1256 SECTION 16  Bacterial Infections
142  Leptospirosis
Delma J. Nieves
Leptospirosis is considered an emerging zoonosis due to increased contact
between animals and humans and the resulting human encroachment
into wildlife habitat. Climate changes of global warming and environ-
mental shifts can affect the degree of transmission. Early detection is
important to prevent morbidity and mortality most effectively. In June
2012, leptospirosis was reinstated as a Nationally Notifiable Condition
in the United States. This has aided in further investigation of incidence,
distribution, and risk factors associated with human cases.112 Worldwide
research efforts continue to dedicate additional resources and improve
understanding of this fascinating organism, which continues to contribute
to human morbidity and mortality. Thus far, leptospirosis is far from
controlled.
HISTORY
Weil286 is credited with providing the first description of leptospirosis
in 1886, although it had likely been previously described under various
names.244 Not until 1915, however, was the causal agent, originally known
as Spirochaeta icterohaemorrhagiae, identified by Inada and associates.126
Eight years earlier, Stimson242 unknowingly had identified the same
organism within sections of kidney obtained from a patient in whom
yellow fever had been diagnosed incorrectly.
Inada and associates demonstrated the role of the rat as a carrier in
1916.126 Noguchi182 first recovered this organism from a Norway rat in
1917. Although the etiology of Weil syndrome in British Army troops
in Flanders was found to be spirochetes and a possible connection with
rats was suspected, the route of acquiring infection was not understood.
In 1922, Leptospira icterohaemorrhagiae was isolated from the blood of
a laboratory worker who developed jaundice after an accidental needlestick
containing the serum of an infected rat.281 For many years the rat was
considered the only animal host of L. icterohaemorrhagiae. Then, in
1944, Randall and Cooper197 isolated this agent from a naturally infected
dog, and L. icterohaemorrhagiae subsequently has been associated with
many animal hosts, including goats, swine, cattle, and hamsters.
In 1938 and 1939, Meyer and associates163 popularized the concept
that infection with L. canicola caused disease in dogs and humans in
the United States. “Canicola fever” first was reported in Great Britain
in 194620; in 1951, 40% of the dogs in Great Britain were noted to be
seropositive.45 Surveys have confirmed the presence of L. canicola infection
in species other than dogs.220,271
In 1950, Gochenour and colleagues103 identified L. pomona as the
agent responsible for leptospirosis in cows. Widespread L. pomona
infection among cattle in the United States was recognized quickly, and
in time this finding stimulated extensive epidemiologic investigations
in livestock. Infections of cattle with L. hebdomidis and L. grippotyphosa
were identified, and, concomitantly, infections of swine and horses with
L. pomona were documented.121 In Europe, L. pomona was identified
as the agent responsible for “swineherd disease,” and it was recovered
from other domestic animals as well. In 1951, the first human cases of
L. pomona infection were identified.
Many new serotypes of leptospires were recognized in the early 1950s
after the establishment of serologic diagnostic services for leptospires by
the Centers for Disease Control and Prevention (CDC) and the Walter
Reed Army Institute of Research. Along with the identification of additional
leptospiral serotypes, the spectrum of clinical disease associated with
infection by leptospires was elucidated. Patients with autumnal fever (a
disease in Japanese peasants and potters) and Fort Bragg fever (a febrile
illness associated with pretibial eruptions described in army recruits)
were shown to suffer from leptospirosis caused by L. autumnalis.102,212
“Mud fever,” “pea-pickers disease,” and “European swamp fever,” names

that were used to describe a disease of undetermined etiology in eastern
Germany, the Far East, and western Poland, respectively, were shown to
be examples of leptospirosis caused by L. grippotyphosa.51 Seven-day fever
in Japan, Wycon fever and Bushy Creek fever in the United States, cane
field fever in Australia, and swineherd disease in Europe were identified
as examples of infection caused by L. hebdomidis, L. canicola, L. pomona,
L. australis, and L. pomona, respectively.26,32,39,63,121
Leptospirosis is a disease now thought to be caused by a single family
of organisms that has multiple serogroups and serotypes4 and is character-
ized by a broad spectrum of clinical findings.190 More than 300 strains,
called serovars (serotypes), which are divided into serogroups on the
basis of common antigens, have been identified. These are divided into
two species: L. interrogans sensu lato, which contains the pathogenic
strains; and L. biflexa sensu lato, which contains the saprophytic strains.118
Using a newer classification based on DNA relatedness, the genus Leptospira
can be classified into 22 species. Identified thus far are 10 species of
pathogenic Leptospira, five intermediate species that may cause mild
clinical manifestations, and seven saprophytic species that are environ-
mental but do not cause disease in humans or animals.38 Serovars of the
same group may be distributed between different species.54 Leptospirosis
is now often described as the most widespread and prevalent zoonotic
disease worldwide. The organism’s ability to survive outside the host as
well as establish a chronic carrier state in rodents promotes its survival
and promulgation. Global warming and increased rainfall are predictors
of increased incidence. In 2003, the World Health Organization (WHO)
reported a worldwide incidence of 0.1 to 1 case per 100,000 population
per year in temperate climates, more than 10 cases per 100,000 population
in humid subtropical region, and more than 100 cases per 100,000
population during outbreaks.118 However, the difficulty in diagnosing
and identifying chronic infection leads to underestimation.3 Traxler and
colleagues analyzed US hospital discharge records for 1998−2009 and
estimated an annual rate of leptospirosis-associated hospitalizations of
0.6 per 1 million population. But this is just the peak of the iceberg in
terms of describing the true incidence of leptospirosis in the United
States, where national surveillance ceased from 1994 to 2013.259 Outbreaks
in poor urban slums with rat infestations have contributed to its title as
a neglected infection of poverty in the United States.122 It is also considered
an infection of adventure seekers with outdoor recreational exposure
and international travel.145,185,215 WHO established the Leptospirosis Burden
Epidemiology Reference Group (LERG), which is focused on better
estimating the global burden of disease.291 In September 2015, LERG
published findings from a systematic literature review of the data on
leptospirosis morbidity and mortality, estimating annually 1.03 million
cases and 58,900 deaths due to leptospirosis worldwide.67
EPIDEMIOLOGY
Animal Reservoirs
Among mammals, rodents are the most important reservoir of leptospires,
but nearly all mammals may be infected and can transmit the disease.257
Leptospires also have been isolated in reptiles and birds, but the epide-
miologic significance of these animals in terms of maintenance of the
organism in nature or transmission of disease to humans is not clear.
For many species, infectivity rates of 10% to 50% have been reported
frequently.121,200 During epizootics, the circulation of leptospires among
many species of animals (including rats, cows, dogs, swine, mongoose)
living within a given biocenosis has been well recognized.19,71,130,166,210,217
The failure of leptospires to elicit a significant systemic antibody
response in certain animal species may be due to the development of
local immunity within their kidney tubules.21,250 Some animals fail to
develop homologous antibody titers but harbor leptospires in their
kidneys for extended periods.250 Thus lack of a positive titer to leptospires,
as determined during the course of serologic surveys of animal popula-
tions, does not indicate absence of infection, leaving the true incidence
of infection unknown.
A particular host species may serve as a reservoir for one or more
serotypes of leptospires, and a particular serotype may be hosted by
many different animal species. Turner264,267 stressed that a particular
animal species commonly serves as a reservoir for selected serotypes
but temporarily may be infected and serve as an incidental host for
other serotypes with which it usually is not infected.
CHAPTER 142  Leptospirosis 1257
Transmission of Leptospires to Humans
Leptospires are transmitted to humans either by contact with blood,
urine, tissues, or organs of infected animals or by exposure to an
environment that has been contaminated by leptospires. Humans usually
represent a dead end in the chain of infection because although person-
to-person transmission is possible theoretically, it is rare. After direct
exposure of humans to infected animals, leptospires may enter breaks
in the skin or may penetrate the mucous membranes, including the
conjunctiva, nasopharynx, and vagina.26,74,211,267 Human-to-human
transmission via human milk has been reported in a breast-fed infant
from a lactating mother who was infected with L. interrogans.36
Indirect transmission of leptospires to humans (from soil or water)
depends on the presence of an environment that favors the survival of
leptospires outside the animal host. A warm climate (25°C [77°F]), the
presence of moisture, and pH values of soil or surface water between
6.2 and 8.0 are optimal conditions for survival of leptospires. These
conditions prevail in many tropical regions throughout the year and
in temperate climates during the late spring, summer, and autumn.
Smith and Self231 demonstrated survival of leptospires in cultures of
infected soil for 43 days. In a survey of leptospirosis in Hawaii from
1999 to 2008, among 177 cases where exposure was determined, 45%
were from recreational exposure (freshwater swimming, hiking, camping),
44% were occupational (taro farming), and 11% were from exposure
around the home (gardening).138 A study of human leptospirosis in the
Caribbean from 1997 to 2005 found that in cases of suspected leptospirosis
the highest seropositivity was found among 1- to 20-year-old and 31- to
40-year-old males, and its occurrence was higher during the wet season.5
Ingestion or exposure from contaminated water sources, such as through
infected rat or cattle urine, is a well-described source of leptospirosis
in humans.47,101,267 The importance of occupation as related to the risk
for leptospirosis was emphasized in 1965.121 Disease appeared to occur
most frequently in people with occupations that required exposure to
cattle or swine or to water contaminated by rat urine. The urine of
infected cows may contain as many as 100 million leptospires per milliliter.
