LipL32 for new early serodiagnosis of human leptospirosis

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Comparative Immunology, Microbiology and Infectious Diseases 33 (2010) 375–387 www.elsevier.com/locate/cimid

Synthetic peptide issued from Hap1/LipL32 for new early serodiagnosis of human leptospirosis F. Aviat a,c,1, S. Rochereau-Roulet a, C. Branger b,2, J.M. Estavoyer c,3, B. Chatrenet d,4, J.L. Orsonneau e,5, C. Thorin a, G. Andre-Fontaine a,* a

Unite´ de Bacte´riologie Me´dicale et Mole´culaire des Leptospires, Ecole Nationale Ve´te´rinaire de Nantes, BP 40 706, 44 307 Nantes Cedex 03, France b Centre for Infectious Diseases and Vaccinology, Biodesign Institute, Arizona State University, AZ, USA c Service des Maladies Infectieuses et Tropicales, Centre Hospitalier Universitaire Saint Jacques, 25000 Besanc¸on, France d Virbac, 13e`me rue LID, BP 27, 06 511 Carros Cedex, France e Institut de Biologie Humaine, Centre Hospitalier Universitaire Hoˆtel Dieu, 44 000 Nantes, France Accepted 31 January 2009

Abstract Leptospirosis is a worldwide zoonosis. Today, serological diagnosis is generally assessed by MAT. We performed ELISA with a synthetic peptide derived from Hap1/lipL32 which is a protein expressed only by pathogenic Leptospira. Repeatability and thresholds were defined with 85 controls sera and 119 hospitalized leptospirosis. The PP-ELISA repeatability and IgM/IgG cut-off values were based on control sera. For these cut-off values, we observed the IgM-PP-ELISA specificity of 89%, whereas it was 100% for the IgG. * Corresponding author. Tel.: +33 2 40 68 76 91; fax: +33 2 40 68 76 94. E-mail addresses: [email protected] (F. Aviat), [email protected] (S. Rochereau-Roulet), [email protected] (C. Branger), [email protected] (J.M. Estavoyer), [email protected] (B. Chatrenet), [email protected] (J.L. Orsonneau), [email protected] (C. Thorin), [email protected] (G. Andre-Fontaine). 1 Present address: Unité de Biologie des Spirochètes, Institut Pasteur, 28, rue du Docteur Roux, 75724 Paris Cedex 15, France. Tel.: + 33 1 45 68 83 66/33 2 40 68 77 77. 2 Tel.: +480 727 8322. 3 Tel.: +33 3 81 66 81 66. 4 Tel.: +33 4 92 08 71 00. 5 Tel.: +33 2 40 08 33 33. 0147-9571/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.cimid.2009.01.001

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Then, we compared PP-ELISA and standard MAT results for leptospirosis patients. The concordance rate for IgM-PP-ELISA and MAT was low (43%), whereas it was 85% for IgG-PPELISA and MAT. During the first 5 days after hospitalization, PP-ELISA gave positive results in 13 out of 16 patients (81%) whereas 8 out of 14 patients (57%) were positive to MAT. ELISA using Hap1/lipL 32-derived synthetic peptide PP is an earlier serological diagnosis of human leptospirosis than MAT. # 2009 Elsevier Ltd. All rights reserved. Keywords: Leptospirosis; MAT; ELISA; Hap1; LipL32; Peptide

