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Plague has been one of the most feared diseases in history, and the third pandemic can be regarded as still ongoing since Yersinia pestis sporadically re-emerges from its reservoir of wild rodents. Plague is a flea-borne disease that may cause large epizootics among rodent population ("rat fall" phenomenon), and humans living in close contact with these rodents may acquire bubonic plague following infective flea bites.1 Most of the time, plague is transmitted without noticeable signs among the natural reservoir in which antibodies to fraction 1 (F1) can be detected.2,3 During the last 15 years, more than 20 countries have reported plague cases to the World Health Organization.4 Yersinia pestis is considered one of the bacteriologic agents that could be used for biological warfare, causing pneumonic plague if spread by aerosol.
Because plague is a fulminating disease and the clinical diagnosis is non-specific, the treatment of suspected cases must be started without delay. A retrospective laboratory confirmation of plague can be obtained by detecting antibodies directed against a capsular protein (the F1 antigen). A simple assay to detect antibodies to the F1 fraction will be useful for epidemiologic surveys and for monitoring the transmission of Y. pestis among the animal reservoir in natural plague foci. We have developed a rapid assay in a half-dipstick format that is able to detect both human and rat IgG antibodies to the F1 fraction.
The test is based on the principle of vertical flow immunochromatography using colloidal gold particles conjugated to protein A. The format of the test is a strip made of specially formulated nitrocellulose (membrane test platform; Whatman, Maidstone, United Kingdom) backed by a protective sheet and flanked at its top by an absorption pad (3MMChr; Whatman). Purified F1 antigen (Institut Pasteur de Madagascar, Antananarivo, Madagascar) at a concentration of 500 µg/mL in phosphate-buffered saline (PBS) was filtered through a 0.22-µ pore size membrane and spread as a thin capture line (Biojet Quanti-3000; Biodot Ltd., Cambridge, United Kingdom) at a concentration of 1µL/cm 10 mm above the bottom of the nitrocellulose strip. The composite was trimmed into 5-mm strips and stored in hermetically closed tubes in the presence of a desiccant (silica gel; Sigma, L’Isle d’Abeau, France) at room temperature. Ten microliters of each serum diluted 1:100 in PBS were added to 15 µL (absorbance at 540 nm = 10) of protein A immunogold conjugate (British Biocell International, Cardiff, United Kingdom) and 45 µl of migration buffer (PBS, 0.5% Tween, 0.5% bovine serum albumin) in the wells of a flat-bottomed enzyme-linked immunosorbent assay (ELISA) microplate (HTS Polysorb; Nunc, Roskilde, Denmark). The strip was dropped into the well and the test could be visually followed as the upward wetting of the strip up to the absorbent pad. After 20 minutes, the assay result was scored positive when a distinct pink line was observed on the F1 capture line. The different parameters of the test were optimized step-by-step using a panel of anti-F1 positive and negative human and rat sera. The anti-F1 IgG indirect ELISA was used as the reference test to assess the sensitivity and specificity of the rapid test.3
The sensitivity of the anti-F1 dipstick test with human sera was assessed using 35 ELISA-positive control sera from convalescent plague patients. These sera were collected in Mahajanga harbor in Madagascar during the outbreak of plague in September 1997.5 The anti-F1 IgG ELISA titers ranged from 50 to 6,400. The specificity was assessed with 37 anti-F1 ELISA-negative control sera collected from healthy subjects in the same city and at the same period of time.
Rats were trapped alive in Antananarivo, Madagascar and individual rat sera (22 ELISA-positive and 24 ELISA-negative sera) were collected from Rattus rattus and R. norvegicus, the two species of rats that serve as reservoirs of Y. pestis in Madagascar. Sera of trapped rats testing positive in anti-F1 ELISA were pooled. The titer of the pool was 104,000. The positive individual controls (ELISA titers ranging from 400 to 416,000) were obtained from rats immunized with purified F1 antigen injected intramuscularly once a week for six weeks (1 mg/mL of F1 antigen in Freund’s complete adjuvant for the first injection and in Freund’s incomplete adjuvant for the subsequent injections). All sera were kept frozen (-20°C) before being tested.
The anti-F1 dipstick assay showed positive results for 33 of 35 positive human sera (sensitivity = 94.3%) and for 22 of 22 positive rat sera plus the pool (sensitivity = 100%). Negative results were obtained for 33 of 37 negative human sera (specificity = 89.2%) and 22 of 24 negative rat sera (specificity = 91.7%). The 95% confidence intervals for these results are shown in Table 1
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As far as we know, this is the first rapid test of its kind that is applicable with both human and animal sera. Due to its good sensitivity and specificity, this dipstick test may be used as a convenient and rapid tool in the surveillance of plague.
Received January 27, 2003. Accepted for publication July 1, 2003.
Financial support: This work was supported by a grant (PEA 803) from the Direction Générale de l’Armement.
Authors’ addresses: Philippe Thullier and Valerie Guglielmo, Immunobiologie, Département de Biologie des Agents Transmissibles, Centre de Recherches du Service de Santé des Armées, 24 Avenue des Maquis du Gresivaudan, PO Box 87, 38702 La Tronche, France, Telephone: 33-4-76-63-69-14, Fax: 33-4-76-63-69-17, E-mails: pthullier{at}yahoo.com and vguglielmo{at}crssa.net. Mino Rajerison, Institut Pasteur de Madagascar, World Health Collaborating Center for Plague, PO Box 1274, Antananarivo, Madagascar, Telephone: 261-20-22-401-64, Fax: 261-20-22-415-34, E-mail: peste{at}pasteur.mg. Suzanne Chanteau, CERMES, PO Box 10 887, Niamey, Niger, Telephone: 227-75-20-40, Fax: 227-75-31-80, E-mail: schanteau{at}cermes.ne.
Reprint requests: Philippe Thullier, Immunobiologie, Département de Biologie des Agents Transmissibles, Centre de Recherches du Service de Santé des Armées, 24 Avenue des Maquis du Gresivaudan, PO Box 87, 38702 La Tronche, France.
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