If conditions are favorable, surface water contaminated by the urine of
infected cattle may remain infectious for several weeks.48 The largest
reported outbreak of leptospirosis in the United States occurred in 1998
in Springfield, Illinois, after a triathlon that was preceded by heavy
rains. There were 66 laboratory-confirmed cases of leptospirosis after
the event, which included a swimming portion in a lake that received
runoff from nearby livestock farms. Fifty-two of these cases occurred
in triathlon participants, and 11 cases involved nonparticipants who
came in contact with lake water near the event.170 In a study performed
in Detroit, 90% of rats carried L. icterohemorrhagiae.77 Strain-specific
tests comparing antibody titers in the sera of inner-city and suburban
children were performed. Thirty-one percent of inner-city children had
antibodies against L. icterohaemorrhagiae; 10% of suburban children
also had antibodies to this organism. Lau and colleagues have studied
the patterns of global climate change, flooding, and urbanization to
predict that an increase in leptospirosis can be expected.144
Venereal transmission of leptospirosis is important in rodents and
can occur in livestock. Leptospires have been recovered from the semen
of bulls and have been transmitted by artificial insemination and by
coitus. The possibility of seminal transmission in humans remains
speculative. Transplacental infection of the fetus in utero is well documented
in livestock and other animals and may occur in humans.50,59,60,64,74,265,267
During the past several decades, the number of cases acquired during
outdoor recreation has increased. In developed countries, an increasing
percentage of cases of leptospirosis are from travelers returning from
areas of high endemicity and are often related to water activities.145 In
California, from 1982 to 2001, 59% of reported cases of leptospirosis
were due to recreational exposure (often to contaminated freshwater),
increasing to 85% in the 1997 to 2001 period.161
The dog, including the immunized pet dog, has been incriminated
as an important vector and as a reservoir of leptospirosis.106,249 Although
immunization of dogs against leptospirosis is possible, it is important
to remember that (1) immunization may not prevent the dog from
having renal carriage and excreting the organism, (2) canine immunity
after immunization may wane after just 1 year, and (3) immunity, when
established, is effective only for serotypes present in the canine vaccine.21
1258 SECTION 16  Bacterial Infections
In one survey of suburban and urban areas, however, between 15% and
40% of dogs were found to be infected.31 Harkin and coworkers found
that irrespective of health status, 8.2% of dogs were shedding pathogenic
leptospires.117 Rojas and colleagues found that 7.05% of dogs had urinary
shedding.204 Dogs were implicated in 58% of the 820 known cases of
leptospirosis reported between 1962 and 1971.53
Asymptomatic renal colonization in humans has been shown. Using
a 16S rDNA hybridization assay technique to identify Leptospira DNA
in urine, Ganoza and associates identified a long-term renal shedder
group (interestingly all women) among persons asymptomatically infected
with pathogenic and intermediately pathogenic Leptospira. Almost 5%
of healthy people living in a rural Amazonian community were urinary
shedders of Leptospira but did not have serologic or clinical evidence
of recent infection.99 It is not understood if this could play a role in
human-to-human transmission or some subtle degree of kidney disease.
A recent review of the literature found that a large proportion of
cases (48%) and deaths (42%) were estimated to occur in adult males
20 to 49 years of age, and the highest estimates of disease morbidity
and mortality were observed in the Global Burden of Disease regions
of South and Southeast Asia; Oceania; Caribbean, Andean, central, and
tropical Latin America; and east sub-Saharan Africa.67 Another study
estimated that globally approximately 2.90 million disability-adjusted
life years are lost per annum from the approximately annual 1.03 million
cases.256 A study reviewing the literature and focusing specifically on
African countries reported that acute human leptospirosis ranged from
2.3% to 19.8% in hospital patients with febrile illness.10
PATHOPHYSIOLOGY
After almost 100 years since the discovery of the causative organism of
leptospirosis, the understanding of mechanisms of pathogenesis has
come a long way. Leptospires are thin and highly motile spirochetes
with two periplasmic flagella responsible for their movement.88 They
have a double-membrane structure, making it unique and similar to
both gram-positive and gram-negative bacteria. Describing the membrane
from inside out, leptospires have an inner cytoplasmic membrane, a
peptidoglycan cell well, periplasm, outer membrane with outer-membrane
proteins (OMPs), phospholipids, and lipopolysaccharide (LPS).69,70
The era of genomics with the data generated by the Leptospira Genome
Project has provided new insights into potential virulence factors as
well as host response to disease.148,155,176 Valuable information has been
obtained by the use of animal models of acute and chronic disease, by
observing the interactions of leptospires with various cell lines, and by
employing molecular genetic tools. Because of their inherent ability to
revolve rapidly in a corkscrew fashion, it had been suggested in early
studies that the organism bores through connective tissue.238 Since then,
the specific factors responsible for invasiveness of leptospires have been
extensively studied and are better understood.85,95,240 In vitro studies
have shown that leptospires bind to a wide range of extracellular matrix
components. They have been shown to bind to several different cells,
including endothelial cells, monocytes, macrophages, kidney epithelial
cells, and fibroblasts.43 They can translocate through polarized MDCK
cells, suggesting a possible intracellular component to its life cycle.27
Goeijenbier and colleagues found that plasma level of soluble E (sE)-
selectin and von Willebrand factor (VWF) was strongly increased in
patients with severe leptospirosis, sE-selectin was significantly elevated
in survivors, endothelial cells exposed to virulent Leptospira showed
increased VWF expression, and soluble Fas-ligand and soluble
interleukin-2 (IL-2) receptor were strongly associated with mortality.105
These findings show that endothelial cell activation and immune activa-
tion were associated with disease severity in leptospirosis patients.
Pathogenic Leptospira may inhibit the activation of the complement
system through the secretion of proteases that cleave and inactivate key
complement proteins.96 The ability of spirochetes to interact with the
host fibrinolytic system may contribute to the degradation of the
extracellular matrix components, immune evasion, and tissue penetra-
tions and invasion.275
Leptospiral immunoglobulin-like proteins (Lig), surface proteins
found in pathogenic leptospires, are believed to be involved in cell
adhesion92 and are among those that have been studied as potential
vaccine candidates.169 Lig-bound plasmin was shown to cleave fibrinogen
and the complement proteins C3b and C5, which may allow for invasion
and complement immune system evasion by Leptosira.52
Specific factors responsible for the virulence of leptospires, although
better understood, still remain unclear. Some of these virulence factors
that continue to be a subject of investigation include LPS, hemolysins,
OMPs, surface proteins, and adhesion molecules.85 An animal model of
disease showed caspase-3 reactive renal epithelial cells, alveolar cells, and
liver cells, suggesting that L. interrogans induce activation of apoptosis
in later phases of the infection process.153 The completion of the Leptospira
genome sequence has revealed several proteins suspected to play a role
in pathogenesis.7,61,198 Comparative analysis of Leptospira proteomes
looking at different serovars of pathogenic organisms, nonpathogenic
organisms, clinical isolates, and acute lethal infection versus chronic
carrier infection has elucidated potential virulence factors and vaccine
candidates.61,69,180,181,254 Hemolysin-like proteins have been identified,
including sphingomyelinases that are present in pathogenic but not
saprophytic leptospires. Surface-exposed proteins that might have potential
roles in adhesion and pathogenesis include OmpL36, OmpL37, OmpL47,
and OmpL54.191 OmpL1 displayed significant adhesin activity binding
to glycosaminoglycans and monolayers of human cells in vitro and may
be a promising component for a subunit vaccine.203 The outer surface
proteins LigA, LigB, and LigC contain immunoglobulin-like domains156
found in virulence factors such as intimin and invasin. Leptospiral
endostatin-like proteins (Len) have been identified that have been shown
to be involved in the organism’s ability to evade the complement system.240
LipL32 is the major OMP of Leptospira shown to bind collagens,
laminin, and fibronectin and is highly conserved in pathogenic
species.114,120,263 It is the most abundant protein in the cell and is highly
immunogenic.280 LipL32 stimulates a strong antibody response during
natural infection, yet vaccination studies using LipL32 have yielded
mixed results.175 A study of Leptospira from urine of infected rats showed
acetylation or trimethylation of the highly abundant LipL32 in com-
parison with culture-grown Leptospira, which did not result in a lysine
modification, suggesting that LipL32 modifications may alter protein
recognition by the immune response, perhaps contributing to bacterial
persistence during infection.290
Loa22 is a lipoprotein that is upregulated during acute infection
and appears to be necessary for virulence in animal models of disease.181,202
However, the specific roles and functions in pathogenesis for many of
these molecules are not entirely understood. A significant amount of
redundancy in functional virulence proteins is suspected.
