Leptospirosis Diagnosis: Competancy of

Various Laboratory Tests

Suman Veerappa Budihal1 and Khalid Perwez2
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Abstract
Leptospira can be found in virtually all tropical and temperate areas of the world and is presumed
to be the most wide spread zoonoses in the world.Humans contact leptospirosis through mucosal
or percutaneous exposure to leptospires in environments contaminated by the urine of
chronically infected animal sources. Despite being common, the diagnosis of leptospirosis is
often not made unless a patient presents with textbook manifestations of the so called Weil’s
disease, such as fever plus jaundice, renal failure and pulmonary haemorrhage. Leptospiral
infection often has minimal or no clinical manifestations; of the cases in which fever develops, as
many as 90% are undifferentiated febrile illnesses. Because of the variety of clinical symptoms
seen in the symptomatic cases, leptospirosis at its onset is often misdiagnosed as aseptic
meningitis, influenza, hepatic disease or fever (pyrexia) of unknown origin. Moreover, clinicians
may fail to recognize that transmission of leptospirosis can occur in the urban setting because it
is incorrectly perceived to be a rural disease. Therefore, diagnosis is based on laboratory tests
rather than on clinical symptoms alone. In developing countries, laboratory facilities may be
inadequate for diagnosis despite a high prevalence of the disease. Of substantial clinical
importance, the syndrome of leptospiral pulmonary haemorrhage has emerged in recent years, in
diverse places around the world.

Keywords: Leptospirosis, Weil’s disease, PCR (Polymearase Chain Reaction), MAT

(Microscopic Agglutination Test)
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Introduction
Leptospirosis is presumed to be the most wide spread zoonoses in the world [1]. The disease
leptospirosis is described as an occupationally transmitted disease. Humans contract leptospirosis
through contaminated urine of chronically infected animal domestic or agricultural rodents, dogs,
pigs and cattle [2]. Environmental conditions are an important influence on the incidence of
leptospirosis; the disease is rare in deserts, common in warm, humid, tropical areas and seasonal
rains and severe weather are associated with increased frequency of disease. Leptospirosis is
found in a wide variety of environmental contexts, in industrialized and developing countries,
and in urban and rural contexts [3]. In India, outbreaks have been reported related to heavy
rainfall in various parts of the country. In South-India, suspected cases are reported between June
and October due to heavy rains and floods. Leptospirosis has been consistently reported from the
Andaman and Nicobar group of Islands (thus called ‘Andaman Haemorrhagic Fever’) West
Bengal, Kerala and Coastal Karnataka, India [4,5].

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Discussion
Leptospirosis at its onset is often misdiagnosed as aseptic meningitis, influenza, hepatic disease
or fever (pyrexia) of unknown origin [6]. Despite being common, the diagnosis of leptospirosis is
often not made unless a patient presents with textbook manifestations of the so called Weil’s
disease, such as fever plus jaundice, renal failure and pulmonary haemorrhage. Leptospiral
infection often has minimal or no clinical manifestations; of the cases in which fever develops, as
many as 90% are undifferentiated febrile illnesses. Moreover, clinicians may fail to recognize
that transmission of leptospirosis can occur in the urban setting because it is incorrectly
perceived to be a rural disease. Therefore, diagnosis is based on laboratory tests rather than on
clinical symptoms alone. In developing countries, laboratory facilities may be inadequate for
diagnosis despite a high prevalence of the disease. Of substantial clinical importance, the
syndrome of leptospiral pulmonary haemorrhage has emerged in recent years, in diverse places
around the world.

Two important issues continue to confront clinicians regarding leptospirosis. The first is how to
reliably establish the diagnosis. The most common way to diagnose leptospirosis is through
serological tests either the Microscopic Agglutination Test (MAT) which detects serovar-specific
antibodies, or a solid-phase assay for the detection of Immunoglobulin M (IgM) antibodies.
Leptospira are present in the blood until they are cleared after 4-7 days following the production
of Leptospira-specific antibodies, initially mainly of the IgM class [7,8]. However, the greatest
drawback of IgM detection assays is that IgM antibodies can persist for many months raising the
questions about whether a positive IgM result accurately identifies a current infection [9].

The MAT is the cornerstone of the serodiagnosis for leptospirosis, because this assay has a high
sensitivity and allows for the detection of group specific antibodies [10]. Two major
disadvantages of this test are that in regions where leptospirosis is common, there may be a
substantial proportion of the population with elevated titres of MAT and secondly, the
performance of MAT is restricted to laboratories that are capable of maintaining strains for the
preparation of live antigens [11]. Therefore, serological tests remain suboptimal for clinical use
in diagnosing leptospirosis as depicted in [Table/Fig-1]. The most promising diagnostic methods
are those that demonstrate the presence of the organisms.

