Bacterial Infections of Humans || Leptospirosis

CHAPTER 21

Leptospirosis

Paul N. Levett and Charles N. Edwards

1. Introduction

Leptospirosis in humans occurs globally(141) as an acuteinfection ranging in severity from unnoticed and subclini-cal to fatal. It is a zoonosis (strictly, an anthropozoonosis)acquired by humans from an animal source. The causal bac-terium is a pathogenic spirochete of the genus Leptospira, ofwhich there are several species and more than 250 serologi-cal varieties (serovars), each of which has some diagnostic,prognostic, or epidemiological significance.(50,70)

The ultimate reservoir of all leptospirosis is anon-human carrier animal that excretes the causal bacteria(leptospires) in its urine. In animals, the leptospires infectprimarily young animals, which may become renal carriersand urinary excreters if they survive an acute initial infection.Human-to-human transmission has been recorded extremelyrarely, congenital infection has been infrequently reported,and laboratory-acquired infections have been documentedrather more frequently.

There are a large number of leptospires identifiedby serology or by genetic relationships. In general, thereis a correlation between animal reservoir, serological type(serovar), and potential severity of the illness. The diseasehas many local and folk names, frequently originating fromhistorical or clinical features or associated occupations, suchas “pretibial fever,” “swineherds’ disease,” “cane-cuttersdisease,” “mud fever”, “pea-pickers’ disease” or “swampfever”.

As it is a zoonosis, leptospirosis is most significant forthose whose work or leisure brings them into contact withinfected animals or their urine. It is highly related to occupa-tion in societies where animal contact is rare except through

Paul N. Levett � Saskatchewan Disease Control Laboratory,Saskatchewan Health, Regina, Saskatchewan, Canada S4S 5W6.Charles N. Edwards � Queen Elizabeth Hospital, St. MichaelBB11155, Barbados, West Indies.

work. In tropical areas especially, it is impossible to avoiddirect or indirect contact with feral carrier rodents or withlivestock. In other areas, only those in selected occupationsare exposed to significant risks. The public health impor-tance of leptospirosis lies in its occupational, seasonal, andsex- and age-related incidence; the acute and sometimes pro-longed incapacity for work; loss of human and livestock pro-ductivity; costs for medical care, workers’ compensation, orsimilar insurance payments; and preventive measures. It canbe epidemic, sporadic, or endemic.

2. Historical Background

The early history of leptospirosis is vague, becauseit was not possible to differentiate the different forms of“malignant jaundice,” which included hepatitis, yellow fever,leptospirosis, and malaria, until scientific clinical and patho-logical knowledge had progressed into the 1880s. The historyof leptospirosis was reviewed by Faine(50) and by Everard.(48)

The clinical entity was described by both Mathieu and Weilin 1886, but Weil’s description became the classical ref-erence. The syndrome of fever, hemorrhage, jaundice, andenlarged liver and spleen, with renal failure, became knownas “Weil’s disease.” Its etiology was unknown until the dis-covery of pathogenic leptospires, first seen in stained tissuesections of liver from a “yellow fever” patient, and later culti-vated independently from Weil’s disease patients among coalminers in Japan in 1914 and German troops in trenches dur-ing World War I in 1915. It was named Spirochaeta ictero-haemorrhagiae by the Japanese; Spirochaeta icterogenes bythe Germans; and later Leptospira icterohaemorrhagiae byNoguchi. Both the Japanese and German workers and theirassociates quickly published much of the basic knowledgeof the microorganisms, as well as the pathogenesis, pathol-ogy, clinical features and diagnosis, and rodent source of lep-tospirosis.

A.S. Evans, P.S. Brachman (eds.), Bacterial Infections of Humans, DOI 10.1007/978-0-387-09843-2 21,C© Springer Science+Business Media, LLC 2009

439

440 P.N. Levett and C.N. Edwards

Less severe illnesses were soon recognized as formsof leptospirosis. Dogs were identified as both reservoirs andsusceptible animals. Once the wider range of clinical mani-festations was recognized, other serovars were isolated frompatients and carrier rodents and were identified, so that bythe mid-twentieth century, 45 serovars significant for humanleptospirosis and their reservoirs had been identified.

A major discovery was that livestock (pigs, cattle) couldbe carriers and could excrete large numbers of leptospires inurine. Both primary (rodent–rodent) and secondary (rodent–livestock–rodent) cycles were identified. The range of knownserovars continued to expand, mainly following researchin tropical countries. Although leptospires could be iso-lated by culturing blood or urine from patients, evidenceof leptospirosis was obtained mainly by serological tests onpatients’ sera. Most culture media required rabbit serum. Theuse of bovine albumin and oleic acid or polysorbate-linkedfatty acids (Tween) to replace rabbit serum was an impor-tant advance, facilitating laboratory cultivation and allow-ing the easier isolation of serovar Hardjo, a major cause ofhuman leptospirosis wherever there are dairy cattle. The sub-type “hardjoprajitno” was originally isolated from an Indone-sian patient, and the subtype “hardjobovis” later, from cattle.Antibodies to Hebdomadis-group leptospires were alreadyknown to be prevalent in both humans and cattle. Genetictyping of leptospires, first done in the 1960s by DNA–DNAhybridization, was a major step that led much later to formalrecognition of genospecies of Leptospira.

Vaccines for control of leptospirosis in dogs were intro-duced in 1939 and in pigs and cattle soon after. These vac-cines would also indirectly assist in protecting humans byreducing excretion of leptospires by animals. Further devel-opments in knowledge of microbiology of leptospires and ofpathology, pathogenesis, immunity, epidemiology, and con-trol of leptospirosis are reflected in improved laboratory anddiagnostic methods during the 1980s. Recent rapid progressin the understanding of the molecular biology of leptospireshas led to the identification of several significant outer mem-brane antigens. The genomes of several strains have nowbeen sequenced and comparative studies are underway.

3. Methodology

The diagnosis of leptospirosis usually depends on lab-oratory confirmatory tests. Laboratory tests may be unavail-able or delayed, because the diagnosis is not considered atan early state or because the patient is not seen until latein the illness. Failure to use appropriate laboratory tests atthe proper time can lead to misdiagnosis in both suspectedand unsuspected patients. Because of diagnostic deficiencies,

mortality, morbidity, and incidence data based on uncon-firmed diagnoses or confirmed diagnoses of a few selectedpatients are fallacious, almost without exception.

3.1. Sources of Data

The World Health Organization (WHO) has recom-mended that leptospirosis be a notifiable disease. Notificationmay be based on hospital admissions and diagnosis, dis-charge data, notification by physicians, or laboratory noti-fication of infections diagnosed by isolation of leptospires,or on seroconversion, or both. Laboratory notification resultsin the recording of higher morbidity rates than practitionernotification. This is partly because patients are discharged,or recover, or die before all laboratory results are returnedto practitioners, who may then see notification as useless.Follow-up of patient contacts has led to detection of cases,not because of the contagiousness of the disease, but becauselocal outbreaks have been detected as a result of inquiries.

Death notifications and autopsy reports have been used.Accurate postmortem diagnosis is impossible where autop-sies are forbidden by custom or belief. Antemortem diag-nosis is required for accurate death notification without anautopsy. Mortality rates may be spuriously high if the diag-nosis is made only in advanced cases who are nearly mori-bund when medical or hospital assistance is sought.

Expression of mortality or morbidity statistics in termsof a total population can give a false index of incidence orprevalence if the disease is not homogeneously distributedin that population, or unless a correction is made for riskof exposure. In a large predominantly urban population, anoverall low morbidity rate may mask a very high rate in aselected group of that population, such as sewer workers,abattoir workers, or meat packers. Statistics should be refinedand expressed in rates relevant to the size of the populationat risk.(50)

Leptospirosis is not a notifiable disease in many juris-dictions, including the USA, where the disease ceased to benationally notifiable after December 1994(24) but remains soin some states.

3.2. Surveys

Serological surveys have been used frequently in studiesof leptospirosis. They can be used on unselected populationsto see whether leptospirosis exists at all. In such studies it isnecessary to use a battery of antigens in agglutination teststo ensure that representative serovars of all serogroups areincluded. Locally prevalent serovars must be included, if theyare known.

Chapter 21 • Leptospirosis 441

Survey information can be used to show which serovarsare likely to be prevalent, by analyzing results of testson batteries of antigens, or by retesting broadly reactivesera against individual serovars. Many sera will react withmore than one serovar, however, because several serovarshave antigens in common, and the highest titer is not nec-essarily against the infecting serovar, especially followingrecent infections. The fact that leptospirosis may not be dis-tributed homogeneously in the population should be borne inmind when planning surveys. The survey should be properlyplanned to request necessary information such as age, sex,occupation, travel, and previous known leptospirosis or simi-lar undiagnosed illness. Adequate statistical planning before-hand is essential to ensure that an appropriate number ofsamples are taken in each group and that results can be ana-lyzed by statistical techniques.

Once the presence of leptospirosis is known or detectedin a survey, the data may be used to determine the populationgroups exposed to a risk or risks and once those are delin-eated, to measure the prevalence of antibodies in one or moreselected subgroups. Another important function of surveys isto ascertain prevalence for evaluating control measures, bothfor a baseline level before their introduction and during andafter implementation as part of their evaluation.

The presence of IgM antibodies may be surveyed usingELISA, allowing large numbers of sera to be tested rel-atively quickly. IgM antibodies usually indicate relativelyrecent infections. Survey information can be important forinterpretation of diagnostic serology in acute cases to estab-lish whether the patient is one of a population or subgroup inwhich there is a known high prevalence of antibodies.

Serological or bacteriological surveys have proved valu-able in investigating outbreaks. Information may be obtainedretrospectively from serum bank specimens collected fromepidemic patients or from patients and their surroundingpopulation in general concurrently with an epidemic. Apartfrom epidemics, surveys of serum banks have been used toascertain overall prevalence rates in certain clinical groups,such as patients with jaundice and fever, undiagnosed asepticmeningitis, or pyrexia of unknown origin.

Prospective surveys offer a better chance of understand-ing the incidence of leptospirosis and the prevalence of anti-bodies in selected groups. These may be carried out onselected groups of hospital admissions or patients in pri-mary healthcare clinics or practices. A suitable protocol isto carry out diagnostic tests for leptospirosis on all clinicallysuspected patients.(41,45,46,88) If serology is tested, a matchedcontrol series, such as nonfebrile patients, must be included.

Collection of specimens for survey purposes requiresprior ethical review and the obtaining of informed consentfrom individual survey subjects. Where surveys are carried

out as part of national or international research programs, itis essential that local customs, traditions, and religious prac-tices of the population are respected and the surveys are car-ried with their cooperation.(50)

3.3. Laboratory Diagnosis

In the absence of clear characteristic clinical fea-tures, especially in mild leptospirosis, laboratory diagnosisis essential. Its importance cannot be overstated, because itis also the basis of epidemiological statistics and preventivepolicies and programs. Not only must the diagnosis be estab-lished as leptospirosis, but the causal leptospire should beidentified, at least by serogroup, and preferably also by iso-lation and identification of serovar and genospecies, for prog-nosis, management, and prevention activities.

3.3.1. Isolation and Identification of Leptospires3.3.1.1. ClassificationandNomenclatureofLeptospires.

All leptospires are similar in appearance and culture. His-torically, they were classified into serovars and serogroups byserology using cross-agglutination with rabbit antisera and amicroscopicendpoint.(37) Allpathogensweregroupedinasero-logically defined species known for years as “L. interrogans,”and nonpathogens were similarly grouped as “L. biflexa.” Nowthe members of the genus Leptospira are classified by geneticrelationships into distinct species (genospecies, see Table 1).Phylogenetic analysis shows the existence of three clades withthe genus, comprised of pathogens, saprophytes and an inter-mediategroupofuncertainpathogenicity (Figure1).GeneraofLeptonema and Turneriella are recognized, comprising mor-phologically similar spirochetes. Many strains are not yet clas-sifiedbygenotype.Severalstrainsofleptospiresclassifiedsero-logically by their antigens as members of a single serovar arefound to belong to different genospecies; that is, more than onespeciesmayexpressthesameserovar-specificantigens.(51,64)

3.3.1.2. Culture. Culture media containing long-chain fatty acids such as oleic acid or Tween with 1%bovine serum albumin as a detoxicant are used widely.A common formulation is the Ellinghausen–McCullough–Johnson–Harris (EMJH) medium. It is usually used in liquidform in screw-cap containers. In tubes of semisolid mediacontaining 0.1–0.2% agar, growth occurs in one or morezones (Dingers’ rings) situated from just below the surfaceto deep in the medium. In fluid media, growth from inoculaof 1–10% of volume can be seen as a birefringent swirl in 3–20 days, depending on the individual strain. Heavier growth,always less than the turbidity seen in cultures of entericbacteria, may be encouraged by shaking during incubation.Optimum temperature for the cultivation of laboratory strainsis 28–30◦C. Most strains grow poorly if at all at 37◦C.


Table 1. Species and Some Serovars and Serogroups of the Genus Leptospira

Species Serovar Serogroup Serovar Serogroup

Pathogens

L. alexanderi Lushui Manhao Nanding HebdomadisL. borgpetersenii Arborea Ballum Jules Hebdomadis

Balcanica Sejroe Mini MiniBallum Ballum Sejroe SejroeHardjo (Hardjobovis) Sejroe Tarassovi TarassoviJavanica Javanica

L. interrogans Australis Australis Icterohaemorrhagiae IcterohaemorrhagiaeAutumnalis Autumnalis Kennewicki PomonaBangkok Australis Kremastos PomonaBataviae Bataviae Lora AustralisBratislava Australis Medanensis SejroeBroomi Canicola Mwogolo Ictero.Bulgarica Autumnalis Naam Ictero.Canicola Canicola Paidjan BataviaeCopenhageni Icterohaemorrhagiae Pomona PomonaDjasiman Djasiman Pyrogenes PyrogenesHardjo (Hardjoprajitno) Sejroe Szwajizak MiniHebdomadis Hebdomadis Zanoni Pyrogenes

L. kirschneri Bim Autumnalis Mwogolo IcterohaemorrhagiaeBulgarica Autumnalis Mozdok PomonaCynopteri Cynopteri Kunming PomonaGrippotyphosa Grippotyphosa

L. noguchii Bajan Australis Louisiana LouisianaFortbragg Autumnalis Panama Panama

L. santarosai Bataviae Bataviae Pyrogenes PyrogenesBorincana Hebdomadis Tabaquite MiniKremastos Hebdomadis Trinidad SejroeNavet Tarassovi Weaveri Sarmin

L. weilii Celledoni Celledoni Mengma JavanicaHainan Celledoni Sarmin Sarmin

Leptospira genomospecies 1 Sichuan Undesignated Leptospira genomospecies 1 Sichuan

Intermediate group

L. broomii undesignated undesignatedL. faineii Hurstbridge HurstbridgeL. inadai Lyme Lyme

Nonpathogens

L. biflexa Patoc SemarangaL. meyeri Ranarum SejroeL. wolbachii Codice CodiceLeptospira genomospecies 3 Holland UndesignatedLeptospira genomospecies 4 Hualin IcterohaemorrhagiaeLeptospira genomospecies 5 Saopaulo Semaranga

Cultures of properly collected clinical material culturedshortly after collection are highly sensitive and capable ofdetecting very small numbers of leptospires after incubationfor from 2 or 3 days to several weeks. Cultures should beexamined at least weekly and incubated for 4 weeks to 2–3months.

