Am. J. Trop. Med. Hyg., 86(3), 2012, pp.

524–527
doi:10.4269/ajtmh.2012.11-0423
Copyright © 2012 by The American Society of Tropical Medicine and Hygiene

Accuracy of a Commercial IgM ELISA for the Diagnosis of Human Leptospirosis in Thailand
Varunee Desakorn,* Vanaporn Wuthiekanun, Vipa Thanachartwet, Duangjai Sahassananda, Wirongrong Chierakul,
Apichat Apiwattanaporn, Nicholas P. Day, Direk Limmathurotsakul, and Sharon J. Peacock
Department of Clinical Tropical Medicine, Department of Tropical Hygiene, Department of Microbiology and Immnunology and
Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand;
Nuffield Department of Clinical Medicine, University of Oxford, United Kingdom;
Medical Department, Udon Thani General Hospital, Udon Thani, Thailand

Abstract. The Leptospira immunoglobulin M enzyme-linked immunosorbent assay (IgM ELISA) has been recom-
mended for the rapid diagnosis of leptospirosis in endemic areas. We conducted a retrospective case-control study of
218 patients (109 leptospirosis cases confirmed by Leptospira culture and/or microscopic agglutination test and 109 control
patients with acute febrile illness) to evaluate the diagnostic accuracy of a commercial IgM ELISA (Panbio) in north-
east Thailand. Paired serum samples taken on admission and at least 10 days after the onset of symptoms were tested.
Using the cutoff value recommended by the manufacturer (11 Panbio units), sensitivity and specificity of IgM ELISA on
paired sera were 90.8% and 55.1%. A receiver operating characteristic curve was used to determine the optimal cutoff
value. This was 20 Panbio units, which gave a sensitivity and specificity of 76.1% and 82.6%, respectively, on paired sera.
We conclude that using either cutoff value, the accuracy of IgM ELISA is limited in our setting.

INTRODUCTION tic accuracy was very low (sensitivity ranged from 36.0% to
60.9% and specificity ranged from 65.1% to 98.0%).9–11 The
Leptospirosis is considered an emerging infectious dis- reason for poor performance in the latter studies may be that
ease in Thailand.1–3 Regular contact with the environment is in countries where leptospirosis is highly endemic, a variable
a major risk factor, with a study in Thailand reporting that lep- proportion of the apparently healthy population may be sero-
tospirosis accounted for around one-third of undifferentiated positive. This would result in variability in the optimal cutoff
fevers in agricultural workers.3 Such infection usually results value in different geographic regions. In this study, we tested
from contact with water or soil contaminated with pathogenic the hypothesis that the optimal cutoff for this assay in Thailand
Leptospira originating from the urine of maintenance (carrier) is higher than that stated by the manufacturer, and determined
hosts such as rodents. The clinical features of leptospirosis are the optimal cutoff value and diagnostic accuracy of the IgM
often nonspecific and severity ranges from a mild flu-like ill- ELISA in this setting.
ness to severe sepsis and septic shock. Signs and symptoms
of leptospirosis may resemble those of other diseases in the
tropics including dengue, scrub typhus, and malaria, resulting MATERIALS AND METHODS
in an inaccurate clinical diagnosis.4 Diagnostic confirmation is
The Standards for the Reporting of Diagnostic accuracy
not attempted across much of the world, however, leading to
testing (STARD) were followed in this study.
sub-optimal patient management and a poor understanding of
Study patients. Patients with laboratory-confirmed lep-
disease epidemiology.
tospirosis (cases) or without leptospirosis (controls) were
In Thailand, the definitive diagnosis of leptospirosis is
identified from a prospective cohort study of consecutive
made by culture of Leptospira spp. from clinical samples such
patients presenting to Udon Thani hospital, northeast Thailand
as blood or urine, or by the reference serological assay, the
with an acute febrile illness between 2001 and 2002.12 In brief,
microscopic agglutination test (MAT). These tests require spe-
patients were recruited into the study during twice daily ward
cialist expertise, are labor-intensive, and provide a retrospec-
rounds. Inclusion criteria were patients who were ³ 15 years of
tive diagnosis.5 Several rapid serological diagnostic tests have
age with fever (> 37.8 °C) of unknown cause who agreed to
been developed as alternatives to MAT, of which a commer-
participate and to attend outpatient follow-up for a convalescent
cial immunoglobulin M enzyme-linked immunosorbent assay
serum sample. Exclusion criteria were patients with a definable
(IgM ELISA) is promising because it can be performed in a
source of infection on admission. Blood samples for Leptospira
greater number of laboratories throughout the tropics, and is
culture and serological testing were taken on admission, and a
inexpensive compared with MAT. The IgM ELISA has been
second serum sample taken 10 or more days after the onset of
recommended by the World Health Organization (WHO) as
symptoms. Leptospira culture and MAT were performed as
a diagnostic test for the serodiagnosis of leptospirosis where
described previously.5,12 Serum was stored at –80 °C until anal-
healthcare resources are limited,4 although its reported accu-
ysis for IgM ELISA and MAT.
racy is variable. A number of studies have reported that IgM
Cases of leptospirosis were defined by isolation of patho-
ELISA has high sensitivity and specificity for the diagnosis
genic Leptospira spp., and/or a 4-fold or greater rise or a
of acute leptospirosis.6–8 However, recent studies evaluating
single serum MAT titer of ³ 1:400. Controls (ratio 1:1) were
this test in Laos, Vietnam, and Hawaii found that its diagnos-
randomly selected from those patients who had convalescent
sample and did not meet the diagnostic criteria for leptospiro-
sis. Overall, 109 cases and 109 controls were recruited in our
* Address correspondence to Varunee Desakorn, Department of
Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol study. A database was constructed in which cases and controls
University, 420/6, Rajvithi Road, Bangkok, 10400 Thailand. E-mail: were entered, randomized, and blinded to the investigators
[email protected] before performing IgM ELISA.

