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JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2002, p. 1037–1043 0095-1137/02/$04.00⫹0 DOI: 10.1128/JCM.40.3.1037–1043.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Vol. 40, No. 3

Enzyme-Linked Immunosorbent Assay Based on Soluble Promastigote Antigen Detects Immunoglobulin M (IgM) and IgG Antibodies in Sera from Cases of Visceral and Cutaneous Leishmaniasis Jeffrey R. Ryan,1* Anthony M. Smithyman,2 G-Halli Rajasekariah,2 Lisa Hochberg,1 John M. Stiteler,1 and Samuel K. Martin1 Entomology Dept., Division of Communicable Diseases and Immunology, Walter Reed Army Institute of Research, Silver Spring, Maryland,1 and Cellabs Pty. Ltd., Sydney, Australia2 Received 31 July 2001/Returned for modification 18 September 2001/Accepted 11 December 2001

Leishmaniasis causes significant morbidity and mortality in areas where it is endemic. In areas where it is nonendemic, global travel and increased incidence of the disease in human immunodeficiency virus and intravenous-drug user populations are also causes for concern. The unavailability of rapid and reliable tests for diagnosis of the various leishmaniases makes patient management difficult. We have developed an enzymelinked immunosorbent assay (ELISA) that can detect immunoglobulin M (IgM) and IgG antibodies in patients with visceral and cutaneous leishmaniasis. These practical assays are based on soluble antigens from promastigotes cultivated in a protein-free medium. In preliminary studies, 129 visceral (Brazil, Italy, North Africa, and Nepal) and 143 cutaneous (Brazil) leishmaniasis patients with controls were tested. Overall, the tests showed a sensitivity of 95.1%. In addition, the ELISA correctly identified 42 sera from Brazilian dogs with canine leishmaniasis and 10 healthy controls. Serological tests for the various clinical manifestations of leishmaniasis could be useful epidemiological and patient management tools in populations of areas of endemicity and nonendemicity. Leishmaniasis is a serious, sometimes fatal disease estimated by the World Health Organization to affect some 12 million people in 88 countries. Recent epidemics in the Horn of Africa, Indian subcontinent, and Brazil have made research in this disease all the more important (7). Attempts at intervention are greatly hindered by the lack of a device to accurately and practically identify patients infected with or exposed to the disease-causing parasite. Many new diagnostic methodologies focus on the patient’s antibody response to make a determination that infection or exposure has taken place. Serological tests for diagnosing visceral leishmaniasis (VL) generally are highly sensitive (⬎90%) (4, 26). However, these tests have experienced problems with their specificity, i.e., false-positive results being obtained with reference samples of other infectious diseases and subclinical leishmanial infections. Modifications of the antigens used for the direct agglutination test (29) and the enzyme-linked immunosorbent assay (ELISA) (2) have recently been reported to be successful in markedly eliminating false-positive results. On the other hand, serological tests are rarely performed to diagnose cutaneous leishmaniasis (CL); the sensitivity and specificity of these tests have been disappointingly low (14, 24). The antigens used as the foundation for past immunodiagnostic tests for leishmaniasis originated from cultured promastigotes (3, 9, 10) or recombinant proteins (2, 22). However, crude antigen preparations from whole-cell lysates lack the metabolic products that promastigotes release into culture medium. These

metabolic products should be included in any diagnostic strategy since their immunogenicity is well established (25, 27). Excreted factor, a component of these antigens, is a negatively charged carbohydrate-like substance which was shown to precipitate antibody from homologous sera of promastigote-infected rabbits (8, 13). The soluble antigens of Leishmania donovani promastigotes are primarily lipophosphoglycan, which is comprised of an albumin binding site, a hydrophylic lipophosphoglycan component, and a repeating phosphorylated saccharide (linked with secreted acid phosphatase [S-AcP]) (15). S-AcP was shown to be the most immunogenic of all the glycoproteins present in L. donovani promastigote-conditioned medium (5, 6). S-AcP from L. donovani promastigote-conditioned medium has been used to immunoprecipitate specific antibody from pooled sera of acutely ill kala-azar patients (12). Recently, Martin and colleagues (20) reported on the use of a soluble antigen preparation from L. donovani that was used to capture specific immunoglobulin G (IgG) antibodies in the sera of kala-azar patients. These findings indicate that the soluble antigens found in conditioned medium can act as the foundation for immunodiagnostic tests for leishmaniasis. The purpose of the present study was to explore the extension of this concept and improve the assay produced by Martin and coworkers (20) so as to be able to detect specific IgG and IgM antibodies in VL and CL patients.

