462^ International Journal of Leprosy^ 1997("). In indeterminate leprosy, in addition toscattered clumps of mononuclear cellsaround skin adnexa there is infiltration of thedermal nerves by chronic inflammatory cellsfollowed by disorganization and partial destruction.AFB may be found inside the dermalnerves or arrector pili muscles (').The other portion of the skin biopsy wasplaced in 70% ethyl alcohol and processedfor PCR studies for M. leprae (").RESULTSOf the 39 patients, 21 were males and 18were females with ages ranging from 8 to45 years (mean 21.5 years). During clinicalexamination 37 patients had a single hypopigmentedskin lesion; of the remaining2, one had 2 skin lesions and the other had3. The peripheral nerves did not show anysignificant change, and the skin smearswere negative in all 39 patients. Althoughall of the 39 were suspected to have leprosy,a definite diagnosis on the basis of clinicalexamination by an experienced leprologist(ii), using the criteria described earlier (I•was offered in only 14 patients who wereclassified as: I indeterminate, 4 TT and 9BT (Table 1). The other 25 were found tohave noncharacteristic hypopigmentedmacules (NHM).Upon histopathological examination 26patients were diagnosed as having leprosyand classified, using the criteria describedearlier, as: 6 indeterminate, 3 TT and 17BT. The other 13 patients were found tohave only NCI (The Table). AFB werefound in only 2 of 26 patients diagnosed ashaving leprosy. All of the 14 clinically diagnosedas having leprosy were confirmedduring histopathological examination.In our PCR study, evidence for the presenceof M. leprae was found in 11 biopsies(The Table); of these, two need specialmention. Although both patients were clinicallysuspected to have leprosy, the histopathologicalexamination revealed onlyNCI. Upon receiving the PCR report showingevidence of M. leprae, a second biopsyfrom the same skin lesion was done forboth patients. One showed typical histologyconsistent with indeterminate leprosy althoughno AFB were found; the other againshowed only NCI. Of the other 9 PCR-positivepatients, 7 were BT, 1 TT and 1 indeterminateleprosy, according to the histo-THE TAfti i ^Clinical and histopatholo,f,,icalclassification and presence ofM. leprae.'` 1 'Classilication.'llistopathologicalAl it inhistopath.sectionsM. lepraein PCRstudyI 6 0 1TT 4 3 I 1BT 9 17 I 7NCI (histopath.)MINI (clinical) 25 13 0 2Total 39 39 2 IINCI = Nonspecific chronic inflammation; NHM =noncharacteristic hypopigniented inacule.pathological study. Both patients, one TTand one BT who showed AFB in histopathologysections, were positive for M.leprae in PCR studies also.DISCUSSIONIn this study of 39 patients with skin lesionssuspected as having early leprosy, 14were diagnosed as leprosy clinically and 26histopathologically. By microscopy Al. lepraewere found in only 2 but using PCRstudies the presence of M. leprae was detectedin 11 specimens. PCR increased byfive to six times the frequency of finding M.leprae in tissue specimens from paucibacillarypatients. This confirms the findings ofother workers who have described high sensitivityand near-perfect specificity of PCRstudies to detect M. leprae ( 2- 5."'").It has been reported that an increase inthe number of AFB-stained sections examinedwill increase the frequency of detectingAFB in tuberculoid leprosy (4). Similarly,an increase in the number of PCRsamples studied increased the frequency offinding M. leprae in tissue specimens ( 13). Itis reasonable, therefore, to suggest that M.leprae are present in detectable numbers inalmost all active leprosy lesions but thatthey are so widely dispersed in tissues thatthe degree of detection will depend uponthe number of different samples examined.It is well known that definite histopathologicdiagnosis of early lesions of leprosydepended entirely upon the finding of oneor more AFB ( 3). Therefore, PCR study,which appears to be more sensitive thanhistopathological examination to detect M.leprae, could be a very good and useful ad-
