64, 4^

Correspondence^ 451

—Noshir H. Antia, F.R.C.S.,F.A.C.S.(Hon.)

Director and TrusteeThe Foundation for Medical Research84-A R.G. Thadani MargWorli, Bombay 400 018, India

Reprint requests to Dr. Birdi.

Acknowledgment. We thank Dr. Elizabeth Bock,University of Copenhagen, for generously providingus with the anti-NgCAM antibodies and Dr. NergisMistry for her assistance in the study. This work wasfunded by grant No. 030074/Z/89/z from The Well-come Trust, London, U.K. N. Singh was a recipient ofa fellowship from the Council of Scientific and Indus-trial Research, Government of India.

REFERENCESI. BtRDI, T. J. and ANTtA, N. II. The macrophage in

leprosy: a review on the current status. Int. J. Lepr.57 (1989) 511-525.

BIRD], T. J., SIIETr•, V. P. and ANTIA, N. H. Differ-ence in AI. /eprae-induced nerve damage in Swisswhite and C57BL/6 mice. (Letter) Int. J. Lepr. 63(1995) 573-574.

3. BRocKEs, J. P., FIEL0s, L. K. and RAFE, M. C.Studies on cultured rat Schwann cells I. Establish-ment of purified populations from cultures of pe-ripheral nerve. Brain Res. 165 (1979) 105-118.

4. EfsafEBER, S., IIANNOCKS, M., METZ, C. N., RIFKIN,D. B. and SALYER, J. L. Transforming growth fac-tor-1/3 regulates axon/Schwann cell interactions. J.Cell Biol. 129 (1995) 443-458.

5. MARI INI, R. and SCHACHNER, M. ImmunOelectronmicroscopic localisation of neural cell adhesionmolecules (LI, N-CAM and myelin-associated gly-coprotein) in regenerating adult mouse sciaticnerve. J. Cell Biol. 106 (1988) 1735-1746.

6. PERRY, V. II. , BROWN, M. C. and GORDON, S.Macrophage response to central and peripheralnerve injury; possible role for macrophages in re-generation. J. Exp. Med. 166 (1987) 1685-1701.

7. SfiETTY, V. P. and AN -nA, N. II. Myelinationaround multiple axons in the peripheral nerve: anunusual ultrastructural observation. Neuropathol-ogy 50 (1980) 147-151.

Low Rates of Detection of Mycobacterial Secretory Antigen85 in Sera of Untreated Leprosy Patients

TO THE EDITOR:Leprosy continues to be one of the major

infectious diseases, affecting about 2.4 mil-lion people worldwide (estimate for De-cember 1993) ( 7 ), despite concerted effortsto control this disease. The World HealthAssembly call for the elimination of leprosyby 2000 A.D. and the World Health Organi-zation (WHO) recommendation of the useof fixed duration multidrug, therapy(WHO/MDT) has made the monitoring oftherapy a matter of importance. In this re-gard, mycobacterial proteins, such as theantigen 85 (Ag85) complex, actively se-creted by growing microbacteria ( 5- "), wereused for the assessment of MDT in leprosy.

Sera of leprosy patients were used for theconcurrent detection of Ag85 (secretoryprotein) and Ag82 (cytoplasmic 65-kDaheat-shock protein) by ELISAs. A ratio ofthe secretory to the cytoplasmic antigen(SCR) indicated secretory efficacy and,hence, viability of Mycobacterium ieprae.

Only 6 of 21 (28.5%) untreated multibacil-lary (MB) and 11 of 34 (32.3%) untreatedpaucibacillary (PB) patients showed signif-icant positivity for Ag85, although amongthe positive cases the SCR was high (> 1)and Ag85 levels between 0.1-10 ng/mlwere detected, indicating bacterial viability.Immune complexes, which have beendemonstrated in MB patients' sera ("• 12 ) andwhich could lower the level of free antigendetection, were suspected to be the cause ofsuch low sensitivity. However, immunecomplex dissociation attempted with MBsera failed to improve sensitivity. Further-more, no correlation was apparent betweenthe bacterial index (BI) in skin smears andthe positivity in ELISA.

