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OUTLINE

 1. Keywords
 2. Introduction
 3. History
 4. Bacteriology
 5. Classification of leptospires
 6. Epidemiology
 7. Clinical presentation
 8. Laboratory diagnosis
 9. References

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CITED BY (134)




TABLES (3)

 1. Table 1
 2. Table 2
 3. Table 3




JOURNAL OF MICROBIOLOGY, IMMUNOLOGY AND INFECTION

Volume 46, Issue 4, August 2013, Pages 245-252

REVIEW ARTICLE
LABORATORY DIAGNOSIS OF LEPTOSPIROSIS: A CHALLENGE

Author links open overlay panelDidierMussoaBernardLa Scolab
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Leptospirosis is caused by pathogenic bacteria called leptospires that are
transmitted directly or indirectly from animals to humans. It occurs worldwide
but is most common in tropical and subtropical areas. It is a potentially
serious but treatable disease. Its symptoms may mimic those of a number of other
unrelated infections such as influenza, meningitis, hepatitis, dengue, or other
viral hemorrhagic fevers. The spectrum of the disease is extremely wide, ranging
from subclinical infection to a severe syndrome of multiorgan infection with
high mortality. Laboratory diagnosis tests are not always available, especially
in developing countries. Numerous tests have been developed, but availability of
appropriate laboratory support is still a problem. Direct observation of
leptospires by darkfield microscopy is unreliable and not recommended. Isolation
of leptospires can take up to months and does not contribute to early diagnosis.
Diagnosis is usually performed by serology; enzyme-linked immunosorbent assay
and the microscopic agglutination tests are the laboratory methods generally
used, rapid tests are also available. Limitation of serology is that antibodies
are lacking at the acute phase of the disease. In recent years, several
real-time polymerase chain reaction assays have been described. These can
confirm the diagnosis in the early phase of the disease prior to antibody titers
are at detectable levels, but molecular testing is not available in restricted
resources areas.

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KEYWORDS

Diagnosis
Laboratory
Leptospirosis
PCR
Serology


INTRODUCTION

The clinical presentation of leptospirosis is unspecific, misdiagnosis is
frequent, and diagnosis is based upon laboratory results. The laboratory
diagnosis of leptospirosis is challenging. The only sensitive and specific test
accurate at the acute phase of the disease is polymerase chain reaction (PCR),
which is not available in most high endemic areas and the serological reference
method by micro agglutination testing (MAT) is restricted to reference
laboratories. In this review we present the advantages and disadvantages of the
laboratory methods for leptospirosis diagnosis and we focus on the rapid tests
currently used in countries with low resources.


HISTORY

Adolf Weil reported the syndrome of icteric leptospirosis with renal failure in
1886 but the disease was recognized earlier as an occupational hazard of rice
harvesting in ancient China.1, 2 Leptospires were first visualized in autopsy
specimen from a patient thought to have had yellow fever. The role of the rat as
a source of human infection was discovered in 1917.


BACTERIOLOGY

Leptospires belong to the order Spirochaetales, family Leptospiraceae, genus
Leptospira.3 They can be pathogenic or saprophytic. Pathogenic leptospires can
be maintained in nature in the renal tubules of animals and saprophytic
leptospires in many types of wet or humid environments.


CLASSIFICATION OF LEPTOSPIRES

Prior to 1989, the genus Leptospira was divided into two species: Leptospira
interrogans (pathogenic strains) and Leptospira biflexa (saprophytic strains).
The species were divided into serovars and the serovars grouped into serogroups.
More than 24 serogroups and 250 serovars of pathogenic leptospires have been
described to date.4 The serovar concept has been widely accepted because it has
some epidemiologic value, but it has no taxonomic standing.5

The serologic classification has been replaced by a genotypic one. The
genomospecies include all L. interrogans and L. biflexa serovars. The genus
Leptospira is divided into 20 species classified into saprophytic, intermediate,
and pathogenic groups. The genomospecies of Leptospira do not correspond to the
previous species L. interrogans and L. biflexa.


