ABSTRACT.
Strongyloides infection may develop into fatal hyperinfection and dissemination syndrome in immunocompromised hosts. Despite suboptimal specificity issues, the detection of IgG antibodies by ELISA has been central in the serodiagnosis of Strongyloides infection. Recently, an IgG4-based lateral-flow test (SsRapid) using recombinant NIE (rNIE) protein with good diagnostic performance has been reported. This study assessed the result concordance between a commercial IgG-ELISA and the SsRapid. Additionally, we determined the Strongyloides seroprevalence and its association with clinical manifestations. Immunocompromised patients (N = 200) were from Hospital Canselor Tuanku Muhriz, Kuala Lumpur, Malaysia, and were diagnosed with HIV/AIDS, hematological malignancy, and solid organ cancers. Their plasma samples were tested using a commercial IgG-ELISA and SsRapid. A fair concordance (κ = 0.27–0.33; P < 0.05) between the tests was demonstrated. The SsRapid exhibited a significantly higher (P < 0.05) seroprevalence (10.5% [21/200]) compared with IgG-ELISA (7.5% [15/200]). After adsorption with rNIE, all SsRapid-positive samples tested negative with the rapid test, thus showing binding specificity. There was no significant association with clinical manifestations. This study revealed that SsRapid is a useful diagnostic tool for Strongyloides infection, and there is a notable seroprevalence among the immunocompromised patients.
The global prevalence of Strongyloides stercoralis is about 613.9 million worldwide,1 but this may be an underestimation due to substantial variability in the disease distribution across countries, lack of prevalence studies, and suboptimal diagnostic methods.2 Strongyloidiasis is a particular problem for patients with impaired cellular immunity. Immunocompromised patients may develop hyperinfection syndrome or disseminated disease, which may have a fatality rate of 90%.3 Recent developments in medical care have resulted in increasing numbers of immunocompromised individuals living longer, which increases their risk of exposure to the infection by S. stercoralis.4
Strongyloidiasis is frequently misdiagnosed because many infections remain asymptomatic due to chronic autoinfection. Most cases of disseminated strongyloidiasis are only diagnosed at autopsy or in the late disease stage.5 Conventional parasitological diagnosis is challenging due to the intermittent larval excretion and low parasite burden. Serological assays have been demonstrated to have a variable diagnostic performance with sensitivity and specificity ranging from 56% to 100% and 29% to 100%, respectively.6 Detection of IgG antibodies has been central for serodiagnosis of Strongyloides infection. A significant drawback with IgG detection is the possibility of cross-reaction with filarial and other helminth antigens.7,8 Thus, researchers have focused on studying the role of the IgG subclasses in the immune response to Strongyloides.9 Recently, there have been two reports on IgG4-based prototype lateral-flow dipstick rapid tests for strongyloidiasis.10,11 One of them, SsRapid, lined with S. stercoralis recombinant NIE (rNIE) protein, showed good diagnostic value.11,12 Hence, the present study assessed the concordance between a commercial IgG-ELISA and SsRapid. We also determined Strongyloides seroprevalence in immunocompromised patients and its association with clinical manifestations.
We used 200 archived plasma samples from immunocompromised individuals at Hospital Canselor Tuanku Muhriz, Kuala Lumpur, Malaysia, collected between 2019 and 2020. The samples were from patients with HIV infection (CD4 count below 200 cells/mm3) (N = 53), hematological malignancy (N = 57), and solid organ cancers (N = 90); the latter two patient groups had completed at least three cycles of chemotherapy at the time of sampling. Stool samples were not available from these patients. Patients’ sociodemographic and clinical data were also retrieved. Ethical clearance for using the archived samples was obtained from the Research Ethics Committee of Universiti Kebangsaan, Malaysia (UKM PPI/111/8/JEP-2021-230).