[Table/Fig-1]:

Advantages and disadvantages of diagnostic tests for the detection of Leptospirosis

Test Advantages Disadvantages
Dark Field Visualize Leptospirosis Lack of sensitivity and specificity. 104
Microscopy (DFM) Leptospires/ml is necessary for one
organism/field to be visible under DFM.
IgM ELISA Most widely used False positive, IgM cannot be detected in early
stages of infection and canpersist in blood for
years.
Microscopic Gold Standard Less sensitive in early phase of disease. Labor
Agglutination Test intensive and complicated procedure as there
(MAT) is a need to maintain Leptospira strain for
preparing liveantigen.
Polymerase Chain Successful in detecting Expensive reagents, Requires large quantity of
Reaction (PCR) Leptospira DNA in serum DNA. Cannot identify the infecting serovar.
and urine samples of
patients
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Culture of Leptospira is difficult for a variety of reasons. The process is very laborious, and can
take up to three months [12]. Therefore, isolation and culture are primarily used for retrospective
diagnosis. Moreover, to culture the organism from tissues or body fluids, knowledge of the stage
of infection is critical. In the acute phase, which lasts for about 10 days, the leptospires can often
be cultured from blood or cerebrospinal fluid (CSF). Usually, when a specific antibody response
is detected (at approximately 10 days), leptospires disappear from the blood. During the second
phase, which may last up to several months, bacteriuria is often intermittent.

Molecular techniques to detect the presence of leptospiral DNA in blood, urine or spinal fluid
have shown to be sensitive and specific; Sensitive assay for the detection of Leptospira DNA that
is based upon amplification of the Leptospirarrs (16S) gene have been developed [13]. The data
suggest that the PCR assay can be used on biological samples such as CSF, urine, or blood as a
diagnostic tool for cases of suspected leptospirosis. The use of this technique is precluded by the
cost and technical factors in non-reference laboratories.

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Historical Aspects
Leptospirosis is an emerging infectious disease caused by pathogenic species of the genus
Leptospira that affects domestic and wild animals worldwide [14]. The classical description of
leptospirosis is that of Weil’s disease, a dramatic acute febrile and sometimes epidemic illness
characterized by jaundice, splenomegaly and nephritis. This discovery by Weil in 1886, though
not the first of its type, antedated the discovery of the infectious agent by about 30 years when it
was discovered and described simultaneously in 1915 by Inada & Ido in Japan and Uhlenhuth &
Fromme in Europe [15]. Stimson demonstrated by silver staining clumps of organisms with
hooked ends and named them Spirochaetainterrogans for their resemblance to a question mark
[16]. It affects more than 160 mammalian species, rodents being the most important reservoir,
though other animals are also affected.

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Taxonomy and Classification

The genus Leptospira belongs to the Leptospiraceae family of the order Spirochaetales. The
nomenclature system used to organize leptospires has been revised, making review of the
literature often confusing. The traditional system divided the genus into two species: the
pathogenic Leptospirainterrogans and the nonpathogenic Leptospirabiflexa. These species were
divided further into serogroups, serovars, and strains, based on shared antigens. L interrogans
included more than 250 serovars that constitute 25 serogroups [17].

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Disease Onset and Progress

Leptospiraare excreted in the urine of the infected host and can survive in soil and infect a
susceptible host by penetration through abraded skin, mucosa, conjunctiva, or intact skin after
prolonged immersion in water, and ingestion through water or food, droplet infection [18].
Animals, including humans can be divided into maintenance host and accidental host. The
disease is maintained in nature by chronic infection of the renal tubules of maintenance hosts
[19]. Incubation period varies from 4-20 days though it usually manifests within 6-8 days.
Occupation is a significant risk factor for humans [20]. Direct contact with infected animals
accounts for most infection in farmers, veterinarians, abattoir workers, meat inspectors, pest
control workers etc. Indirect contact is important for sewer workers, miners, soldiers, rice field
workers etc [21].

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Pathophysiology
Leptospire invasion across the epithelium is followed by proliferation and widespread
dissemination. Every major organ system may be affected, and leptospire antigens can be
detected in affected tissues. Leptospire-mediated injury characterizes the initial phase of the
disease. A host-immune response marks onset of the second phase of symptoms [22].

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Clinical Features of Leptospirosis

Symptoms
Symptom onset often occurs abruptly after the 2- to 20-day incubation period. Direct tissue
injuries from leptospire invasion and toxins, which have been theorized yet never clearly
elucidated, characterize the acute phase. Symptoms then abate with cessation of the systemic
proliferation of leptospires.

The second or immune phase is characterized by increasing antibody titers and inflammatory
infiltration of affected organ systems. Aseptic meningitis and renal dysfunction are hallmarks of
the immune phase. Symptoms may persist for 6 days to more than four weeks, with a mean
duration of 14 days.

Approximately 10% of patients diagnosed with leptospirosis develop signs of Weil disease. The
classic definition of Weil disease is severe leptospirosis presenting with jaundice, renal failure,
and pulmonary hemorrhage. Mortality rates among these patients is 10%, despite care in an
Intensive Care Unit (ICU), and even higher in regions with less sophisticated care. Severe, fatal
cases of leptospirosis may occur without associated jaundice.