Résumé Le diagnostic de la leptospirose, zoonose à répartition mondiale, repose sur la microagglutination (MAT), méthode sérologique tardive. Un peptide (PP) issu d’une protéine spécifique des leptospires pathogènes (Hap1/LipL32) est synthétisé et employé comme antigène pour un ELISA (PP-IgM/IgG). La reproductibilité et les seuils de positivité de l’ELISA-PP sont déterminés à partir de 85 sérums contrôles et 119 leptospiroses confirmées. La spécificité est de 89% pour l’ELISA-PP-IgM et de 100% pour l’ELISA-PP-IgG. Les résultats obtenus en ELISA-PP et MAT sont comparés pour les leptospiroses confirmées. La concordance de l’ELISA-PP-IgG et du MAT est de 85%, celle de l’ELISA-PP-IgM est de 43%. La réponse sérologique vis-à-vis de PP est plus précoce que celle du MAT. Dans les 5 premiers jours d’hospitalisation, l’ELISA-PP est positif pour 13 patients sur 16 (81%) et le MAT pour 8 (57%). Ce travail démontre que l’ELISA-PP est un outil de diagnostic plus précoce que le MAT. # 2009 Elsevier Ltd. All rights reserved. Mots cle´s : Leptospirose ; MAT ; ELISA ; Hap1 ; LipL32 ; Peptide

1. Introduction Leptospirosis occurs in different countries, worldwide. Human beings are often exposed to leptospiral bacteria through contact with either animals or fresh water from rivers and lakes, during occupational or leisure activities and floods. Most mammals infected with Leptospira interrogans sensu lato, are asymptomatic and carry pathogenic leptospiral bacteria in their kidney. Rodents often shed leptospires in their urine, contaminating the human environment with infectious leptospiral germs that can survive in fresh waters for several weeks [1]. Whereas domestic animals (horse, cattle or pig) are affected by clinically milder forms but inducing economic loss, more sensitive species such as man and dog show severe clinical manifestations of the disease. Early clinical diagnosis of leptospirosis is difficult because symptoms are highly variable and common to other acute diseases for instance viral such as dengue, yellow fever, flu, and hemorrhagic fever with renal syndrome. Early diagnosis of leptospirosis is crucial: the sooner the treatment, the faster is the recovery [2]. A delayed diagnosis after the first non-specific symptoms of the acute phase of the disease – fever, myalgia, ocular complaints or meningeal signs, and, more typically, renal and hepatic failures, hemorrhagic disorders or pulmonary diseases – may be fatal.

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Bacterial isolation is inconvenient for clinical diagnosis; therefore, serological tests, approximately 10–15 days after disease onset, are used to confirm clinical suspicion. The microscopic agglutination test (MAT), which detects agglutinating antibodies produced against lipopolyosidic outer antigens, is the gold standard method for diagnosing human leptospirosis [3]. Thus, diagnostic tests are needed to confirm disease in the early stages. PCR based strategies have been developed as an alternative to bacterial isolation, involving amplification of the leptospiral genes, hap1 [4] and rrs [5]. However, serological assays are still cheaper and easier to perform, especially in tropical developing countries. Several tests, such as Lepto Dipstick [6–8], Lepto lateral flow [9], Lepto Dri Dot [10], or ELISA [11,12], have been developed. These tests all detect serum antibodies specific for the highly antigenic lipopolyosidic antigens detected by MAT, most of them using antigens carried by the fully cross-reacting Patoc strain of saprophytic Leptospira biflexa sensu lato [9]. However, many outer membrane proteins (OMPs) are antigenic for the infected host; some of them distinguish between pathogenic and saprophytic strains of leptospires [13]. Hap1/LipL32 [14,15] is an OMP present in pathogenic but absent from saprophytic strains. In a previous study, this protein was produced as a recombinant protein and used in an ELISA test before confirmation by the reference MAT, and used in serological diagnostic tests for humans and animals [16,17]. In protein Hap1/LipL32 [18], we selected a particular amino-acid sequence poly D (residues 160–170) and used this sequence for synthesis of a 26 amino-acid synthetic antigen, herein named ‘‘PP’’. Our purpose was to investigate the specificity and sensitivity of this peptide in leptospirosis diagnosis. Immunoglobulin M (IgM) and Immunoglobilun G (IgG) responses against this peptide PP were compared in human sera from 51 hospitalized patients with no known history of leptospirosis and from 53 patients with confirmed leptospirosis. The sensitivity and specificity of PP-ELISA were assessed; accuracy and concordance were compared with the other tests previously used to assess the leptospirosis diagnosis in the study populations, taking into account the delay since the disease onset whenever available.

