Acta Tropica 176 (2017) 206–223

Contents lists available at ScienceDirect

Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica

Review

Leptospirosis: Molecular trial path and immunopathogenesis correlated with MARK

dengue, malaria and mimetic hemorrhagic infections
Sivan Padma Priyaa, S. Sakinaha, K. Sharmilaha, Rukman A. Hamata, Zamberi Sekawia,
⁎
Akon Higuchib,c,d, Mok Pooi Linge, Syafinaz Amin Nordina, Giovanni Benellif, S. Suresh Kumara,g,
a
Department of Medical Microbiology and Parasitology, Universiti Putra Malaysia, 43400 UPM Serdang Selangor, Malaysia
b
Department of Chemical and Materials Engineering, National Central University, Jhong-li, Taoyuan, Taiwan
c
Department of Botany and Microbiology, King Saud University, Riyadh, Saudi Arabia
d
Department of Reproduction, National Research Institute for Child Health and Development, Tokyo, Japan
e
Department of Biomedical Science, Faculty of Medicine and Health Science Universiti Putra Malaysia, 43400, UPM, Serdang, Selangor, Malaysia
f
Department of Agriculture, Food and Environment, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy
g
Genetics and Regenerative Medicine Research Centre, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

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

Keywords: Immuno-pathogenesis of leptospirosis can be recounted well by following its trail path from entry to exit, while
Leptospira inducing disastrous damages in various tissues of the host. Dysregulated, inappropriate and excessive immune
Leptospirosis responses are unanimously blamed in fatal leptospirosis. The inherent abilities of the pathogen and inabilities of
Arbovirus the host were debated targeting the severity of the disease. Hemorrhagic manifestation through various me-
Co-infection
chanisms leading to a fatal end is observed when this disease is unattended. The similar vascular destructions
Cytokines
and hemorrhage manifestations are noted in infections with different microbes in endemic areas. The simulta-
Dengue
Malaria neous infection in a host with more than one pathogen or parasite is referred as the coinfection. Notably,
Chikungunya common endemic infections such as leptospirosis, dengue, chikungunya, and malaria, harbor favorable en-
Hemorrhagic fever vironments to flourish in similar climates, which is aggregated with stagnated water and aggravated with the
poor personal and environmental hygiene of the inhabitants. These factors aid the spread of pathogens and
parasites to humans and potential vectors, eventually leading to outbreaks of public health relevance. Malaria,
dengue and chikungunya need mosquitoes as vectors, in contrast with leptospirosis, which directly invades
human, although the environmental bacterial load is maintained through other mammals, such as rodents. The
more complicating issue is that infections by different pathogens exhibiting similar symptoms but require dif-
ferent treatment management. The current review explores different pathogens expressing specific surface
proteins and their ability to bind with array of host proteins with or without immune response to enter into the
host tissues and their ability to evade the host immune responses to invade and their affinity to certain tissues
leading to the common squeal of hemorrhage. Furthermore, at the host level, the increased susceptibility and
inability of the host to arrest the pathogens’ and parasites’ spread in different tissues, various cytokines accu-
mulated to eradicate the microorganisms and their cellular interactions, the antibody dependent defense and the
susceptibility of individual organs bringing the manifestation of the diseases were explored. Lastly, we provided
a discussion on the immune trail path of pathogenesis from entry to exit to narrate the similarities and dis-
similarities among various hemorrhagic fevers mentioned above, in order to outline future possibilities of pre-
vention, diagnosis, and treatment of coinfections, with special reference to endemic areas.

1. Introduction Mitsani, 2011). Leptospira interrogans, the causative organism of this

Leptospirosis is a zoonotic disease that claims nearly thousands of
lives annually worldwide. Farmers, fishing industry workers, veter-
inarians, slaughterhouse employees, agricultural workers and sewage
workers are more at risk of leptospirosis infections (Forouhar and
disease, is an aerobic, gram negative bacterium belongs to genus Lep-
tospira, (Phylum- Spirochaetes: Family-Leptospiraceae). It is evolutio-
narily primitive eubacteria having strong survival potential in soil and
water as saprophyte and facultative parasite. BLAST analysis comparing
the protein homologues reveals that the structural similarity with

⁎
Corresponding author at: Department of Medical Microbiology and Parasitology, Universiti Putra Malaysia, UPM Serdang 43400, Selangor, Malaysia.
E-mail address: sureshkudsc@gmail.com (S.S. Kumar).

