lepstospirosis antigen

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Serodiagnosis of human leptorrpirosisemploying immunodominant antM A Ribeiro, C S N Assis, E C RomeroAdolf0 Lutz Institu te, Sao Paulo, Brazil

SummaryIgM-PK-ELISA, an enzyme-linked immunosorbent assay for immunoglobulin M employ-ing enzymatically treated antigen is described for the serodiagnosis of leptospirosis. It isbased on the observation that patients serum samples present IgM antibodies reactingwith a diffuse band of 14-22 kDa proteinase-K resistant. The assay was evaluated inserum samples from patients with leptospirosis (n = 89), typhoid fever (n = 81, malaria(n = 19), syphillis (n = 20). hepatitis (n = 16) and clinically healthy individuals (n = 92). Themicroscopic agglutination tes t (MAT) served as a reference. IgM-PK-ELISA and MATpresented the sens itivi ty of 89.89% and 92.13% and the spec ifici ty of 97.42% and 94.84%respectively . The overall results of the IgM-PK-ELISA are comparable to those o f the MAT.However, the IgM-PK-ELISA detected antibody act ivit y in 38% of acute-phase sera whichwere negat ive by the MAT. Our data suggest that IgM-PK-ELISA can be used as a screen-ing test.Key words: Serodiagnosis, human leptospirosis, ELISA, IgM-PK-ELISA, proteinase-K treated antigen,antileptospira IgMSerodiagn. Immunother. Infect . Disease 1994, Vol. 6, 140-144. September

IntroductionLeptospirosis, a world-wide infection of zoonoticorigin is caused by spirochaetes of Leptospira inter-rogans complex and occurs in Brazil, chiefly duringthe rainy season. In Sao Paulo, Brazil, L. interrogansserovars icterohaemorrhagiae and copenhageni are themost frequently isolated serovarsl. These serovars areusually the cause of Weils syndrome. Leptospirosis isan acute, febrile illness, the severity of which variesfrom mild to rapidly fatal. It may remain undiagnosedbecause the symptoms are non-specific, especially inthe early stages of infection*. Immunity to leptospiro-sis is mainly humorally mediated3-h. Leptospiral infec-tions in humans may be diagnosed in the laboratoryeither by isolating the causal organism or by demon-strating a rise in specific serum antibody. Whereasculture is of undoubted epidemiological importance,the time mediated between the culture and the identi-

Received: 5 May 1994Accepted: 3 June 1994Correspondence and reprint requests to: Dra Maricy A Ribeiro,lnstituto Adolf0 Lutz, Se@o de Imunologia, Av Dr Arnaldo 355, 11andar, CEP: 01246-902 S&o Paulo, SP, Brazil0 1994 Butterworth-Heinemann Ltd0888-0786/94/030140-05

fication of the infecting organism permits only aretrospective diagnosis. The microscopic agglutina-tion test (MAT), for the detection of specif ic antibod-ies, is still the standard reference test for serology. Itremains a specialized test which is not generallyperformed in routine diagnostic laboratories. Someprevious studies showed that IgM was the predomi-nant class of antibody developed during the infec-tion5.8-10. IgM agglutinins may persist for severalyears. Thus, on the basis of MAT, current or recentinfections cannot be readily differentiated from pastinfections. Although the importance of agglutinatingantibodies in immunity has been well established, thenature and identity of the corresponding leptospiralantigens have not so far been greatly studied.Immunoblotting of leptospiral extracts with patientssera revealed antibodies recognizing several compo-nents in the molecular weight range of 14.5-105 kDaof both IgM and IgG response12J3. All patients serumsamples presented IgM antibodies reacting with adiffuse band of 14.8-22 kDa proteinase-K resistantY,13,whereas sera from healthy individuals presented IgMantibodies reacting with several antigens, but lackedresponse against those of the diffuse band of 14.8-22kDa. Chapman et a1.14 eported that the nature of thislow molecular mass antigen is unknown, but warrants