3.3.1.3. Specimens for Culture. Blood cultures takenduring the first few days (up to 10 days, especially in thefirst week) during the fever are frequently positive. Additionof 100 μg/ml of 5-fluorouracil is useful to reduce risks ofovergrowth by bacteria from contaminated specimens. Spe-cial culture media are available for isolating fastidious lep-tospires currently found mainly in veterinary practice.(42)


Pathogens

Intermediate group

Non-pathogens

Leptonema

Turneriella

Figure 1. Phylogenetic relationships based on 16S rRNA genesequences between the genera of the Leptospiraceae. The scale bar rep-resents a 2% difference in sequence. See Table 1 for a list of species inthe three clusters within Leptospira. Adapted from (72) with permissionof the publisher.

Cultures may also be taken from cerebrospinal fluid(CSF), urine, and tissue samples, including autopsy speci-mens. Semisolid medium comprising EMJH with 0.1% agarand 100 μg/ml of 5-fluorouracil is useful for tissues.

3.3.1.4. Microscopy and Examination of Cultures.Dark-field microscopy by an experienced person is usedto recognize leptospires in cultures. Leptospires appear asmotile, bright, beaded, rotating, thin rods, usually with one orboth ends curved, against a black background. Direct exami-nation of clinical specimens by dark-field microscopy is bothinsensitive and nonspecific.(131)

3.3.1.5. Staining. Leptospires are poorly stained byconventional microscopic stains. They may be visualizedby Giemsa or silver deposition methods. Silver deposition byone of several methods can be used for tissue sections, butflawless technique is required to obtain clean, interpretableresults. Immunohistochemical staining has both greater sen-sitivity and specificity than other staining methods.(144) How-ever, the major disadvantage of all immunostains is the needfor specific antiserum. A battery of antisera may be requiredif the serovar is unknown or unsuspected. In extreme cases,where the infection is due to a new serovar, only convales-cent patients’ sera can be used as a source of the primaryantibody for the test until the new organism has been iso-lated and immune sera prepared from it. Results of directmicroscopy or immunostaining can be obtained in minutesto a few hours.

3.3.1.6. Serological Typing. If a leptospire is iso-lated, it must be identified by agglutination with refer-ence antisera or serogroup- and serovar-specific monoclonalantibodies.(37,120) These identification procedures are usuallyperformed only in reference laboratories. A list of species

recognized currently, with the frequently isolated serovarsand their serogroups, is provided in Table 1. A list ofpathogenic serovars and their history(67) is now out of print,but is being revised.

3.3.1.7. Molecular Methods. A number of conven-tional PCR assays were developed for detection ofleptospires, but few were subjected to clinical trials to eval-uate their use in diagnosis of leptospirosis in man(18,87) orin animals.(61,135) The most widely used assays were basedupon different approaches: one detected all pathogens usingtwo quite different targets,(59) while the other used a genus-specific 16S rRNA target.(86)

Recently, real-time PCR assays have been devel-oped, again using 16S rRNA targets.(89,116) However, recentadvances in the understanding of the molecular biologyof leptospires have led to the design of assays that targetvirulence factors expressed by pathogenic leptospires.(75,96)

Real-time PCR assays are potentially more sensitive thanconventional assays. One assay has been evaluated andshown to have sensitivity and specificity comparable toculture.(111)

The genospecies of Leptospira were defined by DNA–DNA hybridization,(16,99,102,143) but this is not a practicalapproach to identification of isolates. Sequence-based iden-tification of Leptospira species offers an alternative, using16S rRNA gene sequencing,(90) but within the nonpathogenicclade in particular the 16S rRNA sequences are highly con-served. Other targets may have better discriminatory powerand are currently being investigated.(69,114)

Molecular methods employed for serovar identificationhave included digestion of chromosomal DNA by restrictionendonucleases (REA), restriction fragment-length polymor-phisms (RFLP), ribotyping, pulsed-field gel electrophoresis(PFGE), and a number of PCR-based approaches.(70) Cur-rently, the most readily applicable molecular method foridentification of serovars is PFGE.(62,63) This approach hasbeen standardized using the PulseNet model.(55)

More recently, sequence-based methods such asamplified fragment-length polymorphism (AFLP) andmultiple-locus variant repeat analysis (MLVA) meth-ods have been developed for the study of leptospi-ral epidemiology.(79,105,112,113,130) The use of multi-locussequence typing is being investigated.(2) The application ofthese tools will enhance understanding of leptospiral epi-demiology at a population level.

3.3.2. Serological Tests. Antibodies in sera frompatients or survey subjects can be detected and measureddirectly by agglutination of leptospires or by ELISA. Testsused for diagnoses of acutely ill patients need to be specificand performed rapidly if they are to aid in management andprognosis, as well as for detection of sources common to


several patients and for prevention. For epidemiological andsurvey purposes, there is less urgency for obtaining testresults, but laboratories should be able to test large num-bers of sera rapidly if necessary. Usually, knowledge of theserovar specificity of reacting antibodies is required, espe-cially where several serovars are endemic, each with a differ-ent reservoir and pattern of transmission.

The microscopic agglutination test (MAT) is the goldstandard test but requires significant expertise to maintainand interpret. IgM detection using ELISA is more widelyused in diagnostic laboratories.

3.3.2.1. Agglutination Tests. The reference methodfor serological diagnosis of leptospirosis is the microscopicagglutination test (MAT), in which patients’ sera are reactedwith live antigen suspensions of leptospiral serovars. Afterincubation the serum/antigen mixtures are examined foragglutination by dark-field microscopy and the titers aredetermined. Appropriate quality control sera of known high-and low-specific reactivity are included as controls. Theauthenticity of strains used in antigen suspensions shouldbe checked periodically against reference antisera. An exter-nal quality control program is organized by the InternationalLeptospirosis Society.(26)

The range of antigens used should include serovars rep-resentative of all serogroups(125) and all locally commonserovars. The highest titer is sometimes recorded against across-reacting serovar. Where several serovars from differ-ent serogroups are prevalent locally, there is a need to testeach patient’s serum against each of them or at least againsta representative serovar of each serogroup. This means thatin many tropical areas, or where the likely infecting serovaris unknown, a battery of up to 30 serovars may be usedfor each serum. There is no satisfactory single group anti-gen with which all patients’ sera will react. The MAT is aserogroup-specific assay. Often, it becomes possible to dis-tinguish a predominant serogroup only months after infec-tion, as cross-reacting titers decline at different rates.(78) Itis thus important if possible to examine serial convalescentspecimens, in order to determine the presumptive infectingserogroup. The ability of convalescent MAT titers to predictthe infecting serogroup may be as low as 40%.(71)

Interpretation of the MAT depends on the stage of ill-ness, the serovar, and local epidemiology. Acute infection issuggested by a single elevated titre detected in associationwith an acute febrile illness. The magnitude of such a titre isdependent upon the background level of exposure in the pop-ulation, and hence the seroprevalence. Thus a titre of ≥200is used to define a probable case with a clinically compatibleillness.(24) This may be appropriate for use in a populationwhere exposure to leptospirosis is uncommon, but in mosttropical countries a higher cut-off titer is necessary for defin-

ing probable cases of leptospirosis. In endemic areas a singletiter of ≥800 in symptomatic patients is generally indicativeof leptospirosis.

Paired sera are required to confirm a diagnosis with cer-tainty. A fourfold or greater rise in titre between paired seraconfirms the diagnosis, regardless of the interval betweensamples. The interval between first and second samplesdepends on the delay between onset of symptoms and pre-sentation of the patient. If symptoms typical of leptospirosisare present then an interval of 3–5 days may be adequateto detect rising titers. However, if the patient presents ear-lier in the course of the disease, or if the date of onsetis not known precisely, then an interval of 10–14 daysbetween samples is more appropriate. Less often, serocon-version does not occur with such rapidity, and a longer inter-val between samples (or repeated sampling) is necessary.MAT serology is insensitive, particularly in early acute-phasespecimens.(32)

MAT interpretation is complicated by cross-reactivitythat occurs between different serogroups, especially in acute-phase samples. Titers following acute infection may beextremely high (≥25,600) and may take months, or evenyears, to fall to low levels.(33,78,104) Rarely, seroconversionmay be delayed for many weeks after recovery, and longerserological follow-up will be necessary to confirm the diag-nosis.

The MAT is the most appropriate test to employ in epi-demiological sero-surveys, since it can be applied to serafrom any animal species, and because the range of antigensutilized can be expanded or decreased as required. Only onespecimen is required for epidemiological and prevalence sur-veys. Serum may be frozen during transport and storage. It isusual to use a titer ≥100 as evidence of past exposure.(50)

However, conclusions about infecting serovars cannot bedrawn without isolates; at best the MAT data can give a gen-eral impression about which serogroups are present within apopulation.(71) Antibodies persist following clinical or sub-clinical infection for long periods.(11,33,78)

The MAT is a complex test to control, perform, andinterpret.(125) Live cultures must be maintained of all theserovars required for use as antigens. This applies equallywhether the test is performed with live or formalin-killedantigens. The labor-intensive nature of the test leads toobserver error and fatigue following prolonged reading oftests. Interpretive errors may result from the use of inappro-priate serovars in the panel of antigens. Direct measurementof specific IgM or IgG in MAT is not feasible. The repeatedweekly subculture of large numbers of strains presents haz-ards for laboratory workers and laboratory-acquired infec-tions have been reported.(4,100) Other drawbacks include thecontinuous risk of cross-contamination of the antigen cul-


tures, necessitating periodic verification of the identity ofeach serovar. MAT titers are also affected by the culturemedium in which the antigens are grown.

3.3.2.2. Enzyme Immunoassay (ELISA). Detection ofIgM antibodies by ELISA has been widely applied(1,121)

for diagnosis of human leptospirosis. ELISA products areavailable commercially in several formats(74,115,140) and arewidely used for screening specimens before testing by MAT.ELISA tests do not measure the same antibodies as those inagglutination tests, so there is little correspondence betweenthe optical densities or titers in ELISA and the agglutinatingtiter in MAT. In pigs and cattle, blood levels of IgM react-ing in ELISA have been correlated with renal excretion ofleptospires.

4. Biological Characteristics of the Organism

Leptospires are tightly coiled spirochaetes, usually0.1 μm × 6–20 μm, but occasional cultures may con-tain much longer cells. The helical amplitude is approx-imately 0.1–0.15 μm and the wavelength approximately0.5 μm.(50) The cells have pointed ends, either or both ofwhich are usually bent into a distinctive hook (Figure 2).Two axial filaments (periplasmic flagella), with polar inser-tions, are located in the periplasmic space.(118) Leptospiresexhibit two distinct forms of movement, either translationalor rotational. Dark-field microscopy of a wet preparationshows leptospires rotating rapidly around their long axis andtranslationally motile in either direction. One or both endsare usually hooked, imparting a looped appearance to the endof the leptospire when it rotates rapidly. In semisolid agar

Figure 2. Scanning electron micrograph of leptospiral cells boundto a 0.2 μm filter. Magnification, approximately ×3,500. (Courtesy ofJanice Carr, Public Health Image Library, Centers for Disease Controland Prevention).

media, movement is slowed. Morphologically all leptospiresare indistinguishable, but morphology of individual isolatesvaries with subculture in vitro and can be restored by pas-sage in hamsters. Leptospiral lipopolysaccharide has a simi-lar composition to that of other gram-negative bacteria.(20)

All members of the genus Leptospira are obligateaerobes or microaerophiles with an optimum growth tem-perature of 28–30

◦C at pH from 7.2 to 7.8. They produce

catalase, oxidase, and urease. Many strains produce lipase.They grow in simple media enriched with vitamins (vitaminsB2 and B12 are growth factors), long-chain fatty acids andammonium salts. Long-chain fatty acids are utilized as thesole carbon source and are metabolized by β-oxidation.(64)

Phenotypic tests are not helpful for identification of lep-tospires, which must be identified by a combination ofmolecular and serological approaches.

Leptospires are easily killed by drying, by moderateheat (above 42–45◦C for a few minutes), by acid conditions(below pH 7.0) or alkaline conditions (above approximatelypH 7.8), and by disinfectants such as phenolics, quaternaryammonium derivatives, halogens, and aldehydes (formalde-hyde, glutaraldehyde).

The leptospiral genome is comprised of two chromo-somes, approximately 3500–4300 kb and 300–350 kb inlength respectively. The genome of L. interrogans is approx-imately 4800 kb in size,(95,103) while that of L. borgpeterseniiis approximately 15% smaller.(21) Leptospires contain vari-able numbers of rRNA genes, which are widely spaced on thelarger chromosome. Numerous repetitive elements have beenidentified; several are insertion sequences, and are found inmany serovars, but the copy number varies widely betweendifferent serovars and among isolates of the same serovar.(14)

Genomic rearrangements resulting from multiple transloca-tions have been identified.(21,95)

A wide range of antibiotics is effective against lep-tospires in the laboratory, although penicillin, cefotaxime,ceftriaxone, and doxycycline are widely used therapeuticagents, and doxycycline is recommended for chemoprophy-laxis.