EPIDEMIOLOGY

Leptospirosis is the most widespread zoonosis in the world and is considered as
an emerging global public health disease.6

It occurs worldwide with a higher incidence in warm than in temperate regions.
The number of severe human cases worldwide is estimated above 500,000.7
Incidences range from 0.1–1/100,000/year in temperate climates,
10–100/100,000/year in the humid tropics to over 100/100,000/year during
outbreaks and in high-exposure risk groups. The endemicity of the disease is
mainly located in the Caribbean, Central and South America, Southeast Asia and
Oceania.8 During the past several years, large outbreaks have occurred in many
countries, particularly in Southeast Asia, Central and South America.
Case-fatality rates range from <5% to 30%.

Effective surveillance systems with appropriate laboratory support exist in
developed countries but are often lacking in the disease-endemic developing
areas. The reported incidence of leptospirosis reflects the availability of
laboratory diagnosis and the clinical index of suspicion as much as the
incidence of the disease. For example, the actual incidence of leptospirosis in
the Asia Pacific region is not well documented9 and leptospirosis is often
underestimated.10 With the hyperendemic Southeast Asia zone, Oceania exhibits a
significant burden of leptospirosis. In the Asia Pacific region, predominantly
in developing countries, leptospirosis is largely a water-borne disease.

Numerous animals, primarily mammals, are sources of human infection. Rodents are
the most important and widely distributed reservoirs of leptospires. Some
serovars are associated with a particular species of natural maintenance host.
In chronic infections, leptospires are localized in the kidneys, usually without
detectable clinical manifestations.

The usual mode of contamination is abrasions or cuts in the skin or via the
conjunctiva through direct or indirect contact with urine or tissues of infected
animals. Other modes of contamination, such as inhalation of water or aerosols,
animal bites, or interhuman transmission, have been rarely demonstrated.

Leptospirosis is an occupational disease for veterinarians, farmers, abattoir
workers, butchers, hunters, rodent control workers, and other occupations
requiring contact with animals. Indirect contact with contaminated wet soil or
water is responsible for the great majority of cases in the tropics, either
through occupational exposure as in rice or taro farming, flooding after heavy
rains, or exposure to damp soil and water during avocational activities.
Contamination due to recreational exposures is increasing, often in association
with adventure tourism in tropical endemic areas.

Three epidemiological patterns have been defined: in temperate climates where
few serovars are involved and human infection occurs by direct contact with
infected animals; in tropical wet areas where there are many more serovars
infecting humans and animals and larger numbers of reservoir species; and in the
urban environment as a rodent-borne infection.11


CLINICAL PRESENTATION

It may range from a flu-like illness to a serious and sometimes fatal disease.
Confusion with other diseases, especially dengue fever and other hemorrhagic
fevers, is frequent in the tropical areas. The mean incubation time is 1–2
weeks, with a range of 2 days to 30 days. The acute or septicemic phase lasting
about 1 week is followed by an immune phase characterized by antibody
production. The great majority of infections are subclinical or of very mild
severity. The most common symptoms are febrile illness of sudden onset, chills,
headache, myalgia, abdominal pain, and conjunctival suffusion. Other clinical
presentations may be predominant, in addition to hepatic or renal dysfunction,
leptospirosis should be seriously considered in patients with pulmonary symptoms
and fever, especially in subtropical and tropical areas, as reported in a
retrospective study conducted in Taiwan.12


LABORATORY DIAGNOSIS


SPECIMEN COLLECTION

Several blood tubes should be collected at the early phase of the disease:
standard blood culture bottle or tube; nonadditive or gel separator tubes for
chemistry and serology; and EDTA tube for blood count.

For blood culture, blood with heparin to prevent clotting is recommended but
ideally blood is inoculated directly into blood culture bottles containing
culture medium for leptospires.

For molecular testing, published studies showed mixed results13: serum was
reported to be inferior to plasma14, 15; serum was reported to be superior to
whole blood; and buffy coat was reported to be superior to plasma and serum.16
Heparin was reported to be inhibitory.17

All blood samples must be conserved for subsequent additional testing. Acute
blood samples are of great importance for serology in order to demonstrate a
seroconversion.


