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      Accuracy of Five Serologic Tests for the Follow up of Strongyloides stercoralis Infection

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          Abstract

          Background

          Traditional faecal-based methods have poor sensitivity for the detection of S. stercoralis, therefore are inadequate for post-treatment evaluation of infected patients who should be carefully monitored to exclude the persistence of the infection. In a previous study, we demonstrated high accuracy of five serology tests for the screening and diagnosis of strongyloidiasis. Aim of this study is to evaluate the performance of the same five tests for the follow up of patients infected with S. stercoralis.

          Methods

          Retrospective study on anonymized, cryo-preserved samples available at the Centre for Tropical Diseases (Negrar, Verona, Italy). Samples were collected before and from 3 to 12 months after treatment. The samples were tested with two commercially-available ELISA tests (IVD, Bordier), two techniques based on a recombinant antigen (NIE-ELISA and NIE-LIPS) and one in-house IFAT. The results of each test were evaluated both in relation to the results of fecal examination and to those of a composite reference standard (classifying as positive a sample with positive stools and/or at least three positive serology tests). The associations between the independent variables age and time and the dependent variable value of serological test (for all five tests), were analyzed by linear mixed-effects regression model.

          Results

          A high proportion of samples demonstrated for each test a seroreversion or a relevant decline (optical density/relative light units halved or decrease of at least two titers for IFAT) at follow up, results confirmed by the linear mixed effects model that showed a trend to seroreversion over time for all tests. In particular, IVD-ELISA (almost 90% samples demonstrated relevant decline) and IFAT (almost 87%) had the best performance. Considering only samples with a complete negativization, NIE-ELISA showed the best performance (72.5% seroreversion).

          Conclusions

          Serology is useful for the follow up of patients infected with S. stercoralis and determining test of cure.

          Author Summary

          Patients infected by S. stercoralis are at risk of fatal complications. It is therefore mandatory to demonstrate complete response to therapy. Post treatment evaluation should be done with highly sensitive diagnostic methods, which can exclude the persistence of the infection. Serology is more sensitive than fecal examination and coproculture. In this study, we compare the post-treatment performance of five serology tests, and suggest that they can be useful for the follow up of patients with S. stercoralis infection, especially in non-endemic areas, where there is no risk of reinfection. In fact, the results of the tests show a progressive decrease, towards negativization, of the values (expressed in different units, depending on the specific test) through time.

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          Most cited references 17

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          A review of solutions for diagnostic accuracy studies with an imperfect or missing reference standard.

          In diagnostic accuracy studies, the reference standard may be imperfect or not available in all patients. We systematically reviewed the proposed solutions for these situations and generated methodological guidance. Review of methodological articles. We categorized the solutions into four main groups. The first group includes methods that impute or adjust for missing data on the reference standard. The second group consists of methods that correct estimates of accuracy obtained with an imperfect reference standard. In the third group a reference standard is constructed by combining multiple test results through a predefined rule, based on a consensus procedure, or through statistical modeling. In the fourth group, the diagnostic accuracy paradigm is abandoned in favor of validation studies that relate index test results to relevant clinical data, such as history, future clinical events, and response to therapy. Most of the methods try to impute, adjust, or construct a reference standard. In situations that deviate only marginally from the classical diagnostic accuracy paradigm, these are valuable methods. In cases where an acceptable reference standard does not exist, the concept of clinical test validation may provide an alternative paradigm to evaluate a diagnostic test.
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            Diagnostic Accuracy of Five Serologic Tests for Strongyloides stercoralis Infection

            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 [1]. For this reason, chronically infected patients are often found even in areas where transmission no longer occurs [2]. 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]) [3]. 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 [4], 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 [5] 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 [6]. 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 [7], [8], [9], [10]. 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 [10], [11], [12], 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 [13], [14]. 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.
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              Patterns of detection of Strongyloides stercoralis in stool specimens: implications for diagnosis and clinical trials.

              Reported efficacies of drugs used to treat Strongyloides stercoralis infection vary widely. Because diagnostic methods are insensitive, therapeutic trials generally require multiple negative posttreatment stool specimens as evidence of drug efficacy. However, only a single positive stool specimen is usually required for study enrollment. To determine the reproducibility of detection of S. stercoralis larvae in the stool, 108 asymptomatic infected men submitted 25 g of fresh stool once a week for eight consecutive weeks for examination by the Baermann technique. During the 8-week study, 239 (27.7%) of 864 stool specimens were positive for S. stercoralis. Rates of detection of larvae in the stool specimens ranged from eight of eight specimens in 3 (2.8%) men to none of eight specimens in 36 (33.3%) men. Of 43 men for whom S. stercoralis was detected in at least two of the first four stool specimens, only 1 (2.3%) man tested negative on all of the next four specimens. In comparison, of 29 men who had detectable larvae in only one of the first four specimens, 22 (75.9%) tested negative on all of the next four samples. Thus, if these 29 men had been enrolled in a therapeutic trial between the first and second sets of four specimens, the efficacy of a drug with no activity against this parasite would have been estimated to be 76%. These data suggest that patterns of S. stercoralis detection vary widely among infected persons and that intermittent larval shedding can lead to inflated estimates of drug efficacy. Before a patient is entered in a clinical trial of drug efficacy, four consecutive stool specimens should be examined for S. stercoralis; only persons with two or more positive specimens should be enrolled.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, CA USA )
                1935-2727
                1935-2735
                10 February 2015
                February 2015
                : 9
                : 2
                Affiliations
                [1 ]Center for Tropical Diseases (CTD), Sacro Cuore Hospital, Negrar, Verona, Italy
                [2 ]Coordinating Resources to assess and improve health status of migrants from Latin America (COHEMI) project study group, European Commission, Health Cooperation Work Programme, FP7 (GA-261495), Milan, Italy
                [3 ]Department of Public Health, IRCCS—Mario Negri Institute for Pharmacological Research, Milan, Italy
                [4 ]National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, United States of America
                [5 ]Instituto de Investigaciones en Enfermedades Tropicales—Universidad Nacional de Salta/CONICET, Oran, Argentina
                [6 ]Barcelona Centre for International Health Research (CRESIB, Hospital Clinic-Universitat de Barcelona), Barcelona, Spain
                Universidade Federal de Minas Gerais, BRAZIL
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: DB ZB AJK MA TBN. Performed the experiments: RM ROC MD ST. Analyzed the data: DB ZB MS AA. Contributed reagents/materials/analysis tools: ZB RM ROC MD ST TBN. Wrote the paper: DB ZB. Critically revised the manuscript: ARM JM.

                Article
                PNTD-D-14-01342
                10.1371/journal.pntd.0003491
                4323101
                25668740

                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

                Page count
                Figures: 6, Tables: 2, Pages: 12
                Product
                Funding
                This work has been partly supported by the EC within the 7th Framework Programme under the COHEMI project - grant agreement n. FP7-GA-261495. The work performed by RM, ROC, and TBN was funded in part by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases (NIAID). We thank Bordier Affinity Products SA for donating the Bordier ELISA kits. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Custom metadata
                All relevant data are within the paper and its Supporting Information Files.

                Infectious disease & Microbiology

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