5. Descriptive Epidemiology

5.1. Prevalence and Incidence

Statistics of prevalence and incidence are influenced fre-quently by the lack of reliability in diagnosis and by failureto recognize the selective risk to population groups. Preva-lence figures derived from planned surveys are much morereliable than those based on hospital records or disease notifi-cations. Antibody prevalence rates in tropical countries with


large feral and peridomiciliary rodent populations are muchhigher than in temperate climates, where leptospirosis tendsto be more occupationally segregated.

Reported statistics are also influenced by technical fac-tors such as the starting dilution for sera, the interpretation oflow titers, and the techniques used for serology. These fac-tors are often not clear from reports. When using prevalencestatistics for epidemiological and control policy planning, itshould be noted that the duration of persistence of antibodiesafter infection is variable.

Second infections with a different serovar have beenreported, indicating that there is little cross-immunitybetween serogroups. Very rare reports of second infectionswith the same serovar are poorly documented.

In the United States, approximately 100 cases of lep-tospirosis are reported each year; however, the disease is notnotifiable in many states and national data have not been col-lected for over 10 years. Cases occur throughout the country,but they are most common in tropical regions (Hawaii andPuerto Rico).

5.2. Epidemic Behavior and Contagiousness

Leptospirosis is not considered to be contagious,because spread from human to human is almost unknownand direct inoculation into the body via skin, mucosal, orconjunctival penetration is required for infection. Laboratoryinfections, a significant occupational problem, have occurredafter inoculation into the eye or through the skin.(100) In nat-ural infections an animal source of infection is required, withthe rare exception of transplacental infection. Animal urineis the most important vehicle of infection for man, but directcontact with infected animals, their tissues or other body flu-ids are also important methods of transmission.

Significant changes in the epidemiology of leptospirosishave occurred in recent years. In developed countries the dis-ease is much less prevalent than it was in the mid-twentiethcentury, for several reasons. Increasing urbanization hasreduced the opportunity for exposure in the rural environ-ment and immunization has largely controlled the disease indomestic animals. However, against this background thereremain foci of infection, such as the recognition of contin-uing rat-associated leptospirosis in inner cities(132) and theemergence of canine infection in Eastern North Americacaused by serovars Grippotyphosa and Pomona,(101,136,137)

presumably acquired from racoons, skunks or possums.There has been a significant rise in travel-associated lep-tospirosis, often associated with adventure tourism in thetropics,(25,109) and invariably associated with exposure tofresh water. By far the greatest epidemiological change glob-ally has been the recognition of large outbreaks of lep-

tospirosis associated with excess rainfall.(68,91,122,142) Theseoutbreaks often occur in regions where diagnostic capacityis least well developed.

Although leptospirosis is a zoonosis, human socialfactors are most important in epidemiology. Occupationalexposure to animals and contaminated environments isimportant, but avocational factors, such as housing con-ditions, sanitation, food storage conditions, livestock anddomestic animals, and social and religious customs all affectthe likelihood of exposure to leptospirosis.

5.3. Geographical Distribution

Leptospirosis is distributed worldwide,(141) whereverfresh water and carrier animals occur. There is a directrelationship with soil and geologic environment, which canaffect the ability of leptospires to survive in surface watersand mud and soil through their effects on acidity andporosity.(65) Environmental changes will affect the magni-tude of the risk of human leptospirosis, either positively ornegatively.(98,133)

5.4. Temporal Distribution

Throughout the world, leptospirosis is related to wetperiods of the year. The wet seasons, whether monsoon,floods, or spring and summer, are associated with surfaceconditions favoring leptospirosis in all parts of the world.In temperate climates the risks increase with fresh springand summer growth of pastures. Risks also increase forharvest workers for many crops but not wheat or corn.Well-documented epidemics have been recorded during rice,sugarcane, and pea harvests.

5.5. Age

Although leptospiral antibodies can be found in peo-ple of all ages, the predominant incidence of acute infectionis found in the 20- to 50-year age range, though in devel-oping countries more cases may be seen in younger peo-ple. Pediatric and congenital infections have been described.The age distribution reflects the relative amount of envi-ronmental exposure to risk factors in the age groupsconcerned

5.6. Sex

Leptospirosis is predominantly a disease of malesbetween the ages of 10 and 59 years. The case-fatality rateis age-dependent, rising to almost 30% in elderly adults.


5.7. Race

There is no evidence for any risk factors determined byrace. Very little attention has been paid to genetic determi-nants of susceptibility to leptospirosis, but one recent studyhas shown that HLA-DQ6-positive triathletes were at greaterrisk of acquiring leptospirosis.(76)

5.8. Occupation

There is a strong occupational trend in all regions. Evenin tropical climates, those whose jobs bring them into indi-rect contact with animals via rodent urine contamination insuch activities as rice planting and harvesting, fish farming,forestry, mining, boating, and military exercises are at spe-cial risk. In all climates those exposed to urine contaminationdirectly or indirectly from livestock and domestic dogs androdents have a greater risk and increased incidence of infec-tion. Occupational hazards involve special considerations ofpreventive measures to protect workers. Some countries haveworkers’ compensation schemes which apply to persons whoacquire leptospirosis at work.

5.9. Occurrence in Different Settings

Leptospirosis poses a special problem after natural dis-asters or in case of civil disruption. Rodent infestation mayincrease and with it risks to humans from leptospirosis aswell as other zoonoses. Military, paramilitary, police, orcivilian emergency workers may be forced into conditionssuch as floods and destroyed buildings that house rodents.Frequently, troops have been exposed to leptospirosis in rat-infested trench warfare or in jungle warfare or exercises.Modern leisure activities have attracted city dwellers to ruralsettings, sometimes in faraway lands, in search of adven-tures in canoeing, rafting, caving, and climbing. The risksof contracting leptospirosis on these vacations need to beemphasized, but it is important to recognize that the activitiesthemselves put the participants at risk in temperate regions aswell as tropical regions.(12,84,91,110,124)

5.10. Socioeconomic Factors

It is difficult to calculate the costs to society of theeffects of leptospirosis. Some of the factors to be consid-ered are the costs of labor to replace sick workers; the costto the self-employed or peasant farmer or villager in moneyto replace his own inability to produce food for his family,and in pain and sometimes chronic illness; costs of medicalattention including hospital and medical services; and costsin insurance for lost work, production, and workers’ com-pensation. The first requirement is adequate diagnosis so that

the actual impact of the disease can be assessed. However,costs of special diagnostic services and surveillance for lep-tospirosis are hard to justify if there is an impression that thedisease is rare or absent, so that the circular logic leads tocontinued underdiagnosis and underreporting. Existing mildillnesses have frequently and easily been attributed to numer-ous other causes, including “influenza” or “viral infections”,but may well be leptospirosis. Moreover, when diagnosticcapacity does exist, the erroneous perception may occur thatleptospirosis is more prevalent than in neighboring regionswhere no cases are diagnosed.(49)

An important socioeconomic consideration is the effectof leptospirosis on productivity of animals used for foodor burden. Leptospirosis can cause abortions, stillbirths,failure to thrive, retarded growth, milk failure or spoilagefor human consumption, and loss of productivity in meatanimals among livestock. These effects can in themselvesplace an additional burden of malnutrition on people alreadythreatened by the risk of illness.

6. Mechanisms and Routes of Transmission

Leptospirosis is transmitted primarily from its animalhost reservoirs by urine. Excretion in urine may be intermit-tent or continuous and may contain very low numbers or verylarge numbers of leptospires; all of the former may occur atdifferent times in a single animal. The reasons for the vari-able output are not known. Urine from carriers frequentlycontains antibodies to the homologous serovar of leptospires.The intermittent shedding, the difficulties of detecting smallnumbers of urinary leptospires in livestock, and the poten-tially enormous load of leptospires excreted into the surfacewater and soil environment from heavy feral rodent environ-mental contamination are the main sources of difficulties incontrol in different situations.

Pathogenic leptospires can survive free in the envi-ronment, depending on soil type and geologic factors inmoist conditions such as in soil, mud, swamps, drains, sur-face waters, streams, and rivers, as long as conditions arenot acid.(58) All of these sites are well known as sources ofleptospirosis.(5,36) The leptospires can infect fresh host ani-mals or humans via broken skin or mucosal surfaces.

Food preparation areas can be contaminated by infec-tious urine from foraging carrier rodents, resulting in infec-tions in food process workers. Urine splash and aerosolscan infect milkers (especially in “herringbone”-pattern milk-ing sheds), veterinarians, and animal handlers. Animal tis-sues and blood are infrequently causes of leptospirosis, moreoften so in workers, inspectors, and transporters of meat.Conception products and autopsy of infected livestock and


separated kidneys used for domestic food can transmit lep-tospirosis to veterinarians, farmers, laboratory workers, andfood-handlers and cooks.

Numerous outbreaks associated with water have beenreported.(70) Spread of leptospirosis is not known to occurby airborne, respiratory, or gastrointestinal routes. However,immersion in, or ingestion of, water is a risk factor in otheroutbreaks(22,91) and it seems probable that leptospires canpenetrate the oral or nasal mucosa. Human-to-human trans-mission is virtually unknown, apart from in utero congenitalinfection.

Laboratory-acquired infections usually have resultedfrom accidents involving direct skin penetration with cul-tures or suspensions of infected tissues via syringe and nee-dle, broken glass, or splash into an unprotected eye, and frombeing bitten by infected animals (usually rats) whose urinecontaminated the bite.

Leptospires die rapidly when dry. Drying out of surfacesor environments contaminated by infective material preventsspread, even after rehydration. Heating above about 42◦C islethal to leptospires, but they survive freezing.

7. Pathogenesis and Immunity

7.1. Incubation Period

The incubation period of leptospirosis varies from 2 to21 days, usually between 3 and 10 days, although longerperiods have been reported. It is relatively shorter withlarger infective doses or more virulent leptospires. Incuba-tion periods can be calculated from a recognized infectionepisode, but they are difficult to define when there is contin-ual or repeated exposure. The relatively sudden onset makesit easier to detect the first clinical symptoms unless theyare very mild or unnoticed. In these cases the incubationperiod may be erroneously dated to the onset of late pre-senting symptoms such as hemorrhage, meningitis, or renalfailure.

7.2. Virulence and Its Attributes

The mechanisms by which leptospires cause disease arenot well understood. A number of putative virulence factorshave been demonstrated, but with few exceptions their rolein pathogenesis remains unclear. Toxin production and adhe-sion have been shown by some strains in vitro and in vivo.Surface antigens clearly play an important role in virulence.The lipopolysaccharide (LPS) of leptospires is only weaklyendotoxic. Early studies demonstrated the importance of LPSas a target of the immune response,(50) and recent data sug-gest that the O antigen content is modulated in acute ver-

sus chronic infections.(93) Much recent work has focusedon the role of surface lipoproteins as potential virulencefactors.(31) Many of the surface exposed lipoproteins such asOMPL1, LipL21, LipL32, LipL41, LipL45, and LipL46 areup-regulated in vivo, while others such as LipL36 are down-regulated.(82,94) LigA and LigB are adhesins, the expressionof which is induced by physiological osmolarity.(27,81) Otheradhesins have also been described recently.(9)

Genomic analysis has shown the existence of severalhemolysins in L. interrogans.(145) Lk73.5 is a host-induciblesphingomyelinase in L. interrogans that is not producedin vitro.(7) LipL32, the predominant surface protein, is ahemolysin.(15) LruA and LruB are inner membrane lipopro-teins that share epitopes with equine corneal proteins.(127)

IgG and IgA antibodies to these proteins are found in uveiticeye fluids. Other proteins have been shown to bind factor Hand this contributes to serum resistance.(85,128)

7.3. The Course of Events in Infection

7.3.1. Entry. Leptospires gain entry through smallabrasions or cuts in the skin or mucosal surfaces. Entry mayoccur via the conjunctiva or by aerosol into the lungs, as wellas by inhalation following immersion in contaminated water.As described above, ingestion of water probably results inpenetration of the oral or nasal mucosa. Laboratory infec-tions have occurred through injections, needle-stick injuriesand cuts, and eyesplash accidents.

7.3.2. Spread. Leptospires that enter the body spreadat once in the lymphatics and bloodstream. There is no ini-tial localizing acute inflammation at the site of entry. Lep-tospires can be found distributed in various tissues within2 h of infection. Both TLR-2 and TLR-4-dependent mech-anisms appear to be important in the activation of the innateimmune system.(134,139) Nonpathogenic leptospires and avir-ulent strains of the pathogenic species of Leptospira areopsonized by innate serum IgM. All leptospires may beopsonized by specific immune IgM or IgG in hosts previ-ously immunized by infection or vaccination. Phagocytosisoccurs rapidly in reticuloendothelial fixed phagocytes in theliver (Kupffer cells) and lung. These phagocytes later migrateto the spleen. The bloodstream is cleared rapidly follow-ing opsonization, which may occur with minimal detectablelevels of immunoglobulin recognized as agglutinating anti-body. Polymorphonuclear and mononuclear phagocytes canbe observed to take up leptospires from the tissues followingthe development of antibody.

In the absence of immunity and consequent phagocy-tosis, leptospires grow in the body in an initial infectionas if in culture. Nothing is known of specific nutritionalrequirements in vivo. Both virulent and avirulent leptospires


have been shown to adhere to fibroblast-derived cells in thelaboratory.(8)

A threshold level of leptospires is required in the bloodbefore lesions occur. In severe leptospirosis approximately104 leptospires per ml blood are associated with a pooroutcome.(107,123) The first and most important lesions to bedetected are in small blood vessels throughout the body.Localized endothelial cell degeneration occurs, followed bysmall localized extravasations that frequently contain lep-tospires.

Fever occurs at an early stage of infection. At this stageit is also possible to demonstrate microscopic focal degen-eration of skeletal muscle fibers, notably in the calf muscles.The extreme pain in muscles and acute tenderness found clin-ically are presumably the result of these lesions. Thus, themain presenting symptoms of severe headache, fever, andmuscle pains can be attributed to the effects of leptospiresgrowing in the body to threshold levels sufficient to causecharacteristic damage to small blood vessels.