NONSPECIFIC LABORATORY FINDINGS

The various nondiagnostic abnormalities are reported in Table 118; these can
only suggest leptospirosis. Specific microbiological tests are required for
confirmation.

Table 1. Nonspecific laboratory findings

1. Blood analysis1.1. Leukocytosis with a shift to the left1.2. Thrombocytopenia
in >50%181.3. Elevated1.3.1. Erythrocyte sedimentation rate1.3.2. Creatinine
(usually <20–80 mg/L)1.3.3. Urea (usually <1000 mg/L)1.3.4. Aminotransferases
(rarely > 200 IU/L)1.3.5. Bilirubin (may rise to 800 mg/L)1.3.6. Alkaline
phosphatase2. Urine analysis2.1. Proteinuria, pyuria, microscopic hematuria,
hyaline, and granular casts3. Cerebrospinal fluid analysis3.1. Normal or
slightly elevated cerebrospinal fluid pressure3.2. Initially a predominance of
polymorphs or lymphocytes (total cell counts generally <500 × 106/L) and
lymphocytes predominance later. Pleocytosis can persist for weeks3.3. Elevated
protein (50–100 g/L)3.4. Glucose is usually normal3.5. Xanthochromia may occur


MICROSCOPIC DEMONSTRATION

Leptospires cannot be observed under the ordinary light microscope but by
darkfield microscopy as thin, coiled, and rapidly moving microorganisms.
Sensitivity of darkfield microscopy is approximately 107 leptospires/L. Direct
examination of blood and urine has both low sensitivity and specificity, it is
subject to misinterpretation of fibrin or protein threads, then is not
recommended as a routine procedure.

Leptospires are not stained by conventional Gram staining. Available staining
methods to increase the sensitivity of direct examination are:
immunofluorescence, immunoperoxidase, silver staining, Warthin-Starry staining,
immunohistochemistry, and in situ hybridization. All of these suffer from the
same drawbacks as darkfield microscopy: a high risk of false-positive and
false-negative results.


ISOLATION OF LEPTOSPIRES

Samples for culture should be collected prior to the administration of
antibiotics. Blood, cerebrospinal fluid and dialysate should be cultured in the
first 10 days of the illness, and urine from the second week of the illness.
Several specific media were described by Fletcher et al. The most used medium is
based on the oleic acid-albumin Ellinghausen-McCullough-Johnson-Harris (EMJH)
medium (Becton Dickinson and Company, Difco™) and is available commercially.

Samples should be stored and transported at ambient temperatures. Survival of
leptospires in human urine is limited so urine should be processed immediately.
Cultures are incubated in the dark at 28–30°C and examined weekly by darkfield
microscopy for up to 13 weeks prior to being discarded.


ANTIGEN DETECTION

Different antigen detection tests have been developed but none of them is
sensitive enough to be routinely used.19


ANTIBODY DETECTION

The MAT, which is the serological reference test, was first described in 1918 by
Martin and Pettit. Live antigens representing different serogroups are reacted
with serum samples and the agglutination is examined by darkfield microscopy.
Panels of live leptospires belonging to different serovars must be maintained in
the laboratory. As a minimum, the panel should include all locally circulating
serovars and, if these serovars are unknown or subject to change, the panel
should include serovars representing all serogroups. An incomplete panel should
be responsible for false negative results. MAT may be positive from Day 10–12
after the onset of illness, sometimes later if specific antibiotics have been
prescribed. MAT was reported to have a sensitivity of 41% during the 1st week,
82% during the 2nd to 4th week, and 96% beyond the 4th week of illness.20 The
cut off value on a single sera depends from the seroprevalence. For the Center
for Disease Control, a probable case is defined as a titer ≥200 associated with
a clinically compatible illness21; in a publication from the Center for Disease
Control of Taiwan, an antibody titer ≥100 was regarded as a probable case of
leptospirosis22; in a study conducted in Thailand a positive MAT was defined as
a single titer ≥400.23 The Leptospirosis Burden Epidemiology Reference Group
consider a single MAT ≥1:400 (or single MAT ≥1:100 in nonendemic regions) to be
consistent with leptospirosis. A low titer is appropriate in a population in
which exposure to leptospirosis is uncommon but, if exposure is frequent, as in
most tropical countries, a higher cut-off titer is necessary. In very high
endemic areas, a single titer of 800 in symptomatic patients is generally
indicative but a 1600 titer has been recommended. In cases of previous infection
with a different serogroup, interpretation is complicated by the “anamnestic
response” (the rise in antibody titer is directed against a previous infecting
serovar). A fourfold or greater rise in titer between paired sera is required to
confirm leptospirosis. MAT detects both class M and class G antibodies, and
cannot differentiate between current, recent, or past infections. It may
identify the presumptive serogroup, and under the best conditions the serovar
because interpretation is complicated by the high degree of cross-reaction that
occurs between different serogroups, especially in acute-phase samples.

