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INTERNATIONAL JOURNAL OF LEPROSY^ Volume 61, Number 4

Printed in the

CURRENT LITERATURE

This department carries selected abstracts of articles published in current med-ical journals dealing with leprosy and other mycobacterial diseases.

Chemotherapy

Chong, P. Y. and Ti, T. K. Severe abdominalpain in low dosage clofazimine. Pathol-ogy 25 (1993) 24-26.

We describe a 41-year-old leprosy patienttreated for 10 years with clofazimine whounderwent laparotomy for severe abdomi-nal pain. At surgery, the only significantfindings were that the celiac lymph nodeswere enlarged and stained purplish-black aswere the omentum and the intraperitonealfat. No other cause of abdominal pain wasidentified. On histological examination,reddish-purple crystals were identified atfrozen section but not in the paraffin sec-tions. —Authors' Abstract

Edstein, M. D. and Rieckmann, K. H. Lackof effect of proguanil on the pharmaco-kinetics of dapsone in healthy volunteers.Chemotherapy 39 (1993) 235-241.

The multiple-dose kinetics of dapsone(DDS) and its principal metabolite mono-acetyldapsone (MADDS) were determinedin six healthy volunteers after daily admin-istration of low-dose dapsone (10 mg).Comparison with a previous study involv-ing the same volunteers on a daily regimenof proguanil (200 mg) plus dapsone (10 mg)revealed no statistically significant differ-ences in the maximum plasma concentra-tions, area under the plasma drug concen-tration curves, and elimination half-lives ofboth DDS and MADDS in the presence ofproguanil. Although these findings suggestthat proguanil does not alter the pharma-cokinetics of DDS and MADDS, the pos-sibility that proguanil affects the dispositionof hydroxylated metabolites of dapsone,which appear to mediate dapsone toxicity,cannot be excluded.— Authors' Abstract

Gonzalez, A. B., Maestre, J. L., HernAndez,0., Columbie, Y., Atrio, N., Martin, M.,Fermindez, A. M. and Rodriguez, J. Sur-

vey for secondary dapsone and rifampicinresistance in Cuba. Lepr. Rev. 64 (1993)128-135.

A total of 1211 Cuban multibacillary lep-rosy patients treated for at least 5 years wereclinically and bacteriologically examined.They were being treated according to a two-phase monotherapy regimen with RMP firstand DADDS afterward. On skin-smear ex-amination 50 patients were found positive,of which 9 showed a BI of 3+ or higher atany site. With regard to the clinical status,the only cases found with clinical signs ofrelapse were 5 out of 7 long-standing pa-tients with BI of 4+ and 5+. A sixth patientof this high BI group who showed good clin-ical condition, except for a heavy infiltra-tion in both earlobes, was receiving a secondRMP course when examined and biopsiedfor this research. These 9 patients werebiopsied and susceptibility tests to RMP andDDS performed. The results showed that in1 case the Mycobacterium leprae were re-sistant to both drugs; the organisms from 2other patients were susceptible to RMP butlow-grade resistant to DDS. Those from an-other patient were susceptible to RMP andfully resistant to DDS. In 3 other cases thebacilli did not multiply in any of the micebut 1 of these strains was from the patienttaking a second RMP course, and, therefore,this strain might also be susceptible to RMPand resistant to DDS. In the last 2 casesmultiplication was only observed in 2 of thecontrols and in 1 of the 0.0001% DDS-treat-ed mice; therefore, these experiments werenot conclusive, and the AFB recovered wereinoculated into fresh mice to repeat the testsbut these failed to multiply.— Authors'Summary

Ji, B. H., Jamet, P., Perani, E. G., Bobin,P. and Grosset, J.-H. Powerful bacteri-cidal activities of clarithromycin and

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648^ International Journal of Leprosy^ 1993

minocycline against Mycobacterium lep-rae in lepromatous leprosy. J. Infect. Dis.168 (1993) 188-190.

Thirty-six patients with newly diagnosedlepromatous leprosy were allocated ran-domly to three groups and treated for 56days with minocycline (100 mg daily), clar-ithromycin (500 mg daily), or clarithro-mycin (500 mg) plus minocycline (100 mgdaily). All groups had rapid and remarkableclinical improvement and significant de-cline of the bacterial and morphologic in-dices in skin smears during treatment. Morethan 99% and > 99.9% of the viable My-cobacterium leprae had been killed by 28and 56 days of treatment, respectively, asmeasured by inoculation of organisms re-covered from skin samples, taken before andduring treatment, into the foot pads of im-munocompetent and nude mice. Clinicalimprovement and bactericidal activity didnot differ significantly among the threegroups. Adverse reactions were rare andmild, and no laboratory abnormality wasdetected during the trial. Both clarithro-mycin and minocycline displayed powerfulbactericidal activities against Al. leprae inleprosy patients, and may be considered im-portant components of new multidrug reg-imens for the treatment of multibacillaryleprosy. — Authors' Abstract

Lim, J. T.-E. and Tan, T. Efficacy and safetyof multidrug therapy in paucibacillaryleprosy in Singapore. Lepr. Rev. 64 (1993)136-142.

A total of 49 patients with pauciba.cillaryleprosy (PB) who completed multidrug ther-apy (MDT) between 1985 and 1990 wereanalyzed retrospectively for efficacy andcomplications; 20 (40.8%) patients had bor-derline-tuberculoid (BT), 13 (26.5%) hadtuberculoid (TT), 1 (2.1%) had indetermi-nate (I), and 15 (30.0%) had pure neural (N)leprosy; 26 patients (76.5% of 34 non-neuralleprosy) were skin biopsied for histologicalcure before MDT was stopped. Of these 26patients, 19 had histological clearance at 6months while the remaining 7 cleared be-yond 1 year (18-36 months). The remain-ing 8 non-neural patients who refused re-biopsy had MDT for 6-8 months and theMDT was stopped when there was clinicalclearance. Of the 15 neural (N) leprosy pa-

tients, 11 were given MDT for 6 monthswhile the rest had 12-18 months of treat-ment; 1 patient with neural leprosy, whowas treated for 6 months, relapsed with BTleprosy 18 months post-treatment. Therewere few complications among the 49 pa-tients-4 (8.2%) patients developed reac-tion to dapsone, 1 (2.0%) had the dapsonesyndrome, 2 (4.1%) had hemolytic anemiaand 1 (2.0%) had dapsone hepatitis; 7(14.3%) patients had type 1 reaction.—Au-thors' Summary

Poitineau, Y., Barthelemy, J., Rouby, D.,Fauron, P., Barran, P. and Beligon, C.Fatal poisoning by rifampicin— a case re-port. Therapie 48 (1993) 271-273.

Successful suicide attempts by rifampinare not commonly reported in literature.Fatal cases and mechanisms of death aremost of the time unexplained. We report asuicidal case in a 33-year-old man with fatalcourse occurring 27 hr after acute overdos-age with 15 g rifampin. Criteria of prog-nostic value are discussed: clinical signs, inparticular the red man syndrome, and bi-ological data. None of them allows us toprevent fatal issue, but since cardiac arrestof unknown origin may rapidly occur, ad-mission in intensive care must be carriedout promptly with a total dose absorbed of12 g and/or evident clinical signs. — Au-thors' Abstract

Singh, R. P., Tiwari, V. D. and Chattopa-dhyay, S. P. Comparative study of shortterm results in two multidrug regimens inmultibacillary leprosy. Indian J. Lepr. 65(1993) 173-180.

Thirty lepromatous and borderline lep-romatous leprosy patients were treated withmultidrug therapy in an open trial. Fifteenof them received the standard WHO mul-tidrug regimen, i.e., rifampin 600 mg andclofazimine 300 mg monthly, supervised,and dapsone 100 mg daily and clofazimine100 mg on alternate days as self adminis-tered; the other 15 received a modified mul-tidrug therapy regimen comprising of rifam-pin 600 mg, clofazimine 100 mg anddapsone 100 mg daily for 21 days as sug-gested by the Indian Association of Leprolo-gists, followed by the standard WHO regi-men. The observation period was 6 months.

61, 4^ Current Literature^ 649

Clinical, bacteriological, histological andimmunological parameters were studied.The fall in morphological index was muchfaster in patients receiving the modifiedmultidrug therapy regimen compared tothose receiving the standard WHO regimen.Otherwise, there was no difference betweenthe two groups of patients. Five patientsdeveloped type 1 (upgrading) reaction, withone developing ulnar nerve paralysis. Nountoward effects of drugs were noted in thestudy subjects except for darkening of skincolor of all the patients.— Authors' Abstract

Thomas, A., Joseph, P. and Prabhakar, R."Flu" syndrome associated with othersystemic manifestations with once amonth rifampicin in the treatment ofmultibacillary leprosy. Indian J. Lepr. 65(1993) 219-224.

Rifampin, a potent bactericidal drug, hasbecome an essential component of multi-drug treatment regimens for tuberculosis andleprosy. One of the adverse reactions de-scribed following twice weekly or onceweekly administration of rifampin is the"flu" syndrome. This syndrome is immu-nological in nature and is often associatedwith the presence of circulating rifampin-dependent antibodies. Initially it wasthought to be uncommon with longer in-tervals between doses of the drug, i.e., oncea month administration, as advocated in thechemotherapy of leprosy. However, there

have been a few reports about the occur-rence of "flu" syndrome with a regimencontaining rifampin once a month. We re-port here three cases of leprosy developing"flu" syndrome, associated with other sys-temic manifestations, during the monthlyphase of their treatment. — Authors' Ab-stract

Venkatesan, K., Chauhan, S. L., Girdhar,A. and Girdhar, B. K. Bioavailability ofdapsone on oral administration of Dap-somine ® —a comparative evaluation. In-dian J. Lepr. 65 (1993) 157-161.

This study describes a comparative eval-uation of dapsone kinetics in humans onadministration of Dapsomine ® , a capsulecontaining dapsone 100 mg dispersed in anoily base suspension of clofazimine 50 mg.Seven untreated lepromatous leprosy pa-tients were given one capsule of Dapso-mine ® a day for 7 days and the pharma-cokinetics parameters in this group werecompared with those from another group ofseven patients who received dapsone 100mg and clofazimine 50 mg separately. Therewere no statistically significant differencesin parameters such as peak dapsone plasmaconcentration (C„,„), basal plasma level(C,,), time to peak level (t„,„), absorptionhalf-life (t,,a), elimination half-life (t„,(3) andareas under plasma concentration-timecurves (AUCo,„ and AUC0_,, h) between thetwo groups.— Authors' Abstract

Clinical Sciences

Avelleira, J. C. R., Vianna, F. R., Coutinho,R. B., Marques Boechat, A. and Gomesde Andrade, V. R. [Distribution of singlecutaneous lesions in paucibacillary lep-rosy.] Acta Leprol. 8 (1993) 127-131. (inFrench)

In this paper the authors study the sitesof single lesions in 317 paucibacillary pa-tients registered at the outpatient units ofthe CMS Jorge Saldanha and the CurupaitiState Hospital in Rio de Janeiro, Brazil. Thepreferential sites of lesions in the populationstudied, their relation with age and sex and

factors likely to influence their distributionare discussed. The findings are comparedwith other similar studies performed in Asiaand Africa. — Authors' English Abstract

Girdhar, B. K., Girdhar, A., Chauhan, S. L.,Malaviya, G. N., Husain, S. and Mu-kherjee, A. Borderline tuberculoid re-lapse in lepromatous leprosy. Lepr. Rev.64 (1993) 157-163.

We report details of two patients who hadbeen treated for a long time by monotherapyand who had remained smear negative for


over 10 years found to have relapsed withborderline-tuberculoid (BT) leprosy.—Au-thors' Summary

Husain, A., Husain, S., Malaviya, G. N. andBahadur, R. R. Myiasis in leprosy. ActaLeprol. 8 (1993) 137-141.

During the 1989-1991 period, leprosypatients with various ulcers attending thesurgical outpatient department at CentralJALMA Institute for Leprosy, Agra, India,were seen. Of these, 64 cases were found tobe infested with maggots. Live maggots werecollected in all cases from different sites, i.e.,nasal cavity, hand, great toe and second toe.It was possible to rear the maggots into fliesin 53 out of 64 cases. In 11 cases the maggotsdid not survive and died in the early partof their life cycle. Four different types offlies were identified: Sarcophaga haemor-rhoidalis, Chrt ,somya befziana, Callitrogaamericana and Musca domestica. — Au-thors' Summary

Jayakumar, J., Aschhoff, M. and Gomathi,A. Reaction in borderline tuberculoidleprosy presenting with multiple subcu-taneous nodules. Indian J. Lepr. 65 (1993)239-242.