The natural history of the further development of lep-tospirosis in either humans or animals is similar. Exponen-tially increasing numbers of leptospires lead to severe bloodvessel damage, local ischemia, and small hemorrhages inmost tissues, and eventually to major organ damage. Depend-ing on the serovar of leptospire, there is greater or lesserhepatocellular degeneration, leading to jaundice; renal tubu-lar degeneration similar to that seen in crush injuries or afterrenal ischemias, leading to an acute, often hemorrhagic inter-stitial nephritis; and pulmonary hemorrhages ranging frominsignificant to gross and fatal, with hemoptysis. Cholecys-titis, symptoms of acute abdominal emergency, meningitis,hemiparesis, myocarditis, adrenal hemorrhage, and placenti-tis have all been reported.

7.3.3. Lesions. Once the infection is fully developed,the typical lesions described in clinical–pathological andpostmortem studies of leptospirosis, usually of the severe andfatal types, appear. Hemorrhage is associated with a suddendrop in platelet numbers, possibly as a result of leptospiraltoxin action or of adhesion of leptospires. The potential roleof disseminated intravascular coagulopathy syndrome is notclear.(39) Anemia is common, usually not as a result of hem-orrhage alone. Evidence of nitrogen retention is found almostuniversally, albeit transitorily, reflecting the renal ischemiaoccurring even in mild types of leptospirosis. In these cases,patients whose kidneys are functioning poorly and at thelimit of compensation due to other causes of renal insuf-ficiency may be precipitated rapidly into renal failure. Insevere cases there is very widespread tubular degeneration,which is the usual main cause of death. Other potentiallyfatal lesions include hepatocellular degeneration, pulmonaryhemorrhages, and myocarditis.(6,34,35)

7.3.4. Transplacental Spread. Transplacental infec-tion can occur at any stage of leptospirosis. If the fetus orplacenta is severely damaged, abortion will occur. Stillbirthor congenital leptospirosis may follow infection late in preg-nancy. Abortion is a well-documented cause of loss of pro-ductivity of livestock. In human congenital infection, IgMantibodies can be demonstrated in the fetus and cord bloodand leptospires in the degenerative lesions in tissues.(50)

7.3.5. Recovery. Recovery from leptospirosis com-mences as soon as the patient produces opsonizing anti-body, which leads to rapid phagocytosis of leptospires inthe circulation and in the tissues. Mopping up phagocy-tosis also follows destruction of leptospires by antibiotics.Opsonizing antibody levels can be correlated with agglu-tinating antibody levels. Virtually any detectable specificagglutinating antibody in MAT can signify opsonic activity.Bacteriologic clearance of live leptospires from tissues byantibody, however, does not ensure clinical recovery, espe-cially in severe leptospirosis, because lesions in organs andtissue damage may have proceeded to stages where vitalfunctions are almost irreversibly impaired. Clinical support-ive measures for renal failure (dialysis), hemorrhage (trans-fusion), myocarditis, and liver failure may be necessary untilthe patient’s tissues regenerate. Complete recovery is the rulein surviving patients, although rare examples of prolongedasymptomatic renal carriage have been recorded in people.

7.3.6. Carrier State. The carrier state occurs veryrarely, if at all, in humans. This is assumed to result fromthe acidic reaction of human urine. In animals it followsrecovery from systemic infection, which may be subclinical.Leptospires are attached to the epithelial surface of proxi-mal renal tubule cells, where they grow in the tubular lumenand are excreted in the urine. Homologous antibody may bedemonstrated in the urine of carriers. Active excretion maybe sporadic or intermittent. The mechanisms of adhesion,immunologic events, and nutritional needs of leptospires inrenal tubules need to be elucidated. There is no inflamma-tion around affected tubules, which appear to be morpholog-ically intact in electron microscopic studies. In some animals(especially dogs and pigs) there may be considerable scar-ring in the kidneys where leptospires occupy renal tubules,apparently related more to the recovery and repair processesfollowing renal damage (acute nephritis) and hemorrhage inthe acute state of infection than to the presence of leptospiresin the tubules. Leptospires are not found in renal tissue out-side renal tubules in carrier animals. Genital carriage of lep-tospires has been described in livestock.

7.3.7. Autoimmunity and Hypersensitivity. Theroles of autoimmunity and hypersensitivity in producinglesions are unclear. The acute nature of infection andabsence of chronic infection precludes consideration of


these immunopathologic mechanisms in the developmentof initial lesions. However, leptospires may be sequesteredin “privileged sites” during acute infection, leading to laterallergic reactions to their presence. A notable exampleis uveitis that has followed either systemic infection oraccidental laboratory inoculation into the conjunctiva.Leptospires may be cultured from the anterior chamberof the eye for long periods after infection. Contact withleptospiral antigen can cause an allergic aggravation ofthe uveitis. A similar condition known as equine recurrentuveitis or “moon blindness” occurs in horses and resultsfrom antigenic mimicry between epitopes on equine corneaand an antigen expressed by some Leptospira serovarsassociated with equine uveitis.(77)

7.4. Immunity

7.4.1. Specific and Nonspecific Immunity. Low lev-els of innate antileptospiral IgM antibodies, found in theserum of humans and other animals not known to have beenexposed to leptospirosis, destroy avirulent leptospires but areinactive against virulent strains. Nothing is known of nonspe-cific immunity to leptospirosis stimulated by infection withother bacteria or their LPS.

Specific immunity to leptospirosis develops rapidly,within a few days of clinical or subclinical infection. Fullyeffective immunity can be transmitted passively naturallythrough the placenta and colostrum and artificially withconvalescent or hyperimmune serum. Both IgM and IgGantibodies are protective, and presumably colostral IgAantibodies are also, although there is no direct evidence.Immunity is associated with agglutinating antibodies whichare also opsonic; moreover, a very low level of agglutinatingantibody is protective. Immunity to reinfection is specificfor the infecting serovar or closely antigenically relatedserovars, almost exclusively within the same serogroupdetermined by MAT.

Some animal species are relatively resistant to lep-tospiral infection with certain serovars; the basis of suchresistance is unknown. It is not known whether humans areincapable of infection with any serovars. Generally, younganimals are much more susceptible than old, provided theyhave no maternally endowed passive immunity. The devel-opment of age-related relative resistance to leptospirosisreflects the rate of maturation of the B-cell immune response.

The humoral immune response induced by immuniza-tion protects against disease but not against infection andsubsequent renal shedding. Cell-mediated responses occurfollowing infection and following immunization with newervaccines(92); there appears to be a lower degree of cell-mediated immunity following natural infection. In contrast

to the traditional vaccines, cattle immunized with these vac-cines are protected against renal colonization and urinaryshedding.(13) Recent evidence suggests that a Th1 immuneresponse also occurs in humans.(66,129)

The duration of immunity in humans is not known.Agglutinating antibodies fall in titer after infection, but therate is very variable in individuals and there are no studiesof the relative rates of fall of IgM and IgG antibodies. Somepeople known to have had leptospirosis are seronegative in1 or 2 years and others are still seropositive after more than10 years, although the possibility cannot be excluded thatthey have had continued exposure to leptospires during thisperiod.(33,44) It is not clear that the loss of detectable aggluti-nating antibody over time following infection is synonymouswith loss of immunity. These deficiencies in knowledge areobviously important for understanding immunity, epidemiol-ogy, and planning for vaccines.

7.4.2. Antibodies. Convalescent sera contain specificIgM, IgG, or both. Usually, IgM is elaborated first, withinapproximately 7–20 days of infection, followed by IgG. Insome patients, seroconversion does not occur until 30 daysor more after onset of infection. IgM persists at low levelsin certain cases, while in others IgG is sometimes the soledemonstrable immunoglobulin. However, information fromthis type of retrospective study is necessarily limited in valuebecause one cannot know for certain beforehand the previ-ous history of exposure to leptospirosis. Cross-reactions withrelated and occasionally with unrelated serovars occur, espe-cially early in infection. These “paradoxical reactions” occurtypically, for example, between serovars Icterohaemorrha-giae and Canicola, and Icterohaemorrhagiae and Pomona.IgG antibodies tend to be more specific. In some patientstreated very early with penicillin whose infections wereproved with blood cultures, seroconversion did not occur atall or was delayed several weeks. Antibodies to the infectingserovar may be found in the urine of carrier animals.(50)

7.4.3. Antigens Related to Pathogenesis andImmunity. Antibodies reacting with antigens in flagellar,subsurface, and outer-envelope locations have been iden-tified in sera from patients who have been proved to haveleptospirosis. Some of the antibodies persist for months andyears.(11,33,78)

7.4.4. Vaccines. Vaccines for leptospirosis wereintroduced soon after leptospires were identified and couldbe cultivated. Preparations effective in protecting animalsand humans have been made by killing the leptospires incultures with heat or formalin and injecting doses subcu-taneously or intramuscularly. Formalin-killed cultures areused widely to vaccinate animals (cattle, pigs, dogs) and arein use in some countries to vaccinate people. The vaccineis usually given subcutaneously in two doses and repeated


annually. There is a high incidence of painful swellings atthe injection site, especially on revaccination.

In countries where leptospirosis is confined to clearlyrecognized occupational groups whose source of infectionis livestock, the vaccination of livestock is recommendedand used widely to decrease shedding by carrier animalswith the twofold purpose of reducing and eliminating animalleptospirosis, thus increasing productivity, and protectinghumans at risk from unavoidable exposure to these animals.Pregnant sows are commonly vaccinated to ensure passiveprotection to growing piglets. However, piglets may acquireasymptomatic leptospirosis after their passive protection hasbeen lost, and they may then become shedders and thus a haz-ard to handlers, slaughtermen, and other abattoir workers.(23)

Similarly, cattle that have been vaccinated are protectedagainst disease but not against infection. The major limita-tion to use of vaccines in dogs is the potential for infectionwith serovars that are not included in the vaccine. Vaccinateddogs may thus represent a source of human infection.(47,53)

More recently a new generation of vaccines havebeen introduced that induce cell-mediated immunity incattle.(19,92) Moreover, immunization with such vaccines pre-vents both disease and urinary shedding.(13)

8. Patterns of Host Response

Several epidemiological features of leptospirosis deter-mine the host responses and clinical features. The disease isalmost uniquely acquired from carrier animals, which cannotbe identified as carriers or shedders without laboratory tests.Inapparent infections in humans are not relevant to clinicaldisease or transmission.

The severity and presenting features of clinical lep-tospirosis result from a complex of climatic, social, and geo-graphic factors influencing the animal host and source, andthus the serovar and the clinical features and in turn the accu-racy of diagnosis and notification. In general, different lep-tospiral serovars are associated with characteristic groups ofanimal species.

Living conditions for most people in tropical countriesfavor contact with rodent sources of leptospires rather thanother animals. Most of the leptospires that clinically resultin the very severe leptospirosis of the classical Weil’s dis-ease type, which may be fatal following renal failure, hem-orrhages, and jaundice, are carried by rats. Serovars of theIcterohaemorrhagiae, Autumnalis, and Bataviae serogroupsare examples.

Conversely, in temperate climates, in addition to rats,there are other carriers of leptospires such as field mice, pigs,dogs, and cattle that shed mainly leptospires of those serovars

causing a milder form of illness. The disease also tends to bemuch more occupationally related and confined to people incontact with these animal sources. Serovars Grippotyphosa,Hardjo, Pomona, and Tarassovi are examples of this group.

Perceptions and statistics of leptospirosis are influencedby whether the disease is seen as the severe or the mild type.Mild infections of either variety have often been overlookedbecause the symptoms are not pathognomonic. Usually, seri-ously ill patients who needed hospital treatment or died wereaccurately diagnosed as leptospirosis. Since they were theonly ones in whom the diagnosis was made, the clinical fea-tures recorded and reported in textbooks have been those ofthe severe type, emphasizing jaundice, hemorrhages, renalfailure, meningitis, and a high case-fatality rate. Overall,these symptoms appear in only a few and late cases of theclassical syndrome of Weil’s disease. Most patients affectedwith any serovar report initial symptoms as described below.The severe and the mild types differ in their subsequentprogress.

8.1. Clinical Features

The clinical features are usually divided into the mildanicteric phase,(10) accounting for about 90% of all cases,and the severe or icteric disease causing the syndrome Weil’sdisease.(41) Characteristically, leptospirosis commences sud-denly with headache, myalgia, fever, and red eyes. Headachedevelops without warning and may be extremely severe.Myalgia is usually felt in the back and especially in the calfmuscles, which may be excruciatingly tender to touch. Thefever is generally greater than 39◦C and the red eyes are theresult of conjunctival suffusion, reflecting a generalized dila-tion of blood vessels, rather than a conjunctivitis. There isfrequently a macular rash on the trunk, lasting only a fewhours. Careful examination of the palate may show a simi-lar petechial rash. In untreated patients with the mild type ofleptospirosis, the symptoms usually persist for 3–10 days,followed by gradual but complete recovery. Occasionally,further acute symptoms may develop. These include oliguriafrom renal failure; acute abdominal pain, resembling an acuteabdominal emergency and sometimes due to acute cholecys-titis or pancreatitis; meningism, hemiparesis, or other neuro-logical signs; and cough, sometimes with mild hemoptysis.Jaundice is rare in mild-type leptospirosis. Aseptic menin-gitis may develop as a later complication, but most patientsrecover fully within 3–6 weeks.

In patients with severe leptospirosis, the symptomsincrease in severity, at times following a 1- or 2-day tempo-rary remission. Delirium and confusion may develop, withincreasing evidence of renal failure, together with hemor-rhages and jaundice. In addition, the spleen may be enlarged.


Pulmonary hemorrhages may be diffuse, resembling multi-ple infarcts or bronchopneumonia, accompanied by cough-ing of blood, but in some cases the pulmonary hemorrhagesand hemoptysis may be gross. Adult respiratory distress syn-drome can also occur and cause death. As liver and renalfailure progress, jaundice and oliguria increase, confusionand delirium give way to unconsciousness, and death occursthrough renal and hepatic failure. Supportive measures suchas dialysis can tide patients over renal failure until renalstructure is regenerated and function restored. Myocarditis,evidenced by arrhythmias, can cause sudden death. Finalrecovery is usually complete unless vital organs are irrepara-bly damaged. The earlier severe phase is described as thesepticemic stage and the later the tissue phase.

