Department of Microbiology, Ibrahim Medical College, Dhaka, Bangladesh
Advanced Medical & Dental Institute, Universiti Sains Malaysia, 13200 Bertam, Kepala Batas, Pulau Pinang, Malaysia
Background and objectives: Leptospirosis is a
zoonotic infection with worldwide distribution caused by the Leptospira species and predominant in
the tropical and subtropical regions. Information on leptospirosis in
Bangladesh is limited. The present study was designed to detect
anti-leptospiral antibodies in human serum samples in Bangladeshi population by
developing an in-house ELISA using recombinant LipL32 (rLipL32) antigen. The
study was conducted from April 2014 to December 2014.
Method: Healthy individuals from two rural
areas and fever cases from one urban healthcare center were enrolled in the
study. Rural health centers were located at Sonargoan and Bajitpur sub-district
(Upozilla) of Narayaganj and Kishorganj districts. Sonargoan health center is
located 26 km south-east and Bajitpur is located 71 km north-east of Dhaka
city. About 1-2 ml of blood was collected with aseptic measure and serum was
separated and stored at -200C until used. Anti-leptospiral IgG antibody was
determined by recombinant LipL32 (rLipL32) antigen based indirect enzyme linked
immunosorbent assay (ELISA). Seropositive cases were further confirmed by
commercial Leptospira IgG ELISA.
Results: The study included 250 febrile cases
and 376 healthy individuals from urban and rural areas, respectively. Out of
total 626 study population, anti-LipL32 specific IgG antibody was detected in
70 individuals (11.2%). The rate of positivity of anti-LipL32 antibody among
the healthy individuals from rural area was 10.6% while the rate was 12.0% in
urban febrile population. The rate of positivity in rural and urban population
was not significantly (p>0.05) different. Among the urban population, the
rate of seropositivity was 9.1% and 16.4% in 21-40 yrs and above 40 years age
group respectively while the rate was 7.2% and 14.0% in rural population respectively.
Out of 70 seropositive cases detected by LipL32 ELISA, 65 (92.9%) were positive
by commercial ELISA.
Conclusion: The present study has
revealed that leptospirosis is prevalent in Bangladesh and should be looked for
in febrile and clinically suspected cases. The study has also demonstrated that
rLipL32 protein may be used as a candidate antigen for the serodiagnosis of
IMC J Med
Sci 2017; 11(2): 50-55
Correspondence:Prof. J Ashraful Haq, Professor of
Microbiology, Ibrahim Medical College, 122 Kazi
Nazrul Islam Avenue, Dhaka, Bangladesh. Email: firstname.lastname@example.org
Leptospirosis is a spirochetal zoonosis that
infrequently causes a wide spectrum of clinical manifestations in humans.
Currently leptospirosis is considered as an emerging global public health
problem, particularly in resource-poor countries in tropics. The burden of
leptospiral disease falls predominantly on people living in poverty and under
inadequate sanitary conditions. Leptospira
spp infect various animals including rat and other rodents. Animal excrete Leptospira through urine, which
contaminate water and soil. People at risk for leptospirosis include farmers,
abattoir workers, sewer workers and others who have contact with soil, water
and animal [1-3]. The risk during the rainy season becomes higher after
flooding when the human population may be exposed to water contaminated with
urine from infected animals. The estimated annual leptospirosis morbidity in
South and South-East Asia is 17.97 and 55.54 per 100,000 populations .
Although data on leptospirosis in Bangladesh is
limited, a serological survey in a rural flood prone district of Bangladesh in
1994 showed 38% seropositivity in 89 samples of human sera tested, indicating
that the rural population was at high risk of leptospiral infection . In
2000, acute-phase serum specimens from 359 dengue-negative patients in Dhaka
were assessed for leptospirosis. Leptospira spp
was detected by polymerase chain reaction (PCR) in 63 (18%) of them .
Another study in 2000, screened people with fever in slum of Dhaka city and
reported leptospirosis in 8.4% cases . Presence of leptospirosis among the
cattle in dairy farms in Chittagong division of Bangladesh had also been
Several diagnostic tests are used to detect
acute and past leptospiral infection. Microscopic agglutination test (MAT) is
considered as gold standard test for the diagnosis of leptospirosis. But the
test requires live organisms. Detection of leptospiral DNA in clinical samples
by polymerase chain reaction (PCR) has been employed to diagnose leptospirosis
[8,9]. Recently, rLipL32 antigen based serodiagnostic tests have been developed
to conduct seroprevalence studies in human leptospirosis [8,10-12]. LipL32 is
an outer membrane protein which is highly conserved among pathogenic Leptospira spp and prominent immunogen
during leptospirosis. A study from Thailand has shown the diagnostic
sensitivity and specificity of the LipL32 dipstick assay as 100% and 98.33%,
respectively when compared to those of MAT. The results suggest that the
recombinant LipL32 is a good diagnostic detection reagent for detection of
antibodies against Leptospira.