65, 4^Job, et al.: PCR in Early Diagnosis^ 463junct in the diagnosis of early leprosy.However, we suggest that the feasibilityand cost-effectiveness of these two methodsto detect M. leprae be evaluated under differentlaboratory conditions.With the availability of numerous effectiveantileprosy drugs, leprosy is beingrapidly controlled throughout the world.The profile of leprosy patients presenting inan outpatient clinic is also radically changing,and more and more early lesions areencountered. Therefore, any additional toolfor use in detecting early lesions will be agreat advance in the field of early diagnosis.Detection of M. leprae using PCR methodologyis a very new test, and has only recentlyemerged as a diagnostic tool. It is, atpresent, too elaborate and expensive to beused in routine diagnostic work. Improvingthe techniques of PCR study and making itcheaper is imperative. Its possible use inleprosy reference laboratories for diagnosisis unquestionable. In this connection, it isworth pointing out that one of our patientsinitially diagnosed as nonspecific chronicinflammation of skin histopathologicallywas later confirmed as indeterminate leprosyonly with the assistance of the PCR results.It is suggested that PCR techniquesfor detecting M. leprae may profitably beused in association with histopathologicalstudies for patients suspected of leprosyfrom very low-endemic areas and fromnonendemic countries.It was feared that the PCR technique is sosensitive that there may be many false-positives.In this study, only one specimen maybe considered as false-positive becauseeven the second biopsy from the PCR-positivelesion came up with negative histopathology.SUMMARYFor 39 patients suspected of early leprosy,skin biopsies of the lesions were doneand bisected. One piece was used forhistopathologic examination and the otherfor polymerase chain reaction (PCR) studiesto detect Mycobacterium leprae. The diagnosisof early leprosy was made clinicallyin 14 patients and by histopathologicstudy in 26 patients. Acid-fast bacilli wereseen in the histopathologic sections of onlytwo patients, and M. leprae were detectedusing PCR techniques in I I patients. In onepatient the diagnosis of leprosy was madeonly because of the detection of M. lepraein the PCR study. Since even in endemiccountries the profile of leprosy is changing,detection of leprosy lesions in their earlystages has become increasingly important.Since the finding of M. leprae is crucial inthe confirmatory diagnosis of early leprosy,it is suggested that PCR studies to detect M.leprae be done wherever possible in conjunctionwith histopathologic examination.It is also recommended that the feasibilityand the cost-effectiveness of both of thesemethods to find M. leprae be evaluated.RESUMENSe tomaron hiopsias de pie! de las lesiones de pacientescon sospecha de lepra incipiente. Las hiopsiasse cortaron en 2 partes. Una pane se 1156 pant estudioshistopittolOgicos y Ia otra para hussar Mycobacteriumleprae por la tecnica de Ia reacciOn en cadena de Iapolimerasit (PCR). El diagnOstico de la lepra tempranitse hizo clInicamente en 14 pacientes y por histopatologiaen 26 pacientes. Se observaron hacilos acid°resistentes en las sceciones histopatolOgicas de solo 2pacientes. Por PCR Af. leprae se detect() en 1I pacientes.En on paciente, el diagnostico de lepra se hizosolo por el resultado positivo de Ia PCR. La detecciOnde las lesiones dc Ia lepra en sus estadlos incipientes escada vez miis importance porque aun en los paisesendemicos el pertil de Ia enfermedad estii cambiando.Puesto que el hallazgo de M. leprae es crucial para eldialmOstico contirmatorio de la lepra temprana, se sogicrcque en los estudios para detectar Al. leprae se incluyantanto PCR como el examen histopittolOgico.Tambien se recomienda quc se evaltie Ia practicabilidady el costo-hencticio de ambos tnetodos