Microbiological Research 207 (2018) 108–115

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Microbiological Research
journal homepage: www.elsevier.com/locate/micres

Leptospirosis in human: Biomarkers in host immune responses T

a,⁎ b,c b,d a,e a a b
Chin VK , Lee TY , Lim WF , Wan Shahriman YWY , Syafinaz AN , Zamberi S , Maha A
a
Department of Medical Microbiology & Parasitology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400, UPM, Serdang, Selangor, Malaysia
b
Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400, UPM Serdang, Selangor, Malaysia
c
School of Foundation Studies, Perdana University, 43400, Serdang, Malaysia
d
Integrative Pharmacogenomics Institute (iPROMISE), Universiti Teknologi MARA Selangor, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia
e
Department of Medical Laboratory Technology, Faculty of Health Sciences, Universiti Teknologi MARA, Cawangan Selangor Kampus Puncak Alam, 42300 Bandar
Puncak Alam, Selangor, Malaysia

A R T I C L E I N F O A B S T R A C T

Keywords: Leptospirosis remains one of the most widespread zoonotic diseases caused by spirochetes of the genus
Leptospirosis Leptospira, which accounts for high morbidity and mortality globally. Leptospiral infections are often found in
Host immune responses tropical and subtropical regions, with people exposed to contaminated environments or animal reservoirs are at
Biomarkers high risk of getting the infection. Leptospirosis has a wide range of clinical manifestations with non-specific signs
Cytokines
and symptoms and often misdiagnosed with other acute febrile illnesses at early stage of infection. Despite being
Immune mediators
Proteomic approach
one of the leading causes of zoonotic morbidity worldwide, there is still a gap between pathogenesis and human
immune responses during leptospiral infection. It still remains obscure whether the severity of the infection is
caused by the pathogenic properties of the Leptospira itself, or it is a consequence of imbalance host immune
factors. Hence, in this review, we seek to summarize the past and present milestone findings on the biomarkers of
host immune response aspects during human leptospiral infection, including cytokine and other immune
mediators. A profound understanding of the interlink between virulence factors and host immune responses
during human leptospirosis is imperative to identify potential biomarkers for diagnostic and prognostic appli-
cations as well as designing novel immunotherapeutic strategies in future.

1. Introduction
Leptospirosis is a globally widespread zoonotic disease caused by
spirochetes Leptospira which has high impact on health consequences in
human and domestic animals (Levett, 2001; Bharti et al., 2003). Human
leptospirosis is transmitted through direct contact of wounds with urine
and tissues of the infected animals or indirectly through the mucous
membranes of the nose, eyes and mouth with contaminated water or
soil (Bharti et al., 2003; Palaniappan et al., 2007). Pathogenic Leptospira
species have their own affinity and special adaptations towards specific
mammals and hence causes clinical manifestations in varying degrees
(Levett, 2001). For example, rodent reservoirs are carriers for Leptospira
species and do not show any diseases. Meanwhile, infected companion
animals or livestock may suffer from abortion and multiple organs in-
juries and infected humans may present with a variety of clinical
manifestations from asymptomatic infection to life threatening disease
(Ashford et al., 2000; Ganoza et al., 2010). With mild infections, pa-
tients typically present with symptoms viz fever, headache, and
myalgia, and less frequently by meningitis, conjunctival suffusion, rash,
renal insufficiency and jaundice. Symptoms may be biphasic and some
can resolve spontaneously. However, these symptoms and signs are
non-specific and could be frequently misdiagnosed with other causes of
acute febrile illness such as dengue fever, influenza or malaria (Bharti
et al., 2003; Wuthiekanun et al., 2007).
The development of severe leptospirosis could be accompanied by
symptoms which include jaundice, pulmonary haemorrhage, hepatic
and kidney failure (Chirathaworn et al., 2016). Of the complications
that arise from leptospiral infection, severe pulmonary haemorrhage
syndrome (SPHS) and Weil’s syndrome (combination of acute renal
insufficiency, haemorrhage and jaundice), are among the well-known
forms of severe leptospirosis. Notably, mortality recorded in Weil’s
disease and severe pulmonary haemorrhage syndrome (SPHS) are >
10% and 50%, respectively (McBride et al., 2005; Gouveia et al., 2008).
High incidences of leptospiral infection are often seen in tropical
and subtropical regions. There are approximately 1.03 million cases
and 59,000 deaths reported globally (Costa et al., 2015; Torgerson
et al., 2015). Hence, the high frequency of leptospiral infection con-
stitutes a serious clinical problem worldwide that needs to be addressed

⁎
Corresponding author.
E-mail addresses: cvk717@gmail.com (C. VK), tzeyan.lee@gmail.com (L. TY), limwaifeng85@gmail.com (L. WF), shahrimanuitm@gmail.com (W.S. YWY),
syafinaz@upm.edu.my (S. AN), zamberi@upm.edu.my (Z. S), maha@upm.edu.my (M. A).

