Avidity as a criterion for diagnosis of human strongyloidiasis increases specificity of IgG ELISA Larissa Rodrigues Bosqui, Henrique Tomaz Gonzaga, Maria do Ros´ario de F´atima Gonc¸alves-Pires, Fabiana Martins de Paula, Ricardo Sergio Almeida, Wander Rog´erio Pavanelli, Ivete Conchon-Costa, Julia Maria Costa-Cruz, Idessania Nazareth da Costa PII: DOI: Reference:

S0732-8893(17)30260-2 doi: 10.1016/j.diagmicrobio.2017.08.008 DMB 14406

To appear in:

Diagnostic Microbiology and Infectious Disease

Received date: Revised date: Accepted date:

10 April 2017 15 August 2017 16 August 2017

Please cite this article as: Bosqui Larissa Rodrigues, Gonzaga Henrique Tomaz, do Ros´ ario de F´ atima Gon¸calves-Pires Maria, de Paula Fabiana Martins, Almeida Ricardo Sergio, Pavanelli Wander Rog´erio, Conchon-Costa Ivete, Costa-Cruz Julia Maria, da Costa Idessania Nazareth, Avidity as a criterion for diagnosis of human strongyloidiasis increases specificity of IgG ELISA, Diagnostic Microbiology and Infectious Disease (2017), doi: 10.1016/j.diagmicrobio.2017.08.008

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ACCEPTED MANUSCRIPT Avidity as a criterion for diagnosis of human strongyloidiasis increases specificity of IgG ELISA Larissa Rodrigues Bosqui1, Henrique Tomaz Gonzaga2, Maria do Rosário de Fátima

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Gonçalves-Pires2, Fabiana Martins de Paula3; Ricardo Sergio Almeida4, Wander

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Rogério Pavanelli1, Ivete Conchon-Costa1, Julia Maria Costa-Cruz2 and Idessania

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Nazareth da Costa1*

1 Departamento de Ciências Patológicas, Laboratório de Parasitologia, Universidade Estadual de Londrina, Paraná, Brasil

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2 Departamento de Imunologia, Microbiologia e Parasitologia, Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Minas Gerais, Brasil

São Paulo, Brasil

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3 Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo –

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Brasil

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4 Departamento de Microbiologia, CCB, Universidade Estadual de Londrina, Paraná,

*Corresponding author Idessania Nazareth da Costa Departamento de Ciências Patológicas – Laboratório de Parasitologia Universidade Estadual de Londrina-UEL-Rodovia Celso Garcia Cid Campus Universitário, Cx. Postal 6001, CEP 86051-990 - Londrina – PR. Tel: +055-43-3371-4539 Email: [email protected]

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ACCEPTED MANUSCRIPT Abstract This study evaluate the inclusion of the IgG avidity index in ELISA to detect antiStrongyloides stercoralis IgG. The ELISA index revealed 70% of specificity. With the

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inclusion of screening AI, specificity increased to 80%. IgG avidity complemented

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traditional IgG ELISA by eliminating some of the suspected or false positive cases.

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Keywords: strongyloidiasis, cross-reaction, avidity-ELISA, Strongyloides sp.

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ACCEPTED MANUSCRIPT Immunodiagnostic tests are considered efficient due to their high sensitivity and specificity; however, they are not always able to distinguish between active S. stercoralis infection in negative results from coprological examinations and positive

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results of immunological examinations (Gutierrez and Maroto, 1996; Gonzaga et al.,

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2011; Levenhagen and Costa-Cruz, 2014; Bosqui et al., 2015).

Avidity has been applied as a complementary tool for conventional ELISA to determine the functional affinity of immunoglobulin G (IgG) in various parasitic

strongyloidiasis (Gonzaga et al., 2011).

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diseases (Bela et al., 2008; Viana et al., 2001; Manhani et al., 2009) and in the

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The determination of IgG avidity can be useful to diagnose active infection, mainly in strongyloidiasis, due to the complexity of the parasitological diagnosis. Therefore, the aim of the present study was to assess IgG AIs in individuals with positive serology for S. stercoralis analyzed by ELISA to detect IgG anti-Strongyloides. Serum samples were obtained from 90 individuals from Londrina, Paraná State,

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Brazil, in the period from January 2015 to January 2016. Stool samples were processed by parasitological methods according to Hoffmann et al. (1934), Faust et al. (1939), Kato and Miura (1954), as modified by Katz et al. (1972) and the method of

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Rugai et al. (1954). Samples were divided into three groups: Group 1 - 30 individuals with confirmed parasitological diagnosis for S. stercoralis; Group 2 - 30 individuals

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copropositive for other parasitic diseases, including Giardia lamblia (n = 10), Ancylostomatidae (n = 6), Ascaris lumbricoides (n = 1), Enterobius vermicularis (n =

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5), Hymenolepis nana (n = 5), and Entamoeba histolytica/E. dispar (n = 3); and Group 3 - 30 apparently healthy individuals with negative coproparasitological tests. The present study was approved by the Research Ethics Committee of the Universidade Estadual de Londrina (1494.2013-56). The saline extract was obtained according to the method proposed by Gonzaga et al. (2011). The protein dosage was analyzed by the method of Lowry et al. (1951). All the criteria for the ELISA method were developed, based on the detection kinetics of IgG antibody. We carried out preliminary experiments to determine the optimal conditions for IgG avidity ELISA by means of block titrations of the reagents. To assess IgG avidity, we started with the previously tested screening dilution (1:80), following the protocol previously described by Gonzaga et al. (2011). Polystyrene microtiter plates were coated with the saline extract overnight. Serum samples diluted in PBS-T (1:80, 1:160, 1:320, 1:640) were added in 3

ACCEPTED MANUSCRIPT quadruplicate. Then, half of the serum samples were treated with urea (6 M) diluted in PBS (PBS-U+). Subsequently, we added enzyme-conjugated antibody diluted at 1:2000 in PBS-T, and the plates were incubated for 45 minutes at 37 °C.