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Ribeiro et al.: Serodiagnosis of human leprospirosis 141

further study as it is clearly a major antigen recognizedby both infected and vaccinated humans. Furthermore,as this immunodominant antigen appears to be broadlyreactive with several serovars and elicits antibodiesearly in the immune response to leptospiral infection,it may be useful as the basis of a defined crossreactivediagnostic test for human leptospirosist4. Undefinedcrossreactive preparations have previously been usedfor this purpose but they are not yet used as a routinetest in diagnostic laboratoriesx~15-1x, in part because theantigens detected by antileptospiral antibodies havenot been defined and because there is no agreementon the standardized methods of producing theseantigens. Bahaman et al.? proposed that furtherinvestigation should use a pure and more specificleptospiral fraction, as the test antigen, to lower thebackground readings of the controls. The mainproblem is in early diagnosis?, where detection ofspecific IgM may be important because of its earlyappearance and long persistence, and because of thefailure of some patients to produce specific IgGantibodiesl. The objective of the present study was todevelop an IgM-PK-ELISA, an enzyme-linkedimmunosorbent assay for immunoglobulin M whichuses enzymatically treated antigen to detect antilep-tospiral antibodies and to evaluate its potential useful-ness in human serodiagnosis.

Materials and methodsSeraThe following four collections of sera were studied:1. Two serial samples from each of 82 leptospirosispatients who had seroconversion by MAT.2. Two samples from seven patients with leptospirosisconfirmed only on the basis of clinical and epidemi-

ological data.3. Serial sets of sera collected from seven patients withWeils syndrome who were followed for 4-8 months.4. Control serum samples obtained from 92 healthyblood donors, 20 patients with syphilis, 19 patientswith malaria, 16 patients with hepatitis and eight

sera from patients with typhoid fever.IgM-PK-ELISAAntigen preparationStrain M20 of serovar copenhageni, was grown for 6-7days in Korthoff medium. The culture was centrifugedfor 20 min at 10 000 X g. The pellet, washed three timeswith sterile physiological saline to remove adherentproteins present in the medium, was resuspended in 2ml of distilled water, successively frozen, thawed threetimes, and sonicated at an output of 1 mA at 4 C for 3min (MSE Sifam Electrical Instrument Co. Ltd.,England). After a centrifugation at 2000 X g, the super-natant had its protein concentration** estimated to be 2mg ml-, with bovine serum albumin (BSA) as standard.

Enzymatic treatmentsThe digestion of the soluble antigen by proteinase-Kfollowed the procedure described by Cambiaso et al.2.Briefly, equal volumes of substrate and enzyme (0.2 mgml- in 0.1 M tris-HCl buffer pH 7.5, containing 20 mMCaCl,) were mixed and incubated overnight at 37 C.The reaction was stopped by the addition of phenyl-methylsulfonyl fluoride (1 InM). After proteinase-Ktreatment, the antigen was treated with 50 ~1 ml-l eachof DNase and RNase (Sigma Chemical Co., St Louis,MO, USA) for 3 h at 37 C. This will be referred to asthe pK-treated antigen. Desiccated aliquots of thisantigen solution were stable, at room temperature. forat least 18 months.

Coating of microtitre platesThirty ul of antigen diluted to 1 : 500 in absoluteethanol was pipetted into one half of the wells ofpolystyrene microtitre plates (Corning, New York,USA). The remaining wells were coated only with 30l~,l of ethanol. The plates were left at 37 C until thefluid had evaporated. The plates were stored at roomtemperature in a dry place until use.

Test performanceBefore use the plates were thoroughly washed fourtimes with 0.01 M phosphate buffered saline (PBS) pH7.2. One hundred l~,l of patients serum was added tothe wells in a dilution of 1 : 400 in skimmed-milk 1%in PBS. The incubation period was 1 h at 37 C. Theplates were washed four times with PBS. One hundredl~,l of peroxidase-labelled anti-human IgM (goat IgGanti-human IgM p-chain specific, Sigma) was added tothe wells in a dilution of 1 : 1000, as determined bycheckerboard titration. After an incubation period of1 h at 37 C the plates were washed as described above.Fifty ~1 of substrate (0.4 mg ml-l o-phenylenediaminein phosphate-citrate buffer, pH 5.0 and 0.012% HZ02)was pipetted into the wells. After 10 min at roomtemperature in a dark chamber, 50 ~1 of 1 M H2SO4containing 0.5% sodium sulphite was added to eachwell to stop the reaction. The intensity of the resultingyellow colour was measured in a spectrophotometer(Titertek Multiskan, MCC Flow Labs. Inc, Finland) at492 nm. The cutoff points were expressed by means ofoptical density (OD) of three negative sera included ineach plate plus three standard deviations, and theseODs were used to signify the lowest level at whichspecific IgM was considered to be present, i.e. apositive result.

