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Diagnostic accuracy of an IgM ELISA and comparison with two PCRs for
early diagnosis of human leptospirosis
N.B. Vanasco, P. Jacob, N. Landolt, Y. Chiani, M.F. Schmeling, C. Cudos, H. Tarabla, J. Lottersberger
PII:
DOI:
Reference:

S0732-8893(16)00003-1
doi: 10.1016/j.diagmicrobio.2016.01.002
DMB 13992

To appear in:

Diagnostic Microbiology and Infectious Disease

Received date:
Revised date:
Accepted date:

29 September 2015
22 December 2015
5 January 2016

Please cite this article as: Vanasco NB, Jacob P, Landolt N, Chiani Y, Schmeling MF,
Cudos C, Tarabla H, Lottersberger J, Diagnostic accuracy of an IgM ELISA and comparison with two PCRs for early diagnosis of human leptospirosis, Diagnostic Microbiology
and Infectious Disease (2016), doi: 10.1016/j.diagmicrobio.2016.01.002

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Diagnostic accuracy of an IgM ELISA and comparison with two PCRs for early diagnosis of

human leptospirosis

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Vanasco NB1,2; Jacob P1; Landolt N1; Chiani Y1; Schmeling MF1; Cudos C1; Tarabla, H3,

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Lottersberger J.1,2

Instituto Nacional de Enfermedades Respiratorias (INER) Dr. Emilio Coni, Administracin

8260, (3000) Santa Fe, Argentina.


2

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Nacional de Laboratorios e Institutos de Salud (ANLIS) Dr. Carlos G. Malbrn, Av. Blas Parera

Facultad de Bioqumica y Ciencias Biolgicas, Universidad Nacional del Litoral (FBCB - UNL).

Santa Fe, Argentina. Ciudad Universitaria, Paraje El Pozo, (3000) Santa Fe, Argentina
Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral (FCV - UNL), Kreder 2086,

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(3080) Esperanza, Santa Fe, Argentina.

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ABTRACT

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Corresponding author: Vanasco NB

ELISA tests and PCR may play a key role for early detection and treatment of human leptospirosis
in developing countries. The aims of this study were to develop and validate an IgM ELISA under
field conditions, and to compare the diagnostic accuracy among IgG, IgM ELISAs, conventional
PCR (cPCR) and real-time PCR (rtPCR) for early detection of human leptospirosis. Overall
accuracy of IgM ELISA was: sensitivity 87.9%, specificity 97.0%, AUC 0.940. When the four
methods were compared IgM ELISA showed the greatest diagnostic accuracy (J=0.6) followed by
rtPCR (J=0.4), cPCR (J=0.2) and IgG ELISA (J=0.1). Our results support the use of IgM ELISA
and rtPCR for early diagnosis of the disease. Moreover, due to their high specificity could be also
useful to replace or supplement MAT as a confirmatory test, allowing more confirmations.

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Highlights
IgM ELISA showed the greatest diagnostic accuracy

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Our results support the use of IgM ELISA and rtPCR for early diagnosis of leptospirosis

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IgM ELISA and rtPCR could be useful to replace or supplement MAT as confirmatory test

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Keywords: Human leptospirosis; Diagnostic methods; IgM ELISA; IgG ELISA; real time PCR,
conventional PCR.

1. INTRODUCTION

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Leptospirosis, caused by spirochetes of the genus Leptospira, is a major public health issue due
to its global distribution in animals and natural environments, and its high potential for causing

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epidemics and death in humans (Picardeau et al., 2014). The disease is often confused with virus

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infections such as dengue, hemorrhagic fever, influenza and hantavirosis (Levett, 2001;).
The diagnosis of leptospirosis on clinical grounds is challenging and the disease is frequently not

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recognized, underreported and hence, neglected. Therefore, laboratory confirmation of the disease
is essential (WHO-ILS, 2003).
During acute phase, leptospires or their components may be detected by blood culture or
polymerase chain reaction (PCR). Blood culture provides proof of infection, but is insensitive,
tedious and has little clinical value for patient management, as it can take weeks or months to
confirm the results (Goris et al., 2012). Several PCRs have been developed for the detection of
leptospires and those targeting pathogenic species are exemplified by the gene LipL32. PCR on
blood samples seems to be useful in the first week of the disease (Ahmed et al., 2009). However,
relatively few of them have been validated for use in human beings (Picardeau et al., 2014).