In view of the above, the present study was
undertaken to determine the leptospiral infection among rural and urban people
of Bangladesh using a rLipL32 based in-house ELISA.
Study population and place: Healthy individuals
from two rural areas and febrile cases from one urban healthcare center were
enrolled in the study. Rural centers were located at Sonargoan and Bajitpur
sub-district (Upozilla) under Narayaganj and Kishorganj districts respectively.
Sonargoan is located 26 km south-east and Bajitpur is located 71 km north-east
of Dhaka city. Individuals with no history of fever in last one month prior to
enrollment were considered as healthy and enrolled in the study. Patients with
fever for more than 5 days attending an urban healthcare center in Dhaka city
were enrolled and were denoted as ‘febrile case’.
In order to determine the cut-off optical
density (OD) value of ELISA test, blood from 105 healthy newborn babies from a
hospital of Dhaka city were included in the study. Samples from neonates were
leftover blood collected for other routine investigations. The mothers of the
neonates were from urban affluent class and had least chance of exposure to
leptospiral infection. About 1-2 ml of blood was collected with aseptic measure
and serum was separated and stored at -200C until used. Informed consent was obtained
from the participants before collection of blood. The study was carried out
from April to December 2014.
Determination of anti-rLipL32 IgG antibody by
Recombinant outer membrane protein of 32 kDa, present in pathogenic Leptospira spp, was used for detecting Leptospira spp. specific IgG antibody by
enzyme linked immunosorbent assay (ELISA) as described by Voller et al .
Recombinant LipL32 protein was over expressed
from the recombinant plasmid pAELipL32 which we obtained from Prof. OA Dellagostin,
Núcleo de Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade
Federal de Pelotas, Brazil. The recombinant plasmid was transformed into
BL21(DE3) plysS strain. For protein purification, positive clone was inoculated
into 100 ml Luria Broth (LB) and allowed to grow at 370C until the
OD reached to 0.5 to 0.6 at 600 nm, induced with 1.0 mM IPTG (isopropyl-b-D-thiogalactopyranoside)
for about 4 hour. Cell pellet was resuspended in BugBuster Protein Extraction
Reagent and purified using His•Bind Purification Kit (Novagen, USA). The
purified antigen (Fig-1) was reconstituted with sterile distilled water to make
a concentration of 1 µg/µl and aliquoted for further use. The expression and
purification of protein was carried out at Advanced Medical & Dental
Institute, Universiti Sains Malaysia, Malaysia.
The 96 well EIA plate (Linbro, USA) was coated
with rLipL32 antigen 5µg/ml in 0.5 M carbonate/bicarbonate buffer (pH 9.6). To
each well 100 µl volume of coating buffer was added and incubated overnight at
40C. The plate was washed three times with PBS-0.05%Tween 20 (PBS-T,
pH 7.4)) and blocked by incubating for 2 hrs with PBS-T containing 2% BSA at 370C.
The plate was then washed three times with PBS-T. A volume of 100 µl serum
(1:40 dilutions) sample was added into each well and incubated for 4 hours at
370C. After washing with PBS-T three times, 100 µl of horseradish
peroxidase conjugated anti-human IgG antibodies (1:4000) was added and
incubated at 370C for 2 hours. After washing three times with PBS-T,
50 µl of TMB substrate was added to each well and incubated at room temperature
for 30 minutes in dark. Then 50 µl of 1M sulfuric acid was added in each well.
The colour developed was measured by EIA plate reader (Human ELISA Reader) at
450 nm. Optimum concentration of the antigen (5µg/ml) and serum dilution
(1:140) was predetermined by checkerboard titrations. A reagent blank and a
positive and negative control wells were included in each plate along with the
Determination of cut-off OD value: A cut off OD values
for rLipL32 specific IgG antibody was determined to find out the exposure rate
to Leptospira spp. in the study
population. ELISA was performed with sera from 105 healthy newborn babies of
Dhaka city who were presumed not to be exposed to Leptospira infection. The
mean OD + 3xSD of these sera were taken as cut-off OD value to determine the
exposure rate. The mean OD±SD of the 105 healthy newborn babies were 0.14±0.08.
