INTERNATIONAL JOURNALOF LEPROSY Volume 72, Number 4 ...ila.ilsl.br/pdfs/v72n4a17.pdf · Eating...

Anonymous. A Romanist View of the Fish-Eating Leprosy Theory. Archives ACen-tury Ago. Arch. Dermatol. 140 (2004)789.

Even in an age of masterful clinicians, SirJonathan Hutchinson was exceptional. Inaddition to his clinical work, Sir Jonathanwas a prolific writer serving as editor for atime of the British Medical Journal . . . and,most incredibly, from 1889 –1900 single-handedly writing every article in his 1-manjournal, the Archives of Clinical Surgery. SirJonathan was a medical polymath, an expertin ophthalmology , neurology , pathology ,surgery, and dermatology. It is in syphilol-ogy where his eponymous fame is most en-during . . . having seen over 1 million cases(1) of syphilis. . .

Unfortunately appended to his illustriouscareer will always be the enduring embar-rassment of his idée fixe —that leprosy iscaused by eating rotten fish. Notwithstand-ing the questionable geography or the less-than-obvious logical dietary leap, for therest of his life Hutchinson espoused his doc-trine despite universal rejection by contem-

porary leprologists. Even the discovery ofthe leprosy bacillus by his good friend Ger-hard Hansen in 1874 couldn’ t dissuadeHutchinson. Hutchinson was mildly non-plused when no one could discover [thebacillus] in any fish. . . Hutchinson con-fessed that one reason he never sought to ex-perimentally test his theory was “the assuredconviction that the general facts were over-whelmingly conclusive and that they neededonly to be clearly set forth.” Indeed the onlyreason Hutchinson’s theory gained any cir-culation at all, if never actual acceptance,was because of his otherwise impeccablereputation.—[Condensed from the essay .DMS] Archives of Dermatology

Al Samie, A. R., and Al Qubati, Y. Leprosycontrol in the Republic of Yemen: co-operation between government and non-government or ganizations, 1989 –2003.Lepr. Rev. 75(2) (2004) 164–170.

Although the prevalence rate of leprosy inthe Republic of Yemen has dropped belowthe WHO elimination level of less than one

INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 4Printed in the U.S.A.

(ISSN 0148-916X)

CURRENT LITERATURE

This department carries selected abstracts of articles published in curr ent medicaljournals dealing with leprosy and other mycobacterial diseases.

General and Historical 509Chemotherapy 513Clinical Sciences 516Immunopathology 525

Leprosy 531Tuberculosis 535

Microbiology 544Leprosy 547Tuberculosis 547

Experimental Infections and Vaccines 551Epidemiology and Prevention 555Rehabilitation and Social Concerns 557Other Mycobacterial Diseases 558Molecular and Genetic Studies 565

General and Historical

509

case per 10,000 of the population, it is stillregarded as a serious public health problemcalling for continued vigilance, notably inthe detection and treatment of hidden andundiagnosed cases. In the past, religiousmisinterpretation has generated adverse be-haviour patterns towards people affected byleprosy, characterized by aggression, negli-gence and isolation. Until about 1982, fol-lowing a visit of a leprologist (Dr . S. K.Noordeen) from the World Health Organi-zation, there was no leprosy control programand attempts to establish one remained inef-fective until in 1989, when an agreementwas signed between the Ministry of PublicHealth and Population and the German Lep-rosy Relief Association. This led to the de-velopment of a leprosy control program infour governorates, later extended to the restof the country . This paper describes theprogress made in the control of leprosy inthe Yemen, 1989–2003, by the Ministry ofHealth and Population and the GLRA, in as-sociation with two local societies.—Au-thors’Abstract

Donoghue, H. D., Spigelman, M., Green-blatt, C. L., Lev-Maor, G., Bar-Gal, G.K., Matheson, C., Vernon, K., Nerlich,A. G., and Zink, A. R. Tuberculosis:from prehistory to Robert Koch, as re-vealed by ancient DNA. Lancet Infect.Dis. 4(9) (2004) 584–592.

During the past 10 years palaeomicrobi-ology, a new scientific discipline, has devel-oped. The study of ancient pathogens by di-rect detection of their DNA has answeredseveral historical questions and shownchanges to pathogens over time. However,ancient DNA (aDNA) continues to be con-troversial and great care is needed to providevalid data. Here we review the most suc-cessful application of the technology, whichis the study of tuberculosis. This has pro-vided direct support for the current theory ofMycobacterium tuberculosis evolution, andsuggests areas of investigation for the inter-action of M. tuberculosis with its host.—Au-thors’Abstract

Hughes, M. S., James, G., Taylor, M. J.,McCarroll, J., Neill, S. D., Chen, S. C.,

Mitchell, D. H., Love, D. N., and Malik,R. PCR studies of feline leprosy cases. J.Feline Med. Surg. 6(4) (2004) 235–243.

16S rRNA gene sequence analysis pro-vided evidence for two different mycobac-terial species, Mycobacterium leprae-murium and a potentially novel species, ascausative agents of “feline leprosy.” Com-parison of 16S rRNA gene sequence dataobtained for M. lepraemurium and the po-tentially novel species indicated 12 nu-cleotide differences over a 446 bp region en-compassing the V2 and V3 hypervariableregions. From available 16S rRNAgene se-quence data, M. lepraemurium shared great-est nucleotide identity with M. avium subsp.paratuberculosis and M. avium. The novelspecies had a long helix 18 in the V3 regionand shared greatest nucleotide identity withM. leprae , M. haemophilum and M. mal-moense. The novel species had an additional‘A’ nucleotide at position 105 of the aligned16S rRNA gene sequence, the only othermycobacterial database sequence havingthis same extra nucleotide being M. leprae.This nucleotide variation was exploited to de-velop specific PCR assays for the two species.These were found to be effective and specificwhen tested against a panel of mycobacteriaincluding species found in feline leprosy le-sions and closely related mycobacteria andalso when applied directly to formalin-fixed,paraffin-embedded tissues from feline lep-rosy cases.—Authors’Abstract

Jianping, S., Wenzhong, L., Meiwen, Y.,Jun, Y., Longchao, Z., Rongmao, W.,Lufang, H., Hongjiang, M., Fuchang,Y., Xinguo, H., and Liangde, P. Analy-sis on the detection of new leprosy casesbefore, during and after the year of lep-rosy elimination campaigns. Lepr. Rev.75(2) (2004) 157–163.

To analyze the impact on of case findingof leprosy elimination campaigns (LECs),data on newly detected leprosy cases in aleprosy endemic area were collected before,during and after the year of LEC. The num-ber of new leprosy cases detected during theyear of LEC was significantly higher thanpreviously. The number of newly detectedcases after the year of LEC was similar to

510 International Journal of Leprosy 2004

that of detected before the year of LEC incounties with persisting case finding activi-ties. However, the number of newly detectedcases after the year of LEC significantly de-creased in counties without active case find-ing activities. The average distance from thehomes of leprosy cases detected during LECto the leprosy control unit at the count townwas 62.8 km, which is farther than that ofother leprosy cases detected before and afterthe year of LEC. The average time from dis-ease onset to diagnosis of leprosy cases de-tected after the year of LEC shortened. Theresults also showed that carrying out LECsis unlikely to have a significant impact onthe trend of case finding within a short timein local areas, but it may improve some in-dicators of leprosy patients and so promoteleprosy control in local areas.—Authors’Abstract

Karthikeyan, K., and Thappa D. M. Mod-ular teaching program on leprosy. IndianJ. Lepr. 75(4) (2003) 317–325.

