60, 3^ Editorials^ 451

Modern Sensitive Techniques for the Detection ofMycobacterium leprae *

When Shepard reported that the leprosybacillus could be grown in the foot pads ofmice in 1960,' it introduced a new era anddimension to research and the understand-ing of disease processes in leprosy. Recentadvances in molecular biology appear to beon the brink of offering highly technical andsensitive implements for the diagnosis ofleprosy. It would appear that some 30 yearsof research and, hopefully, the consequentmanagement of leprosy is on the brink ofanother quantum leap forward. At the mo-ment extensive applications can be envis-aged for research, but applications in thefield for disease control would appear to besome way off The World Health Organi-zation (WHO) has set up trials in some cen-ters with a view to the possibility of adapt-ing some of these interesting high-technologytools for use in the field. 2

This essay discusses these recent devel-opments together with the implications theyhold for the advancement of clinical un-derstanding and treatment of leprosy.

Historical perspective. Since the discov-ery of the leprosy bacillus' by G. H. A. Han-sen in 1873 (a remarkable achievement inits day due to the prevalent moral and re-ligious beliefs associated with the disease),scientific knowledge of leprosy has pro-gressed slowly in the main. However severalimportant individual steps have given extraimpetus.

* This review, written in 1991 by Peter R. Ayliffewhile a student at St. George's Hospital Medical School,London, shared first prize in the annual competitionsponsored by the British Leprosy Relief Association(LEI'RA) for essays on various aspects of leprosy. Wetake pleasure in publishing this review. The author'spresent address is: Peter R. Ayliffe, 4 Dahomey Road,Streatham, London SW l6 6ND, U.K.

The JOURNAL will also publish the other essaysharing LEPRA's 1991 first prize in its December 1992issue.

' Shepard, C. C. The experimental disease that fol-lows the injection of human leprosy bacilli into thefoot pads of mice. J. Exp. Med. 112 (1960) 445-454.

= Techniques for leprosy diagnosis "exciting" but stillexperimental. TDR News 34 (1990) 3, 7.

' Thangaraj, R. H. and Yawalkar, S. J. Leprosy formedical practitioners and paramedical workers, 4th ed.Basle: CIBA-GEIGY, 1989.

Since that time tests for the organism haveinvolved complex and time-consuming his-tological and microscopical techniques. Thecomplex nature of the infective process andthe unpredictable response to the infectionby the host have all contributed to the slowprogress. The practical aspects of identify-ing and culturing the leprosy bacillus havealso contributed to the slow progress. Twomajor advances in the past 30 years havemaintained momentum in research intoleprosy. However, apart from informationon the development of resistance to drugsalready available, their impact on the man-agement of the disease in the field has beenlimited.

The mouse foot pad model for Mycobac-terium leprae infection developed by Shep-ard was important since it provided the startfor laboratory based research into leprosy.It offered a means of assessing the growthof the leprosy bacillus. Growth in the footpads of mice is an indicator of viability; itis not quantitative on its own. Quantifica-tion of viability is determined by the pro-portional bactericidal test. 4 Although onlylimited multiplication occurs in the mousefoot pad, it allows evaluation of chemo-therapeutic regimens; 5 it also serves as thebasic model for testing the efficacy of po-tential vaccine preparations. The use of nudemice, which are congenitally athymic, 6-9

Colston, M. J., Hilson, G. R., Banerjee, D. K. The"Proportional Bactericidal Test": a method for assess-ing bactericidal activity in drugs against M. leprae inmice. Lepr. Rev. 49 (1978) 7-15.

Shepard, C. C. 1976. Experimental chemotherapyin leprosy. Bull. WHO 53 (1976) 425-433.

Chehl, S., Ruby, J., Job, C. K. and Hastings, R. C.The growth of M. leprae in nude mice. Lepr. Rev. 54(1983) 283-304.

'Colston, M. J. and Hilson, G. R. Growth of M.leprae and M. morainal in congenitally athymic (nude)mice. Nature (London) 262 (1976) 736-741.

Fieldsteel, A. H. and Levy, L. Neonatally thymec-tomized rats injected with Mycobacterium leprae: re-sponses to primary infection, secondary challenge, andlarge inocula. Infect. Immun. 14 (1976) 736-741.

Kohsaka, K., Mori, T. and Ito, T. Lcpromatoidlesions developed in nude mouse inoculated with M.leprae. Lepro 45 (1976) 177-187.

452^ International Journal of Leprosy^ 1992

provided an animal model of lepromatousleprosy that considerably expanded theknowledge and defined the importance ofcell-mediated immunity (CMI) in host re-sistance to leprosy, and provided modelsthat more closely mimicked the clinicalfindings seen in lepromatous leprosy.

Kirchheimer and Storrs in 1971 1 " iden-tified the nine-banded armadillo as a sus-ceptible host for the growth of Al. leprae.The armadillo model provided for the firsttime large quantities of homogenous bacillifor immunological, immunotherapeutic andimmunoprophylactic studies.

Important in research and clinical appli-cations over the years has been the Ridley-Jopling classification." This is an importanttest in the clinical setting for determiningthe response to treatment. Over the years ithas provided a reliable and reproducible as-sessment of disease activity based on theZichl-Neelsen staining of histological spec-imens. Specimens are classified by a bac-terial index, where the number of acid-fastbacilli (AFB) in the biopsy is assessed on alogarithmic scale of 0 to 6+, and this in-formation helps to place the patient on thescale within the classification. The morpho-logical index relates the number of solid-staining bacilli to the total number of AFB.This is important in assessment of the vi-ability of bacilli in the biopsy, since someorganisms may be fragmented or stain un-evenly, and this is used in monitoring theefficacy of treatment. Other characteristics,such as type of immune cells present in thebiopsy sample and the nature of the skin-test response, arc also important in classi-fication.

Obstacles to advances in research andclinical progress. Although the discovery ofthe bacillus is nearly 120 years old, M. lep-rue remains uncultivatable. Human biop-sies have served as the only source of theleprosy bacillus. These crude preparationsyielded inadequate numbers of organisms

'" Kirchheimer, W. F. and Storrs, E. E. Attempts toestablish the armadillo (Dasypus novemeinctus) as amodel for the study of leprosy. I. Report of lepromatoidleprosy in an experimentally infected armadillo. Int. J.Lepr. 39 (1971) 693-702.

" Ridley, D. S. and Jopling, W. B. Classification ofleprosy according to immunity: a five-group system.Int. J. Lepr. 34 (1966) 255-273.

for most studies, and the lack of reliablemethods for quantitating viable organismsprevented standardization ofdifferent prep-arations. It seemed that every step forwardwas associated with a despairing obstacle.Despite the dedication of researchers, thesepredicaments have contributed to the lackof' progress and impeded the necessary ad-vances in the field. The study of the epi-demiology of leprosy has been particularlyhampered by the lack of sufficiently reliablequick and efficient tests. Knowledge of thedisease in this area remains particularlyscanty.

It has been known for a long time thatpeople exposed to leprosy do not necessarilycatch the disease. The majority of peopleexposed effectively resist infection with Al.leprae even in highly endemic areas. It isnow thought that as many as 200 individ-uals become infected with AI. leprae for eachcase that is detected or develops overt dis-ease.'' In those people unable to mount anadequate immune response to infection withAl. leprae, the incubation period varies; usu-ally in the range of 2 to 4 years but it maybe 30 to 40 years. 1- ' 5 The histopathologyof the early lesion in leprosy (usually a by-popigmented maculc) may be that of non-specific, chronic dermatitis. The presence ofacid-fast bacilli or infiltrate in nerve bundlesis diagnostic, but indeterminate leprosy re-mains diagnostically difficult with a vari-able course. In approximately 75% of pa-tients the disease heals spontaneously; somecases remain indeterminate for a prolongedperiod of time, and some progress to one ofthe established forms of the disease.

