**5. Diagnosis**

Infection occurred by pathogenic leptospires is divided into two stages, first stage is acute stage or septicaemia (because septicaemia is in this stage), which lasts from 7 to 10 days with headache and myalgia. The second stage is immune stage which is after first week of infection and lasts 4–30 days [34]. During first stage, leptospires are present in blood and can say bacterial count is high in the blood, while when second stage starts, then the level of antibodies IgM and IgG start to increase and this increase in antibodies titer is correlated to elimination of leptospires from blood. *Leptospira* antigens and DNA sometimes may not be detected from the blood; this may be due to late sampling, or sampling in acute stage where proper level of leptospiremia is not developed and due to antibiotic administration, leptospires are eliminated from the blood. False negative results will be there, if we detect antibodies prior to sero-conversion during acute stage. Coagulated blood has serum and clot while non-coagulated blood has plasma, RBCs, WBCs and platelets. That can be collected according to the tests. If you are going to do gene amplification than EDTA, plasma gives the best results [50]. Leptospires can be detected in urine and cerebrospinal fluid samples. Many kits are available in the market for rapid detection of leptospires from blood, urine, and CSF sample; these kits basically detect nucleic acid of leptospires, but for these tests, purification of nucleic acid is required [51].