http://dx.doi.org/10.1016/j.actatropica.2017.08.007
Received 7 July 2017; Received in revised form 3 August 2017; Accepted 4 August 2017
Available online 16 August 2017
0001-706X/ © 2017 Elsevier B.V. All rights reserved.
S.P. Priya et al.
archaeal and eukaryal proteins raising the possibility of the emergence
of this pathogenic bacterium from non-pathogenic one, possibly by
lateral gene transfer for better survival (Ren et al., 2003). This micro-
organism is typically excreted in urine from reservoir hosts, such as
rats, rodents, dogs, cattle, and others. It can enter into the body via
intact mucosa and/or abraded skin (Fig. 1). In the case of severe
flooding, individuals are at greater risk of coming in contact with lep-
tospira in endemic areas. The incubation period for infected individuals
is 7–10 days on average, resulting in clinical manifestations in two
stages: the initial leptospiremic stage and the subsequent immune stage
involving multiple organs. The initial leptospiremic phase reflects the
multiplication of the organism in the blood and tissues upon entry.
Leptospira binds to capillary endothelium, resulting in vasculitis, a
major cause of multisystem involvement (Evangelista et al., 2014b) or
Weil’s syndrome extends to liver, heart, kidney and nervous system
resulting in cardiac and renal failure. Brain involvement and neurolo-
gical manifestations are not common in leptospirosis (Mathew et al.,
2006).
Simultaneous infection of a host with more than one pathogen or
parasite is referred as the coinfection. Coinfection of leptospirosis with
dengue (Dircio Montes Sergio et al., 2012; Wijesinghe et al., 2015;
Perez Rodriguez et al., 2014; Singh et al., 2013; Zaki and Shanbag,
2010; Nunez-Garbin et al., 2015), chikungunya (Nhan et al., 2016) and
malaria (Lindo et al., 2013; Costa Ade et al., 2010; Magalhaes et al.,
2014) incidences are increasing, especially in pacific regions where
these diseases are endemic (Victoriano et al., 2009; Cao-Lormeau and
Musso, 2014). Hemorrhagic manifestation through different microbes
with a fatal end is observed when these diseases were unattended and
share similar endothelial destruction through different routes. The
bioactive molecules are similar but driven through different pathways.
The maximum spread and establishment of lesions are typically ob-
served by the first week of infection. During this crucial time, if timely
control over biomolecules can be exerted to stop the progress of the
microbe initiated damages, then the host-induced damages can be re-
duced. Coinfections in endemic areas and during outbreaks can be
handled together by understanding the similarities and dissimilarities
in immuno-pathogenesis to uniquely designed diagnosis, treatment, and
preventive modalities.
Immune response of a host to any pathogen or parasite either innate
or adaptive at cellular level involves interaction of molecules, which are
dynamic and complex in function, integrated with tissue damages, re-
pair, and resolution. During their signal inducing effector functions,
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Acta Tropica 176 (2017) 206–223
Fig. 1. This schematic diagram explains the trans-
mission cycle of Leptospira in the environment.
Rodents and wild animal act as reservoirs for
Leptospira, contaminating the water, vegetation, and
soil through their urine. Human with skin abrasions
and exposed mucosal surfaces get infected through
direct contact with Leptospira and disseminate the
pathogens throughout the body via the circulatory
system.
they do influence homeostasis, hematopoiesis and nervous system due
to their complicated mechanisms with overlapping functions. Recent
studies conducted within the last two decades have focused on large
variations in laboratory diagnostics, case reports and epidemiological
procedures concerning the identification of leptospirosis and other
hemorrhagic fevers (Wiwanitkit and Wiwanitkit, 2013; Musso and La
Scola, 2013). However, only a few studies have described a systematic
approach to differentiate coinfections with leptospirosis (Dircio Montes
Sergio et al., 2012; Mishra et al., 2013; Levett et al., 2000; Flannery
et al., 2001).
2. Invading the surface barriers/breaching the epidermal barriers
The surface defense such as physical (skin and mucosa), chemical
(antimicrobial secretions) and biological (commensals) barriers of the
body needs to be breached to activate the next level of defenses, the
innate and adaptive immune responses. The fundamentals of immune
interactions in leptospirosis are yet poorly described, in spite of millions
of researches and projects have completed and running (Cassadou et al.,
2016).
2.1. Physical barriers
Human body surfaces and cavities are covered with intact epithe-
lium, which is an effective barrier against microbes. Any breach in the
integrity of epithelial junctions, such as injured skin or through the
mucous membranes, generates an entry route for many microbes, in-
cluding Leptospira species (Adler and de la Pena Moctezuma, 2010)
(Fig. 1). The epithelium covering skin and mucous membrane or-
opharyngeal and ocular region is stratified with multiple layers, and the
thickness of this barrier varies according to the toughness of the in-
tended function (Junqueira and Mescher, 2010). Either complete injury
(ulcer) or partial injury (erosion) to the epithelium with or without
exposure of the connective tissue components enhances the entry of
Leptospira species (Haake and Levett, 2015). Epithelial thickness in skin
varies from thick palm (1.5 mm) to thin eyelid (0.05 mm), the corneal
mucosa having 52 μm (Hogan et al., 1971) and in oral mucosa varies
from 294 μm (buccal mucosa) to 99 μm (floor of the mouth) (Prestin
et al., 2012). The thick epithelial layers of the skin are maintained tight
by keratin and fillagrin bond and physiologically desquamating epi-
thelial cells releasing amino acids, lactic acids, urea' and salts, act as a
repellent coat (named as the natural moisturizing factor) to preserve the
S.P. Priya et al.
skin pliable and water repellent. The epithelium is an avascular struc-
ture with lack of blood vessels delivered defense cells, but themselves
secret cholesterol, free fatty acids and sphingolipids (especially cer-
amides) to stack the surface and intercellular areas to prevent water and
microbe movement (Coderch et al., 2003). Other than wounded skin
and mucosal area, systematic histopathological and histochemical stu-
dies needed to explore whether and how Leptospira invade into the
intact skin of different thickness and soiled skin of vulnerable profes-
sionals.
Leptospira penetrates the undamaged mucosal layer but not the skin.
The differences in preventive barriers are thickness and the moisture
content between them, but the distinguished secretions of skin epithe-
lium preventing their entry need to be explored to initiate the invention
of topical applications to enrich the preventive ability especially for
travelers to the endemic area. Epithelium also secrets eicosanoids
(Brock and Peters-Golden, 2007), an inflammatory mediator and do
possess non-keratinocytes like Langerhans cells (well known as antigen
presenting cells (APC) or dendritic cells (DC) in the basal layer), which
senses the antigens and present it to regional lymph nodes for specific
defense (Junqueira and Mescher, 2010). There are specialized in-
traepithelial γd T cells initiates a rapid defense by recognizing self-
antigens expressed on damaged or stressed cells and maintains tissue
homeostasis (Jameson et al., 2002). Broken skin and thin mucosa lack
theses protection shields render susceptible to leptospiral invasion. Nil
reports about entry through the intact skin strongly recommend the
need for educating personal hygiene awareness in endemic areas as a
crucial factor for primary prevention.
2.2. Chemical barriers
The chemical barrier other than mentioned above secretions in-
cludes lysozyme in sweat, saliva, and tear which is an enzyme that can
break down peptidoglycans present on the microbial cell wall (Asoh
et al., 2014). RNase (Simanski et al., 2012) and DNase secreted by skin
are more powerful that, they might interfere the laboratory procedures
of molecular biology if gloves not worn. There are few research works
explaining the production of chemicals barriers in the presence of
Leptospira species, however, a clear understanding is still needed for
each virulent strain. Defensins (Wu et al., 1992) and cathelicidin
(Sambri et al., 2002) are antimicrobial peptides (AMP), the small ca-
tionic peptides secreted by a variety of cells (Ganz, 1994) including
neutrophils and epithelial cells against many microbes, including Lep-
tospira. AMPs’ secreted by surface barrier cells are effective chemical
barriers at surface level.
2.3. Biological barriers
The biological barrier is by the non-pathogenic microbes on the skin
named as commensals, which are always defending their territory by
fighting with other invading pathogens. The revised ratio of the bac-
terial and human cells is 1:1 according to current report (Sender et al.,
2016) and the difference in the microbiome in each human, each
community and each ethnic group (Hospodsky et al., 2014) is well
versed. The commensals improving resistance to local and systemic
infections in different aspects is a never ending research interest (Zhang
and He, 2015). The need for exploring individual genetic susceptibility
in endemic areas and the difference in susceptibility among the en-
demic and non-endemic areas are to be strengthened regarding lep-
tospirosis (Esteves et al., 2014). Interestingly prophylactic and ther-
apeutic aspects of topical probiotics have been reviewed (Volz and
Biedermann, 2009).
The increased susceptibility resulting from polymorphisms asso-
ciated with the human leukocyte antigen (HLA DQ6) has also been
reported (Lingappa et al., 2004). Individual susceptibility due to de-
fective surface barrier function or due to genetic defects in the barrier
or nutritional imbalance targeting the surface barrier dysfunction has
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Acta Tropica 176 (2017) 206–223
not been explored in Leptospira species. The role of micronutrients and
the risk and severity of leptospirosis has been documented (Herman
et al., 2016) and for dengue (Ahmed et al., 2014). The importance of
malnutrition in underdeveloped countries especially those belong to
endemic areas are to be seriously taken for further researches to prevent
the leptospirosis, because malnutrition is one of the common cause of
immunodeficient state (Keusch, 2003).
Dengue virus (DENV), chikungunya virus (CHIKV) and malarial
parasites escape surface barriers with the help of mosquito vectors,
which deliver them directly to connective tissue or dermal region by
their biting mouthparts (proboscis) (Cox et al., 2012; Pingen et al.,
2016). The inability of all these pathogens or parasites to breach the
surface barriers needs to be strongly highlighted and included in pri-
mary prevention plan. Leptospira species do show their best way of
escaping from all these barriers by different mechanisms. Therefore,
key prevention at this level is highly encouraging to prevent all these
diseases together, if proper personal and environmental hygiene could
be educated to the public.
3. Driving beyond the dermal layer
Stakeholders do rise to understand more about the entry of
Leptospira species. However, the researchers are trying to find out the
mechanisms of immunity after the Leptospira breaches the dermal layer
of the host system. Innate immunity readily recognizes trespassers once
they clear the obstacle of surface barriers, by identifying pathogen
(non-self) related immuno-stimulants like formyl methionine con-
taining peptides, surface molecules like LPS, short sequence bacterial
DNA and CpG motifs, all of them including Leptospiras’, attract the
neutrophils (PMN) (Mandal et al., 2005) The innate immune network
consists of cell population resident to the local tissue including DC
(APC), PMN, mast cells, T- cells and natural killer cells and their cell
secretions namely cytokines. The pattern recognition receptors (PRR)
distributed on these cells recognize the Leptospira specific receptors and
other microbe specific receptors on the pathogens to initiate the pha-
gocytosis and to trigger the genes of the immune response. The PRR can
be soluble as in complement system or attached on to the host cell
membrane (Alberts, 2008). PRRs on the cells are strewn as membrane-
bound receptors including Toll-like receptors (TLRs) (Akira, 2009) and
C-type lectin receptors (CLRs) (Hoving et al., 2014; Geijtenbeek and
Gringhuis, 2009) or unbound in the cytosol like NOD-like receptors
(NLRs) (Proell et al., 2008) and RIG-I-like receptors (RLRs) (Loo and
Gale, 2011). The specificity shown by different PPRs for different pa-
thogen necessitates reconsidering new concept that the PPR in innate
immunity cannot be considered as a non-specific response, but a pa-
thogen specific immediate response.
3.1. Complement system
The complement system consists of about 30 interacting proteins
elicit a cascade of reactions to recruit PMNs to the site of pathogens,
opsonizes the pathogen for better engulfing of PMNs, initiates cell lysis
by membrane attack complexes, regulating antibody effector mechan-
isms and modulating T cell function (Stoermer and Morrison, 2011).
Complements are readily available proteins secreted by the liver in the
inactive form and widely distributed extra (ECM) and intravascularly
(plasma). The Leptospira species interact with few of the complement
proteins of both classical and alternative pathways to evade the im-
munity. This is achieved by the blocking action of the surface proteins
of Leptospira Lig binding protein with factor H (FH), factor H-like 1
(FHL-1), factor H-related 1 (FHR-1), LigB with C3b and C4b, Lsa30 with
C4 bp regulator, Lsa33 with C4 bp and LcpA (leptospiral complement
regulator-acquiring protein A) with C4 binding protein (C4 bp) (da
Silva et al., 2015; Barbosa et al., 2010; Fraga et al., 2014; Domingos
et al., 2012; Choy, 2012; Castiblanco-Valencia et al., 2012; Souza et al.,
2012).
 Table 1
Correlation and common clinical features of leptospirosis with dengue, malaria and chikungunya.

Leptospirosis Dengue Malaria Chikungunya

S.P. Priya et al.

Type of microbe Gram-negative, aerobic bacteria,
Family: Leptospiraceae
Genus: Leptospira
Transmission to host Direct mucosal entry; Reservoir mammals
are rats, dogs and pigs (Adler and de la
Pena Moctezuma, 2010; Andre-Fontaine,
2006)
Incubation 2–30 days (Adler and de la Pena
Moctezuma, 2010)
Mode of Living, Target cells and Extracellular and Facultative intracellular
organs
All these hemorrhagic fevers, Cells: endothelial, hepatocytes and
unanimously alter epithelial cells
microcirculation of entire body.
Organs: liver, lung, kidney, eye and brain
(Adler and de la Pena Moctezuma, 2010)
Cytokines associated with IL-1b,2, 4,6, 8, 10,17A, and TNF α (Goris
pathogenesis et al., 2011; Reis et al., 2013)
Common clinical features for the infections discussed here are fever, myalgia, headache, abdominal pain vomiting, anemia, thrombocytopenia
Single stranded- positive sense RNA virus,
Family: Flaviviridae
Genus: Flavivirus
Via Aedes mosquito vectors (commonly Aedes
aegypti)
4–6 days (Guzman and Kouri, 2004)
Obligate intracellular
Cells: macrophages, Langerhans cells,
monocytes, T cells, dendritic cells,
hepatocytes, endothelial cells, mast cells
Organs: damages primarily in liver, blood
cells and blood vessels, but lung, eye, heart,
pancreas and brain are secondary to bleeding.
(Simmons et al., 2012)
IL-2, 4, 5, 6, 8, 10, 12, 17AIFN α, γ, TNF α, TNF α, IL4, IL10, IL6, IFN- gamma,
(Chaturvedi et al., 1999; Diamond et al.,
2000; Arias et al., 2015; Arias et al., 2014)
Plasmodium falciparum, Plasmodium vivax;
Unicellular Parasites
Family: Plasmodiidae
Genus: Plasmodium
Via Culex mosquito vectors
7–30 days (Bartoloni and Zammarchi, 2012)
Intra and extra cellular stages during their
complex life cycle, Hepatocytes, red blood
cells, endothelial cells (Bousema et al., 2008)
lymphotoxin
Single stranded-positive sense RNA virus
Family: Togoviridae
Genus: Alphavirus
Via Aedes mosquito vectors
4–7 days (Lum and Ng, 2015)
Obligate intracellular
Cells: endothelial, epithelial and fibroblastic primary
cells (Wikan et al., 2012), muscle satellite cells (Ozden
et al., 2007), dermal macrophages (Sourisseau et al.,
2007) and PBMC (Sourisseau et al., 2007)
Organs, muscle joints, connective tissue, spleen, lymph
node, brain, and skin (Sourisseau et al., 2007)
TNF α, IFN γ, IFN α, IL2, 6,7,15,17,18 (Teng et al.,
2015)

