896–902.
- 11. Simpson WG, Gerhardstein DC, Thompson JR (1993) Disseminated Strongyloides stercoralis infection. South Med J 86: 821–825.
- 12. Buonfrate D, Requena-Mendez A, Angheben A, Munoz J, Gobbi F, et al. (2013) Severe strongyloidiasis: a systematic review of case reports. BMC Infect Dis 13: 78.
- 13. Requena-Mendez A, Chiodini P, Bisoffi Z, Buonfrate D, Gotuzzo E, et al. (2013) The laboratory diagnosis and follow up of strongyloidiasis: a systematic review. PLoS Negl Trop Dis 7: e2002.
- 14. Machicado JD, Marcos LA, Tello R, Canales M, Terashima A, et al. (2012) Diagnosis of soil-transmitted helminthiasis in an Amazonic community of Peru using multiple diagnostic techniques. Trans R Soc Trop Med Hyg 106: 333–339.
- 15. Dreyer G, Fernandes-Silva E, Alves S, Rocha A, Albuquerque R, et al. (1996) Patterns of detection of Strongyloides stercoralis in stool specimens: implications for diagnosis and clinical trials. J Clin Microbiol 34: 2569–2571.
- 16. Uparanukraw P, Phongsri S, Morakote N (1999) Fluctuations of larval excretion in Strongyloides stercoralis infection. Am J Trop Med Hyg 60: 967–973.
- 17. Siddiqui AA, Berk SL (2001) Diagnosis of Strongyloides stercoralis infection. Clin Infect Dis 33: 1040–1047.
- 18. Sato Y, Kobayashi J, Toma H, Shiroma Y (1995) Efficacy of stool examination for detection of Strongyloides infection. Am J Trop Med Hyg 53: 248–250.
- 19. Nielsen PB, Mojon M (1987) Improved diagnosis of strongyloides stercoralis by seven consecutive stool specimens. Zentralbl Bakteriol Mikrobiol Hyg A 263: 616–618.
- 20. Jongwutiwes S, Charoenkorn M, Sitthichareonchai P, Akaraborvorn P, Putaporntip C (1999) Increased sensitivity of routine laboratory detection of Strongyloides stercoralis and hookworm by agar-plate culture. Trans R Soc Trop Med Hyg 93: 398–400.
- 21. Carroll SM, Karthigasu KT, Grove DI (1981) Serodiagnosis of human strongyloidiasis by an enzyme-linked immunosorbent assay. Trans R Soc Trop Med Hyg 75: 706–709.
- 22. Sato Y, Kobayashi J, Shiroma Y (1995) Serodiagnosis of strongyloidiasis. The application and significance. Rev Inst Med Trop Sao Paulo 37: 35–41.
- 23. Bon B, Houze S, Talabani H, Magne D, Belkadi G, et al. (2010) Evaluation of a rapid enzyme-linked immunosorbent assay for diagnosis of strongyloidiasis. J Clin Microbiol 48: 1716–1719.
- 24. van Doorn HR, Koelewijn R, Hofwegen H, Gilis H, Wetsteyn JC, et al. (2007) Use of enzyme-linked immunosorbent assay and dipstick assay for detection of Strongyloides stercoralis infection in humans. J Clin Microbiol 45: 438–442.
- 25. Conway DJ, Atkins NS, Lillywhite JE, Bailey JW, Robinson RD, et al. (1993) Immunodiagnosis of Strongyloides stercoralis infection: a method for increasing the specificity of the indirect ELISA. Trans R Soc Trop Med Hyg 87: 173–176.
- 26. Gam AA, Neva FA, Krotoski WA (1987) Comparative sensitivity and specificity of ELISA and IHA for serodiagnosis of strongyloidiasis with larval antigens. Am J Trop Med Hyg 37: 157–161.
- 27. Loutfy MR, Wilson M, Keystone JS, Kain KC (2002) Serology and eosinophil count in the diagnosis and management of strongyloidiasis in a non-endemic area. Am J Trop Med Hyg 66: 749–752.
- 28. Krolewiecki AJ, Ramanathan R, Fink V, McAuliffe I, Cajal SP, et al. (2010) Improved diagnosis of Strongyloides stercoralis using recombinant antigen-based serologies in a community-wide study in northern Argentina. Clin Vaccine Immunol 17: 1624–1630.
- 29. Lindo JF, Conway DJ, Atkins NS, Bianco AE, Robinson RD, et al. (1994) Prospective evaluation of enzyme-linked immunosorbent assay and immunoblot methods for the diagnosis of endemic Strongyloides stercoralis infection. Am J Trop Med Hyg 51: 175–179.
- 30. Ravi V, Ramachandran S, Thompson RW, Andersen JF, Neva FA (2002) Characterization of a recombinant immunodiagnostic antigen (NIE) from Strongyloides stercoralis L3-stage larvae. Mol Biochem Parasitol 125: 73–81.