https://doi.org/10.1016/j.micres.2017.11.015
Received 8 June 2017; Received in revised form 23 November 2017; Accepted 25 November 2017
Available online 28 November 2017
0944-5013/ © 2017 Elsevier GmbH. All rights reserved.
C. VK et al.
properly. Increased incidence of leptospiral infection indicates the dif-
ficulty of diagnosing and treating the disease and thus, a better un-
derstanding of leptospiral infection followed by the development of
sensitive and specific diagnostic methods and appropriate strategies for
leptospirosis treatment are beneficial.
Hitherto, the underlying pathogenesis of leptospirosis remains ob-
scure. It is believed that both pathogen and host factors are responsible
for the development and progression of severe infection. However, it is
still poorly understood whether the final outcome of severe illness is a
result from direct tissue damage caused by the Leptospira, or it is a
consequence of an imbalance of host immune response to infection.
Numerous studies have reported the involvement of immune mediators
such as cytokines and chemokines and their potentials as promising
biomarkers for disease severity prediction or therapeutic efficacy.
However, there are still lots of unsolved questions pertinent to this
disease that need to be addressed by further research. Hence, in this
review, we focus on the leptospiral pathogenesis and host immune re-
sponse during leptospirosis to gain insights and for better strategized
planning in the future to combat this disease.
2. Leptospiral pathogenesis
Based on 16SrRNA phylogeny, Leptospira genus can be categorized
in three large subgroups; Group I (pathogenic), Group II (intermediate
pathogenic) and saprophytes (Lehmann et al., 2014). Hitherto, there
are nine pathogenic Leptospira species in Group I including L. kirschneri,
L. interrogans, L. noguchii, L. borgpetersenii, L. alexendari, L. weilii, L.
santarosai, L. kmetyi and L. alstoni. Group I pathogens can be further
classified into more than 250 distinct serotypes which produce a wide
variety of clinical manifestations, ranging from subclinical infection to
severe and fatal disease, with most of the severe disease arise from
serovars belonging to evolutionarily-related species, L. interrogans, L.
noguchii and L. kirschneri (Brenner et al., 1999; Slack et al., 2009).
Meanwhile, Group II consists of five intermediately pathogenic Leptos-
pira species including L. licerasiae, L. wolffii, L. broomii, L. inadai and L.
fainei, where infection with these species will result in mild and self-
resolving and without fatal complications (Schmid et al., 1986; Brenner
et al., 1999; Petersen et al., 2001; Levett et al., 2006; Matthias et al.,
2008; Slack et al., 2008). There are six saprophytic Leptospira species
including L. meyeri, L. terpstrae, L. biflexa, L. vanthielii, L. yanagawae and
L. wobachii, in which these species are free-living microorganisms in
environment and do not cause any complications in human (Brenner
et al., 1999). In addition, a new non-pathogenic saphroyte, L. idonii has
been discovered and described recently (Saito et al., 2013).
Before leptospiral genome era, the underlying mechanisms on the
pathogenesis of leptospira remains largely unknown (Ko et al., 2009).
With the advancement of next generation sequencing (NGS), the first
genome sequence of Leptospira interrogans was completed on 2003 (Ren
et al., 2003). In-depth analysis on a series of genes responsible for
chemotaxis and lipopolysaccharide (LPS) synthesis has revealed the
unique physiological and pathogenic features of this species con-
tributing towards pathogenesis in human. Furthermore, Leptospira
genome project which was initiated on 2011 has enhanced our under-
standing on the pathogenesis mechanisms of Leptospira and aided us in
distinguishing Leptospira species in terms of gene and pathway in-
volvements (Ricaldi et al., 2012).
Establishment of human leptospirosis lies on the ability of patho-
genic Leptospira to invade, colonise, disseminate and extract host nu-
trients for survival. The persistency in host invasion, colonisation and
dissemination is assisted by the expression of various virulence factors
by pathogenic Leptospira. The severity of leptospirosis can be de-
termined by different virulence factors related to pathogenic Leptospira
(Thaipadungpanit et al., 2007), inoculum size that modifies infecting
pathogen burden (Ganoza et al., 2006) or differences in host immune
response (Wagenaar et al., 2009a). Different Leptospira serotypes confer
different virulence factors that play important roles in leptospiral
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Microbiological Research 207 (2018) 108–115
pathogenesis to adapt to host’s environment. To date, there are a lot of
studies documented the Leptospira virulence factors (Ko et al., 2009;
Adler et al., 2011; Narayanavari et al., 2012; Wang et al., 2012) in-
cluding lipopolysaccharides (LPS) (outer membrane), outer membrane
proteins (OMPs) like lipoprotein, hemolysins, OmpA-like Loa22, lep-
tospiral immunoglobulin-like (Lig) proteins, sphingomyelinases and
adhesion molecules.