The possible role played by animal hosts in determining the virulence
of leptospires for humans remains speculative. Faine86 compared the
fate of virulent and nonvirulent strains of L. icterohaemorrhagiae in
guinea pigs. Both strains behaved similarly after intraperitoneal infection,
but virulent organisms survived and multiplied, whereas avirulent strains
did not. Both virulent and avirulent strains were taken up by fixed
phagocytes in reticuloendothelial tissues in vivo. Faine87 also showed
that the severity of lesions correlated positively with the number of
organisms present and that a discrete number of organisms were required
to cause death. He hypothesized that virulence results from the selective
multiplication of virulent leptospires in vivo. This hypothesis was
supported by the fact that maximum virulence can be regained after a
single animal passage that follows isolation in culture. Virulence may
be lost in culture by mutation to nonvirulent forms.
The humoral response is the main mechanism of resistance against
leptospirosis. Immunoglobulin G (IgG) and IgM antibodies are detected
in patients who have recovered from infection. Antibodies are produced
against leptospiral LPS. LPS vaccines are effective, but immunity is
limited to homologous serovars.85 Wang and associates283 demonstrated
that polymorphonuclear leukocytes are not an efficient defense factor
for pathogenic leptospires in nonimmune hosts. The virulence of
leptospires appears to be related to their ability to resist killing both by
serum and by neutrophils.
A toxic and pathogenic potential in vivo for lipid products of lep-
tospiral metabolism has been suggested.2 The cell wall of the leptospire
is high in lipid content; component fatty acids vary among leptospiral
strains. Lipids are used as a source of energy by leptospires.2 Saprophytic
leptospires invariably possess lipase activity, whereas pathogenic

leptospires may be lipase positive or lipase negative.2 Kasarov and
Addamiano137 investigated the lipolytic activity of leptospires on serum
lipoproteins. On the basis of their ability to attack these lipoproteins,
leptospires can be divided into three groups: (1) strains that degrade
lecithin and sphingomyelin, (2) strains that degrade neither lecithin
nor sphingomyelin, and (3) strains that degrade lecithin but not
sphingomyelin. Virulent leptospires behaved as group 1 and 2 strains,
whereas saprophytic leptospires behaved as a group 3 strain.
Pathophysiology as seen and understood of the clinical disease
processes seen in leptospirosis is outlined in Box 142.1. Hemolytic
anemia, jaundice, hepatic and renal injury, pulmonary injury, and
cardiovascular, nervous system, ocular, and musculoskeletal findings
have been extensively studied and described.
CLINICAL MANIFESTATIONS
Leptospirosis is an acute systemic infection characterized by extensive
vasculitis. Serologic surveys in human populations indicate that a large
number of subclinical infections also occur. Surveys of veterinarians
and packinghouse and abattoir workers reveal positive leptospiral titers
BOX 142.1  Pathophysiology of Specific Clinical Findings in Leptospirosis
Hemolytic Anemia, Jaundice, and Hepatic Injury
• Presence of a hemolysin in the supernate of leptospiral cultures9,30,123,172,206
• Cloned hemolysin of L. interrogans serovar Lai demonstrated cell
membrane pore-forming activity in vitro147
• Thrombocytopenia, depletion of serum prothrombin, depletion of host
vitamin K51,62,81,110,196,201,283
• Capillary injury perhaps by toxin15
• Hemorrhage of skin or mucosal surfaces and rarely gastrointestinal or into
a vital organ110
• Hepatic manifestations, including jaundice, most likely are the result of
hepatocellular injury14,15,34,81,196,281
• Hemolysis possibly contributing to jaundice81,253
• One or more toxins by leptospires or release of various products after lysis
possibly injuring hepatocytes16,109,198
Renal Injury
• Tubular epithelial cell necrosis, acute vasculitis, segmental thickening of
basement membrane, and interstitial edema
• Infiltrates of lymphocytes, monocytes, plasma cells, and neutrophils
• Cells lining the lumen of the renal tubules distended, disorganized, and
contain hyaline, granular, epithelial, and bile casts
• Glomeruli showing mesangial hyperplasia, focal fusion of foot processes,
swelling of the epithelium in Bowman capsule, and thickening of the
basement membrane14,16,59,66,76,125,142,229,292
• Renal potassium wasting by increased secretion of aldosterone and
cortisol1
• Tubulointerstitial nephritis the most common lesion associated with
chronic infection14,125
• Leptospires demonstrated in the liver, renal tubules, and the interstices of
the renal cortex14,81,142
• Leptospires evading the renal immune system, including absence of
complement, downregulation of antigens, delayed lymphocyte infiltration,
and delayed colonization of renal tubules167,168
• Impaired renal blood flow leading to nephropathy15,16,34,66,81,84,146
• Host response to infection possibly inducing damage as B cells and T cells
induce inflammation in the liver and kidneys via a TLR-independent
pathway108,194,287,288
Pulmonary Involvement
• Pulmonary lesions generally the result of hemorrhage rather than acute
inflammation17,189,226,293
CHAPTER 142  Leptospirosis 1259
in 5% to 16% of people tested.131,165,171,255 A low index of suspicion for
this disorder by physicians, coupled with the diversity and nonspecificity
of its manifestations, accounts for the significant number of cases that
go unrecognized. In one series of 483 proven cases, only 17% were
diagnosed initially as leptospirosis.32
The incubation period generally is 7 to 12 days, but a range of 2 to
20 days has been noted.216,238,266 The incubation period does not vary
significantly among serotypes and is not of prognostic significance.
Variability in incubation period may be attributed to the dose of virulent
organisms to which the host is exposed and to the portal of entry of
the organism.216,238,266
The clinical course of leptospirosis varies, but generally it is predict-
able: both anicteric leptospirosis and icteric leptospirosis follow a biphasic
course (Fig. 142.1).
The first stage (septicemic phase) is characterized by acute systemic
infection. The onset of symptoms is abrupt. This phase terminates after
4 to 7 days; symptomatic improvement and defervescence coincide with
disappearance of leptospires from the blood, CSF, and all other tissues,
with the exception of the aqueous humor and renal parenchyma.
Antibody titers to leptospires develop rapidly; this immune response
• Leptospires visualized directly in lung parenchyma226
• Alveolar septal deposition of immunoglobulin and complement76,179,218,293
Cardiovascular Findings
• Hypovolemia or hypotension from dehydration, bleeding, or third
spacing18,81,93,189,293
• Vascular collapse seen with adrenal insufficiency secondary to
hemorrhage147
• Hypoperfusion, focal hemorrhagic myocarditis, acute
coronary arteritis, pericarditis, aortitis, cardiac arrhythmias, congestive
heart failure, hypertension, hypovolemia, electrolyte imbalance, or
uremia14,75,223,234,248
• Epicardium, endocardium, and myocardium all possibly
involved14,15,81,109,253
Nervous System Findings
• Meningeal reaction occurs only after the development of antibody,
leptospiral meningitis suggested to be a reflection of an antigen-antibody
reaction14,17,21,62,110
• Uncommon features of leptospirosis include encephalitis, myelitis,
radiculitis, and peripheral neuritis14,81,100,104,142,164
• Neurologic manifestations attributed to subarachnoid, peripapillary, and
subdural hemorrhages46,48
Ocular Involvement
• Possibly direct Leptospira-mediated injury to eye structures, but suggestion
that cross-reacting antibodies play a role in Leptospira-associated
recurrent uveitis143,274
Musculoskeletal Findings
• Myalgia an early clinical feature concurrent with leptospirosis septicemic
stage, correlating with the timing of the histologic changes in muscle that
often quickly resolves
• Muscle pain that subsides after leptospiral agglutinin titers
develop35,81,129,146,224,235,253
• Bone involvement in leptospirosis not a significant feature127
Other
• No characteristic lesions noted in the adrenal glands, lymph nodes, spleen,
gastrointestinal tract, pancreas, ureter, or bladder
• Interstitial edema with monocytic and lymphocytic infiltrates found in
testicular tissue associated with impaired spermatogenesis104
1260 SECTION 16  Bacterial Infections
Anicteric Leptospirosis
First stage Second stage
3–7 days 0 days–1 month
(Septicemic) (Immune)
Fever
Myalgia Meningitis
clinical findings
Headache Uveitis
Important
Abdominal pain Rash
Vomiting Fever
Conjunctival
suffusion
Fever
Blood
Leptospires
present
CSF
Urine
FIG. 142.1  Clinical course of leptospirosis: anicteric and icteric disease. CSF, Cerebrospinal fluid.
heralds the second, or “immune,” stage of the illness, which lasts 4 to
30 days. Leptospiruria is prevalent and continues for 1 week to 1 month;
generally, it is unaffected by antibiotic therapy. Meningitis or hepatic
or renal manifestations, when present, reach their peak intensity during
this stage of the disease.
There is also a more recently described severe pulmonary form of
leptospirosis that may occur in the absence of jaundice or as an elevated
Weil syndrome and carries a high mortality (see later).152
Leptospirosis manifestations in children are similar and can be very
severe as well.58,134,135,262,273,274, 276 During the 2001 outbreak of leptospirosis
in Mumbai, India, 32% of children admitted in a 2-month period had
leptospirosis.135 A year earlier in the same city, following floods, lepto-
spirosis was found in 34% of children screened who had abrupt onset
of fever, contact with flood water, conjunctival suffusion, abdominal
pain, or rash.134
Anicteric Leptospirosis
Ninety percent or more of all patients with leptospirosis are anicteric.