209
Rash + + ± ±
Jaundice + ± +
Leukocytosis + ± – ±
Leukopenia ± + ± ±
Hyperbilirubinemia + ± + +
BUN (Blood Urea Nitrogen) elevated + ± – –
Tourniquet test positive + + – –
Hepato-splenomegaly ± ± + +
Common treatment for all the microbial infections under discussion are supportive or symptomatic treatment like fluid replacement and adequate rest
Specific treatment 1. Antibiotics: oral or intravenous for mild No specific antiviral treatment (Chawla et al., Antimalarial drugs (e.g. chloroquine and No specific antiviral treatment.
and severe cases respectively (Brett-Major 2014, 2009). artemisinin). However, many drug-resistant
and Coldren, 2012). Plasmodium strains currently reported.
2. Whole blood or platelet transfusion for Patients are advised to get isolated with Patients are advised to get isolated with Patients are advised to get isolated with treated net to
Weil’s syndrome (Wiwanitkit, 2006). treated net to prevent viral spread especially treated net to prevent of spread the protozoa prevent viral spread especially during febrile illness
during febrile illness especially during febrile illness (Biamonte
et al., 2013)
3. Usefulness of steroid to prevent Treatment with corticosteroids is under Corticosteroids contraindicated (Bettadapura et al.,
pulmonary and ocular damages reported controversy (Rajapakse et al., 2014; Chawla 2013)
(Trivedi et al., 2001; Martins et al., 1998) et al., 2014)
Immune evasion 1. Resists complement mediated killing 1. Antibody dependent enhancement (ADE) 1. Antigen diversity/polymorphism, 1. ADE (Stapleford et al., 2014),
(Fraga et al., 2014) (Halstead 2007),
2. Reduced expression of antigenic proteins 2. Inadequate immune response “Original 2. Antigen variation (Scherf et al., 2008) 2. Utilizing apoptotic machinery (Krejbich-Trotot et al.,
(Monahan et al., 2008) antigenic sin” (Mongkolsapaya et al., 2003) 2011),
3. Higher expression of O antigen (Nally 3. Apoptotic mimicry (Amara and Mercer 3. Total immune suppression (Craig and 3. Evade antibody dependent neutralization (Lee et al.,
et al., 2005) Biofilm formation (Ristow 2015) Scherf 2001) 2011)
et al., 2008)
4. Inhibiting IFN signal (Green et al., 2014) 4. Resists complement mediated killing
(Schmidt et al., 2015)
Alters host lipid synthesis (Heaton and
Randall 2010)
Acta Tropica 176 (2017) 206–223
S.P. Priya et al.
Complement system as one of the controlling factor of diseases se-
verity (Yamanaka et al., 2013) as well as contributing factor to en-
dothelial damage in DENV infections which were discussed by several
researchers (Martina et al., 2009; Dalrymple and Mackow, 2012). An-
tibodies of the NS1 protein of DENV triggers several complement pro-
teins and contribute to endothelial damage and vascular leakage
leading to hemorrhage (Avirutnan et al., 2006).
CHIKV infection is less reported with the role of complement (Reddy
et al., 2017). Both the classical and the alternative pathways of the
complement system are strongly activated in malaria (Wenisch et al.,
1997) and causing the depletion of complement proteins which chal-
lenges the host's innate defense (Korir et al., 2014). Malarial parasites,
when exposed to human serum actively recruit FH and its alternative
spliced form FH-like protein 1 to its surface to protect from complement
activation and uses the same during sexual forms to evade complement-
mediated killing in the mosquito host (Schmidt et al., 2015).
The complements’ pathogenic roles in a variety of autoimmune,
ischemic and inflammatory diseases have been documented (Thurman
and Holers, 2006; Holers, 2014). The post infection follow-up records
monitoring the complements’ level and the associated cytokines and the
symptoms of documented complications have never been recounted for
all the mentioned hemorrhagic fevers. Systematic studies to be initiated
to record proper follow-up of the infected patients to analyze the im-
muno-pathogenesis for prevention.
3.2. Toll like receptors
TLR is about 10 in humans (Pandey and Agrawal, 2006) distributed
abundantly on epithelial cells, macrophages, and neutrophils and re-
cognizes non-self-structures including all microbes and synthetic in-
vaders (Kawai and Akira, 2005). They activate nuclear factor (NF)-κB,
AP (Activator protein)-1 and IFN (interferon) regulatory factor for the
effective immune response (Kawai and Akira, 2006) by inducing cyto-
kines and chemokines and also aiding DCs. By upregulating the costi-
mulatory molecules of DC they initiate the adaptive response which is
the top level of the immune defense. Cell surfaces express TLRs 1, 2, 4,
5, and 6 and the TLRs 3, 7, 8, and 9 are present within the cell inside
endosomes (Pandey and Agrawal, 2006). TLR10, a modulatory PRR
with inhibitory properties (Oosting et al., 2014).
TLR 2 and TLR 4 mediated responses can clear the pathogen ef-
fectively through B cells (Chassin et al., 2009), but the leptospiral LPS is
less endotoxic because of the different lipid A moiety than other gram
negative bacteria and stimulate TLR2 instead of TLR4, which weakens
the clearance. Since TLR4-MD2 pathway is not activated in humans, the
Leptospira is located in the cytosol of macrophages, but in murine
macrophages where the pathway is activated, the organism is located
inside the phagosome, which explains the question of human suscept-
ibility (Nahori et al., 2005).
TLR 3 is mainly linked in controlling the cytopathic effect induced
by DENV infections by mediating IFN α/β release to inhibit viral re-
plication (Tsai et al., 2009). Also in CHIKV TLR 3 regulates host im-
munity in human and mice by mediating production of type I IFN (Her
et al., 2015). TLR 2 and TLR6 were also found to be associated with
DENV infection and play a potential role in the immunopathogenesis
(Chen et al., 2015). Malaria parasites are recognized by TLR4, a het-
erodimer of TLR2/TLR1 or TLR2/TLR6 and TLR 9 (Coban et al., 2007;
Gun et al., 2014).
Ability of the microbes to evade the effects of immune system by
modulating the TLR system (Yokota, Okabayashi and Fujii 2010) and
the TLR polymorphism leading to increased individual susceptibility to
infections have been reported and asserts the importance of alternative
approach in treatment modalities (Mockenhaupt et al., 2006; Misch and
Hawn, 2008; Yokota et al., 2010; Pulendran, 2009; Ferwerda et al.,
2008). Moreover, the vaccines arbitrate their efficacy by activating
specific innate immune receptors (Platt and Wetzler, 2013), especially
the TLR, which alarms the importance of validating the immune
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Acta Tropica 176 (2017) 206–223
responses to vaccines. TLR based molecular level studies need to be
simulated in larger units among the different ethnic group to validate
the findings with independent approaches to aim for novel therapeutic
intervention strategies (Buonaguro and Pulendran, 2011).
3.3. Cytokines
Cytokines are potent chemicals in very minute quantity, fastening
changes at the molecular level in many target cells and influencing all
systems of the body. Immune responses and its related cytokine storm
as chemical war has been immensely discussed by research works with
a special focus on leptospirosis and dengue (Rizvi et al., 2011;
Alvarado-Esquivel et al., 2016; Rathakrishnan et al., 2012) as well as in
CHIKV (Lum and Ng, 2015; Chaaitanya et al., 2011) and malaria
(Angulo and Fresno, 2002), yet the clear pathway of Leptospira patho-
genesis never been unanimously concluded. The cytokines involved in
Leptospira, DENV, CHIKV and malaria pathogenesis are summarized in
Table 1. The individual and synergistic effect of cytokines on the vas-
cular endothelial cells increases capillary permeability resulted in
leakage, endothelial dysfunction and hemorrhagic episodes (Burke-
Gaffney and Keenan, 1993; Mathew and Rothman, 2008). Many con-
flicting results have been blamed for the lack of standardization in
many aspects like less number of samples, lack of independent variables
like diseases with mild and severe (Reis et al., 2013), a large panel of
cytokines released during the different period of illness confusing due to
associated diseases. Pro-inflammatory cytokines IL1, IL2, IL6, and TNFα
and the anti-inflammatory cytokines IL10 and IL4 were associated with
all these infections of discussion (Reis et al., 2013; Angulo and Fresno,
2002; Rathakrishnan et al., 2012; Chaaitanya et al., 2011), but the
majority of the reports have agreed with the association of IL10 in-
crease and severity of disease (Reis et al., 2013; Venugopalan et al.,
2014; Zhang et al., 2012; Ferreira et al., 2015; Perez et al., 2004).
Systematic retrospective studies and prospective animal studies
about the common cytokines of endemic diseases at different stages
might bring a simple common solution by intervening cytokine actions
to control the disease process as well for early prediction of damages.
The high serum level of IL6 noticed during post infection period and in
infected joints is correlated with persistent arthralgia (Noret et al.,
2012; Hoarau et al., 2010). Interestingly, the IL6 has the ability to drive
osteoclastogenesis (Hofbauer et al., 1999) and responsible for rheu-
matic symptoms, which are prevented in IL6 neutralized mice (Chen
et al., 2014). This raises the possibility of the modulating the post in-
fection complications by directing inhibitory action on uncontrolled or
over expressed cytokines. More cautious to mention is that there are
almost nil reports about the recovered patients’ follow-up investigations
like blood profiles of mentioned diseases to find the subclinical da-
mages during post infection period similar to arthralgia of CHIKV.
The interferon (IFN) are antiviral cytokines include IFN-I, IFN-II,
and IFN-III classes. The IFN-I class involves IFN-α and IFN-β, the IFN-II
includes IFN-γ and the IFN-III includes IFNλ1, IFNλ2, IFNλ3 and IFNλ4
(de Weerd and Nguyen, 2012; Palma-Ocampo et al., 2015). IFN α and β
are secreted by epithelial cells and fibroblasts, both upregulate the MHC
I receptors and inhibits the viral proliferation. Lymphocytes also secrete
IFN α. NK cells secrete IFN γ which is another potent antiviral protein
upregulates MHC I and II. Since the intracellular phase is very transient,
IFN response is very mild in Leptospirosis. It is believed with current
reports that the DENV might have evolved to reduce the IFN action on
them with the help of nonstructural proteins NS2A and NS4 B by
blocking the IFN receptor- Janus kinase/signal transducer pathway and
activation of transcription signaling pathway in the infected cell (Gun
et al., 2014; Dalrymple et al., 2015; Hsu et al., 2016). IFN-α, IFN-β, and
IFN-γ modulate the course of malarial progression and are associated
with the severity of the infection (Gun et al., 2014).
The pre-existing and/or co-existing systemic conditions including
co-infections do alter the cytokines level. We hypothesize that while the
skin or mucosa at the site of entry of a microbe in in need of pro-
S.P. Priya et al.
inflammatory cytokines, any pre-existing pathological conditions and
co-infections might have already loaded the anti-inflammatory cyto-
kines in blood and the protein inventory, liver might be in stress of
delivering proteins to build cytokines for diseases progression at dif-
ferent phases. Unfortunately, due to the dual circulation in an organ
like liver, cytokines with synergetic and antagonistic actions are loaded
(cytokine storm) and shatter their function. Since the liver is the
common organ of damage in all these diseases, the hypothesis regarding
cytokine storm needs to be explored. The usefulness of cytokine ad-
juvant and anti-cytokine therapies and techniques like cytokine dialysis
in different infections in different stages of the disease can be studied
systematically (Uchino et al., 2002; Rathakrishnan et al., 2012).
3.4. Innate defence cells
Transient or short living first defence phagocytes include the most
common PMN along with basophils, eosinophils and mast cells. The
long living patrolling phagocytes or scavenger cells are the macro-
phages. These cells are attracted to the local area within no time by
chemicals released by the pathogens, activated macrophages, and
complements. PMN attack the pathogenic and non-pathogenic strains of
Leptospira, but the pathogenic strains resist by mechanisms including
catalase expression (Murgia et al., 2002). Neutrophilic extra cellular
trap mechanism is mentioned for the prevention of initial dissemination
(Scharrig et al., 2015). Direct involvement of phagocytes is less re-
ported with DENV infection, but they assist the activation of the com-
plements, coagulation, and fibrinolytic mechanisms and that might lead