SsRapid is a prototype lateral-flow rapid test produced at USM. It detects IgG4 antibodies and uses rNIE as the test line and goat anti-mouse IgG as the control line. The test was performed on serum/plasma samples as reported previously.11,13 Briefly, two drops of buffer A (phosphate-buffered saline, pH 7.2) were placed into well C, and 25 µL of buffer B (Chase buffer; Reszon Diagnostics, Selangor, Malaysia) was pipetted in well B to reconstitute the dried IgG4–gold (Usains Biomics Laboratory, Penang, Malaysia). In well A, serum (10 µL) was mixed with an equal volume of buffer B. After the mixture migrated up the dipstick, it was transferred to well B. When the IgG4–gold conjugate was fully absorbed, the dipstick was transferred to well C for a washing step. The background cleared after approximately 10 minutes, and the dipstick result was read. Two red lines (control and test lines) on the dipstick indicated a positive result, and one red control line denoted a negative result. Samples with positive results were adsorbed with rNIE, then tested with SsRapid to demonstrate they were true positives and not cases of cross-reactions, using a protocol described previously.14
Plasma samples were also assayed for specific IgG antibodies using a commercial IgG-ELISA kit (Euroimmun IgG-ELISA, Lubeck, Germany) to detect specific anti-Strongyloides IgG. The test was performed according to the manufacturer’s instructions. Results were interpreted by calculating the sample optical density (OD) ratio, that is, sample OD/cutoff OD. A sample ratio < 0.8 was considered a negative result; between 0.8 and 1.0 was borderline, and a ratio ≥ 1.1 was positive.
Three data sets were used in the concordance analysis using Cohen’s Kappa statistic. Analysis 1 included the IgG-ELISA borderline results. In Analysis 2, the borderline results were considered positive, and in Analysis 3, borderline results were considered negative. A χ2 test was used to compare seroprevalence by IgG-ELISA and SsRapid and determine the association between Strongyloides seroprevalence and clinical manifestations. A P value of < 0.05 was considered statistically significant.
Prevalence by SsRapid was 10.5% (21/200) and by IgG-ELISA was 7.5% (15/200). Table 1 shows the concordance among results of IgG-ELISA and SsRapid. Based on Analysis 1, a fair concordance was found between the two assays (κ = 0.27; 95% CI 0.11–0.42), with 7/200 (3.5%) of the samples positive by both assays. Analysis 2 and 3 also showed a fair concordance between the two assays (κ = 0.31–0.33; P < 0.05). After the 21 SsRapid-positive sera were adsorbed with rNIE, all showed negative results with the rapid dipstick test. It indicates high specificity and that they were true positive samples and not cases of cross-reactions. Figure 1 shows the dipstick images of pre- and post-adsorbtion of 6 of 21 of the positive samples. All commercial tests for strongyloidiasis detect IgG antibodies, and there is no commercial IgG4-ELISA on the market. Most laboratories would use a commercial test when testing/screening immunocompromised patients. Thus, we had compared the SsRapid IgG4 assay with a commercial IgG-ELISA instead of a laboratory-based IgG4-ELISA.
Table 1.
Concordance of S. stercoralis detection by IgG-ELISA and SsRapid (N = 200)
| SsRapid −ve | SsRapid +ve | Total | Kappa value (95% CI) | |
|---|---|---|---|---|
| Analysis 1 | ||||
| IgG-ELISA borderline | 12 | 3 | 15 | 0.27 (95% CI 0.11–0.42) |
| Negative | 159 | 11 | 170 | |
| Positive | 8 | 7 | 15 | |
| Analysis 2 (IgG-ELISA borderline result considered as negative) | ||||
| Negative | 171 | 14 | 185 | 0.33 (95% CI 0.12–0.54) |
| Positive | 8 | 7 | 15 | |
| Analysis 3 (IgG ELISA borderline result considered as positive) | ||||
| Negative | 159 | 11 | 170 | 0.31 (95% CI 0.12–0.49) |
| Positive | 20 | 10 | 30 | |
Figure 1.