In both children and adults, leptospirosis commonly presents with fever, myalgia, and headache.
Lethargy, emesis, abdominal pain, photophobia, arthralgia, cough, diarrhea, or constipation also
may occur. The differential diagnosis for these symptoms is confounding and ranges from benign
viral syndromes of childhood to meningitis and sepsis [23].

Laboratory diagnosis of leptospirosis

Laboratory diagnosis of leptospirosis is mandatory because the clinical picture is not specific in
either humans or animals, moreover, in endemic regions, existence of similar infections can
cause confusion in the diagnosis.

The various diagnostic tools available for the detection of leptospirosis are enumerated
hereunder.

General Clinical Laboratory Findings

A. Erythrocyte Sedimentation Rate is elevated, WBC counts range from below normal to
moderately elevated.

B. Liver Functions Tests show an elevation in aminotransferases, bilirubin and alkaline

phosphatase, hyperbilirubinaemia is out of proportion to jaundice in cases of icteric leptospirosis.

C. Renal Function Tests are usually impaired as indicated by raised plasma creatinine.

D. Urine Analysis demonstrates proteinuria, pyuria, microscopic haematuria, hyaline and

granular casts.

E. Lumbar Puncture reveals an elevated CSF pressure, predominance of lymphocytes and

polymorphs.
F. Peripheral Blood Smear shows peripheral leukocytosis with shift to left and
thrombocytopenia.

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Direct Diagnostic Methods

Microscopy

Direct Microscopic observation is used to detect leptospires in body fluids, check culture
growths etc. Dark Field Microscopy is the usual method, but immunostaining is useful in certain
special circumstances

Darkfield and phase contrast: Leptospires are seen as thin, bright, actively motile rods, moving
with characteristic rapid spinning and jerking motility. Approximately, 10 leptospires/mL are
necessary for one cell per field to be visible by darkfield microscopy. However, the positivity of
darkfield microscopy decreases from 100% to 90.9% with increase in the duration of infection
for greater than 1 week. Another disadvantage of this technique is that both false positive and
false negative diagnosis can be easily made even in experienced hands [24].

Histochemical stains: A variety of histopathological stains have been used for the detection of
leptospires in clinical specimens. The first to be used were the silver stains. The Wrthin-Starry
stain is widely used now [25].

Immunostaining: It may be used to find leptospires where they are scarce, or where there is
material that precludes the use of darkfield microscopy. But any immunosatin requires a primary
antibody specific for the serovar being sought, on its own or in a pool or composite mixture of
antibodies to different serovars. Too many varieties in a pool will dilute any one, so high titre
antisera conjugates are required. In other words, it may be not be advantageous in early
infections [26].

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Culture
Fluid media are used for primary culture. Greater yields and faster growths are obtained in
Tween (oleate)-albumin media such as EMJH (Ellinghausen, McCullough, Johnson, Harris) than
media with rabbit serum (8-10% v/v). Media with rifampicin, neomycin, actidione are used for
primary isolation from contaminated samples. The culture of these organisms takes almost 3
months and is thus, impractical for immediate diagnosis. The organism has a relatively long
doubling time (6-8 hours or more). Additionally, they are highly infectious organisms requiring
‘Biosafety level II’ facilities [27].

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Molecular Methods
Direct Polymerase Chain Reaction (PCR) on specimens enables rapid and direct diagnosis, at
least in the early and convalescent stages of infection. The reaction detects leptospiral DNA in
the specimen, down to extremely small amounts equivalent to the DNA content of about 10
leptospires or less. A limitation of PCR-based diagnosis of leptospirosis is the inability of most
PCR assays to identify the infecting serovar [28].

A study on 103 patients of meningitis of unknown cause showed that 39.08% were positive by
PCR, 3.88% by ELISA & 8.74% by MAT [29].

Nested PCR and PCR/RFLP for 16S ribosomal RNA gene amplification.

Leptospiral genomic DNA was extracted from suspected human serum samples. The DNA was
air-dried, dissolved in TE buffer (10 mMTris-HCl, pH 8.0, 0.1 mM EDTA), and kept at −20°C
until use.The DNA was quantified by agarose gel electrophoresis and spectrophotometrically by
calculating the A 260 /A 280 ratios and the A 260 values to determine protein impurities and
DNA concentrations. Leptospira DNA was amplified by using the primers.These primers
amplified all pathogenic and non-pathogenic Leptospira species [30].

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Serological and Other Indirect Methods

Most cases of leptospirosis are diagnosed by serology. Antibodies can become detectable by the
6th to 10th day of disease and reach peak levels within three to four weeks. Antibody levels may
then gradually decline but remain detectable for years.

Microscopic Agglutination Test (MAT) [31]

The MAT is a sensitive assay, but because of the antigenic heterogeneity of Leptospira spp.
requires a large number of serovars asantigens. In addition, it would not be useful at the early
stages of the disease when the antibody to Leptospira spp. is not present or, if present, is at a low
level in the CSF. Positive results are defined as a 4-fold rise in titer between acute and
convalescent specimens. A single titer exceeding 1:200 or serial titers exceeding 1:100 suggest
leptospirosis, but neither is diagnostic. Some patients have serological evidence of previous
infection with a different leptospiralsero group. In these cases, serological diagnosis is
complicated further by the “anamnestic response”, in which the first rise in antibody titre is
usually directed against the infecting serovar from the previous exposure.