Reaction in borderline leprosy usuallymanifests with exacerbation of existing le-sions, or with the appearance of new skinlesions, or both. The skin patches becomemore erythematous with distinct edges.There is edema of the lesions and the ex-tremities; even macules become plaques.The new lesions in the skin usually resemblethe pre-existing lesions. In this paper werecord the sudden appearance of numerousfairly large subcutaneous nodules as•a man-ifestation of (upgrading, type 1) reversal re-action in a patient with borderline tuber-culoid leprosy. —Authors' Abstract

Jayakumar, J., Aschhoff, M. and Renuka,G. Mycetomas in leprosy. Indian J. Lepr.65 (1993) 229-233.

Neuropathic plantar ulcer is one of themost common complications of leprosy andis seen in 10% to 15% of leprosy patients.Even with such high prevalence of plantarulcers, mycetomas of the feet in leprosy pa-tients seem to be very rare. Ordinarily my-

cctomas are produced following the intro-duction of these exogenous pathogenicorganisms into the tissues as a result of trau-ma. Yet there seems to be hardly any as-sociation between anesthetic feet in leprosyso prone to trauma and mycetomas. Lep-rosy is a chronic granulomatous disease andis known to be associated with other gran-ulomatous infections like tuberculosis andsyphilis. Recently, there has been a reportof a case of chromoblastomycosis in a pa-tient with borderline tuberculoid leprosy.We report here two instances of leprosy pa-tients having mycetoma of the foot.—Au-thors' Abstract

Mafoyane, N. A., Jacyk, W. K. and Lotz,B. P. Primary neuritic leprosy in a blackSouth African. Lepr. Rev. 63 (1992) 277-281.

A 37-year-old black South African manwas admitted to Kalafong Hospital (SouthAfrica) in November 1989 with an 18-month history of pain in both hands fol-lowed by weakness and loss of sensation.Both his father and sister had leprosy butthe patient himself denied ever having skinlesions. Clinical, neurophysiological andhistopathological examination led to a di-agnosis of primary neuritic leprosy, with all4 limbs involved and 14 nerves affected.Selective involvement of the facial nervebranches, with normal blink reflexes, wasobserved. A biopsy of the rural nerve sug-gested borderline leprosy. The authors com-ment that cases of primary neuritic leprosy(without skin lesions) are not infrequent onthe Indian subcontinent. However, they areunaware of previous formal reports of suchcases in black Africans; they cite a personalcommunication from R. E. Pfalzgraff stat-ing that neuritic leprosy does occur in Africabut in < 1% of leprosy cases (compared with18% of all leprosy cases in Chingleput Dis-trict in India). —C. A. Brown (Trop. Dis.Bull.)

Pavithran, K. Shoreline nails following typeII lepra reaction. Indian J. Lepr. 65 (1993)225-227.

The growing portion of the nail is the nailmatrix, and any disturbance in the growthof the cells of the nail matrix results in mor-phological changes in the nail plate. In lep-


rosy the growth of the nails may be affectedcausing them to become curved, brittle andthin. They may also appear dry, lusterless,shrunken, narrowed and longitudinallyridged. Singh, et al. reported Terry's nailsin a patient with BT leprosy and Patki andMehta noted Beau's illness on the nails ofa patient with BL leprosy who had associ-ated dapsone-induced erythroderma. Wereport a patient with lepromatous leprosywho developed "Shoreline nails" followingtype 2 lepra reaction.— Author's Abstract

Rao, S. P. and Bharambe, M. S. Electro-neuro-physiological studies in early tu-berculoid leprosy. Indian J. Lepr. 65(1993) 181-187.

Electrophysiological studies were carriedout in early tuberculoid type of leprosy inorder to study their utility in detecting nervedamage before the onset of obvious func-tional deficit. Fifty-three cases showing onemixed nerve thickening in one limb wereselected. Nerve conduction studies (bothmotor and sensory) were done using the sin-gle-blind technique. There was no statisti-

cally significant difference between the find-ings obtained from clinically thickened andnonthickened nerves. There was also no di-rect relationship between clinical sensorydeficit and electrophysiological abnormali-ty. Clinical motor power loss was well cor-related with electrophysiological abnormal-ities.— Authors' Abstract

Reddy, B. S. N., Sheriff, M. 0., Garg, B. R.and Ratnakar, C. Solitary neurofibromamimicking nerve abscess of leprosy. In-dian J. Lepr. 65 (1993) 235-238.

Pathological thickening of a peripheralnerve in an area endemic for leprosy shouldmake one suspect this disease as the primarycause, since the other etiological factors arerare. Nevertheless, it is necessary that oneshould also keep in mind these causes in thedifferential diagnosis. We report here an in-teresting instance of solitary neurofibromaarising from the left lateral popliteal (com-mon peroneal) nerve clinically simulatingpure neuritic leprosy with nerve abscess ina 15-year-old girl.— Authors' Abtract

Immuno-Pathology

Chen, Z.-L., Tang, Q.-G., Wang, Z.-M. andChen, J. Pilot study to determine ac-ceptability and ability of heat-killed My-cobacterium leprae plus BCG (HKML +BCG) vaccine to induce skin test conver-sion. Lepr. Rev. 64 (1993) 117-127.

Although local reactions, including ery-thema, induration and ulcers, appeared inevery patient after the injection of the com-bined heat-killed Mycobacterium leprae(HKML) + BCG vaccine, they were ac-cepted by the patients. There was no ten-dency for the local reaction to become ag-gravated after repeated vaccination.However, systemic reactions, mainly iri-docyclitis and complaint of numbness of thefingers and toes, became quite common af-ter the fifth vaccination and, therefore, sig-nificantly reduced the acceptability of vac-cine by injection. It seems that repeatedvaccination might activate the iridocyclitis,but the relationship between the complaint

of numbness and vaccination has not beenwell established. Neither typical erythemanodosum leprosum (ENL) nor reversal re-action had been observed throughout thetrial. A significant proportion of patientsconverted to soluble M. leprae skin test an-tigen (SMLA) positivity after repeated vac-cination. However, it seems the positivestatus was not stable, since many of themreverted to negative after the following vac-cination. After the seventh vaccination, thepositive conversion rate to SMLA-I was 45%and to SMLA-II was 35%. After the eighthvaccination, 66.7% of patients converted toMitsuda reaction positive, which has beenconfirmed by histopathological examina-tion. Nevertheless, further follow up is re-quired in order to determine whether or notsuch conversion will be of a long duration.The reactions to SM LA-I and SM LA-II wereassociated but only correlated at a moderatelevel. Overall, the positive conversion rateto SMLA-I was significantly higher than that

652^ International .Journal of Leprosy^ 1993

to SM LA-II after repeated vaccination. Nei-ther the early reaction nor the late (Mitsuda)reaction of the lepromin test were correlatedto either SM LA reaction. The repeated vac-cination of HKML + BCG vaccine did notaffect the weakly positive anti-PGL-1 Al.leprae antibody level seen in the skin-smear-negative lepromatous patients participatingin this study. — Authors' Summary

Doherty, T. M., Backstrom, B. T., Love, S.G., Harding, D. R. K. and Watson, J. D.Identification of T-cell stimulatory epi-topes from the 18-kDa protein of My-cobacterium leprae. Int. Immunol. 5(1993) 673-680.We have used different mouse strains to

examine in vivo and in vitro responses to the18 kDa protein of Mycobacterium leprae,which appears to be strongly immunogenicin both mice and humans. B- and T-cellstimulatory epitopes recognized by differentstrains of mice have been mapped usingoverlapping peptides that span the entire18-kDa protein. Previous work establishedthat immunization of mice with the 18-kDaprotein results in specific antibody produc-tion to common B-cell epitopes, and im-munization of mice with peptides contain-ing these B-cell epitopes resulted in theinduction of specific IgG to only a limitedsubset of epitopes in each strain. Now wereport that T cells purified from mice im-munized with peptides that stimulate an-tibody production, proliferate in vitro whenrechallenged. The proliferating T cells pro-duce levels of IL-2 and IFN-gamma thatindicate antigen-specific T-helper type 1 cellsare present in significant numbers. Thus, acomparison of in vivo and in vitro data sug-gests that T cells bearing the phenotype as-sociated with potentially protective cell-mediated responses can be primed in vivoby epitopes on small peptides. Since T cellsfrom both strains of mice are capable ofresponding to the immunogenic syntheticpeptides in vitro, but give different re-sponses to the same peptides in vivo, factorsother than epitope structure appear to in-fluence T-cell subset activation. This mayhave important implications for diseasessuch as leprosy where a polarized T-cell re-sponse appears to develop and for the de-velopment of synthetic subunit vaccines. —Authors' Abstract

Esaguy, N., Freire, 0., Van Embden, J. D.A. and Aguas, A. P. Lactoferrin triggersin vitro proliferation of T cells of Lewisrats submitted to mycobacteria-inducedadjuvant arthritis. Scand. J. Immunol. 38(1993) 147-152.

We have recently reported antigenic (B-cell) crossreactivity between the mycobac-terial 65-kDa heat-shock protein (hsp65) andhuman lactoferrin (LF), and we suggestedthat this crossreactivity might have a rolein mycobacteria-associated autoimmunedisease. Here, we have searched for anti-LFT-cell reactivity in Lewis rats submitted toa mycobacteria-triggered autoaggressivedisorder (adjuvant arthritis, AA), an auto-immune disorder characterized by high anti-hsp65 reactivity. We have quantified the invitro proliferative response to LF of lymphnode and spleen cells of Lewis rats killed 9,14 and 21 days after the immunization withthe AA-triggering, mycobacteria-containingadjuvant (complete Freund's adjuvant,CFA). We found that LF induced significantproliferation of lymph node T cells of ratsundergoing AA. This T-cell proliferation wasnot as marked as the one provoked by hsp65;it was, nevertheless, significantly higher (p< 0.05) than that produced by a nonar-thritogenic antigen (i.e., albumin). T cellsfrom naive or mineral oil (incompleteFreund's adjuvant, IFA) injected rats didnot respond to LF or hsp65. These dataindicate that LF may work as an accessorystimulatory factor of the T-cell autoreactiv-ity associated with mycobacteria-inducedarthritis. — Authors' Abstract

Kaur, S., Sharma, V. K., Basak, P., Kaur,I. and Radotra, B. D. Concurrent skin andnerve histology in leprosy and its role inthe classification of leprosy. Lepr. Rev.64 (1993) 110-116.

Concurrent skin and nerve histology wasevaluated in 60 leprosy patients (25 BT, 28BL and 7 LL). The twin aims were to studythe comparative histology and the useful-ness of nerve histology in the classificationof the disease. In BT patients, clinical andhistological classification was in agreementin 11 (44%) skin and 17 (68%) nerve bi-opsies. Concurrent skin and nerve histologywas in consonance in 14 (56%) BT patients,while in 6 (24%) patients only nerve his-


tology was helpful in the classification of thedisease, the skin histology being nonspecif-ic. Nerve histology was classified as BL in3 (12%) BT patients, the skin histology wasnonspecific. In the BL group, the histologyof 23 (82.4%) nerve biopsies correlated withthe clinical classification, in contrast to skinhistology which correlated with clinical as-sessment in 19 (68%) patients only. In theLL patients, the histology of nerve corre-lated with the clinical classification in 5 pa-tients (71.4%), compared to histology of theskin in 4 (57%) patients only. The GF washigher in the nerves than in the skinthroughout the leprosy spectrum (BT, BL,LL); the difference was, however, marginalin BL leprosy. The average bacterial index(BI) was higher in nerves (4+) compared tothat of skin histology and slit-skin smears(3+) in BL leprosy. There was, however, nodifference in the BI of the slit-skin smears,skin and nerve biopsies in lepromatous lep-rosy. It is inferred that the neural histologyis often more useful than skin histology inthe classification of leprosy patients (p <0.01) and it correlates better with clinicalclassification, particularly in the borderlinetuberculoid disease. The neural histologygave a better idea about the bacterial loadin the BT, BL patients. It is proposed thatbacteriologically negative patients clinicallyand histologically classified as BT, but withnerve histology more consistent with BL,should be considered multibacillary for pur-poses of therapy.— Authors' Summary

Launois, P., Niang, M. B. N., Sarthou, J.L., Rivier, F., Drowart, A., Van Vooren,J. P., J. and Huygen, K. T-cellstimulation with purified mycobacterialantigens in patients and healthy subjectsinfected with Alycobacterium leprae-se-creted antigen-85 is another immuno-dominant antigen. Scand. J. Immunol. 38(1993) 167-176.