Leptospiral lipopolysaccharides (LPS) is the major outer membrane
of Leptospira, with varying LPS structure in different serovars and an-
tigenically diverse (Bulach et al., 2000). Leptospiral LPS is a well-
known virulence factor contributing towards the pathogenesis in
human, due to its ability to adhere with host’s extracellular matrix,
including laminin, collagen and fibronectin (Hoke et al., 2008). Lep-
tospiral LPS exhibits similarity to gram-negative-bacterial LPS, both
structurally and immunologically. In human cells, leptospiral LPS ac-
tivates macrophage through a Toll-like receptor 2 (TLR2) pathway, in
the presence of CD14 (Werts et al., 2001). In contrast to mouse cells,
TLR2 and TLR4 pathways were involved, suggesting host-specific ac-
tivation (Nahori et al., 2005). Similarly, both pathogenic and inter-
mediately pathogenic leptospiral LPS confers different carbohydrate
and lipid compositions that undergo different leptospiral pathogenesis
(Patra et al., 2015). Other leptospiral outer membrane proteins (OMPs),
such as lipoproteins LipL32 is only conserved and expressed in patho-
genic species during infection (Haake et al., 2000).
Hemolysins is a toxin made from protein and lipid that can lyse cell
membrane of erythrocytes and other cells (Thompson and Manktelow,
1986). Pathogenic Leptospira interrogans can express leptospiral SphH
that destroys mammalian cell membranes by pore formation, without
the involvements of sphingomyelinase or phospholipase activities (Lee
et al., 2002). However, leptospiral SphA demonstrated sphingomyeli-
nase activity on cell membrane by increased membrane permeability,
aggregation and fusion (Lee et al., 2002; Goni and Alonso, 2002).
During Leptospira interrogans infection, hemolysin Sph1, Sph2, Sph3,
HlpA and TlyA are secreted to stimulate proinflammatory cytokines via
TLR2, TLR4, JNK and NFkB pathways (Wang et al., 2012). Meanwhile,
Leptospiral immunoglobulin-like (Lig) proteins, such as LigA, LigB and
LigC are expressed only in pathogenic Leptospira species, which they are
anchored to the outer membrane to facilitate mammalian host cell in-
vasion or attachment (Matsunaga et al., 2003). Exposure to physiolo-
gical osmolarity leads to high expression of LigA and LigB that interacts
with extracellular matrix proteins, such as collagen I and IV, laminin,
fibronectin and fibrinogen that may be involved in the colonization and
dissemination of leptospirosis (Choy et al., 2007).
A recent study on Leptospira genome revealed that approximately
900 genes in pathogenic Leptospira strains could be involved in the
pathogenicity of leptospirosis. Moreover, the roles of most of the genes
remain obscure and current known proteins failed to explain the viru-
lence mechanisms of leptospira as a whole. Furthermore, mutation
analysis has revealed that some of these genes have pronounced func-
tions in leptospira pathogenesis where mutations in these genes reduce
virulence of leptospira. These genes include Loa22, OmpA-family pro-
tein and several other proteins (Adler et al., 2011).
The latest breakthrough on pathogenesis of Leptospira was demon-
strated by Fouts et al. (2016) where the authors performed comparative
cross-species genomic analysis of the Leptospira genus to identify the
virulence factors associated with the pathogenesis and mammalian host
adaptation during leptospirosis. In the study, the authors have identi-
fied few metabolic mechanisms related to host adaptation by Leptospira,
including pathogen-specific porphyrin metabolism, sialic acid bio-
synthesis and cobalamin (B12) autotrophy as a Leptospira virulence
factor. Besides that, the authors also identified some novel Leptospira
virulence proteins including pathogen-specific adhesins and Virulence
Modifying (VM) proteins. The authors also suggested that CRISPR/Cas
system, which is only present in pathogenic Leptospira, could be the
underlying causes for Leptospira genus refractoriness to gene targeting
(Fouts et al., 2016).
C. VK et al.
3. Biomarkers during severe leptospirosis
Over the past few decades, studies on host immune response and the
search for potential biomarkers during human leptospirosis in both
animals and human have expanded tremendously. Identifying suitable
biomarker during human leptospirosis is indeed important in aiding us
to differentiate the severity of the infection for diagnostic and prog-
nostic applications. Hence, in this review, we summarized the past and
present studies on host immune response during human leptospirosis
and the relevancy of these biomarkers in severe leptospirosis context.
3.1. Cytokines and chemokines
Tumor necrosis factor-alpha (TNF-α) is considered as one of the
widely studied cytokine and is a prominent mediator for severe lep-
tospirosis. TNF-α is a pro-inflammatory cytokine produced by mono-
cytes, macrophages and resident renal cells that plays crucial roles in
various biological events including vasodilatation, tissue development
and death, cell differentiation and immune-mediated diseases
(Balamayooran et al., 2010). Earliest findings from Estavoyer et al.