Though repeated attention has beendrawn to a lack of proper teaching-learningmodules in leprosy endemic countries, nosatisfactory module exists. Keeping in viewthis fact, we attempted to draft a suitablemodule on leprosy that could be used toteach leprosy to undergraduate medical stu-dents in a simple and comprehensive man-ner. We used two different modules, ModuleA and Module B, to teach two dif ferentbatches of students of the pre-final year (VIand VII semesters) of the MBBS course.Both these modules were conducted by theDepartment of Dermatology and STD, withparticipation by the Departments of Micro-biology, Pathology and Preventive and So-cial Medicine. The drafts of the moduleswere discussed before hand in the Depart-ment, keeping in mind the number of daysallotted to us. Both the modules were dif-ferent in certain aspects, but the basic con-cept was the same. Because Module A hadmore time, certain practical aspects werealso discussed. It was interesting to note thatthe percentage of increase in the post-testscore was 17 for Module A and 15 for Mod-ule B, thus proving that both the moduleswere effective in conveying the core mes-sage about leprosy.—Authors’Abstract

Meima, A., Smith, W. C., van Oort-marssen, G. J., Richardus, J. H., andHabbema, J. D. The future incidence ofleprosy: a scenario analysis. Bull. WorldHealth Organ. 82(5) (2004) 373–380.

OBJECTIVE: To investigate the impactof the current strategy for the elimination ofleprosy on its incidence and to assess theconsequences of failure to sustain thisstrategy. METHODS: Scenarios for assess-ing the impact of the elimination strategywere implemented in a computer simulationprogram. The scenarios reflected the as-sumptions made regarding contagiousness,transmission and bacille Calmette-Guerin(BCG) vaccination. The trend in case detec-tion rate for the main countries in which lep-rosy was endemic during 1985 –1998 wasfitted, and incidence up to 2020 was pro-jected. FINDINGS: Owing to the gradualshortening of delays in detection up to 1998,and because of the low relapse rate that oc-curs with multidrug treatment MDT , inci-dence is predicted to decrease beyond 2000in all scenarios. The annual decline was afew per cent higher when favourable as-sumptions were made about protection andcoverage of BCG vaccination. Overall, thepredicted annual decline in incidencesranged from 2% to 12%. CONCLUSION:The elimination strategy reduces transmis-sion, but the decline may be slow . Relax-ation of control after 2005 is unjustifiedgiven the uncertainty about the rate of de-cline and the adverse effects of longer de-lays in detection. A long-term strategy forleprosy control should be adopted.—Au-thors’Abstract

Rao, T. P., Krishnamurthy, P., Vijayaku-maran, P., Mishra, R. K., and Samy, M.S. “Instant” new leprosy case-detection:an experience in Bihar State in India. In-dian J. Lepr. 75(1) (2003) 9–15.

The National Leprosy Eradication Pro-gramme (NLEP) is based on survey, educa-tion and treatment, including coverage of allthe registered cases with multi-drug therapy(MDT). The Government of India intro-duced MDT in all leprosy endemic districtsthrough a vertical set-up, and through mo-bile leprosy treatment units in low endemic

72, 4 Current Literature, General and Historical 511

districts. Anti-leprosy work has not beenuniform in all the states and needed push-start in some, such as Bihar . There havebeen spurts of leprosy elimination activitiesand the entire populations of the regionshave not been covered because of variousadministrative reasons and logistic prob-lems. In Singhbhum district of Bihar, a suc-cessful attempt was made to cover the max-imum population by campaign approach.The strategy was to involve all the fieldworkers of the leprosy program in the dis-trict, supported by a small group of experi-enced personnel. The campaign, lasting for39 working days, resulted in detecting lep-rosy cases equivalent to 64% of cases de-tected during the previous one full year. Theentire operation helped the local staff to gainexperience that would be useful for the fu-ture of the NLEP, and also provide an in-sight into working practices. Similar cam-paign approach can be used in situationswhere case-detection activities are feebleand the implementation of MDT is slow. Ifsuch campaigns are repeated at appropriateintervals, it will be a great support to achiev-ing the goal of leprosy elimination.—Au-thors’Abstract

Subramanian, M., Showkath Ali, M. K.,Thorat, D. M., Muthukumar , M.,Sathiskumar, E., Ramadoss, C., andAli Khan, M. Leprosy situation in en-demic states of India and prospects ofelimination of the disease. Indian J. Lepr.75(4) (2003) 335–345.

In India there is a dramatic fall in theprevalence rate (PR) of leprosy, but the newcase-detection rate (NCDR) has not been re-duced concomitantly. It is the operational ef-ficiency of the National Leprosy EradicationProgramme (NLEP) that has led to a signif-icant reduction in the NCDR in AndhraPradesh and Tamil Nadu. The ratio of PR toNCDR has been declining in these twostates and it reveals that elimination couldbe reached even with the high NCDR levelof 3 to 4 per 10,000 population, particularlyif single skin lesion (SSL) cases are dis-charged through single dose treatment of ri-fampicin, ofloxacin and minocycline (ROM).On the other hand, the significant number ofcases detected in Bihar and Orissa during

modified leprosy elimination campaigns(MLECs) reveals that there are lacunae inoperational activities in new case-detectionresulting in a lar ge number of undetectedcases in the community. Only one-third ofthe cases are reporting voluntarily. Aware-ness of leprosy is not adequate to motivatethe patients to report voluntarily and com-plete their treatment, thus underscoring theneed for relying on active case-detection sothat transmission can be broken and elimi-nation of leprosy achieved. In addition, theinfluence of socio-economic factors on con-tinued occurrence of leprosy cannot be ruledout. The establishment of a sentinel surveil-lance system along with a computerizedsimplified information system to gain in-depth knowledge on the functioning of theNLEP will ensure operational efficiency. Inview of this situation, the NLEP shouldadopt a more realistic approach towardsreaching the elimination goal.—Authors’Abstract

Visschedijk, J., Engelhard, A., Lever, P.,de faria Grossi, M. A., and Feenstra, P.Leprosy control strategies and the inte-gration of health services: an interna-tional perspective. Cad. Saúde Pública19(6) (2003) 1567–1581.

Abstract. Integration of leprosy servicesinto the general health services is regardedas the core strategy to ensure that leprosycontrol remains cost-effective and equitable,and, thus, sustainable in the coming years.In this article an extensive review is pre-sented of the integration of leprosy servicesinto the general health services. After the ra-tionale of integration is discussed, the arti-cle highlights several recent developmentswithin leprosy control and the health sectorthat are in support of the integration process.An overview is presented of recent experi-ences in countries that have already em-barked on the integration process. Based onthese experiences important lessons can belearned and incorporated into a model forthe process of integration. This model,which is presented at the end of the article,will assist countries to successfully integrateleprosy services into the general health ser-vices.—Cadernos de Saúde Pública


Bermudez, L. E., Kolonoski, P ., Seitz, L.E., Petrofsky, M., Reynolds, R., Wu, M.,and Young, L. S.SRI-286, a thiosemicar-bazole, in combination with mefloquineand moxifloxacin for treatment of murineMycobacterium avium complex disease.Antimicrob. Agents Chemother. 48(9)(2004) 3556–3558.

Treatment of Mycobacterium avium dis-ease remains challenging when macrolideresistance develops. We infected C57 beigemice and treated them with mefloquine,SRI-286, and moxifloxacin. SRI-286 (80mg/kg) was bactericidal in the liver. Meflo-quine plus moxifloxacin or mefloquine plusSRI-286 were better than mefloquinealone.—Authors’Abstract

de Savary, N., Lee, R., and Vaidya, B. Severehypothyroidism after thalidomide treatment.J. Roy. Soc. Med. 97 (2004) 443–445.

An important but little recognized side-effect of thalidomide is hypothyroidism.

Three cases of thalidomide-induced hy-pothyroidism were reported during the early1960s. In 2002, Badros and colleagues re-ported a patient who developed severe hy-pothyroidism about three months after start-ing thalidomide for multiple myeloma.Furthermore, they found that 14% of patientson thalidomide treatment were subclinicallyhypothyroid (TSH >10 µ/L) at three months,and suggested that thyroid dysfunction mightcontribute to some of the known side-effectsof the drug, such as fatigue, constipation andbradycardia.—[Abstracted from text] Jour-nal of the Royal Society of Medicine

Douglas, J. T., Cellona, R. V., Fajardo, T.T. Jr., Abalos, R. M., Balagon, M. V.,and Klatser, P. R. Prospective study ofserological conversion as a risk factor fordevelopment of leprosy among house-hold contacts. Clin. Diagn. Lab. Im-munol. 11(5) (2004) 897–900.