* Corresponding author. Mailing address: Department of Entomology, Division of Communicable Diseases and Immunology, Walter Reed Army Institute of Research, Silver Spring, MD 20910-7500. Phone: (301) 319-9532. Fax: (301) 319-9012. E-mail: Jeffrey.Ryan @na.amedd.army.mil.

Sera. Sera collected from human patients admitted to clinics in Brazil, Italy, North Africa, Nepal, and Walter Reed Army Medical Center with either splenic aspirates or skin biopsies from lesions positive for leishmania parasites by culture and/or microscopy were selected from the serum bank. In total, 129 VL (Italy, Brazil, North Africa, and Nepal) and 143 CL patients (Brazil) (136 Leishmania

MATERIALS AND METHODS

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braziliensis-infected and 7 Leishmania panamensis-infected patients) with controls were tested. Human negative controls were from 12 normal patients from areas of nonendemicity with no documented infection or exposure to leishmania parasites. In addition to the human manifestations assayed, sera from 42 Brazilian dogs with a clinical diagnosis for canine leishmaniasis were tested against positive control sera from a commercial source (Bordier Affinity Products, S.A., Crissier, Switzerland) and 10 negative controls from a pathogen-free canine research colony (College of Veterinary Medicine, North Carolina State University, Raleigh, N.C.). Antigen preparation. Washed promastigotes were inoculated into 200 ml of a defined, conditioned protein-free medium to give a final density of 108 cells/ml. The parasites were incubated at 26°C for 72 h in roller bottles. Thereafter, the spent medium was harvested by centrifugation at 9,000 ⫻ g for 30 min and the relative protein concentration of the soluble antigens (exoantigen) was estimated by measuring the optical density (OD) at 280 nm (21). ELISA. Plate sensitization was effected by coating polystyrene, 96-well microtiter plates (Immulon 4; Dynatech Laboratories, Chantilly, Va.) with 100 ␮l of the respective exoantigen solution (5 ␮g of protein per well). L. donovani (Walter Reed reference strain 130, clone E) exoantigen was used to sensitize plates for VL and canine leishmaniasis samples, and Leishmania mexicana (ATCC strain 50157) exoantigen was used to sensitize plates for CL samples. Plates were then blocked with 1.0% casein (Sigma Chemical Co., St. Louis, Mo.) in phosphate-buffered saline (PBS) for 1 h at room temperature. The blocking buffer was removed by aspiration, the serum samples (100 ␮l of 1:1,000 dilution) and appropriate controls were added to the microtiter plate, and the plate contents were incubated at 26°C for 40 min. After the mixture was washed with 0.05% PBS–Tween 20 (PBS-Tween) buffer four times, goat anti-human IgG (whole molecule) conjugated with horseradish peroxidase (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.) was added at a 1:5,000 dilution and the plate contents were incubated at 26°C for 1 h. The plate was then washed four times with PBS-Tween buffer, and 100 ␮l of 3,3⬘,5,5⬘-tetramethyl-benzidene substrate (Kirkegaard & Perry Laboratories) was added to each well. The plate contents were incubated in the dark, and the OD was periodically read at a 650-nm wavelength in an ELISA plate reader (Molecular Devices, Menlo Park, Calif.) until the OD of a reference positive control (S5, kala-azar patient, Nepal) reached 0.8. At this point 100 ␮l of a stop solution (0.1 M phosphoric acid) was applied to the plate, and the final OD reading was taken at 450 nm. A reference positive serum was used in all plates, and only interassay variation of less than 10% was accepted. The lower limit of positivity (cutoff) was determined by the mean of the negative controls subset ⫹ 3 standard deviations (18). SDS-PAGE and Western blot analysis. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was accomplished by use of a MiniProtean II apparatus (Bio-Rad) as previously described (19). Briefly, the corresponding antigen preparation (L. donovani for VL patients; L. mexicana for CL patients) was boiled for 5 min in sample buffer minus reducing agent and was immediately subjected to electrophoresis on 4% stacking, 12.5% separating bisacrylamide gels. A wide-range molecular mass marker (Bio-Rad) was used. An antigen load of 120 ␮g of protein was used in each preparative minigel. Gels were run at 100 V of constant voltage for 1.5 h in Tris-glycine-SDS buffer (pH 8.3). Protein bands from the gel were transferred to nitrocellulose (28). Briefly, antigens from the SDS-polyacrylamide gels were electroblotted onto 0.45-␮mpore-size nitrocellulose membranes (Bio-Rad) with standard transfer buffer (0.02 M Tris, 0.15 M glycine, 0.1% SDS, and 20% methanol) and 250 mA of constant current for 1 h at 4°C. Following the blotting, portions of the membranes containing the protein markers were stained with 0.5% Amido black for 5 min and were destained in distilled water-glacial acetic acid solution. The membranes were immediately blocked with a 2% skim milk 100 mM Tris–0.9% NaCl–0.1% Tween 20 (TTBS) solution and were kept refrigerated until use. Immunoblotting. A minor modification of the immunoblotting procedure outlined by Isaza and colleagues (17) was used to detect antibodies from patient sera bound to the antigens of the Western blot. Briefly, 4-mm-wide strips were cut from previously blotted and blocked membranes. The strips were incubated with diluted sera (1:3,200 in TTBS) for 30 min at room temperature with constant agitation. After incubation with the primary antibody, the strips were washed four times for 10 min each time with TTBS. After the last wash, anti-human IgG–horseradish peroxidase complex (Kirkegaard & Perry Laboratories) was added, and the mixture was incubated for 30 min under the same wash conditions described above. The membranes were developed with a TMB substrate (Kirkegaard & Perry Laboratories) for 5 min. Rinsing the strips with distilled water and the addition of PBS-EDTA (20 mM) stopped the reaction.

