IJMRHS Leptospirosis-Review article

693Mohit et al., Int J Med Res Health Sci. 2015;4(3):693-701

International Journal of Medical Research&

Health Scienceswww.ijmrhs.com Volume 4 Issue 3 Coden: IJMRHS Copyright @2015 ISSN: 2319-5886Received: 1stMay 2015 Revised: 9th June 2015 Accepted: 19th June 2015Review article

DECIPHERING LEPTOSPIROSIS-A DIAGNOSTIC MYSTERY: AN INSIGHT

*Mohit Bhatia1, B L Umapathy2

1Senior Resident, Department of Microbiology, Govind Ballabh Pant Institute of Post Graduate MedicalEducation and Research, New Delhi2 Professor, Department of Microbiology, Sree Mookambika Institute of Medical Sciences, Kulasekharam,Kanyakumari District, Tamil Nadu

*Corresponding author email: [email protected]

ABSTRACT

Leptospirosis is an emerging infectious disease which has been recognized as the most common zoonoticinfection in the world. It affects human beings and many other species of vertebrates . Most commonly, theinfection is acquired by direct or indirect exposure to urine of reservoir animals through contaminatedsoil, mud & water entering via small abrasions or breaches in the skin & mucous membranes duringoccupational, recreational or vocational activities. The signs & symptoms resemble a wide range ofbacterial & viral diseases & sometimes can present as food poisoning, chemical poisoning & snake bitealso due to which the diagnosis is often missed. This review article aims to focus on the role of Dark FieldMicroscopy (DFM), culture, Enzyme Linked Immuno Sorbent Assay (ELISA), Macroscopic Slide Agglutinationtest (MSAT), Microscopic Agglutination Test (MAT) and Faine’s criteria in the diagnosis of leptospirosis.

Keywords: Dark Field Microscopy, Enzyme Linked Immuno Sorbent Assay, Macroscopic Slide Agglutinationtest, Microscopic Agglutination Test, Faine’s criteria

INTRODUCTION

Leptospirosis also known by various names like“Weil’s disease”, “Pretibial fever”, “Fort Braggfever”, “Peapicker’s fever” in different parts ofthe world is an acute bacterial infection causedby spirochetes belonging to the genus Leptospirathat can lead to multiple organ involvement andfatal complications.[1] It has been recognized as themost common zoonotic infection in the world.[2]

Leptospirosis has a wide geographical distributionand occurs in tropical, subtropical and temperateclimatic zones. The incidence is higher in thetropics than in temperate regions.[3] Most countriesin the South East Asia region are endemic toleptospirosis.[2]

Leptospirosis affects human beings and manyother species of vertebrates .[2] Most commonly,

the infection is acquired by direct or indirectexposure to urine of reservoir animals throughcontaminated soil, mud & water entering viasmall abrasions or breaches in the skin & mucousmembranes during occupational, recreational orvocational activities.[4] A small number of organismscan cause infection. The incubation period usuallyranges from 7-10 days. Leptospirosis may follow abiphasic course. During the first 10 days, there is aphase of leptospiraemia when the leptospires multiplyin blood and spread to different organs. The chancesof recovery of leptospires from blood or other tissuesor body fluids is usually high during this stage. Thisphase is followed by immune phase or leptospirureaphase when the organisms are excreted in the urine.In this phase, the chance of recovery of organisms

DOI: 10.5958/2319-5886.2015.00132.0


from the blood is low. The ideal specimen forisolation or demonstration of leptosires duringimmune phase is urine.[5] The signs & symptomsresemble a wide range of bacterial & viraldiseases & sometimes can present as foodpoisoning, chemical poisoning & snake bite alsodue to which the diagnosis is often missed.[6] Themost common clinical syndrome is anictericleptospirosis which is a self limiting illness.[4]

Weil’s disease or icteric leptospirosis, is generallythe most severe illness, with symptoms caused byliver, kidney & vascular dysfunction (jaundice,oliguria/anuria, bleeding & lethal pulmonaryhaemorrages).[4] The case fatality rate of leptospirosisis upto 10%.[4]

The clinical diagnosis of leptospirosis may bedifficult to arrive at. A high index of suspicion isrequired in patients with Pyrexia of UnknownOrigin (PUO) with or without jaundice, especially inpresence of history of animal contact, diagnosismay be established by laboratory investigations.[7]

