Optimisation and application of an indirect immunofluorescence test for the diagnosis of leptospirosis in conventional laboratories

A Armada, Wilmer Fernandes, Teresa Carreira, Vieira ML

Research output: Contribution to conferencePosterpeer-review


Leptospirosis is a re-emerging zoonotic disease with a worldwide distribution, with more than 1,000,000 severe cases reported annually in the human population1. It is caused by pathogenic spirochetes of the genus Leptospira. The definitive diagnosis of leptospirosis, along with compatible clinical findings, is based on the Microscopic Agglutination Test (MAT), a serological reference test
recommended by the World Health Organization (WHO). However, MAT is sometimes not feasible due to the high complexity of its execution since it is a serovar-specific test, requiring a battery of about 20 different serovars. Thus, this study aims to optimize optimize an indirect immunofluorescence test (Lept-IFA) for leptospirosis diagnosis, using three pathogenic serovars, recognized for their high and ubiquitous prevalence in Portugal
Methods: Representative serovars of Leptospira interrogans s.l. (Copenhageni, Ballum and Pomona) and L. biflexa s.l (Patoc) were maintained in EMJH medium. In order to preserve surface leptospiral proteins, spirochetes were harvested by low-speed centrifugation2 and loaded to each spot of the IFA slide coated with lysine and then fixed with formalin. MAT positive and negative sera, negative and positive serum for Lyme Borreliosis were selected for the IFA optimization assay.
To evaluate the specificity of Lept-IFA, sera from patients infected by other spirochetes were also tested. For the sensitivity assessment, leptospirosis positive and negative sera were tested by IFA against other six pathogenic serovars. Fifty human serum samples, selected from the BioBank of IHMT NOVA/Leptospirosis laboratory, with a clinical indication for leptospirosis and analysed by MAT, were tested by Lept-IFA.
Results: Lept-IFA confirmed all the positive samples tested by MAT (36%), 8% were inconclusive (NC) and 56% were negative. In the group of NC sera, both Lyme borreliosis sera cross-reacted with Patoc.
Conclusions: Lept-IFA, a simplified assay performed with only three pathogenic and one saprophytic serovars, presents a high specificity and sensitivity. This study pointed out that Lept-IFA is a useful tool as a first-line screening test for serologic diagnosis of leptospirosis in regions/countries whose laboratories do not have the reference test recommended by the WHO. However, this method lacks reliable standardization and is very dependable on the qualification of the observer.
Original languageEnglish
Publication statusPublished - 16 Apr 2023
Event33rd European Congress of Clinical Microbiology and Infectious Diseases - Copenhagen, Denmark
Duration: 15 Apr 202318 Apr 2023


Conference33rd European Congress of Clinical Microbiology and Infectious Diseases
Abbreviated title33rd ECCMID


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