Article

Rapid tests for diagnosis of Leptospirosis: Current tools and emerging technologies

Institut Pasteur, Unité de Biologie des Spirochètes, National Reference Center and WHO Collaborating Center for Leptospirosis, Paris, France.
Diagnostic microbiology and infectious disease (Impact Factor: 2.46). 10/2013; 78(1). DOI: 10.1016/j.diagmicrobio.2013.09.012
Source: PubMed

ABSTRACT

Leptospirosis is an emerging zoonosis with a worldwide distribution but is more commonly found in impoverished populations in developing countries and tropical regions with frequent flooding. The rapid detection of leptospirosis is a critical step to effectively manage the disease and to control outbreaks in both human and animal populations. Therefore, there is a need for accurate and rapid diagnostic tests and appropriate surveillance and alert systems to identify outbreaks. This review describes current in-house methods and commercialized tests for the rapid diagnosis of acute leptospirosis. It focuses on diagnostic tests that can be performed with minimal training and limited equipment in less-developed and newly industrialized countries, particularly in resource-limited settings and with results in minutes to less than 4 hours. We also describe recent technological advances in the field of diagnostic tests that could allow for the development of innovative rapid tests in the near future.

Full-text

Available from: Rudy A Hartskeerl, Jul 03, 2015
Review
Rapid tests for diagnosis of leptospirosis: Current tools and emerging technologies
Mathieu Picardeau
a
, Eric Bertherat
b
, Michel Jancloes
c
, Andreas N. Skouloudis
d
,
Kara Durski
b
, Rudy A. Hartskeerl
e,
a
Institut Pasteur, Unité de Biologie des Spirochètes, National Reference Center and WHO Collaborating Center for Leptospirosis, Paris, France
b
World Health Organization, Health Security and Environment/Pandemic and Epidemic Diseases, 20 Av Appia, 1211, Geneva 27, Switzerland
c
Health and Climate Foundation, 1425K St NW suite 350, Washington DC 20005, USA
d
Institute for Environment and Sustainability, Joint Research Centre, European Commission, Ispra VA, Italy
e
Royal Tropical Institute, KIT Biomedical Research, WHO/FAO/OIE and National Collaborating Centre for Reference and Research on Leptospirosis, Amsterdam, The Netherlands
abstractarticle info
Article history:
Received 23 July 2013
Received in revised form 9 September 2013
Accepted 15 September 2013
Available online 1 October 2013
Keywords:
Leptospirosis
Diagnostics
Outbreak
Surveillance
Diagnostic test
Early warning
Early diagnosis
Innovation
Leptospirosis is an emerging zoonosis with a worldwide distribution but is more commonly found in
impoverished populations in developing countries and tropical regions with frequent ooding. The rapid
detection of leptospirosis is a critical step to effectively manage the disease and to control outbreaks in both
human and animal populations. Therefore, there is a need for accurate and rapid diagnostic tests and
appropriate surveillance and alert systems to identify outbreaks. This review describes current in-house
methods and commercialized tests for the rapid diagnosis of acute leptospirosis. It focuses on diagnostic tests
that can be performed with minimal training and limited equipment in less-developed and newly
industrialized countries, particularly in resource-limited settings and with results in minutes to less than 4
hours. We also describe recent technological advances in the eld of diagnostic tests that could allow for the
development of innovative rapid tests in the near future.
© 2014 Elsevier Inc. All rights reserved.
1. Introduction
Leptospirosis is of particular public health concern due to its
global distribution, its epidemic potential, its presence in animals
and the natural environment, and its high potential for human
mortality, if left untreated. A World Health Organization (WHO)
lead experts' group estimated the global burden of leptospirosis to
be 873,000 severe annual cases and 49,000 deaths (http://www.
who.int/zoonoses/diseases/lerg/en/index.html). The recent out-
breaks of leptospirosis in the South East Asia have increased the
awareness of the need for improved diagnostic tests for leptospi-
rosis (Agampodi et al., 2011; Amilasan et al., 2012). Leptospirosis is
also one of the most important zoonotic diseases over the world.
Human infection results from contacts with carrier animals or
environment contaminated with leptospires. It is a major environ-
mental endemic disease with increased threat of severe epidemics
often linked with natural disasters such as oods and hurricanes
(Lau et al., 2010; Levett, 2001).
Because there is much overlap in the clinical presentation of
undifferentiated febrile illnesses, which includes leptospirosis, ma-
laria, rickettsioses, and arboviral diseases, it is not possible to reliably
predict the pathogen based on clinical signs and symptoms (WHO,
2003). The lack of adequate and easy-to-use lab diagnostics is key in
leptospirosis: it largely contributes to the under-recognition of its
burden and it is an obstacle to the understanding of the natural history
of the infection. This means that many questions related to the control
strategy remain unanswered, including on case management, partic-
ularly in epidemic situation.
Public health authorities and clinicians have pointed out the
urgent need for developing more effective technologies on case
detection and diagnosis.
Culture and the microscopic agglutination test (MAT), which are
gold standard methods for leptospirosis diagnosis, are not useful for
early diagnosis: culture of Leptospira, which are slow-growing
bacteria, is difcult, and anti-Leptospira antibodies can only be
detected by the second week after symptoms (Levett, 2001).
Moreover, these techniques require specicequipmentand/or
laboratory and highly trained staff. Early and accurate diagnosis, so
that treatment with antibiotics can be started without delay, may
contribute to improved patient outcomes (Bharti et al., 2003). This
may also minimize the cost of hospitalization.