Therefore, the calculated cut-off OD value was 0.38 (0.14+3x0.08). Any sample
showing OD above this cut-off value of 0.38 was considered as positive and
referred to as exposed to Leptospira
of Leptospira specific IgG antibody by commercial ELISA: Leptospira specific
IgG antibody was also determined by commercial ELISA (DRG International Inc,
USA) to compare the results obtained by in-house ELISA using rLipL32 antigen.
The kit used purified Leptospira biflexa
(serovar patoc 1) antigen for detection of antibody in serum. The assay was carried
out as per instruction of the manufacturer.
showing rLipL32 protein expressed from the recombinant plasmid pAELipL32 and
purified using His•Bind Purification Kit (Novagen, USA). Lane 1: Protein
ladder, Lane 2: protein following viva spin, Lane 3: protein following acetone
total of 626 samples were included in the study. Out of which, 376 samples were healthy individuals from rural
area and 250 were febrile cases from urban area. Out of total 626 study population,
anti-rLipL32 specific IgG antibody higher than the cut-off OD value (>0.38)
were detected in 70 individuals (11.2%). The rate of positivity of anti-rLipL32
antibody among the healthy individuals from rural area (Bajitpur and Sonargaon)
was 10.6%, while the rate was 12.0% in urban population. The detail location
wise (urban and rural) rate of seropositivity is shown in Table 1. The rate of
positivity in rural and urban population was not significantly (p>0.05)
Table-1: Seroprevalence of anti-leptospiral
IgG antibody among rural and urban study population by ELISA using rLipL32
Age specific seropositivity rate for
anti-rLipL32 antibody is shown in Table-2. Among the urban population, the rate
of seropositivity was 9.1% and 16.4% in 21-40 yrs and above 40 years age group
respectively; while rate was 7.2% and 14.0% in rural population respectively.
There was no positive case in age group 1-20 years in urban population while
5.4% was positive among rural population. There was an increased rate of
seropositivity with the increase of age. The overall (rural and urban) rate of
leptospira specific rLipL32 antibody was significantly (p<0.05) higher in
people over 40 years of age compared to 21-40 years group (Table-2).
of 70 seropositive cases by rLipL32 in-house ELISA, 65 (92.9%) were found
positive by commercial ELISA.
Table-2: Age specific
seropositivity rate of anti-leptospiral
IgG antibody of study population by rLipL32 antigen based ELISA
Leptospirosis is an
infectious disease with a worldwide distribution. Currently, prevention and
control of leptospirosis have received much attention of public health
authorities. Improved diagnostic test for leptospirosis is needed to aid
clinical diagnosis of acute cases and assess the prevalence. Recently,
recombinant antigen based serodiagnostic assays have been developed to diagnose
human leptospirosis. Among the many candidate antigens, LipL32, an outer
membrane protein, is highly conserved among pathogenic Leptospira spp .
In the present study, we have used rLipL32
antigen based ELISA to determine the seroprevalence of leptospiral infection in
selected urban and rural population. We have used a cut-off OD of >0.38 for
our in-house rLipL32 based ELISA to consider a sample positive for leptospiral
infection. The results when compared with the commercial ELISA was found almost
similar. Positive samples by rLipL32 ELISA was found positive in 92.9% cases by
commercial ELISA. Therefore, rLip32 could be considered as a good candidate
antigen for ELISA to detect Leptospira
spp specific infection in our geographical locations. However, further study is
necessary to optimize the test for detecting anti-leptosipral IgM antibody to
detect acute infection.
Leptospirosis has been reported to be prevalent
in our neighbouring countries and other countries of South and South-East Asian
regions . In India, a 5 year consecutive sero-epidemiological study
conducted in Kerala showed that 29.6% inhabitants possessed anti-leptospiral
antibodies . Previous studies from Bangladesh reported 8% to 38% leptospira
infection among febrile cases from urban and rural areas of the country [4-6].
But there is no study regarding the sero-prevalence of leptospiral infection
among healthy rural Bangladeshi population. In the present study, prevalence of
leptospirosis was found 12% among the febrile urban population and 10.64% among
the healthy individuals from rural areas. The rate of infection was not
significantly different among urban febrile cases and healthy individuals of
rural areas. However, the overall rate of infection increased significantly
with the increase of age. Previous studies reported that older people (20 years
and above) were at a greater risk for leptospirosis than children .