The low sensitivity in ELISA could notbe attributed to the lack of antigen secretionby intracellular bacilli since the presence ofAg85 in the skin and nerve tissue of un-treated leprosy patients was clearly demon-strable by immunocytochemistry. Skin sec-

452^ International Journal of Leprosy^ 1996

tions showed intense staining within foamymacrophages, sweat and sebaceous glandsand endothelial cells with the antigen pack-aged in large macrophage granulomas,leaving the surrounding connective tissuefree of stain. This lack of staining in con-nective tissue lends itself to several inter-pretations: a) lack of diffusion of antigenfrom macrophages, b) breakdown of anti-gen in connective tissue, c) binding of Ag85to components of the connective tissue(e.g., fibronectin) ('), thus altering the con-figuration of its antibody-binding epitopes.

Nerve biopsies from untreated MB casesalso showed diffused straining withinmacrophages in the neural granuloma butnot in the extracellular matrix, Schwanncells or endothelial cells. Although theoverall pattern remains unchanged, the tis-sues of untreated PB patients showed com-paratively less Ag85 than did MB cases.The virtual absence of secretory antigen inSchwann cells was concordant with the lackof antigen detection in M. leprae-infected,murine Schwann cell cultures despite an av-erage 15-20-fold multiplication of intracel-lular bacilli observed in these cultures,( 2 ).

On the other hand, if growth of M. Lep-rae takes place within macrophages in tis-sues, release of mycobacterial secretoryantigens in the extracellular milieu is ex-pected since M. tuberculosis H37Rv-in-fected macrophages secrete significantquantities of Ag85 in culture supernatantsduring the growth phase ( 5 ).

In the case of M. leprae-infected macro-phages in tissue culture, Ag85 remains un-detectable in culture supernatants despiteoverloading of the host cells and the use ofprotease inhibitors, such as aprotonin, tominimize antigen degradation by macro-phage proteases. In such short-term culturesactive growth of M. leprae is not possiblealthough intracellular maintenance ofbacilli and slow growth within macro-phages has been relatively well establishedO. II).

Both armadillos, during experimentalleprosy infections, and leprosy patientsshow the presence of anti-Ag85 antibodies(4.10.

4 ) ,) signifying its functional presence. Itis possible, however, that sensitization isdue to the cell-bound forms of the antigenrather than the secreted form. However, an-tibody detection for the monitoring of ther-

apy has its inherent ambiguities ( 3 . 4 . 1 ") andmonitoring of therapy would he infinitelymore successful if antigens reflecting viablebacteria could be demonstrated. A demon-stration of the process of modification of es-tablished secretory antigens or productionof novel antigens by leprosy bacilli maygive a much needed fillip to this objective.

—Nerges F. Mistry, Ph.D.Senior Research Officer

—Anand lyer, B.Sc.Research StudentThe Foundation for Medical ResearchBombay, India

—Morten Harboe, M.D., Ph.D.Professor Medicine (Immunology)IGRIRikshospitaletOslo, Norway

—Noshir H. Antia, F.R.C.S.,F.A.C.S(Hon.)

Director and TrusteeThe Foundation for Medical Research84-A R.G. Thadani MargWorli, Bombay 400 018, Indict

Acknowledgment. This study was supported by agrant from NORAD. We gratefully acknowledge Ac-worth Leprosy Hospital and Maharastra Lokahita SevaMandal, Bombay, for providing us with patient materi-als for use in this study.

REFERENCESI . ABou-aip, C., RATLIFF, T. L., WIRER, 11. G., HAR-

13o0, M., BENNEDSEN, J. and Rook- , G. A. W.Characterization of fibronectin-binding antigensreleased by Mycobacterium tubenmlosis and M.bolls BCG. Infect. Immun. 56 (1988) 3046-305I.

2. ANTIA, N. 1-1., MISTRY, N. F. and KULKARNI, V. C.Multiplication of armadillo-derived M. leprae inmurine dissociated Schwann cell cultures. Int. J.Lepr. 56 (1988) 632-635.

3. ARAJ, G. F., FAHMAWI, B. II., Cuucn, T. D. andMUSTAFA, A. S. Improved detection of mycobac-terial antigens in clinical specimens by combinedenzyme-linked immunosorbent assays. Diagn. Mi-erobiol. Infect. Dis. 17 (1993) 119-127.

4. LAusaus, P., M'BAYANtr, N. N., DRowART, A., VANVoRFN, J. P., SARTnoti, J. L., LAI.U, T., MILIAN, J.and I ItlYGON, K. IgG response to purified 65- and70-kDa mycobacterial heat shock proteins and to

64,4^ Correspondence^ 453

antigen 85 in leprosy. Int. J. Lepr. 62 (1994)48-54.