524
 ELISA FOR THE DIAGNOSIS OF LEPTOSPIROSIS IN THAILAND 525

Leptospira IgM ELISA. Detection of IgM antibodies to Government of Thailand. Each patient was recruited to the
Leptospira species was determined using a commercially study after written informed consent was obtained.
available Leptospira IgM ELISA (Panbio Pty., Ltd.,
Queensland, Australia). The assay was performed according RESULTS
to the manufacturer’s instructions. Briefly, test sera, cutoff
calibrator, and positive and negative control sera were A case-control study consisting of 109 leptospirosis cases
diluted 1:100 in serum diluent, and 100 µL added to and 109 controls was used to evaluate the accuracy of the
Leptospira antigen-coated microwells and incubated for IgM ELISA. Of 109 leptospirosis cases, laboratory confirma-
30 minutes at 37 °C. After washing with phosphate-buffered tion was made on the basis of being positive by culture and
saline containing 0.05% Tween 20, 100 µL of HRP conjugated MAT in 19 patients (17%), culture positive/MAT negative
anti-human IgM was added and incubated for another in 15 patients (14%), and culture negative/MAT positive in
30 minutes at 37 °C. After further washing, 100 µL of 75 patients (69%). The proportion of study patients who were
tetramethylbenzidine substrate was added and incubated at male was 82% and 57% for cases and controls, respectively
room temperature for 10 minutes, after which the reaction (P < 0.001). Three cases (2%) and four controls (4%) died
was stopped with 100 µL of 1 M phosphoric acid. The during hospital admission (P > 0.1). All patients in the con-
absorbance of each well was read at a wavelength of 450 nm trol group had a convalescent serum sample taken a median of
with a Bio-Tek ELX 808 plate reader (Bio-Tek Instruments, 17 days (interquartile range [IQR] 13–21 days, range 10–43 days)
Winooski, VT). The results were expressed as Panbio units after the onset of symptoms. The discharge diagnoses of con-
calculated by the ratio of sample absorbance to the mean trols were as follows: scrub typhus (N = 51), bacterial septice-
cutoff absorbance multiplied by 10. The recommended cutoff mia (N = 8) (Escherichia coli [N = 2], Klebsiella pneumoniae
for a positive result is a value of ³ 11 Panbio units, and is [N = 2], Acinetobacter baumannii [N = 1], Corynebacterium
interpreted by the manufacturer to indicate recent infection jeikeium [N = 1], Enterococcus spp. [N = 1], and Streptococcus
of leptospirosis. pneumoniae [N = 1]), dengue fever (N = 4), murine typhus
Evaluation of IgM ELISA in healthy individuals. Serum (N = 4), melioidosis (N = 2), HIV-related infections (N = 2),
samples were obtained from 218 healthy blood donors other diagnoses (N = 6), and unknown diagnosis (N = 32).
residing in northeast Thailand and tested using the IgM The sensitivity and specificity of IgM ELISA were deter-
ELISA to evaluate background seropositivity. To determine mined using culture and/or MAT positivity as the gold stan-
the reference range for this population, IgM Panbio units were dard. The diagnostic accuracy of the ELISA based on testing