Images of some dipsticks tested with serum samples pre- and post-adsorbtion with rNIE. Arrows show the positions of the test lines of different intensities. Above the test line is the control line (goat anti-mouse IgG). Dipsticks with various test line intensities became negative after serum adsorption.
Although both assays are indirect immunoassays and detect specific Strongyloides antibodies, their fair (and not high) concordance is not surprising. The two assays use different assay formats, procedures, types of antigen, and detection antibodies. SsRapid was reported to have a sensitivity and specificity of 91.3% and 100%, respectively11; and in another study, 82% and 96%, respectively.13 Recently, a cassette format of SsRapid was developed and showed 97% sensitivity and 94.5% specificity.12 The Euroimmun ELISA is a 96-plate immunoassay that detects IgG antibodies and uses native Strongyloides antigen. It was reported to have a diagnostic sensitivity and specificity of ∼95%.15 A more recent study reported a reduced diagnostic sensitivity and specificity of 90.6% and 87.7%.16
Both IgG and IgG4 antibodies can be detected during chronic strongyloidiasis.17 Nevertheless, IgG4 antibodies were the most prominent parasite-specific response that recognized more antigens than did any other subclass. The continuous antigenic stimulation of chronic strongyloidiasis may result in an elevated IgG4 subclass response.18 This finding supported our results, which indicated that SsRapid detected a significantly higher (P < 0.05) prevalence of IgG4 antibodies (10.5% [21/200]) compared with the IgG antibodies (7.5% [15/200]) by ELISA. A previous study among cancer patients in a hospital in another part of Malaysia reported a seroprevalence of 3.1% and 4.2% for IgG4-ELISA and a commercial IgG-ELISA detection, respectively.19 Their study was on a different population, and the IgG4-ELISA was a modification of the commercial IgG-ELISA.
Therapy with corticosteroids has adverse effects on the immune responses,20 and the impairment may lead to disseminated strongyloidiasis. Since the seroprevalence in these patients by SsRapid was higher than by IgG-ELISA, the former was used to analyze the patients’ characteristics and association of seroprevalence with clinical manifestations. The mean and median for the patients’ age were 53.12 (SD: ±15.30) and 56.00 (range 17–91) years, respectively. As shown in Table 2, there was no significant association between clinical manifestations and Strongyloides seroprevalence. The small number of SsRapid-positive samples can probably be attributed to this finding. Furthermore, strongyloidiasis symptoms may be variable and are dependent on the host’s immune status, and the symptoms may be masked by their underlying diseases.21
Table 2.
Patients’ characteristics and status of Strongyloides infection based on SsRapid test
| Characteristics | Status of Strongyloides infection | P value | |
|---|---|---|---|
| Positive (N = 21) | Negative (N = 179) | ||
| Gender | |||
| Female | 6 | 76 | 0.221 |
| Male | 15 | 103 | |
| Age (years) | |||
| ≤ 56 years | 7 | 84 | 0.087 |
| > 56 years | 14 | 95 | |
| Diagnosis | |||
| Hemato-oncological malignancy | 5 | 52 | 0.486 |
| HIV | 4 | 49 | |
| Solid organ tumor | 12 | 78 | |
| Clinical symptoms | |||
| Gastrointestinal | |||
| Yes | 3 | 40 | 0.295 |
| No | 18 | 139 | |
| Eosinophilia | |||
| Eosinopenia | 5 | 83 | 0.141 |
| Normal | 15 | 89 | |
| Eosinophilia | 1 | 7 | |
| Neutrophilia | |||
| Neutropenia | 1 | 51 | 0.050 |
| Normal | 18 | 107 | |
| Neutrophilia | 2 | 21 | |
| Dermatological | |||
| Yes | 0 | 6 | 0.394 |
| No | 21 | 173 | |
| Respiratory | |||
| Yes | 4 | 38 | 0.812 |
| No | 17 | 141 | |
| Neurological | |||
| Yes | 1 | 17 | 0.473 |
| No | 20 | 162 | |
| Hematological | |||
| Yes | 4 | 24 | 0.481 |
| No | 17 | 155 | |
| Systemic | |||
| Yes | 10 | 93 | 0.707 |
| No | 11 | 86 | |
The present study highlights an advantage of the IgG4-based SsRapid over IgG-ELISA in detecting infections in immunocompromised patients. IgG-ELISA is useful as a high throughput assay in laboratories with adequate facilities and medium to high test requests. However, ELISA requires skilled personnel and cold-chain transportation. Meanwhile, the SsRapid test is useful for laboratories in low-resource areas or with few sample requests; it is also useful for field studies. The rapid test is easy to perform, no equipment is required, and can be transported at room temperature.