2. Diagnostic test needs
The choice for the use of a diagnostic test will depend on a number
of factors, including its diagnostic accuracy, nancial feasibility,
Diagnostic Microbiology and Infectious Disease 78 (2014) 18
Corresponding author. Tel.: +31-20-5665438; fax: +31-20-6971841.
E-mail address: r.hartskeerl@kit.nl (R.A. Hartskeerl).
0732-8893/$ see front matter © 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.diagmicrobio.2013.09.012
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Page 1
technical or practical feasibility, and the need for an early and/or
rapid result.
2.1. Performance of diagnostic tests
The diagnostic accuracy of tests is frequently expressed in terms of
sensitivity, specicity, and predictive values. However, most of the
diagnostic tests for leptospirosis, both commercially available and in-
house tests, have been presented without a solid validation scheme
such as STARD principles (http://www.stard-statement.org), thus
hampering the selection of the test of choice (Goris et al., 2011).
Moreover, especially rapid diagnostic tests and enzyme-linked
immunosorbent assays (ELISAs) have been reported with varying
diagnostic performances in distinct countries or regions. There might
be several explanations for the reported variations in diagnostic assay
performance: i) The retrospective use of selected samples and the
choice of case denition may be a source of bias; ii) it is a
misunderstanding that rapid tests are easy and thus do not require
experience. The subjective judgement of test results by eye, mostly for
serological tests, may also introduce a degree of bias, particularly in
the case of untrained personnel. iii) It may reect population-related
differences such as past exposure to leptospirosis, exposure to
environmental leptospires, or infection with other infectious agents.
This is particularly true for tests based on whole-bacteriaderived
antigens that can lead to false positives due to persistent or cross-
reacting antibodies. This point is not easily solved by using
recombinant antigens. While these will enable more specic test
results with antigen-homologous infections, sequence variation
between Leptospira species and serovars will reduce test sensitivity
in case of many, if not most, other serovars.
The best approach for applying the most optimal test would be to
perform a valid local evaluation prior to introduction or, alternatively,
have tests validated by globally representative serum banks present
on accredited international reference centres that are familiar with
validation schemes of tests.
The need for a high dia gnostic accuracy should always be
considered within the scope of local requirements. While conrma-
tion in expert reference centres requires a high level of diagnostic
accuracy, rapid screening tests might sufce for peripheral health care
centres, certainly when t he local epi dem iol og y has been well
assessed. Moreover, such tests are very useful for monitoring of
outbreaks when a rapid unusual accumulation of cases might give an
early alert, provided that specimens are collected, transported, and
stored in an adequate manner.
2.2. Rapid versus early diagnostic tests
A rapid diagnostic test (RDT) does not equal an early test. While a
rapid test provides a quick test result, an early test conrms a clinical
suspicion at the early acute phase of the disease. For leptospirosis,
early detection is particularly important to eliminate differential
diagnoses and to start the appropriate treatment as soon as possible.
While molecular tests, such as the polymerase chain reaction (PCR),
demonstrating the presence of the causative agent in a clinical sample,
mainly enable conrmation during the rst 5 days post onset of the
disease (DPO), serological tests depend on the accumulation of
detectable amounts of anti-Leptospira antibodies in late acute to
convalescent samples (Ahmed et al., 2009, 2012; Goris et al., 2012).
Hence, serological tests, by denition, conrm the disease afterwards
and thus do not directly contribute to the timely management of the
individual patient.
In summary, novel often expensive early or standard diagnostic
tests are best placed at expert reference centres where conrmation of
RDT results can be performed or at hospitals with the required
expertise. RDTs are highly useful at peripheral facilities and might be
key for early outbreak warning. For all situations, the availability of
baseline data on the local epidemiology is critical for the interpreta-
tion of test results or f or the prediction, prevention, or e arly
intervention of outbreaks. For adequate surveillance of leptospirosis,
the availability of diagnostic tests is pivotal. The higher the diagnostic
accuracy of the test, the better it is for surveillance, although any type
of diagnostic test might be helpful for this purpose, provided that its
performance has been well assessed.
3. What sample(s) for early diagnosis?
Typically, acute symptoms develop 712 days after infection,
although rarely, the incubation period can be as short as 23daysor
as long as 30 days. Infection by pathogenic Leptospira can be divided into
2phases(Fig. 1). The rst phase of leptospirosis is the septicemic or
acute phase, which lasts for 37 days with fever, headache, and myalgia.
During this stage, Leptospira are found in the bloodstream in decreasing
numbers up to 15 days (Agampodi et al., 2012; Bharti et al., 2003). To
detect Leptospira, blood samples have to be collected until 2 days after
the start of antibiotic therapy, si nce antibiotics quickly remove
Leptospira from the blood. The second stage of the disease or immune
phase generally appears during the second week after the onset of
symptoms, and antibodies usually persist for several months (Silva
et al., 1995). During this stage, leptospires are cleared from the
bloodstream as the titres of IgM class antibodies increase (Levett, 2001).
Absence of detection of leptospiral antigen or DNA in conrmed
cases of leptospirosis can be attributed to a low or a short
leptospiremia during the acute phase of the disease, to blood samples
taken late in the disease, or to the administration of antibiotics. For
antibody-based tests, the window of time prior to or during early
sero-conversion may lead to false-negative test results in the early
acute phase of disease.