2. Material and methods All the sera were issued from sera banks systematically built up for potential and later use and controls following the rules of the Ethical Committees. No additional blood collection was performed for this study. 2.1. Study populations A control negative group and a leptospirosis group were studied: - Sera from 51 hospitalized patients without any known infectious disease were used as a negative control to define background and cut-off values for PP-ELISA.

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Table 1 Serovar in leptospirosis cases defined by MAT. Serovars

Number

Australis Canicola Castellonis Cynopteri Grippotyphosa Icterohaemorrhagiae Pomona Pyrogenes Sejroë

7 7 2 9 8 15 1 1 3

Total

53

- Sera from 53 cases: patients showing clinical symptoms of leptospirosis associated to positive PCR or serological criteria to assign a diagnosis of acute leptospirosis. 2.1.1. Control group (n = 51) This group comprised 51 hospitalized patients with C reactive protein in the normal range. These subjects with normal values at the sampling time were considered to have none infectious disease or inflammation. These controls were used to define the serological IgM and IgG background to the peptide used in PP-ELISA. Additionally, two pools of these controls (10 randomized sera) were used to provide a negative internal standard for PP-ELISA performed for leptospirosis cases. 2.1.2. Cases (n = 53) Two groups of cases were used. For each case of one group (n = 34), a single serum was available, whereas for cases of the second group (n = 19), several samples were available for each patient at different sampling times during hospitalization. Serological criteria included one of the following in either acute or convalescent sera: biflexa ELISA IgM positive. A single titre of 1:800 or greater or between acute and convalescent sera, a seroconversion from negative to 1:100 or greater, or a 4-fold rise of titre. Cases exhibited agglutinating antibodies shown in MAT test (with titres between 100 and 12,800) to various pathogenic serovars shown in Table 1. The second group was used for kinetic study of the serological response and to assess test repeatability with two pools of four patients. 2.2. PP-ELISA 2.2.1. PP synthesis The 26 amino-acid peptide (PP) was synthesized by NeoMPS (France). Its experimental molecular weight, determined by mass spectrometry, was 3103.5 (theoretical: 3104.3) and

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its purity, determined by HPLC analysis, was over 94%. Peptide solubility was 2.4 mg/mL in distilled water. 2.2.2. PP-ELISA protocol Flat-bottomed microtiter plates (Immulon 4HBX, NUNC) were coated overnight at 4 8C with 100 mL PP peptide (5 mg/mL) in 0.05 M sodium carbonate in each well (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6). Plates were washed three times with PBS-0.2% Tween 20 (PBST). They were then incubated for 1 h at 37 8C with 100 mL SeaBlock (Interchim) (1 v/25 v) in TNE pH 7.4 blocking solution (150 mM NaCl, 50 mM TRIS, 0.8 mM EDTA). Plates were washed again three times with PBST. Duplicates of 100 mL serial dilutions of the sera in TNE (1:100 to 1:800) were incubated for 1 h at 37 8C. One positive and one negative control were included on each plate as internal standards. The plates were washed three times with PBST. Anti-human rabbit IgM-peroxidase conjugate (100 mL; Jackson ImmunoResearch) or anti-human goat IgG-peroxidase conjugate (Uptima, Interchim), both diluted at 1:5000, were added to each well and plates were incubated for 1 h at 37 8C. Plates were washed three times with PBST. One hundred microlitres of substrate buffer (0.5 mM 2-20 -azino di-ethyl benz-thiazoline-6-sulphonic acid (ABTS), 10 mM of citric acid, 0.03% hydrogen peroxide, pH 4.5) were added to each well. Plates were read at 405 nm with ELISA reader (Labsystem) after 30 min incubation in the dark at 37 8C. 2.2.3. Data analysis The statistical distribution of the IgM and IgG results of sera in PP-ELISA were studied by Kolmogorov–Smirnov test. Comparison between controls and leptospirosis cases results was assessed by Mann and Whitney test.

