INTERNAI Il )7,:. \I. J0i RN',i. (>r LkPitosY^ Volume 67, Number I

l'rinted in the U.S.A.(ISSN OI40-916x1

Serum Markers of Treatment Success in Leprosy 1

Sumner MacLean, Kai Stietenroth, Hilmar Prange, V. V. Pai and R. Ganapati'

The diagnoses of leprosy is usually basedon clinicai examination, supplemented byhistology. There are two polar forms of thedisease, with various less stable stages be-tween the polar forms. At the tuberculoid(TT) pole, cell-mediated immunity (CMI)is strong and there are few bacilli found inthe lesions. At the lepromatous pole, CMI isvery weak and there are many widely dis-persed bacilli. The World Health Organiza-tion (WHO) recommends that leprosy beclassified as either paucibacillary (PB) ormultibacillary (MB).

The bacterial index (BI) counts thebacilli microscopically observed in smearstaken from patients. The test results varywith the technicians involved, and bacilliare usually not seen in PB cases. Conse-quently, a more reliable way of monitoringtreatment success is desirable. A recent ar-ticie ( 1 ) suggested that serum intracellularadhesion molecule-1 (ICAM-1) and E-se-lectin might be useful for monitoring lep-rosy treatment. We herein re-examine thedata used by the authors of that article, andevaluate additional variables from the samefrozen serum.

PATIENTS AND METHODSNew patients presenting to the urban

Botnbay Leprosy Project treatment center,Mumbai, India, were selected for study. In-formed consent was obtained from each pa-tient. There were 20 women and 54 men.The mean age was 36 years (range 10-80),distributed normally except for peaks in the

' Rcccived for publicalion on 2.3 Scptcmbcr 1998.Accepted for publicalion in revised forni on 13January 1999.

' S. MacLean, Ph.D., Prof.; K. Stietenroth, M.D.; H.Prange, M.D., Prof., Department of Neurology, Univer-sity 1lospital, Robert-Koch-Strasse 40, D-37075 Gottin-gen, Germany. V. V. Pai, M.D.; R. Ganapati, M.B.B.S.,D.V.U., Bombay Leprosy Project. 11 VN Puras' Marg,Sion-Chunabhatti, Mumbai 400 022, India.

Reprint reyuests to Prof. MacLean at the abovc ad-dress or FAX 49-551-39-8405.

20-25-year-old age group; PB patients av-eraged about 10 years younger than MB pa-tients. There were 45 MB patients for 14 ofwhom the initial values were available(Table 1), 29 PB for 11 of whom initial val-ues were available (Table 2), and 15 unin-fected controls (Table 3). Duration of ther-apy was usually 6 months for PB and 12months for MB patients. All patients hadserum samples taken before treatment andafter 1 month of MDT. MB patients re-ceived 600 mg rifampin and 50 mg clofaz-imine monthly under supervision, with adaily self-administered 100 mg dose ofdapsone and 50 mg of clofazimine. PB pa-tients received 600 mg rifampin per monthand 100 mg of dapsone daily. Reactionswere treated with tapering doses of steroids,starting at 60 mg.

Recorded data included: condition (re-sponse) on completion as good (N = 56),fair (N = 17), or poor (N = 1), there beingpersonal subjective assessments by experi-enced leprologists of the response to treat-ment compared to the normal response; thenumber of lesions, their distribution andtype; the initial and final BI; date and typeof any reaction, and final total serum pro-tein, albumin, IgG, ICAM-1 and E-sefectin.For 25 patients, the initial values of ICAMand other variables were used; for the oth-ers, only final results were available. Clini-cal and histological examinations were per-formed in Mumbai, India, and serum wasexamined using standard methods in thespecialized Neurochemical Laboratory, De-partment of Neurology, University Hospi-tal, Gottingen, Germany. Statistical analy-ses were performed using the StatisticalPrograms for the Social Sciences (SPSSversion 6) programs, (SPSS, Chicago).

RESULTSPoorer patient response was correlated

with age (r = 0.632, p <0.01) and loweredalbumin (r = 0.427, p <0.01). Albumin val-ues below 41 g/1 were especially associated

19

Units

yearsmg/dIng/nilng/nil

ng/nilng/nilme/1

ntg/cl I

20^ International.luurnal of LeprOS_y^ 1999

TAnl.l: 1^ffiriables o/ n n iltibaeillary patients includcd ia the study (IV = 14).

1.1in. Niax. Meara Mcdian Std. dev.