PCR on serum and plasma from whole blood anticoagulated with
heparine, a natural inhibitor of PCR, was found to be less sensitive
than PCR performed on other fractions for detection of Leptospira DNA
(Bourhy et al., 2011; Kositanont et al., 2007; Levett et al., 2005;
Smythe et al., 2002; Stoddard et al., 2009). Previous studies have
generally found that plasma from EDTA anticoagulated whole blood
gave optimal results for DNA amplication (Ahmed et al., 2009). For
serology assays, the use of serum or plasma, either heparin or EDTA
anticoagulate d, produces equivalent results (Goris et al., 2012).
Alternatively, leptospires can also be detected by culture and PCR
Fig. 1. The kinetics of leptospiral infection in blood. Infection produces leptospiraemia
in the rst few days after exposure, which is rapidly followed by migration of
leptospires to target organs. Anti-Leptospira IgM antibodies are detectable before the
appearance of agglutinating and IgG antibodies. The broken line (———) indicates the
dynamics of the presence of leptospires or leptospiral antigen and DNA in the blood;
the solid line indicates the level of IgM anti-Leptospira antibodies; the dashed line (——)
indicates the dynamics of the anti-Leptospira IgG antibody response. The depicted
dynamics are relative and are not intended to indicate quantitative levels, which vary
between patient species and individuals.
2 M. Picardeau et al. / Diagnostic Microbiology and Infectious Disease 78 (2014) 18
Page 2
from urine 1014 days after the onset of symptoms albeit that this
does not contribute to an early diagnosis (Levett, 2001).
The nucleic acidbased diagnostic tests involve nucleic acid
purication. Commercially av ailabl e kits usually allow a good
recovery of DNA from blood within less than 1 hour (Bourhy et al.,
2011). The use of magnetic beads allows concentration of nucleic acid
or antigens in samples (Schreier et al., 2012; Taylor et al., 1997). To
simplify DNA extraction procedures, use of whole blood spotted on
Whatman FTA lter paper, which is a chemically treated lter paper,
can allow for the rapid isolation of pure DNA. Similarly, serological
studies can be performed from dried whole blood spotted onto
Whatman lter paper (Desvars et al., 2011). These commercially
available reagents are an easy and inexpensi ve means for the
collection and storage of samples in resource-limited settings or for
the shipment of samples to reference centres.
4. Current diagnostic tools
As current diagnostic tools, we consider the direct examination
of blood, the rapid nucleic-acid dia gnosis, and rapid antibody -
based tests.
4.1. Direct examination of blood
The bacterial load in blood ranges from 10
2
to 10
6
Leptospira per
millilitre (Agampodi et al., 2012) in the acute phase. In theory,
leptospirosis can therefore be diagnosed by dark-eld microscopy of
blood taken during the rst week of illness. The limit of detection was
determined as approximately 10
4
leptospires per millilitre of blood or
urine (Table 2). Although it is relatively inexpensive, this test
requires a dark-eld microscope, which is rarely available or
affordable in resource-limited settings. Dark-eld microscopy of
blood is unreliable as Brownian movement of collagen brils, red
blood cell membranes, and other artefacts can resemble viable
leptospires (Vijayachari et al., 2001).
4.2. Rapid nucleic acidbased diagnostic test
4.2.1. Polymerase chain reaction
PCR-based methods are becoming more widely used for the
detection of pathogenic Leptospira strains, in part because of their
superior sensitivity and ability to establish an early diagnosis. Real-
time PCR, either using SYBR Green or Taqman technology, has the
advantage that it gives a result much faster than conventional PCR and
is less prone to contamination. The commercialization of portable PCR
thermocyclers compatible with real-time PCR chemistries may also
allow the rapid detection of pathogens in the eld.
Several conventional (including nested PCR) and real-time PCRs
have been developed for the detection of leptospires, targeting whole
arrays of genes, whether or not conned to pathogenic species,
exemplied by secY and lipL32, respectively. However, relatively few
assays have been validated for use with a variety of human and
canine samples (Ahmed et al., 2012; Ahmed et al.; Slack et al., 2007;
Thaipadungpanit et al., 2011; Villumsen et al., 2012). This is
surprising because a good diagnostic accuracy as revealed by a solid
validation should be the prime criterion of choice for implementing a
diagnostic PCR. The limit of detection of PCR assays was generally
determined as 1001000 bacteria per millilitre of blood or urine
(Bourhy et al., 2011; Slack et al., 2006; Smythe et al., 2002; Stoddard
et al., 2009). Bacterial load may be obtained if quantitative standards
are included in the amplication run and a standard curve has been
produced. However, this may not be informative as the quantitative
leptospiremia was not always correlated with the vital prognosis of
patients (Agampodi et al., 2012; Segura et al., 2005; Truccolo et al.,
2001). A positive PCR usually indicates that one of the members of the
pathogenic Leptospira species is present in the sample but PCR cannot
be used to predict the leptospiral serovar. There are many methods of
post-amplication analysis, but only a few have been described for
the identication of leptospiral genotype or serovar, including melt
curve analysis (Merien et al., 2005) and DNA sequencing (Perez and
Goarant, 2010). Further studies should evaluate the usefulness of
other post-amplication analyses such as high-resolution melt
analysis (which measures the melting temperature of amplicons in
real time, using a uorescent DNA-binding dye) and microarray
analysis for the identication of Leptospira at the subspecies level
(Ahmed et al., 2010
).