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The microscopic agglutination test (MAT) is the international reference standard in
serodiagnosis. MAT has the advantage of being serogroup specific, although cross and paradoxical

reactions among serogroups do occur (Levett, 2003). Conventional diagnostic tests, such as MAT,

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mostly rely on serology and thus, at the best confirm the disease at a late acute phase, when
antibiotic treatment is already less effective (McBride et al., 2005). Moreover, MAT requires

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technical experience to maintain a large number of live Leptospira strains and to read and interpret
the test results (Faine 1982). To overcome these problems, some potentially useful tests have been

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developed, particularly enzyme linked immunosorbent assays (ELISAs) to detect IgM and IgG
antibodies. While the agglutination tests require subjective interpretations that can diminish
repeatability and reproducibility of the test results, ELISAs provide objective numerical outcomes
(Vanasco et al., 2012).

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In Argentina, the National Net of Laboratories of Leptospirosis is coordinated by a reference

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laboratory ("Laboratorio Nacional de Referencia de Leptospirosis-LNRL). The net includes, at


least, one regional or provincial laboratory throughout the country. Most of them perform screening

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tests like the macroscopic agglutination test with thermo-resistant antigen and ELISAs. Positive

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samples are sent to the LNRL to be confirmed by MAT. Since no commercial diagnostic methods
are available in Argentina, PCR, IgM and IgG ELISAs had to be developed and validated within the
country (Vanasco et al., 2007, 2012). These field validations required sample sizes and designs that
could assure the external validity of the studies. ELISAs and PCRs could play a key role for early
case detection and treatment in Argentina as well as in other developing countries.
The aims of this study were to develop and validate an IgM ELISA under field conditions, and to
compare the diagnostic accuracy among IgG, IgM ELISAs, conventional PCR (cPCR) and real-time
PCR (rtPCR) for early detection of human leptospirosis.
2. MATRIALS AND METHODS
2.1. Patients, case definition and samples

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Characteristics of the participants of study are shown in Table 1.
Confirmed cases (n=323): patients with one or more of the following criteria: i) single MAT titer

with a pathogenic strain titer 1/400, (ii) seroconversion four-fold titer rise MAT in paired

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samples (two or more), or (iii) positive culture.


Non Cases (n=554):

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a) Discarded leptospirosis cases (n=297): patients with symptoms compatible with


leptospirosis but having a negative MAT titer (<1/50) in two samples collected in different

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phases of the illness.

b) Confirmed cases of diseases other than leptospirosis (103 patients): to evaluate IgM
ELISA Sp, 148 samples from patients with Argentinian hemorrhagic fever (AHF) (n=53),
dengue (n=48), Hantavirus (n=37), H1N1 Flu A (n=7) and psittacosis (n=3) were studied. They

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were confirmed as follows: AHF by IgG ELISA seroconvertion, dengue by IgG ELISA

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seroconvertion and neutralization, Hantavirus by IgM ELISA detection, IgG ELISA

2014).

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seroconvertion or rtPCR, flu A by rtPCR and psittacosis by direct immunofluorescence (MSAL,

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c) Healthy donors (n=154): healthy blood donors from endemic area. Since they were
not patients, Days Post Onset (DPO) were non-existent.
Samples (serum, whole blood with EDTA for PCR and with heparin for culture) referred from
every Argentina provinces from suspected patients of leptospirosis were received at the LNRL of
the Instituto Nacional de Enfermedades Respiratorias (INER) in Santa Fe, Argentina. Since
samples were collected at different stages of illness, DPO was estimated as the number of days
between the beginnings of symptoms and classified into confirmed and discarded cases. Samples
were stored at -70C until processing.
Six hundred and twenty confirmed and discarded leptospirosis patients (n=1037 serum and blood
samples) were selected by simple random sampling from N=5773 suspected patients referred to the

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LNRL in Santa Fe, Argentina from 1999 to 2014. Serum samples from patients with diseases other
than leptospirosis (n=148) were provided by the National Reference Laboratory for Human Viral

Diseases (Instituto Nacional de Enfermedades Virales Humanas-INEVH). Serum samples from

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healthy donors from endemic area (n=154) were provided by Santa Fe Province blood bank.
In addition to the biological samples, a surveillance form was received with every patient

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sample, containing data of demographic characteristics, clinical presentation, routine clinical


laboratory findings and history of risk exposures prior to onset. Serum samples were collected at

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least seven days apart from each other which included acute-phase illness (0-9 DPO) and
convalescence phase illness (>9 DPO).