The commonly followed case definition, which isalso recommended by the WHO and InternationalLeptospirosis Society prescribes that any personpresenting with acute onset of fever, headacheand body aches associated with severe muscletenderness particularly in calf muscles,haemorrhages including sub-conjunctivalhaemorrhage, jaundice, cough, breathlessness andhaemoptysis, oliguria, signs of meningeal irritationshould be suspected as a case of leptospirosis andinvestigated[8]. A suspect, who tests positive in anyof the screening tests such as dipstick, lateral flowor latex agglutination test should be considered as aprobable case. Successful isolation of Leptospiresfrom clinical specimens, a four-fold or higher risein titre or seroconversion in paired MicroscopicAgglutination Test (MAT) or a positive PolymeraseChain Reaction (PCR) is considered asconfirmatory evidence of current leptospiralinfection [8]. Owing to shortcomings of laboratorytests in establishing early diagnosis of leptospirosis,the World Health Organization (WHO), introducedFaine’s criteria which includes the scoring of clinical,epidemiological and laboratory parameters of patients(Parts A, B and C respectively) [9].This criteria hasbeen simultaneously modified and validated by Bratoet al and Shivkumar et al, who recommended additionof abdominal symptoms, local factors like rainfall and

newer investigations in the total scoring respectively[10,11]. Microbiological diagnosis of leptospirosisaims at demonstrating the leptospires, by culturingthem or by demonstrating an appreciable antibodyresponse to them.[12]

Laboratory diagnostic tests are broadly dividedinto two categories viz., Direct evidence -isolation of organism or demonstration ofleptospires by dark field microscopy oramplification of specific fragment of leptospiralDNA; and Indirect evidence- detection ofantibodies to leptospires.[13]

This review article aims to focus on the role of DarkField Microscopy (DFM), culture, Enzyme LinkedImmuno Sorbent Assay (ELISA), Macroscopic SlideAgglutination test (MSAT), MicroscopicAgglutination Test (MAT) and Faine’s criteria in thediagnosis of leptospirosis.

REVIEW

Dark-Field Microscopy (DFM)DFM has often been employed for examination ofbody fluids such as blood, urine, CSF anddialysate fluid.[14] The specimens should be takenaseptically and sent to laboratory without delay.They must not be frozen.[5] Oxalate, citrate,heparin or EDTA may be used as anticoagulantfor blood or pleural fluid.[5,15] Approximately 104

leptospires/ml of sample are necessary for oneorganism per field to be visible by DFM.[14]

DFM is the procedure of choice for thedemonstration of the organisms in tissue fluids.[16]

It is particularly useful for observing leptospiresin culture, particularly when they are present inlarge numbers. It can be used for observingagglutination in the MAT.[17] However, it istechnically demanding. Recognizing leptospires isdifficult, particularly when only small numbersare present.[17] Reading the results is alwayssubjective as in the majority of the samples thenumber of organism per field ranges from 0-2and there is always doubt about typicalmotility.[5] Double centrifugation of the sample atlow speed to separate the cellular elements, andthen at high speed, help concentrate theleptospires.[12] The usefulness of differentialcentrifugation is limited and the motility of theorganism further reduces after centrifugation athigher g.


The advantage of this technique is that laboratories,where the facilities for the other tests are notavailable, can undertake this technique. But theresults should be confirmed with other standard tests.The various limitations of this technique include thefollowing: Low sensitivity (40.2%) and specificity(61.5%). Serum proteins and fibrin strands in bloodresemble leptospires thereby making it extremelydifficult to diagnose the disease. The concentration oforganism is frequently too low in the specimens. Thistechnique requires expertise. [5]

Culture: Leptospires are obligate aerobes with anoptimum growth temperature of 28° C to 30°C.[14]