to pathogenesis like endothelial damage (Butthep et al., 1993). Acti-
vated PMNs are blamed for the pathogenesis of severe malaria in-
cluding cerebral malaria (Pukrittayakamee et al., 1992; Feintuch et al.,
2016).
The surface barriers efficiently prepared not only for local defence
but to initiate the adaptive immune response with the help of secret
squads, the Langerhans cells (dendritic cell) present in the basal layer of
epithelium, which take the antigens from the invaders including
Leptospira and migrates from epithelium towards regional lymph node
to present the antigen to T-helper cells to proceed with antibody for-
mation by B-cells or to stimulate memory cell, if infection is secondary
(Liu, 2001; Collin et al., 2013). These deeds take almost two to four
days to receive a response from the lymphocytes or more specifically
the action of acquired immunity. Therefore, at this stage, the demon-
stration of Leptospira from the patient either by PCR, Immuno-histo-
chemical and simple microscopic technique including blood and urine
smears is the best way of confirming the diagnosis. Furthermore, DCs
are regulators of adaptive immunity in lymphoid and non-lymphoid
tissues. They do activate the Natural Killer (NK) cells and other resident
immune and non-immune cells for the immediate control of the in-
vaders by their cytokine release (Cooper et al., 2004; Nakayama et al.,
2011; Albert, 2008). They deliver strong signals to arouse the cells of
innate and acquired immunity.
Zuerner et al. (2011) suggested that the quick response from NK
cells to act against Leptospira is important to develop immunity
(Zuerner et al., 2011; Sun et al., 2009). NK cells of the human are a
special type of bone-marrow derived lymphocytes working in innate
immunity with ability to kill virally infected and tumor cells with no
prior sensitization and also without MHC 1 expression (O’Leary et al.,
2006; Vivier et al., 2011). They kill them by secretion of cytotoxic
chemokines and inducing lysis of the cell by antibody dependent cell
mediated cytotoxicity (Robertson, 2002). DCs release chemokines to
recruit NK cells by extravasation of NK (Della Chiesa et al., 2005).
Immunological memory demonstrated in NK cells opens the way for
programming them with the vaccine. The DENV evade their action by
upregulating the MHC I receptors and reduces the susceptibility of the
NK cell induced lysis because the KIR is stimulated which prevents
cytotoxic effect (Yossef et al., 2012). Strong innate immune response
with early clearance in children is reported (Simarmata et al., 2016)
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and also the importance of NK cells in for better cytokine signals for
viral clearance (Chaix et al., 2008). The efficient innate immune re-
sponse against malarial protection and modulating adaptive immune
responses need to be utilized by careful intervention during treatment
(Stevenson and Riley, 2004). NK cells have a prominent role in CHIKV
infections (Teo et al., 2015). Early clearance and less post infection
complaints of arthralgia have ameliorating role of NK cells in children
(Simarmata et al., 2016).
Innate immunity elicited on the recognition of pathogen specific
surface molecules. Leptospira have express proteins/biomolecules on the
outer membrane for adhesion with multifarious structures of the con-
nective tissue (Fig. 2). Because the connective tissue is widely dis-
tributed through the entire body as a binding structure, bacterial
binding ability improves host tissue occupancy. Approximately, 260
membrane-associated proteins have been identified through genomic
studies (Nascimento et al., 2004). Only a few of these proteins have
been well-studied with respect to adhesion, transmission, and viru-
lence. These proteins include: OMPL (outer membrane protein Leptos-
pira proteins) 1 (Oliveira et al., 2013), 36 (Pinne et al., 2010), 37 (Pinne
et al., 2010), 47 (Pinne et al., 2010), 67 and 54; Lig (Leptospira im-
munoglobulin-like proteins) A (Choy et al., 2007; Lin et al., 2009), B
(Lin et al., 2009) and C; Len (Leptospira endostatin-like proteins)
A,B,C,D,E and F (Stevenson et al., 2007); Lsa (Leptospira surface adhe-
sion proteins) 20 (Mendes et al., 2011), 21 (Atzingen et al., 2012), 23
(Siqueira et al., 2013), 25 (Domingos et al., 2012), 26 (Siqueira et al.,
2013), 27 (Longhi et al., 2009), 30 (Oliveira et al., 2013), 33 (Domingos
et al., 2012), 36 (Siqueira et al., 2013), 63 (Barbosa et al., 2006) and 66
(Atzingen et al., 2012); Loa (Leptospira outer membrane protein A-like
protein) 22 (Koizumi and Watanabe, 2003); MFn (Pinne et al., 2012)
(Microarray/membrane Fibronectin-binding proteins) 1,2,6,7 and 9;
and LipL (OM Lipoproteins) 32 (Murray, 2015), 41 (Lin et al., 2013), 46
(Matsunaga et al., 2006), 53 (Oliveira et al., 2010) (Fig. 3). The range of
bacterial adhesive proteins attaching with human tissues is described in
Fig. 3. Among these proteins, only Loa22 fulfills Koch’s postulates
(Ristow et al., 2007; Zhang et al., 2010). These proteins exhibit dif-
ferent binding affinities with connective tissue molecules, namely la-
minin, fibronectin, collagen, tropoelastin, elastin, heparin and other
blood-related molecules, such as plasmin, plasminogen, Factor H and
complement (Vieira et al., 2014). Previous studies have identified
multiple adhesion molecules (Barbosa et al., 2006) in Leptospira species,
which exhibit good adhesion with cells and the extracellular matrix
(ECM), as shown in Figs. 2 & 3. Investigations about the cellular and
extracellular binding proteins and its genetic study might help to design
acellular vaccine (contains only fragments of the cell) to protect and
treat leptospirosis (Vieira et al., 2014; Oliveira et al., 2015).
The ability to invade and survive intracellularly in phagocytic and
non-phagocytic cells with the help of receptor mediated endocytosis has
been correlated with its virulence, but since it is rarely demonstrated
with the characteristics of intracellular pathogens such as cell to cell
spread and intracellular multiplication, the suggestion of transcytosis
(Barocchi et al., 2002) is widely supported and the Leptospira is reported
as extracellular organism.
Unlike leptospiral infections, viral infections strictly survive in-
tracellularly, but they do elicit an innate response because of the lib-
eration of their specific proteins. Although DENV produces the same
clinical manifestations as leptospirosis, the entry of this virus is through
the bite of infected Aedes mosquitoes (Benelli and Mehlhorn, 2016).
After the bite from an infected mosquito, DENV is initially internalized
inside human cells through receptor mechanisms, including clathrin-
mediated endocytosis (Ang et al., 2010) or clathrin-independent en-
docytic pathways (Acosta et al., 2009), and then the single-stranded
positive sense RNA viral genome is introduced into the host cell ma-
chinery to produce viral proteins (Seema and Jain, 2005). The specific
surface proteins for the DENV have not yet been identified, but once
these viruses enter humans, many molecules are employed to gain ac-
cess to different types of tissues (Cruz-Oliveira et al., 2015). These
S.P. Priya et al.
Fig. 2. This schematic figure shows Leptospira having the double plasma membrane, typical of gram negative bacteria, with the endoflagellum between them; the endo-flagella helps their
spiral structure and movement. The array of surface binding proteins has been symbolized.
viruses have three structural proteins, core (C), envelope (E) and
membrane (M) proteins, and seven non-structural proteins (NS 1, 2a,
2b, 3, 4a, 4b, 5) (Rothman 2004; Modis et al., 2004; Kapoor et al., 1995;
Innis et al., 1989). Host proteins play an important role during viral
entry and subsequent virion and envelope assembly. The main attach-
ment factor depends on glycosaminoglycan (Chen et al., 1997), den-
dritic cell adhesion molecules (Tassaneetrithep et al., 2003), macro-
phage mannose receptors (Miller et al., 2008), lipopolysaccharide
receptor CD14 (Chen et al., 1999) and stress-induced proteins, such as
heat shock proteins 90 and 70 (Reyes-Del Valle et al., 2005; Navarro-
Sanchez et al., 2003). DENV enters a cell via direct membrane trans-
location, mediated through C protein (Freire et al., 2013) or receptor-
mediated endocytosis (Acosta et al., 2009). DENV targets monocytes,
macrophages, and dendritic cells (Wu et al., 2000). These cells have a
mannose-specific C-type lectin receptor DC-SIGN (dendritic cell specific
ICAM3-grabbing non-integrin receptor), which mediates the viral entry
into the cell (Navarro-Sanchez et al., 2003), and subsequently, these
cells transfer the virus to T-cells in regional lymph nodes. Although T
cell transferring the invaders to lymph node is part of immune clear-
ance mechanism for adaptive or cell mediated immunity, these cells
unfortunately become accidental culprits for spreading and damaging
the tissues because the intracellular virus evade the immune system,
resulting in widespread tissue damage, which manifests as hemorrhagic
disease (Courageot et al., 2003; Marianneau et al., 1998; An et al.,
2004). Since viruses are in need of intracellular environments for
multiplication, the extracellular matrix attachment of these particles is
not well-studied, unlike leptospira.
Chikungunya (CHIKV) is an enveloped, single-stranded, positive
sense RNA virus, also transmitted to humans by the bite of Aedes
mosquitoes, similar to DENV and Zika virus. CHIKV comprises four non-
structural proteins (nsP1, nsP2, nsP3, and nsP4) and five structural
proteins (C–E3–E2–6K–E1). E1 and E2 are associated with viral at-
tachment and entry into the specific cell of interest (Schwartz and
Albert, 2010; Voss et al., 2010). Following the bite of an infected Aedes
vector, CHIKV locally infects epidermal epithelial and dendritic cells for
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initial replication and entry into lymph nodes or direct entry into blood
circulation to reach the target tissues or cells. The N-linked glycosylated
transmembrane glycoprotein on CHIKV interacts with C-type lectin
receptors in mammalian-like dendritic cell immune receptors (DCIR)
expressed on monocytes, macrophages, dendritic cells, B-cells and
neutrophils (Bates et al., 1999). Target cell entry occurs via clathrin-
independent, Eps15-dependent endocytosis (Bernard et al., 2010). Even
though the classical clinical manifestations of CHIKV is not typical with
other hemorrhagic fevers, the initial symptoms and considerable
emerging reports about the co-infections with leptospirosis applies to
include in this discussion.
Plasmodium falciparum is the most common malaria parasite among
the five human-infecting species identified and is responsible for
600,000 deaths annually (Nilsson et al., 2015). The malarial parasite
enters humans through mosquito bites, and the surface proteins of these
parasites, including the P. falciparum erythrocyte binding protein
(EBV)-175, interact with the surface membrane protein (glycophorin A)
of human erythrocytes for entry. Furthermore, these parasites induce
the infected erythrocytes to express receptors for adherence with en-
dothelial cells, progressing to the erythrocytic phase of the parasite life-
cycle (White et al., 2013). This phase clinically manifests as hemor-
rhage, reflecting the sequestration of infected erythrocytes within blood
vessels (White et al., 2013) and endothelial damage (Brown et al.,
2013).
Leptospira are typically extracellular organisms moving along in-
tercellular junctions (Haake and Levett, 2015), although intracellular
survival has been reported (Merien et al., 1997; Thomas and Higbie,
1990). The motility (Fontana et al., 2016) of the corkscrew shaped
organism is considered as one of the virulent factors at this level, which
enhances their locomotion along the cleaved planes created with their
ability to secrete collagenase (Janwitthayanan et al., 2013). Leptos-
piremia (Leptospira in the blood) reflects the ability of these organisms
to swim effectively along tissues and reach blood vessels by evading the
immune system to survive extra- and intravascular sites within minutes
after surface entry. The entry is achieved through associated surface
S.P. Priya et al.
Fig. 3. Leptospira binding proteins with extracellular matrix molecules. The extensive array of binding proteins helps them early spread and extensive involvement of many organs. The
proteins with the broad spectrum of binding ability with many types of tissue are marked in the center of the figure (brown box). The groups of proteins having the affinity for particular
host proteins are marked around.