See Current Literature, Epidemiology andPrevention, p. 556.

Fernandez-Martinez, E., Morales-Rios,M. S., Perez-Alvarez, V., and Muriel, P.Immunomodulatory ef fects of thalido-mide analogs on LPS-induced plasma andhepatic cytokines in the rat. Biochem.Pharmacol. 68(7) (2004) 1321–1329.

Thalidomide has shown to inhibit, selec-tively and mainly the cytokine tumor necro-sis factor-alpha (TNF-alpha), thus, thalido-mide has inhibitory consequences on othercytokines; this is ascribed as an im-munomodulatory effect. Novel thalidomideanalogs are reported with immunomodula-tory activity. The aim of this work was tosynthesize some of these analogs and to as-sess them as immunomodulatory agents inan acute model of LPS-induced septic chal-lenge in rat. Animal groups received orallytwice a day vehicle carboxymethylcellulose(0.9%), or thalidomide in suspension (100mg/kg), or analogs in an equimolar dose.Two hours after last dose, rats were injectedwith saline (NaCl, 0.9%, i.p.) or LPS (5mg/kg, i.p.). Groups were sacrificed 2 hrafter injection and samples of blood and liverwere obtained. TNF-alpha, interleukin-6, -1beta, and -10 (IL-6, IL-1beta, IL-10) werequantified by enzyme linked immunosorbentassay (ELISA) and studied in plasma andliver. After 2 hr of LPS-induction, differentpatterns of measured cytokines were ob-served with thalidomide analogs adminis-tration evidencing their immunomodulatoryeffects. Interestingly , some analogs de-creased significantly plasma and hepaticlevels of LPS-induced proinflammatoryTNF-alpha and others increased plasmaconcentration of anti-inflammatory IL-10.Thalidomide analogs also showed slight ef-fects on the remaining proinflammatory cy-tokines. Differences among immunomodu-latory effects of analogs can be related topotency, mechanism of action, and halflives. Thalidomide analogs could be used asa pharmacological tool and in therapeuticsin the future.—Authors’Abstract

Makino, K., Nakajima, T., Shikamura,M., Ito, F ., Ando, S., Kochi, C., Ina-

72, 4 Current Literature, Chemotherapy 513

Chemotherapy

gawa, H., Soma, G., and Terada, H. Ef-ficient intracellular delivery of rifampicinto alveolar macrophages using rifampicin-loaded PLGA microspheres: ef fects ofmolecular weight and composition ofPLGA on release of rifampicin. ColloidsSurf B Biointerfaces. 36(1) (2004) 35–42.

Monodispersed PLGAmicrospheres con-taining rifampicin (RFP) have been pre-pared by solvent evaporation method usinga Shirasu porous glass (SPG) membrane.The microspheres were spherical and theiraverage diameter was about 2 microm. Theloading ef ficiency of rifampicin was de-pendent on the molecular weight of PLGA.The higher loading efficiency was obtainedby the usage of PLGAwith the lower molec-ular weight, which may be caused by the in-teraction of the amino groups of rifampicinwith the terminal carboxyl groups of PLGA.PLGA with the monomer compositions of50/50 and 75/25, of lactic acid/glycolic acid,were used in this study . From rifampicin-loaded PLGA microspheres formulatedusing PLGA with the molecular weight of20,000, rifampicin was released with almostconstant rate for 20 days after the lag phasewas observed for the initial 7 days at pH 7.4.On the other hand, from rifampicin-loadedPLGA microspheres formulated usingPLGA with the molecular weight of 5000 or10,000, almost 90% of rifampicin-loaded inthe microspheres was released in the initial10 days. Highly ef fective delivery of ri-fampicin to alveolar macrophages was ob-served by the usage of rifampicin-loadedPLGA microspheres. Almost 19 timeshigher concentration of rifampicin wasfound to be incorporated in alveolar macro-phages when rifampicin-loaded PLGA mi-crospheres were added to the cell culturemedium than when rifampicin solution wasadded.—Authors’Abstract

Maurer, T., Poncelet, A., and Berger, T.Thalidomide treatment for prurigo nodu-laris in human immunodeficiency virus-infected subjects: ef ficacy and risk ofneuropathy. Arch. Dermatol. 140(7)(2004) 845–849.

OBJECTIVE: To evaluate safety and effi-cacy of thalidomide in the treatment of

prurigo nodularis in a group of human im-munodeficiency virus (HIV)-infected pa-tients whose condition was recalcitrant tostandard treatment. DESIGN: Prospectivestudy. SETTING: Outpatient dermatologyand neurology clinic, both referral set-tings.PATIENTS: Eight HIV -infected pa-tients with refractory prurigo nodularis; atotal of 10 met inclusion criteria, but 2 couldnot be followed up. INTER VENTIONS:Treatment with thalidomide, 100 mg/d. Sub-jects were randomized after 1 month to re-ceive 100 or 200 mg/d. If side ef fects werenoted, the drug was reduced to a tolerabledose or discontinued. Subjects were moni-tored at baseline and monthly for degree ofpruritus and total area of body involvementof prurigo nodularis. Sequential neurologicassessments were performed. MAIN OUT-COME MEASURES: Efficacy and toxic ef-fects. RESULTS: The dosage of thalidomideranged from 33 to 200 mg/d. Eight subjectshad a greater than 50% response in reduc-tion of itch over 3.4 months (average).Seven subjects had a greater than 50% re-duction of skin involvement over 5 months(average). Three subjects developed tha-lidomide peripheral neuropathy (TPN).There was no correlation between durationof treatment, daily or cumulative dose, andTPN. A change in the Neuropathy Impair-ment Score of 10 points was a good markerof TPN, as was a greater than 50% decreasein the sural sensory nerve action potentialamplitude. CONCLUSIONS: Thalidomidereduced the signs and symptoms of prurigonodularis in HIV -infected subjects. Onethird of subjects developed TPN, under-scoring the importance of careful neurologicassessment.—Authors’Abstract

Noguchi, T., Sano, H., Shimazawa, R.,Tanatani, A., Miyachi, H., and Hashi-moto, Y. Phenylhomophthalimide-typeNOS inhibitors derived from thalido-mide. Bioorg. Med. Chem. Lett. 14(16)(2004) 4141–4145.

Thalidomide shows moderate inhibitory ac-tivity toward neuronal nitric oxide synthase(nNOS) and inducible NOS (iNOS), but nottoward endothelial NOS (eNOS). Structuraldevelopment studies of thalidomide yieldednovel phenylhomophthalimide-type NOS in-


hibitors with enhanced activity and differentsubtype selectivity.—Authors’Abstract

Seidel, V., and Taylor, P. W. In vitro ac-tivity of extracts and constituents ofPelagonium against rapidly growing my-cobacteria. Int. J. Antimicrob. Agents.23(6) (2004) 613–619.