They can be grown in liquid, semi-solid(containing 0.2-0.5% agar) or solid media.[18] Theonly organic compounds required for their growthare Vitamins B1 and B12, and long chain fattyacids . Fatty acids are the main source of carbonand energy for leptospires. These are also requiredas a source of cellular lipids, as leptospirescannot synthesize fatty acids de novo. Liquidmedium is used for the cultivation of leptospireswhich can later be used for harvesting antigens to beused in various serological tests. Liquid medium canbe converted into semisolid and solid by theaddition of agar or agarose. Semisolid mediacontain 0.2-0.5% agar whereas solid mediumcontain 0.8-1% agar.[18,19] Semisolid medium iscommonly used for the isolation of leptospiresand for maintaining the cultures. In semisolidmedia, growth reaches a maximum density in theform of a discrete zone under the surface of themedium usually within 7-21 days. This growth isknown as Dinger’s ring and is related to theoptimum oxygen tension.[14] Solid medium is notideal for isolation or maintenance of leptospiresand mainly used for the research purpose toclone the leptospires from mixed letospiracultures.[19]

A wide variety of culture media can be used forthe cultivation of leptospires.[19] The routinelyused culture media are described briefly here:a) Media that contain rabbit serum: These includeKorthof’s medium, Fletcher’s medium and Stuart’smedium. Rabbit serum contains nutrients includinghigh concentrations of bound vitamin B12 whichhelps in the growth of leptospires. All thesemedia can be used for the isolation of leptospiresfrom the clinical specimens and for the

maintenance of leptospires but not for thepreparation of antigens for MAT.[19]

b) Fatty acid albumin medium: In this medium longchain fatty acid is used as a nutritional sourceand serum albumin as detoxicant. This medium ispopularly known as Ellinghausen-McCullough-Johnson-Harris (EMJH) medium and widely usedfor isolation, maintenance and preparation ofantigens for MAT and for growing leptospires inbulk.[19]

c) Protein-free medium: In this medium long chainfatty acids are treated with charcoal to detoxifythe free fatty acids which are highly toxic toleptospires.[19]

Culture media can be enriched by addition of 1%fetal calf serum or rabbit serum to cultivate thefastidious leptospiral serovars.[19] Due to the abilityof leptospires of incorporating purine and notpyrimidine bases into their nucleic acids, they areresistant to the antibacterial activity of thepyrimidine analogue,5-Fluorouracil.[12] Selectiveculture media, containing 5-FU 50-1000 μg/ml ora combination of nalidixic acid 50 μg/ml,vancomycin 10 μg/ml and polymixin B sulphate 5units/ml or a combination of actidione 100 μg/ml,bacitracin 40 μg/ml, 5 FU 250 μg/ ml, neomycinsulphate 2 μg/ml, polymixin B sulphate 0.2 μg/mland rifampicin 10 μg/ml can be used to avoid thecontamination.[19] Contaminated cultures may bepassed through a 0.2µm or 0.45µm filter beforesubculture into fresh medium.[20]

The infecting strains can often be isolated inculture, provided that suitable material is obtainedbefore antibiotics have been administered. Early inthe course of illness – during the leptospiraemicphase – the samples of preference are blood orcerebrospinal fluid; later during the phase ofleptospiruria – it is urine.[12]

Blood: Leptospires have rarely been isolated fromblood weeks after the onset of symptoms.[14] Bloodshould be cultured in the first ten days of theillness and before antibiotics are given. Venousblood is collected with aseptic precautions andideally inoculated at the bedside into bloodculture bottles containing culture medium forLeptospira. Only few drops of blood are inoculatedinto several tubes, each containing 5 ml of asuitable medium as large inocula will inhibit thegrowth of leptospires.[17] Cultures are incubated


between 28 to 30°C for 4 to 6 weeks orlonger.[5,14] Culture are examined using dark fieldillumination initially on first, third and fifth daysfollowed by 7-10 days interval up to 6 weeks.[5]

Blood culture is particularly valuable in man, asthe serological response can be slow and may beabsent altogether if antibiotics are given early.Urine: During the leptospiruria phase which ischaracterized by increasing concentrations ofantibodies after about 1 week from onset of clinicalillness, the urine and the renal cortex post mortemare the most suitable inocula for the isolation ofleptospires from humans. Fresh mid-stream urine iscollected and inoculated immediately. One drop ofundiluted urine is inoculated into the first tubecontaining 5 ml of culture medium. Alternatively,urine samples may be centrifuged for 30 minutes at1600 g or 1 min at 10,000 g and the pellet re-suspended in medium, after which ten-fold serialdilutions are made immediately in 1 or 2additional tubes. Since urine is acidic anddecreases the viability of leptospires, it should beinoculated into the medium within 2 hours aftervoiding. Viability is reported to be increased inurine samples neutralized with sodium bicarbonateand by using phosphate-buffered bovine serumalbumin solution. Media containing 5-fluorouracilor appropriate antibiotics that suppress the growthof bacterial contaminants and leave leptospiresunaffected may be beneficial in reducing thecontamination of media inoculated with urinesamples obtained from various patients clinicallysuspected to be suffering from leptospirosis.[17]