log transformed so that data was normally distributed, and the paired (acute and convalescent) sera and using a positive
reference range then calculated using the geometric mean and result on admission and/or during follow-up was deter-
range values (geometric mean ± 2 SD). mined using the manufacturer’s cutoff of > 11 Panbio units.
Statistical analysis. Statistical analyses were performed The sensitivity and specificity of paired serum samples were
using STATA/SE version 11.2 (College Station, TX). 90.8% (95% CI = 83.8–95.5) and 55.1% (95% CI = 45.2–64.6),
Diagnostic sensitivity and specificity of the IgM ELISA was respectively (Table 1). Recalculation of these using results
defined against the combined result for culture and MAT from the admission sample only gave a sensitivity and
(a positive result of either or both being interpreted as specificity of 52.3% (95% CI = 42.5–62.1) and 66.4%
diagnostic for leptospirosis), and expressed as a proportion (95% CI = 56.6–75.2), respectively.
with exact 95% confidence interval (CI). The Fisher’s exact A ROC curve analysis was performed using results based
test was used to compare categorical variables, and the Mann- on paired sera, which demonstrated an area under the curve
Whitney test was used to compare continuous variables. The of 0.85 (95% CI = 0.79–0.89). From this, an optimal cutoff was
optimal cutoff value of IgM ELISA was determined using estimated to be ³ 20 Panbio units. This gave an accurate clas-
receiver operating characteristic (ROC) curves.13 Median sification in 79.4% of patients compared with 72.5% if the low
time to positivity of IgM ELISA in leptospirosis cases was cutoff value was used (Figure 1). Using this higher cutoff value,
estimated by survival analysis. This analysis was censored for the sensitivity and specificity of the ELISA on paired sera
loss to follow-up. were 76.1% (95% CI = 67.0–83.8) and 82.6% (95% CI = 74.1–
Ethical statement. The study protocol was approved by the 89.2), respectively, and those on admission were 30.8% (95%
Ethics Committee of the Ministry of Public Health, Royal CI = 22.2–40.5) and 90.7% (95% CI = 83.5–95.4), respectively.

TABLE 1
Sensitivities and specificities of Panbio IgM ELISA for the diagnosis of leptospirosis*
Gold standard result† % (95% CI)

Cutoff value Sample timing Positive (+) or negative (–) result + – Sensitivity Specificity

³ 11 Panbio units On admission‡ + 56 36 52.3 (42.5–62.1) 66.4 (56.6–75.2)

– 51 71
Paired sera + 99 49 90.8 (83.8–95.5) 55.1 (45.2–64.6)
– 10 60
³ 20 Panbio units On admission‡ + 33 10 30.8 (22.2–40.5) 90.7 (83.5–95.4)
– 74 97
Paired sera + 83 19 76.1 (67.0–83.8) 82.6 (74.1–89.2)
– 26 90
* IgM ELISA = immunoglobulin M enzyme-linked immunosorbent assay; CI = confidence interval.
† Positive gold standard result was isolation of pathogenic Leptospira spp., and/or a 4-fold or greater rise or a single serum MAT titer of ³ 1:400.
‡ Sera on admission of two leptospirosis cases and two controls were not available to test by the IgM ELISA.
526 DESAKORN AND OTHERS