They frequently escape definitive diagnosis because jaundice and azotemia
are absent. The onset of the septicemic phase of anicteric leptospirosis
is abrupt82 and heralded by fever, malaise, headache, myalgia, and
occasionally prostration and circulatory collapse.158 Chills, remittent
fever, headaches, severe myalgia, and abdominal pain are prominent
for 4 to 7 days. Fever defervesces by lysis, and other symptoms resolve.
Death is an extraordinarily rare occurrence in the first stage of anicteric
illness. Some patients with anicteric leptospirosis do not experience a
biphasic illness and remain asymptomatic after the first week.82
The second phase of anicteric disease may be characterized by fever,
uveitis, rash, headache, and meningitis. If present, fever usually is of
brief duration and has a lower peak than that of the septicemic
phase.81,82,121 Maximum temperatures range from 38.2°C to 40.6°C
(100.8°F–105°F), with one or more daily peaks. Recurrence of fever 2
or 3 weeks after leptospirosis resolves is not unusual, but no reports of
the isolation of leptospires from blood document relapse on these
occasions. Relapse generally occurs when the immune response of the
host is peaking and at a time of maximal leptospiruria, which suggests
an allergic or immune basis for the febrile episodes.34 Headache may
be intense and usually is not controlled well by analgesics. Typically, it
is frontal in distribution and characterized as bitemporal or occipital.
It may be associated with retrobulbar pain.65,81,82 Persistence or recurrence
of headache after termination of the septicemic phase of disease generally
indicates the onset of meningitis. The factors responsible for headache
in the septicemic phase of leptospirosis are unknown.
Icteric Leptospirosis
(Weil syndrome)
First stage Second stage
3–7 days 10–30 days
(Septicemic) (Immune)
Jaundice
Hemorrhage
Renal failure
Myocarditis
Blood
CSF
Urine
Restlessness, nocturnal confusion, mood disturbances, and mild
alterations in consciousness usually occur briefly and commonly in
both stages of leptospirosis.81,121 Delirium, hallucinations, psychotic
behavior, and suicidal tendencies have been reported.81,121,222
Anorexia, nausea, vomiting, and abdominal pain may be reported
in both stages of anicteric disease. Constipation, diarrhea, and
gastrointestinal hemorrhage also have been documented.81,158,182 Generally,
hemorrhagic complications are associated exclusively with icteric disease.
Physical examination during the septicemic stage may reveal dehydra-
tion, muscle tenderness, conjunctival suffusion, generalized lymphade-
nopathy, hepatosplenomegaly, and rashes that may be macular,
maculopapular, erythematous, urticarial, petechial, purpuric, hemor-
rhagic, or desquamating. Skin lesions are most prominent over the
trunk, but any area of the body may be affected. Pretibial eruptions
have been noted in patients with infection caused by L. autumnalis,
but other serotypes also may cause disease with pretibial eruptions.
Recurrent, transient urticarial eruptions have appeared for many days
after resolution of the other manifestations of leptospirosis. Pharyngitis,
rales, arthritis, and nonpitting edema occur less commonly.* Tachycardia
is a common occurrence, and cardiac arrhythmias are noted occasion-
ally.188,234 Hypotension rarely occurs in anicteric leptospirosis.82
Muscle pain and tenderness may be generalized, but the muscles of
the calf, lumbosacral spine, and abdomen are affected most frequently.
Tenderness and rigidity of the abdominal wall may suggest the possibility
of an acute surgical abdomen. Tenderness of the muscles adjacent to
the cervical spine often causes nuchal rigidity in patients without
meningeal involvement. The muscle tenderness usually subsides with
termination of the septicemic stage of the disease.
Conjunctival suffusion, photophobia, ocular pain, and conjunctival
hemorrhage are more specifically helpful diagnostic signs. Chemosis
and inflammatory exudates generally are absent despite marked con-
junctival infection. In anicteric disease, conjunctival infection involves
primarily the bulbar conjunctiva only. It appears by the third day of
illness and disappears 3 days to 3 weeks later.
Abdominal pain and tenderness, when associated with vomiting
and hypoactive bowel sounds, clearly suggest the possibility of a surgical
abdomen and present a challenging diagnostic problem because acute
intraabdominal catastrophes may complicate the natural history of this
disease. Nonobstructive toxic dilation of the gallbladder requiring
cholecystotomy has been noted repeatedly in children with leptospirosis
*References 11, 14, 33, 65, 82, 121, 129, 158, 207, 260, 266,

FIG. 142.2  Nonobstructed toxic dilation of the gallbladder in a child with
leptospirosis. Radiograph demonstrates a dilated, opaque gallbladder
protruding from the inferior margin of the liver.
(Fig. 142.2). Pain of this type must be differentiated from myositis,
subperitoneal or subserosal hemorrhage, abdominal wall causalgia, or
pancreatitis, all of which may occur in some children with anicteric or
icteric disease.
Pulmonary involvement may be observed in anicteric patients,
generally during the septicemic phase, and usually is manifested as a
dry, hacking cough, occasionally productive of blood-stained sputum,
or by the finding of infiltrates on a chest radiograph.33,193,226 Hemoptysis,
chest pain, respiratory distress, and cyanosis appear rarely during anicteric
disease.193 Hemoptysis, when present, clears in 3 to 5 days. Physical
examination of the chest may reveal rales, evidence of consolidation,
or a pleural or pericardial friction rub.
Chest radiographs may show (1) confluent infiltrates or massive
consolidation representing larger areas of pulmonary hemorrhage; (2)
small, patchy, snowflake-like lesions in the periphery of the lung fields
that are restricted to a few intercostal spaces or disseminated widely;
and (3) solitary, patchy lesions with ill-defined margins.193,282 Of these
radiographic appearances, the second is most common. Small pleural
effusions are rare occurrences in anicteric disease,282 and hilar adenopathy
has not been described. Although the chest radiograph may help delineate
the extent of pulmonary disease, it does not provide information that
could be considered pathognomonic of leptospirosis.
Other signs and symptoms of the septicemic phase of anicteric
leptospirosis that have been reported include parotitis,29 orchitis,243
epididymitis,121 prostatitis,121 otitis media,33 arthralgia,33,81,121 and
monoarticular or polyarticular arthritis.76
The hallmark of the immune phase of anicteric leptospirosis is
meningitis, and it is reflected by CSF pleocytosis with or without
meningeal symptoms or signs. During the leptospiremic phase, leptospires
may be found in the subarachnoid space unassociated with the presence
of inflammatory cells. As an antibody titer develops, leptospires are
cleared rapidly from CSF, and an inflammatory response develops.81 If
CSF is examined during the second week of illness in all patients with
anicteric leptospirosis, a meningeal reaction can be demonstrated in
more than 80%, but only 50% of these patients have clinical signs and
symptoms of meningitis.49,81 The severity of meningitis varies and does
not correlate with the severity of other clinical manifestations of lep-
tospirosis. Symptoms referable to the nervous system usually subside
within 1 or 2 days but rarely persist for 2 or 3 weeks. The CSF pleocytosis
may persist for 2 to 3 months but generally disappears within 7 to 21
days.81 In some cases, patients do not have symptoms during the
CHAPTER 142  Leptospirosis 1261
septicemic phase of leptospirosis but seek medical attention during the
immune phase because of headache, vomiting, and nuchal rigidity.
Papilledema rarely has been observed in patients with leptospirosis.46
Lumbar puncture may reveal CSF pressures varying from normal
to 350 mm H2O. Mean values generally are less than 200 mm H2O.124
Cell counts within CSF vary from normal to more than 500 cells/mm3;
generally, fewer than 500 cells have been reported.81,121 Polymorphonuclear
leukocytes predominate early in the immune phase, but mononuclear
cells subsequently predominate. Protein concentrations within CSF
range from normal to 300 mg/dL. In some cases, protein values have
been elevated in the absence of pleocytosis. Abnormal values may persist
for several weeks after the clinical symptoms resolve.33,49,121,159,160 Glucose
concentrations within CSF generally are normal.49,82,159
Encephalitis, focal weakness, spasticity, paralysis, nystagmus, periph-
eral neuritis, cranial nerve paralysis, seizures, radiculitis, visual distur-
bances, myelitis, Guillain-Barré syndrome, or acute disseminated
encephalomyelitis may appear with or subsequent to the immune stage
of anicteric disease.12,25,55,81,84,121,159,164,272 Generally, these symptoms resolve,
but complete resolution may require several weeks to months. Neurologic
sequelae secondary to central nervous system hemolysis may occur.46,48
The anterior uveal tract may be affected as early as the third week
of illness, but symptoms may be found up to 1 year after the onset of
leptospirosis. Conjunctival suffusion (characteristic during the septicemic
phase) is not found in the immune stage of the disease. Rather, iritis,
iridocyclitis, and, occasionally, chorioretinitis are noted.32,81,82,121 Uveal
involvement may be unilateral or bilateral and may occur as a single,
self-limited episode, as recurrent episodes, or as a chronic unrelenting
process.41,81 The severity of the uveitis does not correlate with the severity
of other clinical manifestations. When uveitis is transient or self-limited,
complete healing is the rule; however, in some cases, blindness and
cataract formation are noted.