The MAT is complex to control and perform; it cannot be standardized because
live leptospires are used as antigens.

Enzyme-linked immunosorbent assay (ELISA) detects antibodies reacting with a
broadly reactive genus-specific antigen and thus is not suitable for
identification of the causative serovar or serogroup. Commercial kits are
available. The cut-off point is determined on the same considerations as for the
MAT. Serogroups Grippotyphosa and Australis gave false negative results. ELISA
is usually positive from Day 6–8, earlier than the MAT, and it may be negative
earlier. Most of the commercial ELISA kits use as antigen the nonpathogenic
Leptospira biflexa patoc strain. ELISA allows detection of specific IgM class
antibodies. IgM may remain detectable for several months or even years. Positive
ELISA should be confirmed by MAT.

Other serological tests have been developed: complement fixation,
counterimmunoelectrophoresis, indirect fluorescent antibody, indirect
hemagglutination (IHA), sensitized erythrocyte lysis, latex agglutination (LA),
macroscopic slide agglutination, microcapsule agglutination, and Patoc slide
agglutination.

Rapid screening tests based on four immunological principles are used: particle
agglutination (centrifugation of whole blood required, detection of a weak
agglutination is difficult, reagents often require refrigeration); immunodot or
dipstick/comb (results are visualized as a spot, dot, or line, test requires
less than 30 minutes to develop, reagents do not require refrigeration);
immunofiltration or flow-through device (the assay require several steps,
reagents often require refrigeration); immunochromatography or lateral flow (a
visible line at test and control location indicates a positive reaction, no
special equipment required, they are one step tests and are completed within 15
minutes; depending on the assay, whole blood, serum, or plasma can be used,
reagents do not require refrigeration).24


EVALUATION OF RAPID SCREENING TESTS

Rapid tests are easy to use and can be performed by individuals without special
technical training. Some of them can be performed on whole blood and can be
stored for prolonged periods at ambient temperatures, and standard laboratory
equipment is not required. Even though the reading and interpretation of rapid
test reactions is claimed to be simple, some training is required to perform and
interpret them correctly. Interobserver variability in reading and
interpretation of the end points may provide inconsistent results.

These tests are primarily IgM detection assays, but because IgM is not
detectable until the second week after symptom onset, they have low sensitivity
in the early acute phase of illness when patients present for medical
treatment.25

In a large multicenter evaluation of an IgM Leptospira dipstick assay conducted
in areas with high and low leptospirosis endemicity, the mean sensitivity was
60.1% on sera collected within the first 10 days of the illness and the results
were concordant with an ELISA IgM.26

Four rapid tests (ELISA IgM, IHA, IgM dipstick assay, IgM dot-ELISA dipstick
test) were evaluated: the sensitivity ranged from 38.5% (IHA) to 52.7% (IgM
dipstick assay) on acute sera collected prior to 14 days after onset of the
disease, by comparison, the sensitivity was 48.7% with MAT.27 Sensitivity on
convalescent sera ranged from 67.2% to 84.4% and was 93.8% for MAT.