include short duration of follow-up and inadequacy of an

IgM response in some leptospirosis patients. Taking a second
convalescent sample or sampling after a longer duration of
follow-up may be required. IgM may also fail to be elevated
in patients presenting with a second or subsequent episode of
infection, when the antibody response would be predicted to
be largely IgG. Poor specificity of the IgM ELISA is also likely
to be caused by a high background seropositivity rate in the
general population in our setting. Positivity of IgM ELISA
could also represent past exposure of leptospirosis. This is
strongly supported by the high seropositivity rate in healthy
individuals if the low cutoff value is chosen.
We conclude that the Leptospira IgM ELISA is not suffi-
ciently accurate in Thailand. The recommendation by WHO
to use such an assay for the diagnosis of leptospirosis if finan-
cial resources permit requires country-specific evaluation to
determine its clinical use before implementation.
FIGURE 1. Receiver-operator characteristic curve of the Leptospira
immunoglobulin M enzyme-linked immunosorbent assay (IgM ELISA)
versus gold standard test (a combination of culture and MAT) on Received July 1, 2011. Accepted for publication August 30, 2011.
paired sera.
Acknowledgments: We are grateful to the director and staff of the
Medical Department of Udon Thani General Hospital (Udon Thani
Province, Thailand). We thank Lee D. Smythe (WHO/FAO/OIE
Median duration between onset of symptoms and primary Collaborating Centre for Reference & Research on Leptospirosis,
specimen collection were 7 days (IQR 4–10 days) and 10 days Western Pacific Region, Communicable Disease Unit, Queensland
(IQR 7–16 days) for cases and controls, respectively. Using Health Scientific Services, Brisbane, Australia) who undertook the
survival analysis, the median time from onset of symptoms to MAT tests. We thank Boongong Noochan, Amornwadee Sungkakam,
Premjit Amornchai, and Sayan Langla (Mahidol-Oxford Tropical
positivity of IgM ELISA in leptospirosis cases was estimated Medicine Research Unit, Faculty of Tropical Medicine, Mahidol
to be 7 and 14 days for the low cutoff value (³ 11 Panbio units) University), for technical assistance. We also thank Associate Professor
and high cutoff value (> 20 Panbio units), respectively. Pratap Singhasiwanon (Dean of the faculty of Tropical Medicine,
The optimal cutoff value defined here was almost twice Mahidol University) and Professor Punnee Pitisuttithum (Head of the
Department of Clinical Tropical Medicine) for their support.
the cutoff value recommended by the manufacturer, suggest-
ing high background seropositivity. This was evaluated by Financial support: The study was funded by the Wellcome Trust.
testing 218 serum samples from healthy individuals residing Disclaimer: The authors have no conflict of interest. The Panbio
in northeast Thailand. The ELISA results for these samples Leptospira IgM ELISA kits were provided without charge by Panbio
demonstrated a geometric mean value of 5.7 Panbio units, Pty, Ltd., Queensland, Australia. The company has no role in the study
design, data collection and analysis, interpretation of results, and writ-
with a normal range (geometric mean ± 2 SD) of 1.7 to
ing or submission of this manuscript.
19.3 Panbio units. Of 218 healthy individuals, 28 (12.8%) had
an IgM ELISA level equal to or more than 11 Panbio units, Authors’ addresses: Varunee Desakorn, Vipa Thanachartwet, Duangjai
Sahassananda, and Wirongrong Chierakul, Department of Clinical
and 6 (2.8%) had an IgM ELISA level equal to or more than Tropical Medicine, Faculty of Tropical Medicine, Mahidol University,
20 Panbio units. Bangkok, Thailand, E-mails: [email protected], tmvtn@mahidol
.ac.th, [email protected], and [email protected]. Vanaporn
Wuthiekanun and Nicholas P. Day, Mahidol-Oxford Tropical Medi-
DISCUSSION cine Research Unit (MORU), Faculty of Tropical Medicine, Mahidol
University, Bangkok, Thailand, E-mails: [email protected] and nickd@
Reassessment of diagnostic sensitivity and specificity of the tropmedres.ac. Apichat Apiwattanaporn, Medical Department, Udon
Thani General Hospital, Udon Thani, Thailand, E-mail: a-apiwat@
IgM ELISA showed that using either the low or high cutoff
hotmail.com. Direk Limmathurotsakul, Department of Tropical Hygiene,
value, the accuracy of this test is poor in Thailand. Using the Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand,
cutoff value recommended by the manufacturer (11 Panbio E-mail: [email protected]. Sharon J. Peacock, Department of Micro-
units), the sensitivity on paired sera is high (90.8%) and may be biology and Immunology, Faculty of Tropical Medicine, Mahidol Uni-
used to rule in patients with suspected leptospirosis. However, versity, Bangkok, Thailand, E-mail: [email protected].
the poor specificity of the test (55.1%) suggests that using this
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