Exploring diagnostic application of cross-reactivity between Leptospira serovars as used in standard microscopic agglutination test for leptospirosis

Abstract

The microscopic agglutination test (MAT) is widely considered the gold standard techniques for diagnosis of Leptospirosis because of its high specificity, and ability to elucidate the infecting Leptospira species or serogroups. However, the high biosafety risk and operational costs associated with procurement and maintenance of large panels of live fastidious Leptospira cultures makes it difficult for non-specialized laboratories especially in Low and middle-income countries to widely adopt MAT. This has resulted in under diagnosis and underreporting of incidences of the disease. The current study aims at exploring cross-reactivity between Leptospira serovars in an effort to devise a smaller but equally efficient panel of MAT serovars that will in turn minimize laboratory-running costs for the test, reduce the turn-around-time and associated biosafety risk.This was an experimental study in which cross-reactivity was explored to devise and assess the diagnostic efficacy of a shorter MAT panel over a standard 12 serovar MAT panel.Cross reactivity was noted in three pairs of serovars when using commercial anti-Leptospira antibodies. The cross-reactive serovar pairs were Hardjo and Sejroe, Icterohaemorrhagiae and Copenhageni, Bratslava and Australis. The same pairs of serovars were cross-reactive (up to 100%) when tested against highly seropositive sera samples (anti-Leptospira antibody titres ≥800) from cattle and dogs. However, cross-reactivity between serovars Icterohaemorrhagiae and Copenhageni was weaker when tested against serum samples (only 40% reactivity). Of the 300 random serum samples tested, matching results between both panels were registered in 293 samples (97.67%). Overall, there was a very strong agreement between the MAT results of the standard MAT panel and the devised shorter one (Cohen's kappa statistic 0.947±0.058; 95% CI 0.909-0.986). The sensitivity and specificity of the shorter panel were 96.97% (95% CI 0.914-0.994) and 98.01% (95% CI 0.950-0.995), while the positive and negative predictive values were 96.0% and 98.5%, respectively.This study shows that serovars of the same serogroup cross-react in both commercial and non-commercial anti-sera. The shorter panel (9 serovars) is highly sensitive, specific and compares well with the standard MAT, and thus could be used as an alternative for diagnosis of leptospirosis in dogs and cattle in Uganda.