J. CLIN. MICROBIOL.

FIG. 1. Boxplots of levels of Leishmania-specific IgG, as measured by ELISA and shown in OD values in 129 VL patients. The threshold of positivity or cutoff value shown as a horizontal line represents 3 standard deviations (Std Dev) above the mean value for healthy controls. Conf Pos, confirmed positive; Neg Cntrl, negative control.

RESULTS In this study 284 human and 52 canine sera were tested by an antibody capture micro-ELISA technique using a soluble antigen fraction derived from culturing either L. donovani or L. mexicana promastigotes in a conditioned, protein-free medium. The assay was improved by modifying serum sample and conjugate dilution, substrate, incubation times, and temperatures. Presently, the assay is able to detect specific IgG and IgM antibodies with various degrees of success in patient serum samples from known positive cases of VL and CL. Initially, we tested all categories of patient samples with the WR0130E L. donovani antigen. However, the assay was not sensitive to CL patient serum samples (data not shown). This shortcoming led us to retest the CL samples using the L. mexicana (ATCC strain 50157) antigen. Final results by category are outlined below. In Fig. 1 to 6, the performance of each assay for IgG and IgM detection is graphically represented with boxplots and whisker plots (Fig. 1). For each plot, the horizontal line represents the median value, the box indicates the interquartile range, and the vertical lines indicate the range of values.

FIG. 2. Boxplots of levels of Leishmania-specific IgM as measured by ELISA and shown in OD values in 129 VL patients. The cutoff value shown as a horizontal line represents 3 standard deviations above the mean value for healthy controls.

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FIG. 3. Boxplots of levels of Leishmania-specific IgG as measured by ELISA and shown in OD values in 143 CL patients. The cutoff value shown as a horizontal line represents 3 standard deviations above the mean value for healthy controls.

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FIG. 5. Boxplots of levels of Leishmania-specific IgG as measured by ELISA and shown in OD values in 41 canines infected with Leishmania chagasi. The cutoff value shown as a horizontal line represents 3 standard deviations above the mean value for healthy controls.

VL. When the WR0130E L. donovani exoantigen was used as the material to coat the microtiter plate, the negative control serum subset gave a negative cutoff score of approximately 0.225 for the IgG assay. Figure 1 is a boxplot to illustrate specific IgG antibody levels measured in VL patient serum samples. With respect to specific IgG, all 129 clinically confirmed positive VL patient sera gave OD readings above the negative cutoff (100% sensitivity). Figure 2 is a boxplot showing specific IgM antibody levels measured in the same samples. When IgM was measured, the negative control sera tested gave a negative cutoff score of approximately 0.310. The sensitivity for this assay was 94.57% (122 of 129 positive). CL. When the ATCC 50157 L. mexicana exoantigen was used as the material to coat the microtiter plate, the negative control serum subset gave a negative cutoff score of approximately 0.3 for the IgG assay. The boxplot in Fig. 3 illustrates specific IgG antibody levels measured in CL patient serum samples. With respect to specific IgG, 132 of 143 clinically confirmed positive CL patient sera gave OD readings above the negative cutoff (92.31% sensitivity). Figure 4 is a boxplot depicting specific IgM antibody levels measured in the same samples. When IgM was measured, the negative control sera