proteins, particularly on the outer membrane, which improves contact
with host tissue and promotes the mobility of these organisms (Adler
and de la Pena Moctezuma, 2010). The ability of the pathogen to
trespass the surface level of defense within minutes without eliciting
any local immune reaction unlike other pathogenic spirochetes is less
interrogated. The receptors expressed to bind the host plasminogen can
lead to activation of proteases as well as the degradation of ECM and
intercellular junctions. This can be connected to the leptospiral mobility
even after binding with ECM, the transcytosis and transmigration along
the intercellular junctional cleavage, especially in epithelial and en-
dothelial tissues (Barocchi et al., 2002; Vieira et al., 2013; Vieira et al.,
2012; Vieira et al., 2014). Also, the ability to bind with cadherins (i.e.
adhesive proteins made up of the extra cellular domain and a cyto-
plasmic tail to link with the intracellular cytoskeleton, belong to the
family of calcium-dependent transmembrane adhesion proteins)
(Evangelista et al., 2014a) can mutually aid them to reach the blood
circulation within few minutes.
3.5. Acquired immunity
As explained under innate immunity section, the surface barriers
initiate the adaptive immunity at the entry level of pathogens. The
evidence of acquired immunity in leptospirosis was demonstrated with
the antibodies to LPS in animal serum since it was thought that the
humoral immunity alone work against leptospirosis (Adler, 2015). The
leptospiral virulence factors responsible for disease severity and anti-
body formation are associated with lipopolysaccharide (Patra et al.,
2015), haem oxygenase (Murray et al., 2009), flagellar protein
(Fontana et al., 2016) and outer membrane/surface proteins (Vieira
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Acta Tropica 176 (2017) 206–223
et al., 2014; Viratyosin et al., 2008). Once the Leptospira species enters
and creates adhesions, locally available neutrophils are initiated with
phagocytosis and the complement system enhances the action of these
cells through opsonization (Fraga et al., 2011) (Fig. 4).
Leptospira can be effectively controlled by specific antibody medi-
ated immunity. The invading leptospiral antigens are collected and
processed by DCs and presented to helper T cells to process and present
it to B-cells, which then matures into plasma cells for specific anti-
bodies. The antibodies are specific to the antigen expressed by the
serovar and so antigenic shift in the same serovar or different serovar
cannot be embattled. The complication in vaccine preparation and
passive immunity has never prevailed. Diagnostic techniques used IgG
and IgM antibodies for the highly accepted MAT test. The intracellular
phase should initiate the T cell response but it depends on the TLR2,
TLR4 and IFN γ production, which is an acquired immune response or
cell-mediated immune war start with the formation of antibodies
against the Leptospira, which is poorly, reported both in animal and
human studies. The γδ T cells may play an important role in immunity
against bacterial, viral, and parasitic infections including Leptospira
(Truccolo et al., 2001; Johnson et al., 2005). The role of acquired im-
munity on Leptospira in human is less explained. The γδ T cells are
distributed near the epithelial structures and guard against the non-self-
invaders and recognizes distressed epithelial cells without the need of
antigen processing (by DC) and MHC (Major Histocompatibility Com-
plex) intervention, an empathetic prompt play which is not accom-
plished by αβ T cells and NK cells (Hsu et al., 2015; Pang et al., 2012)
Absence of similar action in human adds the answer to conundrum on
human susceptibility. The known combinatorial function of γδ T cells
among immune repertoire strengthens the need to study their effect on
S.P. Priya et al.
Leptospira and to discuss the aspects like inducing immunotherapy
(Yuri, 1993).
Antibody mediated defense is extensively studied in DENV, espe-
cially their transient autoimmunity contributing the disease severity.
Anti NSI antibodies cross react with host hepatocytes, endothelial cells
and platelets aggravating the disease by cellular functional alteration,
cellular damage and triggering apoptosis eventually resulting in in-
creased EC damage, platelet depletion and cytokine storm preventing
clotting and repair (Avirutnan et al., 2007). There are several hy-
potheses explained including antigen-antibody-complement-mediated
vascular permeability, antibody-dependent enhancement, mimetic an-
tibodies, cross-reactive T-cell response, original antigenic sin, directly
infecting cells responsible for vascular leakage myeloid cells and en-
dothelial cells (Halstead, 2015). Strikingly, the NS1 interaction with
TLR4 similar to LPS of gram negative bacteria has been observed on the
surface of endothelial cells, macrophages and monocytes and their as-
sociation with the release of cytokines (Modhiran et al., 2015). TLR4
antagonist LPS- Rhodobactersphaeroides, inhibits this interaction and
prevents the cytokine related damages opens new therapeutic modality
to control the similar interactions (Modhiran et al., 2015). Since the
viral load in blood is very low during the manifestation of DHF, the
viral protein toxicity is the acceptable concept needs to be emphasized
during prevention and treatment of DSS/DHF. The γδ T cells do con-
tribute to the immune protection against DENV infection by providing
an initial source of IFN-γ for antiviral effect and by killing DENV-in-
fected cells (Tsai et al., 2015).
The role of regulatory T cells (Tregs) and the B cell on persistence
arthralgia and their mechanism of action in pathogenesis are under
discussion (Lum et al., 2013; Lee et al., 2015; Teo et al., 2013) for
prevention. The role γδ T cells on regulating inflammatory responses of
CHIKV during the acute stages especially on joint tissues (Long et al.,
2015) is narrated.
Both the cellular and humoral supports of the adaptive immune
system are vital for malarial elimination, but the memory is transient
(Rowe et al., 2009). The γδ T cells play an essential role from the be-
ginning of the infection (Inoue et al., 2013). Evidence on naturally
acquired immunity for surface proteins supported the use of P. falci-
parum erythrocyte membrane protein 1 (PfEMP1), a variant surface
antigens of the parasite suggested for the vaccine development (Chan
et al., 2012). Autoantibodies induced by the malarial parasite reacting
on host cytoskeletal network have been discussed (Mattei et al., 1989),
but yet to be investigated since an extensive sequence homology be-
tween human and the parasite is reported (Faya et al., 2015).
4. Nutritional acquisition, organ invasion and immune evasion for
persistence
Initial leptospiremia manifests within minutes of infection, with
organism counts of 106 to 107 per ml of blood (Truccolo et al., 2001)
and the presence of microbe persists up to 2 weeks. DENV reaches its
maximum load in blood by 103 RNA particles/ml in primary DENV
infections and > 106 RNA particles/ml in secondary DENV infections
from 5 to 8 days (Johnson, Russell and Lanciotti 2005), and malarial
parasite level in the blood reaches up to 2500 parasites per 200 white
cell (Goncalves et al., 2014).
The necessity for Leptospira to swim rapidly towards the blood vessel
is their craving for iron, B vitamins, ammonium, fatty acids and β
oxidation for energy. The nutrition acquisition from RBCs urges them to
reach the nearby blood vessels from the site of entry. The RBCs are the
best source even though many cells of the host can satisfy all but the
rich iron. The RBCs are the chemoattractant for the Leptospira (Yuri
et al., 1993) and to reach them the layers of blood vessels need to be
penetrated by binding with various surface proteins of the host. Pa-
thogenic Leptospira identified with 12 of the TonB- dependent receptors
important for active nutrient import, which is more in number than the
saprophytic ones (Nascimento et al., 2004; Narayanavari et al., 2012b).
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Acta Tropica 176 (2017) 206–223
The predicted sphingomyelinases (Narayanavari et al., 2012a) and
phospholipases have crucial roles not limited to cracking the host cells
for nutrition but facilitates escape from phagosomes, aids for coloni-
zation, immune evasion and persistence (Flores-Diaz et al., 2016). Re-
searches to nullify this tissue drilling bacterial enzymes might lead to
new prospective in primary prevention. Initial leptospiremia might be
effectively removed through the defense action of immune cells, but the
ability of the Leptospira to evade the immune system prevails the per-
sistence of these organisms and subsequent tissue damage in selective
organs with different mechanisms adapt by these organisms in different
tissues (Haake and Levett, 2015).
Leptospiral organisms have been identified in multiple organs by
three days post-infection (Segura et al., 2005) and immune reaction-
provoked inflammatory cytokines are the major culprits underlying the
pathogenesis of all organ damage (Vernel-Pauillac and Merien, 2006)
(Fig. 4). Liver autopsy specimens of leptospirosis demonstrated a large
number of Leptospira in hepatocytes with disturbed intercellular junc-
tions, congested sinusoids and hepatocyte apoptosis (Merien et al.,
1997). The intercellular junctional disturbance between hepatocytes,
hepatocyte damage and elevated bilirubin results in jaundice. The
elevated bilirubin proposed with leptospirosis induces hemolysis
(Avdeeva et al., 2002). The kidney is the target tissue for the persistence
of the infection. The systemic load of an organism can effectively be
eliminated through the immune system, but the immune-privileged
kidney tissue is utilized by Leptospira for survival, multiplication, and
shedding. These organisms escape through the surface of the epithelial
cells of proximal convoluted tubules and colonize the brush border
area, shedding as leptospiruria. Transcytosis, enhanced through a pro-
teolytic mechanism, has been suggested as a mechanism for crossing the
epithelial cell membrane (Vieira et al., 2014; Kassegne et al., 2014).
Transcytosis has been observed in hamsters and sheep, showing that the
Leptospira living in the proximal tubule with close association with the
microvilli of the epithelial surface does not exhibit any cellular pa-
thology and might present a commensal relationship (Haake and Levett,
2015). Leptospira survives in the renal tissues by presenting various
mechanisms, such as biofilm formation (Ristow et al., 2008), reduced
antigenic protein expression (Monahan et al., 2008) and increased LPS
O antigen (O antigen is a polysaccharide, hydrophilic in nature and an
immunodominant domain present in the outermost end of LPS), asso-
ciated with complement resistance (Clay et al., 2008). The pathogenesis
of severe pulmonary hemorrhage syndrome (SPHS) is associated with
vascular damage, the immune complex deposition on the alveolar
membrane and coagulation abnormalities (Silva et al., 2002; Wagenaar
et al., 2007). The pulmonary pathology worsens the patient’s condition
because of these added effects, which reduce oxygen delivery to the
tissues. The hemorrhagic episodes deplete the red blood cells needed to
carry sufficient oxygen to the tissues, and this pulmonary insufficiency
aggravates the situation, leading to central nervous system (CNS)
manifestations (neurological symptoms) such as confusion because the
CNS is highly sensitive to hypoxia.
A severe headache with leptospirosis should be considered a neu-
rological symptom, particularly meningitis, which challenges the di-
agnosis because this disease can manifest as septic and aseptic forms
(Silva et al., 2002). In addition, altered mental status and confusion
were reported during severe stages of the disease (Ko et al., 1999).
During the recovery phase, the ocular manifestation with uveitis is
common in either mild or severe form, with self-limiting capabilities.
The ability to demonstrate bacterial DNA in the aqueous humor of the
eye at this stage using PCR has been reported (Chu et al., 1998;
Rathinam, 2005). Leptospira have evolved various mechanisms to evade
immune reactions. Reportedly, the less reactive LPS of Leptospira trig-
gers Toll-like Receptor 2 (TLR2) instead of TLR4, unlike other more
potent LPS (i.e., Escherichia coli), which reduces the effectiveness of
defensive action (Werts et al., 2001; Werts, 2010). Innate immunity can
be effectively initiated against the LPS and peptidoglycan membranes
only when both (TLR4 and TLR2) receptors are stimulated for these
S.P. Priya et al. Acta Tropica 176 (2017) 206–223