- 31. Ramanathan R, Burbelo PD, Groot S, Iadarola MJ, Neva FA, et al. (2008) A luciferase immunoprecipitation systems assay enhances the sensitivity and specificity of diagnosis of Strongyloides stercoralis infection. J Infect Dis 198: 444–451.
- 32. Borisenko V, Hu W, Tam P, Chen I, Houle JF, et al. (2006) Diffractive optics technology: a novel detection technology for immunoassays. Clin Chem 52: 2168–2172.
- 33. Goh JB, Tam PL, Loo RW, Cynthia GM (2003) A quantitative diffraction-based sandwich immunoassay. Anal Biochem 313: 262–266.
- 34. Karp CL, Neva FA (1999) Tropical infectious diseases in human immunodeficiency virus-infected patients. Clin Infect Dis 28: 947–963.
- 35. Bradley SL, Dines DE, Brewer NS (1978) Disseminated Strongyloides stercoralis in an immunosuppressed host. Mayo Clin Proc 53: 332–335.
- 36. Rubin RH, Ikonen T, Gummert JF, Morris RE (1999) The therapeutic prescription for the organ transplant recipient: the linkage of immunosuppression and antimicrobial strategies. Transpl Infect Dis 1: 29–39.
- 37. Ursini T, Polilli E, Fazii P, Ieraci A, Sindici G, et al. (2013) Late diagnosis of central nervous system involvement associated with lethal dissemination of Strongyloides stercoralis in an advanced HIV patient from Nigeria. Int J Infect Dis 17: e280–e282.
- 38. Kalita J, Bhoi SK, Misra UK (2012) Fatal Strongyloides stercoralis infection in a patient with chronic inflammatory demyelinating polyneuropathy. Pathog Glob Health 106: 249–251.
- 39. Petithory JC, Derouin F (1987) AIDS and strongyloidiasis in Africa. Lancet 1: 921.
- 40. Neto VA, Pasternak J, Moreira AA, Duarte MI, Campos R, et al. (1989) Strongyloides stercoralis hyperinfection in the acquired immunodeficiency syndrome. Am J Med 87: 602–603.
- 41. Koosha S, Fesharaki M, Rokni MB (2004) Comparison of enzyme-linked immunosorbent assay and indirect immunofluorescence assay in the diagnosis of human strongyloidiasis. Indian J Gastroenterol 23: 214–216.
- 42. Mascarello M, Gobbi F, Angheben A, Gobbo M, Gaiera G, et al. (2011) Prevalence of Strongyloides stercoralis infection among HIV-positive immigrants attending two Italian hospitals, from 2000 to 2009. Ann Trop Med Parasitol 105: 617–623.
- 43. Schaffel R, Nucci M, Carvalho E, Braga M, Almeida L, et al. (2001) The value of an immunoenzymatic test (enzyme-linked immunosorbent assay) for the diagnosis of strongyloidiasis in patients immunosuppressed by hematologic malignancies. Am J Trop Med Hyg 65: 346–350.
- 44. Genta RM (1988) Predictive value of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of strongyloidiasis. Am J Clin Pathol 89: 391–394.
- 45. Lindo JF, Atkins NS, Lee MG, Robinson RD, Bundy DA (1996) Parasite-specific serum IgG following successful treatment of endemic strongyloidiasis using ivermectin. Trans R Soc Trop Med Hyg 90: 702–703.
- 46. Muck AE, Pires ML, Lammie PJ (2003) Influence of infection with non-filarial helminths on the specificity of serological assays for antifilarial immunoglobulin G4. Trans R Soc Trop Med Hyg 97: 88–90.
- 47. Grove DI, Blair AJ (1981) Diagnosis of human strongyloidiasis by immunofluorescence, using Strongyloides ratti and S. stercoralis larvae. Am J Trop Med Hyg 30: 344–349.
- 48. Costa-Cruz JM, Bullamah CB, Goncalves-Pires MR, Campos DM, Vieira MA (1997) Cryo-microtome sections of coproculture larvae of Strongyloides stercoralis and Strongyloides ratti as antigen sources for the immunodiagnosis of human strongyloidiasis. Rev Inst Med Trop Sao Paulo 39: 313–317.
- 49. Ramachandran S, Thompson RW, Gam AA, Neva FA (1998) Recombinant cDNA clones for immunodiagnosis of strongyloidiasis. J Infect Dis 177: 196–203.
- 50. Porto AF, Neva FA, Bittencourt H, Lisboa W, Thompson R, et al. (2001) HTLV-1 decreases Th2 type of immune response in patients with strongyloidiasis. Parasite Immunol 23: 503–507.
- 51. Carvalho EM, Da Fonseca PA (2004) Epidemiological and clinical interaction between HTLV-1 and Strongyloides stercoralis. Parasite Immunol 26: 487–497.
- 52. Pays JF (2011) [Combined infection with HTLV-1 and Strongyloides stercoralis]. Bull Soc Pathol Exot 104: 188–199.