Immunochemical research combined withextensive ultrastructural analysis of Al. lep-rae cell walls and associated component

' 2 Abe, M., Minagawa, F., Yoshino, Y., Ozawa, T.,Saikawa, K. and Saito, T. Fluorescent leprosy antibodyabsorption (FLA-BS) test for detecting subclinical in-fection with ,Ify'cobae[erium leprae. Int. J. Lepr. 48(1980) 109-119.

" Levis, W. R., Schuman, J. S., Friedman, S. M. andNewfield, A. An epidemiological evaluation of leprosyin New York City. JAMA 247 (1982) 3221-3226.

'-' Aycock, W. L. and Gordon, J. E. Leprosy in vet-erans of American wars. Am. J. Sci. 214 (1947) 329-339.

Barrett-Connor, E. Latent and chronic infectionsimported from Southeast Asia. JAMA 239 (1978) 1901-1906.

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molecules has demonstrated a surface to thebacilli essentially bereft of proteins andpolypeptides.' ' 8 The major obstacle to thestudy of Al. leprae stems from the inabilityto culture the organism in vitro, limitingworkers to bacteria grown experimentallyin infected mice, rats, or armadillos. Theserelatively low numbers of organisms con-sequently yield only small amounts of pu-rified proteins; this severely limits analysis.

The practical clinical immunology of lep-rosy is at present based almost entirely onreactivity to intradermal skin tests with lep-romin, a heat-killed suspension ofM. lepraeoriginally obtained from homogenized hu-man lepromas" now prepared from infect-ed armadillo tissue. The response to lep-romin has no real diagnostic value becauseindividual patients' immune responses varyimmensely. However, it does indicate theimmune capability of any individual to Al.leprae, and has a prognostic value in clinicalpractice. 20 Typically, a positive reaction isbiphasic. An early reaction'' at 24 to 48hours (Fernandez reaction) is a delayed-typehypersensitivity reaction (probably to sol-uble protein antigens in the preparation) andoccurs in tuberculoid leprosy as well as con-tacts or healthy individuals sensitized to Al.leprae or who are sensitized to crossreactingantigens from other mycobacteria. The late(Mitsuda) reaction to lepromin is measuredat 21 days, and represents the induction ofacquired cell-mediated immunity to M. lep-rae, manifested by an epitheloid cell gran-uloma reaction. 22 Positive Mitsuda reac-

Barksdale, L. and Kim, K. S. Mycobacterium.Bacteriol. Rev. 41 (1977) 217-372.

" Lederer, E. A., Adam, A., Ciorbaru, A., Petit, J.F. and Wietzerbin, J. Cell walls of mycobacteria andrelated organisms: chemistry and immunostimulantproperties. Mol. Cell. Biochem. 7 (1975) 87-104.

18 Schleifer, K. H. and Kandler, 0. Peptidoglycantypes of bacterial cell walls and their taxonomic im-plications Bacteriol. Rev. 36 (1972) 407-477.

Dharmendra, P. Studies of the lepromin test; abacillary antigen standardized by weight. Lepr. India14 (1942) 122-129.

20 Rees, R. J. W. The significance of the leprominreaction in man. Prog. Allergy 8 (1964) 224-258.

Fernandez, J. M. M. The early reaction inducedby lepromin. Int. J. Lcpr. 8 (1940) 1-14.

" Mitsuda, K. On the value of a skin reaction to asuspension of leprous nodules. Hifuka Hinyoka Zasshi.(Jap. J. Derm. Urol.) 19 (1919) 697 [Reprinted in En-glish. Int. J. Lcpr. 21 (1953) 347-358.]

Lions are seen in patients with tuberculoidleprosy. Weakly positive cases may help inthe diagnosis of borderline disease. In lep-romatous leprosy, no response is seen, in-dicating the absence of cell-mediated im-munity to M. leprae. Even after many yearsof chemotherapy, the lepromin test remainsnegative in lepromatous leprosy. 23

Need for sensitive testsResearch applications. Prior to the ap-

plication of monoclonal antibodies and re-combinant DNA techniques, immuno-chemical approaches for the study ofproteins in Al. leprae were tedious and high-ly complex. Most of the early work was per-formed by the immunoprecipitation of pro-tein and other antigens with polyclonalantisera in agarose gels by two-dimensionalimmunoelectrophoresis. 24 Despite sophis-ticated analysis of these profiles, little hasbeen learned about the chemical nature ofthe antigens contributing to these variousimmunoprecipitates.

Diagnostic tools. Established leprosy il-lustrates a continuous spectrum of diseasefrom localized, self-healing, granulomatouslesions with very few demonstrable bacillito a widespread, progressive, anergic dis-ease with massive numbers of M. /eprae. 2 )Borderline leprosy encompasses those typesof the disease between lepromatous leprosyand tuberculoid leprosy. A patient with bor-derline leprosy can develop clinical, bacte-riological, and histopathological features ofmore tuberculoid disease with time this iscalled upgrading. As in tuberculoid leprosy,the signs and symptoms of borderline lep-rosy tend to be due to a mixture of bacterialproliferation and the immunological hostresponse. The numbers of AFI3 in lesionsvary from undetectable to 1 AFB to 100AFB in any oil immersion microscopicfield. 26 Sensitive tests to detect infection with

" Mukerjee. N. and Kundu. S. The late leprominreaction in subsided lepromatous cases. Int. J. Lepr. 29(1961) 14-19.

Harboe, M. The immunology of leprosy. In: Lep-rosy. Hastings, R. C., ed. Edinburgh: Churchill Liv-ingstone, Ltd., 1985, pp. 53-87.

Ridley, D. S. Pathogenesis of Leprasy and RelatedDiseases. Cambridge: Butterworth, 1988.

10 Hastings, R. C., Gillis, T. P., Krahenbuhl, J. L.and Fran/blau, S. G. Leprosy. Clin. Microbiol. Rev. 1(1988) 330-348.

454^ International Journal of Leprosy^ 1992

the lepromatous bacilli will, it is envisaged,help in the prediction of clinical outcome.

Sensitive and specific tests have the po-tential for early diagnosis, detection of sub-clinical infection and the monitoring or pre-dicting of the disease outcome of individualpatients, and the response to chemotherapyand drug screening.

Monitoring response to treatment. Bettertests would provide rapid and reliably sen-sitive means in multibacillary patients ofdistinguishing between relapse due to re-activation of residual leprosy bacilli and re-lapse due to reinfection with a different strainof bacillus. 27 ' 29 More sensitive tests wouldalso be helpful in defining a cure for leprosy.It is known that many mycobacteria in bothleprosy and tuberculosis remain metaboli-cally inactive for considerable periods oftime and, as such, are not killed by antibi-otics. These inactive bacteria, referred to aspersisters, can reactivate many years later.Thus, any test which can detect them willbe very useful clinically.

Development of drug resistance. One ofthe most promising applications of specifictests could be to discriminate between livingand nonliving leprosy bacilli in tissue andblood specimens. 29 . 30 Potential applica-tions in this area arc very exciting and wouldbe useful in determining and monitoring thedevelopment of drug resistance (an increas-ingly worrisome problem in the treatmentof leprosy previously only achieved by in-jecting M. leprae from patients into foot padsof mice, feeding the mice the drugs, andfollowing the course of this localized infec-tion). This is a procedure that is tedious andtime consuming; and development of sig-nificant results might take months. The pro-cedure is costly in materials and resources,so much so that realistically the idea could

" Hirschberg, H. The role of macrophages in thelymphoproliferative response to .1/. leprae in vitro. Clin.Exp. Immunol. 34 (1988) 46-51.

" Beiguelman, B. Leprosy and genetics; a review ofpast research with remarks concerning future investi-gations. Bull. WHO 37 (1967) 461-476.