Enzyme Linked Immunosorbent Assay (ELISA)

This test relies on the detection of IgM antibodies which appear in the blood a day or so earlier
than those used in MAT. There is often poor correlation between MAT and ELISA results on
sera of individuals. The reference standard is MAT, IgM antibodies become detectable during
the first week of illness, allowing the diagnosis to be confirmed and treatment initiated while it is
likely to be most effective though, antibody levels are generally low or absent during very early
infection [32,33].

Though Microscopic agglutination test is considered to be the gold standard in the diagnosis of
leptospirosis, its use as a routine diagnostic test in a clinical laboratory is limited. The test is both
complex and tedious for routine use. Many studies have demonstrated Pan Bio ELISA to be
more sensitive than MAT for detection of cases early in acute illness [34]. IgM antibodies start
appearing during the first week of illness though antibody levels are low or not detectable very
early on in the illness. Leptospirosis can be diagnosed on the basis of the presence of IgM
antibodies by Pan Bio ELISA, in a single serum sample collected during the acute phase of the
illness. A convalescent sample taken after two weeks is required to confirm the results. A
limitation of using a single serum sample in the demonstration of IgM antibodies is the absence
of antibodies very early on in the infection or the persistence of antibodies. IgM antibodies in
leptospirosis persist for a long period with varying rates of decline [35]. A single serum sample
taken during an acute febrile illness with symptoms of leptospirosis is presumptive evidence of
infection, and therefore requires confirmation by further testing.

The bacterial concentration is less in serum than fresh blood. Studies comparing the PCR and
IgM have demonstrated PCR alone to be less sensitive than serological tests over the course of
the disease; it was the most sensitive method in those samples with no demonstrable antibodies
collected during the very early stages of the disease [36,37]. Therefore use of PCR in
combination with IgM ELISA would improve the sensitivity of the diagnosis of leptospirosis in
the first phase of the disease.

Testing an in-house ELISA with formalin-treated and boiled bacteria from the intermediate
species Leptospirafainei as an antigen to detect Leptospira-specific IgM antibodies. The samples,
tested by a MAT as a reference test, were used to evaluate the ELISA. The kappa value was 0.92
(95 % confidence interval 0.88–0.96), which indicated excellent agreement between the MAT
and ELISA. The overall performance of this in-house ELISA suggests applicability as a rapid
screening test for the diagnosis of leptospirosis in resource-limited settings and in hospitals and
laboratories where a MAT is not available [38].

Indirect Haemagglutination Assay (IHA)

IHA testing is a rapid and easily performed method of diagnosis that is based on genus-specific
antibodies. However, contrasting results have been obtained through various studies done to find
the sensitivity and specificity of IHA in early infections. It has been shown to have a sensitivity
of 92% and specificity of 95% compared with MAT. It can be concluded that IHA has a very
limited scope in diagnosing Leptospirainfections before 8days [39].

Leptodipstick Assay

This is an assay that detects Leptospira-specific IgM antibodies in human sera [40].

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Conclusion
When using a single sample collected during the early, acute phase of the disease, results of Pan
Bio IgM ELISA can give us a presumptive diagnosis of leptospirosis. Very early on in the
infection it may even fail to detect the presence of antibodies. PCR is a sensitive and specific
technique which can detect the presence of DNA in the very early stage of the disease, so PCR
together with IgM ELISA can be used to confirm the diagnosis, early on in the acute stage of the
infection.

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Notes
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Financial or Other Competing Interests

None.

