The authors studied 66 wild nine-banded armadillos from Brazil. the ear samples were collected and Ziehl-Neelsen or Fite-Faraco stains were performed, as well as immunostaining using polyclonal BCG antibody, to avaluate the presence of the Mycobacterium leprae. the AFB were not detected by the Ziehl-Neelsen or Fite-Faraco staining, neither immunoexpression of the BCG marker. However, many normal structures from the ears of the nine-banded armadillos, such as condrocytes, condroblasts, fibroblasts and endothelial cells, and Gram positive bacteria cocci, showed false positive reaction by the BCG marker. the authors discuss the use of the immunohistochemical studies with the polyclonal BCG antibody to identify M. leprae antigens in wild armadillos.Fed Univ Espirito Santo, Dept Social Med, Infect Dis Unit, Vitoria, BrazilUniversidade Federal de São Paulo, Dept Pathol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Dermatol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Pathol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Dermatol, São Paulo, BrazilWeb of Scienc

Deps, P. D.

Michalany, N. S.

Tomimori-Yamashita, J.

Fine, Paul EM

LSHTM Research Online

Commentary: is it really M. leprae?

Michalany, Nilceo S. [UNIFESP]

Tomimori-Yamashita, Jane [UNIFESP]

Repositório Institucional UNIFESP

INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 3Printed in the U.S.A.(ISSN 0148-916X)TO THE EDITOR:Current identification of Mycobacteriumleprae is done by Fite-Faraco or Ziehl-Neelsen stains, but low numbers of bacillusin the tissue could make identification diffi-cult. Some authors have reported that im-munohistochemistry techniques demon-strate antigens of M. leprae instead of thewhole bacillus, increasing the sensibility ofthe histopathology diagnose mainly in pau-cibacillary forms (1, 5, 7). Takahashi, et al. (7)identified more then 20% of the AFB fromof the skin biopsy of the indeterminate lep-rosy (paucibacillary form) using immuno-staining with anti-BCG (bacilli of Calmette-False Positive Reaction of the ImmunohistochemistryTechnique Using Anti-BCG Polyclonal Antibodies toIdentify Mycobacterium leprae in Wild Nine-Banded Armadillos1ABSTRACTThe authors studied 66 wild nine-banded armadillos from Brazil. The ear samples werecollected and Ziehl-Neelsen or Fite-Faraco stains were performed, as well as immunostain-ing using polyclonal BCG antibody, to avaluate the presence of the Mycobacterium leprae.The AFB were not detected by the Ziehl-Neelsen or Fite-Faraco staining, neither immuno-expression of the BCG marker. However, many normal structures from the ears of the nine-banded armadillos, such as condrocytes, condroblasts, fibroblasts and endothelial cells, andGram positive bacteria cocci, showed false positive reaction by the BCG marker. The au-thors discuss the use of the immunohistochemical studies with the polyclonal BCG antibodyto identify M. leprae antigens in wild armadillos.RÉSUMÉLes auteurs ont étudié 66 tatous à neuf bandes sauvages du Brésil. Les échantillons d’oreillefurent prélevés et des colorations de Ziehl-Neelsen et de Fite-Faraco, ainsi qu’un immuno-marquage utilisant un sérum polyclonal dirigé contre le bacille de Calmette et Guérin (BCG),afin d’évaluer la présence de Mycobacterium leprae. Des bacilles acido-résistants (AAR) nefurent pas détectés par le Ziehl-Neelsen ou le Fite-Faraco et une immuno-expression du mar-queur BCG ne fut pas observée non plus. Cependant, de nombreuses structures normales desoreilles de tatous à neuf bandes, telles que des chondrocytes, des chondroblastes, des fibro-blastes et des cellules endothéliales, ainsi que des coques bactériennes positives à la colorationde Gram, ont montré une réaction faussement positive au marqueur BCG. Les auteurs discu-tent de l’utilité des études immunohistochimiques avec un anticorps polyclonal dirigé contrele BCG pour identifier des antigènes de M. leprae chez les tatous sauvages.RESUMENSe estudiaron 66 armadillos silvestres de 9 bandas de Brasil. Se colectaron muestras de lasorejas, se seccionaron y se tiñeron por Ziehl-Neelsen o Fite-Faraco, y se evaluó la presenciade Mycobacterium leprae utilizando un anticuerpo policlonal anti-BCG. No se detectaronbacilos ácido-resistentes por las tinciones de Ziehl-Nelseen/Fite-Faraco y tampoco se encon-traron bacilos con el suero anti-BCG. Sin embargo, se observó que muchas estructuras nor-males de las orejas de los armadillos, tales como condrocitos, condroblastos, fibroblastos ycélulas endoteliales, y bacterias Gram negativas, dieron reacciones falsas-negativas con el re-activo anti-BCG. Los