None of the archived samples was collected before the patients’ chemotherapy, thus a limitation of our study. The Strongyloides seroprevalence can be expected to be higher with samples before the immunosuppressive treatment. Comparing the antibody status before and after immunosuppressive therapy may provide a better insight into the reliability of serological diagnosis, particularly in immunocompromised patients. Another concern is the lack of association between clinical manifestations and Strongyloides seropositivity, and this may be addressed in the future by increasing the sample size. The lack of a direct detection method using stool samples to detect Strongyloides infection, such as culture and molecular diagnostic, is also a limitation of this study. The latter is particularly useful as a diagnostic tool in immunocompromised patients.
In conclusion, detection of Strongyloides infection using SsRapid IgG4 assay showed a fair concordance with IgG-ELISA, with the former showing a higher detection rate. This study also highlights a significant seroprevalence of Strongyloides infection among immunocompromised patients. Hence, immunocompromised patients with a history of residing in endemic areas should be routinely screened for Strongyloides infection. This approach will decrease the chance of progression to the highly fatal hyperinfection or disseminated strongyloidiasis.22
REFERENCES
- 1. Buonfrate D. et al. , 2020. The global prevalence of Strongyloides stercoralis infection. Pathogens 9: 468. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Schär F, Trostdorf U, Giardina F, Khieu V, Muth S, Marti H, Vounatsou P, Odermatt P, 2013. Strongyloides stercoralis: global distribution and risk factors. PLOS Negl Trop Dis 7: e2288. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Salvador F, Sulleiro E, Sánchez-Montalvá A, Saugar JM, Rodríguez E, Pahissa A, Molina I, 2014. Usefulness of Strongyloides stercoralis serology in the management of patients with eosinophilia. Am J Trop Med Hyg 90: 830–834. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Roseman D, Kabbani D, Kwah J, Bird D, Ingalls R, Gautam A, Nuhn M, Francis JM, 2013. Strongyloides stercoralis transmission by kidney transplantation in two recipients from a common donor. Am J Transplant 13: 2483–2486. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Byard RW, 2019. Lethal strongyloidiasis. Diagnostic and forensic issues. J Forensic Leg Med 62: 103–106. [DOI] [PubMed] [Google Scholar]
- 6. Arifin N, Hanafiah KM, Ahmad H, Noordin R, 2019. Serodiagnosis and early detection of Strongyloides stercoralis infection. J Microbiol Immunol Infect 52: 371–378. [DOI] [PubMed] [Google Scholar]
- 7. 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. [DOI] [PubMed] [Google Scholar]
- 8. Jin Y, Kim EM, Choi MH, Oh MD, Hong ST, 2017. Significance of serology by multi-antigen ELISA for tissue helminthiases in Korea. J Korean Med Sci 32: 1118–1123. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Norsyahida A, Riazi M, Sadjjadi SM, Muhammad Hafiznur Y, Low HC, Zeehaida M, Noordin R, 2013. Laboratory detection of strongyloidiasis: IgG−, IgG4—and IgE-ELISAs and cross-reactivity with lymphatic filariasis. Parasite Immunol 35: 174–179. [DOI] [PubMed] [Google Scholar]