All these epidemiologic and clinic data plus all leptospiral specific laboratory results and this

2.2. Laboratory methods

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surveillance form were entered into the INER database.

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Culture was initiated only when blood samples were collected within the first 10 DPO. Fletcher
medium and Ellinghausen-McCullough as modified by Johnson and Harris (EMJH) culture medium

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was used (Faine 1982). Inoculated media were incubated for a maximum of 4 months at 30C and

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weekly checked for leptospira growth by dark field microscopy.


MAT was performed with a panel of 12 live leptospires as described elsewhere (WHO-ILS,
2003). The reference strains used as antigens represented the following 9 serogroups: Ballum,
Canicola, Pomona, Grippotyphosa, Icterohaemorrhagiae, Pyrogenes, Tarassovi, Sejroe, Bataviae
and Patoc, evaluated to be used in Argentina (Jacob et al., 2015).
Serum was defined as positive for MAT titer 1:50, in concordance with national surveillance
guidelines for Argentina (AAVLD, 1994). The presumed infecting serogroup in confirmed cases
was considered to be the serogroup against which highest MAT titers were directed (WHO-ILS,
2003).

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In the development of the IgM ELISA the optimal sample and conjugate dilution and all the
assay conditions were evaluated based on the sensitivity and specificity of the test. According with

these results IgM ELISA was performed with antigen prepared as described by Adler et al. (1980)

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from cultures of the serovar Hardjo (strain Hardjoprajitno) and coated on polystyrene plates (Costar
EIA Microplates, USA, cat #2580) as follows. Microplates were coated with 100 l of sonicated

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leptospiral suspension (protein concentration: 1.0 g/ml) and incubated overnight at 4C. Thereafter
they were washed 6 times with phosphate buffer saline (PBS) (pH 7.2) containing 0.05% vol/vol of

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Tween 20 (PBS-T). They were dried and conserved sealed in plastic bags with desiccants at 4C
until use. Samples were diluted 1:200 in protein buffer, PBS with 1% casein (Sigma, USA). A
volume of 100 l of diluted serum was added to each antigen-coated well and the plates were

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incubated for 1h at 37C. As conjugate goat anti-human, IgM Fc specific, labelled with peroxidase
was used (Jackson Immuno Research Laboratories, Inc.; USA), diluted in PBS pH 7.4. After

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washing 6 times in PBS-T, 50 l of diluted (1:5000) conjugate was added. After incubation for
30min at 37C the plates were washed 6 times in PBS-T. Finally, 100 l of a solution containing

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3,3,5,5-tetramethylbenzidine (TMB) and H2O2 was added. After 10 min, the reaction was stopped

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by adding 100 l of 1N H2S04 and read immediately at 450/630 nm using an ELISA microplate
reader (MRX II, Dynatech). Known positive and negative pool controls were included in each plate.
Each sample was tested in duplicate, and an average optical density (AOD) for each was obtained.
Results were expressed as checked optical density (COD), obtained by dividing the AOD by the
optical density of the negative control pool of samples. Samples were considered positive when
observance readings as a COD result were higher than the cut-off point.
IgG ELISA was performed as described by Vanasco et al. (2007).
Leptospira DNA was extracted from 200 l of serum or whole blood samples, according to the
instructions of the QIAamp DNA Blood mini kit extraction kit (Qiagen Inc., Basel, Switzerland).

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TaqMan rtPCR targeting the lipL32 of pathogenic leptospires gene was performed as described
previously by Stoddard et al. (2009). A positive result was considered any exponential curve with a

cycle threshold (CT) prior to cycle 40.

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cPCR was performed using the same primers used for rtPCR (LipL32-45F and LipL32-286R)
and the method described by Slack et al. (2007).

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In both PCR methods indeterminate results were regarded as negatives.


For DNA typing 16S rRNA amplification products were sequenced according to the methods

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described by Mrien et al. (1992). Ten samples of non-cases with positive result to rtPCR were
typified by this method.

