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.

Usefulness of C-Reactive Protein in Differentiating Acute Leptospirosis and Dengue Fever in French Guiana

“…To note, promising tests with potential interest for early distinction between leptospirosis and DF recently have been developed in urine [30, 31] and serum [32]. Still, they seem far from being routinely used, especially in resource-limited countries where the diagnostic dilemma remains the most problematic.…”

ELISA for leptospiral 3-hydroxyacyl-CoA dehydrogenase in urine is a promising screening tool for acute leptospirosis

“…Then, 0.25 ml of urine was added to each well (2 replicates) of a 96-well microtitre plate (Nunc, Roskilde, Denmark). Further, 1 : 1000 and 1 : 2000 dilutions of purified recombinant 3-HADH enzyme [ 11 ] were used as positive controls. Skimmed milk (5 %) (prepared by mixing 5 g of non-fat milk powder in 100 ml distilled water) was used as a negative control.…”

“…One gramme of BSA was diluted in 100 ml of 1× phosphate-buffered saline (PBS) solution to prepare the ELISA buffer (EB). Rabbit anti-3-HADH [ 11 ] antibody was diluted at a ratio of 1 : 3000 in EB, and 50 µl of the dilution was added in each well of the ELISA plate and incubated at 37 °C. After 1 h, the rabbit anti-3-HADH antibody solution was discarded and then the plate wells were washed thrice with TBST solution.…”

“…Based on the findings of Segawa et al ., leptospiral 3-HADH was detected by Toma et al . in urine samples from patients with suspected leptospirosis submitted to the Japanese Reference Laboratories [ 11 ]. This enzyme was detected in the urine at the acute phase of infection when leptospiral DNA in the urine was still negative [ 11 ].…”

“…in urine samples from patients with suspected leptospirosis submitted to the Japanese Reference Laboratories [ 11 ]. This enzyme was detected in the urine at the acute phase of infection when leptospiral DNA in the urine was still negative [ 11 ]. Given that blood and urine PCR sensitivities are time-dependent, 3-HADH offers an expanded detection window and permits the early diagnosis of leptospirosis when other laboratory tests are negative.…”