Peripheral blood leukocytes from 9 pau-cibacillary and 12 multibacillary leprosy pa-tients, from 18 healthy controls and from34 healthy leprosy contacts were stimulatedwith three mycobacterial heat-shock pro-teins with respective molecular weights of70, 65 and 18 kDa and with the secreted30-32-kDa protein, also called antigen 85.Antigen 85 was found to be the most pow-erful T-cell antigen (as measured by lym-

phoproliferation and IFN-gamma secre-tion), eliciting a positive response in all(100%) paucibacillary patients and in alllepromin-positive controls and contacts.The three heat-shock proteins (hsp) wereless active T-cell stimuli. Reactivity to the70-kDa hsp was found in only 44% of thepaucibacillary patients, in 80% of the lep-romin-positive controls and in 60% of thelepromin-positive leprosy contacts. The 65-kDa hsp stimulated T cells in 89% of thepaucibacillary patients and in 80% of thelepromin-positive controls and contacts.Responsiveness to the 18-kDa hsp, finally,was clearly more frequent in tuberculoidleprosy patients (78%) than in lepromin-positive controls (40%) or lepromin-posi-tive leprosy contacts (4%). T-cell reactivityof 8 lepromin-negative controls, of 9 lep-romin-negative contacts, and of 12 multi-bacillary leprosy patients was low to all theantigens tested. Although proliferative andIFN-gamma responses were generally close-ly related, some subjects demonstrated adissociation of these two immune param-eters. Our data confirm previous findingson the powerful T-cell stimulatory proper-ties of antigen 85 during AL leprae infection,and suggest that this antigen is indeed a po-tentially protective T-cell immunogen. —Authors' Abstract

Launois, P., Van den Bussche, P., Niang,N. M., Drowart, A., Van Vooren, J.-P.,Sarthou, J.-L., Millan, J. and Huygen, K.IL-6 production in response to purifiedmycobacterial heat-shock proteins and toantigen 85 in leprosy. Cell. Immunol. 148(1993) 283-290.

IL-6 production was examined in PBMCcultures from healthy leprosy contacts andfrom leprosy patients stimulated with thepurified mycobacterial 18-, 65-, and 70-kDaheat-shock proteins (hsp) and the secretedfibronectin-binding antigen 85 (Ag85). Inlepromin-negative contacts, the 70-kDa hspwas the only antigen capable of eliciting sig-nificant IL-6 production. In lepromin-pos-itive contacts, Ag85, the 65- and the 70-kDa hsp induced substantial IL-6 titers. IL-6levels induced with the 70-kDa antigen wereabout fourfold higher than with the 65-kDahsp or with Ag85. The 18-kDa antigen didnot induce any IL-6 in these healthy con-tacts. PBMC from tuberculoid leprosy pa-


tients produced even more elevated levelsof IL-6, and PBMC from lepromatous lep-rosy patients produced extremely high lev-els of IL-6. All antigens were capable of in-ducing IL-6 in leprosy patients. Highestlevels were found in cultures stimulated withthe 65-kDa lisp, and lowest levels were incultures stimulated with the 18-kDa —Authors' Abstract

Mendez-Samperio, P. Antigen presentationof mycobacterial peptides to human T cellclones can be immunomodulated by add-ing an MHC-specific inhibitor. Cell. Im-munol. 148 (1993) 1-9.

The immunomodulation of T-cell rec-ognition by mycobacterial antigens was in-vestigated using T-cell clones activated withpeptide-pulsed EBV-B cells. An H LA-DR I -restricted T-cell clone from a patient withtuberculosis responded to peptide 65-85from the 65-kDa protein ofMycobacteriumtuberculosis in a dose-dependent manner,while no significant response was inducedby antigen-nonpulsed EBV-B cells or EBV-Bcells pulsed with an unrelated antigen(streptokinase/streptodornase). The ob-served binding to HLA-DR I could be in-hibited when the EBV-B cells were culturedin the presence of an excess of an HLA-DR 1-restricted T-cell epitope (residues 1-20) from the 19-kDa protein of Al. tuber-culosis. This inhibition was dose-depen-dent. In other experiments, proliferation ofa DR 1-restricted T-cell clone from a healthyindividual which responded to peptide 1-20 was inhibited by an excess of peptide65-85, confirming that these peptides areable to compete for the same DR 1-bindingsite. Nevertheless, the T-cell clone from , thehealthy individual showed a relatively low-er percentage of inhibition compared withthe T-cell clone from a patient with tuber-culosis. Furthermore, the intensity of thisinhibition was reversed as the concentrationof stimulatory peptide was increased. Theexperiments described in this paper dem-onstrate the immunomodulation of myco-bacterial antigen presentation by peptidecompetition at the level of MHC-bindingsites. These data may be important for anunderstanding of the interactions involvedin the mycobacterial cell-mediated immunerecognition.—Author's Abstract

Negesse, Y., Beinmet, K., Miko, T., Won-dimus, A. and Berhan, T. Y. In leprosythe presence of mycobacteria in the nerveis an essential factor in the cycle and spec-trum of Alycobacterium leprae infection.Lepr. Rev. 64 (1993) 104-109.

A total of 220 untreated leprosy patientswho underwent parallel skin and nerve bi-opsies are included in this study, which isintended to evaluate the extent of previ-ously reported differences in bacillary loadbetween skin and nerve lesions in leprosyand to describe the response of peripheralblood lymphocytes to Mycobacterium lep-raeantigens in such patients. In 161 patientsout of the 220, the skin and nerve biopsieswere diagnostic for leprosy. When patientswere grouped according to their skin andnerve lesions, the three groups observed were(I) paucibacillary skin and nerve lesions; (2)multibacillary skin and nerve lesions; and(3) paucibacillary skin and multibacillarynerve lesions. There was no observation ofa group of patients with multibacillary skinand paucibacillary nerve lesions. In all pa-tients with multibacillary nerve lesions, re-gardless of the type of skin lesions, a lowresponse of peripheral blood lymphocytesto Al. leprae was consistently noted. Theseresults suggest that the bacillary load in thenerve is certainly one of the factors deter-mining the immunological spectrum ob-served in leprosy.—Authors' Summary

Prakash, K., Aggarwal, R. and Sehgal, V.N. Significance of antibodies to phenolicglycolipid-I in leprosy diagnosis. J. Der-matol. 19 (1992) 953-958.

A gelatin particle agglutination assay forthe detection of anti-PGL-I antibodies in40 clinically diagnosed and variously clas-sified groups of leprosy cases revealed ele-vated PGL-I antibody titers in 85% of cases.In contrast, the slit-skin smear examinationwas positive in only 30% of cases. It wasfurther observed that out of 28 cases withbacterial index (BI) of 0, 22 cases (78.5%)had significant levels of PGL-I antibodies.There was no case in which the slit-skinsmear was positive and the PGL-I antibodytiter was not significant. The elevated titersof PGL-I antibody better correlated (84%)with histopathological findings than did BI.


Thus, it was concluded that estimation ofPGL-I antibody titer is a better supplementto clinical diagnosis than BI. Significant lev-els of PGL-I antibody were seen in 85% ofcases who had no earlier chemotherapy orwere treated for less than 2 months. Similarfindings were observed in 12 patients whowere on MDT for more than 5 months butfor less than 2 years. In order to determinethe significance of anti-PGL-I antibodies inmonitoring the response of patients to che-motherapy, a longer follow up with a greaternumber of cases should be contemplated.Authors' Abstract

Shende, R. K., Bardapurker, J., Patil, V.and Gaikwad, A. Adenosine deaminaseactivity in leprosy. Indian J. Lepr. 65(1993) 201-205.

Serum adenosine deaminase (ADA) wasstudied in 60 patients of different types ofleprosy and 50 healthy control subjects.ADA levels in patients with tuberculoid(50.50 ± 5.22 U/L), borderline (41.14 ±3.89 U/L) and lepromatous leprosy (30.10± 0.03 U/L) were higher than that in con-trols (17.84 ± 2.78 U/L), thus correlatingwith the immunological status of patients.Patients with lepra reaction showed de-creased ADA levels and higher grade of lep-romin test positivity was associated withincreased ADA activity.— Authors' Ab-stract

Sieling, P. A., Abrams, J. S., Yamamura,M., Salgame, P., Bloom, B. R., Rea, T.H. and Modlin, R. L. Immunosuppres-sive roles for IL-10 and IL-4 in humaninfection—in vitro modulation of T-cell

responses in leprosy. J. Immunol. 150(1993) 5501-5510.IL-10 and IL-4 have been shown to exert

an inhibitory effect on cell-mediated im-mune responses. Our previous studies ofleprosy demonstrated that IL-10 and IL-4mRNA were preferentially expressed in le-sions from lepromatous patients, those im-munologically unresponsive individuals whomanifest widespread infection. To definemore precisely the regulatory roles of thesetwo cytokines in the immune response toinfection, we studied in vitro responses toMycobacteriunz leprae. M. leprae triggeredIL-10 release from PBMC of patients andhealthy donors; the predominant source ofthe IL-10 was found to be monocytes/mac-rophages. Stimulation of PBMC in the pres-ence of neutralizing anti-IL-10 mAb indi-cated that endogenous IL-10 productioninhibits PBMC proliferation and release ofTNF-alpha, GM-CSF, and IFN-gamma.Paradoxically, studies using neutralizinganti-IL-4 mAb indicated that endogenousIL-4 production enhances PBMC prolifer-ative responses most strikingly in lepro-matous patients. We found that rIL-4 ex-panded CD8+ T cells from lepromatouspatients in vitro. CD8+ T cells from lep-romatous patients have been shown to sup-press CD4+ T-cell responses, in part by therelease of IL-4. Our study indicated thatendogenous IL-4 production inhibited IL-10 secretion and, concomitantly, increasedTNF-alpha and GM-CSF release. The pres-ent data suggest that, on balance, IL-4 andIL-10 contribute to immunosuppression inhuman infectious disease.—Authors' Ab-stract

Microbiology

Bhatia, V. N. and Thawani, G. Observa-tions on attempted leprosy cultures in twomedia. Indian J. Lepr. 65 (1993) 163-171.

Suspensions of skin-tissue material col-lected from lepromatous leprosy patientsand material from mouse foot pad harvestswere inoculated into two media, i.e., a bi-

phasic medium and a minimal basal me-dium. The cultures were incubated at 37°Cand 15°C. Small oval (or round) cells ap-peared in these cultures around the tenthday along with a few cystic structures; andthey increased in number later, reaching themaximum around 6-7 weeks. The abovecells appeared acid-fast in some cultures andsome of them appeared to split into pairs


of acid-fast bacilli. The cells were most oftenseen in the biphasic medium at 37°C. Theidentity of these structures is not known atthis stage.— Authors' Abstract

Godard, C. M. Attempts to cultivate My-cobacterium leprae in fat tissue. Acta Lep-rol. 8 (1993) 133-135.

The behavior of Mycobacterium leprae infat tissue was studied. Preadipocyte cellswere infected with Al. leprae and injectedintradermally (i.d.) into nude mice. Adiposenodules obtained by in vivo differentiationof infected cells were maintained in vivo for3 months and subsequently incubated invitro for 3 months. Counts of bacilli showedno increase over this 6-month period. It isconcluded that undifferentiated preadipo-cyte and mature fat cells are not permissivefor Al. leprae. The morphological changesobserved following passage ofM. leprae intoadipose nodules might be related to the pro-cess of adipose cell differentiation. — Au-thor's Summary

Sareen, M., Kaur, H. and Khuller, G. K.Regulation of phospholipid synthesis inMycobacterium smegmatis by cyclicadenosine monophosphate. J. Biosci. 18(1993) 207-212.

Forskolin, an adenylate cyclase activator,and a cyclic AMP analog, dibutyryl cyclicAMP, have been used to examine the re-lationship between intracellular levels of cy-clic AMP and lipid synthesis in Mycobac-terium smegmatis. Total phospholipidcontent was found to be increased in for-skolin-grown cells as a result of increasedcyclic AMP levels caused by activation ofadenylate cyclase. Increased phospholipidcontent was supported by increased [C-14]acetate incorporation as well as increasedactivity of glycerol-3-phosphate acyltrans-ferase. Pretreatment of cells with dibutyrylcyclic AMP had similar effects on lipid syn-thesis. Taking all these observations togeth-er, it is suggested that lipid synthesis is beingcontrolled by cyclic AMP in mycobacte-ria. — Authors' Abstract

Epidemiology and Prevention

Arain, G. M., Alam, S. E., and Chiang, T.Leprosy in Karachi. J. Pakistan Med. As-soc. 42 (1992) 160-161.

