Diagnostic accuracy of an IgM enzyme-linked immunosorbent assay and comparison with 2 polymerase chain reactions for early diagnosis of human leptospirosis

Enzyme-linked immunosorbent assay (ELISA) tests and polymerase chain reaction (PCR) may play a key role for early detection and treatment of human leptospirosis in developing countries. The aims of this study were to develop and validate an IgM ELISA under field conditions and to compare the diagnostic accuracy among IgG, IgM ELISAs, conventional PCR (cPCR), and real-time PCR (rtPCR) for early detection of human leptospirosis. Overall accuracy of IgM ELISA was sensitivity of 87.9%, specificity of 97.0%, and area under the curve of 0.940. When the 4 methods were compared, IgM ELISA showed the greatest diagnostic accuracy (J=0.6) followed by rtPCR (J=0.4), cPCR (J=0.2) and IgG ELISA (J=0.1). Our results support the use of IgM ELISA and rtPCR for early diagnosis of the disease. Moreover, due to their high specificity, they could be also useful to replace or supplement microscopic agglutination test as a confirmatory test, allowing more confirmations

14-3-3 proteins regulate exonuclease 1-dependent processing of stalled replication forks

Replication fork integrity, which is essential for the maintenance of genome stability, is monitored by checkpoint-mediated phosphorylation events. 14-3-3 proteins are able to bind phosphorylated proteins and were shown to play an undefined role under DNA replication stress. Exonuclease 1 (Exo1) processes stalled replication forks in checkpoint-defective yeast cells. We now identify 14-3-3 proteins as in vivo interaction partners of Exo1, both in yeast and mammalian cells. Yeast 14-3-3-deficient cells fail to induce Mec1-dependent Exo1 hyperphosphorylation and accumulate Exo1-dependent ssDNA gaps at stalled forks, as revealed by electron microscopy. This leads to persistent checkpoint activation and exacerbated recovery defects. Moreover, using DNA bi-dimensional electrophoresis, we show that 14-3-3 proteins promote fork progression under limiting nucleotide concentrations. We propose that 14-3-3 proteins assist in controlling the phosphorylation status of Exo1 and additional unknown targets, promoting fork progression, stability, and restart in response to DNA replication stress