(1991) revealed that TNF-α was involved with renal impartment and
high TNF-α levels were prominent in severe non-fatal leptospirosis
(Estavoyer et al., 1991). Following study from Tajiki and Salomao
(1996) also revealed that TNF-α expression was associated with liver,
kidney and lungs involvement as well as bleeding. In addition, high
level of TNF-α was associated with higher mortality seen in patients
suffering from severe leptospirosis (Tajiki and Salomao, 1996). Similar
findings from Kyriakidis et al. (2011) also reported high level of TNF-α
was associated with pulmonary haemorrhage (Kyriakidis et al., 2011).
This haemorrhaging condition could be due to secretion of exotoxin-
haemolysins by Leptospira (Wang et al., 2012) which induces the pro-
duction of TNF-α that acts on both endothelium and macrophages,
causing inflammation of blood vessels, cell and fluid leakage and ulti-
mately leads to severe haemorrhages seen in patients with Weil’s syn-
drome (Diament et al., 2002). Recent studies also showed TNF-α was
significantly higher in severe leptospirosis as compared to the mild
leptospirosis (Chirathaworn et al., 2016; Reis et al., 2013).
However, in contrast to the above findings, Rizvi et al. (2011) re-
ported that TNF-α level was greatly suppressed in most of the leptos-
pira-positive patients with acute liver hepatitis (Rizvi et al., 2011,
2014) and there were no significant differences in TNF-α level between
the mild and severe group of patients with leptospirosis (Chirathaworn
et al., 2016; Mikulski et al., 2015). Possible explanations on the dis-
crepancies between these findings could be due to the measurement of
TNF-α level at different stages of infection, the involvement of multiple
organs dysfunction and the number of sample size studied.
Interleukin 10 (IL-10) is another notable cytokine involved in the
leptospirosis severity. IL-10 is an anti-inflammatory cytokine and has
multiple roles in mediating immune response including regulation of
inflammation in sepsis (Standiford, 2000), down-regulating the ex-
pression of monocyte-derived interleukin 1 (IL-1) and TNF-α (Cassatella
et al., 1994; Bonfield et al., 1995), inhibits nuclear factor kappa B (NF-
kB), regulating nitric oxide synthesis and down-regulation of TNF-α
receptors after lipopolysaccharide (LPS) stimulation (Wang et al.,
1995). IL-10 level is one of the independent determinant in fatal lep-
tospirosis (Reis et al., 2013). Moreover, high fatality was observed in
severe leptospirosis patients with high levels of IL-10 and elevated IL-10
levels were observed in severe leptospirosis patients than in those with
mild disease (Kyriakidis et al., 2011; Reis et al., 2013). Overall, these
findings indicated that IL-10 is a severe mediator during leptospirosis,
however, the immunopathogenic role of IL-10 during severe leptos-
pirosis remain unclear. It is postulated that overproduction of IL-10
may inhibit Th1 response which causes ineffective inflammatory re-
sponse which subsequently leads to immune pathology (Haake and
Levett, 2015). This postulation is supported by animal study where IL-
10 increased the mortality of animals infected with leptospires
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Microbiological Research 207 (2018) 108–115
(Lowanitchapat et al., 2010). However, another study by Volz et al.
(2015) reported that sufficient amount of IL-10 can confer protection to
host by limiting tissue damage induced by inflammatory cytokines and
prevent severe symptomatic infection (Volz et al., 2015). This warrants
further investigations in future to elucidate the role of IL-10 in the
pathological events during leptospirosis.
On the other hand, conflicting data on the IL-10/TNF-α ratio as a
marker for leptospirosis severity have been published. High ratio of IL-
10 to TNF-α was linked with lower disease severity (Tajiki et al., 1997)
while studies from Reis et al. (2013) and Kyriakidis et al. (2011) sug-
gested that high ratio of IL-10 to TNF-α was associated with severe or
fatal outcome. The latest study by Chirathaworn et al. (2016) reported
that there was no significant difference in the IL-10/TNF-α ratio in
leptospirosis patients with or without organ involvement
(Chirathaworn et al., 2016).
Another cytokine involved in disease progression is interleukin 6
(IL-6). IL-6 is an independent predictor of death in leptospirosis as
suggested by Reis et al. (2013). IL-6 possesses both pro-inflammatory
and anti-inflammatory properties. It stimulates hypothalamus and in-
hibits IL-1 and TNF-α (Papa and Kotrotsiou, 2015). High level of IL-6
was often seen in patients with severe leptospirosis and fatal cases
(Wagenaar et al., 2009a; Reis et al., 2013). Moreover, it is often asso-
ciated with severe pulmonary haemorrhage syndrome (SPHS), one of
the fatal outcomes in severe leptospirosis (Reis et al., 2013). In addi-
tion, Papa and Kotrotsiou (2015) reported that IL-6 levels were elevated
at the early stages during severe cases, returning to normal levels at late
stages. Therefore, these findings suggest that overproduction of IL-6
could lead to exacerbation of disease and become potentially fatal. This
indicates that IL-6 might have direct pathophysiological effect on the
severe pulmonary haemorrhage syndrome. In a study by Parsons et al.