Age 20 62 38 36 16Aìbuntin 28 50 40 41 6.2Initial ICA1r1-1 315 998 590 561 218.5Initial 1:-selectin 31 162 89 82 40Initial^III 4 2.79 2.3 1.19Final ICAN1-1 269 464 354.6 356 57.5Final 1?-selectin 38 195 80 68 4112G 15 25 19.5 18 3Final III O 4 1.14 0.3 1.51'final protein 80 116 99 99 9.5

with poorer patient condition. When cor-rected for age, the correlation of albuminwith condition was still significant (r =0.25, p = 0.035). Table 4 shows the correia-tions of the variables. A discriminationfunction using the variables of age, albuminand E-selectin was able to identify 93% ofthe patients in good condition and 65% ofthose ira poor condition. When ICAM wassubstituted for selectin in the regression,discriminating ability for patients in poorcondition dropped about 5%. A logistic re-gression which considered age, E-selectinand albumin of <41 g/1 identified 85% ofthe patients in poorer condition (p = 0.003).In MB patients, the IgG correlated with theBI (p = 0.014) and inversely with albumin(p = 0.011); lowered albumin correlatedweakly with the BI (p = 0.06). With PB pa-tients, these correlations were not signifi-cant. Ali PB patients had a final BI of zero.

The Figure shows the predicted probabil-ity of the patients having a fair or poor re-sponse after treatment according to a jointregression of age, albumin and E-selectin(with no cut-off levei). Note that 7 patientsof 72 were misclassified despite high prob-abilities. For the individual regressions, re-sponse correlated (r = 0.6) with age, with

albumin (r = —0.37), and with E-selectin (r=0.28,p=0.06).

DISCUSSIONThe difference in age between the PB

and MB groups may tend to confound theinfluence of age and type of disease. Bothalbumin and IgG are nonspecific findings,so their use as a specific measure of diseasestatus in leprosy is not warranted. However,the clinician may find them useful in the ab-sence of more specific mensures.

A T cell which has been previously ex-posed to a specific antigen may cause therelease of tumor necrosis factor-alpha(TNF-a) and chemoattractants when itagain encounters the antigen. Cells that at-tach to the skin particularly express cuta-neous lymphocyte antigen and may bind toE-selectin. Adhesion molecules on theirsurface control the binding of T lympho-cytes to other cells. E-selectin (CD26E,ELAM-1) is expressed on vascular en-dothelium a few hours after exposure to thecytokine TNF-ot or to lipopolysaccharide,attracts neutrophils and macrophages, andwanes within about 24 hr. This results ia arolling of circulating leukocytes along anendothelium which has inflammatory cy-

TABLE 2. Variables of paucibacillarv patients included i1 the stucly (N = 11).

Min. Max. Mean Mediar Std. dcv.

Age I5 72 34 26 17.6Albumin 36 51 45 44 5Initial ICAN1-1 274 760 471 445 128Initial E -selectin 26 127 77 75 29Final ICAM-1 290 413 342 327 43Final E-selectin 23 84 63 62.5 18.5IgG 12 31 17 16 5.5Total protein 81 119 105 l01 13

Units

yearsrng/d Ing/ntlng/mlng/nilng/nilmg/1mg/dl

67, 1^ MacLean, et al.: Serrim Markers^ 21

TABLE 3. Variables o/ . normal controls incluclecl in Ilw slucly (N = 15).

Min. Nlac. N 1ean Median Std. dev. Units

Age 25 50 36 35 7 yearsSerem albumin 38 48 44.6 45 2.7 nlg/dlICAM-1 233 398 301 281 52 ng/mlIgG 9 19 14.7 14 2.7 agi!E-selectin 34 76 55 56 14 ng/m1

Total protein 78 104 91 90 9 mg/dl

tokines. ICAM-1 is also induced on en-dothelial cells by TNF-a although later andover a longer period (24-96 hr irr nitro)than E-selectin. ICAM-1, aided by cy-tokines, enables the firm attachment ofleukocytes to the endothelium. With the as-sistance of integrins, the phagocytes canthen cross the endothelial wall to the site ofinfection. More leukocytes and monocytesare then attracted to the site by further re-lease of chemokines and adhesion to E-se-lectin ('•'). Since ICAM-1 had been foundto increase in step with the degree of in-flammation ( 2) and decrease rapidly in theabsence of cytokines, the paper by Rieck-mann, et al. (') concluded that ICAM-1 andE-selectin were promising treatment suc-

cess markers for patients with leprosy.However, that paper used overal1 averages.When the results for individual patientswere examined, there was no useful corre-spondence (data submitted).

Age and serum albumin are useful inmonitoring therapy. To combine the consid-eration of E-selectin with there two factorswould, for practical purposes, require theuse of a computer and software applied to abase of local data and would imply the re-examination of misclassified patients.

CONCLUSIONSerum albumin correlated best with both

patient response and the BI. Patient age wasan important negative factor. E-selectin cor-

TABLE 4. Cerre/titio/is at final sampling.