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The analyses were performed using GraphPad Prism 5.0 statistical package.

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The cut-off point was calculated by the receiver operating characteristic (ROC) curve for each dilution (Greiner et al., 1995). The reactivity of serum samples were expressed using ELISA index. IgG avidity was expressed as AI: at 1:80 dilution,

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screening AI - mean OD of wells treated with urea/mean OD of untreated wells) x 100%; and then mean AI, i.e., mean of AI obtained from different positive dilutions (EI

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>1.0) (Wiuff et al., 2002). IgG ELISA for strongyloidiasis group was used as definitive diagnosis criterion (S. stercoralis larvae found in feces) in combination with the results of AIs for groups 1 and 2 as a complementary tool. Thus, the following criteria were established to assess the AI: cut-off selection for obtaining 100% IgG sensitivity and maximum specificity; and AI calculation for the positive samples of groups 2 and 3,

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considering the lower positive IgG dilution (EI>1.0). The criterion proposed by Gonzaga et al. (2011) i.e., AI >75% to exclude active infection was considered. Diagnostic parameters of sensitivity and specificity of ELISA were calculated

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considering IgG ELISA (EI >1.0) and IgG ELISA + AI (AI>75%). The combination of different methods improves the sensitivity and specificity in

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clinical trials and in situations that require high-precision diagnosis (Requena-Méndez et al., 2002). It is possible to observe that the ELISA index exhibited 100% positivity in

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all dilutions of group 1 according to the cut-off; whereas group 2 exhibited crossreaction in the dilution 1:640 for Ancylostomatidae (n = 2), E. vermicularis (n = 1), and H. nana (n = 1) (Figure 1A). Calculating screening AI and mean AI at different positive serum dilutions can reduce potential differences in avidity due to antibody response intensity and IgG concentration in each sample, pointing toward an individual status (Butler, 2000). These variations may be explained by differences in infection severity, larval excretion, host-parasite interaction, and environmental factors (Uparanukraw et al., 1999). The result can be observed in chart 1B, where the calculation of AI in the first dilution (1:80) was minimized with the best diagnostic performance of screening AI in the fourth dilution (1:640). The AI for IgG was greater in group 3 four dilutions tested. At this point, the AI ranged from 42 to 86% (mean = 58%). The combination of negative fecal test for S. stercoralis and positive serology, associated with avidity data 4

ACCEPTED MANUSCRIPT (AI > 75%), suggests that the AIs observed in groups 2 and 3 indicated possible exclusion of active infection. This statement agrees with Gonzaga et al. (2011). The diagnostic sensitivity and specificity parameters revealed 100% sensitivity

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according to the ELISA index (EI). Regarding AI, in groups 2 and 3, specificity reached

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80% and 85% for screening AI and mean AI, respectively (Table 1). This demonstrated that the higher specificity exhibited by ELISA results association suggests that S. stercoralis infection is unlikely to occur in individuals with EI > 1 and

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AI > 75%.

The present study tested for the first time the use of IgG avidity in copropositive

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individuals for other parasitic diseases. The specificity of avidity test was discussed by De Ory et al. (1993) in Epstein-Barr virus infections. According to them the specificity of the avidity test is hampered by serological criteria in recent or past cases classification. The parasitological and serologic evidences to indicate strongyloidiasis cases can fail, from setting time of infection to the cross-reactivity possibility. The G2 proved

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infection/detectable cases by other parasites with possible history of infection with S.

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stercoralis, increasing the specificity of the test.

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FINANCIAL SUPPORT This study was supported by grants from the Coordenação de Aperfeiçoamento de

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Pessoal de Nível Superior (CAPES – Grant No. 40002012026M9 (L.R.B.) and

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Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG – Grant No.

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CBB-PPM-00396-13) (J.M.C.C).

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Requena-Méndez A, Chiodini P, Bisoffi Z, Buonfrate D, Gotuzzo E, Muñoz J. The laboratory diagnosis and follow up of strongyloidiasis: a systematic review. PLoS Negl Trop Dis 2013: 7: e2002.

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ACCEPTED MANUSCRIPT Legend Figure Figure 1. Anti-IgG Strongyloides detection by ELISA and/or combined with avidity. (A) ELISA-IgG in groups of patients with strongyloidiasis (G1) with other parasitic

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diseases (G2) and healthy individuals (G3). ELISA index (EI) calculated from the cut-

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off established by ROC curves for 100% sensitivity. % + = percentage of positive samples (EI >1). (B) IgG avidity index (AI) calculated for samples from G2 and G3 positive ELISA-IgG. % AI >75% = percentage of samples excluding the active

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infection by S. stercoralis (AI >75%). n = sample size.

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Fig. 1

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ACCEPTED MANUSCRIPT Table 1.Diagnostic parameters of ELISA with selected cut-off, to attend the maximum sensitivity definitive diagnostic standard (against Strongyloides stercoralis larvae), and / or combined with avidity IgG antibodies in the patient groups with other parasitic diseases and healthy individuals, for specificity increment.

80 % 85 %

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EI – ELISA Index; AI – Avidity Index

70 %

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100 %

Screening AI 100 % Mean AI 100 %

Specificity

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Sensitivity

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Serum dilution 1:80 ELISA IgG (EI >1) ELISA IgG + AI (AI >75%)

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Avidity as a criterion for diagnosis of human strongyloidiasis increases specificity of IgG ELISA.

This study evaluates the inclusion of the IgG avidity index in ELISA to detect anti-Strongyloides stercoralis IgG. The ELISA index revealed 70% of spe...
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