Death from renal failure may occur at any state of theillness. The autopsy appearances will depend on the dura-tion and degree of development of lesions. Usually, there isgeneralized jaundice and widespread hemorrhage in the skin,muscles, peritoneal surfaces, and lungs. The kidneys areenlarged, bile stained, and have subcapsular hemorrhages.Bleeding into all viscera including the adrenals is common.The liver shows signs of hepatitis and hemorrhages. Deathis so rare in mild cases that there is no record of significantand characteristic autopsy appearances. Uveitis may developat any time after the second week, and as late as 6 months. Itmay affect one or both eyes.(80)

8.1.1. Congenital Leptospirosis. Leptospirosis inpregnant women can be transmitted to the developing fetusat any state of pregnancy.(28,52) The fetus is actively infected.If it is severely damaged, even by serovars causing the mildtype of leptospirosis in the mother, it may die.(3,29) If infec-tion occurs near the time of birth, the child may developcongenital neonatal leptospirosis, whose manifestations aresimilar to those of adult leptospirosis. Complete recoveryfollows satisfactory treatment. The diagnosis of intrauterineleptospirosis depends on the correct diagnosis in the motherand evidence that the fetus is infected as shown by fetal dis-tress. Antenatal proof is usually only available if the fetusdies. If the fetus recovers before birth, IgM antibodies willbe present in the cord blood. The diagnosis of neonatal con-genital leptospirosis involves the same methods as used inadults.

8.1.2. Clinical Laboratory Findings. Laboratorytests, other than bacteriologic or serological, may help sup-port the clinical diagnosis. In the absence of bleeding, theerythrocyte count and hemoglobin may be normal unlessthe patient is jaundiced, and then they are both reduced.Leukocytosis of 11–20,000/mm3 (11–20 × 109/liter) is usualin icteric patients. The platelet count is frequently reduced,especially in severely jaundiced patients. The erythrocytesedimentation rate is generally raised.

Blood urea and creatinine are frequently raised, at leasttransiently, even in mild cases. Liver function tests are use-ful diagnostically. The serum aminotransferases can be nor-mal or mildly elevated. The aminotransferases are usuallybelow 250 I.U./L and are seldom greater than 500 I.U./L. Theserum bilirubin level is always raised in icteric or preictericleptospirosis. The combination of raised blood urea, raiseddirect bilirubin, and normal or slightly elevated aminotrans-ferases serves to differentiate leptospirosis from hepatitis inwhich the serum aminotransferases are always significantlyelevated.(41)

Proteinuria is a constant but sometimes transient find-ing, even in mild cases. In severe cases, or wherever thereis renal failure, granular and hyaline casts will be found inurine, together with bile.

Examination of CSF reveals a leukocytosis andincreased protein with normal glucose.

8.1.3. Prognosis. Recovery is usually completewithin a few weeks in mild-type patients. However, residualdepression, irritability, and psychosis have been reported,lasting up to 6 months and preventing a return to normal lifeand work. Occasional reports of prolonged headache andbackache following mild leptospirosis are hard to evaluate.Patients who survive the severe type of leptospirosis usuallyshow no further signs of illness within a few weeks of fullrecovery of function in vital organs. Prolonged neurologicalsymptoms and signs have been recorded, but in some ofthese patients there is room for doubt about the validity ofthe original diagnosis. In the severe disease mortality rangesfrom 5 to 25%.

8.1.4. Treatment. Penicillin (or erythromycin inallergic patients) is the recommended treatment of choice asearly as possible in the illness.(38) Doxycycline (83) or a third-generation cephalosporin are also used.(97,117) Given on thefirst day or two in patients diagnosed in the acute stage ofinfection, it can abort the symptoms and progress of eitherthe mild or the severe form abruptly, so that the patient isfrequently clinically well within 24 h.

Antibiotics given in the late (“tissue”) stages may makeno difference to the outcome,(30,40) because the disease pro-cess is then a consequence of established lesions, the lep-tospires usually having been eliminated by the patient’sdevelopment of antibodies. However, most clinicians wouldgive patients the benefit of the doubt and administer intra-venous penicillin, cefotaxime, or ceftriaxone. A Jarisch–Herxheimer reaction (temporary aggravation of symptomsand fever) may occur when antibiotic treatment is com-menced in well-advanced patients,(54,126) but this should notprevent the initiation of antibiotic treatment.(138)

In severe cases, symptomatic and supportive systemictreatment is required for hemorrhage and shock. Meticulous


attention to fluid and electrolyte balance is critical. Renalfailure may be treated by peritoneal dialysis, hemodialysis orcontinuous venovenous hemofiltration. There is no specifictherapy for the hepatic insufficiency. Patients with arrhyth-mias and myocarditis should be monitored in an intensivecare environment and the various arrhythmias treated. Severepulmonary hemorrhage may require assisted positive pres-sure ventilation.

8.2. Diagnosis

8.2.1. Clinical Diagnosis. The clinical features inearly leptospirosis of either the mild or the severe type are notpathognomonic and give no indication of the diagnosis to aninexperienced clinician. In areas where the disease is com-mon and has been recognized using laboratory confirmation,fresh cases can be diagnosed with a high degree of accuracy,provided that there is not a concomitant outbreak of a simi-larly presenting disease at that time e.g., dengue fever.(73,106)

Epidemiological factors such as occupational or domes-tic exposure to risk of infection from carrier animals, recentexposure to fresh water with immersion, or recent travelwill influence the interpretation of clinical findings, pendingresults of confirmatory laboratory tests.

8.2.2. Laboratory Bacteriologic Diagnosis. Theprinciples of diagnosis are isolation of leptospires fromblood, urine, CSF, or other sites during the acute illness,demonstration of leptospires in tissues and body fluids, andserological tests. Because of the low index of suspicion com-bined with the perceived difficulty of culturing leptospires,most cases are diagnosed by serology.

8.2.2.1. Diagnosis by Direct Detection of Leptospires.Leptospires can be seen occasionally in body fluids bydark-field illumination. Inexpert observers looking at bodyfluids can mistake fibrin threads for leptospires. Dark-fieldmicroscopy is neither sensitive nor specific enough for diag-nostic use.(131) Leptospires may also be identified directly bystaining of smears, or of frozen or wax-embedded histolog-ical sections of autopsy or biopsy material. Molecular diag-nosis of leptospirosis is discussed above.

8.2.2.2. Culture. Diagnostic cultures are usuallymade from specimens of blood, urine, CSF, other body fluidsand exudates, and occasionally from tissues obtained atbiopsy or autopsy. Tween–albumin fluid culture media suchas EMJH are most generally useful, because more exactingstrains grow better in it on primary isolation. Special mediaare required for primary isolation and growth of fastidiouspathogens.

Blood cultures are made by inoculating blood drawnaseptically by venipuncture directly into culture media. Inoc-ulation of several tubes, each containing 5–10 ml of medium,

with one or two drops of blood per tube (up to 250 μl pertube), is recommended. The blood should be diluted in theculture medium by a factor ofat least one in ten in order todilute antileptospiral activity in the blood. If blood cannotbe inoculated at the bedside, it can be collected in a sterileammonium oxalate or heparin anticoagulant tube or even asa last resort in a plain tube and transported to a laboratory,where the blood or serum can be inoculated into medium.If possible, multiple cultures should be prepared from eachspecimen. Penicillinase and sodium polyanetholesulfonate(SPS) can be added if required. The inoculated media areincubated at 30◦C and examined for growth by dark-fieldmicroscopy daily for the first week and weekly thereafter,up to 3 months, and ideally they should be subculturedweekly. The value of blood culture is definitive identificationof the etiological agent rather than speed. Isolates may beidentified by serological or molecular methods as describedabove.

Culture of urine requires an alkalinized fresh mid-stream or catheter specimen taken with care to minimizecontamination. A selective medium containing 100 μg/ml5-fluorouracil as an inhibitor of contaminant microorgan-isms and semisolid medium containing 0.1% agar are rec-ommended, using at least a tenfold dilution of urine in themedium. Multiple cultures should be prepared from eachspecimen.

Leptospires may be found in CSF in the first 10 days ofillness and can be cultured from it by inoculating 0.1–0.5 mlinto 5–10 ml of fluid or semisolid culture medium.

8.2.2.3. Serological Diagnosis. Even if cultures areset up, recovery of leptospires requires weeks, rather thandays, of incubation. Moreover, the diagnosis of leptospiro-sis is often not suspected, or the patient does not seek med-ical attention until the illness is well advanced, when lep-tospires be difficult to recover. Thus, serological tests maybe the most reliable means by which a retrospective, if notconcurrent, diagnosis can be made.

Both IgM detection by ELISA and detection of agglu-tinating antibodies by MAT are useful for diagnosis. Serumspecimens for diagnosis by serological tests should be takenimmediately at the outset of investigations, preferably on thefirst day of illness, and tested as soon as possible. A fur-ther specimen should be taken on the fifth to seventh days,and further tests if required on the 10th and 12th days. IgMantibodies are detectable 5–7 days after onset of symptoms,thus sensitivity of IgM detection is low during the first weekof symptoms, rising rapidly after that.(32) Early in the ill-ness, detection of IgM is significantly more sensitive thanthe MAT. A positive IgM should be followed by MAT test-ing of acute and convalescent specimens for confirmation byrising antibody titers.


8.2.3. Differential Diagnosis. At the onset of eithermild or severe leptospirosis, there is little to distinguishit from any other acute febrile illness. The very severeheadache, fever, and myalgia are characteristic, but atthis stage they may be confused with severe influenza,poliomyelitis, dengue, Q fever, and viral meningitis. Themain differentiating criteria in leptospirosis are raised bloodcreatinine and urea, leukocytosis, and in some cases, an earlyrise in titer of leptospiral antibodies. At a later stage the maindifferential diagnoses are with viral meningitis and acuteabdominal emergencies in mild-type leptospirosis and withmalaria, blackwater fever, viral meningitis, hepatitis, yellowfever, hemorrhagic fever with renal syndrome (HFRS) andacute glomerulonephritis in severe leptospirosis. The maindiagnostic criteria are high serum bilirubin with low or nor-mal levels of serum aminotransferases, thrombocytopenia,and renal insufficiency with significant and rising titers ofspecific antileptospiral antibodies. The isolation and identifi-cation of leptospires from blood, urine, or CSF, or a positivePCR assay, confirm the diagnosis. At autopsy, differentialdiagnosis depends on characteristic pathological changes ofjaundice and widespread hemorrhages with pale, enlargedhemorrhagic kidneys showing histological evidence of renaltubular degeneration and hemorrhage, liver cell degenera-tion, myocarditis, and pulmonary hemorrhages. Postmortemserum specimens may be tested for antibodies. PCR andIHC testing of autopsy specimens are sensitive, particu-larly in patients who die of fulminant disease before theyseroconvert.(17)

In all patients, an epidemiological history of occupa-tional or accidental exposure to risk and animal contact ortravel should increase the index of suspicion and indicate theneed for specific diagnostic tests.

9. Control and Prevention

9.1. General Concepts

As is the case in all zoonoses, control of leptospirosisin humans is complex, because the primary sources of thedisease are in animals, in some of which control is impos-sible. Regulation if not elimination of leptospirosis may befeasible in livestock and domestic animals, even though car-riers are clinically normal and shedders hard to detect, butit cannot be envisaged in wildlife. Control is further compli-cated by the general and nonspecific clinical picture of mildor early severe leptospirosis, indistinguishable from a varietyof other incapacitating fevers common in tropical areas, sothat an animal source is often not suspected. The essentialsof control and prevention lie in awareness and recognition

of the disease and in containment in livestock and domesticsources.

Awareness and recognition require a knowledge ofepidemiological facts acquired by selected surveillance,reporting, and notification, which in turn need specializeddiagnostic laboratory services and education of physicians,veterinarians, administrators, and the public. Althoughcontainment of animal sources may be seen as a veterinarypublic health and hygiene problem, it is essential to the pro-tection of humans. The main measures are veterinary surveil-lance, treatment, and prevention; occupational hygiene;rodent control; engineering; laboratory safety; and evaluationof preventive measures. In addition, containment extends tothe prevention of human disease by the judicious avoidanceof risk, by immunization, and by chemoprophylaxis.

9.1.1. Education, Laboratory Services, andNotification. Ordinary routine microbiological diag-nostic laboratories are not usually equipped with skilled staffand specialized techniques for leptospirosis. If specializeddiagnosis is not available locally, there should be a centralleptospirosis laboratory available for referral of specimensand consultation. Reference laboratories staffed by expertsare required only on a regional international basis for stockcultures, standard sera, research, education, and consul-tation. New methods of molecular diagnosis may makediagnostic facilities more readily available.

Evaluation of diagnostic laboratory results provides auseful form of surveillance. In addition, prevalence surveysfor antibodies in selected groups can reveal problems or eval-uate control measures. The most effective form of informa-tion gathering should be notification by health practitioners.Usually, this is of limited value because of inadequate diag-nosis, failure to use appropriate laboratory tests, and fail-ure to notify. Statistics of practitioner notifications frequentlyindicate an incidence much lower than diagnosed by labora-tories.

9.1.2. Control Measures in Animals9.1.2.1. Rodent Control. Removal of rodents from the

domestic environment effectively reduces the infection rateof leptospirosis. Standard measures include control of litterand garbage disposal, protecting food sources, trapping, andpoisoning. When they share the environment with crops, pre-cautions can be taken, such as burning off sugarcane beforecutting by hand or mechanization of cutting.(50)

9.1.2.2. Environmental Control. Any measure toreduce the load of leptospires in soil, water, or mudwill reduce the risk to humans. Simple drainage canprevent seepage of urine into waterlogged soils or yardswhere livestock are herded. Environmental engineeringcan also increase the risk by flooding previously dryareas.


9.1.2.3. Immunization, Chemotherapy, and Culling ofLivestock. Excreter rates in cattle and pig herds frequentlyreach levels of 65–90%. If shedders can be identified andremoved from herds, the risks to the herd and to attendanthumans can be reduced, provided the remaining animals areimmunized, the herd is restocked with immunized animals,fresh excreter animals are not introduced, and the herd isprotected from fresh infection from environmental sources.Immunization alone can reduce both animal and indirectlyhuman leptospirosis, but it is hard to evaluate, becausemost studies have been specially set up under artificialconditions accompanied by education and improvementsin occupational hygiene and farming practice. Culling orvaccination is both expensive and their use by small holdingfarmers will depend on the relative values and costs ofanimals, vaccines, and health (or illness). The associatedcosts of transport, skilled attention, and equipment may alsobe relatively high. The control of leptospirosis in stud stock,where leptospires may be found in semen samples used forartificial insemination, requires special methods for testingand transport of semen.