" Fallen, M. J. and Butcher, P. D. New strategies inmicrobiological diagnosis. J. Hosp. Infect. 18 Suppl A(1991) 147-158.

'" Woods, S. A. and Cole, S. T. A rapid method forthe detection of potentially viable ,Ifycobacteriunt lep-rae in human biopsies: a novel application of PCR.FEMS Microbiol. Lett. 65 (1989) 305-310.

only be entertained in the research labora-tory. Furthermore, an effective clinical testwould have tremendous applications forsensitivity testing of Al. leprae prior to ini-tiating chemotherapeutic measures in thefield.

Epidemiology and targeting resources.More sensitive tests have important possi-bilities in epidemiological studies; specificsensitive tests will find applications in mon-itoring the course of control measures inendemic areas. More fundamental researchcan be focused on the immunological mech-anisms that contribute to the protective aswell as the pathological aspects of host re-sponses to M. leprae. Eventually, exploita-tion of this fundamental knowledge shouldallow development of an effective vaccine.

A number of research groups have foundmolecular biological techniques useful fordistinguishing between the different variantstrains of Al. tuberculosis. If similar meth-ods could be used to subtype different strainsof Al. leprae, as many people hope and asit seems likely due to the extraordinary sen-sitivity of these techniques, they could turnout to be very useful tools in epidemiolog-ical studies.

New strategies and recent discoveriesMany new advances have been made in

the last few years in the study of infectiousdiseases at the biochemical and molecularbiological levels. With the inherent diffi-culties in studying the leprosy bacillus, mucheffort has been put into adapting many ofthese techniques to research into leprosy.Furthermore, it would appear that molec-ular biology is all set to enter the diagnosticmicrobiology laboratory. 2 By looking atspecific unique components of Al. leprae, itmay be possible to circumvent the problemsassociated with the growing and studying ofthe leprosy bacillus.

Immunology. Infection with M. leprae ischaracterized by a broad spectrum of hostresponses; there is great variability in thehistopathology and clinical course of the in-fection. There is also a spectrum in the pa-tient's humoral and cell-mediated immuneresponse. Although specific reactivity oflymphocytes can routinely be demonstratedin vitro in peripheral blood, the reactivityof the cells in the leprosy lesions is probablymore relevant to the cell-mediated immune

60, 3^ Editorials^ 455

response of the host to Al. leprae. Mod lin,et al. have investigated the cellular makeupof the various lesions in leprosy, using im-munopathological techniques, 31-33 and theirresults show that the T4/T8 ratio is alteredand the relative number of helper-T cells ismarkedly lower (0.5:1) in lepromatous le-sions compared to a normal 2:1 ratio inperipheral blood. More importantly, theyshowed that the cells are arranged in a dis-tinct architecture within the epithclioidgranulomatous lesions. It is difficult to seethe development of these recent findings intoclinically useful tests, but many of these ex-perimental findings will be useful in assess-ing the response of patients to various an-tigenic preparations in the quest for a vaccinethat will be effective against AI. leprae.

Extensive chemical analysis has shownthe cell wall of M. leprae to be a highlycomplex, lipid-rich, macromolecular struc-ture.' 7 Some cell-wall components areunique to mycobacterial cells, and are foundin most of the different species, for instance,peptidoglycan is present in all mycobacteriawith only minor variations. The most no-table advances in the immunology of lep-rosy have come from the isolation and char-acterization of two Al. leprae antigens —PGL-I and LAM-B.

In the study of Al. leprae an importantcell-wall-associated glycolipid molecule hasbeen of considerable interest—phenolic gly-colipid I (PGL-I)—shown to be species spe-cific and immunogenic during infectionswith Al. leprae. 34 ' 35 In a series of experi-

Modlin, R. L., Kato, H., Mehra, V., Nelson, E.,Fan, X. D., Rca, T. H., Pattengale, P. K. and Bloom,B. R. Genetically restricted suppressor T-cell clonesderives from lepromatous leprosy lesions. Nature(London) 322 (1986) 459-461.

Modlin, R. L., Mehra, F., Jordan, R., Bloom, B.R. and Rca, T. H. In situ and in vitro characterizationof the cellular immune response in erythema nodosumleprosum. J. Immunol. 136 (1986) 883-886.

" Modlin, R. L., Mehra, V., Wong, L., Fujimiya, Y.,Chang, W. C., Horwitz, D. A., Bloom, B. R., Rca, T.and Pattengale, P. K. Suppressor t lymphocytes fromlepromatous leprosy skin lesions. J. Immunol. 137(1986) 2831-2834.

" Brennen, P. J. and Barrow, W. W. Evidence forspecies-specific lipid antigens in Mycobacterium lep -

rae. Int. J. Lepr. 48 (1980) 382-387.Young, D. B. and Buchanan, T. M. A serological

test for leprosy with a glycolipid specific for Mycobac-terium leprae. Science 221 (1983) 1057-1059.

meats, Brennan and co-workers establishedthe chemical structure and the immunolog-ical specificity of PGL-I. 3'. 37 The generalstructure of PGL-I can be described as atrisaccharide moiety linked to a phthiocerollipid core through a phenolic group.

The discovery of PGL-I in armadillo-derived Al. leprae 36 ' 38-4" was importantsince this is immunologically specific to theleprosy bacillus and has subsequently beenshown to be located on the surface of theorganism by electron microscopic and im-munochemical studies. This unique trisac-charide structure accumulates in the tissuesof the armadillo in quantities equal to halfthe total weight of the leprosy bacilli pres-ent. 38 . 4" The ability to produce purifiedpreparations of antigens specific to Al. lep-rae allowed immunologists to use advancedtechniques to further the knowledge of theimmunology of leprosy.

PGL-I appears to be associated with theouter surface of M. leprae" and, signifi-cantly, has been isolated from both purifiedbacteria and Al. /eprae-infected tissues inrelatively high concentrations. 35 PGL-I mayact as a virulence factor in infection. Theapplication of these studies in clinicallymonitoring the response to chemotherapyhas already been demonstrated.''- The assay

" Brennan, P. J. The plithiocerol-containing surfacelipids of M. leprae—a perspective of past and presentwork. Int. J. Lepr. 51 (1983) 387-396.

Gaylord, H. and Brennan, P. J. Leprosy: antigensand host-parasite interactions. In: Parasite Antigens:Toward New Strategies for Vaccines. Pearson, T. W.,ed. New York: Marcel Dekker, Inc., 1986, pp. 49-89.

" Hunter, S. W. and Brennan, P. J. A novel phenolicglycolipid from Mycobacterium leprae possibly in-volved in immunogenicity and pathogenicity. J. Bac-teriol. 147 (1981) 728-735.

Hunter, S. W. and Brennan, P. J. Further specificextracellular phenolic glycolipid antigens and a relateddiacylphthiocerol from Mycobacterium leprae. J. Biol.Chem. 258 (1983) 7556-7562.

4" Hunter, S. NV., Fujiwara, T. and Brennan, P. J.Structure and antigenicity of the major specific gly-colipid antigen of Mycobacterium leprae. J. Biol. Chem.257 (1982) 15072-15078.

Young, D. B., Khanolkar, S. R., Barg, L. L. andBuchanan, T. M. Generation and characterization ofmonoclonal antibodies to the phenolic glycolipid ofAlycobacterium leprae. Infect. Immun. 43 (1984) 183-188.