para Ia deteccitinde M. leprae.RESUMEOn a preleve et coupe en fragments des biopsies cutaneesdes lesions de 39 patients presentant tine lepredebutante. Un fragment a ete utilise pour [examenhistopathologique, et l'autre pour des examens aVCC Iareaction de polymerase en chaine (PCR) pour &teeterle Mycobacterium leprac. Lc diagnostic de lepre debutantea ete etabli cliniquement chez 14 patients, et parexamen histopathologique chez 26 patients. Desbacilles acido-resistants ont ete vus dans les coupeshistopathologiques de deux patients settlement, et duleprae a ete detecte chez 11 patients par Ia techniquede PCR. Chez on patient, le diagnostic de leprea ete etahli seulement it cause de Ia detection de Al.leprae dans l'examen par PCR. Puisque menu dans lespays endemiques le profit de la lepre est changcant, Iadetection des lesions lepreuses dans leurs stades preepeesa pris one importance croissante. Puisque la decouvertede Al. leprae est essentielle dans Ia continua-
464^ International Jot irnal of Leprosy^ 1997Lion du diagnostic de lepre debutante, nous suggeronsque des examens de PCR soient realises, pour &teeterdu Al. leprue, partout on c'est possible, en conjonctionavec l'histopathologie. Nous recommandons egalementque la faisabilite et le rapport cont-efficacite de ces deuxmethodes de detection du M. leprue soient evalues.Acknowledgment. We acknowledge with gratitudethe financial support received from AmericanLeprosy Missions International, Greenville, SouthCarolina, U.S.A. We are grateful to Mr. K. Rajannaand Mrs. Naoko Robbins for technical help and toMiss K. Jayanthi for secretarial assistance.REFERENCES1. BRYCESON, A. and PIALTZGRAFF, R. E. Leprosy.3rd edn. New York: Churchill Livingstone, 1990,pp. 29-43,57.2. DE WIT, M. Y. L., FABER, W. R., KRIEG, S. R.,DOUGLAS, J. T., LUCAS, S. B., ASUWAT, N. M., PAT-TYN, S. R., HUSSAIN, R., PONNIGIIAUS, J. M.,HARTSKEERI., R. A. and KLASTER, P. R. Applicationof PCR for detection of M. leprue in skin tissues.J. Clin. Microbiol. 29 (1991) 906-910.3. FINE, P. E. M., Jon, C. K., LUCAS, S. B., MEYERS,W. M., PONNIGIIUAS, J. M. and STERNE, J. B. C.Extent, origin and implications of observer variationin histopathological diagnosis of suspectedleprosy. Int. J. Lepr. 61 (1993) 270-282.4. FLEURY, R. F. and ARANDA, C. M. Detection ofAFB in tuberculoid biopsies. (Letter) Int. J. Lepr.63 (1995) 103.5. Jon, C. K. and CHACKO, C. J. G. A simplified 6group classification of leprosy. Lepr. India 54(1982) 26-32.6. Jon, C. K. and CIIACKO, C. J. G. A modificationof Fite's stain for demonstration of Al. leprue intissue sections. Indian J. Lepr. 58 (1986) 17-18.7. MISRA, N. RAMESH, V., MISRA, R. S., NARAYAN, N.P. S., Coi.s .roN, M. J. and NA II, I. Clinical utilityof LSR/A 15 gene for Al. leprue detection in leprosytissues using PCR. Int. J. Lepr. 63 (1995)35-41.8. RAH, A., DONOGHUE, H. D. and STANFORD, J. L.Application of PCR for detection of M. leprueDNA in specimens from treated patients. Int. J.Lepr. 63 (1995) 42-45.9. RIDLEY, D. S. and Jon, C. K. The pathology ofleprosy. In: Leprosy. Hastings, R. C., ed. NewYork: Churchill Livingstone, 1985, p. 110.10. SANTOS, A. R., Di: MIRANDA, A. B., SARNo, E. N.,SUFFYS, P. N., and DI:GRACE, W. M. Use of PCRmediatedamplification of M. leprue DNA in differenttypes of clinical samples for the diagnosisof leprosy. J. Med. Microbiol. 39 (1993) 298-304.11. WILLIAMS, D. L., GILLIS, T. P., Bourn, R. J.,LOOKER, D. and WATSON, J. D. The use of specificDNA probe and PCR for detection of M. leprue. J.Infect. Dis. 162 (1990) 193-200.P. WORLD HEALTI I ORGANIZATION. A guide to leprosycontrol. 2nd edn. Geneva: World Health Organization,1988, p. 19.13. YOON, K., CHAO, S., LEE, S. M., ABALOS, R. M.,CELLONA, R. V., FAJARDo, T. T., JR., GUIDO, L. S.,DELA CRUZ, E. S., WAI.SII, G. P. and KIM, J. Evaluationof PCR amplification of M. leprue-specificrepetitive sequence in biopsy specimens from leprosypatients. J. Clin. Microbiol. 31 (1993)895-899.