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References
[1] Wu W, Bao L, Wu Q, Li S, Huang W. World Health Organization. Leptospirosis worldwide.
Wkly Epidemiol Rec. 1999;74:237–242.647. [PubMed]
[2] Rebecca P, Deborrah D. Overview of the epidemiology, microbiology, and pathogenesis of
Leptospira spp. in humans. Microbes Infect. 2000;2:1265–76. [PubMed]
[3] Everard JD, Everard COR. Leptospirosis in the Caribbean. Rev. Med. Microbiol.
1993;4:114–22.
[4] John JT. Emerging and reemerging bacterial pathogens in India. Indian J Med Res.
1996;103:4–18. [PubMed]
[5] Roy S, Biswas D, Vijayachari P, Sugunan AP, Sehgal SC. A clone of
Leptospirainterroganssensustricto is the major cause of leptospirosis in the Archipelago of the
Andaman & Nicobar Islands, India. Letters in Applied Microbiology. 2005;41:179–85.
[PubMed]
[6] Turner LH. Leptospirosis I. Trans R. Soc Trop Med Hyg. 1967;61:842–55. [PubMed]
[7] Chernukha YG, Shishkina ZS, Baryshev PM, Kokovin IL. The dynamics of IgM and IgG
antibodies in leptospiral infection in man. Zentralbl. Bakteriol.Mikrobiol. Hyg. 1976;236:336–
43. [PubMed]
[8] Silva MV, Camargo ED, Batista LA, et al. Behaviour of specific IgM, IgG and IgA class
antibodies in human leptospirosis during the acute phase of the disease and during
convalescence. J. Trop. Med. Hyg. 1995;98:268–72. [PubMed]
[9] Cumberland PC, Everard COR, Wheeler JG, Levett PN. Persistence of anti-leptospiralIg M,
IgG and agglutinating antibodies in patients presenting with acute febrile illness in Barbados
1979-1989. Eur J Epidemiol. 2001;17:601–08. [PubMed]
[10] Brandão AP, Camargo ED, da Silva ED, Silva MV, Abrão RV. Macroscopic agglutination
test for rapid diagnosis of human leptospirosis. J ClinMicrobiol. 1998;36:3138–42. [PMC free
article] [PubMed]
[11] Turner LH. Leptospirosis II. Serology. Trans. R. Soc. Trop. Med.Hyg. 1968;62:880–89.
[PubMed]
[12] Pike RM. Laboratory-associated infections: summary and analysis of 3921 cases. Health
Lab. Sci. 1976;13:105–14. [PubMed]
[13] Amouriaux Merièn P, Perolat P, Branton G, Saint IG. Polymerase Chain Reaction for
detection of Leptospira spp. In clinical samples. Journal of Clinical Microbiology.
1992;30:2219–24. [PMC free article] [PubMed]
[14] World Health Organization. Leptospirosis worldwide. Epidemiol. Rec. 1999;74:237–42.
[PubMed]
[15] Uhlenhuth P, Fromme W. Experimentelle Untersuchungenüber die sogenannte Weilsche
Krankheit (ansteckende Gelbsucht) Med Klin. 1915;44:1202–03.
[16] Stimson AM. Note on an organism found in yellow-fever tissue. Public Health Rep.
1907;22:541–542a.
[17] Kmety E, Dikken H. Classification of the species Leptospirainterrogans and history of its
serovars. Groningen, The Netherlands: University Press Groningen; 1993.
[18] Diesch SL, McCulloch WF. Isolation of pathogenic leptospiresv from waters used for
recreation. Public Health Rep. 1966;81:299–304. [PMC free article] [PubMed]
[19] Babudieri B. Animal reservoirs of leptospirosis. Ann NY Acad Sci. 1958;70:393–413.
[PubMed]
[20] Waitkins SA. Leptospirosis as an occupational disease. Br J Ind Med. 1986;43:721–25.
[PMC free article] [PubMed]
[21] Terry J, Trent M, Bartlett M. A cluster of leptospirosis among abattoir workers. Commun
Dis Intell. 2000;24:158–60. [PubMed]
[22] Kelley PW. Infectious diseases. 2nd ed. Philadelphia, Pa: W. B.Saunders; 1998.
Leptospirosis, p. 1580-1587. In S. L. Gorbach, J. G.Bartlett, and N. R. Blacklow (ed.)
[23] Edwards GA, Domm BM. Human leptospirosis. Medicine. 1960;39:117–56. [PubMed]
[24] Turner LH. Leptospirosis III. Maintenance, isolation and demonstration of leptospires.
Trans R Soc Trop Med Hyg. 1970;64:623–46. [PubMed]
[25] Ellis WA, Brien JJO, Neill SD, Ferguson HW, Hanna J. Bovine leptospirosis:
microbiological and serological findings in aborted fetuses. Vet. Rec. 1982;110:147–50.
[PubMed]
[26] Uip DE, Amato Neto V, Duarte MS. Diagnosticoprecoce da leptospirosepordemonstracao
de antigenosatravés de exameimuno-histoquíminoemmúsculo da panturrilha. Rev Inst Med Trop
Sao Paulo. 1992;34:375–81. [PubMed]
[27] Johnson RC, Harris VG. Differentiation of pathogenic and saprophytic leptospires. 1.
Growth at low temperatures. J Bacteriol. 1967;94:27–31. [PMC free article] [PubMed]
[28] Bal AE, Gravekamp C, Hartskeerl RA, MezaBrewster JD, Korver H, Terpstra WJ.
Detection of leptospires in urine by PCR for early diagnosis of leptospires. J Clin Microbiol.
1994;32:1894–98. [PMC free article] [PubMed]
[29] Romero EC, Billerbeck AEC, Lando VS, Camargo ED, Souza CC, Yasuda PH. Detection of
leptospira DNA in patients with Aseptic meningitis by PCR. J Clin Microbiol. 1998;36:1453–55.
[PMC free article] [PubMed]
[30] Sedigheh Z, Neda S, Mandana A. Molecular epidemiology of leptospirosis in northern iran
by nested polymerase chain reaction/restriction fragment length polymorphism and sequencing
methods. Am J Trop Med Hyg. 2010;82(5):899–903. [PMC free article] [PubMed]
[31] Galton MM, Powers DK, Hall AM, Cornell RG. A rapid microscopic-slide screening test
for the serodiagnosis of leptospirosis. Am J Vet Res. 1958;19:505–12. [PubMed]
[32] Terpstra WJ, Ligthart GS, Schoone GJ. ELISA for the detection of specific IgM and IgG in
human leptospirosis. J. Gen. Microbiol. 1985;131:377–85. [PubMed]
[33] Terpstra WJ, Ligthart GS, Schoone GJ. Serodiagnosis of human leptospirosis by enzyme-
linked-immunosorbent-assay (ELISA) Zentbl. Bakteriol. 1980;247:400–05. [PubMed]
[34] Levett PN, Branch SL, Whittington CU, Edwards CN, Paxton H. Two methods for rapid