3. Results 3.1. Background in negative control samples Serial dilutions of 51 control samples were tested by PP-ELISA. The optimal dilution for serum analysis, taking into account background optical density (OD) in controls without antigen or without sera, was 1:100 (data not shown). All the results shown in this study are for 1:100 dilution. 3.1.1. PP-IgM-ELISA The background of control population was defined for sera of 51 controls. The results are shown in Fig. 1 and Fig. 3A. The median absorbance of the 51 controls was 0.231 (minimum = 0.100 and maximum = 1.394). Using the Kolmogorov–Smirnov test, as shown in Fig. 2, IgM absorbance did not show a normal distribution (P < 0.001). As the median absorbance was 0.231, a possible cut-off for IgM was arbitrarily designed as 0.500 absorbance.

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Fig. 1. Box plots of IgM and IgG absorbances: controls versus cases.

Fig. 2. Histogram and density line for IgM (A) and IgG (B) distribution in control group.

3.1.2. PP-IgG-ELISA The background of control population was defined for sera of 51 controls. The results are shown in Fig. 1 and Fig. 3B. The median absorbance of the 51 controls was 0.105 (minimum = 0.056 and maximum = 0.244). Using the Kolmogorov–Smirnov test, IgG absorbance did not show a normal distribution (data not shown). As the median absorbance was 0.105, a possible cut-off for IgG was arbitrarily designed as 0.250 absorbance (Fig. 2B). 3.2. ELISA repeatability Test repeatability was estimated by repeating PP-ELISA for each secondary antibody, with positive and negative control pools (sera of the second group of cases and sera of controls), with and without PP antigen. Repeatability was expressed as coefficient of variation (cv = sd/m) (Table 2).

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Table 2 PP-IgM and IgG-ELISA repeatability. Antibody class

Reagent

Number of repeated tests

OD (mean)

OD (sd)

cv (sd/m)

IgM

Negative pool Positive pool No serum

40 24 64

0.199 1.032 0.071

0.051 0.042 0.008

0.256 0.041 0.112

IgG

Negative pools Positive pools No serum

8 8 28

0.113 0.704 0.063

0.005 0.034 0.006

0.044 0.048 0.095

3.3. Analysis of sera from leptospirosis cases Sera were analyzed using the arbitrary cut-off values 0.500 for PP-IgM-ELISA and 0.250, for PP-IgG-ELISA. 3.3.1. PP-IgM-ELISA 3.3.1.1. PP-IgM-ELISA results were obtained for 53 cases. The results are shown in Fig. 1A. The median absorbance of the 53 cases was 0.424 (minimum = 0.101 and maximum = 2.408). Taking in account the non normal distribution of the absorbance, comparison of cases versus controls results was illustrated by spots (Fig. 3) and performed by Mann and Whitney test. There is a significant difference within IgM absorbance of the controls and IgM absorbance of the cases (P < 0.0002). Taking in account an OD: 0.500 as IgM cut-off, 23/53 patients were IgM positive. 3.3.2. PP-IgG-ELISA 3.3.2.1. PP-IgG-ELISA results were obtained for 53 cases. The results are shown in Fig. 1B. The median absorbance of the 53 cases was 0.477 (minimum = 0.176 and maximum = 1.967). Taking in account the non normal distribution of the absorbance, comparison of cases versus controls results was illustrated by spots (Fig. 3) and performed by Mann and Withney test. There is a significant difference within IgG absorbance of the controls and IgM absorbance of the cases (P < 0.0001). Using an OD: 0.250 as IgG cut-off, 50/53 patients were IgG positive. 3.3.3. PP-ELISA efficacy compared to MAT Comparison between PP-ELISA and MAT could be done only for the same day of collection (Table 3). Forty-eight samples for which MAT was previously performed were available for ELISA. Only 15 over 48 (31%) were PP-IgM positive, whereas 37 over 48 (77%) were PP-IgG positive. Over 38 samples with MAT titre 400, 12 were PP-IgM positive but 34/38 (89%) were PP-IgG positive. For 14/48 cases MAT was performed during the first 5 days of admission to hospital, 8 of 14 (57%) gave MAT titres 100 (Fig. 3). During the same delay, sera from 16 cases could