Age Albumin Cond. EndICAM

EndSele.

FinalBI IgG Les.

type

AgeAlbumin

1.000-0.360

-0.3601.000

0.597-0.375•

0.035-0.224

0.344'-0.235 ,

-0.317'-0.232

0.170-0.360'

-0252' ,

0.147Spearman's Cond. 0.597° -0.375' 1.000 t).009 0.223 -0.064 0.119 -0.228correlation End ICAM 0.035 -0.224 0.009 1.000 0233' 0.235 0.320' 0.045cocfficient

(r)End Sele.Final BI

0.344'-0.317 ,

-0.235-0.232

0.223' ,

-0.0640.2330.235

1.0000.041

0.0411.000

0.470'0.244

-0.172-0.022

IgG 0.170 -0.360 0.119 0.320 0.470' 0.244 1.000 -0.177Les. type -0252' , 0.147 -0.228 0.045 -0.172 -0.022 -0.177 1.000

Age 0.002 0.000 0.770 0.003 0.034 0.147 0.031Albumin 0.002 0.001 0.055 0.047 0.126 0.002 0.212

Significance Cond. 0.000 0.001 0.940 0.060 0.677 0.311 0.050(2-tailed) End ICAM 0.770 0.055 0.940 0.049 0.121 0.005 0.704p value End Sele. 0.003 0.047 0.060 0.049 0.792 0.000 0.149

Final BI 0.034 0.126 0.677 0.121 0.792 0.106 0.888IgG 0.147 0.002 0.311 0.005 0.000 0.106 0.130Les. type 0.031 0.212 0.050 0.704 0.149 0.888 0.130

Age 74 74 74 74 72 45 74 74Albumin 74 74 74 74 72 45 74 74Cond. 74 74 74 74 72 45 74 74

N End ICAM 74 74 74 74 72 45 74 74End Sele. 72 72 72 72 74 44 72 72Final BI 45 45 45 45 44 74 45 45IgG 74 74 74 74 72 45 74 74Les. type 74 74 74 74 72 45 74 74

Correlation is significant at the 0.01 levei (2-tailed).'' Correlation is significant at the 0.05 levei (2-tailed).

111

1111 111 111 111 111 111111111111

2*12"1111111

11111

2 r1 111

•2 1 22122

2222 1"

X i

1 1 2 2 22

2i 2

22^ Inte rnation al .1011171(11 of Lepros.v^ 1999

16

FR^12EQuE^8NcY

4

Probability^o^.25^.5^ .75^ 1Group: 111111111111111111111111111111222222222222222222222222222222

Tio: FIGURE. Prediction of condition hy logistic regression of age, albumin and [-selectin at the end of treat-ment; ohscrved groups and predicted probabilities. * = Predicted probability is for memhership in group 2 (lessthan good condition).

related weakly with patient condition. IgGcorrelated with the BI. Serum albumin, ageand E-selectin, considered jointly, wereuseful as predictors of therapy success.

SUMMARYIntercellular adhesion molecule-1 (ICAM-

1) and E-selectin and other variables wereevaluated as possible markers of the suc-cess of multidrug therapy (MDT) in lep-rosy. Multibacillary (MB, N = 45) and pau-cibacillary (PB, N = 29) leprosy patientswere examined during MDT, which typi-cally lasted 12 months for MB and 6months for PB patients. Serum values fortotal protein, albumin, immunoglobulingamma (IgG), ICAM-1, and E-selectin (se-lectin) were recorded, as were lesion type,number, and distribution. Response at theend of therapy was assessed as good, fair,or poor. The bacterial index (BI) of lesionswas measured at the beginning and end oftherapy. The earlier reported lindings of thisinvestigation are herein re-examined. Re-sults: age and lowered serum albumin cor-related with the poorer condition of the pa-tients, as did elevated selectin. Albuminwas inversely correlated with the BI (p =0.008) in MB patients, and IgG was posi-tively correlated (p = 0.009). ICAM andE-selectin alone were not useful markers ofindividual patient condition. A regressioncombining serum albumin under 41 g/1, age

and E-selectin was able to identify 85% ofthe patients in poorer condition. Conclu-sion: serum albumin was a useful nonspe-cific marker of both patient condition andinfection. Age is an important negative fac-tor in patient response. Albumin and IgGcorrelate with the BI and with each other (p= 0.011) in MB patients, but net in PB pa-tients.