2.3. Statistical analyses and ethics statements

All techniques were performed using a blinded design. Data of selected patients was obtained

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from the LNR Database, de-identified and no attributable to individual patients.

t test.

test. Age and DPO were compared on confirmed/discarded cases by means of Student

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means of

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Associations between sex and confirmed/discarded cases of leptospirosis were performed by

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2.4. IgM ELISA validation


Diagnostic accuracy was estimated by testing sensitivity and specificity with 95% confidence
intervals (CI) using MedCalc (Schoonjans et al., 1995). The optimal cut-off point was determined
plotting sensitivity vs. 1- specificity by means of Receiver Operating Characteristic (ROC) using
MedCalc. Diagnostic accuracy was defined by the Area Under the ROC Curve (AUC).
Youndens index (J) of positive and negative results was also calculated. ELISA results were
compared against case definition.
2.5. Accuracy comparison of PCRs and ELISAs assays for early diagnosis
Inclusion criteria were: acute phase (1-9 DPO) serum samples received in the LNRL of the INER
from 2004 to 2014, with all necessary data to define a case and enough sample volume to run all

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diagnostic tests. Two hundred and thirty four samples were included from acute confirmed cases,
discarded cases and confirmed cases other than leptospirosis for the four tests accuracy comparison.

Samples were selected from 2004 due their better conservation.

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To analyse the performance of each method, one hundred and seven samples were stratified into
three categories: 0-3, 4-6 and 7-9 DPO.

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Diagnostic accuracy was estimated by test sensitivity and specificity with 95% confidence
intervals (CI) using Epidat, 2014. Test results were compared against case definition.

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3. RESULTS
3.1. Cases characteristic

The majority of cases were referred from the Central (74.9%) and North eastern (12.7%) regions
of Argentina. Confirmed cases were more prevalent in males than in females (p<0.05). No

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significant differences were found between age and DPO distributions on cases and discarded cases.

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Maximum DPO was 174 days. Icterohaemorrhagiae (24.7%) was the most prevalent presumptive
infecting serogroup by MAT, followed by Canicola (22.8%) and Sejroe (16.6%) (Table 1).

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Samples from confirmed and discarded cases of leptospirosis on the acute phase were taken

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before antibody peak (Me= 5 days), while on the convalescent phase samples were taken after
antibody peak (Me= 18 days).
3.2. IgM Validation
IgM ELISA results according to case definition criteria are shown in Figure 1.

Table 2 shows the overall IgM ELISA validation sensitivity and specificity on acute and
convalescent phases.
IgM ELISA showed a high area under the ROC curve (AUC= 0.940). Overall specificity was
97.0%. Among non cases, specificity increased from 95.1% in suspected cases without leptospirosis
to 99.4% in healthy donors and 100% in patients with diseases other than leptospirosis (data not

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shown). While sensitivity was higher in convalescent (98.4%) than acute phase (76.6%), specificity
remained high in both phases (96.9 and 94.9%, respectively).

3.3. Accuracy comparison of PCRs and ELISAs assays for early diagnosis.

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As shown on Table 3, IgM ELISA showed the highest overall sensitivity (63.5%), followed by
rtPCR (43.9%), IgG ELISA (30.8%) and cPCR (24.3%) in samples from 0-9 DPO. IgM ELISA

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showed the greatest diagnostic accuracy at 1-9 DPO (J=0.6) followed by rtPCR (J=0.4). On the
whole acute period (0-9 DPO), 45 cases were only detected by IgM ELISA, 24 only by rtPCR, 23

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by bough tests and 15 by none of them. If these results are interpreted in parallel, sensitivity would
be 85.9% (77.9%-91.9%).

Seven confirmed diseases other than leptospirosis (three Hantaviruses, three H1N1 and one FHA
case) were also rtPCR positive. However, six of them showed homology with L. interrogans in the

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analysis of the 16S rRNA sequencing and two of the Hantavirus cases had positives titters to

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Icterohaemorrhagiae in MAT. Only one Hantaviruses case showed homology with Acidobacteria.
Meanwhile, four discarded cases resulted IgM ELISA positive. Although in this study only first

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not shown).

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samples of these four cases were included, second samples were IgM ELISA positive as well (data

As shown on Figure 2, while DPO increased sensitivity diminished on both PCRs and increased
on both serological methods (particularly IgM ELISA).
During the first 0-3 DPO, rtPCR (51.4%) and IgM ELISA (45.7%) showed the highest Se. On
this first period, eleven cases were only detected by rtPCR, nine only by IgM ELISA, seven by both
tests and eight by none of them. On the contrary, on the second period (4-6 DPO), thirty cases were
only detected by IgM ELISA, ten only by rtPCR, 13 by bough tests and 7 by none of them. On the
third period (7-9 DPO) all cases were detected by at least one test, 6 only detected by IgM ELISA, 3
only by rtPCR and 3 by bough tests.