Extracts of the roots of plants of the Gera-niaceae family have been used for many yearsin South Africa as native herbal remedies andthere is circumstantial evidence for efficacyin the treatment of pulmonary tuberculosis.We have examined dried roots of Pelargo-nium reniforme and P. sidoides for antibac-terial activity against rapidly growing my-cobacteria. Fractions with activity againstMycobacterium aurum and M. smegmatiswere obtained from both plant species bybioassay-guided fractionation of n-hexaneextracts and were found to contain mixturesof straight-chain fatty acids. Analysis by gaschromatography-mass spectrometry (GC-MS) of the corresponding fatty acid methylesters revealed structures with chain lengthsranging from C12 to C26. Unsaturated com-pounds were analyzed as the correspondingdimethyl disulfide adducts to determinedouble-bond positions. Active mixtures dif-fered in the relative abundance of their com-ponents, but all contained 16:0 (palmitic),Delta9-18:1 (oleic) and Delta9,12-18:2(linoleic acid) as the major components.When tested against M. aurum , M. smeg-matis and other rapidly growing mycobac-teria ( M. fortuitum , M. abscessus and M.phlei), all saturated compounds except 12:0were devoid of antimycobacterial activity,whereas unsaturated compounds showed an-timycobacterial activity related to their de-gree of unsaturation, their chain length andthe bacterial species tested. The most potentcompound was linoleic acid, with MIC of 2mg/l against M. aurum.—Authors’Abstract

Shimizu, S., Suzuki, M., Tomoda, A.,Arai, S., Taguchi, H., Hanawa, T., andKamiya, S. Phenoxazine compounds pro-duced by the reactions with bovine hemo-globin show antimicrobial activity againstnon-tuberculosis mycobacteria. Tohoku J.Exp. Med. 203(1) (2004) 47–52.

We studied the anti-microbial ef fects ofphenoxazines produced by the reaction of o-aminophenol or its derivatives with bovinehemoglobin, on seven species of mycobacte-ria such as Mycobacterium tuberculosis, My-cobacterium marinum , Mycobacterium in-tracellulare, Mycobacterium scrofulaceum,Mycobacterium fortuitum , Mycobacteriumkansasii and Mycobacterium smegmatis andsome bacteria such as Escherichia coli ,Pseudomonas aeruginosa, Salmonella enter-ica ser ovar Typhimurium, Staphylococcusaureus, Listeria monocytogeneses . Thesephenoxazines, including 2-amino-4, 4alpha-dihydro-4alpha, 7-dimethyl-3H-phenoxzine-3-one (Phx-1), 3-amino-1, 4alpha-dihydro-4alpha, 8-dimethyl-2H-phenoxazine-2-one(Phx-2), and 2-aminophenoxazine-3-one(Phx-3), prevented the proliferation of fournon-tuberculosis mycobacteria including M.scrofulaceum, M. kansasii, M. marinum, andM. intracellulare dose-dependently, thoughthe inhibitory effects of these phenoxazinesdiffered according to the species of myco-bacteria. However these phenoxazines failedto prevent the proliferation of M. tuberculo-sis, M. fortuitum, and M. smegmatis, and theconcerned bacteria other than mycobacteria.The present results may contribute to devel-opment of novel antibiotics against non-tuberculolsis mycobacteria.—Authors’ Ab-stract

Ziglam, H. M., Daniels, I., and Finch, R.G. Immunomodulating activity of ri-fampicin. J. Chemother . 16(4) (2004)357–361.

It has been shown that some antibioticscan modify cytokine production. We haveexamined the effect of rifampicin on secre-tion of interleukin-1beta (IL-1beta), IL-6,IL-10, and tumor necrosis factor alpha(TNF-alpha) by lipopolysaccharide (LPS)-stimulated or heat killed staphylococci (Pan-sorbin) stimulated monocytes. Secretion ofIL-1beta and TNF-a were significantly in-hibited (p <0.002) whereas secretion of IL-6and IL-10 were significantly increased (p<0.003) by rifampicin treated mononuclearcells. Rifampicin had immunomodulatoryeffects through its capacity to alter the se-cretion of tested cytokines by human mono-cytes.—Authors’Abstract

72, 4 Current Literature, Chemotherapy 515

Arruda, A. P., Marques, W. Jr., Foss, N.T., Garbino, J. A., Virmond, M., andBarreira, A. A. Near nerve potential ofsural nerve in leprosy . Arq. Neuropsi-quiatr. 62(3A) (2004) 571–574. Epub2004 Aug. 24.

Leprosy neuropathy is characterized byinitial involvement of the small nerve fibers,later followed by involvement of the largefibers, when routine nerve conduction stud-ies become abnormal. To increase the diag-nostic yield and precocity of these studies,we applied the near nerve technique to thesural nerve of 8 leprosy patients. Contrary toour expectations, the main component of thesural nerve sensory action potential was ab-normal in all patients, but the minimum con-duction velocity originating from small 3–6mm fibers was normal or only mildly in-volved in three patients. Also, althoughSchwann cells are the first to be involved inleprosy, the results are suggestive of axonaldegeneration instead of demyelination. Tobetter understand the neurophysiology andphysiology of leprosy and to increase the ac-curacy and precocity of the diagnosis, it willbe necessary to investigate patients in thevery early stages of the disease and to cor-relate these findings with the correspondingnerve pathology.—Authors’Abstract

Bhatia, A., Nanda, S., Gupta, U., Gupta,S., and Reddy, B. Topical phenytoin sus-pension and normal saline in the treat-ment of leprosy trophic ulcers: arandom-ized, double-blind, comparative study. J.Dermatolog. T reat. 15(5) (2004)321–327.

OBJECTIVE: To evaluate and comparetwo strengths of topical phenytoin sodiumsuspension (2% and 4%) with normal salinein the healing of acute trophic ulcers in lep-rosy patients. METHODS: A prospective,parallel, double-blind, randomized studywas conducted in 45 leprosy inpatients withacute trophic ulcers. Patients were random-ized to receive 2%, 4% or normal salinedressing on their ulcers once daily for 4weeks. Efficacy parameters such as a reduc-

tion in the surface area of the ulcer, bacterialculture of the ulcer swab, appearance ofhealthy granulation tissue, cessation of ulcerdischarge and overall gradation of clinicalhealing and safety were assessed at weeklyintervals. RESULTS: The ulcer area reduc-tion was greater in the 2% and 4% pheny-toin groups compared with the normal salinegroup (p <0.001). Appearance of healthygranulation tissue and cessation of ulcer dis-charge was also observed earlier in the twophenytoin groups. At the end of 4 weeks, 11ulcers each had healed completely in boththe 2% and 4% phenytoin groups comparedwith none in the control group. There wereno statistical dif ferences between the 2%and 4% phenytoin groups. No side ef fectswere reported by any patient. CONCLU-SION: Topical phenytoin appears to be aneffective, safe and cheap therapeutic optionfor the healing of trophic ulcers in leprosypatients.—Authors’Abstract

Boggild, A. K., Correia, J. D., Keystone,J. S., and Kain, K. C. Leprosy inToronto: an analysis of 184 importedcases. Canadian Med. Assn. J. 170(1)(2004) 55–59.

Background: Leprosy is a rare but seriousmycobacterial infection. Immigration fromareas where the disease is endemic has re-sulted in the importation of leprosy intocountries where it is not endemic and wherephysicians and health care workers havelittle or no experience in diagnosis and ther-apy. In this study we characterized leprosypatients seen in a tropical disease unit thatmanages most of the reported leprosy casesin Canada. Methods: We reviewed the clin-ical records of all 184 leprosy patients whowere referred to the tropical Disease Unit atToronto General Hospital, Toronto, Ontario,Canada, between 1979 and 2002 and ab-stracted demographic and clinical informa-tion. Results: Patients were more likely to bemale (122 or 66.3%) and of Indian (44 or23.9%), Filipino (49 or 22.6%) or Viet-namese (37 or 20.1%) origin. Patients expe-rienced symptoms for a mean of 4.8 yrs be-fore referral to the Tropical Disease Unit.


Clinical Sciences

Most had no family history of leprosy(152/172 or 84%). Most patients presentedeither with borderline tuberculoid (80 or43.5%) or borderline lepromatous (37 or20.1%) disease. On average, patients pre-sented with 5.8 skin lesions. Upper - andlower-extremity nerve dysfunction wascommon at presentation, with up to one-third of patients demonstrating either senseor motor loss. A significantly greater lagtime to presentation was observed in pa-tients who emigrated from low-prevalenceregions (p <0.001). Interpretation: Leprosyis a chronic infectious disease that is associ-ated with serious morbidity if left untreated.Leprosy is uncommon in developed coun-tries, but it is important for physicians tohave a high index of suspicion when aforeign-born patient presents with chronicdermatitis and peripheral nerve involve-ment.—Tropical Disease Bulletin

Burman, K. D., Rijall, A., Agrawal, S.,Agarwalla, A., and Verma, K. K. Child-hood leprosy in eastern Nepal: a hospital-based study. Indian J. Lepr. 75(1) (2003)47–52.