The precise incidence of involvement of the uveal tract is unclear
because symptoms may be minimal or may not appear until after other
clinical manifestations have resolved completely. The generally benign
course of uveitis may be attributable to the capacity of leptospires to
survive in the aqueous humor without eliciting an intense inflammatory
response.82 Despite the presence of high titers of specific antibodies to
leptospires in serum, antibodies to leptospires are absent or found in
low titer in the aqueous humor.
Leptospiruria is the rule during the immune stage of anicteric
leptospirosis, and it is not associated with impaired renal function. In
contrast to many animal species, humans do not serve as a reservoir
for leptospires; leptospiruria is transient. In anicteric patients, proteinuria,
pyuria, microscopic hematuria, and mild to moderate azotemia may
be observed.33
The white blood cell count may be low, normal, or elevated. Neu-
trophilia is the rule, regardless of the total white blood cell count.
Leukocytosis generally is associated with hepatic involvement. Anemia
is an inconsistent finding; when present, it may be attributable to blood
loss, vascular damage, or hemolysis. In the absence of blood loss, sig-
nificant anemia is not a manifestation of anicteric cases. The erythrocyte
sedimentation rate is elevated consistently.
Icteric Leptospirosis (Weil Syndrome)
The term Weil syndrome should be applied to define a form of lepto-
spirosis that is distinctive in clinical expression but nonspecific
with respect to serotypic etiologic agents. In addition to having the
symptoms and signs of anicteric leptospirosis, Weil syndrome is set
apart by the presence of impaired hepatic and renal function, vascular
collapse, hemorrhage, severe alterations in consciousness, and a high
mortality rate.
Weil syndrome may be heterogeneous in its manifestations, and the
course may be dominated by symptoms of renal, hepatic, or vascular
dysfunction. Jaundice and azotemia may be so severe that the biphasic
course of illness is not observed. Fever may persist without defervescence
between the septicemic and immune stages and is more prominent and
of longer duration during the immune stage than in anicteric cases. The
mortality rate, despite adequate supportive care, is between 5% and 10%.
Jaundice remains the hallmark of Weil syndrome. The intensity of
jaundice varies; a maximum total serum bilirubin concentration in the
1262 SECTION 16  Bacterial Infections
range of 60 to 80 mg/dL has been reported.159 Usually, the bilirubin
concentration is less than 20 mg/dL. Both direct- and indirect-reacting
bilirubin levels increase, but an increase in the direct fraction usually
accounts for most of the elevation in bilirubin.239 Jaundice may occur
as early as the third day of illness or may not appear until the second
week.17,82 The serum bilirubin concentration peaks within the first 7
days after the onset of jaundice in 85% of cases.196
Modest elevations in serum alkaline phosphatase level and depressed
activity of plasma prothrombin are noted occasionally.196 Hypopro-
thrombinemia responds uniformly to parenteral administration of
vitamin K. The serum albumin level may be depressed; concentrations
of 2.0 to 2.5 g are not uncommon.239 Aspartate aminotransferase and
alanine aminotransferase values are elevated minimally, rarely exceeding
100 and 200 units, respectively. The urine may contain bilirubin and
urobilinogen.
Hepatomegaly is found in approximately 24% of patients, a frequency
that is no greater than that in anicteric cases.81 Transient biliary obstruc-
tion, probably intrahepatic, may occur, but no evidence that obstructive
phenomena are the primary mechanism of impaired hepatic function
exists. Even in severely icteric cases, acholic stools generally are not
observed.81,121 Pruritus has been reported rarely in patients with lepto-
spirosis.81 The presence of abnormal urinary urobilinogen values in the
absence of acholic stools suggests patency of the biliary tract in most
cases.196
In some reports of children with leptospirosis, acalculous cholecystitis
has been seen in 55% of cases.241 In these patients, right upper quadrant
pain, tenderness, and a palpable mass were present. Abdominal radiographs
confirmed the presence of a mass in the region of the gallbladder. When
cholecystotomy was performed, a massively distended gallbladder contain-
ing colorless bile was noted. Routine aerobic and anaerobic cultures of
bile were negative, but cultures for leptospires were positive.
Hepatic dysfunction is not an important cause of death in patients
with leptospirosis. It is present, however, in most patients who die of
this disease;, conversely, a fatal outcome is extremely rare in the absence
of hepatic dysfunction.33 Renal dysfunction, cardiovascular collapse,
and hemorrhagic complications occur most often in patients whose
icterus is most prominent.
Renal dysfunction may be observed in all forms of leptospirosis,
regardless of the severity of disease or the serotype causing infection.33,81,121
Symptoms attributable to functional renal impairment generally are
observed only during icteric leptospirosis.17,33,81,82,266 In the leptospiremic
phase, abnormal urinalysis results are noted in as many as 80% of
cases.33,81,241 Proteinuria is the most frequent abnormality and generally
is mild. Hyaline or granular casts and cellular elements (red and white
blood cells) may be found in the urinary sediment. Microscopic or
gross hematuria is seen in many patients and most likely reflects the
presence of a hemorrhagic diathesis rather than glomerular injury.272
Fatal cases of icteric leptospirosis in which urinalysis results were normal
have been reported.17 The abnormalities in urinary sediment and proteinuria
may persist for weeks in patients without significant azotemia.28
Oliguria or anuria may be noted as early as the third day of illness
but occurs more commonly after the first week. Generally, blood urea
nitrogen values remain below 100 mg/dL, but values may exceed 300 mg/
dL in some cases.121,183 The height of the blood urea nitrogen value is
not of prognostic value in individual cases, although in groups of patients
it correlates well with outcome.253
Azotemic patients with leptospirosis can be divided into two groups:
(1) those with decreased renal perfusion (ratio of urine osmolality [UOsm]
to plasma osmolality [POsm] of about 2 : 1) and a good response to fluid
administration; and (2) those with a UOsm/POsm ratio close to 1 : 1, with
impaired resorption of sodium and water from the renal tubules and
no response to administration of fluids. The manifestations of the second
group of patients are those of acute tubular necrosis. The factors responsible
for oliguria in the first group of patients (those with prerenal azotemia),
including hypotension, shock, and volume depletion, if uncorrected,
ultimately may progress to acute tubular necrosis as well.
Anuria is an ominous sign, whereas diuresis is a good prognostic
omen.17 The impairment in renal function may persist, and fatalities
have been recorded after the onset of diuresis.183 Hyposthenuria can
persist for months in some cases.81 Some evidence of renal disease has
been demonstrated by renal function tests and renal biopsies for as
long as 6 months after the onset of leptospirosis.23 Renal failure is the
principal cause of death in patients with leptospirosis, but it generally
is reversible in time. Usually, renal function improves by 6 months after
acute infection.72
Cardiac involvement is a relatively infrequent occurrence, but when
it is present, congestive heart failure and cardiovascular collapse may
occur.82,129 Electrocardiographic changes are seen in all forms of lepto-
spirosis.188 In one series of patients, electrocardiograms obtained during
the first week of illness were abnormal in 90% of patients at a time
when none had signs or symptoms of congestive failure, pericarditis,
or hypotension.188 The electrocardiographic abnormalities disappeared
by 10 days in most cases. The electrocardiographic changes that have
been described often are nonspecific findings common to many infectious
diseases or attributable to fever alone.188,208
Cerebrovascular accidents may occur in patients with leptospirosis.149
In a study of 21 cases in which postmortem examination was performed,
subarachnoid hemorrhage was described in 1 case, cerebral hemorrhage
in 2, and recent cerebral infarction in 1.
Hyponatremia is a rather consistent finding in patients with severe
icteric leptospirosis. The hyponatremia appears to be the result of (1)
failure of the sodium pump, which causes sodium to move intracellularly
in exchange for potassium; and (2) redistribution of fluid such that the
extracellular fluid space is expanded at the expense of the intracellular
space. Hyponatremia in these patients may be unresponsive to either
sodium replacement or fluid restriction. It is treated best by fluid
restriction, which can be continued unless systemic blood pressure falls.
Clinical improvement in the patient generally follows a spontaneous
increase in serum sodium level, which may occur before any other
evidence of clinical improvement is noted.
Most patients with leptospirosis recover without long-term sequelae.214
However, given the evidence of prolonged urinary shedding, it has been
speculated that there may be a chronic form of leptospirosis.122 A prospec-
tive study in São Paulo, Brazil, followed patients who had been hospital-
ized with leptospirosis236: two of 47 patients reported continuing
symptoms, one with profound malaise without a defined reason and
one with new diagnosis of panic disorder. A study294 in a Leptospira-
endemic town reported that subjects with a microscopic agglutination
test (MAT) titer of 400 or greater showed a decreased renal function
compared to those with lower titers. And, two subjects with persistently
high MAT titers and positive urine Leptospira DNA had worsening renal
function during the study period of 2 years.