(2005), the authors observed increased levels of interleukin 8 (IL-8) and
IL-6 were associated with increased risk of death in patients with acute
respiratory distress syndrome (Parsons et al., 2005). Ahuja et al. (2012)
demonstrated that in a mouse model of acute lung injury, IL-6 may have
direct role in increasing lung inflammation and resulting in poor out-
come (Ahuja et al., 2012).
The study by Reis et al. (2013) has provided us with new insights on
host immunity during severe leptospirosis. In the study, the authors
suggested ‘cytokine storm’ which comprises of both pro- and anti-in-
flammatory cytokines (IL-2, IL-1b, IL-4, IL-6, IL-17A, IL-8, TNF-α and
IL-10) were significantly higher in severe cases compared to mild cases.
In addition, the authors reported high levels of IL-6, IL-8 and IL-10 that
were observed in fatal cases compared to non-fatal cases during severe
leptospirosis. Moreover, through the study, the authors surmised that
specific cytokine responses were varied with clinical outcomes, in-
cluding the need of hospitalization, development of severe pulmonary
haemorrhage syndrome and death. However, no robust conclusion can
be drawn from this study regarding the role of ‘cytokine storm’ as
mediators of pathogenesis during leptospirosis or they are just bio-
markers of disease severity or progression. The authors also suggested
that investigation on temporal evolution of the cytokine response
should be carried out to further elucidate the role of cytokines in the
pathogenesis of severe leptospirosis.
By using suspension array system, Papa and Kotrotsiou (2015) re-
ported clusters of cytokines, chemokines and colony-stimulating factors
(CSF) were elicited during acute phase of leptospirosis. In addition, the
authors speculate some of these cytokines have specific association with
sampling time, the presence of haemorrhagic manifestations and the
pulmonary involvement during leptospirosis. These cytokines include
IL-8, IL-6, granulocyte-macrophage CSF (GM-CSF), vascular endothelial
growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1)
and interferon-inducible protein 10 (IP-10) where MCP-1, IP-10 and IL-
6 levels were increased in most of the cases. On the other hand, the
authors reported that although elevated MCP-1 levels were observed in
both mild and severe patients, however, only the severe cases showed
significantly higher levels of MCP-1 as compared to the control group.
C. VK et al.
Findings from animal study also showed that increased in the MCP-1
expression was well associated with the severity of the disease (da Silva
et al., 2009). Meanwhile, VEGF levels were significantly higher in se-
vere as compared to non-severe cases. The similar findings also ob-
served in hamsters infected with Leptospira interrogans (Coutinho et al.,
2014) and patients with Hantavirus infection, suggesting the role of
VEGF in vascular damage repair (Tsergouli and Papa, 2013).
Based on the transcriptomic analysis of severe leptospirosis cases,
Lindow et al. (2016) concluded that the failure of host immune system
to mount effective innate and adaptive immune response against lep-
tospires will result in the mortality seen in patients with severe lep-
tospirosis. In addition, through the study, a few unique biomarkers
including RANTES and CH3L1, could serve as potential diagnostic and
therapeutic candidates for leptospirosis patients at high risk of dying.
The function of RANTES as a chemokine is to recruit immune cells to
the infection site. This suggests that aberrant cell trafficking will lead to
poor and slower adaptive immune adaptive immune responses parti-
cularly in the fatal cases (Lindow et al., 2016). Pro-inflammatory cy-
tokines will induce CHI3L1 expression which is normally associated
with increased patient mortality in sepsis and other infectious or in-
flammatory diseases (Prakash et al., 2013). Lindow and her colleagues
has even suggested that CHI3L1 could well represent a new potential
prognostic and therapeutic strategy for leptospirosis (Lindow et al.,
2016). Hence, both CHI3L1 and RANTES serum levels are potential
candidates for diagnostic markers, which could help identify patients at
risk for developing severe leptospirosis (Lindow et al., 2016).
In contrast to most of the findings published, Mikulski et al. (2015)
reported there was no significance in most of the cytokines studied
during severe and mild leptospirosis (Mikulski et al., 2015). In another
study by Chirathaworn et al. (2016), where the authors compared the
cytokine levels between two groups, with or without organ involvement
in two stages, acute and convalescent. IL-6, IL-10 and IL-8 levels in
acute stage were significantly higher in organ involvement group than
in without organ involvement group, however, only IL-8 level was
significant higher with organ involvement when comparing the cyto-
kine levels in convalescent stage (Chirathaworn et al., 2016). Taken
together, these findings raise particular concerns. Firstly, the im-
portance of sampling time when considering cytokine levels as bio-
markers for leptospirosis severity. Second, the possibilities of an un-
known dilution of serum due to patients who were treated with
antimicrobial therapy or massive fluid infusion may interfere with the
cytokine levels measured. Therefore, in future, investigation of cytokine
levels should be carried out in larger groups of patients and with dif-
ferent sampling intervals as this will enable us to capture the most
suitable timing for specimen collection and to determine the most ap-
propriate cytokine as biomarker in disease monitoring.