Eight rapid tests (IHA, 2 IgM dipstick assay; indirect fluorescent antibody, 3
ELISA IgM, LA) have been evaluated in Hawaii and the authors concluded that all
tests were insensitive for diagnosis within the first week of the disease while
it is during this time that important therapeutic decisions are likely to be
made.28 Evaluation of two rapid tests at the acute visit for leptospirosis (IgM
dipstick assay, LA) and dengue (IgM dipstick assay, Dengue duo rapid strip) in a
tropical field setting yielded sensitivity from 13% to 22 % for leptospirosis
(positive predictive value range, 15–18%) and from 8% to 19% for dengue with the
conclusion that their utility at the acute phase of dengue and leptospirosis is
limited.29

Because of their low sensitivities, use of these tests for the initial
management of acute mild leptospirosis in adults was inferior to empirical
treatment in a study conducted in Thailand.30

The low sensitivity of these tests at the acute phase of the disease is not
related to the rapid test format but is due to the fact that the tests detect
IgM antibodies.


MOLECULAR DIAGNOSIS

The need for rapid diagnostics at the time of admission has led to the
development of numerous PCR assays. Their advantage lies in the ability to
obtain a definitive diagnosis during the acute stage of the illness prior to
antibodies are detectable, while treatment may be effective.

PCR detects DNA in blood in the first 5–10 days after the onset of the disease
and up to the 15th day. The bacterial load in serum/blood ranges from 105 to 109
leptospires/L.

PCR allows detection of leptospires in culture negative blood if the patient has
received an effective antimicrobial drug but have not cleared nonviable
organism.31

PCR is based on the detection of genes universally present in bacteria as
gyrB,32 rrs (16S rRNA gene),33 secY34; or genes restricted to pathogenic
Leptospira spp. as lipL32, lfb1,35 ligA, and ligB2.36

Conventional PCR assays have not been well evaluated, leaving its diagnosis
value unclear.37, 38 It has been replaced by real-time quantitative PCR (qPCR),
which combines amplification and detection of amplified product in the same
reaction vessel with excellent sensitivity and specificity and low contamination
risk.39 Detection can be performed using SYBR Green, which provides sensitive
detection but is less specific than detection using fluorescent probe technology
such as TaqMan probes.

A number of qPCRs have been introduced: SYBR Green qPCR targeting secY or
lipL32; TaqMan qPCR targeting lipL32; rss (16S); and a multiplex assay for
simultaneous detection and differentiation of pathogenic and nonpathogenic
leptospires.40

Four qPCR, SYBR green, and TaqMan assays targeting the secY, lfb1, and lipL32
genes have been recently evaluated. They detected from 105 bacteria/L to 106
bacteria/L of pure culture, whole blood, plasma, and serum samples. The authors
recommend a continual evaluation and, if necessary, modification of the primers
and/or probes used to ensure effective detection of the circulating leptospires
isolates. Lyophilized reagent-based PCR assay for the detection of leptospires
have been developed.41


TYPING METHODS

Severe cases can be due to all infective serovar. Identification is not required
for clinical care but is of particular interest from the public health
perspective. It may indicate the sources of infection and reservoirs and thus
contribute to the choice of methods for prevention and control.

Antigen–antibody reactions, such as MAT, can be used to identify strains, but
are laborious and time-consuming, which restricts their use to specialized
laboratories. In serogroup determination the antigen suspension of the unknown
strain is used in titrations with several antisera representing all recognized
serogroups; in the cross-agglutination-absorption test, the reaction of the
unknown strain and its antiserum is compared with reference strains and their
antisera, typing by monoclonal antibodies is based on the recognition of antigen
patterns of serovars by panels of monoclonal antibodies.

As serotyping is complex and can only be performed in reference laboratories, a
number of molecular techniques have been developed as alternatives to or in
complement to serotyping including: DNA–DNA hybridization, restriction fragment
length polymorphisms, pulsed-field gel electrophoresis, ribotyping, PCR-based
typing, insertion sequences based typing, amplification with specific primers,
variable number of tandem repeats, low-stringency single specific primer PCR,
PCR restriction endonuclease analysis, arbitrarily primed multiple locus
sequence typing, random amplification of polymorphic DNA, and determination of
sequences of PCR products.42

The usual target for sequence-based identification of Leptospira species is the
16S rRNA gene.43 Other genes can be used, such as rpoB encoding the β-subunit of
RNA polymerase44, 45, 46 or gyrB encoding the β-subunit of DNA gyrase.