RESUMENSe evaluaron las moléculas de adhesión intercelular

(ICAM-1) y las selectinas E como posibles mar-cadores del éxito de la poliyuimioterapia (PQT) en lalepra. Los pacientes con lepra multibacilar (MB, N =45) o paucibacilar (PB, N = 29) se examinaron durantela PQT, la cual fue de 12 meses para los pacientes MBy de 6 meses para los PB. Se registraron los valores deproteínas totales en el suero, de alhúmina, de in-munoglobulina gamma (1gG), de ICAM-1, y de se-lectina E, así como el número, tipo y distribución delas lesiones. La respuesta al final del tratamiento secaliiicó como huena, regular, o pobre. El índice bacte-riano (B1) de las lesiones se ntidió al inicio y aI final dela PQT. Resultados: la edad avanzada y los niveles ba-jos de alhúmina correlacionaron con la pobre condi-ción de los pacientes; esto tamhién ocurrió con losniveles elevados de selectina. La alhúmina correla-cionó en forma inversa con el BI (p = 0.008) en los pa-cientes MB, y la IgG correlacionó positivamente (p =0.009). Los niveles de ICANI-1 y de selectina E, solos,no fueron marcadores titiles de la condición individualde los pacientes. Una regresión, combinando los nive-les de alhúmina por debajo de 41 JI. la edad y losniveles de selectina E, permiti() identificar aI 85% de

67, 1^ MacLean, et al.: Senti,, Markers^ 23

los pacientes en Ia condición más deplorable. Con-clusiún: Ia albtímina dcl surro ftic un marcador no es-pecífico útil para establecer tanto la condición de lospacientes como la infeccibn. La cdad es un importantefactor negativo en la respuesta dc los pacientes. La al-húmina y Ia IgG correlacionan coa el BI y entre si (p =0.011) en los pacientes MB poro no cai los pacientes PB.

RÉSUMÉLes molécules d'adhésion intercellulaire de type 1

(ICAM-1), les sélectines E et d'autres variahles ont étéévaluées en tant que inarqueurs potentiels de réponse àla polychintiothérapie (PCT) efficace contre Ia lèpre.Des patients atteints de lépre multibacillaire (MB, N =45) et paucibacillaire (PB, N = 29) ont été exantinéspendant leurs PCT, laquelle dura enviam 12 mois pourles patients MB et 6 mois pour les patients PB. 11 futenregistré, à partir du sérum, les valcurs des protéincstotales, de 1'albumine, des immunoglobulines gamma(IgG), des ICAM-1 et des sélectines E, de mênte que letype, le nombre et ht distribution des lésions. Laréponse en tin dc traitement fut estintéc comme bonnc,adéquate ou mauvaise. L'indice bactéridien (IB) deslésions f ut mesuré au déhut et à la fin du traitement.Les premiéres données rapportécs à partir de cetteétude sont ré-éxaminées ici. Résultats: l'âge et une al-buntinc sériquc basse étaicnt corrélés avec un mauvaisétat général du patient, comme 1'indiquait égalententune quantite élcvée de sélectine. Chez les patients MB,1'albumine était inversement corrélée avec l'lB (p =0,008), et la quantité d'IgG était positivement corréléeavec l'IB (p = 0,009). Les ICAM-I et les sélectines E,

isolées, n'étaient pas de bon indicatcurs de l'étatgénéral des patient à l'échelle individuellc. Une ré-gression contbinant une albumine sériquc dc moins de41 gll, l'âgc et les sélectines E fut capable d'identifier85% des patients avec un mauvais état général. Con-clusion: 1'albumine sériquc est un marqucur non-spé-cifique utile à la fois pour prédire l'état général et l'in-fection du patient. L'âge est un factcur négatif impor-tant pour prédire Ia réponse au traitement du patient.L'albuntinc et les IgG corrèlcnt avec l'IB et entre cux(p = 0.011 ) chez les paticnts MB, mais pas chez lespatients PB.

Acknoccledgment. These investigations were par-tially supported by a grant troar the Krupp Foundation.

REFERENCES1. JANEw,w, C. A. and TRAVtazs, P. Inununologv. 3rd

cdn. Oxford: Blackwell, 1997, S.9-7 ff.2. NAKANnR:v, S., Onr,s i, tI. and Fui auixtA , K. lo

.cila expression of the cell adhcsion molecules in in-tla ntntatory bowel disease: evidente of immuno-logic activation of vascular endothelial cclls. Lab.Invest. 69 (1993) 77-85.

3. Rii:c tANN, P., SciioLzE, G., Wtacttsta.ntcAuN, I.,GANAi',m, R. and Pznnci:, 11. W. Soluhlc adhcsionmolecules in sera of patients with leprosy: leveis ofsoluble intercellular adhcsion molcculc-1 (sICAM-1) rapidly dccreasc during multi-drug thcrapy. Clin.Exp. Immunol. 105 (1996) 65-68.

4. Rorn, 1., BArsrorr, J. and MATE, D. Inununology.London: Mosby, 1994, pp. 148 ff.