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Fig. 3. Distribution of IgM and IgG absorbances: controls versus cases.

be tested with PP-ELISA. Thirteen of these sera (81%) were tested positive for either PPIgM or PP-IgG. For patient 2 shown in Fig. 4, for instance, MAT gave negative results on day D11 after hospitalization, however, reached a titre of 1:12,800 three weeks later, confirming the leptospirosis diagnosis. For this case the PP-IgG-ELISA showed positive results as soon as at time of admission to hospital (D0). For patient 8 (Fig. 5), 2 days (D2) after hospitalization a PCR assay was positive and was confirmed on Day 39 by MAT performed in convalescent serum. For this patient PP-IgG-ELISA was positive for the sample collected 5 days (D5) after hospitalization (Fig. 5). The efficiency of PP-ELISA was then compared to widely used Patoc-ELISA, the strain L. biflexa Patoc is well-known for its wide cross-reactivity with pathogenic leptospires. Forty-six sera from cases tested by Patoc-ELISA were tested by PP-ELISA (Table 4). Twenty-four (52.2%) samples were positive for the Patoc-ELISA and 39 (84.8%) were positive for the PP-ELISA. Negative results of Patoc-ELISA were observed in 17 out of the 39 PP-ELISA-positive samples, whereas 2 of the 24 Patoc- ELISA positive sera were negative for the PP-ELISA.

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Table 3 Concordance of MAT and PP-ELISA and in leptospirosis samples (n = 48).

Fig. 4. Patient 2, serological tests: MAT, PP-ELISA (IgM and IgG) after admission to hospital.

4. Discussion The IgM and IgG results did not show a normal distribution because of few outlier high values. In such a situation the medians were more robust and more informative than the means. Natural background of the serological response to PP peptide in healthy patients was estimated using leptospirosis-free sera and two cut-off values (IgM = 0.500 and IgG = 0.250) were designed for subsequent experiments in leptospirosis samples. Nevertheless, one subject from this group exhibited a very high optical density (OD = 1.394) for the PP-IgM-ELISA; this was not consistent with clinical data at that time. Thus, for these cut-off values, we observed the IgM-PP-ELISA specificity of 89%, whereas

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Fig. 5. Patient 8, PCR and serological tests: MAT and PP-ELISA (IgM and IgG), after admission to hospital.

Fig. 6. Cumulative proportion of patients with positive results for PP-ELISA (16 sera tested) and MAT (14 sera tested).

Table 4 Patoc-ELISA versus PP-ELISA in 46 cases. PP-ELISA (IgM or IgG)

Patoc-ELISA Negative Positive

Negative

Positive

5 2

17 22

22 24 (52.2%)

7

39 (84.8%)