serological diagnosis of acute leptospirosis. Clin Diagn Lab Immunol. 2001;8:349–51. [PMC
free article] [PubMed]
[35] Ahmed SN, Shah S, Ahmed FMH. Laboratory diagnosis of leptospirosis. J Postgrad Med.
2005;51:195–200. [PubMed]
[36] Ooteman MC, Vago AR, Koury MC. Evaluation of MAT, IgM ELISA and PCR methods
for the diagnosis of human leptospirosis. Journal of Microbiological Methods. 2005;65(2):247–
57. [PubMed]
[37] Fonseca de A C, Teixeira de Freitas VL. Polymerase chain reaction in comparison with
serological tests for early diagnosis of human leptospirosis. Trop Med Inst Health.
2006;11(11):1699–707. [PubMed]
[38] Pascale B, Muriel V, Mathieu P. Evaluation of an in-house ELISA using the intermediate
species Leptospirafainei for diagnosis of leptospirosis. J Med Microbiol. 2013;62(Pt_6):822–27.
[PubMed]
[39] Imamura S, Matsui H, Ashizawa Y. Indirect hemagglutination test for detection of
leptospiral antibodies. Jpn J Exp Med. 1974;44:191–97. [PubMed]
[40] Gussenhoven GC, Menno AWG van Der Moorn, Marga GA, Goris WJ, Terpstra RA, et al.
LEPTO Dipstick, a dipstick assay for the detection of Leptospira-specific IgM antibodies in
human sera. Journal of Clinical Microbiology. 1997;35:92–97. [PMC free article] [PubMed]
 Leptospirosis Workup
Updated: Jun 13, 2018
 Author: Sandra G Gompf, MD, FACP, FIDSA; Chief Editor: Michael Stuart Bronze, MD more...
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Approach Considerations
Leptospires grow slowly in culture, and recovery rates are low. Serologic tests
are available only in specialized laboratories, and the sensitivity of acute
serologic tests is low. Consequently, those tests should not be the basis on
which treatment is initiated. In a patient with compatible symptoms and a
plausible exposure history, empiric therapy should be started.
Laboratory studies are used for two purposes: to confirm the diagnosis and to
determine the extent of organ involvement and severity of complications.
Laboratory confirmation of leptospirosis can be accomplished through
isolation of the pathogen or by serologic testing.
Isolation of the leptospires from human tissue or body fluids is the criterion
standard, but culture is not routinely available; thus, molecular assays such as
DNA PCR are more commonly used, if available. Consultation with the local
microbiology laboratory is essential, because processing requires specialized
techniques. Urine is the most reliable body fluid to study because the urine
contains leptospires from the onset of clinical symptoms until at least the third
week of infection.
Other body fluids contain the organism, but the window of opportunity to
isolate them is shorter. Blood and CSF may produce positive PCR or cultures
during the first 7-10 days of symptoms.
Tissues (ie, liver, muscle, kidney, skin, eyes) are also sources of identification
of the leptospires but are obviously more complicated to acquire.
Most often, paired acute and convalescent serum specimens are used to
confirm the diagnosis. Again, this is a delayed means of confirmation because
the acute sera are collected 1-2 weeks after onset of symptoms, and the
convalescent sera are collected 2 weeks afterward.
Antileptospire antibodies in these samples are detected using the microscopic
agglutination test (MAT). The Centers for Disease Control and Prevention
(CDC) laboratory in Atlanta, Georgia, performs the MAT using 23 leptospire
antigens. A 4-fold rise in MAT titer between acute and convalescent sera with
any of these antigens confirms the diagnosis of leptospirosis.
Faster laboratory methods may strongly suggest the diagnosis of
leptospirosis, but they may be no more readily available than the CDC
laboratory in Atlanta. A single MAT titer of 1:200 on any sera or identification
of spirochetes on dark-field microscopy, when accompanied by the
appropriate clinical scenario, is strongly suggestive.
In suspected leptospirosis, further laboratory studies should be routinely
performed to determine the extent and severity of organ involvement after the
acute phase of illness. A complete blood cell count (CBC) is necessary.
Findings on general laboratory studies are as follows:
 In patients with mild disease, elevated erythrocyte sedimentation rates
and peripheral leukocytosis (3,000-26,000 x 109/L) with a left shift are
noted
 Significant anemia due to pulmonary and gastrointestinal hemorrhage
can occur
 The platelet count may be diminished as a component of disseminated
intravascular coagulation (DIC)
 levels of blood urea nitrogen and serum creatinine may be profoundly
elevated in the anuric or oliguric phase
 Serum creatine kinase levels (MM fraction) are often elevated in patients
with muscular involvement.
 Coagulation times may be prolonged in patients with hepatic dysfunction
and/or DIC On liver function testing, serum bilirubin levels elevate as part
of the obstructive disease due to capillaritis in the liver. Levels of
hepatocellular transaminases are elevated less often and less
significantly (usually < 200 U/L). Jaundice and bilirubinemia
disproportional to hepatocellular damage is common in leptospirosis;
alkaline phosphatase levels may be elevated 10-fold.
On urinalysis, proteinuria may be present. Leukocytes, erythrocytes, hyaline
casts, and granular casts may be present in the urinary sediment.
Analysis of the CSF is useful only in excluding other causes of bacterial
meningitis. When the CNS becomes involved in leptospirosis,