9.1.3. Treatment of the Carrier State. Carrier ani-mals have been cured by streptomycin treatment. The costis relatively high and the results so difficult to evaluate infield conditions that it is considered impractical for generaluse.(43,56)

Humans do not become chronic carriers or excretersfor any length of time. Elementary domestic, personal, andhospital hygiene ensures little risk from patients and con-valescents still excreting leptospires. Urine specimens fromconvalescent patients are not recognized sources of labora-tory infection, but care should be taken nonetheless.

9.1.4. Occupational Hygiene. Preventive measuresfor people who cannot avoid exposure to risk are centeredaround methods for physical protection in their work orleisure. Waterproof footwear, watershedding aprons wornoutside and over the boots or other clothing, and clear plas-tic facemasks have all been recommended for milkers andslaughterhouse workers. In hot climates or working condi-tions, impermeable protective clothing is uncomfortable andpoorly accepted. Nevertheless, rubber boots provide obviousprotection. Wearing of gloves by field workers, such as canecutters, is also protective. Most infections appear to followpenetration of cuts and damaged or abraded skin by lep-tospires. It is therefore important to protect such injuries, butoften not practicable to do so in daily working conditions.

Good drainage of floors and herding yards and controlof rodent infestation in food preparation areas are impor-tant occupational hygienic measures, but no measures willbe effective unless the people at risk understand the disease,the means of infection, and the need for precautions so that

they are prepared to implement recommended practices. Therisk for field workers can be reduced by attempting to controlthe environmental hazards.

9.2. Antimicrobial Prophylaxis

Chemoprophylaxis with doxycycline has been usedto prevent leptospirosis in soldiers exposed to jungleconditions(119) and could be used similarly to protect anyoneexposed to a significant risk for a brief defined period. Peo-ple in such categories could include those about to undertakerafting or canoeing trips, cavers, grain farmers and workersat harvest time when risks are serious for a short period only,and field ecologists trapping small wild rodents. Doxycyclineprophylaxis of large populations after severe flooding has notbeen shown to be effective for preventing infection,(57,108) butmay reduce the incidence of clinical disease.

9.3. Immunization

Immunization of humans and animals with killed cul-tures of leptospires has been used almost since leptospireswere discovered. Vaccines are serovar specific and currentlyadministered subcutaneously in two or more doses 2 or 4weeks apart and repeated annually. Vaccines for human useare licensed in France, Cuba, and several Asian countries.

Local reactions of pain, redness, and swelling rangingfrom mild to very severe occur, especially on revaccination.Washed or whole culture vaccines have been employed inseveral countries, mainly in Asia, where the risks to life andhealth from severe leptospirosis could justify the side effectsof the vaccine.

10. Unresolved Problems

There is a series of interconnected questions that needanswers before great improvements in control of leptospiro-sis can be achieved. Neither the duration of postinfectionimmunity nor its specificity is known. Conventional wisdombased mainly on animal experimentation states that immu-nity is specific for antigens that are serovar specific, or at bestshared within a serogroup. Is there any underlying genus-or species-specific immunity in humans mediating a level ofresistance to all serovars of pathogenic leptospires?

There are genetic differences in the susceptibility ofpigs to primary infection. At least one study from an outbreaksuggests that human susceptibility may be under geneticcontrol.(76) Can this study be generalized, and if so, are thedifferences reflected in the response to particular serovars orto all serovars, and thus to the pathogenicity of particularserovars for humans?


There are over 250 serovars described among thepathogenic leptospires, based on antigenic surface charac-teristics mediated by LPS-related polysaccharide epitopes.Although part of the LPS gene has been characterized, thegenetic basis for serovar specificity and variation is stillunknown. This problem becomes acutely relevant withthe recognition that the various species of Leptospira eachcontain serovars also found in other species; that is, serovarspecificity overlaps the genetic groupings. Moroever,mosaicism within several genes suggests horizontaltransfer.(60) The effects of these observations on classificationof leptospires have implications for diagnosis, epidemiology,and prognosis. Many more isolates need to be genotyped,but to do this will require more easily applied methods.

Rapid bedside tests are required that will enable a clini-cian to identify whether or not a patient has leptospirosis, andif so, which serovar, often at a time in the illness before anti-bodies have appeared in blood. Direct tests for leptospiresor their antigens would overcome the serious difficulty ofevaluating the significance of a positive serological test ina patient in an endemic area where most of the populationhave antibodies, even residual IgM, to one or more serovars,or where the patient may have residual antibodies from aninfection with a serovar unrelated to the present illness. Sim-ilar direct diagnostic methods would allow easier and morepositive detection of carrier animals than is possible now.

Most people at risk from leptospirosis live in developingcountries without ready access to specialized diagnostic ortreatment facilities. There is seldom a capacity for new scien-tific research into the problems identified here. On the otherhand, leptospirosis is rarely diagnosed in urban agglomer-ations in developed countries where most researchers andinstitutes capable of high technology are found and thereare relatively few among them interested in leptospirosis.Knowledge and research in leptospirosis are thus not avail-able in most developed countries, to which developing coun-tries turn for scientific assistance. The situation is aggravatedwhen inadequate statistics in developed countries indicate aspuriously low rate of leptospirosis, leading to an impressionthat the disease is nonexistent or unimportant and does notjustify allocation of funds for the maintenance of existingresearch and surveillance or for new studies toward solutionsto the problems outlined above.

11. References

1. Adler, B., Murphy, A. M., Locarnini, S. A., & Faine, S. (1980). Detec-tion of specific anti-leptospiral immunoglobulins M and G in humanserum by solid-phase enzyme-linked immunosorbent assay. Journalof Clinical Microbiology, 11, 452–457.

2. Ahmed, N., Devi, S. M., Valverde Mde, L., Vijayachari, P.,Machang’u, R. S., Ellis, W. A., et al. (2006). Multilocus sequencetyping method for identification and genotypic classification ofpathogenic Leptospira species. Annals of Clinical Microbiology andAntimicrobials, 5, 28.

3. Aker, N., James, E. B., Johnston, A. M., & Pasvol, G. (1996). Lep-tospirosis in pregnancy: an unusual and relatively unrecognised causeof intrauterine death. Journal of Obstetrics and Gynaecology, 16,163–165.

4. Alexander, A., Baer, A., Fair, J. R., Gochenour, W. S., King, J. H., &Yager, R. N. (1952). Leptospiral uveitis. Report of a bacteriologicallyconfirmed case. Archives of Ophthalmology, 48, 292–297.

5. Alexander, A. D., Evans, L. B., Baker, M. F., Baker, H. J., Ellison,D., & Marriapan, M. (1975). Pathogenic leptospiras isolated fromMalaysian surface waters. Applied Microbiology, 29, 33–33.

6. Arean, V. M. (1962). The pathologic anatomy and pathogenesisof fatal human leptospirosis (Weil’s disease). American Journal ofPathology, 40, 393–423.

7. Artiushin, S., Timoney, J. F., Nally, J., & Verma, A. (2004). Host-inducible immunogenic sphingomyelinase-like protein, Lk73.5, ofLeptospira interrogans. Infection and Immunity, 72, 742–749.

8. Ballard, S. A., Williamson, M., Adler, B., Vinh, T., & Faine, S.(1986). Interactions of virulent and avirulent leptospires with primarycultures of renal epithelial cells. Journal of Medical Microbiology, 21,59–67.

9. Barbosa, A. S., Abreu, P. A., Neves, F. O., Atzingen, M. V., Watanabe,M. M., Vieira, M. L., et al. (2006). A newly identified leptospiraladhesin mediates attachment to laminin. Infection and Immunity, 74,6356–6364.

10. Berman, S. J., Tsai, C. C., Holmes, K. K., Fresh, J. W., &Watten, R. H. (1973). Sporadic anicteric leptospirosis in South Viet-nam. Annals of Internal Medicine, 79, 167–173.

11. Blackmore, D. K., Schollum, L. M., & Moriarty, K. M. (1984). Themagnitude and duration of titres of leptospiral agglutinins in humansera. New Zealand Medical Journal, 97, 83–86.

12. Boland, M., Sayers, G., Coleman, T., Bergin, C., Sheehan, N.,Creamer, E., et al. (2004). A cluster of leptospirosis cases in canoeistsfollowing a competition on the River Liffey. Epidemiology and Infec-tion, 132, 195–200.

13. Bolin, C. A., & Alt, D. P. (2001). Use of a monovalent leptospiralvaccine to prevent renal colonization and urinary shedding in cattleexposed to Leptospira borgpetersenii serovar hardjo. American Jour-nal of Veterinary Research, 62, 995–1000.

14. Boursaux-Eude, C., Saint Girons, I., & Zuerner, R. L. (1998). Lep-tospira genomics. Electrophoresis, 19, 589–592.

15. Branger, C., Sonrier, C., Chatrenet, B., Klonjkowski, B.,Ruvoen-Clouet, N., Aubert, A., et al. (2001). Identification ofthe hemolysis-associated protein 1 as a cross-protective immunogenof Leptospira interrogans by adenovirus-mediated vaccination.Infection and Immunity, 69, 6831–6838.

16. Brenner, D. J., Kaufmann, A. F., Sulzer, K. R., Steigerwalt, A. G.,Rogers, F. C., & Weyant, R. S. (1999). Further determination ofDNA relatedness between serogroups and serovars in the family Lep-tospiraceae with a proposal for Leptospira alexanderi sp. nov. andfour new Leptospira genomospecies. International Journal of Sys-tematic Bacteriology, 49, 839–858.

17. Brown, P. D., Carrington, D. G., Gravekamp, C., van de Kemp, H.,Edwards, C. N., Jones, S. R., et al. (2003). Direct detection of lep-tospiral material in human postmortem samples. Research in Micro-biology, 154, 581–586.


18. Brown, P. D., Gravekamp, C., Carrington, D. G., Van de Kemp, H.,Hartskeerl, R. A., Edwards, C. N., et al. (1995). Evaluation of thepolymerase chain reaction for early diagnosis of leptospirosis. Jour-nal of Medical Microbiology, 43, 110–114.

19. Brown, R. A., Blumerman, S., Gay, C., Bolin, C., Duby, R., &Baldwin, C. L. (2003). Comparison of three different leptospiral vac-cines for induction of a type 1 immune response to Leptospira borg-petersenii serovar Hardjo. Vaccine, 21, 4448–4458.

20. Bulach, D. M., Kalambaheti, T., de La Pena-Moctezuma, A., & Adler,B. (2000). Lipopolysaccharide biosynthesis in Leptospira. Journal ofMolecular Microbiology and Biotechnology, 2, 375–380.

21. Bulach, D. M., Zuerner, R. L., Wilson, P., Seemann, T.,McGrath, A., Cullen, P. A., et al. (2006). Genome reductionin Leptospira borgpetersenii reflects limited transmission poten-tial. Proceedings of the National Academy of Sciences USA, 103,14560–14565.

22. Cacciapuoti, B., Ciceroni, L., Maffei, C., Di Stanislao, F., Strusi, P.,Calegari, L., et al. (1987). A waterborne outbreak of leptospirosis.American Journal of Epidemiology, 126, 535–545.

23. Cargill, C. F., & Davos, D. E. (1981). Renal leptospirosis in vacci-nated pigs. Australian Veterinary Journal, 57, 236–238.

24. Centers for Disease Control and Prevention. (1997a). Case definitionsfor infectious conditions under public health surveillance. Morbidityand Mortality Weekly Reports, 46 (No. RR-10), 49.

25. Centers for Disease Control and Prevention. (1997b). Outbreak ofleptospirosis among white-water rafters – Costa Rica, 1996. Morbid-ity and Mortality Weekly Reports, 46, 577–579.

26. Chappel, R. J., Goris, M. G., Palmer, M. F., & Hartskeerl, R. A.(2004). Impact of proficiency testing on results of the microscopicagglutination test for diagnosis of leptospirosis. Journal of ClinicalMicrobiology, 42, 5484–5488.

27. Choy, H. A., Kelley, M. M., Chen, T. L., Moller, A. K., Matsunaga, J.,& Haake, D. A. (2007). Physiological osmotic induction of Lep-tospira interrogans adhesion: LigA and LigB bind extracellu-lar matrix proteins and fibrinogen. Infection and Immunity, 75,2441–2450.

28. Chung, H.-L., Ts’ao, W.-C., Mo, P.-S., & Yen, C. (1963). Transpla-cental or congenital infection of leptospirosis. Chinese Medical Jour-nal, 82, 777–782.

29. Coghlan, J. D., & Bain, A. D. (1969). Leptospirosis in human preg-nancy followed by death of the foetus. British Medical Journal, 1,228–230.

30. Costa, E., Lopes, A. A., Sacramento, E., Costa, Y. A., Matos, E. D.,Lopes, M. B., et al. (2003). Penicillin at the late stage of leptospiro-sis: a randomized controlled trial. Revista do Instituto de MedicinaTropical de Sao Paulo, 45, 141–145.

31. Cullen, P. A., Haake, D. A., & Adler, B. (2004). Outer membraneproteins of pathogenic spirochetes. FEMS Microbiology Reviews, 28,291–318.

32. Cumberland, P. C., Everard, C. O. R., & Levett, P. N. (1999). Assess-ment of the efficacy of the IgM enzyme-linked immunosorbent assay(ELISA) and microscopic agglutination test (MAT) in the diagnosisof acute leptospirosis. American Journal of Tropical Medicine andHygiene, 61, 731–734.

33. Cumberland, P. C., Everard, C. O. R., Wheeler, J. G., &Levett, P. N. (2001). Persistence of anti-leptospiral IgM, IgG andagglutinating antibodies in patients presenting with acute febrile ill-ness in Barbados 1979–1989. European Journal of Epidemiology, 17,601–608.

34. de Brito, T., Machado, M. M., Montans, S. D., Hosino, S., &Freymuller, E. (1967). Liver biopsy in human leptospirosis: a light

and electron microscopy study. Virchows Archiv A, PathologicalAnatomy and Histopathology, 342, 61–69.