A hospital-based retrospective study onchildhood leprosy was carried out at B.R.Koirala Institute of Health Sciences, Dha-ran, covering the period April 1998–April2002. 20 (4.45%) leprosy patients were de-tected in children aged 6 –14 years. Themale:female ratio was 4:1. History of con-tact was found in 10% of the patients. Thecommonest type of leprosy was borderlinetuberculoid leprosy (550%), followed byborderline lepromatous leprosy (301%).Most of the patients had more than one le-sion. Nerve involvement and grade 2 defor-mity were noted in 55% and 20% of the pa-tients, respectively . Slit skin smear waspositive in 30% of patients.—Authors’ Ab-stract

Cortés, S. L., and Rodríguez, G. Leprosyin children: association between clinicaland pathological aspects. J. Trop. Pediatr.50(1) (2004) 12–15.

Leprosy among children is a public healthproblem reflecting the disease’ s transmis-

sion in the community and the efficiency ofcontrol programs. To evaluate some clinical,epidemiological and histopathological crite-ria, as well as the level of agreement be-tween clinical and histopathological diag-noses, 207 biopsies were studied frompatients less than 15 years old who wereclinically diagnosed with leprosy betweenMarch 1994 and September 2000. Leprosywas confirmed by histopathology in 1 19cases (57.5 per cent). Forty-seven per centof children were 10 years old or more; 28.5per cent shared their dwellings with leprosypatients; 35 per cent had only one lesion,and 43 per cent were multibacillary cases.Agreement between clinical and histo-pathological classification was 36 per cent;hypochromic chronic eczema and post-inflammatory incontinence of melanin pig-ment were the clinical lesions most fre-quently mistaken with leprosy . Leprosyamong children represents 7 per cent of newleprosy cases in Colombia and the high per-centage of multibacillary cases suggests thatdiagnosis is being made late. The diseasemust be investigated in all children livingwith leprosy patients and skin biopsy is rec-ommended to avoid false-positive diag-noses.—Tropical Disease Bulletin

Couppié, P., Abel, S., Voinchet, H., Rous-sel, M., Hélénon, R., Huerr e, M.,Sainte-Marie, D., and Cabié, A. Im-mune Reconstitution Inflammatory Syn-drome Associated with HIV and Leprosy.Arch. Dermatol. 140 (2004) 997–1000.

Background: Immune reconstitution in-flammatory syndrome (IRIS) is an unusualinflammatory reaction to an opportunisticinfection that occurs in human immuno-deficiency virus (HIV)-positive patientswith profound immunosuppression duringthe reconstitution of the immune system inthe initial months of highly active anti-retroviral treatment.

Observations: We describe 3 cases ofleprosy occurring in patients treated with acombination of 3 antiretroviral drugs whofulfilled the criteria for IRIS. A reactionalstate occurred in all 3 cases. Two of the 3 pa-tients presented an unusual ulcerous pro-gression of the lesions not generally ob-served in cases of leprosy. The outcome was

72, 4 Current Literature, Clinical Sciences 517

favorable in all 3 cases. The frequency ofIRIS associated with leprosy in FrenchGuiana and Martinique is estimated at 3cases per 1000 HIV-positive patients receiv-ing highly active antiretroviral treatment.

Conclusions: Leprosy should be recog-nized as an IRIS-associated infection withpossibility of atypical presentation.—Ar-chives of Dermatology

Dave, S., Nori, A. V., Thappa, D. M., andSiddaraju, N. Leprous osteitis present-ing as bone cyst and erosions. Dermatol.Online J. 10(1) (2004) 17.

A 30-year-old man presented to theHansen outpatient department with swellingand ulceration of toes for 2 months andswelling of the right fifth and fourth fingersand the left second finger for 1 month. In ad-dition to skin lesions of lepromatous leprosy(subpolar type), there was nontender, non-fluctuant swelling of the right fifth andfourth fingers and left second finger . Skinover the right fifth finger showed sinus-likeopenings with associated purulent dis-charge. He also had swelling and ulcerationof second left toe. Slit-skin smear (SSS)showed a bacterial index of 6+ from the earlobes and cutaneous nodules, 4+ from thepatch, and 3+ from normal skin. ModifiedZiehl-Neelsen staining of the discharge ex-truding from the sinuses on the right fifthfinger also showed abundant acid-fastbacilli. Radiography of the hands and feetshowed lytic lesions in the distal epimeta-physeal region of proximal phalanx of theright fifth finger and left second finger anderosion of distal end of proximal phalangesof both second toes. Histopathological ex-amination of biopsy specimen from thepatch (back) showed features of leproma-tous leprosy, and Fite-Faraco stain for tissueacid-fast bacteria (AFB) was strongly posi-tive. Fine-needle-aspiration cytology (FNAC)from the lytic lesion in the bone also showedpredominantly foamy macrophages withstrongly positive staining for AFB with afew interspersed lymphocytes, epithelioidcells and Langhans giant cells. On the basisof these features, a clinical diagnosis of sub-polar lepromatous leprosy with leprous os-teitis was made. In today’ s clinical era of

improved case detection and prompt treat-ment with effective multidrug regimens, ad-vanced bone changes are rarely encountered.We describe this case of lepromatous lep-rosy that developed cavitating lesions of thephalanges of the hand, seen on x-ray aswell-defined bone cyst and erosions.—Au-thors’Abstract

Dawe, S., Lockwood, D. N., and Creamer,D. A case of post-partum borderline tu-berculoid leprosy complicated by a me-dian nerve abscess, peptic ulceration andrifampicin-induced haemolytic renal fail-ure. Lepr. Rev. 75(2) (2004) 181–187.

We report a case of borderline tuberculoidleprosy complicated by a median nerve ab-scess, acute renal failure secondary torifampicin-induced haemolysis and duode-nal ulceration secondary to steroid use. Ri-fampicin induced hameolysis is a rare andprobably under -reported complication ofleprosy multi-drug therapy. It should be con-sidered when patients complain of flu-likesymptoms after taking their monthly ri-fampicin.—Authors’Abstract

de Freitas, M. R., Nascimento, O. J., deFreitas, M. R., and Hahn, M. D. Iso-lated superficial peroneal nerve lesion inpure neural leprosy: case report. Arq.Neuropsiquiatr. 62(2B) (2004) 535–539.

Patients with leprosy may have onlynerve involvement without skin changes.These cases are known as pure neural lep-rosy and can be seen in 10% of leprosy pa-tients. Most patients have mononeuritic ormultiple mononeuritic neuropathy patterns.The isolated lesion of the superficial pero-neal nerve is uncommonly seen. We report apatient with involvement of this nerve inwhich there was no thickening of superficialnerves. The performed nerve biopsy showedinflammatory infiltration, loss of fibers andpresence of Mycobacterium leprae. We be-lieve that in prevalent leprosy countries weshould take in mind the possibility of iso-lated pure neural leprosy in some patientswithout skin lesion. In these cases the diag-nosis of leprosy is impossible on clinical


grounds and nerve biopsy is mandatory.—Authors’Abstract

Helmer, K. A., Fleischfr esser, I.,Kucharski-Esmanhoto, L. D., Neto, J.F., and Santamaria, J. R. The Lucio’sPhenomenon (necrotozing erythema) inpregancy. An. Bras. Dermatol. 79(2)(2004) 205–210. [Article in Spanish].