Presence of rLipL32 specific IgG among our
study population has shown that leptospiral infection is prevalent both in
rural and urban areas of Bangladesh. Anti-leptospiral IgG remains persistent
for several years following acute infection [15-16]. Persistence of antibody
may create problem in interpreting the serological tests detecting IgG
antibodies in acute cases unless rising IgG antibody or Leptospira specific IgM is detected.
The present study has demonstrated that a large
proportion of people residing in both rural and urban areas of Bangladesh are
exposed to leptospiral infection and rLipL32 antigen is a potential candidate
for the serodiagnosis of leptospirosis.
1. Jackson LA, Kaufmann
AF, Adams WG, Phelps MB, Andreasen C, Langkop CW, et al. Outbreak of leptospirosis associated with swimming. Pediatr Infect Dis J. 1993; 12:
2. Costa F, Hagan
JE, Calcagno J, Kane M, Torgerson P, Martinez-Silveira MS, et al. Global morbidity and
mortality of leptospirosis: a systematic review. PLoS Negl Trop Dis. 2015; 9(9):
e0003898. doi:10.1371/ journal. pntd.0003898.
3. Whitney EAS,
Ailes E, Myers LM, Saliki JT, Berkelman RL. Prevalence of and risk factors for
serum antibodies against Leptospira
serovars in US veterinarians. J Am Vet
Med Assoc. 2009; 234: 938–944.
4. Morshed MG, Konishi H, Terada Y, Arimitsu Y,
Nakazawa T. Seroprevalence of leptospirosis in a rural flood prone
district of Bangladesh. Epidemiol Infect.
1994; 112: 527–31.
5. LaRocque RC,
Breiman RF, Ari MD, Morey RE, Janan FA, Hayes JM, et al. Leptospirosis during dengue outbreak, Bangladesh. Emerg Infect Dis. 2005; 11(5): 766-769.
6. Kendall EA,
LaRocque RC, Brooks WA. Leptospirosis as a cause of fever in urban Bangladesh. Am J Trop Med Hyg. 2010; 82: 6 1127-1130.
7. Parvez MA,
Prodhan MAM, Rahman MA, Faruque MR.
Seroprevalence and associated risk factors of Leptospira interrogans serovar Hardjo
in dairy cattle of Chittagong, Bangladesh. Pak
Vet J. 2015; 35(3): 350-354.
8. Musso D, Scola
BL. Laboratory diagnosis of leptospirosis: A challenge. J Micro Immu Infec. 2013; 46:
9. Ahmed SA, Sandai
DA, Musa S, Hoe CH, Riadzi M, Lau KL, Tang TH. Rapid diagnosis of leptospirosis
by multiplex PCR. Malays J Med Sci.
2012; 19(3): 9-16.
10. Boonyod D,
Poovorowan Y, Bhattarakosol P, Chitrathaworn C. LipL32, an outer membrane protein of Leptospira, as an antigen in a dipstick assay for diagnosis of
leptospirosis. Asian Pac J Allergy
Immunol. 2005; 23: 133-141.
11. Flannery B, Costa D, Carvalho FP, Guerreiro H,
Matsunaga J, da Silva ED, et al.
Evaluation of recombinant leptospira
immunosorbent assays for the serodiagnosis of leptospirosis. J Clin Microbiol. 2001; 39: 3303-10.
12. Haake DA, Chao G,
Zuerner RL, Barnett JK, Barnett D, Mazel M, et
al. The leptospiral major outer membrane protein LipL32 is a lipoprotein
expressed during mammalian infection. Infect
Immun. 2000; 68: 2276- 2285.
13. Voller A,
Bartlett A, Bidwell DE. Enzyme immunoassays with special reference to ELISA
techniques. J Clin Path. 1978; 31: 507-520.
14. Kuriakose M, Paul
R, Joseph MR, Sugathan S and Sudha TN. Leptospirosis in a midland rural area of
Kerala State. Indian J Med Res. 2008;
15. Cumberland P,
Everard CO, Wheeler JG, Levett PN. Persistence of anti-leptospiral IgM, IgG and
agglutinating antibodies in patients presenting with acute febrile illness in
Barbados 1979-1989. Eur J Epidemiol.
2001; 17(7): 601-608.
16. Finsterer J,
Stöllberger C Sehnal E, Stanek G. Mild leptospirosis with three-year
persistence of IgG- and IgM-antibodies,
initially manifesting as carpal tunnel syndrome. J Infec. 2005; 51(2):