35. de Brito, T., Morais, C. F., Yasuda, P. H., Lancellotti, C. P.,Hoshino-Shimizu, S., Yamashiro, E., et al. (1987). Cardiovascularinvolvement in human and experimental leptospirosis: pathologicfindings and immunohistochemical detection of leptospiral antigen.Annals of Tropical Medicine and Parasitology, 81, 207–214.

36. Diesch, S. L., & McCulloch, W. F. (1966). Isolation of pathogenicleptospires from waters used for recreation. Public Health Reports,81, 299–304.

37. Dikken, H., & Kmety, E. (1978). Serological typing methods of lep-tospires. In T. Bergan & J. R. Norris (Eds.), Methods in microbiology(Vol. 11, pp. 259–307). London: Academic Press.

38. Edwards, C. N., & Levett, P. N. (2004). Prevention and treat-ment of leptospirosis. Expert Review of Anti-infective Therapy, 2,293–298.

39. Edwards, C. N., Nicholson, G. D., Hassell, T. A., Everard, C. O. R., &Callender, J. (1986). Thrombocytopenia in leptospirosis: the absenceof evidence for disseminated intravascular coagulation. AmericanJournal of Tropical Medicine and Hygiene, 35, 352–354.

40. Edwards, C. N., Nicholson, G. D., Hassell, T. A., Everard, C. O. R., &Callender, J. (1988). Penicillin therapy in icteric leptospirosis. Amer-ican Journal of Tropical Medicine and Hygiene, 39, 388–390.

41. Edwards, C. N., Nicholson, G. D., Hassell, T. A., Everard, C. O. R.,& Callender, J. (1990). Leptospirosis in Barbados: a clinical study.West Indian Medical Journal, 39, 27–34.

42. Ellis, W. A., McParland, P. J., Bryson, D. G., Thiermann, A. B., &Montgomery, J. (1986). Isolation of leptospires from the genital tractand kidneys of aborted sows. Veterinary Record, 118, 294–295.

43. Ellis, W. A., Montgomery, J., & Cassells, J. A. (1985). Dihy-drostreptomycin treatment of bovine carriers of Leptospira inter-rogans serovar hardjo. Research in Veterinary Science, 39,292–295.

44. Everard, C. O. R., & Bennett, S. (1990). Persistence of leptospiralagglutinins in Trinidadian survey subjects. European Journal of Epi-demiology, 6, 40–44.

45. Everard, C. O. R., Edwards, C. N., Everard, J. D., & Carrington, D. G.(1995). A twelve year study of leptospirosis on Barbados. EuropeanJournal of Epidemiology, 11, 311–320.

46. Everard, C. O. R., Fraser-Chanpong, G. M., Hayes, R.,Bhagwandin, L. J., & Butcher, L. V. (1982). A survey of lep-tospirosis in febrile patients mainly from hospitals and clinics inTrinidad. Transactions of the Royal Society of Tropical Medicine andHygiene, 76, 487–492.

47. Everard, C. O. R., Jones, C. J., Inniss, V. A., Carrington, D. G., &Vaughan, A. W. (1987). Leptospirosis in dogs on Barbados. IsraelJournal of Veterinary Medicine, 43, 288–295.

48. Everard, J. D. (1996). Leptospirosis. In F. E. G. Cox (Ed.), The Well-come Trust Illustrated History of Tropical Diseases (pp. 111–119,416–418). London: The Wellcome Trust.

49. Everard, J. D., & Everard, C. O. R. (1993). Leptospirosis in theCaribbean. Reviews in Medical Microbiology, 4, 114–122.

50. Faine, S., Adler, B., Bolin, C., & Perolat, P. (1999). Leptospira andleptospirosis (2 ed.). Melbourne: MedSci.

51. Faine, S., & Stallman, N. D. (1982). Amended descriptions of thegenus Leptospira Noguchi 1917 and the species L. interrogans (Stim-son 1907) Wenyon 1926 and L. biflexa (Wolbach and Binger 1914)Noguchi 1918. International Journal of Systematic Bacteriology, 32,461–463.

52. Faine, S., & Valentine, R. (1984). Leptospirosis hardjo in pregnancy.Medical Journal of Australia, 311–312.


53. Feigin, R. D., Lobes, L. A., Anderson, D., & Pickering, L. (1973).Human leptospirosis from immunized dogs. Annals of InternalMedicine, 79, 777–785.

54. Friedland, J. S., & Warrell, D. A. (1991). The Jarisch-Herxheimerreaction in leptospirosis: possible pathogenesis and review. Reviewsof Infectious Diseases, 13, 207–210.

55. Galloway R., & Levett, P. N. (2008). Evaluation of a modified pulsed-field gel electrophoresis approach for the identification of Leptospiraserovars. American Journal of Tropical Medicine and Hygiene, 78,628–632.

56. Gerritsen, M. J., Koopmans, M. J., & Olyhoek, T. (1993). Effect ofstreptomycin treatment on the shedding of and the serologic responsesto Leptospira interrogans serovar hardjo subtype hardjobovis in exper-imentally infected cows. Veterinary Microbiology, 38, 129–138.

57. Gonsalez, C. R., Casseb, J., Monteiro, F. G., Paula-Neto, J. B.,Fernandez, R. B., Silva, M. B., et al. (1998). Use of doxycycline forleptospirosis after high-risk exposure in Sao Paulo, Brazil. Revista doInstituto de Medicina Tropical de Sao Paulo, 41, 59–61.

58. Gordon Smith, C. E., & Turner, L. H. (1961). The effect of pH on thesurvival of leptospires in water. Bulletin of the World Health Organi-zation, 24, 35–43.

59. Gravekamp, C., van de Kemp, H., Franzen, M., Carrington, D.,Schoone, G. J., van Eys, G. J. J. M., et al. (1993). Detection of sevenspecies of pathogenic leptospires by PCR using two sets of primers.Journal of General Microbiology, 139, 1691–1700.

60. Haake, D. A., Suchard, M. A., Kelley, M. M., Dundoo, M., Alt, D. P.,& Zuerner, R. L. (2004). Molecular evolution and mosaicism of lep-tospiral outer membrane proteins involves horizontal DNA transfer.Journal of Bacteriology, 186, 2818–2828.

61. Harkin, K. R., Roshto, Y. M., Sullivan, J. T., Purvis, T. J., &Chengappa, M. M. (2003). Comparison of polymerase chain reac-tion assay, bacteriologic culture, and serologic testing in assessmentof prevalence of urinary shedding of leptospires in dogs. Journal ofthe American Veterinary Medical Association, 222, 1230–1233.

62. Herrmann, J. L. (1993). Genomic techniques for identification of Lep-tospira strains. Pathologie Biologie, 41, 943–950.

63. Herrmann, J. L., Bellenger, E., Perolat, P., Baranton, G., & SaintGirons, I. (1992). Pulsed-field gel electrophoresis of NotI digests ofleptospiral DNA: a new rapid method of serovar identification. Jour-nal of Clinical Microbiology, 30, 1696–1702.

64. Johnson, R. C., & Faine, S. (1984). Leptospira. In N. R. Krieg &J. G. (Eds.), Bergey’s Manual of Systematic Bacteriology (Vol. 1,pp. 62–67). Baltimore: Williams & Wilkins.

65. Kingscote, B. F. (1970). Correlation of bedrock type with the geog-raphy of leptospirosis. Canadian Journal of Comparative Medicine,34, 31–37.

66. Klimpel, G. R., Matthias, M. A., & Vinetz, J. M. (2003). Leptospirainterrogans activation of human peripheral blood mononuclear cells:preferential expansion of TCR γδ+ T cells vs TCR αβ+ T cells. Jour-nal of Immunology, 171, 1447–1455.

67. Kmety, E., & Dikken, H. (1993). Classification of the species Lep-tospira interrogans and history of its serovars. Groningen: UniversityPress Groningen.

68. Ko, A. I., Galvao Reis, M., Ribeiro Dourado, C. M., Johnson, W. D.,Riley, L. W., & Salvador Leptospirosis Study Group. (1999). Urbanepidemic of severe leptospirosis in Brazil. Lancet, 354, 820–825.

69. La Scola, B., Bui, L. T. M., Baranton, G., Khamis, A., & Raoult, D.(2006). Partial rpoB gene sequencing for identification of Leptospiraspecies. FEMS Microbiology Letters, 263, 142–147.

70. Levett, P. N. (2001). Leptospirosis. Clinical Microbiology Reviews,14, 296–326.

71. Levett, P. N. (2003). Usefulness of serologic analysis as a predictorof the infecting serovar in patients with severe leptospirosis. ClinicalInfectious Diseases, 36, 447–452.

72. Levett, P. N. (2004). Leptospirosis. In G. L. Mandell, J. E. Bennett &R. Dolin (Eds.), Principles and Practice of Infectious Diseases (6thedn., pp. 2789–2795.). Philadelphia: Elsevier.

73. Levett, P. N., Branch, S. L., & Edwards, C. N. (2000). Detection ofdengue infection in patients investigated for leptospirosis in Barba-dos. American Journal of Tropical Medicine and Hygiene, 62, 112–114.

74. Levett, P. N., Branch, S. L., Whittington, C. U., Edwards, C. N., &Paxton, H. (2001). Two methods for rapid serological diagnosis ofacute leptospirosis. Clinical and Diagnostic Laboratory Immunology,8, 349–351.

75. Levett, P. N., Morey, R. E., Galloway, R. L., Turner, D. E., Steiger-walt, A. G., & Mayer, L. W. (2005). Detection of pathogenic lep-tospires by real-time quantitative PCR. Journal of Medical Microbi-ology, 54, 45–49.

76. Lingappa, J., Kuffner, T., Tappero, J., Whitworth, W., Mize, A.,Kaiser, R., et al. (2004). HLA-DQ6 and ingestion of contaminatedwater: possible gene-environment interaction in an outbreak of lep-tospirosis. Genes and Immunity, 5, 197–202.

77. Lucchesi, P. M., Parma, A. E., & Arroyo, G. H. (2002). Serovar dis-tribution of a DNA sequence involved in the antigenic relationshipbetween Leptospira and equine cornea. BMC Microbiology, 2, 3.

78. Lupidi, R., Cinco, M., Balanzin, D., Delprete, E., & Varaldo, P. E.(1991). Serological follow-up of patients in a localized outbreak ofleptospirosis. Journal of Clinical Microbiology, 29, 805–809.

79. Majed, Z., Bellenger, E., Postic, D., Pourcel, C., Baranton, G.,& Picardeau, M. (2005). Identification of variable-number tandem-repeat loci in Leptospira interrogans sensu stricto. Journal of ClinicalMicrobiology, 43, 539–545.

80. Mancel, E., Merien, F., Pesenti, L., Salino, D., Angibaud, G., & Per-olat, P. (1999). Clinical aspects of ocular leptospirosis in New Cale-donia (South Pacific). Australian and New Zealand Journal of Oph-thalmology, 27, 380–386.

81. Matsunaga, J., Sanchez, Y., Xu, X., & Haake, D. A. (2005). Osmolar-ity, a key environmental signal controlling expression of leptospiralproteins LigA and LigB and the extracellular release of LigA. Infec-tion and Immunity, 73, 70–78.

82. Matsunaga, J., Werneid, K., Zuerner, R. L., Frank, A., & Haake, D. A.(2006). LipL46 is a novel surface-exposed lipoprotein expressed dur-ing leptospiral dissemination in the mammalian host. Microbiology,152, 3777–3786.

83. McClain, J. B. L., Ballou, W. R., Harrison, S. M., & Steinweg, D. L.(1984). Doxycycline therapy for leptospirosis. Annals of InternalMedicine, 100, 696–698.

84. Meites, E., Jay, M. T., Deresinski, S., Shieh, W. J., Zaki, S. R.,Tompkins, L., et al. (2004). Reemerging leptospirosis, California.Emerging Infectious Diseases, 10, 406–412.

85. Meri, T., Murgia, R., Stefanel, P., Meri, S., & Cinco, M. (2005). Reg-ulation of complement activation at the C3-level by serum resistantleptospires. Microbial Pathogenesis, 39, 139–147.

86. Merien, F., Amouriauz, P., Perolat, P., Baranton, G., & SaintGirons, I. (1992). Polymerase chain reaction for detection of Lep-tospira spp. in clinical samples. Journal of Clinical Microbiology, 30,2219–2224.

87. Merien, F., Baranton, G., & Perolat, P. (1995). Comparison ofpolymerase chain reaction with microagglutination test and culturefor diagnosis of leptospirosis. Journal of Infectious Diseases, 172,281–285.


88. Merien, F., & Perolat, P. (1996). Public health importance of humanleptospirosis in the South Pacific: a five-year study in New Cale-donia. American Journal of Tropical Medicine and Hygiene, 55,174–178.

89. Merien, F., Portnoi, D., Bourhy, P., Charavay, F., Berlioz-Arthaud,A., & Baranton, G. (2005). A rapid and quantitative method for thedetection of Leptospira species in human leptospirosis. FEMS Micro-biology Letters, 249, 139–147.

90. Morey, R. E., Galloway, R. L., Bragg, S. L., Steigerwalt, A. G.,Mayer, L. W., & Levett, P. N. (2006). Species-specific identificationof Leptospiraceae by 16S rRNA gene sequencing. Journal of ClinicalMicrobiology, 44, 3510–3516.

91. Morgan, J., Bornstein, S. L., Karpati, A. M., Bruce, M., Bolin, C. A.,Austin, C. C., et al. (2002). Outbreak of Leptospirosis amongTriathlon participants and community residents in Springfield, Illi-nois, 1998. Clinical Infectious Diseases, 34, 1593–1599.

92. Naiman, B. M., Alt, D., Bolin, C. A., Zuerner, R., & Baldwin, C. L.(2001). Protective killed Leptospira borgpetersenii vaccine inducespotent Th1 immunity comprising responses by CD4 and γδ T lym-phocytes. Infection and Immunity, 69, 7550–7558.

93. Nally, J. E., Chow, E., Fishbein, M. C., Blanco, D. R., &Lovett, M. A. (2005). Changes in lipopolysaccharide O antigendistinguish acute versus chronic Leptospira interrogans infections.Infection and Immunity, 73, 3251–3260.