4.2.2. Isothermal methods
An increasing number of isothermal amplication techniques,
including nucleic acid sequencebased amplication, loop-mediated
isothermal amplication (LAMP), helicase-dependent amplication,
rolling circle amplication, and strand displacement amplication,
have been recently developed, some of which have been applied to
leptospirosis (Colenbrander et al., 1994; Koizumi et al., 2012; Lin et al.,
2009; Sonthayanon et al., 2011). Isothermal amplication is an
attractive alternative to PCR-based methods since thermocyclers are
not required. It simply requires a heating device to maintain a
constant temperature of 6065 °C, making it particularly suited to
resource-limited settings. In LAMP, specic and efcient amplication
of DNA is performed within 1 hour by Bst DNA polymerase using 6
primers under isothermal conditions. The amplied DNA can be
detected by visual inspection of uorescence or turbidity, without the
need for gel electrophoresis (Mori and Notomi, 2009). The method
may also enable direct amplication from clinical specimens, thereby
eliminating the need for an additional nucleic acid purication step.
LAMP assays targeting the lipL41 or rrs genes were recently developed
for the rapid detection of pathogenic Leptospira spp. (Koizumi et al.,
2012; Lin et al., 2009; Sonthayanon et al., 2011). The specicity of
LAMP assays was moderate to low, and the limit of detection was
determined between 2 and 100 leptospires per reaction mixture
(Ahmed et al., 2009; Slack et al., 2007;Sonthayanon et al., 2011).
Besides, current costs of an LAMP assay are similar to those of a real-
time PCR and it is as yet unclear whether LAMP will become
economically competitive. Despite its advantages, the usefulness of
LAMP for the diagnosis of leptospirosis needs to be further evaluated
in endemic area with resource-limited settings.
4.3. Rapid leptospirosis antibody-based tests
4.3.1. ELISA
Traditional serological methods such the ELISA are widely used for
the diagnosis of leptospirosis. ELISA can be performed with minimal
training and typically provides results in 24 hours. Numerous
commercial IgM ELISAs have been developed based on detection of
antibodies against whole c ell Leptospira (Table 1), usually, the
saprophyte Leptospira biexa, which shares many surface antigens
with pathogenic strains. In-house IgM ELISA based on a whole cell
antigen extract obtained from a local isolate can also be used (Goris
et al., 2012).
Recombinant surface proteins or lipoproteins of Leptospira have
also been used as antigens. In general, the antigens used for ELISA may
not recognize the diversity of circulating strains, and the overall
sensitivity of these tests is poor (Levett, 2001; McBride et al., 2005). A
conclusive serological diagnosis of leptospirosis cannot be made by
ELISA alone but needs laboratory conrmation through MAT, PCR, or
culture. The results of ELISA are usually obtained in a few hours
(Table 2), and it may require several samples to decrease the cost of
the assay.
Despite the varying performance of ELISA, studies have
generally found that the assay detects anti-Leptospira antibodies
earlier in the course of the disease than MAT (Aviat et al., 2010;
Bajani et al., 2003;Cumberland et al., 1999; Doungchawee et al.,
3M. Picardeau et al. / Diagnostic Microbiology and Infectious Disease 78 (2014) 18
Page 3
2008; Levett, 2001; Signorini et al., 2013). Anti-Leptospira IgM
antibodies are not detectable before 45 days after onset of
symptoms (Fig. 1) but appear earlier than IgG and agglutinating
antibodies (Silva et al., 1995).
4.3.2. Other rapid antibody detection methods
Other rapid antibody detection methods include macroagglutina-
tion, immunouorescence assay, indirect hemagglutination assay
(IHA), latex agglutination, lateral ow assays (LFA), and IgM dipstick
(McBride et al., 2005).
Immunouorescence makes use of antibodies con jugated to a
uorescent dye as detection reagent. The method can detect
specic antib odies in body uids or antigen in tissue sections.
The sensitivity and specicity of the indirect uorescent antibody
(IFA) test correspond to those of the ELISA (Appassakij et al., 1995).
However, this test is not commercially available, and it requires a
uorescence microscope.
The macroscopic slide agglutination test (MSAT) uses dense
suspensions of killed Leptospira serovars (Wolff and Bohlander,
1966). The antigen should consist of all locally prevalent strains or,
alternatively, the saprophyte L. biexa serovar Patoc, which shares
many surface antigens with pathogenic strains. A drop of serum is
mixed on a glass plate with the antigen, briey incubated at ambient
temperature and inspected by naked eye for presence of agglutina-
tion. The MSAT is relatively insensitive for diagnosis but may be useful
for epidemiological screening (Marin-Leon et al., 1997).
IHA uses red blood cells sensitized with an extract of an
erythrocyt e-sensitizing substance from L. biexa serovar Patoc
(Chang et al., 1957; McComb et al., 1957). IHA detects both IgM
and IgG antibodies. Heat-inactivated serum is mixed with sensitized
red blood cells, and agglutination is examined by the naked eye.
Estimates of the sensitivity of the IHA in populations in which
leptospirosis is endemic have varied from good (Levett and
Whittington, 1998) to poor (Efer et al., 2000), possibly because
of differences in case ascertainment and study design, including
inclusion of epidemiological distinct populations and the unavail-
ability of prospective unbiased samples (Hull-Jackson et al., 2006;
McBride et al., 2007).
Table 1
Commercial tests used for the diagnosis of acute leptospirosis.