SUSCEPTIBILITY TESTING

Susceptibility testing is not routinely performed due to the long incubation
time required and the difficulty in quantifying growth accurately.


SAFETY PROCEDURE

Standard microbiological laboratory safety procedures are required when working
with leptospires (Biosafety Level II facilities).


DEFINITION OF LEPTOSPIROSIS

The Leptospirosis Burden Epidemiology Reference Group definitions of
leptospirosis are reported in Table 2.

Table 2. Leptospirosis Burden Epidemiology Reference Group definitions of
leptospirosis

1. Definitive case: symptoms consistent with leptospirosis and any one of the
following:1.1. 4-fold increase in MAT titer between acute and convalescent serum
samples1.2. Single MAT ≥1:400 (or single MAT ≥1:100 in nonendemic regions)1.3.
Isolation of Leptospira spp. from a normally sterile site1.4. Detection of
Leptospira spp. in clinical samples using histological, histochemical, or
immunostaining techniques1.5. Leptospira DNA detected by PCR2. Presumptive case:
symptoms consistent with leptospirosis and any one of the following:2.1.
Presence of IgM antibodies, as shown by ELISA or dipstick2.2. Presence of IgM or
IgG antibodies, as shown by immunofluorescence assay


THE MOST RELEVANT TESTS

Laboratory testing depends on the temporal stage of the disease, its prevalence,
the presence of a laboratory and if present the availability of specific tests.
Advantages and disadvantages of common diagnostic tests for leptospirosis are
reported in Table 3.

Table 3. Advantages and disadvantages of common diagnostic tests for
leptospirosis

Empty CellMicroscopic demonstrationCultureSerology MATSerology ELISA IgMSerology
rapid tests IgMMolecular testingSpecimen collectionBlood, urine, CSFBlood,
urine, CSF, tissuesBloodBloodBloodBlood, urine, CSF, tissuesWindow of
positivity1st wk: blood, CSF1st 10 dFrom Day 10–12From Day 6–8From Day 6–8From
Day 5–10 in blood2nd wk: urineProcessing timeAvailable in 1 h2 wk to 4 moSeveral
wks if not locally availableAvailable in 1 dAvailable in 15–30 minAvailable in 1
dEarly diagnosisNoNoNoNoNoYesDefinitive diagnosis if positiveNoYesYes
(seroconversion)Yes (seroconversion)Yes (seroconversion)YesIdentificationNoYes
(if MAT or molecular testing available)YesNoNoYes (by additional molecular
tests)RemarkLow sensitivity and specificityLow sensitivity, slow, difficultGold
standard but very difficultNeeds confirmation by MATNeeds confirmation by MATThe
only sensitive test at the acute phaseNot recommended for diagnosisEquipment
requiredDark field microscopeSpecific culture mediaReference laboratory
onlyStandard laboratoryLaboratory equipment not requiredSpecial equipment,
dedicated laboratory space, highly skilled personnel

Within the first days of the disease, the only sensitive and specific test is
PCR. At this stage, rapid diagnosis is only possible if quick and easy molecular
testing is possible. In resource-restricted countries, the cost and requirement
for special equipment and technical expertise remain as barriers limiting its
use. As in most endemic area, molecular testing is not available in general
practice, confirmation of the diagnosis cannot be obtained rapidly.

From the second week on the disease, serological diagnosis is based on the
detection of specific IgM. If a laboratory is present, all serological tests can
be performed, expect MAT; in the absence of laboratory, rapid tests can be
performed.