Summary: The Lucio’s phenomenon, atype 2 reactional condition in leprosy prob-ably mediated by immune complexes, is asevere necrotizing skin reaction that occursmainly in patients with non-nodular lepro-matous leprosy. This report presents a 27-year-old woman, in her 32nd week of preg-nancy, with a one-week history of painfulskin lesions in extremities, reddish-purple,sharply delineated, confluent, with bullaeand occasional necrosis and ulceration. Theoatient also referred fever . Bacilloscopyshowed acid-fast bacilli and globi, and thehistopathologic findings of a skin biopsywere consistent with lepromatous leprosyand Lucio’s phenomenon. Prednisone andmultidrug therapy with rifampin, clofaz-imine and dapsone were given, with remis-sion. Pregnancy has been associated with ahigh incidence of first diagnosis of leprosyor with an exacerbation of symptoms in pa-tients with the established disease becausehormonal alterations cause immunologicalimbalance, particularly between the lastthree months of pregnancy and the first threemonths of lactation, when immunosuppre-sion is higher. Despite the recommendationnot to take drugs during pregnancy, the mul-tidrug therapy regimen must be used, sincethe benefits achieved with the treatment sur-pass the risks.—Anais Brasileiros de Der-matologia

Jain, R., Dogra, S., Kaur, I., and Kumar,B. Leprosy and herpes zoster; an associ-ation or dissociation? Indian J Lepr. 75(3)(2003) 263–264.

Nerve involvement is common to thepathogenesis of both leprosy and herpeszoster. We report two cases of borderlineleprosy in which the skin lesions character-

istically spared the healed zoster scar. Pos-sible mechanisms and relationship are dis-cussed.—Authors’Abstract

Karthikeyan, K., and Thappa, D. M.Squamous cell carcinoma in plantar ul-cers in leprosy: a study of 1 1 cases. In-dian J. Lepr. 75(3) (2003) 219–224.

The objectives of our study were to de-scribe and analyze the malignancies that oc-curred in plantar ulcers of leprosy patients.The possible predisposing conditions, dura-tion and extent of the spread of the tumourwere also studied. All patients with trophiculcer of the foot attending the urban leprosyclinic in our hospital from January 1998 toJanuary 2003 were screened for change tomalignancy. During the study period, 79cases of plantar ulcers in leprosy were seen.The mean age of these cases was 39.9 yearswith male-to-female ratio of 4:1. Elevencases with plantar ulcers and malignantchange were diagnosed in our hospital dur-ing the study period. The male-to-femaleratio was 4.5:1. The mean age of these pa-tients was 60.6 years. Their age ranged from46 to 75 years. Nine of the cases weretreated cases of borderline tuberculoid lep-rosy, while two had treated lepromatous lep-rosy. In our study, two distinct morphologi-cal types of malignant changes were seen.Histopathologically, all cases, except one,were of well-dif ferentiated squamous cellcarcinoma variation; one case had verrucouscarcinoma. Though trophic ulcers are com-mon in leprosy cases, only long-standingand neglected ones undergo malignancy.—Authors’Abstract

Katoch, K., Katoch, V. M., Natrajan, M.,Sreevatsa, Gupta, U. D., Sharma ,V. D.,and Shivanavar , C. T. 10–12 yearsfollow-up of highly bacillated BL/LLlep-rosy patients on combined chemotherapyand immunotherapy. Vaccine. 22(27–28)(2004) 3649–3657.

This study reports the follow-up results of36 highly bacillated untreated BL/LL caseswho were serially allocated to three treat-


ment groups. Group I patients received amodified WHO regimen (Rifampicin 600mg once a month supervised, 50 mg of Clo-fazimine and 100 mg of Dapsone daily un-supervised) and BCG 0.1 mg per dose 6monthly; group II patients received the samemulti-drug treatment (MDT) and Mw (2 ×10(8) killed bacilli per dose) 6 monthly:group III patients received the same MDTwith 0.1 ml of distilled water 6 monthly andacted as a control. Treatment was continuedtill smear negativity. All these three groupswere comparable by their initial clinicalscore, bacteriological index (BI), viablebacilli as assessed by the mouse footpad(MFP), bacillary adenosine triphosphate(ATP) content and also histologically at thetime of starting treatment. All these param-eters were evaluated every 6 months. Thevaccines were well tolerated. All the patientsin group I became smear negative by 3.5years, in group II in 3 years whereas thosein group III took 5 years. The incidence ofreactions was the same in all the groups dur-ing the first 2 years, however , patients ofgroup III (MDT + placebo) continued tohave reactions up to 3 years. No viablebacilli could be detected in the local and dis-tal sites as estimated by MFP and bacillaryATP after 12 months in both the im-munotherapy groups. These could be de-tected in patients on MDT alone up to 24months of therapy. Histologically patients inboth the immunotherapy groups (groups Iand II) showed accelerated granuloma clear-ance, histological upgrading and non-specific healing without granuloma forma-tion both at the local and distal sites and thiswas achieved much earlier compared to theMDT + placebo group. Thus, by the additionof immunotherapy the ef fective treatmentperiod of achieving bacteriological negativ-ity could be reduced by about 40%, time pe-riod of reactions reduced by 33% and therewere no reactions and/or relapses in the10–12 years post-treatment follow-up.—Authors’Abstract

Lomonte, C., Chiarulli, G., Cazzato, F .,Giammaria, B., Marchio, G., Losurdo,N., Antonelli, M., Casucci, F ., andBasile, C. End-stage renal disease in lep-rosy. J. Nephrol. 17(2) (2004) 302–305.

BACKGROUND: Leprosy or Hansen’ sdisease (HAD) undoubtedly remains anemergency in certain countries. It is an an-cient deforming disease caused by Myco-bacterium leprae . The countries with thehighest endemic leprosy rate in 2000 wereBrazil, India and Madagascar. In Italy, theold epidemic has been defeated and there areapproximately 400 patients under constantmonitoring with three to four new cases peryear involving Italian residents. The kidneyis one of the tar get or gans during thesplanchnic localization of leprosy . Thehistopathological renal lesion spectrum in-cludes glomerulonephritis (GN), renal amy-loidosis (RA) and interstitial nephritis (IN).Both proteinuria and chronic renal failureare the main clinical expressions of renaldamage in leprosy. To the best of our knowl-edge, very little is reported concerning end-stage renal disease (ESRD) in leprosy pa-tients both in the most important nationaland international renal registries and in theavailable literature. This study aimed to re-port the long-term experience of our depart-ment in this field. METHODS: To achievethis, we analyzed retrospectively the HADCenter (Gioia del Colle) database at our hos-pital. RESUL TS: Eight leprosy patientswere dialyzed from 1980 to June 2003 (sixmales and two females), with a mean age of61.0 ± 8.9 S.D. yrs (range: 51–76) and amean HAD duration of 36.1 ± 5.1 yrs. Thefirst clinical nephropathymanifestationswere non-nephrotic proteinuria associatedwith chronic renal failure in four patients,and nephrotic proteinuria in four patients.Kidney biopsies performed in three patientsshowed two had RA, and one had IN. Twopatients were treated initially by peritonealdialysis; they were then switched to he-modialysis (HD) after 3 and 10 months be-cause of recurrent peritonitis. HD treatmentlasted 40.6 ± 31.4 months (range: 9 –101).Six patients died, one due to hyperkalemia,one because of a technical dialysis accident,and the remainder due to causes unrelated tothe dialysis treatment. Two patients are stillalive, treated with HD for 17 and 44 months.CONCLUSIONS: Uremia represents a latecomplication of leprosy and has a multifac-torial genesis, although RA is among themost frequent causes, conventional bicar-bonate HD appears to offer good results in


the treatment of uremia in leprosy pa-tients.—Authors’Abstract

Malaviya, G. N. Palmaris longus—a musclewith multiple uses in leprosy-af fectedhands. Indian J. Lepr. 75(4) (2003) 327–334.