5. MISTRY, N. F., MuRTY, S., SMINA, K. B., KUL-KARNI, V. C., ANTIA, N. II. and HARBOE, M. Im-munodiagnosis of tuberculosis and leprosy. (Let-ter) Natl. Med. J. India 6 (1993) 297.

6. MottAtv, C. J., TURK, J. L., RYDER, G. and WATERS,M. F. R. Evidence for circulating immune com-plexes in lepromatous leprosy. Lancet 2 (1972)572-573.

7. ota S. K. Elimination of leprosy as a pub-lic health problem: progress and prospects. Bull.WHO 73 (1995) 1-6.

8. PiissoLANI, M. C. V. and BRENNAN, P. J. My-cobacterium leprae produces extracellular ho-mologs of the antigen 85 complex. Infect. Immun.60 (1992) 4452-4459.

9. RAMASESH, N., HASTINGS, R. C. and KRAIIENBUI11.,J. L. Metabolism of Al. leprue in macrophages.Infect. Immun. 55 (1987) 1203-1206.

10. RUMSCHLAG, H. S., SIIINNICK, T. M. and COHEN,M. L. Serological responses of patients with lep-romatous and tuberculoid leprosy to 30-, 31-, and32-kilodalton antigens of Mycobacterium tubercu-losis. J. Chit. Microbiol. 26 (1988) 2200-2202.

11. SATISII, NI. and NATII, I. The uptake of 'H thymi-dine in Al. leprae-inoculated mouse macrophagesas a rapid indicator of bacterial viabilitiy. Int. J.Lepr. 49 (1981) 187-193.

12. SEHGAI., S. and KUMAR, B. Circulating and tissueimmune complexes in leprosy. Int. J. Lepr. 49(1981) 294-301.

13. WIRER, H. G. and HARBOE, M. The antigen 85complex: a major secretion product of M. tubercu-losis. Microbiol. Rev. 56 (1992) 648-661.

14. WIKta II. G., HARBOE, NI., NAGAI, S., PATAR-ROYO, M. E., RAMIREZ, C. and CRUZ, N. MI313 59,a widely cross-reacting protein of Mycobacteriumboris BCG. Int. Archs. Allergy Appl. Immun. 81(1986)307-314.

Prevalence of Antibodies to Hepatitis C Among RecentlyTreated Leprosy Patients in Senegal Parallels Those in

Normal Populations

To THE EDITOR:Recent reports focused on a higher

prevalence of hepatitis C (HCV) antibodies(Abs) in leprosy patients than in matcheduninfected individuals ( 3 . 5 ). Having fol-lowed a 175-patient cohort under field con-ditions, we do not confirm a higher carriageof hepatitis C among leprosy patients inSenegal, and we suggest that the high levelspreviously observed might rather be associ-ated with epidemiological conditions withinthe populations studied.

Infection with Mycobacterium leprue isaccompanied by a profound alteration ofcellular immunity leading to the differentaspects of the leprosy spectrum. Little isknown about the exact influence of the lackof T-cell immunity observed in leprosy andthe susceptibility to viral infections. In thisline, we investigated the seroprevalence toHCV using sequentially 2 third-generationELISAs (Ortho HCV 3.0°°; Chiron Corp.,Emeryville, California, U.S.A.; Murex anti-HCV version III, Templehill, England) andan immunoblot assay (Ortho RIBA 3.0.®;Chiron). Genotyping of positive HCV serawas carried out using an ELISA revealingcirculating antibodies to recombinant NS4

proteins (Murex HCO2) ('). Two othermarkers of viral infection were also exam-ined, i.e., the presence of hepatitis B virusantigen (HBs Ag) and HIV antibodies usingcommercially available kits; Monolisa' andElavia mixte", respectively (Sanofi-Pasteur,Marne la Coquette, France).

Subjects consisted of 147 newly diag-nosed patents and 28 previously treated(monotherapy) patients entering a new sur-vey for the efficacy of a fully supervised,intermittent, single-dose poly-antibiother-apy. Enrollment was done from March toDecember 1995; patients received full in-formation regarding this investigation andan initial blood sample was taken at thetime of the enrollment visit. During the sur-vey, serum was kept in a dry sterile tube at4°C (for approximately 12 hr) in the fieldand then frozen at —20°C until further assays.

A similar prevalence of the viral markerswas found in sera from leprosy patients anda normal population. The HIV antibodyprevalence reported here is in line with pre-vious studies conducted on West Africanpopulations ( 21. Secondly, the HBs antigencarriage found in this study was not higherthan that found in recent determinations

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