If the time from the onset of the illness is not indicated, we strongly suggest
that the laboratory contacts the physician in order to perform the most relevant
test: in the 1st week the negative predictive value of serology is very low, and
from the 2nd week the negative predictive value of PCR is very low. In our
laboratory we perform both tests, and despite frequent information of the
clinicians, inappropriate tests are prescribed and are responsible for
misdiagnosis.

The low level of concordance between PCR, MAT, and ELISA IgM reflects the phases
of the disease suggesting that molecular and serological methods may be used in
different periods.47

Treatment should be initiated as soon as the diagnosis of leptospirosis is
suspected and preferably prior to the fifth day after the onset of illness.
Clinicians should not wait for the results of laboratory tests prior to starting
treatment.

Even tests with high sensitivity and specificity may have limited utility in
general use because of low predictive values. The predictive value of a test
varies with the prevalence of the disease in the target population The positive
predictive value of a rapid diagnostic tests for leptospirosis was poor at both
acute and convalescent visits because of the low prevalence of the disease in
the population of febrile patients in Thailand.

In many developing countries, including most of the leptospirosis endemic areas,
laboratory capabilities to detect pathogenic microorganisms are often
inadequate. Sometimes, basic necessities and equipment are missing such as
electricity, refrigerators, and trained laboratory personnel. Because of their
ease of use, even in primary health centers, rapid tests are often used in
routine practice in many clinical settings. However, these rapid diagnostic
tests may not reach optimal sensitivity until at least a week after onset of
fever, well after the time when patients first present to medical care. As the
sensitivity of the tests is low at the acute visit, these rapid diagnostic tests
should be used with caution to rule out leptospirosis, the same restriction
should be considered when using ELISA IgM tests.

Recommended articles



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CITED BY (134)


 * EVALUATION OF ANTI-LIPL32 CARBON NANOTUBE IMMUNOFLUORESCENCE PROBE
   (CARBO-LIP) AND COMPARISON WITH MAT, IGM ELISA, IGM SPOT TEST AND CULTURE FOR
   EARLY DETECTION OF LEPTOSPIROSIS AT LOCAL HOSPITAL
   
   2022, Journal of Microbiological Methods
   Show abstract
   
   Leptospirosis is an emerging public health problem affecting people mainly
   from tropical and subtropical regions. Therefore, there is a need for rapid
   and sensitive tests for proper and prompt treatment. Recently we have
   demonstrated Carbo-Lip probe, which was fabricated through immuno recognition
   method with fluorescent dye functionalized LipL32 monoclonal antibodies,
   secondary antibody and Leptospira for rapid and accurate diagnosis. In an
   effort to validate Carbo-Lip, we collected clinical samples from a cohort of
   104, consisting of 26 positive, 40 negative and 38 unconfirmed cases of
   Leptospirosis. Subsequently, the test was also compared and validated with
   the gold standard method microscopic agglutination test (MAT), IgM ELISA, IgM
   spot test, and culture. Carbo-Lip exhibited a sensitivity of 75% with
   specificity of 92.3% for Leptospirosis in comparison with MAT. The fabricated
   Carbo-Lip sensor could be used as a potential diagnostic tool for early
   detection of Leptospirosis in patients from endemic areas.