94. Nally, J. E., Timoney, J. F., & Stevenson, B. (2001). Temperature-regulated protein synthesis by Leptospira interrogans. Infection andImmunity, 69, 400–404.

95. Nascimento, A. L., Ko, A. I., Martins, E. A., Monteiro-Vitorello, C. B., Ho, P. L., Haake, D. A., et al. (2004). Compara-tive genomics of two Leptospira interrogans serovars reveals novelinsights into physiology and pathogenesis. Journal of Bacteriology,186, 2164–2172.

96. Palaniappan, R. U., Chang, Y. F., Chang, C. F., Pan, M. J.,Yang, C. W., Harpending, P., et al. (2005). Evaluation of lig-basedconventional and real time PCR for the detection of pathogenic lep-tospires. Molecular and Cellular Probes, 19, 111–117.

97. Panaphut, T., Domrongkitchaiporn, S., Vibhagool, A.,Thinkamrop, B., & Susaengrat, W. (2003). Ceftriaxone com-pared with sodium penicillin G for treatment of severe leptospirosis.Clinical Infectious Diseases, 36, 1507–1513.

98. Patz, J. A., & Kovats, R. S. (2002). Hotspots in climate change andhuman health. British Medical Journal, 325, 1094–1098.

99. Perolat, P., Chappel, R. J., Adler, B., Baranton, G., Bulach, D. M.,Billinghurst, M. L., et al. (1998). Leptospira fainei sp. nov., isolatedfrom pigs in Australia. International Journal of Systematic Bacteri-ology, 48, 851–858.

100. Pike, R. M. (1976). Laboratory-associated infections: summary andanalysis of 3921 cases. Health Laboratory Science, 13, 105–114.

101. Prescott, J. F., McEwen, B., Taylor, J., Woods, J. P., Abrams-Ogg, A.,& Wilcock, B. (2002). Resurgence of leptospirosis in dogs in Ontario:recent findings. Canadian Veterinary Journal, 43, 955–961.

102. Ramadass, P., Jarvis, B. D. W., Corner, R. J., Penny, D., &Marshall, R. B. (1992). Genetic characterization of pathogenic Lep-tospira species by DNA hybridization. International Journal of Sys-tematic Bacteriology, 42, 215–219.

103. Ren, S. X., Fu, G., Jiang, X. G., Zeng, R., Miao, Y. G., Xu, H.,et al. (2003). Unique physiological and pathogenic features of Lep-tospira interrogans revealed by whole-genome sequencing. Nature,422, 888–893.

104. Romero, E. C., Caly, C. R., & Yasuda, P. H. (1998). The persistence ofleptospiral agglutinins titers in human sera diagnosed by the micro-

scopic agglutination test. Revista do Instituto de Medicina Tropicalde Sao Paulo, 40, 183–184.

105. Salaun, L., Merien, F., Gurianova, S., Baranton, G., & Picardeau, M.(2006). Application of multilocus variable-number tandem-repeatanalysis for molecular typing of the agent of leptospirosis. Journalof Clinical Microbiology, 44, 3954–3962.

106. Sanders, E. J., Rigau-Perez, J. G., Smits, H. L., Deseda, C. C.,Vorndam, V. A., Aye, T., et al. (1999). Increase of leptospiro-sis in dengue-negative patients after a hurricane in Puerto Rico in1996. American Journal of Tropical Medicine and Hygiene, 61,399–404.

107. Segura, E. R., Ganoza, C. A., Campos, K., Ricaldi, J. N., Torres, S.,Silva, H., et al. (2005). Clinical spectrum of pulmonary involvementin leptospirosis in a region of endemicity, with quantification of lep-tospiral burden. Clinical Infectious Diseases, 40, 343–351.

108. Sehgal, S. C., Sugunan, A. P., Murhekar, M. V., Sharma, S., &Vijayachari, P. (2000). Randomized controlled trial of doxycyclineprophylaxis against leptospirosis in an endemic area. InternationalJournal of Antimicrobial Agents, 13, 249–255.

109. Sejvar, J., Bancroft, E., Winthrop, K., Bettinger, J., Bajani, M.,Bragg, S., et al. (2003). Leptospirosis in “Eco-Challenge” ath-letes, Malaysian Borneo, 2000. Emerging Infectious Diseases, 9,702–707.

110. Self, C. A., Iskrzynska, W. I., Waitkins, S. A., Whicher, J. W., &Whicher, J. T. (1987). Leptospirosis among British cavers. Cave Sci-ence, 14, 131–134.

111. Slack, A., Symonds, M., Dohnt, M., Harris, C., Brookes, D., &Smythe, L. (2007). Evaluation of a modified Taqman assay detectingpathogenic Leptospira spp. against culture and Leptospira-specificIgM enzyme-linked immunosorbent assay in a clinical environment.Diagnostic Microbiology and Infectious Disease, 57, 361–366.

112. Slack, A., Symonds, M., Dohnt, M., & Smythe, L. (2006a). Animproved multiple-locus variable number of tandem repeats analy-sis for Leptospira interrogans serovar Australis: a comparison withfluorescent amplified fragment length polymorphism analysis and itsuse to redefine the molecular epidemiology of this serovar in Queens-land, Australia. Journal of Medical Microbiology, 55, 1549–1557.

113. Slack, A. T., Dohnt, M. F., Symonds, M. L., & Smythe, L. D.(2005). Development of a multiple-locus variable number of tandemrepeat analysis (MLVA) for Leptospira interrogans and its applicationto Leptospira interrogans serovar Australis isolates from far NorthQueensland, Australia. Annals of Clinical Microbiology and Antimi-crobials, 4, 10.

114. Slack, A. T., Symonds, M. L., Dohnt, M. F., & Smythe, L. D. (2006b).Identification of pathogenic Leptospira species by conventional orreal-time PCR and sequencing of the DNA gyrase subunit B encodinggene. BMC Microbiology, 6, 95.

115. Smits, H. L., Ananyina, Y. V., Chereshsky, A., Dancel, L., Lai, A. F.R. F., Chee, H. D., et al. (1999). International multicenter evaluationof the clinical utility of a dipstick assay for detection of Leptospira-specific immunoglobulin M antibodies in human serum specimens.Journal of Clinical Microbiology, 37, 2904–2909.

116. Smythe, L. D., Smith, I. L., Smith, G. A., Dohnt, M. F.,Symonds, M. L., Barnett, L. J., et al. (2002). A quantitative PCR (Taq-Man) assay for pathogenic Leptospira spp. BMC Infectious Diseases,2, 13.

117. Suputtamongkol, Y., Niwattayakul, K., Suttinont, C., Losuwanaluk,K., Limpaiboon, R., Chierakul, W., et al. (2004). An open, random-ized, controlled trial of penicillin, doxycycline, and cefotaxime forpatients with severe leptospirosis. Clinical Infectious Diseases, 39,1417–1424.


118. Swain, R. H. A. (1957). The electron-microscopical anatomy ofLeptospira canicola. Journal of Pathology and Bacteriology, 73,155–158.

119. Takafuji, E. T., Kirkpatrick, J. W., Miller, R. N., Karwacki, J. J.,Kelley, P. W., Gray, M. R., et al. (1984). An efficacy trial of doxycy-cline chemoprophylaxis against leptospirosis. New England Journalof Medicine, 310, 497–500.

120. Terpstra, W. J. (1992). Typing leptospira from the perspective of areference laboratory. Acta Leidensia, 60, 79–87.

121. Terpstra, W. J., Ligthart, G. S., & Schoone, G. J. (1980). Serodiagno-sis of human leptospirosis by enzyme-linked-immunosorbent-assay(ELISA). Zentralblatt fur Bakteriologie, Mikrobiologie und Hygiene:I Abt. Orig. A, 247, 400–405.

122. Trevejo, R. T., Rigau-Perez, J. G., Ashford, D. A., McClure, E. M.,Jarquin-Gonzalez, C., Amador, J. J., et al. (1998). Epidemic lep-tospirosis associated with pulmonary hemorrhage-Nicaragua, 1995.Journal of Infectious Diseases, 178, 1457–1463.

123. Truccolo, J., Serais, O., Merien, F., & Perolat, P. (2001). Followingthe course of human leptospirosis: evidence of a critical threshold forthe vital prognosis using a quantitative PCR assay. FEMS Microbiol-ogy Letters, 204, 317–321.

124. Tunbridge, A. J., Dockrell, D. H., Channer, K. S., &McKendrick, M. W. (2002). A breathless triathlete. Lancet,359, 130.

125. Turner, L. H. (1968). Leptospirosis II. Serology. Transactions of theRoyal Society of Tropical Medicine and Hygiene, 62, 880–889.

126. Vaughan, C., Cronin, C. C., Walsh, E. K., & Whelton, M. (1994). TheJarisch-Herxheimer reaction in leptospirosis. Postgraduate MedicalJournal, 70, 118–121.

127. Verma, A., Artiushin, S., Matsunaga, J., Haake, D. A., &Timoney, J. F. (2005). LruA and LruB, novel lipoproteins ofpathogenic Leptospira interrogans associated with equine recurrentuveitis. Infection and Immunity, 73, 7259–7266.

128. Verma, A., Hellwage, J., Artiushin, S., Zipfel, P. F., Kraiczy, P.,Timoney, J. F., et al. (2006). LfhA, a novel factor H-bindingprotein of Leptospira interrogans. Infection and Immunity, 74,2659–2666.

129. Vernel-Pauillac, F., & Merien, F. (2006). Proinflammatory andimmunomodulatory cytokine mRNA time course profiles in hamstersinfected with a virulent variant of Leptospira interrogans. Infectionand Immunity, 74, 4172–4179.

130. Vijayachari, P., Ahmed, N., Sugunan, A. P., Ghousunnissa, S.,Rao, K. R., Hasnain, S. E., et al. (2004). Use of fluorescentamplified fragment length polymorphism for molecular epidemiol-ogy of leptospirosis in India. Journal of Clinical Microbiology, 42,3575–3580.

131. Vijayachari, P., Sugunan, A. P., Umapathi, T., & Sehgal, S. C. (2001).Evaluation of darkground microscopy as a rapid diagnostic pro-cedure in leptospirosis. Indian Journal of Medical Research, 114,54–58.

132. Vinetz, J. M., Glass, G. E., Flexner, C. E., Mueller, P., &Kaslow, D. C. (1996). Sporadic urban leptospirosis. Annals of Inter-nal Medicine, 125, 794–798.

133. Vinetz, J. M., Wilcox, B. A., Aguirre, A., Gollin, L. X.,Katz, A. R., Fujioka, R. S., et al. (2005). Beyond disciplinary bound-aries: leptospirosis as a model of incorporating transdisciplinaryapproaches to understand infectious disease emergence. EcoHealth,2, 1–16.

134. Viriyakosol, S., Matthias, M. A., Swancutt, M. A., Kirkland, T. N., &Vinetz, J. M. (2006). Toll-like receptor 4 protects against lethal Lep-tospira interrogans serovar Icterohaemorrhagiae infection and con-tributes to in vivo control of leptospiral burden. Infection and Immu-nity, 74, 887–895.

135. Wagenaar, J., Zuerner, R. L., Alt, D., & Bolin, C. A. (2000). Compar-ison of polymerase chain reaction assays with bacteriologic culture,immunofluorescence, and nucleic acid hybridization for detection ofLeptospira borgpetersenii serovar hardjo in urine of cattle. AmericanJournal of Veterinary Research, 61, 316–320.

136. Ward, M. P., Guptill, L. F., Prahl, A., & Wu, C. C. (2004). Serovar-specific prevalence and risk factors for leptospirosis among dogs:90 cases (1997–2002). Journal of the American Veterinary MedicalAssociation, 224, 1958–1963.

137. Ward, M. P., Guptill, L. F., & Wu, C. C. (2004). Evaluation of envi-ronmental risk factors for leptospirosis in dogs: 36 cases (1997–2002). Journal of the American Veterinary Medical Association, 225,72–77.

138. Watt, G., Padre, L. P., Tuazon, M., & Calubaquib, C. (1990). Limu-lus lysate positivity and herxheimer-like reactions in leptospirosis: aplacebo-controlled study. Journal of Infectious Diseases, 162, 564–567.

139. Werts, C., Tapping, R. I., Mathison, J. C., Chuang, T. H., Kravchenko,V., Saint Girons, I., et al. (2001). Leptospiral lipopolysaccharide acti-vates cells through a TLR2-dependent mechanism. Nature Immunol-ogy, 2, 346–352.

140. Winslow, W. E., Merry, D. J., Pirc, M. L., & Devine, P. L. (1997).Evaluation of a commercial enzyme-linked immunosorbent assay fordetection of immunoglobulin M antibody in diagnosis of human lep-tospiral infection. Journal of Clinical Microbiology, 35, 1938–1942.

141. World Health Organization. (1999). Leptospirosis worldwide, 1999.Weekly Epidemiological Record, 74, 237–242.

142. World Health Organization. (2000). Leptospirosis, India. Report ofthe investigation of a post-cyclone outbreak in Orissa, November1999. Weekly Epidemiological Record, 75, 217–223.

143. Yasuda, P. H., Steigerwalt, A. G., Sulzer, K. R., Kaufmann, A. F.,Rogers, F., & Brenner, D. J. (1987). Deoxyribonucleic acid related-ness between serogroups and serovars in the family Leptospiraceaewith proposals for seven new Leptospira species. International Jour-nal of Systematic Bacteriology, 37, 407–415.

144. Zaki, S. R., Shieh, W.-J., & The Epidemic Working Group. (1996).Leptospirosis associated with outbreak of acute febrile illness andpulmonary haemorrhage, Nicaragua, 1995. Lancet, 347, 535.

145. Zhang, Y. X., Geng, Y., Bi, B., He, J. Y., Wu, C. F., Guo, X. K., etal. (2005). Identification and classification of all potential hemolysinencoding genes and their products from Leptospira interrogansserogroup Icterohaemorrhagiae serovar Lai. Acta PharmacologicaSinica, 26, 453–461.

Date post:	21-Dec-2016
Category:	Documents
Upload:	elias
View:	212 times
Download:	0 times

Bacterial Infections of Humans || Leptospirosis

Documents