Validation of a Microsphere Immunoassay for Serological Leptospirosis Diagnosis in Human Serum by Comparison to the Current Gold Standard
March
Validation of a Microsphere Immunoassay for Serological Leptospirosis Diagnosis in Human Serum by Comparison to the Current Gold Standard
Sarah J. Wynwood 0 1
Mary-Anne A. Burns 0 1
Glenn C. Graham 0 1
Steven L. Weier 0 1
David B. McKay 0 1
Scott B. Craig 0 1
0 1 Faculty of Science , Health and Education , University of the Sunshine Coast , Sippy Downs, Queensland , Australia , 2 WHO/OIE/FAO Collaborating Centre for Reference and Research on Leptospirosis, Queensland Health Forensic and Scientific Services , Archerfield, Queensland , Australia , 3 Chemical Analysis Unit, Queensland Health Forensic and Scientific Services , Archerfield, Queensland , Australia , 4 School of Biomedical Sciences, Queensland University of Technology , Brisbane , Australia
1 Editor: L. C. Pamela , Small , University of Tennessee, UNITED STATES
A microsphere immunoassay (MIA) utilising Luminex xMap technology that is capable of determining leptospirosis IgG and IgM independently was developed. The MIA was validated using 200 human samples submitted for routine leptospirosis serology testing. The traditional microscopic agglutination (MAT) method (now 100 years old) suffers from a significant range of technical problems including a dependence on antisera which is difficult to source and produce, false positive reactions due to auto-agglutination and an inability to differentiate between IgG and IgM antibodies. A comparative validation method of the MIA against the MAT was performed and used to determine the ability of the MIA to detect leptospiral antibodies when compared with the MAT. The assay was able to determine samples in the reactive, equivocal and non-reactive ranges when compared to the MAT and was able to differentiate leptospiral IgG antibodies from leptospiral IgM antibodies. The MIA is more sensitive than the MAT and in true infections was able to detect low levels of antibody in the later stages of the acute phase as well as detect higher levels of IgM antibody earlier in the immune phase of the infection. The relatively low cost, high throughput platform and significantly reduced dependency on large volumes of rabbit antisera make this assay worthy of consideration for any microbiological assay that currently uses agglutination assays.
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Funding: This study was funded internally by the
Research and Development Office, Department of
Health, Queensland. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
and require specialist expertise and equipment. The current gold standard diagnostic assay
for leptospirosis (MAT) cannot determine IgG from IgM antibodies and relies on live
cultures, which presents problems in the way of maintenance and attenuation. Development of
a new diagnostic assay for serological diagnosis of leptospirosis that is specific, sensitive and
able to discriminate between IgG and IgM classes of antibodiesas well as being more cost
effectivewill significantly improve the capabilities for detecting leptospirosis infections. It
will provide medical professionals with more valuable diagnostic information and public
health professionals with improved epidemiological information.
Leptospirosis is considered to be the most widespread zoonotic disease in the world [1] with
clinical diagnosis proving challenging due to the non-specific nature of symptoms associated
with the disease. There are some 300 leptospiral serovars belonging to a number of different
serogroups. Currently there are 24 sero-groups of pathogenic leptospires based on their
antigenic relatedness [2]. Leptospirosis was first reported in Australia in 1933 in the state of
Queensland and has since been isolated Australia wide [3] with Queensland reporting the
majority of these cases (57.6%) [4]. In 2011 the reported incidence of leptospirosis in Queensland
was 3.4 cases per 100,000 people and overall in Australia the incidence was 0.84 cases per
100,000 people [5]. At present, 24 serovars of Leptospira spp are recognised in Australia and in
recent years a dramatic increase in the incidence of leptospirosis cases in Australia (particularly
Queensland) has been noted with environmental factors believed to be the main influence on
this increase [6].
Diagnosis of leptospirosis occurs at two stagesduring the acute phase the live organism
can be detected by two methods. Polymerase chain reaction (PCR) testing is a useful molecular
detection tool for rapid qualitative diagnosis of leptospirosis in its earliest stage [7]. Serum or
blood samples provided for PCR testing must be collected within a precise timeframe (08
days post onset) to enable diagnosis. Blood culture isolation can also be utilised in the early
stages of leptospiral infection (010 days post onset), however this method is time consuming,
requires specialised media and equipment and can take months for a serovar specific r (...truncated)