Fig. 4. This schematic figure focuses on the pathogenesis of dengue, leptospirosis, and malaria, showing common aspects of invasion and cellular events leading to diagnostic confusion.
Dengue: During primary dengue virus infection, the immune cells release cytokines and other factors which lead to viremia and loosen the endothelial layer. Later, the virus activates
memory T cell, in addition to the viral infecting endothelial cells, which finally lead to endothelial damage and inflammation at surrounding area. Leptospirosis: during Leptospira
infection, the immune cells release pro-inflammatory cytokines and chemokines which contribute to inflammation and damage of the endothelial layer. Malaria: In malaria, Plasmodium
parasites invade the blood cells. Infected red blood cells express the surface marker that binds to the receptors of endothelial cells activating them. Later, cytokines are released and fibrin
is deposited into the blood vessel causing obstruction, plasma leakage, and inflammation.

Gram-positive and Gram-negative components (Chassin et al., 2009).
Leptospira species bind and evade or down regulate the action of com-
plement regulatory proteins Factor H and C4b binding Protein (C4BP)
(Meri et al., 2005), thereby preventing the opsonization and formation
of the membrane attack complex on the bacterial surface and increasing
the persistence of these microbes. The less virulent LPS and evasion of
the complement system promote infection by rejecting the effective
elimination of these components (Chassin et al., 2009).
The success of any viral infection is accomplished by the capability
of entering the host cell and taking over cellular functions to direct
them toward the effectual production of new virus (Bahir et al., 2009).
A proteome based analysis on viral adaptation reported that the viral
structural proteins exhibit similarity to the host-preferred codon (Lobo
et al., 2009). The probable explanations may be related to the devel-
opment of humans and the co-evolution of the viruses or may be due to
the hypothesis that major portions of the human genome are actually of
viral origin. DENV has tropism for the immune system, liver and en-
dothelial cell (EC) linings of blood vessels. A review based on autopsy
specimens of 160 cases revealed demonstration of DENV in liver,
spleen, lymph node, lungs, kidney, thymus, and brain (Martina et al.,
2009).
The ability of DENV to attach to an array of molecules has been