polymorphonuclear leukocytes initially predominate and are later replaced by
monocytes. CSF protein may be normal or elevated, whereas glucose levels
remain normal. CSF pressure is normal, but a lumbar puncture can relieve the
headache. Leptospires are routinely isolated from the CSF, but this finding
does not change management of the disease.
Imaging studies are useful in determining the extent and severity of organ
involvement. This may include chest radiography to evaluate lung disease and
biliary tract ultrasonography in suspected acalculous cholecystitis.
Electrocardiographic (ECG) abnormalities are common during the
leptospiremic phase of Weil syndrome. In severe cases, congestive heart
failure and cardiogenic shock may occur.
Culture
Isolating the organism by culture allows definitive diagnosis. Leptospires
remain viable in anticoagulated blood for as long as 11 days; hence,
specimens can be mailed to a reference laboratory for culture. The infecting
serovar can be isolated only by culture.
Blood cultures may be negative if drawn too early or too late. Leptospires may
not be detected in the blood until 4 days after the onset of symptoms (7-14 d
after exposure). Once the immune system is activated, blood cultures may
again become negative. Leptospires may be isolated from the cerebrospinal
fluid (CSF) within the first 10 days.
Leptospires may be isolated from the urine for several weeks after the initial
infection. In some patients, urine cultures may remain positive for months or
years after the onset of illness. Positive urine cultures may take as long as 8
weeks to grow.
Microscopic Agglutination Testing
Microscopic agglutination testing (MAT) uses a battery of antigens taken from
common (frequently locally endemic) leptospire serovars. MAT is available
only at reference laboratories, such as the Centers for Disease Control and
Prevention (CDC).
In a patient with clinical findings consistent with the disease, a single titer
exceeding 1:200 or serial titers exceeding 1:100 suggest leptospirosis;
however, neither is diagnostic. A 4-fold rise in titer between acute and
convalescent specimens is considered a positive result. The antibody
response does not reach detectable levels until the second week of illness,
and it can be affected by treatment.
False-negative MAT findings may result from testing a single specimen
obtained before the immune phase of disease. Test accuracy is also affected
by appropriate selection of antigens for the battery, necessitating discussion
with the laboratory about which serovars are suspected or predominate in the
region where the case originated. False-positive MAT results may occur with
cases of Legionella infection, Lyme disease, and syphilis.
Other Tests
Screening tests for leptospirosis, which are easy to perform and provide
results relatively rapidly, include the macroscopic slide agglutination test, the
Patoc-slide agglutination test, the microcapsule agglutination test, latex
agglutination tests, dipstick tests, and the indirect hemagglutination test.
Confirmation of screening test results (positive or negative) is advisable,
however, preferably with MAT. [40]
An immunoglobulin M (IgM) enzyme-linked immunoabsorbent assay (ELISA)
has been developed. The ELISA uses a broadly reactive antigen and is a
standard serologic procedure, as is the MAT. [41] Because it detects IgM, it may
be useful for diagnosis of new infections within 3-5 days. Positive results
should be referred for confirmatory testing.
Nucleic acid amplification (polymerase chain reaction [PCR])–based
techniques have been developed to diagnose leptospirosis. PCR can confirm
the diagnosis rapidly during the early phase of the disease, when leptospires
may be present and before antibody titers are detectable, but it requires
adequate infrastructure such as appropriate equipment, laboratory space, and
skilled personnel. In addition, PCR-based techniques are unable to identify
the infecting serovar, which reduce their epidemiologic and public health
value.
Dark-field examination of blood or urine has been used to identify leptospires.
However, this technique cannot be recommended, as it frequently leads to
misdiagnosis.
Chest Radiography
The most common abnormality on chest radiography is bilateral diffuse
airspace disease. Chest radiography may also reveal cardiomegaly and
pulmonary edema due to myocarditis. In patients with alveolar hemorrhage
due to pulmonary capillaritis, the lung parenchyma may contain multiple
patchy infiltrates.
Histologic Findings
Shortly after inoculation and during the incubation period, leptospires actively
replicate in the liver. The leptospires then disseminate throughout the body
and infect multiple tissues.
Silver staining and immunofluorescence can identify leptospires in the liver,
spleen, kidney, CNS, muscles, and heart. During the acute phase of
leptospirosis, histology reveals these organisms without much inflammatory
infiltrate. In addition to the finding of leptospires during histologic examination,
the pathologic effects of leptospiral toxins are also apparent. See the image
below.
 Silver stain, liver, fatal human
leptospirosis. (This image is in the public domain and thus free of any copyright restrictions.
Courtesy of the Centers for Disease Control/Dr. Martin Hicklin)
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Leptospirosis may be seen as an infective systemic vasculitis. [18] Leptospiral