autores discuten el uso de los estudios inmunohistoquímicos con elsuero policlonal anti-BCG para identificar antígenos de M. leprae en los armadillos silvestres.1 Received for publication on 12 February 2004. Ac-cepted for publication on 26 April 2004.Reprint requests to: Address: Patrícia D. Deps,M.D., Ph.D., Av. Marechal Campos 1468, Núcleo deDoenças Infecciosas, Centro Biomédico. Vitória, ES,Brasil. Email: pdeps@ndi.ufes.br / pdeps@uol.com.br327328 International Journal of Leprosy 2004Guérin) antibodies. Using the same tech-nique, Barbosa Jr., et al. (1) found acid-fastbacilli (AFB) in 50% of the skin biopsieswhere they did not find AFB before by Fite-Faraco stainning. The antigens were de-tected in several sites of the dermis, mainlyin the perivascular region. Kutzner, et al. (4)described the anti-BCG immunostainingtechnique as a promising screening tool forthe detection of common infectious micro-organisms in humans. The anti-BCG anti-bodies do not react with the normal structuresfrom human skin nor with inflammatorycells, and produce little background, al-though Schettini, et al. (6) observed thatmelanophages and mast cells could bestained. Some authors have demonstratedcross reaction between polyclonal antibod-ies and a large spectrum of fungi and bacte-ria (2, 3, 4, 9). Bonenberger, et al. (2) estab-lished that some microorganisms such asMycobacteria and Nocardia were stronglystained by immunostaining with anti-BCG;Criptococcus, Pythium and Prototheca werepartially stained; spirochetes and protozoa(Leishmania) were not stained.We analyzed 66 wild nine-banded ar-madillos, Dasypus novemcinctus species,from the State of Espírito Santo, localizedin Southern region of Brazil. The purposeof this research was to evaluate if im-FIG. 1. Immunohistochemistry findings in ear fragment. Nucleous of the condrocytes, condroblasts, fibro-blasts, endothelial cells presenting immunoexpression to the anti-BCG marker. (Objective 40×).FIG. 2. Immunohistochemistry findings in earfragment. Inflammatory infiltration of the lympho-histiocytes and neutrophils, and Gram positive bacteriaas coccus presenting immunoexpression to the anti-BCG marker (Objective 20×).72, 3 Deps, et al.: Correspondence 329FIG. 3. Immunohistochemistry findings in human skin fragment from leprosy multibacillary patient, the hu-man positive control (Objective 40×).drochloride 60 mg% (SIGMA, EUA),DMSO 1 ml, 6% H2O2 20 volume and PBS100  ml for five minutes at room tempera-ture. Negative controls were prepared byomission of the primary antibody and sub-stitution with BSA. The readings were car-ried out on coded, “blinded” slides.The histopathological examination of theear biopsies showed an inflammatory infil-trate of the lympho-histiocytes and neu-trophils, but no AFB was found by Ziehl-Neelsen or Fite Faraco staining, or byimmunohistochemical study. However, nu-clei of many normal cells, such as condro-cytes, condroblasts, fibroblasts, endothelialcells (Fig. 1), and Gram positive bacteriacocci produced immunostaining with theanti-BCG marker (Fig. 2). In all the mate-rial, the primary antibody was replaced byBSA to demonstrate that there are no reac-tions due to the secondary antibody or slidebackground. The human controls showedmacrophages immunostained with the anti-BCG marker (Fig. 3).The immunohistochemical technique us-ing polyclonal anti-BCG antibody for de-tection of M. leprae antigens in ears frommunostaining with a polyclonal anti-BCGantibody was an appropriate diagnostic toolfor M. leprae detection in these animals.After the armadillos had been captured, theears, margins were collected and fixed in10% neutral buffered formalin.The paraffin-embedded samples were sec-tioned and slides were stained with Ziehl-Neelsen or Fite-Faraco stains and immunohis-tochemistry. We used the immunostainingwith the anti-BCG on human positive-controlbiopsies from multibacillary patients, tocompare with the samples from the armadil-los. After blocking of endogenous peroxi-dase, the slides were incubated with the poly-clonal rabbit antibody (anti-Mycobacteriumbovis, DAKO A/S, Denmark), Dilution of1:100,000 was done in PBS buffered pH 7.4with bovine serum albumin (BSA). Afterthis, the slides were incubated with the sec-ondary antibody, biotinylated goat anti-mouse/rabbit Ig (StrepABComplex/HRPDuet mouse/rabbit, DAKO A/S, Denmark),diluted 1:200 in PBS and with the strepavidin-Biotin-peroxidase complex 1:200 in PBS.The antigens were visualized after incuba-tion with the diaminobenzidine tetrahy-330 International Journal of Leprosy 2004wild armadillos, or other armadillo