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4. DISCUSSION
There is a strong global need for methods to confirm leptospirosis at an early acute phase (WHO,

2011). Diagnostic test performances vary among different regions and countries because of the

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diversity of epidemiological features, leptospiral strains, and patient socio-demographic


characteristics. In this paper, the development and validation of a new IgM ELISA, and its

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comparison with three other tests, two for the detection of leptospira DNA, was performed on a
panel of samples representing the human population of Argentina.

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In previous ELISA developments we used other serovars as antigen. No significant variations


between the different serovars used in the lysate were found, probably due to its genus specific
characteristics (Adler et al. 1980; Vanasco et al. 2007; Vanasco 2012, 2013).
High concentration of serum samples did not improve the sensitivity but diminished specificity

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of the assay. These results are in agreement with previous studies carried out by our group (Vanasco

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2012, 2013)

Although Icterohaemorrhagiae (24.7%) remained the most frequent presumptive infecting

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serogroup, Canicola showed a higher proportional rate than in previous studies (Vanasco et al.,

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2008; Cuds et al., 2014). This could reflect a change in the epidemiology situation of the country,
probably conditioned by the reservoirs dynamic and human- environmental interactions. For this
reason it is crucial to validate leptospirosis tests on representative samples from the target
population.
The overall accuracy of the IgM ELISA was high (AUC: 0.940), being lower on the acute (AUC:
0.892) than on the convalescent phase (AUC: 0.986). The latter may reflect the increase of
sensitivity on the convalescent phase (98.4%), since specificity was high, not only on the overall
analyses (97.0%), but also on both phases (96.9 and 94.9%). A recent meta-analysis had already
shown that ELISAs may be almost two times more efficient when applied in convalescent than in

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acute patients (Signorini et al., 2013). This rise in sensitivity is consistent with the highest peak of
IgM antibodies that are reached at approximately 15 DPO (Levett, 2001).

Finally the high specificity of IgM ELISA in all stages in patients with diseases other than

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leptospirosis and in healthy patients supports the idea of using this method as a supplement or even
as a replacement test for MAT to confirm the disease (Goris et al., 2012). These authors validated

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an IgM ELISA based on the determination of a titer from serial dilutions yielding robust results.
Although our study was based on single well determinations, the results were very similar. The use

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of a single genus specific antigen in these ELISAs increased sensitivity and allowed the detection
of all varieties within a given region (Vanasco et al., 2007), while the use of specific antigens like
LipL32 will diminish sensitivity (Vanasco 2012, 2013; Signorini et al. 2013).
From the comparative evaluation among two serologic ELISAs methods (IgM and IgG) and two

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PCRs targeting LipL32 emerges that surprisingly a serological method, the IgM ELISA, have the

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greatest diagnostic accuracy (J= 0.6, Se=63.5% and Sp= 96.8%) for the early detection (1-9 DPO)
followed by rtPCR (J= 0.4, S=43.9% and Sp= 91.4%). Conventional PCR (J= 0.2) and the IgG

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ELISA (J= 0.1) showed very lower efficiencies.

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The low efficiency of the IgG ELISA (Se=30.8% and Sp=77.1%) in comparison to the IgM
ELISA (Se= 63.5% and Sp=96.8%) agrees with previous reports where IgM ELISA was seven
times more efficient than those detecting IgG (Signorini et al., 2013).
Low sensitivity (43.9% and 24.3% on rtPCR and cPCR, respectively) could reflect the absence
of leptospiral DNA. The latter could be attributed to low/short leptospiremia during the acute phase
of the disease, to antibiotic administration, or to late collection of blood samples (Picardeau et al.,
2014). Other possible explanation includes a very low count of leptospiral DNA in the initial
sample associated with long time of sample storage. Moreover Slettler and Brodard (2015)
suggested that the performance of the rtPCR may be largely influenced by the causative serovar. In

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Argentina, the lower limit of detection with Icterohaemorrhagiae and Harjoe serovars has been
frequently associated with humans and animals cases (AAVLD, 1994; Vanasco et al., 2008).