Severe Pulmonary Form of Leptospirosis
A recently described severe pulmonary form of leptospirosis may occur
in the absence of jaundice or as an elevated Weil syndrome. It carries
a mortality rate of greater than 50%152 and manifests as dyspnea, chest
pain, and cough with hemoptysis.
The chest radiograph shows small nodular opacities that may progress
into larger coalescent areas of consolidation.78 High-resolution computed
tomography shows bilateral ground-glass opacities, areas of consolidation,
airspace nodules, and small pleural effusions. On gross pathology,
extensive pulmonary hemorrhage with numerous bleeding foci of
different sizes can be seen. Histologic sections usually demonstrate
pulmonary congestion, pulmonary edema, and several foci of interstitial
and intraalveolar bleeding, with varying degrees of severity.152 Treatment
involves maintaining hemodynamic stability and respiratory support.
Corticosteroids and platelet transfusions have been used.225 Because
the pathogenesis of the severe pulmonary form of leptospirosis is thought
to likely be immune mediated, immunosuppression has been used in
its treatment, including plasma exchange, cyclophosphamide, desmopres-
sin, dexamethasone, and recombinant factor.152
INDICATORS OF PROGNOSIS
Attempts have been made to identify prognostic factors associated with
leptospirosis. Such studies, which aimed to evaluate the prognostic
significance of certain clinical and laboratory variables at different stages
of disease, have produced variable results. Hypotension, oliguria,
hyperkalemia, the development of pulmonary rales or rhonchi, and

electrocardiographic repolarization abnormalities each have been associ-
ated with a poor prognosis,73,79,80,151,186,252 although conflicting data exist
about the implications of hypotension.80 In a small series of 12 patients,
Truccolo and colleagues261 reported death of all patients with a leptospiral
load greater than 104 organisms/mL as determined by quantitative
polymerase chain reaction (PCR). Chang and colleagues,57 in a series of
11 patients with late leptospirosis, reported mortality in all patients with
hepatitis and a disproportionate aspartate aminotransferase level, as
defined by a ratio of peak aspartate aminotransferase to peak alanine
aminotransferase greater than 3. Respiratory failure, hemoptysis, metabolic
acidosis, and thrombocytopenia also have been associated with increased
mortality rates.252 In Marotto and coworkers’ prediction model, serum
potassium, serum creatinine, respiratory rate, presenting shock, and
Glasgow Coma Scale score less than 15 were independently associated
with development of leptospirosis-associated pulmonary hemorrhage
syndrome.154 Advancing age, renal involvement, pulmonary involvement,
and multiple organ dysfunction have been repeatedly found to be associated
with increased mortality.195 A case of severe leptospirosis complicated
by Epstein-Barr virus reactivation has been described.136 A case has also
been described of an HIV-infected man on antiretroviral therapy and
with a history of hepatitis C who developed fever and hepatitis and was
found to have leptospirosis and acute or recurrence of occult hepatitis
C.90 These cases point to the need to consider concomitant infections in
severe cases of leptospirosis.
LABORATORY DIAGNOSIS
Whenever possible, the physician should use laboratory facilities in
which culture and serologic tests for leptospirosis are performed routinely.
It is recommended that specimens be sent to the standard reference
laboratory, the National Leptospirosis Laboratory at the CDC in Atlanta.
Despite proper collection and handling of specimens, obtaining labora-
tory confirmation of cases of leptospirosis may be difficult, even for
facilities with skill in this area.
A confirmed case of leptospirosis, as defined by the US Department
of Health and Human Services, fulfills one of the following criteria: (1)
clinical specimens that are culture positive for leptospires or (2) clinical
symptoms compatible with leptospirosis and either seroconversion or
a fourfold or greater rise in the MAT titer between acute and convalescent
serum specimens obtained 2 or more weeks apart and studied at the
same laboratory. PCR has been shown to be effective for detecting
pathogenic Leptospira but may not be commercially available.
Presumptive leptospirosis is defined as the presence of clinical
symptoms that are compatible with leptospirosis and an MAT titer of
1 : 100 or greater, a positive slide MAT reaction on a single serum specimen
obtained after the onset of symptoms, or a stable MAT titer of 1 : 100
or greater in two or more serum specimens obtained after the onset of
symptoms.
Identification by Culture
Leptospires can be recovered from blood or CSF obtained from patients
during the septicemic stage of illness or from urine during the immune
stage. Other than these body fluids, only tissue sections obtained by
biopsy or at necropsy are sources from which organisms can be recovered.
Rarely, organisms are isolated from intraocular fluid obtained during
convalescence.8
Media for the cultivation of leptospires generally contain a buffered
solution, with or without peptone and with or without 0.1% to 0.2%
agar to which rabbit serum has been added to provide a final concentra-
tion in the medium of 5% to 10%. In addition, a pH between 7.2 and
7.8 appears to be essential. Clinical material obtained for culture fre-
quently is contaminated; antimicrobial agents, including neomycin,
vancomycin, or bacitracin, added to leptospiral media in low concentra-
tion have been found to be effective in reducing contamination and
exert little if any effect on leptospires.
For routine use, Fletcher semisolid medium94 or EMJH semisolid
medium132,238 is recommended. Stuart medium245 has been used to prepare
and maintain antigens for serologic tests. Tween 80–albumin medium
(OAC) was developed not long ago and is available commercially. This
medium seems to be superior for primary isolation of leptospires.
CHAPTER 142  Leptospirosis 1263
Several solid media are available but appear to be most useful for
isolation and purification of leptospires from contaminated natural
material such as water.246,265 Preparation, use, and maintenance of these
solid media and other media are described in other works.162,246,265
Multiple cultures should be obtained from patients with leptospirosis
because the concentration of organisms in blood at any point in time
is low.132 Freshly drawn blood is most desirable, but leptospires may
remain viable in anticoagulated blood for as long as 11 days.245 Blood
should be inoculated into several tubes of semisolid media. The number
of drops of blood placed into each tube should be varied (one to four
drops). Excessive amounts of blood inhibit the growth of leptospires;
hence a small inoculum yields the best results.94 Cultures are incubated
at 28°C to 30°C in the dark for 6 weeks or longer.
In semisolid media, leptospires grow in a concentrated ring about
0.5 to 1 cm below the surface. Growth may not be detected in Fletcher
semisolid medium for several weeks but may occur earlier in polysorbate
medium.
Contaminated specimens or suspensions of primary cultures in
which contamination is suspected may be inoculated into hamsters.
When an animal dies, phlebotomy or necropsy is performed, and
sections of liver, kidney, and brain then are recultured in appropriate
semisolid media.
If collected during the septicemic phase, CSF may be cultured in
the same manner as blood.
Urine serves as the main source from which leptospires can be isolated
during the immune and convalescent phases of leptospirosis. Prolonged
urinary shedding after infection is possible in humans. Chow and
coworkers reported a 10-year-old girl who shed leptospires in urine for
6 weeks after exposure.58 Clean-voided urine may be inoculated directly
into an appropriate semisolid medium. Urine specimens must be diluted
with sterile, buffered saline solution to ensure growth.253 Best results
are obtained by adding 0.1 mL of urine to 0.9 mL of buffered saline
before inoculation into 5 mL of semisolid medium. This procedure can
be continued with four additional dilutions. Other bacterial contaminants
that may be present in undiluted urine cultures generally do not survive
in these cultures after dilution.246
Identification by Means Other Than Culture
The morphologic appearance of all members of the genus Leptospira
is similar. They are slender, threadlike organisms about 0.1 µm in
diameter and 6 to 12 µm in length, tightly coiled on their long axis.
Like other spirochetes, they cannot be seen in wet preparations by
lightfield microscopy, but on darkfield examination they may be observed
readily. For the detection of one leptospire per high-power field by
darkfield examination, a concentration of 10,000 to 20,000 leptospires
per milliliter of fluid is needed.265 At best, darkfield examination should
be considered an aid that may suggest but not establish a diagnosis of
leptospirosis.
Leptospires can be stained by several silver impregnation tech-
niques.44,140,162,246,265 The modified method of Van Orden has been used
at the CDC for demonstrating organisms in sections of liver, kidney,
or other tissues. Infecting serotypes cannot be differentiated by silver
impregnation techniques. Leptospiral antigen also has been detected
with the use of an immunoperoxidase staining procedure.91
Fluorescent antibody techniques may be applied successfully to the
detection of leptospires in urine or tissue.173,174,246,265 This test is based
on specific antigen-antibody reactions using fluorescence-tagged antisera.
In theory, the fluorescent antibody reaction should demonstrate distorted
and fragmented, as well as whole, organisms, but caution is required.
Control specimens that have been treated with unlabeled antiserum
before the addition of fluorescein-labeled antiserum should be used.162
The control specimen should not fluoresce. The fluorescent antibody
technique may provide the physician with useful information in the
course of the disease in some patients. Positive results, however, are
considered only presumptive evidence of infection.
In addition to this technique, DNA hybridization techniques or
nucleic acid amplification procedures, including PCR protocols using
Leptospira-specific cDNA probes or oligonucleotide primers, can be
used to detect the presence of leptospires in body fluids or culture
supernatants.37 These techniques are being developed in the laboratories
1264 SECTION 16  Bacterial Infections
of the Leptospirosis Branch at the CDC, but proof of their superiority
in terms of sensitivity or specificity in detecting leptospiral organisms
in body fluids or other clinical samples has not been established.