Taken together, the severity of leptospiral infection could be due to
deranged immune response, especially innate immunity to mount spe-
cific innate immune responses against leptospires, which leads to in-
effective early control of infection and followed by diminished activa-
tion of adaptive immune responses. As a result, leptospires keep
proliferating and grow in host tissues which subsequently promotes
host systemic inflammation, followed by tissue damage and mortality
seen in leptospirosis patients. Numerous immune mediators including
cytokines are involved in either exacerbation or resolving leptospiral
infection. Certain studies have postulated that cytokine patterns elicited
may be differing depends on different clinical outcomes, disease se-
verity and sampling time which could serve as potential predictors in
disease monitoring or immunotherapeutic targets. However, more in-
vestigations are warranted to investigate the actual role of cytokines in
the pathogenesis of leptospirosis, either as predictors of severity of
disease or it is just a reflective of immune activation in general. The
relationship between excessive inflammatory response and the severity
of leptospirosis is therefore of utmost importance and ought to be fur-
ther investigated.
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3.2. Serum mediators as potential biomarkers during severe leptospirosis
A majority of the cytokines and chemokines studied during leptos-
pirosis are also implicated in other bacterial infections, making it dif-
ficult to project them as biomarkers during leptospirosis. Hence, in this
review, we also focus on the potential of serum mediators as biomarkers
during severe leptospirosis, including sST2, long pentraxin, nitric oxide
and copeptin as these serum mediators are not implicated much in
other bacterial infections and could serve as better biomarkers during
leptospirosis.
3.2.1. Nitric oxide
Endothelial dysfunction is one of the possible mechanisms partici-
pating in the pathogenesis of severe leptospirosis. Endothelial dys-
function leads to vascular damage in affected organs and could result in
organ damage (De Brito et al., 1979; Nicodemo et al., 1997). Nitric
oxide (NO) is a crucial mediator of endothelial dysfunction in sepsis and
there are similarities in the clinical spectrum of severe sepsis and severe
leptospirosis. Pro-inflammatory cytokines including TNF-α, IL-6 and IL-
1 can stimulate the expression of inducible endothelial nitric oxide
synthase (iNOS) and in turn increased levels of NO in sepsis (Cohen,
2002). In leptospirosis, production of pro-inflammatory cytokines can
increase NO level as well (Yang and Hsu, 2005). The clear picture of NO
in the pathogenesis of leptospirosis remains elusive.
A study by Prêtre et al. (2011) has shown that expression of in-
ducible endothelial iNOS is important in eliminating the leptospires
where faster functional deterioration was observed in animals given
with 4-aminopyridine, which is an iNOS inhibitor than the untreated
animals (Prêtre et al., 2011). Meanwhile, several studies have docu-
mented the involvement of NO or serum nitrate during severe leptos-
pirosis (Maciel et al., 2006; Avdeeva et al., 2008; Gunaratna et al.,
2012; Kalugalage et al., 2013). These studies indicate high levels of NO
and serum nitrite that were observed in patients with severe leptos-
pirosis as well as leading to severe pathological condition. Another
possible explanation is NO can drive oxidative stress, which is likely to
be involved in tissue and organ damage in leptospirosis, although
current evidence is still lacking.
3.2.2. Soluble serum stimulation-2
Soluble serum stimulation-2 (sST2) is a receptor presented in so-
luble secreted form (sST2) (Tominaga, 1989) or in a membrane-an-
chored form (ST2L) (Yanagisawa et al., 1993). They are members of the
IL-1 receptor family. sST2 is imperative mediators in stimulating host
inflammatory response. Increased secretion of sST2 often involved pa-
tients with inflammatory disorders linked with abnormal Th2 mediated
responses, including, autoimmune diseases (Kuroiwa et al., 2001),
asthma (Oshikawa et al., 2001), idiopathic pulmonary fibrosis (Tajima
et al., 2003), and in patients with sepsis (Brunner et al., 2004).
An important discovery from Wagenaar et al. (2009a) is the asso-
ciation of sST2 with bleeding during severe leptospirosis. In the study,
the authors demonstrated that sST2, IL-6, IL-10 and IL-8 levels were
increased upon patients’ admission, with sST2 levels well correlated
with IL-6, IL-8, and IL-10. Furthermore, the authors showed that soluble
ST2 levels were significantly associated with mortality, severity and
overall bleeding in patients. The association of sST2 with bleeding seen
in severe leptospirosis could be due to disruption of the endothelial cell
barrier, which consequently leads to haemorrhages and exposure of
underlying fibroblasts and secretion of sST2 (Wagenaar et. al., 2009a).
