Leptospiral 3-hydroxyacyl-CoA dehydrogenase as an early urinary biomarker of leptospirosis

Abstract

Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonotic disease. The currently used diagnostic tests are time-consuming, require technical expertise or require the use of sophisticated equipment. Clinicians have pointed out the urgent need to develop a rapid test for the diagnosis of acute leptospirosis with a non-invasive and easy sampling method. In this study, we have focused on a leptospiral enzyme, 3-hydroxyacyl-CoA dehydrogenase (3-HADH), as a urinary biomarker of acute leptospirosis. A specific antiserum for pathogenic Leptospira spp. was produced, targeting a peptide corresponding to amino acids 410 to 424 of 3-HADH. The antiserum was used to investigate whether 3-HADH is excreted in the urine by Western blotting. Among 70 suspected leptospirosis patients, 40 were laboratory confirmed by microscopic agglutination test (MAT) using paired sera samples and/or polymerase chain reaction (PCR). In the acute phase of the laboratory-confirmed leptospirosis cases, sensitivity for 3-HADH, blood PCR and urine PCR were 52.5%, 57.5% and 12%, respectively. 3-HADH was detected from 2 days post-onset of illness (p.o) and could be detected at least until 9 days p.o. The combination of PCR and 3-HADH detection increased sensitivity of diagnosis to 100% in samples collected between 1 and 3 days p.o., and to 82% in samples collected between 4 and 9 days p.o. Our results suggested that the detection of 3-HADH can support a clinical diagnosis of leptospirosis, especially when serological methods are negative during the acute phase.

Keywords: Infectious disease; Microbiology.

Fig. 1

Peptide design to prepare pathogenic Leptospira species-specific antiserum. Predicted antigenic value (relative) in each amino acid position is shown. Pathogenic Leptospira specific regions are showed in red. Yellow circles show putative glycosylation sites.

Fig. 2

Detection of 3-HADH in bacterial whole cell lysates. Lanes: (M) Potein molecular weight markers (1) L. biflexa, (2) L. borgpetersenii ser. Javanica (3) to (8) L. interrogans. Serovars (3) Manilae, (4) Lai, (5) Ratnapura, (6) Icterohaemorrhagiae, (7) Hebdomadis, and (8) Pyrogenes. Lane 9: purified His-tagged rHADH.

Fig. 3

Analysis of urine samples by Western Blotting. Representative urine samples collected up to 3 days p.o. are shown. Lanes 1 to 6: urine samples of laboratory confirmed leptospirosis patients. Lanes 1: patient 12, lane 2: patient 4, lane 3: patient 6, lane 4: patient 16, lane 5: patient 7, lane 6: patient 13. Lanes 7 and 8: whole cell lysates of in vitro cultivated L. interrogans serovar Manilae. Lane 7: culture-attenuated strain (3-HADH-positive, LigA-negative) . Lane 8: virulent strain (3-HADH-positive, LigA-positive). Lane 9: purified His-tagged rHADH (10 ng). Lane 10: human albumin (125 ng).

Fig. 4

Sensitivity of 3-HADH detection and PCR in laboratory confirmed cases. Sensitivity for each method (PCR blood, PCR urine and 3-HADH WB) or sensitivity when several methods are combined (PCR blood and PCR urine, PCR + 3-HADH) are shown based on the days post-onset of illness. PCR + 3-HADH: combination of PCR (blood and/or urine) with 3-HADH detection as the criteria for laboratory confirmation of leptospirosis.