In Karachi, most of the 18,300 registeredcases of leprosy are in former Indian refu-gees who came to Pakistan at the time ofpartition from India. Between 1981-1985,a total of 8779 new cases, of whom about60% were Indians, were registered in thecity. The remainder were mostly from Balu-chistan, the North-west Frontier Province(NWFP), Punjab, Sindh, and Afghanistan.Tuberculoid leprosy prevailed except in theAfghani, 60% of whom had lepromatousleprosy (LL). LL was also relatively com-mon (40% of cases) in individuals from theNWFP. As a rule, leprosy presented in thesecond decade in Indians and Baluchis andin the third decade in Afghanis. Controlmeasures should be concentrated in thosedistricts of Karachi where leprosy is undulyprevalent, e.g., in the Afghan district. —E.M. Scrimgeour (Trop. Dis. Bull.)

Chantraprachoom, C. and Kittampol, K.Leprosy control program in Thailand. Jpn.J. Lepr. 60 (1991) 85-96.

This paper describes the status of leprosyin Thailand under the following main head-ings: historical background, National Lep-rosy Control Program, training, research,multiple drug therapy (MDT), and epide-miological evaluation. The National Lep-rosy Control Program started in 1953; in1971 leprosy control activities were inte-grated into the general health services, withthe exception of six hyperendemic prov-inces which remained specialized (vertical).Detailed information is given on totals ofregistered patients, newly detected patients,those released from control between 1975and 1989, and on the prevalence rate per1000 of the population between 1959 and1989. The prevalence fell from nearly 5 to0.9 per 1000 during a long period of"villagesurvey and domiciliary treatment with dap-sone monotherapy" in 1959-1984. From


1984, when MDT based on WHO recom-mendations was introduced, prevalence hasfallen to 0.31 per 1000. Newly detected casesnumbered 2000 in 1975, rose to about 6000in 1982, and have steadily declined sincethat year to 1594 in 1989. Around 92% ofall patients in Thailand have been treatedwith MDT. — A. C. McDougall (Trop. Dis.Bull.)

Global estimates of the number of peopleneeding medical treatment and care as aresult of leprosy. Lepr. Rev. 63 (1992)375-376.

This Special Article, endorsed by the ILEPMedical Commission in June 1992, com-ments that the WHO's revised estimates ofthe size of the leprosy problem globally needcareful interpretation. It is considered thata distinction should be made between theestimates of those leprosy patients who re-quire chemotherapy and those who havedisabilities as a result of the disease and alsoneed treatment and care. For better com-parisons with earlier estimates of the lep-rosy problem the estimated number of 2-3million people with disabilities (probablyan underestimate) should be added to therevised figure of 5.5 million leprosy patientsrequiring chemotherapy. Multidrug therapyhas been partly responsible for the declinein numbers of active leprosy cases. How-ever, some 40% of leprosy patients are prob-

ably not being reached by multidrug ther-apy. Not until the number of new casesarising each year (currently estimated byWHO as 600,000-800,000) shows a steadydecline can it be said that leprosy has beenbrought under control. —C. A. Brown (Trop.Dis. Bull.)

Kamaludin, F. Leprosy control programmein Malaysia. Jpn. J. Lepr. 59 (1990) 169-182.

The author gives an overview of the sta-tus of leprosy in Malaysia under the mainheadings: historical background, NationalLeprosy Control Program, training, multi-ple drug therapy (MDT), prevalence survey,epidemiological evaluation, clinical andbiomedical research. The National LeprosyControl Program started in 1969 and MDT,based on WHO recommendations, was in-troduced in 1985. The author describes sig-nificant reductions since that date in thenumber of dapsone-resistant cases, reactioncases, the prevalence rate, the number ofpatients admitted to the National LeprosyControl Center at Sungai Buluh and the de-faulter rate. The case detection rate has de-clined from 2.50% in 1981 to 1.27% in 1988and the number of institutionalized patientshas been reduced by 55% in a period of 19years (1970-1989).—A. C. McDougall(Trop. Dis. Bull.)

Other Mycobacterial Diseases and Related Entities

Abbot, N. C., Beck, J. S., Harrison, D. K.and Wilson, S. B. Dynamic thermograph-ic imaging for estimation of regional per-fusion in the tuberculin reaction in healthyadults. J. Immunol. Methods 162 (1993)97-107.

A sensitive method for measurement ofthe volume of blood flow through the skin,based on the kinetics of reheating after lo-calized cooling, is described in this paper.This method has been used to study thetuberculin reaction as a model of cutaneousdelayed-type hypersensitivity (DHS) in man.

Over the positive reaction there is acceler-ated reheating similar in kinetics and extentto that seen after maximal hyperemia in-duced by intradermal injection of histamineor prostaglandin E2. The earlier phase ofreheating (10-100 sec) is more dependenton blood flow; whereas the later phase (100-300 sec) is apparently more dependent onnonperfusion heat exchange mechanisms,including conduction. The reheat kineticmethod is largely dependent on blood flowin the deep dermal vessels (diameter > 50Am); whereas the alternative approach ofmeasurement of the velocity of flow of


erythrocytes in the microcirculation by laserDoppler (LD) flowmetry gives results biasedtoward the most superficial dermal circu-lation. Previous studies with LD flowmetryhave shown that the blood velocity is great-est at the center of weak and strong reac-tions, while in the most intense reactions itis raised at the center but maximal at theperiphery (central relative slowing, CRS)raising the possibility of central ischemia.The reheat kinetics approach has now in-dicated that the deep dermal circulation isnot impaired in CRS reactions. It is con-cluded that there must be partial obstruc-tion of the parts of the microcirculationcommunicating between the deep and su-perficial dermal plexuses, presumably fromthe accumulation of exudate edema in themost intense tuberculin reactions.— Au-thors' Abstract

Alugupalli, S., Olsson, B. and Larsson, L.Detection of 2-eicosanol by gas chroma-tography-mass spectrometry in sputa frompatients with pulmonary mycobacterialinfections. J. Clin. Microbiol. 31 (1993)1575-1578.A total of 96 sputum specimens from

patients with suspected or known myco-bacterial and nonmycobacterial pulmonaryinfections were analyzed by gas chromatog-raphy-mass spectrometry for the presenceof 2-eicosanol. This secondary alcohol wasdetected in all of the 25 sputum specimensculture positive for Mycobacterium tuber-culosis, in 7 of the 9 sputum specimens cul-ture positive for M. cilium complex, and inall 3 of the studied sputum specimens as-sociated with M. malmoense. The alcoholwas not detected in any of the 45 culture-negative sputum specimens or in 14 sputumspecimens culture positive for Staphylococ-cus aureus, Pseudomonas aeruginosa, Mae-mophilus influenzae, and Streptococcuspneumoniae. The ratio of tuberculostearicacid to 2-eicosanol was much lower in spu-tum samples culture positive for mycobac-teria than in the corresponding in vitro-grown cultures. The present findings indi-cate that 2-eicosanol may be useful as achemical marker for rapid diagnosis of pul-monary infections caused by the Al. aviumcomplex, Al. malmoense, and M. tubercu-losis. —Authors' Abstract

Arbeit, R. D., Slutsky, A., Barber, T. \V.,Maslow, J. N., Niemczyk, S., Falkinham,J. 0., O'Connor, G. T. and Von Reyn, C.F. Genetic diversity among strains of My-cobacterium avium causing monoclonaland polyclonal bacteremia in patients withAIDS. J. Infect. Dis. 167 (1993) 1384-1390.

To define the genetic diversity amongMycobacterium avium isolates from humanimmunodeficiency virus-infected patients,specimens were cultured prospectively, andisolates obtained from 14 patients (4 withpositive blood, stool, and sputum; 6 withpositive blood and stool; 3 with positiveblood only; and 1 with positive stool only)were studied. Both serotyping and ribotyp-ing had limited ability to discriminateamong isolates from different patients;whereas the distinctive restriction fragmentprofiles resolved by pulsed-field gel electro-phoresis indicated that each patient was in-fected by a unique strain. Of the 13 bacte-remic patients, 2 were bacteremicconcurrently with two distinct strains. Thefact that Al. avium isolates from AIDS pa-tients exhibit considerable genetic diversitysupports the hypothesis that the infection isacquired from various environmentalsources. Further, individual patients are notinfrequently bacteremic with more than onestrain simultaneously, which may need tobe considered in protocols for the diagnosisand management of Al. avium disease.—Authors' Abstract

Barradell, L. B., Plosker, G. L. and Mc-Tavish, D. Clarithromycin —a review ofits pharmacological properties and ther-apeutic use in Mycobacterium avium in-tracellulare complex infection in patientswith acquired immune deficiency syn-drome. Drugs 46 (1993) 289-312.

Results from noncomparative and pla-cebo-controlled studies demonstrate the ef-ficacy of clarithromycin in the treatment ofdisseminated Mycobacterium avium - intra-cellulare complex (MAC) infection in pa-tients with acquired immune deficiencysyndrome (AIDS). Whether given alone orin combination with other antimycobacteri-al treatments, doses of 500 to 2000 mg (typ-ically 1000 mg) administered twice daily areeffective in controlling bacteremia in these


patients. Clarithromycin has also beenshown to improve clinical symptoms of in-fection and may improve the quality of lifein AIDS patients with MAC infection. Clar-ithromycin is generally well tolerated whenused in the doses typically required for thetreatment of MAC infection (1000 or 2000mg/day). Gastrointestinal disturbances arethe most commonly occurring adverseevents, and occur most frequently at dos-ages of 4000 mg/day. Thus, clarithromycin,as monotherapy or in combination withother antimycobacterial agents, is well tol-erated and effectively eradicates MAC fromthe blood in the short term in patients withAIDS; however, short term monotherapymay lead to bacterial resistance, underscor-ing the importance of long-term treatmentwith a combination of antimycobacterialagents. While the optimal combination reg-imen to prevent the development of resis-tance to antimycobacterial agents by MACremains to be determined, clarithromycinwill almost certainly be a valuable agent inany such combination.—Authors' Abstract

Bermudez, L. E. and Champsi, J. Infectionwith Mycobacterium avium induces pro-duction of interleukin-10 (IL-10), and ad-ministration of anti-IL-10 antibody is as-sociated with enhanced resistance toinfection in mice. Infect. Immun. 61(1993) 3093-3097.

Organisms of the Mycobacterium aviumcomplex are associated with disseminatedinfection in patients with AIDS. The mech-anisms that account for the survival of theintracellular bacteria are unknown. We doc-ument here that infection of C57BL/6 blackmice with Al. avium 101 triggered interleu-kin-10 (IL-10) production. The synthesis ofIL-10 peaked after 2 weeks of infection andremained elevated throughout the period ofinfection. Treatment of M. arium-infectedperitoneal macrophages with recombinantIL-10 suppressed the stimulatory effect oftumor necrosis factor-alpha and granulo-cyte-macrophage colony-stimulating factor.To confirm the possible role of IL-10 in theinfection in vivo, mice were infected withM. avium 101 and simultaneously receivedtreatment with neutralizing anti-11, 10 an-tibody. After 4 weeks the animals were har-vested and the numbers of viable bacteria

were quantitated in the liver, spleen, andblood. The liver and spleen of animals re-ceiving anti-IL-10 antibody had 2 to 3 logunits fewer bacteria than did those of con-trol animals. These results suggest a role forIL-10 in the pathogenesis of Al. avium in-fection. —Authors' Abstract

Birnbaum, G., Kotilinek, L. and Albrecht,L. Spinal fluid lymphocytes from a sub-group of multiple sclerosis patients re-spond to mycobacterial antigens. Ann.Neurol. 34 (1993) 18-24.