tested gave a negative cutoff score of approximately 0.15. Only a few samples (n ⫽ 6) were dramatically above the negative cutoff score, the majority of values for positive samples were at or near the median value of the negative control subset. This assay failed to consistently detect specific IgM in CL patient serum samples (37.9%; 22 of 58 positive). Canine leishmaniasis. When the WR0130E L. donovani exoantigen was used as the material to coat the microtiter plate, the negative control serum subset gave a negative cutoff score of approximately 0.1 for the IgG assay. Figure 5 shows specific IgG antibody levels measured in canine leishmaniasis serum samples. With respect to specific IgG, all 41 clinically confirmed positive canine leishmaniasis serum samples gave OD readings above the negative cutoff (100% sensitivity). Note the relatively large degree of separation between the positive and negative control subsets. The boxplot in Fig. 6 displays specific IgM antibody levels measured in the same samples. When IgM was measured, the negative control sera tested gave a negative cutoff score of approximately 0.25. The sensitivity for this assay was 97.56% (40 of 41). SDS-PAGE and Western blot analyses. The Western blot results for both VL and CL patients and canine and all negative controls are in agreement with the ELISA data. Repre-

FIG. 4. Boxplots of levels of Leishmania-specific IgM as measured by ELISA and shown in OD values in 143 CL patients. The cutoff value shown as a horizontal line represents 3 standard deviations above the mean value for healthy controls.

FIG. 6. Boxplots of levels of Leishmania-specific IgM as measured by ELISA and shown in OD values in 41 canines infected with L. chagasi. The cutoff value shown as a horizontal line represents 3 standard deviations above the mean value for healthy controls.

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FIG. 7. Western blot demonstrating seropositivity and reaction to L. donovani exoantigen with sera from 10 VL patients (V1 to V10) versus healthy human controls (N1 to N4), who show no reaction to the same antigen profile.

sentative samplings of patient (10 patient samples each for VL and CL) results and negative control (n ⫽ 4) Western blot results appear in Fig. 7 and 8, respectively. A representative portion of canine Western blot results is pictured in Fig. 9. The banding patterns of the patient samples used in this study to these antigens seem to be consistent within the clinical manifestations. Specifically, VL patient samples gave a consistent pattern where immunodominant bands with estimated mobility appear at the molecular weights of 130,000, 108,000, 104,000, 96,000, 78,000, 37,000, and 30,000, whereas CL patient samples also gave a fairly consistent pattern. In this instance, immunodominant bands with estimated mobility appear at molecular weights of 220,000, 145,000, 132,000, 108,000, and 78,000. Canine samples gave the strongest bands, with estimated mobility consistently at molecular weights of 132,000, 108,000, 96,000, 41,000, 37,000, and 30,000. Finally, there was no significant banding noted for any of the negative control serum samples used in this assaying. DISCUSSION We have developed ELISAs that can detect IgM and IgG antibodies in patients with VL and CL. These assays are based on soluble antigens from promastigotes cultivated in a protein-

free medium. They are practical because of the low amount of serum needed to perform the assay and the short running time of 4 h. In preliminary studies 129 VL (Brazil, Italy, North Africa, and Nepal) and 143 CL (Brazil) patients with controls were tested. The test showed an overall sensitivity of 95.1% when leishmania-specific IgG was measured against that of healthy, North American negative controls. As previously reported by Martin et al. (20), no cross-reactivities were noted when this assay was used to look for cross-reacting antibodies in patient samples from other parasitic diseases (malaria, echinococciasis, Africa trypanosomiasis, and filariasis). In addition to human leishmaniasis, the ELISA correctly identified 42 sera from Brazilian dogs with canine leishmaniasis and 10 healthy controls. The difference between negative and positive was clearer cut in the case of dog sera than of human sera. This same observation was made in a previous study (16). Prior research attempts employing nonrecombinant antigens in the design of serological tests for leishmaniasis were limited by problems with sensitivity, specificity, and test reproducibility (1, 11, 23). Reasons for these limitations remain elusive but are likeliest attributable to physical and chemical techniques used in antigen preparation. Sensitizing plates with soluble antigens from conditioned media appears to ameliorate this condition.

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FIG. 8. Western blot demonstrating seropositivity and reaction to L. mexicana exoantigen with sera from 10 CL patients (C1 to C10) versus healthy human controls (N1 to N4), who show no reaction to the same antigen profile.