Microscopic agglutination testThe microscopic agglutination test (MAT) wasperformed as described by Sulzer and Jones24. Livecultures of strains o f the following 20 serovars:australis, autumnalis, bataviae, butembo, canicola,castellonis, celledoni, copenhageni, cynopteri, djasiman,


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grippotyphosa, hebdomadis, icterohaemorrhagiae,javanica, Panama, pomona, pyrogenes, shermani,tarassovi and wolffi were used as antigen; theseserovars represent all the serogroups known to beprevalent in Stio Paula, Brazil. Agglutination at aserum dilution of 1 : 200 was considered positive.Gel electrophoresis and immunoblottingAntigenic extracts were subjected to sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunostained with serum samples asdescribed in Ribeiro et al I3.Statistical analysisThe data obtained from the MAT and IgM-PK-ELISAwere analysed using a McNemars test as described byFleiss25.

ResultsImmunoblotting of the PK-treated antigenOur data from the immunoblot analysis indicated thatDNase and RNase treatment after the PK digestionwas important to reduce the background, and themarked reaction of serum samples of the control groupwith the 4@-42 kDa band (Figure 1).

Reactivity of sera in MAT and IgM-PK-ELISATable 1 presents the results obtained with serumsamples from 89 leptospirosis patients and 155 controlsera, analysed by MAT and IgM-PK-ELISA andexpressed as a positive or negative diagnosis.

Figure 1. lmmunoblot of the PK-digestedpreparations of Leptospira serovar copenhageni strainM20 before (I) and after (21 DNase and RNasetreatment for the detection of the IgM antibodies withthe following human sera: leptospirosis patients, lanesa and g; healthy blood donors, lanes b, c and d;patients with syphilis , lanes e and f. Numbers on theleft indicate molecular mass markers (kDa).

Table 1. Comparison between MAT and IgM-PK-ELISA for serodiagnosis of human leptospirosisMAT

Pos. Neg.IgM-PK-ELBA

Pos. Neg. ToralPatients* 82 7 80 9 89Controls 8 147 4 151 155Sensit ivity 82189 = 92.1%Specificity 147/l% = 94.8%*Collections 1 and 2

80189 = 89.9%1511155 = 97.4%

Table 2. Resul ts o f the MAT and the IgM-PK-ELBA inthe first serum samples collected from patients withleptospirosis*MAT

PositiveIgM-PK-ELISA

Negative TotalPositive 23 3 26Negative 24 39 63Total 47 42 89*Collections 1 and 2McNemar x2 = 14.81; kO.01.

IgM-PK-ELISA results did not differ from MATresults when paired sera were assayed. However, IgM-PK-ELISA differed statistically (x2 > 3.77) from MATon the positivity of the first sample (Table 2) throughwhich 38% (24 out of 63) of the patients withleptospirosis had the infection detected earlier than byMAT.From the nine leptospirosis patients who had IgM-PK-ELISA-negative results (see Table l), four werepositive with the MAT for the serovar austrafis and theother five had the infection confirmed only on the basisof clinical and epidemiological data. Three cases hadtheir first sample positive for leptospira antibodies onlyby MAT (see Table 2). However, their subsequent serabecame positive in the IgM-PK-ELISA. Three controlsera were positive by both procedures.