Immune responses to heat shock or stressproteins are observed in several chronic au-toimmune diseases. Such proteins are majorantigens of many bacteria, especially my-cobacteria. To determine whether immuneresponses to stress proteins occur in chronicinflammatory diseases of the central ner-vous system such as multiple sclerosis (MS),we measured proliferative responses of lym-phocytes from spinal fluids and bloods ofpatients with MS and other neurologicaldiseases to a sonicate of Al. tuberculosis, anacetone extract of M. tuberculosis, a recom-binant 65-kDa heat shock protein of Al.leprue, and tetanus toxoid as a control recallantigen. Significantly increased spinal fluidlymphocyte responses to mycobacterialsonicate, relative to responses from pairedperipheral blood lymphocytes, were presentin 14 of 20 specimens from patients withMS (p < 0.025) and 2 of 9 specimens frompatients with other neurological diseases.Spinal fluid lymphocytes also responded totetanus toxoid, but differences betweenblood and spinal fluid were not statisticallysignificant. Lymphocytes from one patientwith MS responded only to Al. leprae. Therewere no proliferative responses to the M.tuberculosis acetone extract. When patientswith MS were classified according to du-ration of disease (< 2- or > 2-yr duration)9 of 10 patients with recent onset had ce-rebrospinal fluid cells that responded to Al.tuberculosis compared with 5 of 10 with lon-ger duration symptoms (p < 0.012). Ourdata suggest a selective recruitment and/orexpansion of mycobacterial reactive cells tothe central nervous system of a subpopu-lation of patients with MS. Immune re-sponses to antigens present in mycobacte-ria, possibly stress proteins, may be


important in perpetuating and amplifyingchronic inflammatory diseases of the centralnervous system such as MS.—Authors' Ab-stract

Drowart, A., Cambiaso, C. L., Huygen, K.,Serruys, E., Yernault, J. C. and Van Voor-en, J. P. Detection of mycobacterial an-tigens present in short-term culture mediausing particle counting immunoassay.Am. Rev. Respir. Dis. 147 (1993) 1401-1406.

Particle counting immunoassay (PACIA)was compared with the BACTEC® systemfor detecting mycobacterial growth aftershort-term culture, and was used to identifyMycobacterium tuberculosis. The latex par-ticles were coated with polyclonal anti-BCGor with specific 2A 1-2 monoclonal antibod-ies. Bottles containing nonradioactive Mid-dlebrook 7H9 liquid medium and BAC-TEC® 12B vials were inoculated with equalamounts of mycobacteria from four refer-ence strains (M. tuberculosis, M. kansasii,M. avium, and M. xenopi). Using anti-BCG,PACIA detected mycobacterial antigens 3to 6 days before the BACTEC system. M.tuberculosis was differentiated from the oth-er mycobacteria using 2A1-2. Seventeenclinical samples were also studied. In thesame 10, the two techniques detected my-cobacteria, PACIA with anti-BCG after 9days and BACTEC® 1 to 5 days later. For9 of the 10 samples, PACIA with 2A1-2detected M. tuberculosis after 20 days, a re-sult confirmed with the AccuProbe ® sys-tem. M. xenopi was biochemically identi-fied in Specimen 10. Nonmycobacterialdiseases were diagnosed in the 7 remainingunreactive specimens. We conclude thatPACIA detects mycobacterial growth ear-lier than BACTEC® and that Al. tubercu-losis can be distinguished from other my-cobacteria in PACIA performed with specificmonoclonal antibodies. —Authors' Ab-stract

Forbes, B. A. and Hicks, K. E. S. Directdetection of Mycobacterium tuberculosisin respiratory specimens in a clinical lab-oratory by polymerase chain reaction. J.Clin. Microbiol. 31 (1993) 1688-1694.

The emergence of epidemic multiple-drug-resistant (MDR) strains of Alycobac-

terium tuberculosis in conjunction with anincrease in the number of reported cases oftuberculosis (TB) represents a major publichealth problem. In light ofa recent outbreakof MDR M. tuberculosis at our center, webegan the development of a polymerasechain reaction (PCR) assay for the rapid di-agnosis of pulmonary TB using two sets ofprimers, one based on the IS6110 repeatedsequence of Al. tuberculosis and the otherbased on the protein antigen b (PAB). Re-action conditions were first optimized as tothe appropriate extraction protocol and theconcentrations of primer pairs, nucleotides,and MgCl2. Following a preliminary eval-uation of the assay with clinical specimens,extraction and amplification procedureswere further modified. PAB and IS6110primers detected between 2 and 23 and 0.023and 0.23 CFU of Al. tuberculosis, respec-tively, in pooled, M. tuberculosis-negativesputa by our optimized PCR assay. Afterroutine processing for mycobacteria, 734specimens were subsequently amplified.DNA for amplification was obtained byboiling and beating the sediments withTween 20. For each reaction, DNA (10 kil)was added to an amplification mixture con-taining 12 pmol of IS6110 primers, 20 pmolof PAB primers, 2 mM MgC1,, 200 ,uM nu-cleotides, and 2.5 U of Taq polymerase andthe mixture was then amplified for 40 cy-cles. The sensitivity and specificity of ourPCR assay were 87.2% and 97.7%, respec-tively. We were unable to interpret the re-sults for seven specimens (1%). In our ex-perience, PCR proved to be a useful, rapiddiagnostic test for TB in a clinical settingand a valuable epidemiological tool for de-termining exposure groups in the hospitalsetting. Our findings also underscore theneed for the systematic optimization of PCRassay conditions. — Authors' Abstract

Godeau, B., Oksenhendler, E. and Bierling,P. Dapsone for autoimmune thrombo-cytopenic purpura. Am. J. Hematol. 44(1993) 70-72.

Twenty-one human immunodeficiencyvirus (HIV)-free and six HIV-infected adultswith autoimmune thrombocytopenic pur-pura (AITP) were treated with dapsone (100mg/day). A response was observed in 13patients (median platelet count before 25 x


10 9 /L, range 3-49; after 109 x 10 9 /L, range69-241). Thrombocytopenia recurred infour of the responders in whom dapsonewas discontinued. No response was ob-served in 12 patients. Dapsone had to bewithdrawn after 2 weeks of treatment in theremaining two patients and after 6 to 8 weeksin three other patients due to intolerance.No serious hematological complicationswere observed. These results confirm thatdapsone is a safe, inexpensive, and effectivetreatment of AITP.— Authors' Abstract

Godfrey-Faussett, P. and Stoker, N. G. As-pects of tuberculosis in Africa. 3. Genetic"fingerprinting" for clues to the patho-genesis of tuberculosis. Trans. R. Soc.Trop. Med. Hyg. 86 (1992) 472-475.

In a study of 117 isolates of Mycobacte-rium tuberculosis collected in Malawi andKenya most paired isolates from individ-uals were identical. Two exceptions werefound in HIV-infected patients: discordantisolates were obtained from a single episodeof disease. Thus, multiple strains may existwithin a single patient with active tuber-culosis.— D. W. FitzSimons (Trop. Dis.Bull.)

Harris, D. P., Vordermeier, H. NI., Friscia,G., Roman, E., Surcel, H. NI., Pasvol, G.,Moreno, C. and Ivanyi, J. Geneticallypermissive recognition of adjacent epi-topes from the 19-kDa antigen of My-cobacterium tuberculosis by human andmurine T cells. J. Immunol. 150 (1993)5041-5050.

The specificity of the T-cell-immune rep-ertoire at the level of individual antigenicdeterminants could play a fundamental rolein the immunopathogenesis of tuberculousinfections. Therefore, we analyzed the im-munogenicity, genetic restriction, and epi-tope core structure of two adjacent, yet dis-tinct, immunodominant T-cell determinantsfrom the 19-kDa antigen of Mycobacteriumtuberculosis. After immunization with twopeptides comprising residues 45 to 64 and61 to 80, vigorous in vitro proliferative re-sponses to the homologous peptide wereelicited in five different strains of C57BL/10 mice (H-2b,k,d,s,f), indicating that bothepitopes were recognized in a geneticallypermissive manner. When immunized with

intact 19-kDa protein, lymph node cellsfrom the same mouse strains responded toboth peptides, with the exception of H-2bmice which did not respond to p45-64. De-layed-type hypersensitivity responses inC57BL/ 10 (H-2b) mice were elicited by p61-80 only; whereas in H-2d mice both pep-tides were delayed-type hypersensitivitynegative, despite eliciting pronounced pro-liferative responses. Analysis of the prolif-erative responses of human PBMC in pu-rified protein derivative-positive healthysubjects and tuberculosis patients revealedsignificant differences in the antigenicity tothe two peptides. All purified protein deriv-ative-positive healthy and diseased individ-uals manifested strong responses to p45-64, indicating HLA permissive recognition.In contrast, pronounced responses to p61-80 were detected only in patients with lym-phatic tuberculosis. Epitope core structures,composed of 6 or 7 residues within eachpeptide, have been mapped with peptidesof overlapping sequence. Significantly, forboth epitopes, the core sequences recog-nized by both human and murine T cellswere almost identical. We conclude that de-spite many similarities between murine andhuman T-cell cpitopc recognition, distinctdifferences in the responsiveness of the in-fected host could play a role in pathogenesis.Furthermore, the genetically permissive na-ture of the identified epitopes is a potentiallyimportant attribute for the development ofpeptide-based diagnostic reagents and vac-cines.— Authors' Abstract

Heym, B., Zhang, Y., Poulet, S., Young, D.and Cole, S. T. Characterization of thekatG gene encoding a catalase-peroxidaserequired for the isoniazid susceptibility ofMycobacterium tuberculosis. J. Bacteriol.175 (1993) 4255-4259.

The isoniazid susceptibility of M•cobac-terium tuberculosis is mediated by the prod-uct of the katG gene which encodes theheme-containing enzyme catalase-peroxi-dase. In this study, the chromosomal lo-cation of katG has been established and itsnucleotide sequence has been determined sothat the primary structure of catalase-per-oxidase could be predicted. The M. tuber-culosis enzyme is an 80,000-dalton proteincontaining several motifs characteristic of

662^ International ,Journal of Leprosy^ 1993

peroxidases, and shows strong similarity toother bacterial catalase-peroxidases. Ex-pression of the katG gene in Al. tuberculosis,

smegmatis, and Es-cherichia coli wasdemonstrated by Western blotting (immu-noblotting). Homologous genes were de-tected in other mycobacteria, even thosewhich are naturally insensitive to isonia-zid.— Authors' Abstract

Huygen, K., Drowart, A., I Iarboe, NI., Ten-berg, R., Cogniaux, .1. and Van Vooren,J. P. Influence of genes from the majorhistocompatibility complex on the anti-body repertoire against culture filtrate an-tigens in mice infected with live M•co-bacterium boris BCG. Infect. lmmun. 61(1993) 2687-2693.C57BL/ I 0 and C57BL/6 mice (H-2b);

BIO congenic mice with f, k, p, q, r, and sH-2 haplotypes; 1310 mice with recombi-nant g2, o2, a, h2, h4, i5, and bql H-2 hap-lotypes; and B6 mice with major histocom-patibility complex (M HC) mutant bml andbm13 (class 1) and bm 12 (class 11) haplo-types were infected intravenously with 4 x10 6 CFU of live Alycobacterium boris BCGand examined by Western immunoblotanalysis for serum antibodies against BCGculture filtrate antigens, following a boostinjection with live BCG or with I3CG cul-ture filtrate. Parental 1310 and 136 mice re-acted very intensely with three culture fil-trate protein bands with estimated molecularmasses of 37, 38, and 40 kDa. Responseagainst the 40-kDa protein was stronger fol-lowing a boost injection with live BCG thanfollowing a boost with culture filtrate. Serafrom mice with f, p, i5, bm 1, and bm 13haplotypes reacted strongly, with both the37-38- and 40-kDa antigens, and sera frommice with q and bql haplotypes showed asomewhat weaker reaction. Sera from micewith r, s, and bm 12 haplotypes reactedagainst the 37-38-kDa antigen but notagainst the 40-kDa antigen, and sera frommice with the h2 haplotype reacted onlywith the 40-kDa antigen but not with the37-38-kDa antigen. Sera from mice with thek, g2, o2, a, and h4 haplotypes showed, atmost, a very weak reaction with the 37-38-and 40-kDa antigens. These results dem-onstrate that MHC genes profoundly affectthe antibody repertoire used against culture

filtrate antigens in mice infected with liveM. bolls BCG. In particular, as shown inmice with the recombinant 1-1-2 haplotypeand in class II mutant 1)11112 mice, the I-Aheterodimer controls the recognition of theimmunodominant 40-kDa antigen. By us-ing crossed immunoelectrophoresis, this 40-kDa antigen was identified as antigen 88according to the reference system of Closs,of al. for I3CG antigens.—Authors' Abstract

Iralu„1. V., Sritharan, V. K., Pieciak, W.S., Wirth, D. F., Maguire, J. II. and Bar-ker, R. II. Diagnosis of Mycobacterium

bacteremia by polymerase chainreaction. J. Clin. Microbiol. 31 (1993)1811-1814.