The reference method for serovar identification is considered to be
the cross-agglutinin absorption test (CAAT) but requires maintenance
of large panels of reference antisera and live antigens. Multilocus sequence
typing has been applied to provide strain information but is limited to
a few species.178 Pulse-field gel electrophoresis can be used to identify
Leptospira to the serovar level, is applicable to all pathogenic species,
and can rapidly identify new serovars.97
Serologic Tests
Evaluation of serologic findings to supplement clinical and epidemiologic
information generally is recommended as a first step in establishing a
diagnosis of leptospirosis. One of the most widely used specific serologic
tests for leptospirosis has been the MAT, in which live antigen is used.
This test is time-consuming and potentially hazardous to the technician
but is considered the reference test against which all other tests are
evaluated. Formalinized antigens can be used for the MAT, and they
are preferred in some laboratories, but the titers obtained are lower
than those obtained with live antigen, and more cross-reactions with
heterologous serotypes occur. Generally, serum is used for the MAT or
other agglutination tests, but CSF, urine, bile, or aqueous humor may
be used as well.162
The CDC uses a standard panel of commonly occurring serovars
for routine performance of the MAT. However, because of geographic
differences in serovar distribution, serovars are added or changed
depending on the region where exposure occurred and the location
from which specimens were obtained. Killed antigens remain stable for
at least 12 months and are available commercially either individually
or in pools. Sulzer and associates246 have provided detailed descriptions
of methods for performance of the MAT. Modifications of the MAT
have been developed and include the semi-micromethod and microtiter
techniques.98
A newer serologic test involving diffusion in gel, the enzyme-linked
immunosorbent assay (ELISA), has been compared with the MAT for
the serologic diagnosis of leptospirosis.71,279 The results suggest that this
test is a viable alternative to the MAT because of its sensitivity, potential
for standardization, and simplicity. Variations of this test have been
developed; rapid serodiagnosis of leptospirosis with the use of an IgM-
specific dot ELISA has proved to be as sensitive and specific as MAT.277,284
The dot ELISA is inexpensive, simple to perform, and uses minute
volumes of leptospiral antigens.
The IgM-PK ELISA, an assay for IgM using a proteinase K–treated
antigen, was compared with the Leptoteste-S macroagglutination test
and with the MAT for the diagnosis of leptospirosis. All three tests were
comparable in their ability to detect the presence of leptospirosis. Both
the IgM-PK ELISA and the Leptoteste-S differed statistically from the
MAT in terms of the positivity of acute phase sera. Thirty-eight percent
of patients with leptospirosis were identified earlier with either test
than when the MAT was used. The IgM-PK ELISA, which had a sensitivity
of 89.9% and a specificity of 97.4%, has been suggested as the test of
choice for laboratories that are equipped to perform ELISA.199
A slide agglutination test available commercially has been compared
with the ELISA IgM and has yielded equivalent results for the diagnosis
of leptospirosis. The slide agglutination test is inexpensive and can be
performed more quickly and more easily than ELISA, thus rendering
it a useful test for laboratories that are less well equipped than those
in which IgM ELISA currently is performed.42
The Lepto Dipstick, a dipstick assay for detection of Leptospira-specific
IgM antibodies in serum, has been studied as a method for the diagnosis
of leptospirosis in situations in which laboratory facilities may not be
available.232 This test’s results correlated well (80−96.7% observed
agreement; κ value, 0.62−0.92) with results obtained by the IgM ELISA
leptospiral antigen detection method.24,113 A 93.2% agreement (κ value,
0.66) was reported between the Lepto Dipstick and MAT, along with a
high number of false-positive results as well.24 The dipstick test is a
valuable and useful tool for rapid screening for leptospirosis and may
be useful in the field for detecting and monitoring outbreaks of lepto-
spirosis. Leptocheck, a simple and rapid commercially available
immunochromatographic test (which can be used on serum or urine)
has also been proved helpful in early diagnosis of leptospirosis cases,
which should then be confirmed with other serologic methods later.192
Other tests that may be used for the serologic diagnosis of leptospirosis
include a complement fixation assay, a hemolytic test, an indirect immu-
nofluorescent test, an erythrocyte-sensitizing substance test, countercurrent
immunoelectrophoresis, and flow cytometry light scatter analysis.56,177,258,295
These tests are genus specific and may yield positive results earlier in the
course of leptospirosis than the agglutination tests do. Their results also
revert to negative earlier; therefore these tests are of little value for serologic
surveys. They may be of value in distinguishing current from past infections
when agglutination test results are equivocal.245,246
An indirect hemagglutination test offers the advantage of detecting
antibodies as early as the fourth day after the onset of illness. It is genus
specific, is less time-consuming, and requires just one antigen in the test
system. It has excellent sensitivity and specificity, and some investigators
have suggested that it may replace the MAT as the screening test of choice.246
Effler and colleagues,83 however, reported discouraging results when this
test was used for the diagnosis of leptospirosis in Hawaii.
Agglutination tests have been considered to be serotype specific. Because
of the antigenic complexity of leptospires, however, cross-agglutination
reactions occur; serotypes that belong to the same serogroup cross-react
at high titers. Early in the course of leptospirosis, heterologous reactions
may be stronger than homologous reactions. Because of these paradoxical
cross-reactions, one should not depend on serologic determination alone
to define the infecting serotype. When agglutination tests are performed
on serial specimens over the course of time, the homologous reaction
becomes the dominant one in most cases. Performance of agglutination
absorption studies may be necessary to define the infecting serotype in
some cases. The antigen (serotype) that absorbs out agglutinin to all the
serotypes in a serogroup most likely is the infecting serotype.162,239,268
A passive microcapsule agglutination test that uses chemically stable
microcapsules instead of sheep erythrocytes has been developed.17 When
compared with the MAT, the passive microcapsule agglutination test
showed a relatively greater degree of genus specificity and 4- to 32-fold
higher titers. The sensitized microcapsules were stable for at least 1
year. This test is simple to perform and reproducible and can be used
readily in the routine laboratory. Moreover, the test appears to be more
sensitive than is the MAT in the early stages of leptospirosis.221
A positive leptospiral agglutination reaction generally is not found
until the 6th to 12th day of illness, and maximal levels are reached
between 21 and 28 days. After recovery, low titers may persist for many
years. One blood sample should be obtained early in the course of
illness, and a second one should be obtained at the end of 1 month.
Negative reactions in serial samples do not exclude the possibility of
leptospirosis because patients may be infected with a serotype not
included in the battery of test antigens or with a previously unrecognized
serotype. Moreover, the titer may have peaked before the acute-phase
specimen was collected. Antibiotic therapy also may suppress the
development of positive titers or delay their appearance.111,162 Peak MAT
titers of 1 : 3000 to 1 : 100,000 usually are reached during the third week
of illness.81,268 An unchanging titer of 1 : 100 on two successive serum
specimens has been defined as sufficient for making a presumptive
diagnosis of leptospirosis. A fourfold increase in titer between acute
and convalescent sera is indisputable evidence of active leptospirosis.
The global criterion for laboratory confirmation of a current Leptospira
infection is usually defined as seroconversion or a fourfold rise in titer
in paired serum samples or set at a single MAT titer greater than 1 : 400
in the presence of clinical signs and appropriate history of animal
contact.88 Goris and colleagues have recently evaluated the performance
of MAT and ELISA in a population in the Netherlands, where positive
culture was used as a reference. They also concluded that the sensitivities
of MAT and IgM ELISA are low at the acute stage of illness and require
a follow-up sample for confirming seroconversion or a significant titer
rise, but rather than waiting 2 to 4 weeks for convalescent titers, these
could be done much sooner in urgent cases because significant antibody
rise was seen in some cases as early as 2 days later, allowing for earlier
diagnosis and proper treatment.107
Leptospira DNA may be detected by PCR at a minimum detection
limit of two to three cells per sample.150,233 Nonradioactive, arbitrarily

primed PCR assays can be used to discriminate species of Leptospira.
Romero and colleagues reported that PCR was more likely to facilitate
the early diagnosis of leptospiral aseptic meningitis than was either the
IgM ELISA or the MAT test.205 Another recent study exploring the use
of 16S ribosomal RNA for development of PCR-based diagnostics for
human leptospirosis found promising results, perhaps superior to
traditional DNA-based PCR.22
PCR, IgM ELISA, latex agglutination, the Lepto Dipstick assay, and
an antibody-based urine antigen assay each may be more sensitive than
the MAT early in the course of disease.139,184,209
Unbiased next-generation sequencing aided in diagnosing a case of
neuroleptospirosis in a 14-year-old boy with severe combined immu-
nodeficiency who had gone undiagnosed for a period of 4 months with
fever and headache worsening to hydrocephalus and seizures. PCR and
serologic testing at the CDC subsequently confirmed L. santarosai
infection, which he had likely contracted on a trip to Puerto Rico.289
TREATMENT
To be of maximum therapeutic benefit, an antimicrobial agent would
have to be administered before invading organisms damage the endo-
thelium of blood vessels and various organs or tissues. One of the
problems in evaluating the efficacy of treatment is the fact that, generally,
leptospirosis is a self-limited disease with a favorable prognosis. Even
patients with severe icteric leptospirosis may recover without specific
treatment.