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S.P. Priya et al.
associated with the susceptibility of the vectors and vertebrates. The
DENV C-protein has been implicated in the transport of the viral
genome directly from one cell to another without requiring complete
assembly (Zhong et al., 2013; Cruz-Oliveira et al., 2015). Following the
incubation of the virus, the clinical presentation is presented as three
phases: febrile, critical and recovery. The febrile phase is manifested
from mild petechial bleeding in the mucous membrane of the oral and
nasal cavities to massive bleeding in the gastrointestinal tract. Liver
enlargement and blood count abnormalities are subsequently initiated
(Kalayanarooj et al., 1997). The critical phase is manifested with
plasma leakage, reflecting extensive endothelial injury and depleted
blood constituents, leading to hemorrhage and clotting, metabolic
acidosis and disseminated intravascular coagulation, which is pre-
carious (Martinez Torres, 2006). The recovery phase is complicated
when the administration of intravenous fluid as a treatment during the
early phases is overzealous, leading to circulatory overload or hy-
pervolemia (Malavige et al., 2004). The pathogenesis is primarily as-
sociated with the damage and consequences of viral-infected cells and
targeted immune responses. Neurological complications associated
with DENV have been reported in up to 6.2% of cases (Hendarto and
Hadinegoro, 1992) and are categorized into three groups according to
the severity as mild with less specific manifestations, such as headache
and dizziness, moderately severe with seizures, encephalitis and pa-
ralysis, and extremely severe with seizures, memory impairment and
psychiatric symptoms (Garcia-Rivera and Rigau-Perez, 2002).
The Plasmodium life cycle is complex, with different stages in hu-
mans and mosquitoes. These parasites have sexual and asexual forms
requiring both humans and mosquitoes to complete the cycle. The
asexual stage in humans is highly pathogenic compared with sexual
gamete formation, which is primarily important for the mosquito in-
fected by humans. The initial step of gametogenesis is completed in
humans and transmitted to mosquitoes during blood meals, and the
sexual cycle is completed with zygote formation and sporozoites, which
infect the next human through a bite. The complicated developmental
processes and non-correlation of the gametocyte density with para-
sitemia end with poorly explained parasite load and infectiveness
(Orjih, 2014), thereby complicating intervention through elimination
based on the infectiveness detected, even after several advanced studies
have been conducted. Therefore, this disease is still confused with many
other diseases widely spread and tropical and subtropical endemic
areas (Nilsson et al., 2015).
Viral infections are managed differently by the body system com-
pared with bacterial infections, once these particles are internalized
inside the target cells. In vertebrate hosts, CHIKV virion are produced
through replication from the infected cells within 8 h post-infection
(Sourisseau et al., 2007). The target tissues, such as the joints, muscles,
liver, spleen, bone, and brain, and related clinical manifestations are
widespread. CHIKV-infected osteoblasts stimulate interleukin 6 (IL6)
and receptor activator of the nuclear factor kappa-B ligand (RANKL),-
thereby promoting further osteoclast activity, resulting in rheumatic
symptoms (Noret et al., 2012). Monocyte chemotactic protein (MCP)-1,
−2, and −3 are chemo-attractants for the signaling of monocytes/
macrophages to the target area, and the expression of these molecules
increases during CHIKV infection. The virus uses these recruited cells as
transport vehicles and reservoirs (Labadie et al., 2010). The relative
increase in dendritic cells (Colonna et al., 2004), CD4+ T cells (Hoarau
et al., 2010) and B cells (Poo et al., 2014), has also been reported, and
the pathogenesis, particularly associated with the joints, has been dis-
cussed. The type of cells and the increase in associated inflammatory
cytokines has been associated with the pathogenesis.
Notably, as outlined above, most hemorrhagic fevers of different
pathogens share common mechanisms, particularly endothelial da-
mage, immune responses and blood cell loss resulting from hemorrhage
and other related events. However, the treatment is not always the
same. The confusion in diagnosis is more complicated, particularly
when the endemic area has an outbreak of the same types of infections,
216
Acta Tropica 176 (2017) 206–223
reflecting similar climatic affinities. These infections might simulta-
neously present in different patients or in the same patient exhibiting
coinfection with both diseases. The treatments for viral infections are
primarily associated with symptomatic support and the prevention of
further damage. However, for Leptospira, or any bacterial infection, the
treatment primarily involves antibiotics and symptomatic support. The
precocious administration of antibiotics for all hemorrhagic fevers in
endemic areas with leptospirosis is not advisable because the over-
loading of antibiotics might lead to hepatic insufficiency. The early
diagnosis/separation of specific infections during the acute phase
would promote therapeutic success. Fever is initiated through a body
mechanism that defends against any microbe. The affinity of hemor-
rhagic infections towards the endothelium and the resulting damage
lead to rashes, reflecting capillary damage and bleeding. The liver
controls body metabolism. The cascade of events and reactions during
any infection are continuously increasing microbial load, cell damages
(of both bacterial and host), cell injury-released materials and loaded
immune cell clearance and their secretions, and the host’s reaction to
these burdens the liver. The acute phase proteins are released from the
liver, suggesting that increased production indirectly increases work-
load. The direct pathogenic action and liver burden lead to abdominal
pain. Multiple small hemorrhages result in the depletion of thrombo-
cytes, leading to thrombocytopenia, bleeding disorders, and a reduction
in circulating red blood cells (RBCs), which leads to anemia. The active
immune defense leads to leukocytosis and subsequently results in the
loss of leukocytes, leading to leukopenia. Taken together, these effects
lead to hypoproteinemia and aggravated endothelial damage with
leaking capillaries and the consequent loss of volume inside the blood
vessels. Liver damage leads to protein deficiency and jaundice (with
hyperbilirubinemia), and the spleen is under stress for the removal of
the damaged RBCs, which leads to hepatosplenomegaly. Ineffective or
unnecessary antibiotic load can worsen these conditions in any un-
trained medical setup. Pulmonary damage adds to hypoxic cell injury,
particularly to vulnerable CNS tissues. Thus, these signs and symptoms
are common for all hemorrhagic fever-inducing diseases under discus-
sion, but some symptoms are more common and more prominent for
particular diseases. Leptospirosis and DENV have nearly similar clinical
presentations, but myalgia is more prominent in CHIKV and rigor is
more prominent in malaria.
5. Genetic susceptibility
Genetic influence on long time protection for reinfection and ge-
netic susceptibility related genes likeIL1b, IL12RB1 and CISH have been
reported for Leptospira (Esteves et al., 2014). The association of human
leukocyte-like antigen DQ-6 (HLA-DQ6) (Lingappa et al., 2004) and
alleles from HLA-A (*24 and *31) and HLA-B*08, interleukin 4 (IL-4)
and IL-4Ra genes (Fialho et al., 2009) towards the risk of leptospirosis
has been also reported.
Human HLA class I and II alleles associated with the development of
DHF have been summarized by Matina et al. (Martina et al., 2009).
They reviewed the polymorphism in genes of TNF-α, vitamin D re-
ceptor, TGF β, FcγRIIa receptor, CTLA-4,DC-SIGN,HLA class I alleles
A*01, A*0207, A*24, B*07, B*46, B*51 HLA class II alleles DQ*1,
DR*1, DR*4 having association with development of DHF/DSS
(Martina et al., 2009). Malarial infection susceptibility and resistance
have been highly probed with genome wide linkage (GWL) and asso-
ciation studies (GWAS) and various controlling pathways and genetic
origins of diseases suggested among different populations (Driss et al.,
2011). CHIKV has been also reported with genetic predisposition in a
study among 100 families showing that no Rh negative blood group was
affected (Lokireddy et al., 2009). Recent advances in genetic studies
have urged many publishers to discuss on genetic susceptibility to many
diseases. However, sample size and methodology and their specificity
and sensitivity for wide populations, genetic diversity even among the
small population, co-infections and pre-existing underlying pathologies
S.P. Priya et al.
of relevant pathologies, have not been corroborated.
6. Clinical presentation of leptospirosis and co-infections
The reductions in morbidity and mortality are dependent on the
early diagnosis of single, co- or mixed infections. Previous evidence
suggests that the pathogens that colonize a human body infected with
leptospirosis are human immunodeficiency virus (HIV) (Biggs et al.,
2013; Ganoza et al., 2005) Malaria (Baliga et al., 2011; Gurjar et al.,
2011; Srinivas et al., 2007; Wongsrichanalai et al., 2003), hepatitis A
(Alves et al., 2011), melioidosis (Hin et al., 2012; Sapian et al., 2012),
scrub typhus (Mahajan et al., 2012), Orientia tsutsugamushi
(Sonthayanon et al., 2013b), and DENV (Sharp et al., 2012; Mohammad
et al., 2008). Multiple co-infections with viruses, such as Hantaviruses,
Leptospira spp., and Babesia spp., have also been observed in rodents
(Tadin et al., 2012).
The coinfection of other infectious agents with Leptospira con-
tributes to the confusion in the diagnosis and treatment of this disease
due to many similarities in early signs and symptoms (Table 1). The
virulence properties of different pathogens indirectly alter or increases
host immunity (Behera et al., 2010). During co-infections with Leptos-
pira, significantly increased immune cell proliferation and damages
multiple organs primarily liver, kidneys, lung, heart, and brain (Lokida
et al., 2016; Zaki and Shanbag, 2010; Romero et al., 2010).
Patients with co-infections are highly loaded with different patho-
gens and need to be handled carefully. Early and rapid diagnosis and
proper interventions should be quantified (Yang et al., 2012). The