Our data demonstrate that uncharacterized soluble factors in spent medium can be used as a foundation for an immunodiagnostic ELISA for VL and CL. When attempting to detect specific IgM, normally one must consider the effect of IgG in the sample and compensate for its presence or eliminate it altogether. To do so, we took a fraction of samples tested (n ⫽ 100) and subjected them to a Rapi-Sep spin column (INDX) before testing for specific IgM. There was no significant change in the IgM values derived for any sample (data not shown). Use of a defined protein-free culture medium reduces antigen production to a simple and inexpensive centrifugation step. The improvements made to this assay greatly increased its sensitivity and practicability. In areas where this disease is endemic, a simple, accurate, and practical test is needed. The use of conditioned media in antibody capture serological tests for leishmanial infections is promising. More testing is needed to validate this assay for all clinical manifestations involving all human-affecting Leishmania species and areas of endemicity. Indeed, one might argue about the limitations of the assay for CL since we demonstrate here the performance of the assay using L. braziliensis case patient samples. It is generally understood that this parasite is “more aggressive,” often leading to mucosal involvement.

Therefore, the B-cell response in CL cases from this parasite may be different from cases of CL due to other, less aggressive parasites. Moreover, if these assays are to be used in areas of endemicity, they will have to be validated with a good sample size of normal patients from areas where the disease is endemic. Regardless of the limitations pointed out above, this concept, once validated, may demonstrate the extreme sensitivity of detecting leishmania-specific antibodies to circulating antigens. Indeed, what is more desirable is an antigen capture assay to detect the exoantigens released by the parasite in not only the vertebrate host but also in the sand fly vector. A serological test for this disease would be a useful epidemiological and patient management tool in populations from areas of endemicity and nonendemicity of leishmaniasis. In summary, we have developed an ELISA that can detect IgM and IgG antibodies in human patients with VL and CL and in dogs with canine leishmaniasis. When using leishmaniaspecific IgG antibodies as a marker for active disease, the test showed an overall sensitivity of 95.1% (261 of 272). The ELISA is simple and inexpensive, is based on soluble antigens from promastigotes cultivated in a protein-free medium, and requires fewer than 4 h to complete.

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FIG. 9. Western blot demonstrating seropositivity and reaction to L. donovani exoantigen with sera from 10 canines infected with L. infantum (D1 to D10) versus healthy canine controls (N1 to N4), which show no reaction to the same antigen profile.

ACKNOWLEDGMENTS We thank L. Thuita-Harun for technical assistance in the initial preparation of the exoantigen material. We thank Reynaldo Dietze and Luis Valli for donating the samples from Brazil and express our sincere appreciation to J. Levine for donating the canine negative control sera. This work is supported in part by funding received from the U.S. Department of Defense Gulf War Illnesses Research Program (PE 0601105D8Z). REFERENCES 1. Anthony, R. L., H. A. Christensen, and C. M. Johnson. 1980. Micro enzymelinked immunosorbent assay (ELISA) for the serodiagnosis of New World leishmaniasis. Am. J. Trop. Med. Hyg. 29:190–194. 2. Badaro, R., D. Benson, M. C. Eulalio, M. Freire, S. Cunha, E. M. Netto, D. Pedral-Sampaio, C. Madureira, J. M. Burns, R. L. Houghton, J. R. David, and S. G. Reed. 1996. rK39: a cloned antigen of Leishmania chagasi that predicts active visceral leishmaniasis. J. Infect. Dis. 173:758–761. 3. Badaro, R., S. G. Reed, A. Barral, G. Orge, and T. C. Jones. 1986. Evaluation of the micro enzyme-linked immunoabsorbent assay (ELISA) for antibodies in American visceral leishmaniasis: antigen selection for detection of infection-specific responses. Am. J. Trop. Med. Hyg. 35:72–78. 4. Bagchi, A. K., S. Tiwari, S. Gupta, and J. C. Katiyar. 1998. The latex agglutination test: standardization and comparison with direct agglutination and dot-ELISA in the diagnosis of visceral leishmaniasis in India. Ann. Trop. Med. Parasitol. 92:159–163. 5. Bates, P. A., M. Gottlieb, and D. M. Dwyer. 1988. Leishmania donovani: identification of glycoproteins released by promastigotes during growth in vitro. Exp. Parasitol. 67:199–209. 6. Bates, P. A., M. K. Kurtz, M. Gottlieb, and D. M. Dwyer. 1987. Leishmania

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