Cerebrospinal Fluid (CSF): Leptospires may beobserved in CSF by DFM and can be isolatedby inoculating 0.5ml CSF into 5 ml semi-solidculture medium during the first week of illness(usually upto 10 days of onset of clinical signs &symptoms).[17,21]

The various advantages of this technique include thefollowing: Isolation in culture is a definite proof ofinfection. Circulating serovars can be identified.Local isolates can be used as antigens in MAT andvaccine development. [5]

The various disadvantages of this technique includethe following: Leptospires are fastidious organismswhich require special medium for isolation.Leptospires grow slowly and therefore, isolation ofleptospires from clinical specimens takes several days

to several weeks. The technique is laborious, timeconsuming and is not possible in small laboratories.Contamination of culture media by other micro-organisms or by saprophytic leptospires is common inroutine practice. The successful isolation rate is lessdue to prior use of antibiotics, imperfectly cleanedglass ware or wrong incubation temperature and pH.[5]

The various serological tests used for the diagnosisof leptospirosis can be divided into two groups,those, which are Genus specific, and those,which are Serogroup specific.(13) The various Genusspecific tests are Macroscopic Slide Agglutinationtest (MSAT), Indirect Fluorescent Antibody Test(IFAT), Indirect Hemagglutination Test (IHA),Counter Immuno Electrophoresis (CIEP),Complement Fixation Test (CFT), Enzyme LinkedImmuno Sorbent Assay (ELISA), MicrocapsuleAgglutination Test (MCAT) and Lepto-Dipstick. TheSerogroup/serovar specific tests are MicroscopicAgglutination Test (MAT) and Serovar specificELISA.[12]

Leptospires have a complex antigenic structure.[12]

Within the two species L.interrogans and L.biflexa,there are many serotypes (now referred to assero-varieties or serovars) that are distinguishedby cross-agglutinin-absorption tests or byantigenic factor analysis. Some of the serovars areclosely related because of common antigens andform clearly defined serogroups.[18] The somaticantigen is genus specific. The surface antigen whichis a polysaccharide is serovar specific.[12] The outermembrane is lipopolysaccharide (LPS) in natureand is a potent immunogen. It is the major antigenand the target of antibody and complement-mediatedbactericidal activity. Antibodies directed against thisantigen are protective in nature. Flagellar antigen iscomposed of both genus and serotype specificantigens. Some serovars, e.g. L. icterohaemorrhagie,have an additional Vi antigen associated withvirulence.[12] The leptospiral outer membranecontains both transmembrane proteins, such as theporin OmpL1 and lipoproteins such as LipL41 andLipL36 .[22] Outer membrane proteins (OMPs) thatare exposed on the leptospiral surface are potentiallyrelevant in pathogenesis because of their location atthe interface between leptospires and the mammalianhost. Results of surface immunoprecipitation studies,immunoelectron microscopy & topological


considerations suggest that OmpL1 and LipL41 arepresent on the surface. Unlike leptospiral LPS,OmpL1 and LipL41 are antigenically conservedamong pathogenic Leptospira species. OmpL1 andLipL41 are expressed during infection of themammalian host.[22] LipL32, the 32-kDalipoprotein is also a prominent immunogen duringhuman leptospirosis. The sequence and expressionof LipL32 is highly conserved among pathogenicLeptospira species. LipL32 may be important inthe pathogenesis, diagnosis, and prevention ofleptospirosis.[23]

Enzyme linked Immuno Sorbent Assay (ELISA)This is now widely used as a genus specificscreening test in man.[12] It is a very sensitive andspecific test for the biological diagnosis ofleptospirosis.[17] It is of particular value as aserological screening test because of its relativesimplicity in comparison with the MAT.[17] Bothperoxidase and urease labelled conjugates havesatisfactorily been used. Stable reagents areavailable and form the basis of bedside tests,which are read visually. The use of computerassisted automated readers and the appropriatecontrols has improved the reproducibility andpredictive value of this test. They can beperformed with commercial kits or with antigenproduced "in house".[12] Antigen preparation forELISA is done cultivating Leptospira interrogansserovar Copenhageni strain Wijnberg or L. biflexaserovar Patoc strain Patoc I in EMJH medium for 10-12 days at 30ºC in shaking incubator. Abundantgrowth is necessary to produce a good antigen.[5]