3.2.3. Long pentraxin
Pentraxins are a superfamily of multifunctional conserved proteins
that are characterized by a pentameric structure. They are usually in-
volved in acute immunological reactions and often known as acute
phase proteins (Wagenaar et. al., 2009b). Long pentraxin (PTX3) is
secreted by a variety of cells such as dendritic cells, mononuclear
phagocytes, dendritic cells, epithelial and endothelial cells (Jumat and
C. VK et al.
John, 2017). Wagenaar et al. (2009b) demonstrated that high levels of
long pentraxin, IL-6 and IL-8 in patients with severe leptospirosis were
associated with mortality. In addition, PTX3 was well correlated with
IL-8 and to a lesser extent with C-reactive protein (CRP) and IL-6 levels.
The authors further revealed that high PTX3 levels were significantly
linked with disease severity, suggesting that PTX3 could be used as an
indicator for monitoring the severity of leptospirosis and to predict the
outcome of the infection (Wagenaar et. al., 2009b). However, larger
cohort studies inclusive of mild, moderate and severely ill patients
should be adopted to evaluate the efficacy of PTX3 as biomarker in
leptospirosis.
3.2.4. Copeptin
Hyponatraemia is one of the common clinical manifestations seen in
severe leptospirosis with renal impairment (Gancheva, 2012). Hypo-
natraemia arises from dysregulated vasopressin release (Rai et al.,
2006). Copeptin is a glycopeptide of the C-terminal part of the arginine
vasopressin (AVP) precursor which can be utilised to measure arginine
vasopressin (AVP) release (Struck et al., 2005; Morgenthaler et al.,
2006). Limper et al. (2010) surmised that copeptin could be a great
marker in predicting the survivals of patients suffer from sepsis or se-
vere sepsis arises from leptospirosis. The author reported that copeptin
levels were reduced during the course of the infection in survivors, and
its value was returned to baseline within 7 days. Meanwhile, higher
copeptin levels were associated with disease severity. Furthermore, the
author concluded that, unlike other biomarkers like C-reactive protein
(CRP) or procalcitonin (PCT), copeptin level seems to be stable and
does not vary much throughout the progression of leptospirosis. Hence,
it is a suitable candidate to discriminate between survival and patients
at high risk of dying. Besides that, copeptin was used as a marker to
monitor patients with leptospirosis during treatment. It is rather useful
especially in endemic areas with limited resources as copeptin may
serve as additional tool for health practitioners to decide on the treat-
ment in patients admitted with leptospirosis (Limper et al., 2010).
4. Cathelicidin and defensins as potential therapeutic agents
during human leptospirosis
Cathelicidin and defensins belong to large group of cationic anti-
microbial peptides with amphipathic properties. They are conserved
component in the innate immune system in all organisms, including
animals, plants together with human and exhibit antimicrobial activity
against many bacteria, fungi, and enveloped viruses (Sambri et al.,
2002; Isogai et al., 2004; Linde et al., 2013). Cathelicidin plays crucial
roles in stimulating phagocytosis, activating neutrophils, reducing LPS-
driven TLR-dependent proinflammatory responses and diminishing
apoptosis (Linde et al., 2013). In leptospirosis context, Lindow et al.
(2016) reported that low serum levels of cathelicidin (LL-37) observed
in fatal cases is responsible for high leptospires loads and therefore
suggests that cathelicidin is a potential therapeutic for leptospirosis.
This is due to the involvement of cathelicidin in killing of leptospires in
vitro (Sambri et al., 2002) and decreased circulating cathelicidin will
contribute to higher bacterial loads and greater tissue damage, which
ultimately result in poor outcome of the infection. The authors further
tested the efficacy of LL-37 (the only human cathelicidin) in a hamster
model of lethal leptospirosis and reported that administration of LL-37
can significantly protect hamsters from lethal infection by controlling
the bacteria loads. Hence, it is postulated that cathelicidin could serve
as potential therapeutic molecule during fatal leptospirosis.
On the other hand, defensins, which shows broad spectrum anti-
microbial activity, is involved in the oxygen-independent microbicidal
mechanisms of neutrophils. An earliest study from Wu et al. (1992) has
demonstrated the capability of the mixture of human defensins to kill
leptospires in vitro by neutrophils (Wu et al., 1992). However, there is
no further testing or subsequent study apart from to test the therapeutic
efficacy of human defensins in animal nor human studies. Meanwhile, a
112
Microbiological Research 207 (2018) 108–115
recent study by Chagan-Yasutan et al. (2016) has unravelled the po-
tential of defensins as potential biomarker during kidney injury in
leptospirosis patients where the authors suggest that urine defensin α1
(uDA1) level may reflect kidney injury in leptospirosis patients due to
their correlation with the serum creatinine level (Chagan-Yasutan et al.,
2016).