Test/kit Manufacturer Technology
Serion ELISA Classic Leptospira IgM Test kit Institut Virion/Serion GmbH (Germany) ELISA
Leptospira IgM ELISA Panbio Ltd (Australia) ELISA
Leptospira IgM Diagnostic Automation/Cortez Diagnostics, Inc. (USA) ELISA
Leptospira IgM Microwell serum ELISA IVD Research Inc. (USA) ELISA
Leptospira IgG/IgM Microwell serum ELISA IVD Research Inc. (USA) ELISA
ELISA (IgM/IgG) Standard Diagnostics (Korea) ELISA
Mouse Leptospira IgG ELISA Kit
b
My-Bio-Source (USA) ELISA
Leptospira IgG/IgM Combo rapid Test CTK Biotech, Inc. (USA) IHA
IHA Mayo medical Laboratories (USA) Indirect hemagglutination test
Leptospira-MC Test Japan Lyophilization Laboratory (Japan) Microcapsule agglutination test
LeptoTek Lateral Flow
a
BioMerieux (the Netherlands) LFA
Test-it Life Assays (South Africa) LFA
Leptocheck-WB Zephyr (India) LFA
SD Leptospira LF Standard Diagnostics (Korea) LFA
IgM lateral ow test Omega Diagnostics (United Kingdom) LFA
LeptoTek Dri Dot BioMerieux (the Netherlands) Latex card-agglutination test
Leptorapide Linnodee, Northern (Ireland) Latex card-agglutination test
Primergen Lepto LipL32 PrimerDesign (France) Real-time PCR
Adiavet
TM
Lepto RealTime
b
Adiagen, BioMerieux (France) Real time-PCR
FastPanel® PCR Canine Leptospirosis Prole
b
Antech Diagnostics (USA) Real time-PCR
a
Expected to be available soon by IMACCESS.
b
Veterinarian use.
Table 2
Performance of rapid diagnostic tests during the acute phase of leptospirosis.
Test Approximate
cost
a
Execution time Equipment Sensitivity
b
Specicity Optimal detection
window
c
Direct examination b 115 min Dark eld microscope 10
4
/mL
d
(analytical)
low Early acute
Commercialized
IgM ELISA
816 12 h ELISA washer and reader N90% 8895% Late acute to
convalescent
In house IgM ELISA 224 h ELISA washer and reader 93% 98-100% Late acute to
convalescent
LFA 251520 min Cold room/refrigerator
e
81% 96% Late acute to
convalescent
Conventional PCR 68 5 h (including DNA
extraction with a kit)
Thermal cycler and gel
electrophoresis system
60100% 90100% Early acute
Real-time PCR 68 2 h (including DNA
extraction with a kit)
Real Time thermal cycler 60100% 90100% Early acute
Isothermal method 1015 2 h (including DNA
extraction with a kit)
Heating device and, in most
cases, gel electrophoresis system
Less than PCR Less than
PCR
Early acute
The generalized data on costs and diagnostic accuracy listed in this table, in part, summarize the data presented by Hartskeerl et al. (2011).
a
Direct costs, not including use of DNA extraction kit, salaries, equipment, etc., but dependent on local import taxes.
b
First 10 days after the onset of symptoms, depending on the stage of disease.
c
Optimal sensitivity expressed in phase of disease: early acute is 5 DPO, late acute is 510 DPO, convalescent is N10 DPO.
d
From Levett, 2001.
e
Several novel LFAs can be stored for months at ambient temperature.
4 M. Picardeau et al. / Diagnostic Microbiology and Infectious Disease 78 (2014) 18
Page 4
The microcapsule agglutination test is using a synthetic polymer in
place of red blood cells (Arimitsu et al., 1982) coated with a mixture of
whole cellderived antigens from pools of serovars, for example,
Australis, Autumnalis, and Hebdomadis. The test has been evaluated
at various reference centres (Arimitsu et al., 1994). It showed
sensitivity similar to the MAT and IgM-ELISA in early acute phase
samples but failed to detect infections caused by some serovars.
The complement xation (CF) test is used as a serological
technique but can also be applied for antigen detection. The test is
based on the fact that if the serum contains antibodies against the
antigen of interest, they will form antigen-antibody complexes,
leading to reaction with the complement system and cell lysis. In the
test, antigen of the suspected infectious agent is added to the heated
serum together with complement proteins. Causative agent-specic
antibodies bind to the antigen, and the resulting complex binds the
complement, thus inhibiting the haemolysis of the erythrocytes.
Absence of haemolysis constitutes a positive test. CF is a tedious in-
house test that has not found a widespread application and lacks
valid evaluations of its diagnostic accuracy (Andreescu, 1990).
LFA can be performed at the bedside of the patient, as whole blood
(nger prick) can be used for testing, and results can be obtained in
10 minutes. The mobile phase is usually made of colloidal gold-
labelled anti-IgM antibody, specic for the human or animal patient.
An LFA based on whole cell antigen extract from the saprophyte L.
biexa was developed by the Royal Tropical Institute (Amsterdam,
The Netherlands) and enables the rapid detection of Leptospira-
specic IgM antibodies in human sera (Smits et al., 2001). This assay,
which is available as a commercial test, showed performances that
are similar to ELISA and has been tested in the eld (Sehgal et al.,
2003). A recent prospective evaluation of 3 RDTs, including 2 LFAs,
revealed a changing diagnostic performance of these tests through
time, and it was argued that companies might need to apply more
strict quality checks at the production procedures (Goris et al., 2013).
An LFA based on recombinant leptospiral immunoglobulin-like (Lig)
proteins, which are surface-exposed proteins specic to pathogenic
Leptospira strains, demonstrated high performance in identifying
leptospirosis during urban epidemics in Brazil (Nabity et al., 2012).