Our study found lower sensitivity and specificity for PCR and specially rtPCR in blood samples

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than previously reported (Slack et al., 2007; Villumsen et al., 2012). Those studies compared PCR
methods only against positive culture. Obviously, chances of being PCR positive are higher when

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leptospirosis is confirmed by culture than when the disease is diagnosed by seroconversion.


Moreover sensitivity can vary according to the sample being tested: whole blood, blood serum or

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plasma (Bourhy et al., 2011).

As DPO increased, so did the sensitivity of serological methods, especially IgM ELISA, as
expected. On the contrary, sensitivity of PCRs decreased. The high proportion of cases with IgM
antibodies, even in early acute stages of the disease, may explain the relatively low sensitivity of

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PCR. However, on the acute phase (0-9 DPO), 45 cases were only positive to IgM ELISA and 24

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were only detected by rtPCR. If the results of both tests are combined and interpreted in parallel,
sensitivity rises up to 85.9%.

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The highest diagnostic sensitivity 0-3 DPO belonged to rtPCR (51.4%), but it was closely

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followed by IgM ELISA (45.7%). The difference between both tests was lower than the most
optimistic estimations (Slack et al., 2007; Villumsen et al., 2012). A least two factors may have
influenced this finding. On one hand, patient memory bias could affect DPO calculations. On the
other hand, analytical sensitivity and specificity from controlled laboratory studies are always
higher than diagnostic sensitivity and specificity from observational studies that include other
factors affecting the outcomes under field conditions (Tarabla and Signorini, 2013). As expected,
our results are in agreement with those reported in Thailand by Thaipadungpanit et al., (2011) for
LipL32 rtPCR under similar study design and number of cases and non cases. However, as
previously discussed on the whole acute phase (0-9 DPO), when results of both tests are interpreted

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in parallel 0-3 DPO, sensitivity rises up to 77.1%. Similar observation can be made for the second
and third periods.

Se for rtPCR 7-9 DPO was good and two cases were only detected by rtPCR, this supports recent

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reports that described a wider window of positivity than previously thought for this test (Agampodi
et al., 2012).

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Overall specificity was high, with the only exception of IgG ELISA (77.1%). The high
specificity of IgM ELISA (96.8%) agrees with previous results (Vanasco et al., 2007; Signorini et

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al., 2013). Four discarded leptospirosis cases IgM ELISA positives on first and second samples did
not show MAT seroconvertion. The latter could be attributed to IgM response to agents not
included in this study, inefficient MAT sensitivity for some case detection (Goris et al., 2012;
Limmathurotsakul et al., 2012) or late MAT seroconvertion (Levett, 2001).

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The specificity of rtPCR was also high (91.4%; 85.2, 95.7). Due to its intrinsic characteristics

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(specific probes and isolated wells), rtPCR usually shows higher specificity than cPCR (Picardeau,
2014) and has been proposed as a confirmatory test in early acute phases (Ahmed, and P. Grobusch,

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2012; Goris et al., 2012; Picardeau et al., 2014). Our results support this view. Moreover, six of the

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seven Non cases (two Hantavirus, three H1N1 and one FHA positive) rtPCR positives showed
homology with L. interrogans in the analysis of the 16S rRNA sequencing so they could be double
infection. Similarly the other two non cases (discarded) who were rtPCR positive could not have
been detected by MAT.

5. CONCLUSION
IgM ELISA could be useful not only as a screening but also as a confirmatory test. It would be
of great relevance in regions where it is not possible to perform techniques such as MAT. On site
confirmation of cases on small regional labs could enhance local and global awareness of
leptospirosis.

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Our results support the use of IgM ELISA and rtPCR in conjunction for early diagnosis.
Moreover due to their high Sp, they could be also useful to replace or supplement MAT as a

confirmatory test, allowing more confirmations. For example, when only one single sample with

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short evolution time (<9 DPO) is available, like fatally cases, the use of this methods could allow
the confirmation. But when the second convalescent serum sample is available, the best strategy

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should include the MAT to know the presumptive infecting serogroup because of its relevance for
epidemiological surveillance.