5. Proteomic approaches in identifying potential biomarkers
during human leptospirosis
Host transcriptomic and proteomic studies are deemed to be im-
portant in both the septicemic and immune phases of leptospirosis in
patients. However, proteomic study on the host-leptospira interaction is
still in its infancy. Srivastava et al. (2012) has first reported on the
alterations of human proteomic profiles induced by Leptospira. The
authors performed comparative proteomic analysis of leptospirosis
patients with the aid of two dimensional gel electrophoresis (2DE) and
2D-fluorescence difference gel electrophoresis (2D-DIGE) as well as
MALDI-TOF/TOF mass spectrometer. From the findings, the authors
documented that 22 serum proteins were differentially expressed in
leptospirosis patients as compared to healthy controls, with 8 candidate
proteins showing different patterns when compared with febrile con-
trols. From the study, the authors deduced that these differential ex-
pressed proteins are important mediators in physiological pathways,
such as complement, acute phase response, hemostasis and coagulation
cascades. In addition, among these differential expressed proteins, the
authors have identified several putative proteins viz α-1-antitrypsin,
ceruloplasmin, vitronectin, apolipoprotein A-IV and G-protein signaling
regulator which could serve as potential diagnostic or prognostic can-
didates for leptospirosis.
On the other hand, Ting et al. (2017) performed proteomic analysis
during mild and severe leptospirosis through 2DE analysis in combi-
nation with LC–MS/MS. The authors discovered five putative proteins
viz apolipoprotein A-I (APOA-I), transferrin (TF), serum amyloid A
(SAA), transthyretin (TTR) and haptoglobin (HP) were differentially
expressed between mild and severe leptospirosis. With the aided of
Ingenuity® Pathway Analysis software, the authors further speculated
that acute-phase signaling pathway was involved in modulating the
expression of these proteins as well as functional network of lipid me-
tabolism, small molecule biochemistry and molecular transport. The
authors further suggested that these serum proteins could be potential
biomarkers in differentiating between mild and severe leptospirosis
(Ting et al., 2017).
Further comprehensive investigation into the proteome dynamics of
leptospirosis infected patients should take into account of proteome
patterns that may be affected by the outcome and complications of the
patients, duration of the infection and also serogroup type of lepto-
pirosis infected patients.
6. Variation of host genetic
The study on host immune responses and in search of potential
biomarkers during leptospirosis remains challenging. Variation in host
genetic makeup may lead to differences in susceptibility towards lep-
tospirosis. Fialho et al. (2009) investigated the association of poly-
morphisms in human leukocyte antigen (HLA), cytokine genes and
killer-cell immunoglobulin-like receptors (KIR) between patients with
past history of leptospirosis and healthy controls. The authors reported
that alleles in the HLA-A and B loci plus several HLA haplotypes were
significantly associated with susceptibility towards leptospiral infec-
tion. Polymorphisms in IL-4 and IL-4Rα genes were also significantly
higher in patients with past history of leptospirosis. Meanwhile, there
was no significant association between KIR gene profile and leptos-
pirosis. On the other hand, Esteves et al. (2014) conducted genetic
susceptibility study in the population of São Miguel Island and reported
that genetic polymorphism in the IL12RB1, IL1β and CISH genes were
C. VK et al.
susceptible to leptospirosis infection. Recent studies also documented
the gene polymorphisms in TLR1 Ile602Ser and TLR-2 Arg753Gln
which have considerable effects on developing severe leptospirosis with
jaundice and hepatic insufficiency (Cédola et al., 2015). Taken to-
gether, these findings highlighted the importance of genetic poly-
morphisms in the host immune response towards leptospiral infection.
It is still remains elusive to what extent genetic variations contribute
towards different susceptibility of hosts towards leptospiral infection. In
addition, there is lack of current evidence to explain why certain in-
dividuals are susceptible to severe leptospirosis while others only de-
velop mild infections.
7. Conclusion
The host-pathogen interaction remains a very complicated process;
concerted effort is much needed to fully dissect the different aspects of
Leptospira virulence and to unravel its interactions with host immune
system. Understanding the role of immune mediators involved during
leptospirosis is crucial and needs to be highlighted as this will enable us
to elucidate the pathological events that occurs during leptospirosis and
also serve as a basis for diagnostic and prognostic applications as well
as vaccine development. Extensive research on leptospirosis should be
carried out to aid us in attaining more holistic understanding on the
complexity between Leptospira and humans and for a better design of
improvised therapeutic regimens in future.
Author contributions
Chin Voon Kin, Lee Tze Yan, Lim Wai Feng and Wan Shahriman,
YWY wrote the manuscript. Chin Voon Kin, Maha Abdullah, Syafinaz
Amin Nordin and Zamberi Sekawi conceptualized the idea and revised
the manuscript. All authors approved the final manuscript.
Conflict of interests
The authors declare that there is no conflict of interest that could be
perceived as prejudicing the impartiality of the review reported.
Acknowledgement
We would like to express our appreciation to the management and
support staffs of the Faculty of Medicine and Health Sciences,
University Putra Malaysia and Long Term Research Grant Scheme
(LRGS) by Ministry of Higher Education Malaysia (UPM/700-2/1/
LRGS/5526400) for providing financial and infrastructure support for
literature search.
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