46

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it was maximal for the IgG (100%). Taking in account this overall specificity, all leptospirosis sera were analyzed using these cut-off values of 0.500 and 0.250 for PP-IgMELISA and PP-IgG-ELISA, respectively. The IgM median of controls was 0.231, whereas it was 0.424 for the cases. The IgG median of controls was 0.105 whereas it was 0.477 for the cases. By Mann and Withney test, IgM and IgG values of the cases were significantly higher than IgM (P < 0.0002) and IgG of the controls (P < 0.0001). Most of the previous data on leptospirosis serodiagnosis compared a new more convenient method to the standard reference test (MAT) for the detection of leptospiral specific antibodies [19,20]. But, for example, the microcapsule agglutination test failed to identify some serovars identified by MAT [21]. Sensitivity, specificity and predictive values for each new method were compared with those for MAT. One study reported 96% sensitivity and 93% specificity for IgM ELISA in confirmed leptospirosis [22], but PCR sensitivity ranged from 36% to 60% in confirmed or probable cases. Since PCR showed leptospires in blood and MAT agglutinating antibody response, the disease stage needs to be taken into account when samples are taken to compare several tests. The sensitivity of the Dip Stick for example, has been observed to be low (32%) in one study [8], but high (80% and 98%) in patients studied by Cermeno-Vinas et al. [7] and Levett and Branch [23], respectively. An effect of the timing of sampling was highlighted in the study of Cohen et al. [24], in which sensitivity ranged from 7% to 81% when samples were taken during either the acute or convalescent stage. IgM dot tests [20] and IgM lateral flow [9] show similar results. de Abreu-Fonseca et al. [19] observed a high level of variation among the results when of the results when MAT or IgM ELISA and PCR were used on samples taken at different time intervals. In this study, we found a low concordance rate 19/48 (40%) between MAT and PP-IgMELISA but higher concordance 41/48 (85%) between MAT and PP-IgG (Table 3). However, serological results with PP-ELISA (IgM or IgG) and MAT appeared to be correlated (Fig. 6). The main difference between the two tests is the stage at which the specific antibodies for each antigen appear. During the first 5 days after admission to hospital, 13 out of 16 samples tested yielded positive results using PP-ELISA and 8 out of 14 showed agglutinating antibodies in MAT (minimal titre = 100). Agglutinating antibodies in MAT are detected later than the anti-PP response. Such differences in the kinetics of the agglutinating or Elisa antibody responses may thereby underlie the low concordance rate observed between MAT and PP-IgM-ELISA (Table 3). Agglutinating antibody production increased during the sampling period, whereas IgM response to Hap1/ Lipl32 started to decrease. Hap1/Lipl32 is early produced by pathogenic leptospires at high level during the first stage of infection [15,25] and is liberated in the medium [26]. Therefore, it is possible that a specific response against Hap1/Lipl32, involving PP, is activated during the incubation stage, as soon as the infectious strain starts dividing in the blood and produces high level of Hap1/Lipl32; whereas, the agglutinating antibody response towards lipopolyosidic antigens is delayed, the immune processing starting after the intensive growth and early death of some infectious bacteria. Our findings show that the antibody response against pathogenic leptospires is very complex, depending of the epitopes presentation. The triggering of the immune response towards the peptide derived from Hap1/Lipl32 is faster than the agglutinating antibody response. The immune response

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to Hap1/Lipl32 ends when the infectious invading process of the virulent strain is stopped by treatment. Therefore, as for PCR, a direct comparison between results from PP-ELISA and MAT is not valid, highly depending of sampling time. Moreover, the sera studied here were collected when the patients were hospitalized meaning that the disease was developed enough some days after the invading process of virulent leptospires. For Lyme borreliosis, the serological response against a peptide (C6) used, was studied. The duration of the serological response to this peptide, studied by Tjernberg et al. [27], was early but transient. Our data show that this synthetic peptide, which is a short sequence from Hap1/Lipl32, is a very useful antigen for accurate and early serological diagnosis of human leptospirosis. Experiments in progress have to optimize the technical conditions of its use for a best efficiency for leptospirosis diagnosis.

Acknowledgements The French Ministry of Agriculture (DGER) supported this work. We are grateful to Prof Guy Baranton and Dr Daniele Postic from the Pasteur Institute, Paris for their interest in our work and for their kindly purchase of sera from leptospirosis patients. We would also like to acknowledge J. Bellin and C. Lefur for their technical assistance.

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