Several attempts have been made to developserotype specific ELISA tests with a variety ofextracted antigens. Tests based on boiled wholecell antigens tend to be genus specific but thosebased on ultrasound-disintegrated or phenol-extracted preparations show considerable serotypespecificity.[12]

The ELISA test gives a positive response in thediagnostic evaluation of leptospirosis a little earlierthan the MAT because it is more sensitive toIgM antibodies. A response 6–8 days followingthe appearance of the first clinical signs isgenerally observed. On the other hand, the testmay become negative more quickly than MAT,although low levels of specific IgM may persist.A potential advantage of the ELISA test is that

it may help to differentiate between currentleptospirosis and previous leptospirosis sinceantibodies from past infection or immunizationmay not be detectable. However, if a total humananti-Ig or IgG conjugate is used instead, thepositivity of the test may be extended, allowingthe detection of residual antibodies in recoveredor immunized patients. The level of positivityobserved with a total anti-Ig conjugate is thenalways equal to or higher than the maximumobserved with anti-IgG or anti-IgM antibodies.[17]

Other advantages include the following: Singleantigenic preparation can be used for ELISA; Heatstable antigens which are stable at roomtemperature for long periods are generally used;ELISA allows rapid processing of large number ofsamples.A limitation to the use of single serum samplesfor serodiagnosisis the persistence of antibodies.Anti-leptospiral IgM antibodies are also persistent,but the rate of decline shows marked variation.Thus, a single IgM positive sample taken duringan acute febrile illness with symptoms suggestiveof leptospirosis is presumptive evidence ofinfection, but this finding requires confirmation bytesting a convalescent sample, preferably by theuse of an alternative method.[24] Infecting serovarcannot be assessed which makes it comparativelyless specific.[12] Another disadvantage of thisprocedure is that it usually requires calibration ofcut-off values and significant titres.[5]

Macroscopic Slide Agglutination Test (MSAT)A rapid macroscopic slide agglutination test canbe used to screen human and animal serumsamples. This test is carried out with a densesuspension of leptospires, which agglutinate intoclumps visible to the naked eye. It can beperformed on slides or plates. There can be differentways to prepare the antigens. One may either use asingle serovar or multiple serovars to prepare theantigens. When multiple serovars are used asantigens, antigens can be pooled. Since the techniquewill require lot of laboratory work, generally a singlenon-pathogenic serovar patoc (strain Patoc 1) isused.[12] The basic principle of the test is similar toother slide agglutination tests used in other infectiousdiseases such as enteric fever or brucellosis. Thereaction is recorded as ++ when the clumps are largeand definite, + when the smaller clumps are well


defined but the suspension is not clear, +/- when fineclumps are visible but the suspension is not clear andnegative when the mixture in the drop is unchanged.Agglutination of + and ++ is considered as positive.[5]

It is a simple, rapid, and sensitive diagnostic testfor active leptospirosis and allows a provisionaldiagnosis of acute leptospirosis to be madewithin a few minutes.[12] The antigen is broadlyreactive and stable for six months at 4ºC to 8ºC.[5] Itis more sensitive than Microscopic AgglutinationTest (MAT) in the early stage of the disease. Thesensitivity of this test can be enhanced by addingthe locally-prevalent serovars. MSAT has showngood correlation with both IgM ELISA and MATin various studies, and therefore can be used as avaluable and simple screening test. It is nothowever suitable for retrospective or survey work.[12]

A high percentage of false positive reactions areobserved, probably due to lack of standardization andquality control of the antigen preparation. Thenumber of false negative reactions are comparativelylow.[5] Positive reactions should therefore beconfirmed by complement fixation or microscopicagglutination tests.[12]