42 Douglas, J. T., Steven, L. M., Fajardo, T., Cellona,R. V., Madarang, NI. G., Abalos, R. M. and Steenber-gen, G. J. The effects of chemotherapy on antibody

456^ International Journal of Leprosy^ 1992

of whole cell antigens of Al. leprac in thescrodiagnosis of leprosy has always beencomplicated by the extensive crossreactionsamong mycobacterial species. Changes inpatients' sera may give early indications ofchange in disease status, and a sensitive testto detect the changing levels and type ofantibody response could, therefore, be ofvalue in managing patients. Specific, syn-thetic antigenic components of PGL-1 of Al.leprac can be used in an enzyme-linked im-munosorbent assay of patients' sera; thismay extend the knowledge of the immuneresponse to leprosy with exciting implica-tions in the development of immunother-apeutic and immunoprophylactic treat-ments . 43

Arabinose- and mannose-containingphosphorylated lipopolysaccharides fromAl. leprac have been isolated and charac-terized. 44 A detailed study by Hunter, et a1. 44

established that the serologically activecomponent, lipoarabinomannan 13 (LAM-B), is acetylated, contains substituents ofphosphatidylinositol, and may be mem-brane associated. LAM-B has proven to behighly immunogenic in most mycobacterialinfections, inducing strong humoral anti-body responses. Since LAM-B is a commonantigen among mycobacteria, it cannot beused effectively for serological tests to detectearly infection with Al. leprac. However,there is evidence to suggest that high levelsof anti-LAM-B are associated with highbacterial loads in leprosy. 45 This may proveto be a useful test for estimating bacterialclearance during antimicrobial chemother-apy by monitoring serum antibody levels toLAM-B.

levels in lepromatous patients. Lepr. Rev. 59 (1988)127-135.

" Convit, J., Aranzazu, N., Ulrich, M., Zuniga, NI.,de Aragon, M., Alvarado, J. and Reyes, 0. Investi-gations related to the development of a leprosy vaccine.Int. J. Lepr. 51 (1983) 531-539.

" Hunter, S. W., Gaylord, H. and Brennan, P. Struc-ture and antigenicity of the phosphorylated lipopoly-saccharide antigens from the leprosy and tubercle ba-cilli. J. Biol. Chem. 261 (1986) 12345-12351.

4 ' Levis, W. R., Meeker, H. C., Schuller-Levis, G.,Serson, E., Brennan, P. J. and Schwerer, B. Mycobac-terial carbohydrate-antigens for serological testing ofpatients with leprosy. J. Infect. Dis. 156 (1987) 763-769.

Another helpful advance with significantresults was the construction of a genomiclibrary in bacteriophage Agtl I, which wasdesigned for regulated expression of Al. lep-rac' proteins in Escherichia co/i. 46 So far, liveprotein antigens of Al. leprac have been ex-pressed in E. coil and analyzed in detail. 46 • 47

Cloned segments of A/. leprac DNA fromthe Xgt 11 library have provided DNA forsequencing of two proteins (18 and 65 kDa)from Al. leprae, both of which have beenshown to produce a humoral immune re-sponse subsequent to vaccination with Al.frprat,:48.

4`' work with T-cell clonesin tissue cultures should provide informa-tion on epitopes recognized by T cells onthese and other proteins, allowing for de-tailed immunochemical analysis of proteinsimportant in the immunological interac-tions of AL leprae and the host. DNA-de-rived peptide sequences will provide the re-agents needed to analyze the relationshipsof antigenic peptides and class II major his-tocompatibility complex (MHC II) mole-cules on antigen-presenting cells. Such stud-ies will be very helpful in unraveling themolecular aspects to the triggering of reac-tive T cells in response to Al leprae, im-portant in mediating protective cell-medi-ated immunity.

The use of epitope libraries 50 • 5 ' for themolecular cloning and precise identification

4' Young, R. A., Mehra, V., Sweetser, D., Buchanan,T., Clark-Curtiss, J., Davis, R. W. and Bloom, B. R.Genes for the major protein antigens of the leprosyparasite Mycobacterium leprac. Nature (London) 316(1985) 450-452.

4' Mehra, V., Sweetser, D. and Young, R. A. Efficientmapping of protein antigenic determinants. Proc. Natl.Acad. Sci. U.S.A. 83 (1986) 7013-7017.

48 Mustafa, A. S., Gill, H. K., Nerland, A., Britton,W. J., Mehra, V., Bloom, B. R., Young, R. A. andGodal, T. Human T-cell clones recognize a major Al.leprae protein expressed in E. coli. Nature (London)319 (1986) 63-66.

'" Mustafa, A. S., Oftung, F., Gill, H. K. and Natvig,I. Characteristics of human T-cell clones from BCGand killed M. leprac-vaccinated subjects and tuber-culous patients. Lepr. Rev. 57 Suppl. 2 (1986) 123-130.

'" Scott, J. K. and Smith, G. P. Searching for peptideligands with an epitope library. Science 249 (1990) 386-390.

si Miles, M. A. and Wallace, G. R. Cloning of mi-crobial epitopes relevant for T and B-cells. BehringInstitute Nlitteilungen 88 (1991) 133-141.

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of T-cell and B-cell subsets is the latest ma-jor advance that is likely to have implica-tions throughout the field of immunology.If and when epitope library studies are usedin the study of leprosy, they will provide thefacility to look at millions of short peptidesequences that can be surveyed for their tightbinding to antibodies, receptors or otherbinding proteins. The library consists of avast mixture of filamentous phage clones,each displaying one peptide sequence on thevirion surface. Any peptide that shows af-finity to the protein being investigated canbe purified and propagated using E. coli,and then sequenced by looking at the codingregion in the viral DNA. It will be possibleto investigate the peptide MHC II interac-tion in leprosy and the pairing of a largenumber of epitopes with their functional re-striction elements and, thus, to study T-cellinteractions in great detail.

Molecular biology. Original ideas in sci-ence usually have more far-reaching effectsthan is initially envisaged, often providingnew insight on unresolved questions or al-ternative approaches to circumvent longstanding problems in basic and applied re-search. Such is the outcome of the devel-opment of DNA amplification by the poly-merase chain reaction (PCR) first describedby Mullis in California. 52 His invention wasstunningly simple and yet awesome in itspotential. Research applications of thistechnique of DNA amplification using com-mercially available equipment and reagentshave already transformed molecular biol-ogy."

The major strength of PCR is the abilityof the reaction to produce incredibly largeamounts of specific DNA sequences, usingtwo oligonucleotide primers that hybridizeto opposite strands and flank the region ofinterest in the target DNA. A repetitive se-ries of cycles involving template denatur-ation, primer annealing, and the extensionof the annealed primers by DNA polymer-ase, results in the exponential accumulationof a specific fragment whose termini are de-

" Mullis, K. M. The unusual origin of the polymer-ase chain reaction. Sci. Am. 262 (1990) 36-46.

" Erlich, H. A., ed. PCR Technology. Principles andApplications for DNA Amplification. New York: Stock-ton Press, 1989.

fined by the 5' ends of the primers. Becausethe products of one cycle can serve as tem-plates in the next, the number of DNA cop-ies of the target approximately doubles atevery amplification cycle. Thus 20 cycles ofPCR yield about a million-fold (2 20) am-plification. 54, " With appropriate hybridiza-tion for the detection of the target DNAsequence, PCR has the theoretical capabil-ity ofdetecting one target sequence in a sam-ple preparation. 5 ". 57

The introduction of the thermostableDNA polymerase (Taq polymerase) trans-formed PCR into a simple and robust re-action which can be automated by a thermalcycling device, instead of having to add freshenzyme at every cycle when using the heat-labile E. coil DNA polymerase." The re-action components (template, primers, Taqpolymerase, deoxyribonucleotides, andbuffer) are all easily available, and all canbe assembled within the reaction tube andthe amplification reaction carried out bysimply cycling the temperature. The speci-ficity and yield of the PCR could be in-creased by changing the reaction parame-ters, for example primer, Mg 2 + concentrationas well as the temperature cycling profile.