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from: http://whqlibdoc.who.int/publications/2011/9789241501521_eng.pdf

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* Seroconvertion in two or more samples

L. wolfii and L. interrogans (Canicola serogroup)

L. interrogans (Icterohaemorrhagiae and Canicola serogroups)

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Figure 1. Fulfillment of the Case definition criteria and IgM ELISA validation results

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Figure 2. Sensitivity and IC 95% of each of the two PCR and ELISAs methods by 3 DPO groups in

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the 107 samples from confirmed cases, Argentina (2004-2014). DPO: Days Post Onset

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Table 1. Characteristics of participants of the evaluations, Argentina (1999-2014)
With Leptospirosis

Without Leptospirosis (Non-Cases)

(Confirmed Cases)

Discarded leptospirosis Confirmed diseases

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Patients characteristic

other than leptospirosis

n=297 (510 samples)

n=103 (148 samples)

Male n (%)

291 (90.1)

207 (70.0)

42 (75.0)

Mean Age males -

32.9 (16.4)

38.5 (18.1)

35.7 (12.5)

Mean Age females -

32.1 (16.7)

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Years (SD)

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n=323 (527 samples)

36.0 (19.7)

42.0 (18.8)

198

16

29

40

17

30

85

DPO known - n (%)

502 (95.2)

498 (97.6)

30 (20.3)

Single serology

120 (37.1)

97 (32.7)

64 ( 62.1)

203 (62.9)

200 (67.3)

39 (37.9)

5 (3)

5 (4)

6 (4)

Years (SD)

242

North east

41

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Central

20

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North west

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South
Unknown

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Source Argentinian Region*

sample- n (%)
Multiple serology
samples - n (%)
DPO in acute phase
patients samples median (IQR)

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DPO in convalescent

18 (11)

18 (11)

22 (21)

phase patients samples

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- median (IQR)

Probable infecting serogroup: Icterohaemorrhagiae (24.7%), Canicola (22.8%), Sejroe (16.6%),

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Tarassovi (7.5%), Ballum (5.3%), Pomona (3.1%), Pyrogenes (1.3%), Grippothyphosa (0.3%),
Coaglutination (18.4%)

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Gender was registered for 675 participants: 323 leptospirosis patients, 297 patients with discarded

leptospirosis and 56 patients with other confirmed disease.

Age was registered for 565 participants: 282 leptospirosis patients, 267 patients with discarded

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leptospirosis and 16 patients with other confirmed disease


*

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DPO: Days Post Onset.

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Familiar residence in data base

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Table 2. IgM ELISA overall validation and grouping by DPO of the samples results in Argentina
(1339 samples, 527 from Confirmed cases and 812 Non cases)

Positive/

Sensitivity

Negative/

Specificity

Tested

% (95% CI)

Tested

% (95% CI)

463/527

87.9 (84.8 -

786/812

190/248

Acute

76.6 (70.8 -

phase 0-9

81.7)

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DPO*

253/257

99.6)

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(n=592)

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nce phase
>9 DPO*

98.4 (96.1 -

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(n=441)
Convalesce

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90.5)

validation

187/193

318/335

Non Cases

CI)

Index J

97.0 (95.6 -

0.940

0.8

98.1)

(0.926 0.950)

96.9 (93.4 -

0.892

98.9)

(0.859 -

Area Under the ROC Curve

0.7

0.919)

94.9 (92.0-

0.986

97.0)

(0.972 0.994)

*Days Post Onset (DPO) was registered in 505 confirmed cases and 528 non cases

Youden

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Overall

AUC (95%

Confirmed cases

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Group

0.9

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Table 3. Comparison of two ELISAs and two PCRs test in samples from 0-9 DPO, for early
detection (n=234 samples, from 224 individuals), Argentina (2004-2014)

Positives

Sensitivity

Non-cases@(n=127)

Negatives

68

63.5 (53.7 -

IgG ELISA

123

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72.6)
33

30.8 (22.3 -

98

40.5)
Real time

43.9 (34.3

47

PCR

24.3 (16.5

26

125

33.5)

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Conventional

116 discarded leptospirosis and 11 confirmed other diseases

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118*

53.8)

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PCR

* Include four Non Interpretable (NI) Real Time results as Negative

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Youden
Index J

Specificity
(%) 95% CI

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(%) 95% CI
IgM ELISA

Confirmed Cases@(n=107)

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Test

96.8 (92.1

0.6

99.1)
77.1 (68.9

0.1

84.1)
91.4 (85.2

0.4

95.7)
98.4 (94.4
99.8)

0.2