DiscussionMAT is the current WHO standard reference test forserology. The cumbersome nature of the MATprecludes its use in most clinical settings due to theneed to use live cultures of virulent leptospires, the useof microscope endpoint determinations and of itshazardous potential to laboratory sta ff. The biggestdrawback is the insensitivity of the test. High antibodytitres are usually not detected before the second weekafter the onset of the clinical symptoms, the maximumbeing reached during the third week7. To confirm theclinical diagnosis it is necessary to analyse a secondserum sample taken a fe w days later, to demonstrate asignificant rise of the antibody titre. Administration of


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Ribeiro et al.: Serodiagnosis of human leptospirosis 143antibiotics, as usually practised, results in the develop-ment of low antibody titres. Nevertheless, these veryoften observed low antibody titres can also be theresult of a past infection. These difficulties have led tothe development of alternative tests for the diagnosisof leptospirosis in recent years. In this study, thediagnostic potential of the IgM-PK-ELISA wascompared with the MAT. We have used PK antigenprepared from strain M20 serovar copenhageni and wehave employed it to detect specific antibodies in thesera of leptospirosis patients in S5o Paulo, Brazil,infected by various serovars/serogroups. This leptospi-ral treatment yielded a purified fraction recognized byIgM antibodies of leptospirosis patients. Immunoblotanalysis (see Figure 1) confirmed the purity of the PKantigen.

The overall results of the MAT are comparable tothose of the IgM-PK-ELISA. However, the IgM-PK-ELISA was capable of detecting antibody activity in38% of acute-phase sera which were negative by theMAT.It was observed that the sensitivity for serogroupaustralis was low. This fact was already reported byCinco et al.y, who suggested that strains of uustralisshould be used in immunoenzymatic assay in order toensure the detection of antileptospira antibodies moreeffectively.Blackmore et al. detected agglutinating antibodiesup to 20 vr after infection, with a marked variationoccurring -in the maximum titres between individuals.These authors indicated that it is therefore not usuallypossible from a single MAT result to estimate retro-spectively the time at which infection occurred. Withthe IgM-PK-ELISA, we also found that antibody activ-ity persists in serum long after seroconversion. Serialsets of sera obtained from patients with Weilssyndrome and followed up for 4-8 months had signifi-cant antibody activity detected by the IgM-PK-ELISAfrom the fifth day of illness, which remained up to theeighth month when its reactivity is usually decreasedfourfold (data not shown). Nevertheless, the observa-tion that 52.4% of patients who seroconverted in MAThad an elevated activity in the IgM-PK-ELISA (OD 20.800) indicates a strong initial antibody response toinfection and suggests that the magnitude of thisresponse in a single serum sample may be indicative ofrecent infection (data not shown). This is important incases of fatal outcomeZh when it is not possible toobtain subsequent serum samples. We tested a samplefrom an individual whose death was attributed toleptospirosis only on the basis of clinical and epidemi-ological data. The MAT titre was negative but theIgM-PK-ELISA detected an OD of 1.091, indicative ofrecent leptospiral infection (data not shown).Sera from the control group showed a positivereaction rate of 5.2% and 2.6% using respectively theMAT and the IgM-PK-ELISA. However, asleptospirosis is widespread in Brazil it is possible thatsome of these reactions were due to subclinical recentor past infection with leptospires.

In conclusion, the IgM-PK-ELISA for the detectionof antileptospira IgM is an easy procedure andachieves adequate sensitivity, specificity and repro-ducibility. The use of a single test serum dilution candiscriminate between the presence or absence of anantibody response which enables the testing of 36 testsamples together with the positive and negativestandards on each microplate. Results can be readvisually or automatically. The IgM-PK-ELISA isespecially suitable for laboratories that do not havefacilities for continuous culture of leptospira since itsdesiccated antigen is stable for at least 18 months atroom temperature. The broad spectrum of serovarsdetected by the IgM-PK-ELISA could enable generalclinical laboratories to perform reliable screening testsfor leptospirosis, including atypical and milder formssuch as influenza-like leptospirosis. which frequentlyescape diagnosis and remain unnoticed as fevers ofunknown origin.

AcknowledgementsThe authors are grateful to Prof Thales De Brito andDra Tiyo Sakurai for their suggestions in the prepara-tion of this manuscript.

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lepstospirosis antigen

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