Development of a sensitive and specific molecular assay for the rapid detection of
Leptospira DNA from human clinical samples
Master of Science in Medical Laboratory Science
2004
Trevor Paul Anderson
Abstract
A hemi-nested PCR assay was developed for the diagnosis of human leptospirosis during
the acute phase of infection. The PCR used a combination of the newly designed primer
Lept-F4 and primers developed by Merien, Amouriaux, Perolat, Baranton and Saint
(1992) J. Clin Microbiol. 20, 2219-2224. This ‘F4 PCR’ was optimised and then
evaluated using clinical samples from two different study groups, one from New Zealand
and the other from Nepal.
The New Zealand study had 66 samples collected from 23 patients from the Canterbury
District Health Board region with suspected leptospirosis. A total of 12 (52.2%) patients
were positive for Leptospira, 9 by PCR, 7 positive by serology and 5 positive by PCR
and serology. The adapted Merien PCR assay detected 14 (21.2%) positive samples and
the F4 PCR detected 15 (22.7%). All 15 F4 PCR amplified products were confirmed as
true positives by DNA sequencing. The single round G1G2 PCR assay detected only 5
out of the 9 positive patients and these results indicate the importance for the increased
sensitivity of the nested procedures. The difference in comparison of PCR and serology
results from the New Zealand study highlights the need for both PCR and serology
testing for the reliable diagnosis of leptospirosis.
The second study group was from Nepal where patients were selected based on initial
symptom of fever on admittance to hospital. The Nepal study group was further separate
based on the time of year that they were collected. Nepal study 1 samples were collected
during the drier winter period and Nepal study 2 samples collected during the monsoon
season. Ninety-four samples were collected from 50 patients for the Nepal study 1 group
but no PCR positive patients were detected; although there were 23 (24.5%) adapted
Merien PCR positives. Sixty-seven samples were collected from 35 patients for the
Nepal study 2 group and 6 (17.1%) PCR positive patients were detected by the F4 PCR
and the Merien PCR. The adapted Merien PCR also detected an extra 13 PCR positive
samples and as in Nepal study 1 these were confirmed as non-specific PCR products by
DNA sequencing. The F4 PCR had better specificity than the adapted Merien assay as all
7 F4 PCR positive samples were confirmed as true positives by DNA sequencing. The
serology results from the Nepal studies were of limited value due to the high number of
potential false positive IgM results due to cross reactions with other diseases; the positive
IgM results from the Nepal studies are unable to be confirmed.
The improved sensitivity and specificity of the leptospiral F4 PCR will provide acute
phase detection of leptospirosis before traditional diagnostic tests, and provide a result
that will allow appropriate early treatment of patients.