Introduction Strongyloides stercoralis (S. stercoralis) is a nematode widely distributed all over the world, in areas where poor hygienic conditions permit the maintenance of its transmission. In the human host the infection is characterized by an autoinfective cycle, that can lead to life-long carriage of the parasite if left untreated . For this reason, chronically infected patients are often found even in areas where transmission no longer occurs . Chronic infection is often clinically silent. It is crucial, however, to detect and treat the infection in order to avoid the risk of the life-threatening complications (hyperinfection and dissemination) that can develop in the face of immunosuppression (e.g. underlying medical conditions and/or iatrogenic [steroids, other immunosuppressive agents]) . Proper diagnostic testing is crucial both to identify S. stercoralis-infected individuals and to evaluate the prevalence of the infection among populations. One of the main problems with S. stercoralis is that its overall prevalence is probably underestimated , mostly due to the lack of sensitivity of fecal – based tests that are the most commonly used assessments for S. stercoralis infection. Serologic tests are also very useful, but their specificity is variable  and more difficult to assess because of the unreliability of the used reference test, i.e. microscopy. Discordant (fecal negative – serological positive) samples cannot be clearly defined. Furthermore, specificity is likely to be variable in different population groups and to be better in environments where other intestinal parasites are rare or absent, while sensitivity may be sub optimal in immunosuppressed patients . An ideal diagnostic tool for S. stercoralis should have a very high sensitivity when used for screening (i.e. candidates for transplantation, chemotherapy, systemic corticosteroids) as well as to detect persistence of infection after treatment (therapeutic failure). Ideally the test should become negative or consistently show a marked decrease in titer in a predictable time after successful treatment. Although some studies document a decline of antibody titer after effective treatment, a clear cut-off value has yet to be defined , , , . For a clinical trial, however, a very high specificity is needed in order to avoid inclusion of false positive subjects. The main objective of the present study was to assess the accuracy of five serologic methods for the diagnosis of S. stercoralis infection in different patient populations. The serologic tools are intended for use both in highly endemic settings (screening of subjects at risk for complications, prevalence studies, clinical diagnosis in adequately equipped laboratories) and in areas of low or no endemicity (screening and diagnosis of immigrants, travelers, and autochthonous infection in elderly patients in countries previously endemic such as in Southern Europe). Methods Conduct of the study The study was carried out in two reference laboratories for parasitic diseases (CTD Negrar - Verona, Italy and NIAID-NIH, Bethesda, US) by well-trained staff members. Samples were selected from a composite study population that is described in detail below. As fecal based methods are virtually 100% specific but lack sensitivity , , , a composite reference standard was also used (see below) as a suggested procedure for the evaluation of diagnostic tests when there is no gold standard , . Study design The study was designed as a retrospective comparative diagnostic study on archived, anonymized serum samples. Sensitivity, specificity and positive and negative predictive values (PPV, NPV) of the index tests calculated against the primary reference standard (direct demonstration of Strongyloides larvae in stools by microscopy or culture) was used as the primary endpoint. A secondary endpoint was a test's sensitivity, specificity and predictive values when compared to a composite reference standard (as defined below). Study samples The study was carried out on fully anonymized, coded serum samples already available at CTD that were selected randomly, within each study group outlined below. The archived specimens were kept frozen at −80°C from the day of the sample collection and tests were executed within 24 hours of unfreezing. Inclusion criteria Serum specimens were selected from a composite patient population including: Group I - Subjects of all ages with S. stercoralis larvae in fecal specimens, identified by microscopy and/or culture (primary reference standard) Group II - Subjects with no previous exposure to S. stercoralis: healthy blood donors and patients of all ages, born and resident in non-endemic areas of Europe and with no travel history to endemic countries. Group III - Subjects with potential, previous exposure to S. stercoralis but with negative fecal tests for strongyloidiasis: a) subjects routinely screened for parasites, with no known parasitic infections. b) patients with other parasitic infections (see below for details). Exclusion criteria Group I - Hyperinfection syndrome (HS) or disseminated strongyloidiasis (DS). HIV patients with CD4+ cells 50 years; previous residence in areas where Strongyloides transmission was known to occur in past decades Group III - HIV patients with CD4+ cells 70% sensitivity. Such standard and available tests could be used both in clinical and public health practices. It must be mentioned, however, that tests based on crude antigen may be difficult to ensure optimal reproducibility among different batches. We strongly recommend laboratories using these tests to put into place clear quality control methods. Study limitations This study has the potential limitations inherent to a retrospective study design. Some quite relevant data were missing for some of the control subjects (i.e. the continent of exposure when/if it did not coincide with the continent of origin). Moreover, as parasitological methods are not 100% sensitive, also for other parasitic infections, it may well be that some infections were missed in control subjects exposed, which may have caused cross reactivity. While we believe that subjects were better classified using the composite reference standard, we cannot exclude a possible misclassification of some of them. Conclusion and further research needs The issue of serology as a marker of cure remains an open question. If we were to rely on fecal-based diagnosis alone, we may wrongly consider cured a patient whose parasite load after treatment is too low to be detected. Thus, an evaluation of serologic tests to assess cure is currently underway. A prospective study that will include PCR on fecal samples is also planned. The ultimate aim is to identify the optimal diagnostic strategy for S. stercoralis for clinical and epidemiological purposes. Supporting Information Figure S1 STARD flow chart. (DOC) Click here for additional data file. Figure S2 ROC curve for IVD ELISA (primary reference standard). (JPG) Click here for additional data file. Figure S3 ROC curve for Bordier ELISA (primary reference standard). (JPG) Click here for additional data file. Figure S4 ROC curve for NIE-LIPS (primary reference standard). (JPG) Click here for additional data file. Figure S5 ROC curve for IFAT (primary reference standard) (numbers correspond to titers, 3 = 1/20 to 9 = 1/1280). (JPG) Click here for additional data file. Figure S6 ROC curve for NIE-ELISA (primary reference standard). (JPG) Click here for additional data file. Table S1 STARD checklist for reporting of studies of diagnostic accuracy. (DOC) Click here for additional data file. Table S2 Test accuracy (composite reference standard) at different cut-off levels of the index tests. (DOC) Click here for additional data file. Table S3 Positive and negative predictive values (PPV, NPV) for different theoretical prevalence levels. (DOC) Click here for additional data file. Table S4 Positive and negative predictive values (PPV, NPV) for different theoretical prevalence levels. (DOC) Click here for additional data file. Table S5 Concordance between pairs of index tests (Kappa test). (DOC) Click here for additional data file.