We describe a rapid polymerase chain re-action (PCR)-based test for diagnosing My-cobacterium avium directly from bloodspecimens. Blood was collected in antico-agulant (EDTA) from patients who also hadblood cultures performed by the lysis-cen-tri fugation method. Blood samples werecentrifuged on a Ficoll-Hypaque gradient topurify peripheral blood mononuclear cells.The purified cells were washed and incu-bated in the presence of Chelex- 100 (a di-valent cation-binding resin), boiled to re-lease mvcobacterial DNA, and thenamplified with M. avium -specific PCRprimers. Amplification was detected by hy-bridization with radiolabelled probe, andthe results were compared with the cultureresults. The PCR assay gave positive resultsfor 12 of 15 specimens that were taken frompatients with positive cultures for Al. aviumcomplex (sensitivity, 80%). The three PCR-negative specimens in this group showedevidence of PCR inhibition. The PCR assaygave positive results for 32 of 228 speci-mens taken from patients with negative cul-tures (specificity, 86%). Of these 32 PCR-positive culture-negative specimens, 27 werealso positive when amplified with primersspecific for the genus .11 ycobacterium, sug-gesting that PCR may be more sensitive thanculture. —Authors' Abstract

Klopman, G., Wang, S., .Jacobs, M. R., Ba-jaksouzian, S., Edmonds, K. and Ellner,J. J. Anti-Mycobacterium avian activityof quinolones: in vitro activities. Antimi-


crob. Agents Chemother. 39 (1993) 1799-1806.The minimal inhibitory concentrations

(MICs) of 88 quinolones against 14 selectedreference and clinical strains of Mycobac-terium avium-M intracellu/arc complexwere determined. Agents tested includedciprofloxacin, sparfloxacin (PD 131501),and 86 other experimental quinolones. Teststrains were selected to represent varioussusceptibilities to ciprofloxacin and otherdrug resistance profiles. MICs were deter-mined by the microdilution method in 7HSFbroth, with incubation for 14 days at 35°C.The results showed 25 of the quinolones tobe active against the strains, with MICs for90% of the strains (MIC,„,^) of 2 to 32 pg/ml. Ten of these compounds had activitiesequivalent to or greater than that of cipro-floxacin. The most active compound wasPD 125354, with an MIC,„ of 0.5 pg/ml andan MIC.„, of 2 pg/ml; comparable values forciprofloxacin were 4 and 8 pg/ml, respec-tively. The next most active compounds,with MIC,„ ) s of 4 pg/ml, were sparfloxacin(PD 131501), PD 123982, PD 135144, andPD 119421. MIC,„,s of PD 131575, PD126889, PD 122642, PD 139586, and PD143289 were 8 pg/ml. Further evaluation ofthe most active agents is warranted, as isassessment of structure-activity relation-ships of active and inactive agents to elu-cidate the active portions of the compoundsand to lead to the development of com-pounds with enhanced activity.—Authors'Abstract

Kocagoz, T., Yilmaz, E., Ozkara, S., Koca-goz, S., Hayran, M., Sachedeva, M. andChambers, H. F. Detection of Mycobac-terium tuberculosis in sputum samples bypolymerase chain reaction using a sim-plified procedure. J. Clin. Microbiol. 31(1993) 1435-1438.

A repetitive sequence of .Ilycobacteriumtuberculosis DNA was amplified by poly-merase chain reaction (PCR), from sputumsamples, for the diagnosis of pulmonary tu-berculosis. The method of heating the sam-ple in a boiling water bath to break downthe bacterial cell wall and to release the DNAwas compared with that of enzymatic lysisof bacteria and then phenol-chloroform ex-traction of DNA. Heating the sample was

the better method with a sensitivity of ap-proximately 10 microorganisms. A total of78 sputum specimens prepared by heatingwere examined by PCR, and the results werecompared with the results of acid-fast-stained smears, cultures, and clinical data.Al. tuberculosis was detected by PCR in allsmear- and culture-positive and smear-neg-ative, culture-positive cases. Additionally,PCR was capable of detecting 4 of 9 caseswhich were smear and culture negative butclinically suspected of tuberculosis. DNAamplification by PCR is a sensitive and spe-cific method for the diagnosis of tubercu-losis, and with this simplified DNA isola-tion procedure it can be used in routineclinical practice.—Authors' Abstract

Lazard, T., Perronne, C., Grosset, J.-H.,Vilde, J. L. and Pocidalo, J. J. Clarith-romycin, minocycline, and rifabutintreatments before and after infection ofC5713L/6 mice with Mycobacterium av-ium. Antimicrob. Agents Chemother. 37(1993) 1690-1692.

C57BL/6 mice were pretreated with ri-fabutin or clarithromycin alone or com-bined with minocycline 3 days before in-travenous challenge (day 0) withMycobacterium avium. Treatment was con-tinued until sacrifice at days 1, 8, 15, and21. Rifabutin or clarithromycin decreasedthe level of infection in both the lungs andthe spleen. Rifabutin was as effective as clar-ithromycin in the lungs but was more ef-fective in the spleen. The clarithromycin-minoc■..cline combination was as effectiveas clarithromycin alone.—Authors' Ab-stract

Limb, D. I., Wheat, P. F., Spencer, R. C.,Harris, G. S., Rayner, A. B. and Watt, B.Comparison of techniques for antimicro-bial susceptibility testing of mycobacte-ria. J. Clin. Pathol. 46 (1993) 403-407.

Aims—To evaluate adenosine triphos-phate (ATP) bioluminescence as a rapidtechnique for antimicrobial susceptibilitytesting of Mycobacterium spp. by compar-ing it with conventional and radiometricmethods, and to assess its potential for usein clinical microbiology laboratories.


Methods-115 clinical isolates from awide range of mycobacterial species and fourcontrol organisms of known susceptibilitywere tested against six antimicrobial agents.Minimum inhibitory concentrations (M ICs)were determined after 4-6 weeks' incuba-tion on Middlebrook 7H10 agar. Suscepti-bility was also determined radiometricallyusing a BACTEC* 460, and by biolumi-nescent assay of ATP using a 1250 lumi-nometer (LKB-Wallac).

Results—Susceptibility results after 7 daysshowed excellent correlation with conven-tionally determined M ICs; 714 susceptibil-ity tests were performed by both techniques,with seven major discrepancies between thetwo systems. For pyrazinamide, agreementwas 100%, but five strains of Al. lithe/vie-1osis, including one control, and 11 myco-bacteria other than Al. tuberculosis (MOTT)failed to grow on Middlebrook agar at pH5.5; 606 tests were performed by radiom-etry, with four major discrepancies betweenthis technique and ATP bioluminescence.No particular species of Mycobacterium gaveaberrant results. Contamination was a prob-lem; 12 of the 119 strains tested were con-taminated at day 1 and had to be repeatedbefore results were obtained. Contamina-tion of individual tests increased signifi-cantly after 7 days of incubation.

Conclusions—ATP bioluminescence canbe used to monitor mycobacterial growth influid culture media; the technique has con-siderable potential for rapid susceptibilitytesting. Advantages include lower initial costof analytical equipment, lower reagent costper test, and the use of nonradioactive sub-strates. —Authors' Abstract

Mackall, J. C., Bai, G. H., Rouse, D. A.,Armoa, G. R. G., Chuidian, F., Nair, J.and Morris, S. L. A comparison of theT-cell delayed-type hypersensitivity epi-topes of the 19-kD antigens from Myco-bacterium tuberculosis and Mycobacteri-11111 intracellulare using overlappingsynthetic peptides. Clin. Exp. Immunol.93 (1993) 172-177.

Mycobacterial disease remains a seriousinternational public health concern. Im-proved methods to rapidly and specificallydetect mycobacterial infections wouldgreatly enhance clinical management of these

diseases. To define species-specific T-cellepitopes that may be useful for the immu-nodiagnosis of mycobacterial infections,polymerized synthetic peptides from the 19-kD Mycobacterium tuberculosis and M. in-tracellulare protein homologs were tested inguinea pig DTH assays. Five Al. tuberculosisand eight M. intracellulare peptides evokedskin test responses. Although all of the ac-tive Al. tuberculosis and seven of the Al.intracellulare peptides elicited nonspecificDTH reactions, the peptide IN13 induceda M. intracellulare -specific skin test reac-tion, and thus represents a specific Al. in-tracellulare T-cell DTH epitope. This resultsuggests that the development of monospe-cific peptide-based immunodiagnostic re-agents may be feasible for future clinicaluse. —Authors' Abstract

Marin, L. M. L., Laneelle, M. A., Prome,D. and Daffe, M. Structures of the gly-copeptidolipid antigens of two animalpathogens—Mycobacterium senegalenseand Mycobacterium porcinum. Eur. J.Biochem. 215 (1993) 859-866.

The structures of the major glycolipid an-tigens of two animal pathogens Mycobac-terium senegalense and Al. porcinum wereelucidated by a combination of fast-atombombardment mass spectrometry, nuclearmagnetic resonance spectroscopy, chemicalanalyses and radiolabeling experiments. Fiveglycoconjugates belonging to the class ofC-mycoside glycopeptidolipids were char-acterized in each species. They shared withthose recently described in Al. peregrinumthe same unusual distribution of the disac-charides on the alaninol end of the mole-cules. Both species showed the presence ofthe novel sulfated glycopeptidolipid. In ad-dition, some acetylated forms of the gly-colipids were also present in the species ex-amined. Identical seroreactivities wereobserved between the glycolipid antigensextracted from M. senegalense, M. porcin-um and M. peregrinum and an antiserumraised against the whole lipid antigens of M.peregrinum. These data reinforce the closetaxonomic relationships between the threemycobacterial species and demonstrate theantigenicity of the new variants of myco-bacterial glycopeptidolipids.— Authors'Abstract

61, 4^ Current L iterature^ 665

Matoba, A. Y., Lee, B. L., Robinson, N. M.,Penland, R. and Osato, M. S. Combina-tion drug testing of Mycobacterium che-lonae. Invest. Ophthalmol. Vis. Sci. 34(1993) 2786-2789.

Medical therapy of Mycobacterium che-lonae keratitis is difficult because there arcso few effective antimicrobial agents andsingle-agent therapy frequently fails clini-cally. To identify more effective medicaltreatment regimens, the in vitro antimicro-bial efficacy ofamikacin, the most frequent-ly used single agent, was investigated incombination with four antibiotics previ-ously reported to have activity against .11.chelonae: erythromycin, imipenem, cipro-floxacin, and vancomycin. The drug com-binations were tested by the checkerboardmethod against seven corneal isolates of Al.chelonae. The combination of amikacin witherythromycin or vancomycin consistentlyled to synergistic or additive effect; howeverthe minimum inhibitory concentrations forvancomycin were very high. The combi-nation of amikacin with imipenem or cip-rofloxacin led to results ranging from an-tagonism to additive effects. Of theantibiotics tested, erythromycin showed themost activity against AI. chclonae in com-bination with amikacin. In vitro combina-tion drug testing of Al. chelonae by thecheckerboard method should be furtherevaluated for clinical relevance in microbialkeratitis. —Authors' Abstract

McDonough, K. A., Kress, Y. and Bloom,B. R. Pathogenesis of tuberculosis—in-teraction of Mycobacterium tuberculosiswith macrophages. Infect. Immun. 61(1993) 2763-2773.

Central to understanding the pathogene-sis of tuberculosis is the interaction betweenthe pathogen and mononuclear phagocytes.A key question about that interaction iswhether Mycobacterium tuberculosis exertsan effect on phagolysosome fusion. We havereexamined the dynamics of phagolyso-some fusion and its effect on intracellularbacterial replication in Al. tuberculosis-in-fected macrophages by performing an ex-tensive study at the electron microscopiclevel. Thoria-labeled murine and humanmacrophages were infected with a virulent(H37Rv) or avirulent (H37Ra) strain of Al.

tuberculosis or with Al. bovis BCG vaccinefor times ranging from 2 hr to 7 days. In allcases, by 2-hr postinfection, approximately85% of the bacteria clearly resided in fusedvacuoles. However, at 4 days postinfection,fusion levels for viable H37Rv and H37Rawere reduced by half; whereas the fusionprofiles of BCG and of heat-killed H37Rvand H37Ra were unchanged. A comparisonof the numbers of bacteria per fused andnonfused vacuoles suggests both a net trans-fer of bacteria out of fused vacuoles andpreferential bacterial multiplication in non-fused vacuoles. H37Rv and H37Ra ap-peared to bud from the phagolysosomes intotightly apposed membrane vesicles that didnot fuse with secondary lysosomes. In somecases, no such membrane was seen and thebacteria appeared to be free in the cyto-plasm. Only viable H37Rv showed a sig-nificant increase in bacterial counts duringthe course of infection. Thus, both of theattenuated strains we examined differedfrom the virulent strain H37Rv in theirabilities to replicate successfully withinmacrophages, but each diverged fromH37Rv at a different point in the process.Viable tubercle bacilli H37Rv and H37Rahad the capacity to escape from fused ves-icles as the infection progressed; BCG didnot. After extrusion from the phagolyso-some, H37Rv, but not H37Ra, was able tomultiply. These results suggest a novelmechanism by which virulent Al. tubercu-losis eludes the microbicidal mechanisms ofmacrophages by escaping from fused phago-lysosomes into nonfused vesicles or the cy-toplasm.— Authors' Abstract

Nikaido, H., Kim, S. H. and Rosenberg, E.Y. Physical organization of lipids in thecell wall ofil/ycobacteriumchelonae. Mol.Microbiol. 8 (1993) 1025-1030.