However, this assay does not appear to be generalizable because of its
limited ability to detect antibodies against serovars not belonging to
the serogroup Icterohaemorrhagiae, which is the pred ominant
serogroup in urban Brazil (Nabity et al., 2012). Another immuno-
chromatographic rapid diagnostic test for the early detection of anti-
Leptospira human IgM is also currently under evaluation at the
Institutes Pasteur in Paris and Nouméa. In this assay, whole killed
bacteria of the intermediate species Leptospira fainei, which may
share common antigenic features with saprophytes and pathogens,
were used as an antigen (Bourhy et al., 2013; Goarant et al., 2013).
Although some of the a forementione d tests are commercially
available, these tests are usually restricted to specic areas (for
example, IFA is mostly used in Thailand), and they are not widely
used in the diagnosis of acute leptospirosis.
5. Tests and operational applications
Early testing for effective case management and early case nding
for outbreak management are critical. This requires the use of early
tests during the 5 DPO. To date, PCR and, more particularly, real-time
PCR, are the most appropriate because of its early diagnostic accuracy.
Further operational research, however, has to be developed in
different epidemiological and operational conditions for making its
use more cost-effective and ensuring early registration at the national
reference centre. In particular, the usefulness of isothermal ampli-
cation techniques needs further evaluation in endemic areas with
resource-limited settings.
Diagnosis should be done at the peripheral level, using RDTs for
rapid, but preliminary, determination, while at a referral laboratory
level, denitive conrmation can be done, using more sophisticated
and expensive tests, such as isolation by culturing, MAT, or real-time
PCR. MAT would remain the best choice if a high early diagnostic
accuracy is not feasible and/or if presumptive information on infecting
serogroups is needed. It is stressed that notably for sero-diagnostics,
local validation is required given the varying performances of such
tests within different epidemiological conditions.
The development of conrmation algorithms has to take into
consideration undifferentiated acute fevers, including malaria, den-
gue, rickettsiosis, yellow fever, and typhoid (WHO, 2003). Some
operational research on the use of multiplex tests is underway.
6. Challenges towards improvement of rapid diagnostic tests
6.1. Sample collection
It is a challenge to determine which type of sample to collect in
order to achieve the greatest diagnostic accuracy. Sample collection
should preferably be non-invasive or be limited to small intervention
(nger prick).
Urine presents a good non-invasive diagnostic sample. The use of
urine has a disadvantage, that is, leptospires are only reliably shed
from the late acute phase and claims on early diagnostic potential of
antigen detection tests using urine still wait for valid evaluation of the
diagnostic performance (Saengjaruk et al., 2002). However, it is
conceivable that Leptospira derived breakdown components or early
immune response molecules are present in the urine at an early stage.
This hypothesis needs to be investigated.
Blood is an excellent diagnostic sample since leptospires are
present in the rst 45 days after onset of disease, which enables early
diagnosis. In the near future, non-invasive blood tests with micro-
portable devices will become available.
Saliva: the interest of this specimen is doubtful as presence of
leptospires, leptospiral antigens, or anti-Leptospira antibodies in saliva
is largely unclear or unknown. As potential non-invasive specimens,
saliva and sputum would deserve further investigations.
Breath has been investigated as a diagnostic sample for several
diseases, including TB. The idea is that the composition of breathed air
changes by infection and may contain components of the causative
agent or body degradation products (Kolk et al., 2012). Breath might
present an interesting diagnostic sample, certainly with the pulmo-
nary syndrome.
Furthermore, one should consider the use of the test-upon-
treatment principle (treatment will increase the concentration of
degradation products that can be more easily detected for conrma-
tive diagnosis than the whole bacterium) as an attractive approach
that, basically, could be combined with any of these clinical samples.
6.2. Multiplex tests for differential diagnosis
In tropical climates, leptospirosis must be differentiated from
other clinically similar acute febrile illnesses that could be prevalent in
the same regions. Leptospirosis PCR- or antibody-based tests could be
combined with other febrile illnesses to assess a specimen for a wide
variety of microorganisms. A multiplex real-time PCR assay, which
allows the sensitive detection of different DNA targets in a single
reaction, could also be developed for the simultaneous detection of
agents of febrile illnesses (e.g., leptospirosis, rickettsioses, dengue,
and other viral haemorrhagic infections). Alternatively, one might
choose for a multiplex system that separates bacterial from viral
diseases, which is of major importance for taking decisions on
treatment. Novel, innovative platforms such as the circular multi-
analyte point-of-care devices present valuable approaches for
multiplex serological detection (http://www.kit.nl/kit/Sylis-en-
HIV-test) and might be adaptable for molecular detection.
5M. Picardeau et al. / Diagnostic Microbiology and Infectious Disease 78 (2014) 18
Page 5
6.3. Antigen capture and/or amplication
Despite intensive investigations, a major challenge remains to
discover surface-exposed antigens or genetic targets that are
conserved across the major leptospiral strains. The genus Leptospira
comprises 21 species subdivided in 9 pathogenic species that are the
etiological agents of leptospirosis, 7 saprophytic species, and 5
intermediates species (Cerqueira and Picardeau, 2009). Pathogenic
Leptospira spp. are classied into more than 250 serovars on the basis
of structural heterogeneity in the carbohydrate component of the
lipopolysaccharide (LPS). The genomes of 5 Leptospira spp. (L. biexa,
L. interrogans, L. santarosai, L. borgpetersenii, and L. licerasiae) have
been sequenced (Bulach et al., 2006; Chou et al., 2012; Picardeau et al.,
2008; Ren et al., 2003; Ricaldi et al., 2012), and an on-going project
should allow the sequencing of about 200 leptospiral strains by the J.