toxins break down endothelial cell membranes of capillaries. This toxin-
mediated process allows for extravasation of blood and leptospires from blood
vessels into the supported parenchyma. Secondarily, because the capillaries
are no longer functional, ischemia and cell death can occur. Later in infection,
mononuclear cells predominate in the areas of this focal cell necrosis.
Leptospires can be identified in immunologically privileged sites, such as renal
tubules, CNS, and the anterior chamber of the eyes, for weeks to months after
the initial infection. In nonhuman animals, the intended hosts of infection, the
leptospires establish residence in these immunologically privileged sites.
Provided that the animal survives the initial infection, a chronic carrier state is
then established, and histology reveals leptospires at these sites for years
after initial infection.
Approach Considerations
Antimicrobial therapy is indicated for the severe form of leptospirosis, but its
use is controversial for the mild form of leptospirosis. A Cochrane Review
found insufficient evidence to advocate for or against the use of antibiotics in
the therapy for leptospirosis. [42]
If antibiotics are used, they should be initiated as soon as the diagnosis of
leptospirosis is considered and should be continued for a full course despite
initial serologic results, because most patients are diagnosed only through
acute and convalescent testing. Early treatment has been shown to offer the
best clinical outcomes; results from controlled studies of treatment during the
immune phase have yielded mixed results. [43, 44]
Mild leptospirosis is treated with doxycycline, ampicillin, or amoxicillin. For
severe leptospirosis, intravenous penicillin G has long been the drug of
choice, although the third-generation cephalosporins cefotaxime and
ceftriaxone have become widely used. Alternative regimens are ampicillin,
amoxicillin, or erythromycin. Several other antibiotics may be useful—for
example, broth microdilution testing has shown sensitivity to macrolides,
fluoroquinolones, and carbapenems [45] —but clinical experience with these
agents is more limited.
Severe cases of leptospirosis can affect any organ system and can lead to
multiorgan failure. Supportive therapy and careful management of renal,
hepatic, hematologic, and central nervous system complications are
important.
Patients should be managed in a monitored setting because their condition
can rapidly progress to cardiovascular collapse and shock. Access to
mechanical ventilation and airway protection should be available in the event
of respiratory compromise. Continuous cardiac monitoring should be attained;
arrhythmias, including ventricular tachycardia and premature ventricular
contractions, as well as atrial fibrillation, flutter, and tachycardia, can occur.
Renal function should be evaluated carefully and dialysis considered in cases
of renal failure. In most cases, the renal damage is reversible if the patient
survives the acute illness. Early initiation of hemodialysis or peritoneal dialysis
considerably reduces mortality risk. A few cases in the literature have reported
that plasma exchange, corticosteroids, and intravenous immunoglobulin may
be beneficial in selected patients in whom conventional therapy does not elicit
a response. [46, 47, 48]
Corticosteroid therapy is controversial. A 2014 review suggests lack of benefit
and possible increased risk of nosocomial infection. [49] As with severity of
illness, response may vary depending on host immune factors or the particular
virulence of the leptospire. Treatment with high-dose pulsed
methylprednisolone (30 mg/kg/d, not to exceed 1500 mg) has been used
successfully to treat patients with leptospiral renal failure without dialysis. This
approach may have be beneficial in resource-poor areas where dialysis is
unavailable and would involve lengthy medical transport. The use of renal-
dose dopamine in conjunction with steroids or diuretics has also been
described. [23]
Pulse-dose steroids may also play a role early in the management of severe
pulmonary disease. [50, 47] Patients with Weil syndrome may need transfusions of
whole blood, platelets, or both. Ophthalmic drops of mydriatics and
corticosteroids have been used for relief of ocular symptoms. [51]
Patients with severe disease should remain hospitalized until adequate
resolution of organ failure and clinical infection. Outpatient follow-up may
include an assessment of renal function to ensure ongoing reversal of any
damage. A cardiac assessment may be indicated in patients with symptoms
suggestive of heart involvement.
Diet and Activity
In mild cases, patients should be encouraged to maintain adequate fluid
intake to avoid volume depletion. In more severe cases, diets appropriate for
the clinical picture should be ordered (eg, electrolyte and protein restriction in
cases of renal insufficiency). Patients with hypotension or clinical shock
should not be fed enterally until adequate perfusion is restored.
Patients with severe disease should be placed on bed rest until adequately
resuscitated and treated. Those with mild disease can pursue activity as
tolerated.