The specificity of the PCR can be ana-lyzed by carrying out gel electrophoresis onthe product, evaluating the production ofthe target fragment, and comparing this tothe other products of the reaction."' 59 Theinitial PCR method using the heat-labile en-

Oste, C. Polymerase chain reaction. Biotechniques6 (1988) 162-167.

" Innis, M. A., Gelfand, D. H., Snivinsky, J. N. andWhite, T. J., eds. PCR Protocols: .1 Guide to Methodsand Applications. San Diego: Academic Press, 1990.

Saiki, R. K., Gelfand, D. H., Stollel, S., Scharf, S.J., Higuchi, R., Horn, G. T., Mullis, K. B. and Erlich,H. A. Primer-directed enzymatic amplification of DNAwith a thermostable DNA polymerase. Science 239(1988) 487-491.

" Li, H. H., Gyllensten, U. B., Cui, X. F., Saiki, R.K., Erlich, 11. A. and Arnheim, N. Amplification ofDNA sequences in a single human sperm and diploidcells. Nature 335 (1988) 414-417.

" Williams, D. L., Gillis, T. P., Booth, R J., Looker,D. and Watson, J. D. The use of a specific DNA probeand polymerase chain reaction for the detection of My-cobacterium leprae. J. Infect. Dis. 162 (1990)193-200.

" Plikaytis, B. B., Gelber, R. H. and Shinnick, T.M. Rapid and sensitive detection of Mycobacteriumleprae using a nested-primer gene amplification assay.J. Clin. Microbiol. 28 (1990) 1913-1917.

458^ International .Journal of Leprosy^ 1992

zyme was not very specific; although a spe-cific target fragment could be amplified upto a million-fold, most of what was synthe-sized was not the target sequence. By clon-ing a (3-globin amplification reaction andscreening the individual clones with a 0-glo-bin probe to detect the target sequence and,with one of the primers, to detect any am-plified sequence, the specificity of the PCRwas estimated to be — 1%. 59 The use of theTag polymerase not only simplified the PCRprocedure, it significantly increased thespecificity and the overall yield. Taq poly-merase also allows the amplification of muchlonger fragments, up to 10 Kb (with somereduction in efficiency); whereas using theKlenow fragment of E. co/i DNA polymer-ase allows amplification of less than 400base pairs. 6() The higher optimum temper-ature of around 75°C for Taq polymeraseallows for a higher temperature to annealthe primers and extension; this increases theoverall stringency of the procedure andminimizes the extension of mismatchedprimers. This increase in specificity resultsin an improved yield of the amplified targetfragment by reducing the competition forenzymes and primers by nontarget productsat each cycle.

The development ofa highly specific PCRtest is dependent upon the selection sof anappropriate target sequence. 54-56, 61 DNAsequence data from which specific PCR testsare based come from recombinant DNA li-braries for M. leprae62 • 63 and other myco-bacteria. Genetic studies have detailed nu-merous DNA sequences suitable fordeveloping species-specific PCR tests."' 65

''" Scharf, S. J., Horn, G. T. and Erlich, H. A. I)irectcloning sequence analysis of enzymatically amplifiedgenomic sequences. Science 223 (1986) 1076-1078.

" Mullis, K. B. and Faloona, F. A. A specific syn-thesis of DNA in vitro via a polymerase-catalysed chainreaction. Meth. Enzymol. 155 (1987) 335-350.

62 Clark-Curtiss, J. E., Jacobs, W. R., Docherty, M.A., Richie, L. R. and Curtiss, R. Molecular analysis ofDNA and construction of genomic libraries of Myco-bacterium leprae. J. Bacteriol. 161 (1985) 1093-1102.

64 Young, R. A., Mehra, V., Sweetser, D., Buchanan,T., Clark-Curtiss, J., E., Davis, R. W. and Bloom, B.R. Genes for the major protein antigens of the leprosyparasite Mycobacterium leprae. Nature 316 (1985) 450-452.

64 Mehra, V., Sweetser, D. and Young, R. A. Efficientmapping of the protein antigenic determinants. Proc.Natl. Acad. Sci. U.S.A. 83 (1986) 7013-7017.

Eisenach, K. D., Crawford, J. T. and Bates, J. H.

Three well-defined antigenic proteins haveregularly been used so far in PCR. Two ofthe proteins (18 kDa and 36 kDa) were foundexclusively in IL /eprae.° 6-68 while the thirdprotein (65 kDa) has been shown to be ahighly conserved heat-shock protein thoughtto be expressed in all prokaryotes. PCR testshave been developed based on the ampli-fication of species-specific target regions ofthe genes encoding these proteins. Thus,PCR tests do not have to be limited to Al.leprae-specific genes, but can be developedfrom sequences found within a highly con-served gene within the mycobacterial genuscontaining species-specific regions. It is notclear at this point which variation will bemost universally applied to PCR.

Sensitivity requirements of PCR tests todetect AI. leprae designed so far mean thatthey must surpass detection levels achievedby conventional standard microscopic ob-servation (10 4AFB/m1)69 • 70 and maintainspecificity. Ideally, this should be directlyfrom clinical specimens without other ma-nipulations.

Mycobacterial identification tests devel-oped from nucleic-acid hybridization tech-niques preceded PCR; they have becomeimportant tools for the identification of my-cobacterial groups. 71-74 These tests are de-

Repetitive DNA sequences as probes for Mycobacte-rium tuberculosis. J. Clin. Microbiol. 26 (1988) 2240-2245.

6" Booth, R. J., Harris, D. P., Love, J. M. and Wat-son, J. D. Antigenic proteins of A/Ivo/mu-icy/um leprae:complete sequence of the gene for the 18-kDa protein.J. Immunol. 140 (1988) 597-601.

" Lamb, F. I., Singh, N. B. and Colston, M. J. Thespecific 18-kilodalton antigen of Mycobacterium lepraeis present in Mycobacterium habana and functions asa heat shock protein. J. Immunol. 144 (1990) 1922-1925.

''" de Wit, M. Y. L. and Klatser, P. R. Purificationand characterization of a 36 kDa antigen of Mycobac-terium leprae. J. Gen. Microbiol. 134 (1988) 1541-1548.

^" Shepard, C. C. and McRae, D. H. A method forcounting acid-fast bacteria. Int. J. Lepr. 36 (1968) 78-82.

'" Bates, J. H. Diagnosis of tuberculosis. Chest 76(1979) 757-763.

" Gonzalez, R. and Hanna, B. A. Evaluation ofgene-probe DNA hybridization systems for the identifica-tion of Mycobacterium tuberculosis and Mycobacteri-um avium - itztracellulare. Diagn. Microbiol. Infect. Dis.8 (1987) 69-77.

60, 3^ Editorials^ 459

signed for identification of the organismswhen sufficient quantities can be grown invitro, and cannot be used directly for detec-tion from clinical specimens. The Clark-Curtiss group developed a DNA hybridiza-tion assay for the identification of AI. lepracin biological tissues, the theoretical lowerlimit of sensitivity was 4 x 10 3 for detectingthe bacillus. 75 The DNA probe used in thetest contained a repetitive sequence specificto Al. leprac, enhancing the theoretical sen-sitivity. However, tests showed the practicallower limit of sensitivity for detecting Al.leprae in the skin of multibacillary patientswas I x 10 5 .

PCR tests with or without the use of DNAhybridization for quality control are chal-lenging direct DNA hybridization assays forthe detection of mycobacteria producingsimilar levels of specificity with claims ofincreased levels of sensitivity. No study us-ing PCR to date has reported a sensitivitylevel worse that 100 organisms, and claimshave been made for the technique being ableto detect a single AI. leprac bacillus. Mostreports show lower levels of detection in thepresence of prokaryotic 6° or cukaryo-tic 59 ' 6" ' 76 DNA as well as normal, unin-fected human skin biopsies." Even if thedetection limit of PCR proved to be a thou-sand times poorer in clinical use comparedto controlled research laboratory condi-tions, it would still be far more sensitivethan any other available test. The utility of

" Ellner, P. D., Kichn, T. E., Cammarata, R. andHosmer, M. Rapid detection and identification ofpathogenic mycobacteria by combining radiometric andnucleic acid probe methods. J. Clin. Microbiol. 26(1988) 1349-1352.