The present paper reviews the anatomy ofpalmaris longus muscle and also the situa-tions where palmaris longus muscle hasbeen used as an independent motor or as adonor of tendon graft material. Its relevancein leprosy-affected hands is also discussedbecause the muscle is usually spared in handpalsies consequent to leprotic neural dam-age. The advantages and disadvantages ofits use in dif ferent operative procedureshave been analyzed. The author ’s experi-ence with this muscle in the correction ofhand deformities in leprosy is described.—Author’s Abstract

Ooi, W. W., and Srinivasan, J. Leprosyand the peripheral nervous system: basicand clinical aspects. Muscle Nerve. 30(4)(2004) 393–409.

Leprosy is one of the most commoncauses of nontraumatic peripheral neuropa-thy in the developing world. The causativeagent, Mycobacterium leprae , has apredilection for Schwann cells, where theorganism multiplies unimpeded by organism-specific host immunity, resulting in destruc-tion of myelin, secondary inflammatorychanges, and destruction of the nerve archi-tecture. The cardinal diagnostic features ofleprosy are anesthetic skin lesions, neuropa-thy, and positive skin smears for the bacilli.However, patients may rarely present with-out skin lesions in pure neuritic leprosy .Electrodiagnostic findings early in the dis-ease reveal demyelinating features, such asslowing of conduction velocity and pro-longation of latencies, but as the diseaseprogresses secondary axonal damagecommonly ensues. Electrodiagnostic studiesare also useful to monitor for toxicity sec-ondary to therapy, particularly thalidomide-associated neuropathy. Nerve biopsy of asensory cutaneous nerve is sometimes es-

sential to confirm a diagnosis of leprosy .Significant advances in understanding of thepathogenesis, mapping of the genome, andother advances in molecular biology may re-sult in better preventive and therapeuticmodalities, and the goal of eradicating lep-rosy as a global problem may yet be real-ized.—Authors’Abstract

Pais, A. V., Pereira, S., Garg, I., Stephen,J., Antony, M., and Inchara, Y. K.Intra-abdominal, crystal-storing histiocy-tosis due to clofazimine in a patient withlepromatous leprosy and concurrent car-cinoma of the colon. Lepr . Rev. 75(2)(2004) 171–176.

We report a case with abdominal compli-cations of clofazimine treatment which in-cluded blackish discolouration of the lymphnodes, omentum and peritoneum. A 44-year-old female with lepromatous leprosy and ahistory of adverse reaction to clofazimine 2years previously, presented with rectosig-moid junction adenocarcinoma. Laparotomyrevealed an inoperable tumour with pig-mentation of the bowel, serosa and peri-toneum. A second operation had o be per-formed for transverse loop colostomy and amesenteric lymph node biopsy sent for frozensection showed typical clofazimine crystals.Despite widespread use for many years in thetreatment of leprosy, this drug is not knownto be carcinogenic and this case provides noevidence for an association or link betweenits use and the patient’s cancer. Apart from itsuse in leprosy, clofazimine may be used in thetreatment of disseminated Mycobacteriumavium-intracellulare infection, Buruli ulcerdue to M. ulcerans and occasionally in othermycobacterial infections. An awareness ofthe rare side-ef fect described above mayhelp in the clinical assessment and manage-ment of such cases, including the avoidanceof unnecessary laparotomy.—Authors’ Ab-stract

Pimentel, M. I., Nery , J. A., Borges, E.,Goncalves, R. R., and Sarno, E. N. Im-pairments in multibacillary leprosy; astudy from Brazil. Lepr . Rev . 75(2)(2004) 143–152.


This is a retrospective cohort study of 103multibacillary leprosy patients (18% BB,48% BL and 34% LL) followed during andafter treatment, in a tertiary referral centrewith an outpatient clinic in an endemic areain Brazil, for an average period of 65months since the start of multidrug therapy(24-dose MDT). The objective of the studywas to identify the role of overt neuritis(presence of pain in a peripheral nervetrunk, with or without enlargement or neu-ral function damage), in the development ofimpairments. They were evaluated using theWorld Health Organization disability gradebefore treatment, at the end of the treatment,and at the end of the follow-up period.Thirty-four percent of patients presentedovert neuritis during MDT , and 45% hadovert neuritis episodes during the follow-upperiod; the most commonly affected nerveswere ulnar , fibular and posterior tibialnerves, and the neuritic episodes were care-fully treated with steroid therapy and phys-iotherapy. Impairments were associatedwith: affected (painful and/or thick) nervesat diagnosis (p <0.005); delay in diagnosis(p = 0.010); impairments already present atthe start of treatment (p = 0.00041 at the endof MDT, and p = 0.000013 at the end of fol-low-up); occurrence of overt neuritisepisodes during MDT (p = 0.0016) or thewhole follow-up (p = 0.015). These datadraw attention to the importance of early di-agnosis and of good neurological examina-tion throughout the follow-up, as well assuggest the importance of neuritis in the in-duction of impairments in multibacillaryleprosy.—Authors’Abstract

Rath, N., and Kar, H. K. Leprosy in HIVinfection: a study of three cases. Indian J.Lepr. 75(4) (2003) 355–359.

The course of leprosy in patients with HIVinfection has been a controversial issue fora long time. It is still a matter of debatewhether the HIV status of an individual hasany impact on the natural history of leprosyand response to anti-leprosy treatment. Wereport here three HIV-positive leprosy cases(two BT and one BB) along with their CD4counts and HIV staging with anti-leprosytherapeutic response. Both BT cases re-sponded well to conventional WHO MDT

(PB) for 6 months, whereas the BB case re-lapsed 3 months after completion of MDT(MB) for one year. However, he became in-active again following a further one-yearcourse of MDT (MB).—Authors’Abstract

Richardus, J. H., Withington, S. G., An-derson, A. M., Croft, R. P., Nicholls, P.G., van Brakel, W. H., and Smith, W.C. S.Adverse events of standardized reg-imens of corticosteroids for prophylaxisand treatment of nerve function impair-ment in leprosy: results from the “TRI-POD” trials. Lepr . Rev . 74(4) (2003)319–327.

Reactions in leprosy causing nerve func-tion impairment (NFI) are increasinglytreated with standardized regimens of corti-costeroids, often under field conditions.Safety concerns led to an assessment of ad-verse events of corticosteroids, based on dataof three trials studying prevention of NFI (theTRIPOD study). A multicenter, randomized,double-blind placebo-controlled trial wasconducted in leprosy control programs inNepal and Bangladesh. Treatment was withprednisolone according to fixed schedulesfor 16 weeks, starting in one trial with 20mg/day (prophylactic regimen: total dosage1.96 g) and in the other two trials with 40mg/day (therapeutic regimen: total dosage2.52 g). Minor adverse events were definedas moon face, fungal infections, acne, andgastric pain requiring antacid. Major ad-verse events were defined as psychosis, pep-tic ulcer, glaucoma, cataract, diabetes andhypertension. Also, the occurrence of in-fected plantar, palmar, and corneal ulcera-tion was monitored, together with occur-rence of TB. Considering all three trialstogether, minor adverse events were ob-served in 130/815 patients (16%). Of these,51/414 (12%) were in the placebo group and79/401 (20%) in the prednisolone group.The relative risk for minor adverse events inthe prednisolone group was 1.6 (p = 0.004).Adverse events with a significantly in-creased were acne, fungal infections andgastric pain. Major adverse events were ob-served in 15/815 patients (2%); 7/414 (2%)in the placebo group and 8/401 (2%) in theprednisolone group. No major adverseevents had a significantly increased risk in


the prednisolone arm of the trials. No casesof TB were observed in 300 patients whocould be followed-up for 24 months. Stan-dardized regimens of corticosteroids forboth prophylaxis and treatment of reactionsand NFI in leprosy under field conditions indeveloping countries are safe when a stan-dard pre-treatment examination is per-formed, treatment for minor conditions canbe carried out by field staff, referral for spe-cialized medical care is possible, and suf fi-cient follow-up is done during and aftertreatment.—Tropical Disease Bulletin

Rijal, A., Agrawal, S., Agarwalla, A., andLakhey, M. Bullous erythema nodosumleprosum: a case report from Nepal. Lepr.Rev. 75(2) (2004) 177–180.