Craig Venter Institute using next-generation DNA sequencing ap-
proaches (http://gsc.jcvi.org/projects.php). This new sequencing
information will make it possible to identify novel diagnostic targets
for specic antigen or nucleic acid detection.
Immunomagnetic capture is use d in a number of tests to
concentrate nucleic acid or antigens in clinical specimens. Magnetic
beads could be conjugated with specic antibodies, which can
recognize and capture target antigen. Immunomagnetic capture
systems could, for example, be made serogroup or serovar specic.
Magnetic beads can also be used to concentrate pure nucleic acid
extracted from clinical samples. It should be stressed that DNA extraction
is the less efcient step in the amplication process. Concentration of
DNA with maximal removal of inhibitors thus should provide an
improvement of the sensitivity for amplication-based methods.
Otherwise, the value of less inhibitor sensitive methods like LAMP or
enzyme free amplication methods needs further investigation.
For isothermal methods that present a promising alternative to
PCR in resource-limited setti ngs, more studies are required to
evaluate their performance characteristics and determine their
ideal application.
6.4. Detection systems
LFAs have successfully been used to detect antigens from bacterial
pathogens in clinical samples. These assays are usually based on
monoclonal antibodies selected to have broad reactivity against
antigen(s) of the pathogen. One limitation for the development of
such a test for the diagnosis of leptospirosis is the relatively low
number of bacteria (b10
6
Leptospira/mL) found in blood and the
duration of time in which the bacterium can be detected. Moreover,
our knowledge of the expression of leptospiral antigens during the
infection remains limited. LPS constitute the main antigen for
Leptospira, but this antigen is serovar specic. Antibodies directed
against leptospiral LPS are therefore restricted to the detection of
antigenic related serovars (or serovars with serologically related LPS).
Although based on in vitro data, it has been shown, for example, that
the conserved lipoprotein LipL32 is the most abundant protein of L.
interrogans at 38,000 copies per cell (Malmstrom et al., 2009).
Monoclonal antibodies targeting this highly expressed protein will
then need to be evaluated for the ability to react with the wide
diversity of leptospiral serovars.
For DNA detection, multiplex real-time PCR assays for the
diagnostic of leptospirosis, together with other agents of febrile
illnesses will be a major improvement (see above). Microarray tests
remain expensive for routine use, and data interpretation usually
requires an adequate training in the use of analysis software.
6.5. Incorporating new technologies
For antigen detection, one of the most promising areas in
molecular diagnostics is the use of nanomaterials. Nanobiosensors,
detecting changing electronic, optic, or other physical parameters
upon capturing the antigen, could detect low levels of antigen in a
biological sample. Moreover, we speculate that the use of quick
response codes on RDTs combined with data and location recording
by a smart phone or even more innovative lab-on-a-chip approaches
that enable online transmission of data to a central point in a country
or region might provide effective tools for surveillance and early
outbreak monitoring. In turn, this will contribute to the understand-
ing of the dynamics of outbreaks and hence to the rational design of
cost effective prevention and early intervention measures.
Such types of biosensors could be suitable not only for the
diagnosis of the disease on humans; it could also become an
important instrument for environmental monitoring as part of
network of sensors for water pollution monitoring (Rickerby and
Skouloudis, 2011). Sensors and biosensors are useful analytical tools
for environmental monitoring because they provide cheap and rapid
analysis of data. Biosensors consist of a biological sensing element,
which may be an antibody, antigen, enzyme or whole cell, interfaced
to a transducer, such as an electrode or optical bre. It was found that
that the afnit y and sensit ivity of imprinted polymers were
comparable to those of polyclonal antibodies. Biosensors integrated
with wireless telecommunication systems could revolutionise envi-
ronmental monitoring due to the following advantages. The nano-
biotechnology-based diagnostic techniques can potentially detect a
wide range of aquatic contaminants and toxins that are of relevance
for leptospiral survival outside the host and, hence, in effect, expose
risks. It will improve the geographical resolution and furnish higher
sensitivity for local detection of sources. It provides a multi-parameter
analysis allowing simultaneous measurements and provides remote
surveillance and control capability for continuous real-time monitor-
ing, early warning, and fast response.
7. Conclusion
Rapid diagnostic tests should ideally be accurate, simple to use,
relatively inexpensive, easy to interpret, stable under extreme
conditions, with little or no processing, and give the results within
12 hours (Banoo et al., 2010). None of the available tests described
above for the diagnosis of acute leptospirosis meet all these criteria.
Diagnostic tests are often sold and used in low- and middle-
income countries without any evidence of effectiveness; this is
particularly true for IgM ELISAs and RDTs that show variable
performance for the diagnosis of acute leptospirosis (Goris et al.,
2013). These differences may be inuenced by the characteristics of
the population, the circulating strains, or the methodology (Banoo
et al., 2010). Therefore, test evaluations should be performed under
the range of conditions in which they are likely to be used in
practice. This will help to choose for the most appropriate test in
case of an outbreak. Participation in a program of inter-laboratory
comparison or prociency testing of rapid tests such as the one
established for MAT (Chappel et al., 2004) should help in producing
reliable and accurate results in endemic countries.
As described above, recent technological advances in the eld of
diagnostic tests could allow for the development of innovative rapid
tests in the near future, enabling online surveillance and early
outbreak warning. However, there may be few organizations
interested in investing in the development of RDTs for neglected
tropical diseases because of a perceived lack of return of investment
from low- and middle-income countries; these are not considered a
viable commercial market.