7 ' Drake, T. A., Herron, R. M., Jr., Hindler, J. A.,Berlin, 0. G. W. and Bruckner, D. A. A DNA probereactivity of Mycobacterium avium complex isolatesfrom patients without AIDS. Diagn. Microbiol. Infect.Dis. 11 (1988) 125-128.

74 Saito, H., Tomioka, H., Soto, K., Tasaka, H., Tsu-kamura, M. Kuzi, F. and Asano, K. Identification andpartial characterization of Mycobacterium avium andMycobacterium intracellulare by using DNA probes. J.Clin. Microbiol. 27 (1989) 994-997.

7 ' Clark-Curtiss, J. E. and Docherty, M. A. A speciesspecific repetitive sequence in Mycobacterium lepraeDNA. J. Infect. Dis 159 (1989) 7-15.

7 ' Hance, A. J., Grandchamp, B., Levy-Frebault, V.,Lecossier, D., Rauzier, J., Bocart, D. and Gicquel, B.Detection and identification of mycobacteria by am-plification of mycobacterial DNA. Mol. Microbiol. 3(1989) 843-849.

these assays for improving current diagnos-tic criteria for leprosy remains undefined.

ConclusionsProblems: criteria for tests to be useful.

Many problems have been unearthed by therapid advances in scientific knowledge ofthe leprosy bacillus in the last few years, andit seems that for every advance there is stilla new obstacle around the corner. At themoment, the full capabilities of most of thesetechniques have only been briefly touchedon, and advances in this area are self fuelingin their possible implications. However spe-cific problems exist, so much so with im-munological tests that it is difficult to seethem as being of much use outside the re-search laboratory. For a new test to be ofany use in the clinical setting, it must bebetter or cheaper than methods currentlyavailable. Apart from very specific and lim-ited applications, the diagnostic value of se-rological tests for leprosy would appear tobe limited. Leprosy diagnosis is usually notdifficult to establish in most cases by thetraditional methods of clinical examinationtogether with skin smears and histopathol-ogy when needed. Indeed, in most cases, thehistological examination oflesions using themicroscope is very sensitive, more so thanmany of the immunological tests so far of-fered. At the moment, sensitivity levels ofimmunological tests may be appropriate forresearch purposes when experimental infec-tions are studied. If one organism can bedetected per microliter of test sample, whenthis is applied to the clinical situation a 70kg patient would have to be infected by 70x 10 6 organisms, assuming erroneously thatthe bacteria were uniformly spread through-out the body before it could be detected (thiscompares with infection by about 10' 3 bac-teria in the far-advanced lepromatous pa-tient). However, currently available testsusing traditional techniques do not give in-formation on the viability of the organismsince dead bacteria will be stained positivetogether with live organisms. Although thisis not a problem in the diagnosis, it is aproblem in the management of patients in-fected with M. leprac resistant to drugs, forexample.

The varied immune responses of patientsinfected with Al. leprae severely limit thevalue of immunological tests. In the study

460^ International Journal of Leprosy^ 1992

be Douglas, et 01. 42 there was no correlationbetween quantitative changes in antibodylevels measured by immunodi (fusion withvalues obtained by ELISA. In individualpatients periods of abnormally high levelsof one class of antibody could be associatedwith normal values for others; this variedwith time and unfortunately did not nec-essarily correlate with antileprosy chemo-therapy. Touw, et a1. 77 also reported ele-vated levels of immunoglobulins with nocorrelation with bacillary load or other clin-ical parameters in patients they studied.

The ELISA for PG L-1 has a sensitivity ofonly about 30% for paucibacillary leprosy,which does not make it a useful field testfor the early stages of paucibacillary leprosy.Even if the specificity were 98%, and evenin a population with a theoretical preva-lence rate of 1% (ten times higher than mostendemic countries), for every thousand testscarried out PGL-I serology would detect only3 of the 10 patients who actually has leprosyif the test was used "blind" with no addi-tional clinical information. At the same time,the test would "identify" 20 people withoutleprosy as being infected with Al. leprae.

Antigenic analysis of proteins is only thefirst step in investigating the complex im-mune interactions subsequent to the infec-tion of humans with M. leprae. Althoughimmunogenic proteins might provide suit-able components for the development ofvaccines against leprosy, their relevance tothe immune response remains unanswered.Detailed studies are needed to define the B-and T-cell stimulatory capacity of thesemolecules. This will require large amountsof purified proteins which are not obtain-able by extraction from native purified ba-cilli.

The extraction of DNA from Al. leprae isnotoriously difficult since the cell is refrac-tory to most cell lysis techniques. It has beenestimated that only 20% of the genomic ca-pability of a microorganism is representedwhen applying detailed phenotypic analy-

Touw, J., Langcndijk, E., Stoner, G. and Belchu,A. Humoral immunity in leprosy: immunoglobulin Gand M antibody responses to Mycobacterium leprae inrelation to various disease patterns. Infect. Immun. 36(1982) 885-892.

ses. Thus, major areas of the genome, pos-sibly encoding important functional orregulatory genes not demonstrable as quan-tifiable phenotypic characteristics, may goundetected. Furthermore, when extractingDNA from M. leprae the number of DNAcontaining, and thus potentially viable, or-ganisms represents only a small fraction ofthe total sample. 78

The major problem with PCR at the mo-ment is the occurrence of false-positive re-actions due to contamination of samples orreagents with DNA. This is not usually dueto biological contamination with test organ-isms, but is often due to the phenomenonof product carry over due to minute quan-tities of DNA created in previous stagescontaminating the reaction mix, and theyare amplified by the PCR to give a false-positive result or raise control backgroundlevels, reducing sensitivity. To avoid theproblems of contamination, strict aseptictechniques must be used in handling PCRreagents and DNA samples. A laboratorydesign implementing physical separation ofcertain procedural aspects of PCR is essen-tial. Apart from the use of strict asepsis,specialized equipment and regular cleaningin detergents and hydrochloric acid are nec-essary. Negative controls, excluding analyteDNA, must be tested alongside the un-known sample being targeted to monitorcontamination of buffers and PCR reagents.

Despite contamination being a problemin some laboratories, this technique is stillin its early days. There is already a list ofrelatively simple measures to reduce the riskof false-positives, with new ideas being pro-posed all the time. 29• 61• 62, 79, 8() A proceduralvariation of the standard PCR, called nestedPCR, has been suggested as a way to reducefalse-positive results produced by the con-tamination of samples by the products ofprevious amplifications. 8 ' In the nested PCR

78 Kwok, S. and Higuchi, R. Avoiding false positivesin PCR. Nature 339 (1989) 237-238.

79 Cone, R. W., Hobson, A. C., Huang, M-L. W. andFairfax, M. R. Polymerase chain reaction decontami-nation: the wipe test. Lancet 2 (1990) 686.

8" Furrer, B., Candrian, U., Wieland, P. and Luthy,J. Improving PCR efficiency. Nature 346 (1990) 324.

8 Kitchin, P. A., Szotyori, Z., Fromhole, C. and Al-mond, N. Avoidance of false positives. Nature 344(1990) 201.

60, 3^ Editorials^ 461

technique, two sets of primers are used foramplification. The first pair of primers (out-side primers) amplifies a portion of the ge-'tome, and the nested primers (inside prim-ers) direct the amplification of sequencescontaining the product. The number of cy-cles required for each pair of primers is muchsmaller (15-25 cycles) than that requiredfor high levels of sensitivity using a singleset of primers (30-50 cycles), so that back-ground or nonspecific amplification shouldbe reduced."