Mycobacterial cell wall functions as aneffective permeability barrier, making thesebacteria resistant to most antibacterialagents. It has been assumed that this lowpermeability was due to the presence of alarge amount of unusual lipids in the cellwall, but it was not known how these lipidsare able to produce such an exceptional bar-rier. We report here the first experimentalevidence on the physical arrangement ofthese lipids based on X-ray diffraction stud-

666^ International Journal q/ Leprosy^ 1993

ies of purified Mycobacterium chelonae cellwall, a result suggesting that the hydrocar-bon chains of the cell-wall lipids are ar-ranged predominantly in a direction per-pendicular to the cell-wall surface, probablyproducing an asymmetric bilayer struc-ture.— Authors' Abstract

Nolte, F. S., Metchock, B., McGowan, J.E., Jr., Edwards, A., Okwumalma, 0.,Thurmond, C., Mitchell, P. S., Plikaytis,B. and Shinnick, T. Direct detection ofMycobacterium tuberculosis in sputum bypolymerase chain reaction and DNA hy-bridization. J. Clin. Microbiol. 31 (1993)1777-1782.

A polymerase chain reaction (PCR) assayfor the rapid diagnosis of pulmonary tuber-culosis was developed by using oligonucle-otide primers to amplify a fragment of1S6110, an insertion sequence repeated mul-tiple times in the chromosome of Mycobac-terium tuberculosis. Sediment obtained fromsputa processed by the N-acetyl-L-cysteine-NaOH method was suspended in a simplelysis buffer and was heated at 100°C for 30min prior to amplification. A dUTP-uracilN-glycosylase PCR protocol was used to pre-vent false-positive test results because of thecarryover of products from previous ampli-fication reactions. The 317-bp amplicon wasdetected by direct gel analysis and Southernblotting and then hybridization with a bi-otin-labeled internal probe. Hybrid mole-cules were detected by using a commerciallyavailable avidin-alkaline phosphatase-chemiluminescent substrate system (Tro-pix, Inc., Bedford, Mass.). The analyticalsensitivity of the assay was 10 fg of purifiedmycobacterial DNA. The limits of detec-tion by culture (Middlebrook 71-111 agar andLowenstein-Jensen medium) and by PCRwere equivalent in terminal dilution exper-iments for organism suspensions and pos-itive sputa. An internal control was used todetect the presence of amplification inhib-itors in each negative reaction mixture. DNAwas purified from inhibitory specimens byphenol-chloroform extraction and ethanolprecipitation. PCR results were comparedwith results of microscopy and convention-al culture for the detection of M. tubercu-losis in 313 sputum specimens. There were124 specimens that were positive for Al.tuberculosis by conventional methods and

113 (91%) that were positive by PCR. PCRdetected 105 of 110 (95%) of the smear-positive and 8 of 14 (57%) of the smear-negative specimens. There were no false-positive results by PCR (specificity, 100%).This PCR assay incorporates several inno-vations that make application of this newtechnology feasible in clinical microbiologylaboratories.— Authors' Abstract

Orme, I. M., Andersen, P. and Boom, W.H. T-cell response to Mycobacterium tu-berculosis. J. Infect. Di s. 167 (1993) 1481-1497.

The T-cell-mediated, acquired immuneresponse to infection with Mycobacteriumtuberculosis, both in humans and in exper-imental models in the mouse, is a complexevent believed to involve a variety of T-cellsubsets that manifest themselves in numer-ous functions, including protection, de-layed-type hypersensitivity, cytolysis, andthe establishment of a state of memory im-munity. These functions in turn involve thesecretion of an array of cytokines, severalof which direct cells of the monocyte/mac-rophage axis to contain and destroy the in-vading bacilli. This article reviews the de-velopment of these ideas, both from clinicalexperience and from basic research in ani-mal models. In addition, the newly emerg-ing hypothesis that the secreted or exportproteins of M. tuberculosis are the key pro-tective antigens leading to the initial ex-pression of acquired specific resistance tothis organism is examined.—Authors' Ab-stract

Orme, I. M., Roberts, A. D., Griffin, J. P.and Abrams, J. S. Cytokine secretion byCD4 T-lymphocytes acquired in responseto Mycobacterium tuberculosis infection.J. Immunol. 151 (1993) 518-525.

The results of this study in which CD4T-cells were harvested from mice at varioustimes during the course of a virulent Myco-bacterium tuberculosis infection and exam-ined for their secretion of cytokines duringculture in vitro with bone-marrow-derivedmacrophages presenting mycobacterial anti-gen, provide evidence for the existence of twoseparate waves of cytokine-producing CD4cells. The first, which peaks at the time atwhich protective immunity was maximally


expressed, was characterized as a IFN-gam-ma-secreting cell population that preferen-tially recognized macrophages presentingmycobacterial culture filtrate proteins, orthat were infected with the live organism.A second population, which emerged 20 to40 days later at a time when the infectionhad been contained, secreted IL-4 in re-sponse to the filtrate proteins, but also re-acted particularly strongly to the 65-kDa(hsp60) heat-shock protein molecule of .1/.tuberculosis. These data indicate that ac-quired immunity to tuberculosis infectioninvolves the production of both Th I - andTh2-like cell populations that difkr in termsof their kinetics of emergence and loss, andin terms of their antigen specificity.— Au-thors' Abstract

Pervin, K., Childerstone, A., Shinnick, T.,Mizushima, V., Vanderzee, R., Hasan, A.,Vaughan, R. and Lehner, T. T-cell epi-tope expression of mycobacterial and ho-mologous human 65-kilodalton heatshock protein peptides in short term celllines from patients with Behcet's disease.J. Immunol. 151 (1993) 2273-2282.

T-cell epitopes of the 65-kDa heat-shockprotein (HSP) were mapped in patients withBehcet's disease (BD) by stimulating T cellswith the overlapping synthetic peptides de-rived from the sequences of the .1Iycobac-terium tuberculosis 65-kDa HSP. Significantlymphoproliferative responses were stimu-lated with four HSP peptides in BD, as com-pared with the related disease (recurrent oralulcers), unrelated disease, and healthy con-trols (p < 0.05 to 0.005). In order to assessthe relative frequency of sensitized lym-phocytes by these peptides, 7353 short-termcell lines (STCL) were generated from thelymphocytes of patients and controls. Pep-tides 111-125, 154-172, and 311-325 (p< 0.001) and peptide 219-233 (p < 0.02)yielded significantly greater frequency ofSTCL in BD than in healthy and diseasecontrols. All but peptide 154-172 stimu-lated only the CD4+ subset of T cells, al-though there was no evidence that reactivityto the selected peptides is restricted by DR2to DR7 antigen. HLA-B51 is significantlyassociated with BD, but there was no evi-dence that B51 was a restricting element,when B51+ patients were compared with

B51— patients with BD, and with B51+healthy control subjects. A comparative in-vestigation was then carried out between thecorresponding mycobacterial and humanHSP peptides. Similar or higher lympho-proliferative responses were stimulated bythe human peptides compared with the my-cobacterial peptides. These results suggestthat the four peptide determinants withinthe 65-kDa HSP might be involved in thepathogenesis of 13D. Whereas the high mi-crobial load and associated stress proteinsfound in oral ulceration of BD may initiatean immune response to these conserved ep-itopes, expression of autoreactive T-cellclones might be stimulated by immuno-dominant T-cell epitopes of endogenousHSP which may induce immunopathologicchanges.—Authors' Abstract

Pourshafie, M., Ayub, Q. and Barrow, W.W. Comparative effects of Mycobacteri-um avium glycopeptidolipid and lipopep-tide fragment on the function and ultra-structure of mononuclear cells. Clin. Exp.Immunol. 93 (1993) 72-79.

Among the various lipids associated withthe cell envelope of the Mycobacterium av-ium complex, the species-specific glycopep-tidolipids (GPL) are responsible for distin-guishing one serovar from another. In acontinuing effort to study the immunomod-ulatory capabilities of these mycobacteriallipids, we have examined and compared theeffects of the GPL and its lipopeptide frag-ment (beta-lipid) on mononuclear cell func-tion. It was observed that the lymphopro-liferative response of murine splenicmononuclear cells to mitogen stimulationwas reduced by both the GPL and its li-popeptide fragment. Although the respon-siveness appeared to be down-regulated toa greater degree by the beta-lipid, treatmentwith either GPL or beta-lipid resulted in therelease of soluble factors from peritonealmacrophages that caused suppression of thelymphoproliferative responsiveness ofsplenic mononuclear cells. Flow cytometricanalysis of peritoneal macrophages revealedthat treatment with the beta-lipid fragmentcaused a marked decrease in expression ofthe C3bi complement receptor, Mac-1, onmacrophages; whereas treatment with GPLresulted in a marked increase in the ex-

668^ International Journal q/ Leprosy^ 1993

pression of Mac-2 receptor on macro-phages. Treatment of peritoneal macro-phages with either GPL or beta-lipid resultedin the release of tumor necrosis factor (TNF),as determined by an L929 biological cyto-toxicity assay. Perturbation of macrophagemembrane ultrastructure by both GPL andbeta-lipid was confirmed by electron mi-croscopy, and may be a possible explana-tion for the resulting alterations in mono-nuclear cell function observed in thisstudy.—Authors' Abstract

Radhakrishnan, V. V., Mathai, A. and Sun-daram, P. Diagnostic significance of cir-culating immune complexes in patientswith pulmonary tuberculosis. J. Med. Mi-crobiol. 36 (1992) 128-131.

A polyethylene glycol (PEG) precipitationmethod was used to examine sera of pa-tients [in India] with active pulmonary tu-berculosis (PT), leprosy and nontuberculouspulmonary diseases, and of healthy controlsubjects for immune complexes (ICs). My-cobacterium tuberculosis antigen 5 was de-tected in the ICs in 80% of patients with PTby the indirect (sandwich) enzyme-linkedimmunosorbent assay (ELISA). Detectionof mycobacterial antigen in ICs has diag-nostic potential as an adjunct in the labo-ratory diagnosis of PT, particularly whenrepeated bacteriological investigations forM. tuberculosis in clinical specimens arenegative. Levels of ICs tend to decrease withthe duration of antituberculosis chemo-therapy and their detection can also be usedto assess the clinical response to therapy inpatients with PT.—Authors' Summary

Raviglione, M. C., Sudre, P., Rieder,.1-I. L.,Spinaci, S. and Kochi, A. Secular trendsof tuberculosis in Western Europe. Bull.WHO 71 (1993) 297-306.

Deaths due to tuberculosis have de-creased uniformly in all countries in West-ern Europe, and most have occurred amongthose aged 65 years. In recent years, tu-berculosis case notifications have continuedto decline in Belgium, Finland, France, Ger-many, and Spain, and have levelled off inSweden and the United Kingdom; increaseshave, however, been recorded in Austria,Denmark, Ireland, Italy, The Netherlands,Norway, and Switzerland. In Denmark, The

Netherlands, Norway, Sweden, and Swit-zerland an increasing number of cases oftuberculosis among foreign-born residentshas resulted in a change from the expecteddownward trend. Human immunodeficien-cy virus (HIV) infection appears to contrib-ute only marginally to the overall tubercu-losis morbidity; however, it appears to beimportant in Paris and its surrounding ar-eas, and tuberculosis is very common amongHIV-infected persons in Italy and Spain.Despite these recent changes in the inci-dence of tuberculosis, there is currently noevidence of its increased transmissionamong the youngest age groups of the in-digenous populations. Properly designeddisease surveillance systems are critical formonitoring the tuberculosis trends so thateach country can identify its own high-riskgroups and target interventions to prevent,diagnose, and treat the disease. Tubercu-losis remains a global disease and becauseof increasing human migrations, its elimi-nation in Western Europe cannot be envis-aged without concomitant improvements inits control in high-incidence, resource-poorcountries.—Authors' Abstract

Ratnaker, P. and Murthy, P. S. Trifluoper-azine inhibits the incorporation oflabeledprecursors into lipids, proteins and DNAof Mycobacterium tuberculosis H37Rv.FEMS Microbiol. Lett. 110 (1993) 291-294.