majority of leptospirosis cases (90%) are noticed during anicteric phase,
which presents as acute influenza or dengue like symptoms, such as
fever, headache and myalgia in the thighs and calf. The important di-
agnostic clues during this acute stage are conjunctival congestion and
sub conjunctival bleeding (Gupta et al., 2007). In the first 5–7 days of
anicteric, the organism is isolated from all tissues where the leptos-
piremic stage occurred. Typically, the patient becomes asymptomatic
after this phase, and the symptoms will reappear with a mild fever and
myalgia after 3 days if it progresses to icteric phase, which can be fatal
(Wysocki et al., 2014). The effects of hepatic involvement include in-
trahepatic cholestasis, centrilobular necrosis and Kupffer cell hyper-
plasia, lung tissue manifest with pulmonary hemorrhage and acute re-
spiratory distress syndrome (ARDS) and cardiac involvement result as
myocarditis (Sondergaard et al., 2016; Wysocki et al., 2014;
Pushpakumara et al., 2015). In most patients, the immune phase is not
manifested, while for other patients, a few neurological symptoms, such
as meningitis, iridocyclitis, and choroid, manifest (Rathinam, 2005;
Romero et al., 2010; Wang et al., 2016). For approximately 15% of
anicteric forms, the neurological manifestations are described as
symptomatic meningitis, while many patients experience asymptomatic
pleocytosis (abnormal, large number of lymphocytes in cerebrospinal
fluid) (Wang et al., 2016). Neurological involvement is purely an im-
munological reaction, as meningism corresponds to the appearance of
antibodies in the serum and cerebrospinal fluid (CSF), which regularly
manifest aseptic meningitis, myelitis, meningoencephalitis, poly-
radiculoneuritis and seizures (Panicker et al., 2001; Olmer et al., 1950).
By days 2 and 3, the CSF abnormality slowly resolves, and the signs of
meningism disappear. In a study involving 100 consecutive patients
with meningoencephalitis, 5 patients had high levels of IgM antibodies
for leptospirosis, suggesting that the leptospiral infection is an un-
noticed cause of meningoencephalitis in endemic areas (Watt et al.,
1989). Another prospective study involving more than 1000 cases of
CNS infections reported 33% of conventional bacterial infections in-
cluding Leptospira (Dittrich et al., 2015). Hence, it has been suggested
that leptospirosis should be measured when diagnosing the cause of
meningoencephalitis in a susceptible person in an endemic area
(Dittrich et al., 2015; Romero et al., 2010). The co-infection of leptos-
pirosis and scrub typhus significantly amplifies macrophage activation
syndrome (Sonthayanon et al., 2013a). The sudden activation and
217
Acta Tropica 176 (2017) 206–223
increased load of immune cells might lead to the hemophagocytosis of
most of the blood cells. The early administration of appropriate anti-
biotics, distinguishing different organisms through proper diagnosis,
and the faultless evaluation of patients are key strategies to reduce the
mortality rate of individuals co-infected with leptospirosis and other
microbial agents (Biggs et al., 2013). Patients with co-infections are
highly loaded with different pathogens and need to be handled care-
fully. Early and rapid diagnosis and proper interventions should be
quantified (Yang et al., 2012). Leptospirosis being misidentified as
DENV and presenting together as co-infections are increasingly re-
ported which require early and proper treatment. If any one of the in-
fections is wrongly diagnosed, then the patient falls into immense death
conditions. To avoid this type of burden during co-infection, improve-
ments in the diagnostics and proper treatment of multiple coinfections
are mandatory (Yang et al., 2012).
During the incubation period, almost all pathogens establish
themselves through multiplication and dissemination into the different
parts of the body using various methods. These processes are achieved
through the ability of the pathogen to invade tissues by succeeding and
evading the human immune system (Table 1), particularly the initial
humoral response (Finlay and McFadden, 2006). The extended estab-
lishment in different organs results in extensively damaged clinical
presentations. Also, during coinfection different pathogens in different
stages might provoke untoward effects for the initiated treatment. The
high microbial count in the body tissues from the initial stage of the
infection is an important factor for treatment. During the high leptos-
piremic phase, antimicrobial treatment might result in a Jarisch-Herx-
heimer reaction, as broken organisms suddenly release their contents,
particularly endotoxins that elevate cytokine levels through normal
responses in the immune system (Friedland and Warrell, 1991). This
effect is fatal, particularly in young or old patients, pregnant women,
and patients with preexisting diseases, such as diabetics. Dengue and
Chikungunya are airborne viral infections with similar clinical pre-
sentations to leptospirosis, and confusion exists worldwide (Behera
et al., 2009). All of these diseases have common initial symptoms and
are also endemic in nature. Local practitioners should note early clinical
presentations with similar signs and symptoms, endemic presentation,
and common vectors (such as mosquitoes or common environmental
risks). Any diagnostic procedure that can identify the disease during the
incubation period is particularly useful in the endemic areas with an
outbreak and might be the best strategy to overcome these microbes
and prevent extensive damage in infected individuals. Molecular deli-
neations of the pathogenesis, from attachment to immune evasion,
might shed light on the prevention and control of these diseases.
Discussions on the pathogenesis of the mentioned hemorrhagic
diseases made evident that it is complicated by excessive immune ac-
tivation and the cytokines involved are summarized in Table 1. As
mentioned earlier, the association of IL10 and the severity of disease in
fatal outcome is well documented. IL 10, the master regulator of im-
munity to almost all pathogenic infections (Table 1) and regulates both
cell mediated and humoral response (Couper et al., 2008). The ex-
cessive, prolonged and untimed release of this cytokine may influence
the persistence of the immunopathogenesis by allowing the pathogen to
escape the immunity by its potent inhibition of pro-inflammatory action
and anti-inflammatory effects. The natural process is actually aimed to
help the host to reduce the tissue damages and fibrosis, but the pa-
thogens are also benefited to escape the immune surveillance. It is
worth mentioning that the wide array of cells having the ability to se-
cret IL10 including non-immune cells and their wide range of action
and control complicates the issue (Couper et al., 2008). Added to that
the multiple organs invading pathogens involve them at different stages
and the immune reactions vary as mentioned. This is to be considered
during the designing of IL10 blocking therapy and other im-
munomodulation therapies (Schulte et al., 2013). The careful analysis
of individual cases added with antimicrobials and/or vaccine to control
the escaping pathogens and untoward effects.
S.P. Priya et al.
7. Diagnostic confusion and global burden
The hemorrhagic fever associated with leptospirosis, dengue, chi-
kungunya, and malaria is confusing, and unfortunately, all of these
diseases share common endemic areas, reflecting the favorable climates
in these regions. Timely diagnosis is most important for clinicians to
provide adequate treatment for these patients. Concurrent infections
with similar clinical manifestations remain a challenge for physicians to
diagnose and treat these infections.
Since, Leptospirosis is equally dangerous to other mammals like the
cow, horse, dog, and pigs, it can cause a huge economic impact on
livestock and milk production. Studies identified the possible spread of
disease from human to animal vice versa is alarming. In spite of many
decades of eminent researches and implementations, the burden of the
disease has never reached insignificant level. The WHO established
LERG (Leptospirosis Burden Epidemiology Reference Group) and
GLEAN (Global Leptospirosis Environmental Action Network) for to
address the issue from different countries and implement preventive
and treatment modalities systematically. 4th GLEAN meeting
(November 2014) reported leptospirosis as a neglected disease high-
lighting the existence of a knowledge gap in controlling the disease.
Their recommendations included ‘One Health Approach’ for human,
animal and agricultural level, establishing the algorithms for laboratory
and treatment modalities, improving the knowledge of the local
spreading pattern among the health workers and evaluating specific
areas periodically and encouraging joint researches by students to focus
on local issues.
8. Conclusions
Leptospirosis is a zoonosis typically transmitted through contact
with water or soil contaminated with urine from infected animals.
Severe flooding increases the risk for leptospirosis in endemic areas.
Rapid testing for diseases and large-scale interventions are necessary to
identify and monitor Leptospira infections. Overall, early diagnosis and
effective treatment will reduce the extensive damage to the organs and
prevent or reduce the permanent damage of post-infection. The detailed
discussion about the pathogen triggered molecular pathways leading to
tissue damage, and the immune response might facilitate the design of a
common protocol to effectively manage these diseases during early
intervention.
Author contributions
Sivan Padma Priya and Sakinah Syed collected the data, designed
figures and co-wrote the manuscript. Mok Pooi Ling, Akon Higuchi,
Rukman Awang Hamat, Syafinaz Amin Nordin, Giovanni Benelli and
Zamberi Sekawi analyzed and edited the data. Suresh Kumar designed
this study, co-wrote, and edited the manuscript.
Conflict of interests
The authors declare that there is no conflict of interest regarding the
publication of this paper.
Acknowledgments
This research received a full financial support by Long Term
Research Grants Scheme (LRGS), Ministry of Educations, Malaysia, with
Grant No. 5526407. Furthermore, this work was financially supported
by Putra Grant, Universiti Putra Malaysia, Malaysia (9436400) as well
as by International Scientific Partnership Program ISPP at King Saud
University (ISPP# 0062).
218
Acta Tropica 176 (2017) 206–223
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