Evaluation of electro-eluted antigens in the serological diagnosis of cutaneous leishmaniasis

Despite efforts to control the disease, human cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) braziliensis remains a major public-health problem in South America. Several parasitological methods, in which the parasites can be visualized, isolated and identified, form the ‘gold standard’ for diagnosis (Gontijo and de Carvalho, 2003). Although very specific, such methods are relatively slow, have low sensitivity and, in the case of culture, require the skills and facilities associated with the aseptic collection of samples and their incubation in complex media. Recently, PCR-based assays have been used for the detection and identification of Leishmania-specific DNA (Pirmez et al., 1999). Such assays can be used to test samples that have been collected non-invasively and are less time-consuming than most other diagnostic methodologies (Garcia et al., 2007). The molecular techniques for the diagnosis of CL (and many other infectious diseases) still, however, require standardization and optimisation before their use becomes routine (Reithinger and Dujardin, 2007). There are also immunological tests for CL, the most frequently used being the Montenegro skin test and various ELISA. Although these tests are not able to identify parasites to species level and may give falsepositive results in cross-reactions with Trypanosoma species and non-target Leishmania species (Vexenat Ade et al., 1996), their ease of use, low cost and sensitivity in detecting active or past cases of leishmanial infection often make them the first choice for screening in endemic areas. ELISA based specifically on the IgG subclass or IgG1 and IgG3 isotypes have proven to be of great value in the diagnosis of active CL, and the titres recorded in such assays may also be useful in the follow-up of treated patients (Rodriguez et al., 1996; Romero et al., 2005; Vidigal Cde et al., 2008; Barroso-Freitas et al., 2009; Castellano et al., 2009). One goal remaining in research on L. braziliensis is the identification of the best parasite antigens for use in diagnostic ELISA. Electrophoresis has already been employed to separate wholeparasite extracts into antigenic fractions, for use in ELISA and other immunological reactions (Legrand et al., 1987; Kutner et al., 1991; Goncalves et al., 2002). The aim of the present study was to evaluate the reactivity in ELISA of various antigens — that had been recovered from L. braziliensis promastigotes, separated by electrophoresis and electro-eluted — with sera from the inhabitants of an area of Brazil (Buerarema, Bahia) where CL is endemic (Salhi et al., 2008). The sera tested came from individuals with active (seven sera) or healed (seven sera) CL lesions. The study protocol was approved by the Ethical Committee of the Universidade Federal do Triângulo Mineiro (protocol # 744). The antigens tested were obtained as previously described (Castellano et al., 2009). Briefly, promastigotes of L. braziliensis strain Lb2904 were grown in supplemented Schneider’s medium (Sigma) within plastic culture flasks at 26uC and harvested after 96 h, when in stationary growth phase. They were then washed three times in RPMI medium (Life Technologies, Carlsbad, CA) and resuspended in Tris-HCl buffer (pH 7.2) containing 1% (v/v) Nonidet P-40 (Sigma) and 0.1 mM Na-p-tosyl-L-lysine chloromethyl ketone (TLCK; Sigma). The mixture was centrifuged (40006g for 30 min Annals of Tropical Medicine & Parasitology, Vol. 104, No. 4, 347–350 (2010)