One major goal is to develop rapid multiplex tests (PCR- or
antibody-based methods) that will differentiate between, for exam-
ple, leptospirosis and dengue or other similar major infectious
diseases. Use of multiplex tests will be very helpful in tropical
countries and could lead to improved patient outcomes. Supporting
the development of rapid tests has been dened as a priority by the
6 M. Picardeau et al. / Diagnostic Microbiology and Infectious Disease 78 (2014) 18
Page 6
Global Leptospirosis Environmental Action Network (GLEAN). GLEAN
was developed by WHO and the Health and Climate Foundation to
strengthen current public health strategies and to mitigate the risk
and impact of leptospirosis outbreaks in populations at high risk
through a cross-sectorial, multidisciplinary approach (http://www.
glean-lepto.org). GLEAN aims include the denition of the challenges
and needs in terms of research and development for rapid testing as
well as the evaluation of the performance of the tests currently
available or under development with the setup of a globally
represent ativ e bank of sera. GLEAN also pr omote s innovative
mechanisms to support research and development of new diagnostic
tools suitable for various aspects of leptospirosis from environmental
monitoring to diagnosis and public health impact assessments.
Acknowledgments
The authors would like to thank all participants of the GLEAN
meetings for their fruitful discussions.
This work is part of the deliverables of the working group Detect
of GLEAN, which main purpose is to help countries in developing and
implementing policies and tools for early outbreak detection, with a
focus on surveillance and diagnostics.
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8 M. Picardeau et al. / Diagnostic Microbiology and Infectious Disease 78 (2014) 18
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    • "A positive PCR generally demonstrates a pathogenic member. However, it is not used to predict the leptospira serotype in the sample [14]. Kucerova et al. investigated 216 leptospirosis suspect patient during 2010 – 2011 using real time PCT to determine the gene coding leptospira surface lipoprotein LipP32 to confirm acute form of leptospirosis and achieved positive results in total 14 biological material (9 urine, 4 blood, 1 bronchoalveolar) obtained from 8 (3.70%) patients [15]. "
    Preview · Article · Jan 2016
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    • "From a total of 85 clinical samples, 53 sera and 5 whole blood samples (68.2%) were positive to 16S rRNA. The difference between LipL32 qPCR positivity and that of 16S rRNA PCR could be related to the limitations of the conventional PCR used in the 16S rRNA (Merien et al., 2005; Picardeau et al., 2014). Fifty five samples were grouped within L. interrogans and one sample (serum 99) within L. kirschneri. "
    [Show abstract] [Hide abstract] ABSTRACT: Leptospira typing is carried out using isolated strains. Because of difficulties in obtaining them, direct identification of infective Leptospira in clinical samples is a high priority. Multilocus sequence typing (MLST) proved highly discriminatory for seven pathogenic species of Leptospira, allowing isolate characterization and robust assignment to species, in addition to phylogenetic evidence for the relatedness between species. In this study we characterized Leptospira strains circulating in Argentina, using typing methods applied to human clinical samples and isolates. Phylogenetic studies based on 16S ribosomal RNA gene sequences enabled typing of 8 isolates (6L. interrogans, one L. wolffii and one L. broomii) and 58 out of 85 (68.2%) clinical samples (55L. interrogans, 2L. meyeri, and one L. kirschneri). MLST results for the L. interrogans isolates indicated that five were probably Canicola serogroup (ST37) and one was probably Icterohaemorrhagiae serogroup (ST17). Eleven clinical samples (21.6%), provided MLST interpretable data: five were probably Pyrogenes serogroup (ST13), four Sejroe (ST20), one Autumnalis (ST22) and one Canicola (ST37). To the best of our knowledge this study is the first report of the use of an MLST typing scheme with seven loci to identify Leptospira directly from clinical samples in Argentina. The use of clinical samples presents the advantage of the possibility of knowing the infecting strain without resorting to isolates. This study also allowed, for the first time, the characterization of isolates of intermediate pathogenicity species (L. wolffii and L. broomii) from symptomatic patients.
    Full-text · Article · Nov 2015 · Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases
    • "Leptospira spp. appears in blood during the acute phase (3–10 days) of the disease and is eliminated from blood in the immune phase with rise of antibody (Picardeau et al., 2014). Thus, Group I sera represented samples from acutely infected patients within 10 days post-infection, since there were circulating bacteria detectable by PCR as well as presence of adequate level of agglutinating antibodies as evidenced by the MAT results. "
    [Show abstract] [Hide abstract] ABSTRACT: This study evaluated two rapid leptospirosis serological tests, Leptorapide® (Linnodee, Northern Ireland) and VISITECT®-LEPTO (Omega Diagnostics, Scotland, UK), which are commonly used in Malaysia A total of 183 samples comprised 113 sera from leptospirosis patients and 70 sera from other infections and healthy controls were used. The leptospirosis sera were grouped into two serum panels i.e. Group I (MAT+, PCR+) and Group II (MAT+). When inconclusive results were interpreted as positives, both tests showed lower diagnostic sensitivities (≤34%) with Group I sera, as compared to Group II sera (Leptorapide®, 93%; VISITECT®-LEPTO, 40%). When inconclusive results were interpreted as negatives, the two tests showed ~20% sensitivity with both serum panels. The diagnostic specificity of VISITEC®-LEPTO (94%) was superior to Leptorapide® (69%). Since both tests had misdiagnosed a large proportion of Group I patients, and showed many inconclusive results among Group II patients, they have limited diagnostic value in detecting acute leptospirosis.
    No preview · Article · Sep 2014 · Diagnostic Microbiology and Infectious Disease
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