Microscopic Agglutination Test (MAT) : This testis considered the gold standard for serodiagnosisof Leptospirosis.[25] MAT is carried out withsuspensions of live cultures or with cultureskilled by the addition of neutralizedformaldehyde.[12] There is a difference in appearancebetween the clumps of agglutinated livingleptospires and those of killed cultures. Livingleptospires are agglutinated into highly refractilespheroids of various sizes which may join toproduce elongated masses of confluent spheroids.By contrast, the agglutinated killed leptospiresform looser masses with an irregular oftenangular, outline; these appear flattened,resembling small piles of threads, or snowflakes,or pieces of cotton wool.[12]

Agglutinating antibodies can be of both IgM andIgG classes.[17] The degree of agglutination canonly be assessed in terms of the proportion offree leptospires. The accepted endpoint of anagglutination reaction is the final dilution ofserum at which 50% or more of the leptospiresare agglutinated. Preparations for MAT requiremeticulous culture of a collection of the livestrains used as antigen suspensions, their regular

subculture and quality control for authenticity andpurity and also skilled educated personnel.[11]

A recent advance is the use of standardizedpreparations of dried leptospires available toaccredited diagnostic laboratories from a centralreference laboratory. Multi-antigen MAT uses abattery of strains giving comprehensive coverageof all serogroups and provides an alternative to theso-called ‘genus-specific’ tests as a means ofdiagnosing leptospirosis. However, the necessity tomaintain large number of live strains of L.interrogans limits its use to reference laboratories.Wherever possible, local isolates of knownidentity should be included in the battery ofstrains in order to increase both the sensitivity andthe specificity of this widely accepted gold standardtest.[12]

Interpretation of diagnostic MAT: The MAT isusually positive 10–12 days after the appearanceof the first clinical symptoms and signs butseroconversion may sometimes occur as early as5–7 days after the onset of the disease.[17] Theantibody response may be delayed if antibiotictherapy was given before the test. A patient with acompatible history may be considered to have currentleptospirosis if he has IgM antibodies to leptospiraand a MAT titre of >/=80.[26]

The optimal cut-off titre is assessed by carryingout a baseline study on distribution of titres inthe community as well as among confirmedpatients. Using this data it is possible to estimatethe sensitivities and false positivity rates atdifferent cut-off titres. The titre that gives thelowest number of false results is then chosen asthe optimal cut-off titre.[5]

In a non-endemic area, even a low level of antibodiesmay signify leptospirosis in the first week of aclinically compatible illness. The titre will rise ina second specimen taken after 3 to 7 days. If thetitre remains below 100, even on repeated testingthen it could probably be due to previousleptospirosis, and not current illness. In endemicareas, the diagnosis will be confirmed if the titrerises on retesting, but will be negated if it isunchanged, assuming that the infecting serovarwas included among the antigens used for MAT.[12]

MAT is the serological test used in referencelaboratories, because of its high degree ofsensitivity and specificity.[27] MAT remains very


useful for epidemiologic studies, identification ofstrains, assessment of the probable infectingserogroup and confirmation of illness for publichealth surveillance.[28]

The disadvantages of MAT are that 14–21 strainshave to be maintained in culture, which is oftenvery difficult. Procedure is complex and timeconsuming. Reading results requires experiencedpersonnel.[5] It cannot be standardized as liveantigens are often used and various factors, suchas the age and density of the antigen culture, caninfluence the agglutination titre.[17] Co-agglutinins(cross-reactions) are frequently present in the seraof patients with leptospirosis. Antibodies whichcause cross-reactions are often the first to appearbut they disappear rapidly. Homologous antibodies,although they appear slightly later, persist muchlonger, thus allowing the presumptiveidentification of the serogroup responsible for theinfection and also the detection of tracesindicating previous infections.[17] Some patientshave serological evidence of previous infectionwith a different leptospiral serogroup. In thesepatients, serological diagnosis is complicatedfurther by the “anamnestic response,” in which thefirst rise in antibody titre is usually directedagainst the infecting serovar from the previousexposure. Only later does it become possible toidentify the serovar or serogroup responsible forthe current infection, as the titre of specificantibody rises. Paradoxical reactions also occur inpatients who have such infections, andinterpretation of serology is furthercomplicated.[14] The MAT cannot differentiatebetween agglutinating antibodies due to current,recent or past infections.[17] Therefore, the use ofMAT is only restricted to some of the specialisedlaboratories.[29]

Faine’s criteria for diagnosis of leptospirosisFaine had formulated a criteria for diagnosis ofleptospirosis on the basis of clinical,epidemiological and laboratory data (Parts A, B andC respectively). A presumptive diagnosis ofleptospirosis may be made if:(i) Parts A and B score = 26 or more (Part Claboratory report is usually not available beforefifth day of illness; thus it is mainly a clinical andepidemiologic diagnosis during early part ofdisease).