 * COMPARISON OF ELISA USING RECOMBINANT LIPL32 AND SONICATED ANTIGEN OF
   LEPTOSPIRA FOR DETECTING BOVINE LEPTOSPIROSIS
   
   2022, Acta Tropica
   Show abstract
   
   Leptospirosis is one of the most widely distributed zoonosis in the world.
   Bovine leptospirosis is a serious problem in bovine production, causing
   reproductive losses. The aim of this work was to compare recombinant LipL32
   with sonicated antigen for detecting anti-Leptospira IgG antibodies in bovine
   serum using ELISA. The Microscopic Agglutination Test (MAT) is used as the
   gold standard. Sonicated antigen from cultures of Leptospira interrogans
   serogroup Icterohaemorrhagiae serovar copenhageni (strain M20) was used for
   the eELISA and rLipL32 for the rELISA. The performance of these assays was
   evaluated using serum samples from 166 bovines, 69 MAT positive and 97 MAT
   negative. At the optimal cut-off point recommended by the receiver operating
   characteristic (ROC) curve analysis, the sensitivity and specificity values
   were 98.6% and 97.9%, respectively, for eELISA, and 85.5% and 86.6%
   respectively, for rELISA. The value for the area under the ROC curve was
   0.998 (0.994-1.0) (CI 95%) for eELISA and 0.929 (0.891-0.968) (CI 95%) for
   rELISA. The ROC curves for rLipL32 and sonicated antigen showed statistically
   significant differences (z = -3.826; p = 0.000). A three-way comparison
   showed statistically significant differences in the sensitivity and
   specificity of rELISA and eELISA. Our results showed that eELISA was more
   specific and sensitive than rELISA. The difference in performance
   (eELISA-rELISA) was 13.4% (4.03-23.28) (CI 95%) for sensitivity and 11.34 %
   (4.07-19.56) (CI 95%) for specificity. Our results show that the eELISA has a
   better diagnostic performance than rELISA for the detection of
   anti-Leptospira IgG antibodies in bovine serum.


 * LEPTOSPIRAL INFECTIONS IN HUMANS
   
   2021, Clinical Microbiology Newsletter
   Show abstract
   
   Leptospirosis is a globally widespread spirochetal infection spread from
   animals to humans. Infections are common in settings of endemicity, primarily
   in tropical regions of the world. Leptospirosis is typically a self-limited
   febrile illness but may progress to potentially fatal multiorgan system
   failure. Patients often present with a nonspecific acute febrile illness that
   is clinically difficult to distinguish from other similarly presenting
   infections endemic to tropical regions, including dengue fever, influenza,
   and malaria. A high index of suspicion is essential to early identification
   of patients who may benefit from antimicrobial therapy. Diagnostic testing is
   key to both recognition of early infection and outbreak investigation,
   typically in the setting of water exposure after heavy rainfall and flooding.
   This review focuses on the epidemiology, clinical manifestations, and
   laboratory diagnosis of leptospirosis, including nucleic acid amplification
   tests, culture, direct detection, and serological approaches.


 * A GRAVEYARD KEEPER WITH SEVERE HYPERBILIRUBINEMIA CAUSED BY LEPTOSPIROSIS
   
   2021, Journal of Microbiology, Immunology and Infection
   
   


 * DEVELOPMENT OF MONOCLONAL ANTIBODIES AGAINST RECOMBINANT LIPL21 PROTEIN OF
   PATHOGENIC LEPTOSPIRA THROUGH PHAGE DISPLAY TECHNOLOGY
   
   2021, International Journal of Biological Macromolecules
   Show abstract
   
   Leptospirosis is a potentially fatal zoonosis that is caused by spirochete
   Leptospira. The signs and symptoms of leptospirosis are usually varied,
   allowing it to be mistaken for other causes of acute febrile syndromes. Thus,
   early diagnosis and identification of a specific agent in clinical samples is
   crucial for effective treatment. This study was aimed to develop specific
   monoclonal antibodies against LipL21 antigen for future use in leptospirosis
   rapid and accurate immunoassay. A recombinant LipL21 (rLipL21) antigen was
   optimized for expression and evaluated for immunogenicity. Then, a naïve
   phage antibody library was utilized to identify single chain fragment
   variable (scFv) clones against the rLipL21 antigen. A total of 47 clones were
   analysed through monoclonal phage ELISA. However, after taking into
   consideration the background OD405 values, only 4 clones were sent for
   sequencing to determine human germline sequences. The sequence analysis
   showed that all 4 clones are identical. The in silico analysis of scFv-lip-1
   complex indicated that the charged residues of scFv CDRs are responsible for
   the recognition with rLipL21 epitopes. The generated monoclonal antibody
   against rLipL21 will be evaluated as a detection reagent for the diagnosis of
   human leptospirosis in a future study.


 * CIRCULATING SEROGROUPS OF LEPTOSPIRA IN SWINE FROM A 7-YEAR STUDY IN FRANCE
   (2011–2017)
   
   2022, Porcine Health Management
   
   

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