Most claims of the beneficial value of antimicrobial agents in human
leptospirosis are based on the response of individual patients rather
than on controlled studies. Hall and associates116 compared the effects
of penicillin, chloramphenicol, chlortetracycline, and oxytetracycline
with placebo in 67 confirmed cases of leptospirosis. No appreciable
effect of antibiotics could be demonstrated on the duration or severity
of illness or on the prevention or amelioration of central nervous system,
hepatic, renal, or hemorrhagic complications of this disease. Moreover,
the duration of leptospiremia and the persistence of organisms in CSF
were not altered by treatment. Kocen141 compared the effects of penicillin
administered on the fourth day of illness to 28 patients with a control
group of 33 who were given only supportive care and reported that the
duration of fever was shorter and the incidence of jaundice, meningismus,
renal involvement, and hemorrhagic manifestations was diminished in
the treated group.141 None of these controlled studies was entirely satisfac-
tory with respect to randomization of patients.
McClain and associates157 studied the therapeutic efficacy of doxy-
cycline in military recruits who contracted leptospirosis while training
at the Jungle Operations Training Center in Panama. Twenty-nine patients
with anicteric disease were treated in a randomized, double-blinded
fashion with doxycycline 100 mg orally twice a day or with placebo.
Therapy was administered for 7 days in the hospital, after which patients
were monitored for 3 weeks. The duration of illness before therapy and
the severity of illness were similar in both study groups. Doxycycline
shortened the duration of illness by 2 days and favorably influenced
fever, malaise, headache, and myalgia. Treatment also prevented lepto-
spiruria, and no significant adverse effects of doxycycline administration
were observed.
In another randomized, double-blinded, placebo-controlled field
trial at the same military training site, Takafuji and associates251 dem-
onstrated that doxycycline in a 200-mg oral dose given weekly or at
the completion of jungle training was highly effective in preventing the
onset of clinical leptospirosis. Twenty cases of disease were documented
in the placebo group (attack rate, 4.2%) as opposed to only one case
in the treatment group (attack rate, 0.2%), findings supporting the
prophylactic utility of doxycycline in this setting.
Watt and associates285 reported the results of a trial in which a 7-day
course of intravenous penicillin with 6 million units per day was
compared with placebo in a randomized, double-blinded trial involving
42 patients. All the patients had severe, advanced disease. Every measur-
able aspect of the disease was affected favorably by penicillin. The
duration of fever was shortened significantly (P < .005) in the group
receiving penicillin. Penicillin therapy decreased the number of days
of hospitalization and prevented the development of leptospiruria. These
CHAPTER 142  Leptospirosis 1265
investigators concluded that intravenous penicillin should be given to
patients with severe leptospirosis, even if therapy can be initiated only
late in the course of their disease. Subsequently, Costa and associates68
reported the contrary. Two hundred fifty-three patients with leptospirosis
and longer than 4 days of symptoms were randomized to receive 6
million units of penicillin for 7 days or placebo (mean pretreatment
duration of symptoms of 6.6 days and 6.5 days, respectively). Neither
the number of days of hospitalization nor the in-hospital case-fatality
rate differed significantly between the two groups.
Limited data exist comparing the use of alternative antimicrobial
agents with penicillin. Panaphut and colleagues187 reported no difference
in time to resolution of fever, mortality, or duration of organ dysfunction
between patients randomized to receive a 7-day course of penicillin or
intravenous ceftriaxone. A subsequent study247 comparing penicillin,
doxycycline, and cefotaxime reported no significant differences among
the three groups with regard to mortality, time to defervescence, or
time to resolution of abnormal laboratory test results.
Empiric treatment with penicillin or a tetracycline (to be avoided in
children younger than 9 years) early in the course of disease can be used
if a diagnosis of leptospirosis is suspected. Parenteral aqueous penicillin
G 6 to 8 million units/m2 per day in six divided doses should provide
optimal blood and tissue concentrations of penicillin. For patients sensitive
to penicillin, tetracycline 25 to 50 mg/kg per day orally in four divided
doses (up to 3 g/day) or doxycycline 5 mg/kg per day orally in two divided
doses (up to 200 mg/day) for 1 week, should be provided.
Management of leptospirosis requires careful attention to supportive
care. Fluid and electrolyte balance requires meticulous attention.
Dehydration, cardiovascular collapse, and acute renal failure may
necessitate prompt and specific treatment. In some cases, acute renal
failure may be prevented by ensuring adequate renal perfusion and
appropriate fluid administration early in the course of disease, when
prerenal azotemia and shock may be seen.28,227 If prerenal azotemia is
suspected, diuresis should be attempted promptly with administration
of a fluid or colloid load designed to expand extracellular volume and
replace extracellular fluid deficits.28 In patients who do not respond to
such therapy, acute tubular necrosis may be suspected, and appropriate
fluid restriction should be initiated. If azotemia is severe or prolonged,
peritoneal dialysis or hemodialysis should be instituted.13,89,270 The use
of exchange transfusion has been suggested in patients with marked
hyperbilirubinemia and acute renal failure and may be associated with
lower mortality.174,183,195,228
The use of corticosteroids in the treatment of severe cases has not
been evaluated critically, but their use in patients with impending hepatic
coma has been suggested.82 Anecdotal reports also suggest that
corticosteroids may be of value in patients with profound hypotension
or shock.
PREVENTION
Prevention of leptospirosis, as with many other infections, invokes hygienic
practices, immunizations, and prophylaxis after potential exposures.
Benches in rat-infested, fish-gutting sheds and sewers may be decon-
taminated. Hygienic conditions should be encouraged in slaughterhouses,
farmyard buildings, and bathing pools. Public health prevention campaigns
in Australia emphasize the “cover-wash-clean up” strategy for reducing
the incidence of leptospirosis. “Cover” exposed skin and mucous
membranes that might come in contact with animals or animal secretions,
“wash” hands or shower after a potential contamination, and “clean up”
areas that might be contaminated or might attract rodents.230 Given the
rise of leptospirosis acquired during recreation or international travel,
travelers should be advised about preventive measures such as avoiding
flood waters, wearing protective clothing and boots, and covering up
cuts and abrasions on their skin when engaging in outdoor activities.145
Monitoring water for infection, whether for consumption or recreation,
could help prevent exposure. Zhou and colleagues describe a PCR-based
DNA microarray system for waterborne pathogen detection in water
samples, including L. interrogans.296 Identifying contaminated water
sources can aid in the prevention of further spread.
In addition to hygiene, prevention of leptospirosis primarily depends
on immunization of animals. Human immunization has generally been
1266 SECTION 16  Bacterial Infections
restricted to individuals with high-risk occupations and in response to
epidemics or following natural disasters. Immunization of workers at
high risk for acquiring leptospirosis has been used successfully in those
working in mines in Japan and Poland, in rice fields in Italy and
Spain, and in sewers in Paris.115,267 Leptospire bacterins are available
commercially and have been evaluated for safety and efficacy in laboratory
animals and domestic livestock.40,133,213,237 The degree of protection attained
depends largely on the antigenic potential of the immunizing agent.
Requirements for the L. pomona vaccine used in cattle are such that not
more than 1 800 of the dose recommended for cattle must protect 80%
of hamsters challenged intraperitoneally 14 to 18 days after vaccination
with a dose of 100 hamster LD50. In contrast, most dogs are immunized
with a vaccine that is but 110 of the potency of that used for cattle. Most
dogs thus immunized have been protected against disease but not neces-
sarily from carrying and excreting leptospires in their urine. Trends
documenting that many cases of leptospirosis in children have been
associated with contact with dogs suggest that more stringent requirements
for the immunization of pet dogs are needed. The challenge in vaccine
efforts has been production of a safe and efficacious vaccine that promotes
cross-protection against various pathogenic serovars that will eliminate
infection and prevent chronic renal carriage.297 Much of leptospirosis
research efforts continue to focus on identification of potential vaccine
candidates.6 The availability of the genome sequence of Leptospira has
facilitated this search and provided grounds for continued attempts at
finding the ideal vaccine.119,169 A detailed review of the literature on vaccines
against leptospirosis was recently published. Adler6 concludes that despite
extensive research and increased knowledge of leptospirosis, the only
vaccine licensed for use in animals and humans are inactivated bacterins
not very different from those first used 90 years ago. There have been
and continue to be active attempts at identifying leptospiral proteins
that are able to elicit cross-protective immunity, but true success has yet
to be reached. Promising candidates under judicious study include LipL32
and LigA.6 The search for the perfect vaccine continues.128,269,278
A randomized trial of doxycycline versus placebo was undertaken
to assess the efficacy of doxycycline prophylaxis in the prevention of
infection and disease caused by leptospires during outbreaks in North
Andaman.219 Leptospiral infection was not prevented, but the patients
who received doxycycline and in whom disease subsequently developed
had lower morbidity and mortality rates.
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