tissues,has not been reported before. We found sev-eral structures presenting immunoexpres-sion to the anti-BCG marker in the epider-mis and the dermis of the ear biopsies,without the morphologic features presentedby the M. leprae immunostained antigenspreviously described. Probably in wildnine-banded armadillo, microorganismsother than Mycobacteria could be responsi-ble for false positive results in these tissues.These findings agree with previous reports(2, 4). Another interesting feature observed isthat the polyclonal anti-BCG antibodystains Gram positive bacteria. Some ani-mals showed very similar structures to themorphology of coccus. This similarity cancreate doubt about the use of the immuno-histochemical methods using the polyclonalanti-BCG alone to detect the M. leprae in-fection in wild armadillos. We do not knowif this technique would be useful to detectmycobacterial antigens in tissue other thanears, such as skin, lymphnodes, or liver. Inconclusion, the immunohistochemical tech-nique, using anti-BCG antibodies, is a veryuseful tool for the diagnosis of human pau-cibacillary or indeterminate forms of lep-rosy, but in wild nine-banded armadillosimmunostaining of normal structures of theear and cross reaction with different infec-tious agents could difficult its utilization forMycobacterial antigen detection difficult.Acknowledgment. The authors would like to thankDr. Carlos Faria from Santa Casa de MisericordiaMedical School, Vitória, Brazil; Dr. Fausto EdmundoLima Pereira from Federal University of EspíritoSanto, Vitória, Brazil for precious critical suggestions;Dr. Charles K. Job from the Department of Pathology,St. Thomas Hospital and Leprosy Center, Chettupattu,India for the histopathologic review, and Ms. SuelyNonogaki from Adolpho Lutz Institute, São Paulo,Brazil for technical assistance.—Patrícia D. Deps, M.D., Ph.D.Department of Social Medicine, InfectiousDisease Unit,Federal University of Espirito Santo,Vitória, Brazil.—Nilceo S. Michalany, M.D.Department of Pathology, Federal University of São Paulo,São Paulo, Brazil.—Jane Tomimori-Yamashita, M.D., Ph.D.Department of Dermatology—Federal University of São Paulo, São Paulo, Brazil.REFERENCES1. BARBOSA, JR. A. A., SILVA, T. C., PATEL, B. N., SAN-TOS, M. I. R., WAKAMATSU, A., and FERREIRA, V. A.Demonstration of mycobacterial antigens in skinbiopsies from suspected leprosy cases in the ab-sence of bacilli. Path. Res. Pract. 190 (1994)782–785.2. BONENBERGER, T. E., IHRKE, P. J., NAYDAN, D. K.,and AFFOLTER, V. K. Rapid identification of tissuemicro-organisms in skin biopsy specimens fromdomestic animals using polyclonal BCG antibody.Vet. Dermatol. 12 (2001) 41–47.3. BYRD, J., MEHREGAN, D. R., and MEHREGAN, D.A. Utility of anti-bacillus Calmette-Guérin anti-bodies as a screen for organisms in sporotrichoidinfections. J. Am. Acad. Dermatol. 44 (2001)261–264.4. KUTZNER, H., ARGENYI, Z. B., REQUERA, L., RUT-TEN, A., and HUGEL, H. A new application of BCGantibody for rapid screening of various tissue mi-croorganims. J. Am. Acad. Dermatol. 38 (1998)56–60.5. MSHANA, R. N., BEHELU, A., STONER, G. L., HAR-BOE, M., and HAREGEWOIN, A. Demonstration ofmycobacterial antigens in leprosy tissues. Int. J.Lepr. 50 (1982) 1–10.6. SCHETTINI, A. P. M., FERREIRA, L. C. L., MILAGROS,R., SCHETTINI, M. C. A., PENNINI, S. N., and RE-BELLO, P. B. Enhancement in the histological diag-nosis of leprosy in patients with only sensory lossby demonstration of mycobacterial antigens usinganti-BCG polyclonal antibodies. Int. J. Lepr. OtherMycobact. Dis. 69 (2001) 335–340.7. TAKAHASHI D. I., ANDRADE, JR. H. F., WAKAMATSU,A., MANINI, M., and DE BRITO, T. Treated-curedindeterminate leprosy: a search for predictivehistopathological and immunohistochemical param-eters in skin biopsies taken from patients at admis-sion and at clinical discharge. Acta Leprol. (Gen-eve) 8 (1992) 95–102.8. TAKAHASHI, D. I., ANDRADE, JR. H. F., WAKA-MATSU, A., SIQUEIRA, S., and DE BRITO, T. Indeter-minate leprosy: histopathologic and immunohisto-chemical predictive parameters involved in its pos-sible change to paucibacillary or multibacillaryleprosy. Int. J. Lepr. Other Mycobact. Dis. 59(1991) 12–19.9. WILEY, E. L., BECK, B., and FREEMAN, R. G. 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International Journal of Leprosy and Other Mycobacterial Diseases

False positive reaction of the immunohistochemistry technique using anti-BCG polyclonal antibodies to identify Mycobacterium leprae in wild nine-banded armadillos

Commentary: is it really M. leprae?

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