Quantifying the results of PCR is verydifficult at the moment. Although the finalproduct of a PCR amplification can be as-sessed quantitatively, relating this to the ab-solute number of microorganisms presentin a given sample can be difficult. Quanti-tative PCR tests have been developed andrely on either internal or external controlsfor standardization. It is difficult to compareamplification between unknown concentra-tions of sample DNA and known concen-trations of control DNA. 82-84 An error canalso be introduced in reading the results,since it is possible to get signals on the gelelectrophoresis from segments of DNA thatare of the same length as the target. Thiscan be prevented by using specific radiola-beled DNA hybridization probes, a prob-lem in the study of other mycobacteria. Inreviewing the literature many studies usingPCR do not use any methods of internalcontrol, such as this use of specific probes.

It is likely that free DNA from Al. lepraeis present in infected tissues, and this is like-ly to confound standardization of PCR tests.Portions of DNA at varying degrees of dis-integration will be capable of providing suit-able template DNA for PCR. It has beenshown" that the removal of M. leprae fromfresh, human biopsy homogenates by cen-trifugation can yield bacilli-free superna-

Dickover, R. E., Donovan, R. M., Goldstein, E.,Dandekar, S., Bush, C. E. and Carlson, J. R. Quanti-tation of human immunodeficiency virus DNA by us-ing the polymerase chain reaction. J. Clin. Microbiol.28 (1990) 2130-2133.

" Abbott, M. A., Poiesz, B. J., Byrne, B. C., Kwok,S., Sninsky, J. and Ehrlich, G. D. Enzymatic gene am-plification: qualitative and quantitative methods fordetecting proviral DNA amplified in vitro. J. Infect.Dis. 158 (1988) 1158-1169.

" Gillis, T. P. and Williams, D. L. Polymerase chainreaction and leprosy. Int. J. Lepr. 59 (1991) 311-316.

tants capable of generating strong PCR sig-nals. This is further evidence that PCR maynot he a useful test for assessing the viabilityofAl. leprae, and the development of mRNAPCR is likely to be the solution to this prob-lem.

The transfer of PCR technology from theresearch laboratory to the clinical diagnosticlaboratory is still a long way off. The costof using PCR is likely to fall progressively.Before the time arrives that it could be con-sidered economically viable for use in thefield, several technical obstacles will haveto be overcome, including test formats, re-ducing contamination and the use of simplenonradioactive binding assays. Also, the costis quite likely to fall in the future as thepatent runs out and open commercial com-petition starts. At the moment the patent isheld by one company, Perkin-Elmer-Cetus.Already a PCR kit is in use in the U.S.A.,marketed by IDEXX for the detection ofM. paratuberculosis. 85 It is used to diagnoseJohne's disease in cattle by the disease ref-erence laboratory.

Future possibilities: research toys or clin-ical tools? With the emergence of ever-in-creasing numbers of dapsone-resistant Al.leprae infections, the importance of moni-toring chemotherapy has been established.Chemotherapy has been further compro-mised by poor compliance, especially bylight-skinned patients treated with clofaz-imine, the necessity of daily treatment, andby toxic side effects. Newer drugs, such asrifampin, may alleviate some of these prob-lems, but the need for careful monitoring isstill very important. A sensitive assay whichcan detect quantitatively and specifically ac-tive, viable Al. teprae could reflect the bac-terial load and, hence, the clinical progres-sion of the disease.

Studies have been carried out usingchanges in patients' sera42 77 using enzyme-linked immunosorbent assays of immuno-globulin levels in response to specific anti-gens (especially PGL-I) and whole cellantigens of Al. leprae in patients treated with

" Vary, P. H., Anderson, P. R., Green, E., Hermon-Taylor, J. and McFadden, J. J. Use of highly specificDNA probes and the polymerase chain reaction to de-tect Mycobacterium paratuberculosis in Johne's dis-ease. J. Clin. Microbiol. 28 (1990) 933-937.

462^ International Journal of Leprosy^ 1992

various antimycobacterial agents. Many ofthese studies showed potential in the ap-plication of ELISA techniques to the mon-itoring of individual patient's antibody re-sponses during the course of treatment. Theymay prove to be useful in measuring theeffectiveness of chemotherapy. This cer-tainly is a tremendous advance on previousmethods of analyzing material for viabilitystudies that involved the inoculation of aconsiderable number of mice and followingthem up for a least a year, as in the pro-portional bactericidal test of Colston, et a/. 4

A rapid and sensitive technique for as-sessing growth and metabolism has been de-scribed by Franzblau, 8" but it does not seemto have been widely used. This test (BAC-TEC 460 test) would appear to be particu-larly suitable for assessing the susceptibilityof M. /clime to antimicrobial drugs in vitroand can give results in 1 week. The systemuses a radiorespirometric method of as-sessing cell metabolism. Nude-mouse-de-rived Al. leprae are grown in a ' 4C palmiticacid labeled medium and the production of' 4CO, is measured by using a Buddemeyer-type detection device. The measurement ofCO, gives the test quantitative capabilitiesand Franzblau suggests that since activitycan he readily detected with 10" bacilli, thetest has potential for use in clinical suscep-tibility testing. PCR techniques will prob-ably eventually make this area of study evenmore simple and speedier. Immunologicaltests are fraught with problems and, as yet,are very disappointing since the assay ofPGL-I is highly specific but unfortunatelyits sensitivity is very poor at the moment.

The development of simple, sensitivePCR tests to detect Al. leprae using specificDNA sequences as primers is a major de-velopment in leprosy research. Immediateapplications with the existing technologymay include epidemiological studies to de-termine the distribution of M. leprae in thevarious subpopulations in endemic areasand within the surrounding environment. 84

PCR could give useful information on thetransmission of the disease and the natureof infective reservoirs. Studies are needed

Franzblau, S. G. Drug susceptibility testing ofMycobacterium leprae in the BACTEC 460 system.Antimicrob. Agents Chemother. 33 (1989) 2115-2117.

in order to establish the accuracy and rele-vance of information obtained by new mo-lecular biological techniques correlated toclinical disease.

Other immediate applications for PCRmay include complementing current clini-cal and histopathological diagnostic criteriafor leprosy if useful information from PCRcan be obtained on the relationship of tar-geted DNA sequences in PCR to the currentclassification of leprosy, as proposed byRidley and Jopling, and currently used forclinical and research purposes." This willbe particularly useful when signs and symp-toms are equivocal, such as often is the casein indeterminate leprosy and in suspectedcases where no AFB can be detected in tis-sue samples. PCR could also he used in thearchival analysis of paraffin-embeddedsamples, since samples of this type may besuitable for PCR87 ." but unsuitable for mi-crobiological assessment by conventionalmeans. Preliminary studies indicate that notall fixatives are good for maintaining DNAintegrity for PCR based on the Al. leprae18-kDa protein gene. 84 Studies still remainto be performed to define the appropriatefixatives for clinical tissues so that both his-tological analysis and testing by PCR canbe performed. A large multicenter study us-ing the PCR based on the selective ampli-fication of a 530-bp fragment of the gene,encoding the proline-rich antigen of /1/.rue. was applied on sections of variouslyfixed or frozen biopsy samples from patientswith leprosy resident in Malawi, Pakistan,Thailand, Belgium and The Netherlands. Asimple procedure for the extraction of DNAfrom Al. leprae in clinical specimens whichprovided a suitable template DNA for am-plification was developed. Previously, DNAextraction involved purification and anelaborate enzymatic treatment in manystages. The simplified method uses the ex-

Shibata, D., Martin, W. J. and Arnheim, N. Anal-ysis of DNA sequences in forty-year-old paraffin-em-bedded thin-tissue sections: a bridge between molec-ular biology and classical histology. Cancer Res. 48(1988) 4564-4566.