- 10. Boonroumkaew P, Sadow L, Sanpool O, Rodpai R, Thancho-nang T, Phupiewkham W, Intapan PM, Maleewong W, 2020. Effectiveness of Strongyloides recombinant IgG immunoreactive antigen in detecting IgG and IgG4 subclass antibodies for diagnosis of human strongyloidiasis using rapid immunochromatographic tests. Diagnostics (Basel) 10: 615. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Yunus MH, Arifin N, Balachandra D, Anuar NS, Noordin R, 2019. Lateral flow dipstick test for serodiagnosis of strongyloidiasis. Am J Trop Med Hyg 101: 432–435. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Noordin R, Osman E, Kalantari N, Anuar NS, Gorgani-Firouzjaee T, Sithithaworn P, Juri NM, Rahumatullah A, 2022. A point-of-care cassette test for detection of Strongyloides stercoralis. Acta Trop 226: 106251. [DOI] [PubMed] [Google Scholar]
- 13. Noordin R, Anuar NS, Juri NM, Wongphutorn P, Ruantip S, Kopolrat KY, Worasith C, Sithithaworn J, Sithithaworn P, 2021. Evaluation of a rapid IgG4 lateral flow dipstick test to detect Strongyloides stercoralis infection in northeast Thailand. Am J Trop Med Hyg 105: 688–691. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Noordin R, Osman E, Anuar NS, Juri NM, Rahumatullah A, Hilmi NAA, 2021. Serum adsorption study to validate the specificity of a rapid test to detect Strongyloides stercoralis infection. Am J Trop Med Hyg 105: 1214–1217. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Warnacke JM, 2019. Sensitive and specific ELISA for the serological diagnosis of Strongyloides infection. J Trop Dis Public Health 7: 26. [Google Scholar]
- 16. Buonfrate D, Marrone R, Silva R, Mirisola C, Ragusa A, Mistretta M, Perandin F, Bisoffi Z, 2021. Prevalence of strongyloidiasis in a cohort of migrants in Italy and Accuracy of a novel ELISA assay for S. stercoralis infection, a cross-sectional study. Microorganisms 9: 401. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Arifin N, Yunus MH, Nolan TJ, Lok JB, Noordin R, 2018. Identification and preliminary evaluation of a novel recombinant protein for serodiagnosis of strongyloidiasis. Am J Trop Med Hyg 98: 1165–1170. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Genta RM, Lillibridge JP, 1989. Prominence of IgG4 antibodies in the human responses to Strongyloides stercoralis infection. J Infect Dis 160: 692–699. [DOI] [PubMed] [Google Scholar]
- 19. Zueter AM, Mohamed Z, Abdullah AD, Mohamad N, Arifin N, Othman N, Noordin R, 2014. Detection of Strongyloides stercoralis infection among cancer patients in a major hospital in Kelantan, Malaysia. Singapore Med J 55: 367–371. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20. Yasir M, Goyal A, Bansal P, Sonthalia S, 2021. Corticosteroid adverse effects. [Updated 2021 Jul 8]. In StatPearls [Internet]. Treasure Island, FL: StatPearls Publishing. Available at: https://www.ncbi.nlm.nih.gov/books/NBK531462/. Accessed December 29, 2021.
- 21. Hassanudin NS, Wahab ZA, Ibrahim K, Nor FM, 2017. Disseminated strongyloidiasis in an immunocompromised host: a case report. Asian Pac J Trop Biomed 7: 587–590. [Google Scholar]
- 22. Requena-Méndez A, Buonfrate D, Gomez-Junyent J, Zammarchi L, Bisoffi Z, Muñoz J, 2017. Evidence-Based Guidelines for Screening and Management of Strongyloidiasis in Non-Endemic Countries. Am J Trop Med Hyg 97: 645–652. [DOI] [PMC free article] [PubMed] [Google Scholar]