(ii) Part A+B+C = 25 or more A score between 20and 25: Suggests a possible but unconfirmeddiagnosis of leptospirosis.[9]

Shivakumar et al from Chennai have suggestedmodification on Faine’s criteria to include localfactors like rainfall and newer investigations in thetotal scoring.[11] As per this, epidemiological andlaboratory criteria (Parts B and C) are modifiedonly; no modification is made in the clinical criteria(Part A).The reasons for modification suggested are asfollows:(i) Most of the leptospirosis is reported inmonsoon and post-monsoon period. Therefore theyhave suggested rainfall separately to be adjustedin the scoring criteria of Part B.(ii) Microscopic agglutination test (MAT) is theGold Standard test, but is complicated and lesssensitive compared to newer tests like IgMELISA and Slide Agglutination Test (SAT). IgMELISA and SAT are simple, sensitive tests andcan be used to diagnose current leptospirosis.Thus, they have been included with appropriatescore being assigned to each one of these.The difficulties in utilizing MAT are due to thefollowing reasons:[30]

(a) The antibody titres rise and peak only insecond or third week. Thus, paired sera are requiredto demonstrate four-fold rise of titre.(b) The test is complicated requiring dark-fieldmicroscopy and cultures of various serovars, whichmay not be available in smaller laboratories.

The advantage of including simple diagnostictests (IgM ELISA or SAT ) in modified Faine’scriteria is that it helps in diagnosing milderforms of leptospirosis which are associated withlow clinical score (Part A). Suggestion ofmodification of existing Faine’s criteria appearsjustified; however further evaluation is required.[30]

Table 1shows comparison between Faine’s &Modified Faine’s criteria.


Table: 1. Table showing comparison between Faine’s & modified Faine’s criteria[9,11]

Faine’s criteriaScore

Modified Faine’s criteriaScorePART A: Clinical data PART A: Clinical data

HeadacheFeverIf fever, temperature 39°C or moreConjunctival suffusion (bilateral)MeningismMuscle pain (especially calf muscle)Conjunctival suffusion + meningism +muscle painJaundiceAlbuminuria or nitrogen retention

22244410

12

10

Diagnosiscertain

210

51525

HeadacheFeverIf fever, temperature 39°C or moreConjunctival suffusion (bilateral)MeningismMuscle pain (especially calf muscle)Conjunctival suffusion + meningism +muscle painJaundiceAlbuminuria or nitrogen retention

22244410

12

541

Diagnosiscertain

15151525

PART B: Epidemiological factors PART B: Epidemiological factorsContact with animals or contact withknown contaminated water

RainfallContact with contaminated environmentAnimal contact

PART C: Bacteriological and laboratoryfindings

PART C: Bacteriological andlaboratory findings

Isolation of leptospires in culture:

Positive serology (MAT)Leptospirosis (endemic)Single positive low titreSingle positive high titre

Leptospirosis (non-endemic)Single positive low titresSingle positive high titresRising titres (paired sera)

Isolation of leptospires in culture:

Positive serologyELISA IgM positive*

MSAT positive*

MAT single high titre*

MAT rising titres (paired sera)*

*Only one of these tests to be scored

CONCLUSION

Leptospirosis is probably an under diagnosedinfectious disease which mysteriously mimics severalclinical conditions. It is an emerging infectiousdisease, the current incidence of which onlyrepresents tip of an iceberg. Although, severalmodalities are currently available for diagnosing thisdeadly infectious disease, but each one of these hascertain limitations. Newer diagnostic tests forleptospirosis are need of the hour which would aidclinical diagnosis during the initial phase of thedisease and rapid case confirmation during outbreaksurveillance.Acknowledgment : Dr Bibhabati Mishra, Director,Professor and Head, Department of Microbiology,Govind Ballabh Pant Institute of Post GraduateMedical Education and Research, New Delhi.

Conflict of interest: NilREFERENCES

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IJMRHS Leptospirosis-Review article