' Lai-Goldman, M., Lai, E. and Grody, W. W. De-tection of human immunodeficiency virus (HIV) in-fection in formalin-fixed, paraffin-embedded tissues byDNA amplification. Nucl. Acids Res. 16 (1988) 8191.

60, 3^ Editorials^ 463

traction of DNA from clinical samples withthe enzyme proteinase K and the detergentTween without any further DNA purifica-tion. This simplified technique showed noloss in amplification efficacy with subse-quent PCR using heat-stable Tad polymer-ase for the specific detection of Al. lepraeY'Simpler techniques may prove to be equallyas effective: boiling has been successfullyused to extract DNA from other mycobac-teria in clinical samples.

Further, if PCR is to be a useful tool inthe management of leprosy correct proto-cols for the handling of tissues in the fieldwill need to be established. No doubt therewill he a rush of clinical testing kits on themarket in the not too distant future. Butbefore these techniques can be usefully ap-plied to the field, preliminary tests on clin-ical material and cost effective analysis oftheir value as leprosy control tools must beestablished.

Studies need to be performed to deter-mine the relationship between the viabilityof AI. leprac and PCR. If PCR results canbe shown to reflect the viability status of ;ILleprac, then the potential of PCR for mon-itoring antileprosy drug therapy and to dif-ferentiate reaction from relapse of the dis-ease in paucibacillary patients may be animportant advance in the management ofthe disease. The monitoring of more highlylabile bacterial components may fulfill thisrole better. As has already been mentioned,DNA is a very stable and resistant moleculeand despite even fixation with Formalin canbe detected by PCR. It is not likely to be agood indicator of viability since it obviouslyis present in dead bacilli. The monitoringof highly labile components of bacterial ac-tivity will probably be far more sensitive inestablishing the viability of Al. lep•ac inclinical biopsy specimens and, thus, morereliable in assessing the response of patientsto treatment. Many workers are looking intothe possibility of using components asso-ciated with transcription and translation, forexample, mRNA. This is particularly ap-propriate since the half-life of mRNA is ten

deWit, M. Y., Faber, W. R., Kreig, W. R. Appli-cation of a polymerase chain reaction for the detectionof Ilycobacterium leprac in skin tissues. J. Clin. Mi-crobiol. 29 (1991) 906-910.

minutes at the most in the prokaryotic cell.In using mRNA as the target for analysis, acomplementary strand of DNA is made byusing reverse transcriptase before amplifi-cation using the PCR.

One of the special features that PCRshould bring to leprosy research is the levelof accuracy in diagnosis. Since A/. leprac-specific sequences of DNA have been char-acterized, these can be used to differentiateAl. leprac from other species. Tests lookingfor AFB in biopsy specimens as used pre-viously in the clinical diagnosis of difficultor ambiguous cases had no specificity.Should stable isolates containing specificDNA or RNA sequences be characterized,as they have been for other human patho-gens, 9°. 9 ' PCR tests could be developed andused for taxonomic purposes with realisticpossibilities in the study of disease trans-mission.

One of the biggest claims made for thefuture of PCR is that it could be used indetecting drug resistance. It is conceivablethat sequences related to drug resistance orsensitivity could be characterized and usedfor developing PCR tests for use in clinicallymonitoring resistance in individual pa-tients. For PCR to be used for this requiresidentification and sequencing of drug-resis-tant genes and, also, that these genes havespecific sequences suitable for primers. Forinstance, it is known that a point mutationin the gyrase gene gives resistance to thequinolones, but this is probably not the onlymechanism since changes in the cell mem-brane, for instance, could confer drug resis-tance. With such a small percentage of thebacterial genome being expressed, it may besome time before the appropriate target se-quences for PCR can be found.

Already immunological tests have beenapplied to this problem but PCR shouldoffer the potential for greater sensitivity.`''

"" Olive, D. M., Atta, A. I. and Setti, S. K. Detectionof toxigenic Eschcrichiacoli using biotin-labelled DNAprobes following enzymatic amplification of the heatlabile toxin gene. Mol. Cell. Probes 2 (1988) 47-57.

"' Kashani-Sabet, M., Rossi, J. J., Lu, Y., Ma, J. X.,Chen, J., Miyachi, H. and Scanlon, K. J. Detection ofdrug resistance in human tumors by in vitro enzymaticamplification. Cancer Res. 48 (1984) 5775-5778.

Melsom, R., Harbor, M. and Naafs, B. Class spe-cific anti-Abwhacterizon leprac antibody assay in lep-

464^ International Journal of Leprosy^ 1992

Combining a PCR test for drug resistancewith an AI. leprae-specific test may providesimultaneous analysis of pathogen identi-fication and drug sensitivity testing directlyfrom infected host tissues. New approachesare needed for studying larger areas of allbacterial genomes and particularly noncul-tivable bacteria, such as AI. leprae. Analysisof DNA restriction fragments after restric-tion endonuclease digestion of genomicDNA and the related techniques of frag-ment length polymorphism analysis holdpromise. As a more complete understand-ing of the M. leprae genome and its relatedfunctional capacity develops, it is antici-pated that most areas of study related toleprosy will be enriched and, thereby, ad-vance the understanding of Al. leprae, itspathogenicity, and the response of the hu-man to its infection.

It is difficult to say with any certainty atall that any of the sensitive techniques re-viewed in this essay will become part of theroutine clinical management of leprosy, butthey certainly seem to address many of theproblems with the study of the disease. In-deed, modern techniques are very sensitive,very useful, and are beginning to be, usedmore widely as research tools. Serologicaltests based on the detection of the Al. leprae-specific antigen PGL-I have been in use forsome time. A Japanese firm has even de-veloped a kit to detect this antibody. Theybelieve that PGL-I serology could clinch thediagnosis in suspected leprosy, detect sub-clinical infection with M. leprae, and pos-sibly indicate the disease outcome in indi-viduals.

Things are not so simple in the field, andexpensive serological tests will probably beof limited use. In the vast majority of lep-rosy cases, is not difficult to diagnose by the

romatous leprosy (BL-LL) patients during their firsttwo to four years of DDS treatment. Int. J. Lepr. 50(1985) 271-281.

traditional clinical examination, with orwithout skin smears and histopathology. Se-rology for leprosy therefore has a limiteddiagnostic value; furthermore PGL-I serol-ogy has a low level of sensitivity of around30% (i.e., 70% false negatives) for pauci-bacillary leprosy.

It can be argued that the treatment of lep-rosy is no longer a scientific problem but alogistical one. Antibacterial chemotherapyfor leprosy, in general, has been both avail-able and adequate since the early 1940s."The problems in disease control have comefrom inadequacies in health care deliverysystems due to the inherent mountainousobstacles with the disease being endemic insome of the world's most geographically re-mote and economically poor nations. It isalso a feature of the disease that it carries ahuge social stigma which results in poor uti-lization of health care resources that are pro-vided, since patients do not want to be la-beled with a condition unacceptable to theirculture.

After all, leprosy is treatable. The devel-opment of resistance to drugs, especiallydapsone, is a worrying problem, but modernsensitive tests will probably play a very im-portant role in the field for the managementof this. They are already proving invaluablein the research laboratory, but if they are tobe of any use at all in the diagnostic micro-biological laboratory they must be easy andcheap to use. Therefore, they must be rel-evant to the areas in which leprosy is en-demic. To steal a phrase from the marketingworld, they must be "user friendly." Themost likely users need sensitive tests thatare cheap, easy to use, and simple forscreening vast numbers of patients.

—Peter R. Ayliffe

" Faget, G. H., Pogge, R. C., Johansen, F. A., Dinan,J. F., Prejean, B. M. and Eccles, C. G. The Promintreatment of leprosy: a progress report. Public HealthRep. 58 (1943) 1729-1741.