We have recently demonstrated that thecalmodulin antagonist trifluoperazine hasantitubercular activity in vitro against My-cobacterium tuberculosis H37R(v) suscep-tible and resistant to isoniazid. It is nowshown that trifluoperazine at a concentra-tion of 50 pg/m1 when added to the cellsalong with the labeled precursors inhibitedthe incorporation of [C-14]acetate into lip-ids (63%) and uptake of [C-14]glycine (74%)and [H-3]thymidine (52%) by whole cells ofM. tuberculosis H37R by 6 hr of exposure.After 48 hr, the inhibition was 87%, 97%and 74%, respectively. However, when thedrug was added to cells taking up and me-tabolizing the labeled precursors at a laterpoint (3 hr for [C-14]acetate and [H-3]thymidine and 12 hr for [C-14]glycine), itinhibited completely the uptake of all theprecursors, at least up to 24 hr. The onset


of inhibitory action was very rapid, i.e., 3hr. It is suggested that trifluoperazine hasmultiple sites of action and acts probablyby affecting the synthesis of lipids, proteinsand DNA.— Authors' Abstract

Riviere, M., Auge, S., Vercauteren, J., Wis-ingerova, E. and Puzo, G. Structure of anovel glycopeptidolipid antigen contain-ing a 0-methylated serine isolated fromMycobacterium xenopi complete H - 1-NMR and C-13-NMR assignment. Eur.J. Biochem. 214 (1993) 395-403.

GPL X-1, a novel glycopeptidolipid(GPL) isolated from Mycobacterium xenopi(CIPT 140 35004), has recently been foundto typify a new class of mycobacterial gly-copeptidolipids devoid of C-mycoside corestructure, the so-called serine-containingglycopeptidolipid [Riviere, M. and Puzo, G.(1991) J. Biol. Chem. 266, 9057-9063].Here we report the purification and char-acterization of a novel serine-containingGPL termed GPL X-IIb, isolated from theM. xenopi strain NCTC 10042. On thin-layer chromatography, this GPL was foundto be present in some other Al. xenopi strainsisolated from patients with pulmonary in-fections. The sugar and amino-acid com-positions of this GPL were elucidated fromthe native form using a combination of two-dimensional homonuclear and heteronu-clear scalar coupling NMR. The peptide andsugar sequences, as well as the methoxylgroup locations on the C-3 of the 6-deoxy-alpha-L-talopyranoside (6dTalp) and on aSer, were unambiguously determined byheteronuclear multiple-bond correlationexperiments. GPL X-IIb was found to becomposed of a lipotetrapeptide of the fol-lowing structure C-12-Ser-OMe-Ser-Phe-aThr-OMe (aThr = allothreonine). The sug-ar part is made up of 3OMe-alpha-L-6dTalpand the following disaccharide: alpha-L-Rhap-(1-3)-2-0-Lau-alpha-L-Rhap (Rhap= rhanmopyranose). Unlike GPL X-I, thesugar attachment sites on the tetrapeptidewere successfully determined from hetero-nuclear three-bond coupling correlation ob-served in the heteronuclear multiple-bondcorrelation spectrum between the anomericcarbon resonances and the beta protons ofaThr-OMe and Ser. It was established thatthe 3OMe-6dTalp glycosylates the Ser while

the disaccharide is linked to the aThr-OMe.Thus, both GPL X-1 and GPL X-IIb sharea common lipotetrapeptide core [with theexception of Ser(OMe)] but drastically differin their oligosaccharide appendage. Thus,by analogy with the M. avium complex, thepresent report suggests that M. xenopi spe-cies can be divided in various serovars char-acterized by the unique structure of theirC-mycoside GPL oligosaccharide append-age, enhancing the interest for this new typeof serine-containing glycopeptidolipid. —Authors' Abstract

Thibert, L. and Lapierre, S. Routine appli-cation of high-performance liquid chro-matography for identification of myco-bacteria. J. Clin. Microbiol. 31 (1993)1759-1763.

Mycolic acid analysis by high-perfor-mance liquid chromatography (HPLC) wasintroduced in our laboratory as the routinetechnique for identifying all clinical isolatesof mycobacteria referred to us. HPLC iden-tified 96.1% of the 1103 strains analyzed;whereas the biochemical procedures and/orthe commercial DNA probes identified98.3% of strains, for an overall agreementof 94.4%. Compared with the probes, therewas 100% specificity and 98.9% sensitivityfor Mycobacterium tuberculosis identifica-tion. HPLC allowed early detection andidentification of the rare mycobacterial spe-cies Al. haemophilum, M. malmoense, M.shimoidei, and M. fallax as well as unchar-acteristic strains of M. simiae. After 18months of routine use, HPLC proved to bereliable, easy to perform, rapid, and lesscostly than other identification methods. —Authors' Abstract

Venisse, A., Berjeaud, J.-M., Chaurand, P.,Gilleron, M. and Puzo, G. Structural fea-tures of lipoarabinomannan from Myco-bacterium bolls BCG; determination ofmolecular mass by laser desorption massspectrometry. J. Biol. Chem. 268 (1993)12401-12411.

It was recently shown that mycobacteriallipoarabinomannan (LAM) can be classifiedinto two types (Chatterjee, D., Lowell, K.,Rivoire, B., McNeil, M. R., and Brennan,P. J. (1992) J. Biol. Chem. 267, 6234-6239)according to the presence or absence of


mannosyl residues (Manp) located at thenonreducing end of the oligoarabinosyl sidechains. These two types of LAM were foundin a pathogenic Mycobacterium tuberculosisstrain and in an avirulent M. tuberculosisstrain, respectively, suggesting that LAMwith Manp characterizes virulent and "dis-ease—inducing strains." We now report thestructure of the LAM from M. boris bacilleCalmette-Guêrin (BCG) strain Pasteur,largely used throughout the world as vaccineagainst tuberculosis. Using an up-to-dateanalytical approach, we found that the LAMof Al. boris BCG belongs to the class ofLAMs capped with Manp. By means of two-dimensional homonuclear and heteronu-clear scalar coupling NMR analysis andmethylation data, the sugar spin system as-signments were partially established, re-vealing that the LAM contained two typesof terminal Manp and 2-0-linked Manp.From the following four-step process: (i)partial hydrolysis of deacylated LAM(dLAM), (ii) oligosaccharide derivatizationwith aminobenzoic ethyl ester, (iii) HPLCpurification, (iv) FAB/MS-MS analysis; itwas shown that the dimannosyl unit a-D-Manp-(1 ---)2)-a-D-Manp is the major resi-due capping the termini of the arabinan ofthe LAM. In this report, LAM molecularmass determination was established usingmatrix-assisted UV-laser desorption/ion-ization mass spectrometry which reveals thatthe LAM molecular mass is around 17.4kDa. The similarity of the LAM structuresbetween M. boris BCG and Al. tuberculosisH37Rv is discussed in regard to their func-tion in the immunopathology of mycobac-terial infection. — Authors' Abstract

Verbon, A., Weverling, G. J., Kuijper, S.,Speelman, P., Jansen, H. M. and Kolk,A. H. J. Evaluation of different test forthe serodiagnosis of tuberculosis and theuse of likelihood ratios in serology. Am.Rev. Respir. Dis. 148 (1993) 378-384.

A serologic test for the diagnosis of tu-berculosis was evaluated in 91 newly di-agnosed tuberculosis (TB) patients of whom15 were HIV positive, in 17 TB patientsduring treatment, and in 220 control sub-jects (including individuals from endemicareas and patients with sarcoidosis orCrohn's disease). Purified proteins of My-

cobacterium tuberculosis with molecularweights of 10,000, 16,000, 24,000, 30,000,38,000, and 70,000 were tested by ELISA.In addition, monoclonal antibody TB72 wastested by competition ELISA. The cutoffvalues were set at the mean plus three stan-dard deviations of the values obtained in100 healthy Dutch army recruits. Only theELISA with the 10,000, 16,000, and 24,000antigens and the TB72 assay discriminatedbetween patients with TB who were not HIVpositive and control subjects. Specificityvaried from 95% to 98% and sensitivity from29% to 51% with the different antigens.Combination of the test results of the ELISAwith the 16,000 antigen and the TB72 assayhad a sensitivity of 65% (95% confidenceinterval, 53% to 75%) and a specificity of96% (95% confidence interval, 92% to 98%).The assay was useful for the diagnosis ofboth pulmonary and extrapulmonary TB.Optimal use of the serologic assay could beobtained when likelihood ratios for each testvalue are calculated instead of using the testdichotomized (positive or negative). Highpost-test probabilities indicate the presenceof TB; low post-test probabilities do not ex-clude the disease and should lead to addi-tional investigations. —Authors' Abstract

Wheeler, P. R., Besra, G. S., Minnikin, D.E. and Ratledge, C. Inhibition of mycolicacid biosynthesis in a cell-wall prepara-tion from Mycobacterium smegmatis bymethyl 4-(2-octadecylcyclopropen- l -Y 1)butanoate, a structural analog of a keyprecursor. Lett. Appl. Microbiol. 17(1993) 33-36.

[C-14]acetate was incorporated into my-colic acids by a cell-free, cell-wall fractionfrom Mycobacterium smegmatis. This ac-tivity was inhibited by methyl 4-(2-octa-decylcyclopropen-l-y1) butanoate which wasdesigned as a structural analog of cis-tetra-cos-5-enoate, a precursor of mycolic acidbiosynthesis. Other fatty acids and theirmethyl esters failed to inhibit mycolic acidbiosynthesis at the concentration 1-2 mg/m1-1, at which methyl 4-(2-octadecylcyclo-propen-l-y1) butanoate was effective. Thus,a novel agent was shown to act against anenzyme activity or target involved specifi-cally in biosynthesis of a characteristic, my-cobacterial, cell-wall component.—Au-thors' Abstract


Witzig, R. S. and Franzblau, S. G. Suscep-tibility of Mycobacterium kansasii toofloxacin, sparfloxacin, clarithromycin,azithromycin, and fusidic acid. Antimi-crob. Agents Chemother. 37 (1993) 1997-1999.

The MICs of ofloxacin, sparfloxacin, clar-ithromycin, azithromycin, and fusidic acidfor clinical isolates of Mycobacterium kan-sasii were determined by the radiometric(BACTEC ) method. All drugs exceptazithromycin elicited MICs for 90% of thestrains tested that were lower than previ-ously reported achievable maximum con-centrations in serum. Ofloxacin, sparfloxa-cin, and clarithromycin had the largestmaximum concentration in serum/MIC for90% of strains ratio of the drugs tested. —Authors' Abstract

Zhang, Y. and Rom, W. N. Regulation ofinterleukin- 1 -beta (IL-1-beta) gene bymycobacterial components and lipopoly-saccharide is mediated by two nuclearfactor-IL6 motifs. Mol. Cell. Biol. 13(1993) 3831-3837.

The cytokines interleukin- 1 beta (IL-1 beta) and tumor necrosis factor alpha(TNF-alpha) are released by mononuclearphagocytes in vitro after stimulation with

mycobacteria and are considered to mediatepathophysiologic events, including granu-loma formation and systemic symptoms. Wedemonstrated that the Mycobacterium tu-berculosis cell-wall component lipoarabi-nomannan (LAM) is a very potent inducerof the IL-1 beta gene expression in humanmonocytes, and investigated the mecha-nism of this effect. We localized the LAM-,lipopolysaccharide (LPS)-, and TNF-alpha-inducible promoter activity to a — 131/ +15(positions —131 to + 15) DNA fragment ofthe IL-1 beta gene by deletion analysis andchloramphenicol acetyltransferase assay.Within this DNA fragment, there were twonovel 9-bp motifs (-90/-82 and —40/-32)with high homology to the nuclear factor-IL6 (NF-IL6) binding site. Site-directedmutagenesis demonstrated that the two NF-IL-6 motifs could be independently acti-vated by LAM, LPS, or TNF-alpha, andthat they acted in an orientation-indepen-dent manner. DNA mobility shift assay re-vealed specific binding of nuclear protein(s)from LAM-, LPS-, or TNF-alpha-stimulat-ed THP-1 cells to the NF-IL6 motifs. Weconclude that the two NF-IL6 sites mediateinduction of IL- 1 beta in response to thestimuli LAM, LPS, and TNF-alpha. —Au-thors' Abstract

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