A patient with lepromatous leprosy, whileon WHO multidrug therapy (MDT) formultibacillary disease, was diagnosed ashaving dapsone syndrome with recurrentepisodes of bullous lesions on the lower ex-tremities for 4–5 years. The lesions were as-sociated with high-grade fever. Examinationrevealed multiple hypopigmented maculeson the limbs. Multiple atrophic scars werealso found on the buttocks and lower limbs.Bilateral ulnar, radial cutaneous and lateralpopliteal nerves were thickened. On day 10of WHO-MB-MDT he developed a flaccidbulla on the lower leg. Skin slit smearshowed a bacterial index (BI) of 3+ and thehistopathology was consistent with type IIreaction. High dose corticosteroid therapywas started but he continued to have new le-sions, and was therefore referred to a centrewhere thalidomide was available. Clinicalresponse was good and he remained symp-tom-free after gradual reduction in dosage.ENL should be differentiated from bullousdrug reactions, pemphigus vulgaris, bullouspemphigoid and other blistering diseases.—Authors’Abstract

Senturk, V., and Sagduyu, A. [Psychiatricdisorders and disability among leprosy pa-tients; a review.] Turk. Psikiyatri. Der g.15(3) (2004) 236–243. [Article in Turkish]

As a gross estimate, leprosy currently af-fects 1 1–16 million patients worldwide

There are currently 3600 registered patientsin Turkey. The social stigma connected toleprosy makes this disease completely dif-ferent from others. Even nowadays peopleaffected by leprosy have to leave their vil-lage or are socially isolated. The physicaldeformity ratio is approximately 25% inother countries whereas it is more than halfin Turkey. The prevalence of mental disor-ders among leprosy patients is higher thanthat among the general population. Depres-sion is the most common psychiatric disor-der among leprosy patients. Another impor-tant finding is that the long duration of theillness and physical handicaps raise the riskof psychiatric disorders. Nevertheless, theresults of two studies conducted in Turkeyon this subject contradict the results of in-ternational studies. Leprosy patients experi-ence functional disabilities that limit theirlives and ability to establish relation shipswith others both in social and occupationalfields. The physical disability rate is high(75%) in Turkey. A review of the literaturerevealed several papers on the psychosocialaspects of illness but few references to thedegree or pattern of psychiatric disordersamong leprosy patients. The main purposeof this paper is to review psychiatric disor-ders and disabilities in leprosy patients andto obtain concrete results.—Authors’ Ab-stract

Soysal, A., Atay, T., Ozu, T., and Arpaci,B. Electrophysiological evaluation of pe-ripheral and autonomic involvement inleprosy. Can. J. Neurol. Sci. 31(3) (2004)357–362.

OBJECTIVE: Motor and sensory nerveconductions, F responses, sympathetic skinresponses and R-R interval variations(RRIV) were studied to determine the typeof peripheral neuropathy among patientswith leprosy. METHODS: Twenty-nine con-secutive patients with leprosy (25 male, 4female) hospitalized in the “Istanbul Lep-rosy Hospital” between January–December,1999 were included in this study . Ten pa-tients had borderline lepromatous leprosy,and 19 had lepromatous leprosy. None of thepatients studied had the tuberculoid form.The mean age was 55 ± 12 years. The con-trol group consisted of 30 (26 male, 4 fe-


male) healthy volunteers (mean age: 58.1 ±7.8 years). All subjects included in the studyunderwent neurological examination andelectrophysiological evaluation. Standardprocedures were performed for evaluatingsensory and motor conduction studies.Motor studies were carried out on both leftand right median, ulnar, tibial and commonperoneal nerves while median, ulnar, suraland superficial peroneal nerves were exam-ined for sensory studies. Sympathetic skinresponse recordings on both hands andRRIV recordings on precordial region weredone in order to evaluate the autonomic in-volvement. RESULTS: The lower extremitywas found to be more severely affected thanthe upper, and sensory impairment predom-inated over motor. Of 58 upper limbs exam-ined, no sympathetic skin responses wasrecorded in 46 (79.3%). Compared with thecontrols, the RRIVs of the leprosy patientswere found to be reduced during both rest-ing and deep forced hyperventilation. CON-CLUSION: Our results indicate that leprosycauses a predominantly axonal polyneuropa-thy that is more severe in the lower extremi-ties. Sensory nerve damage is accompaniedby autonomic involvement.—Authors’ Ab-stract

Taylor, N. L., Raj, A. D., Dick, H. M., andSolomon, S. The correction of ulnar clawfingers: a follow-up study comparing theextensor-to-flexor with the palmarislongus 4-tailed tendon transfer in patientswith leprosy. J. Hand Surg. [Am]. 29(4)(2004) 595–604.

PURPOSE: The extensor to flexor 4-tailedtendon transfer (EF4T) and the palmarislongus 4-tailed tendon transfer (PL4T) are 2surgical procedures used to correct intrinsicparalysis of the hand in leprosy. The EF4Ttraditionally is the more common procedureand requires the transfer of a wrist extensormuscle. The PL4T requires the transfer ofthe palmaris longus and morbidity is ex-pected to be lower. A follow-up study wasperformed to determine whether the clinicaloutcome of the PL4Tis superior to the EF4Tprocedure in leprosy patients with ulnarclaw fingers that are considered mobile be-fore surgery. METHODS: Fifty-five patientspresented 65 af fected hands, of which 40

hands had the PL4T and 25 had the EF4Tprocedure. Each hand was assessed beforesurgery and at follow-up evaluation by pre-determined angle measurements, standard-ized photographs, mechanical function, andpatient satisfaction. Each hand was given anoverall technical grade according to previ-ously published standards. RESULTS: Afteran average follow-up period of 33 monthsthere was no statistically significant differ-ence in the technical outcome or patient sat-isfaction between the 2 tendon transfer pro-cedures. CONCLUSIONS: Whenever thepalmaris longus is available it may be con-sidered to be the motor tendon of choice toundertake a many-tailed procedure for clawfinger reconstruction in mobile hands para-lyzed by leprosy . The palmaris longusshould be considered as a possible motortendon when correcting intrinsic muscleparalysis of the hand.—Authors’Abstract

van Brakel, W. H., Anderson, A. M.,Withington, S. G., Cr oft, R. P .,Nicholls, P. G., Richardus, J. H., andSmith W. C. S. The prognostic impor-tance of detecting mild sensory impair-ment in leprosy: a randomized controlledtrial (TRIPOD 2). Lepr . Rev . 74(4)(2003) 300–310.

This study was designed to investigatewhether leprosy patients diagnosed with mildsensory impairment have a better prognosiswhen treated with steroids than similarly im-paired patients treated ith placebo. A multi-center, randomized, double-blind, placebo-controlled trial was conducted in Nepal andBangladesh [date not given]. Patients wereeligible if they had a confirmed leprosy diag-nosis, were between 15 and 50 years old, hadmild sensory impairment of the ulnar or pos-terior tibial nerve of less than 6 months du-ration and did not require steroids for otherreasons. ‘Mild impairment’ was defined as‘impaired on the Semmes-Weinstein monofil-ament test, but testing normal on the ballpensensory test.’Subjects were randomized to ei-ther prenisolone treatment starting at 40 mgper day, tapering over 4 months, or placebo.Nerve function was monitored monthly. Anypatient who deteriorated was taken out of thetrial and was put on full-dose steroid treat-ment. Outcome assessment was done at 4, 6,


9 and 12 months from the start of the treat-ment. Outcome measures were the proportionof patients needing full-dose prednisoloneand the Semmes-Wenstein sum scores. Eachpatient contributed only one nerve to theanalysis. Seventy-five patients had nerves el-igible for analysis, of whom 41 (55%) and 34(45%) were allocated to the prednisolone andplacebo arms, respectively . At 4 months,three patients in the prednisolone arm (7%)and six in the placebo arm (18%) had an out-come event requiring full-dose steroids. At 12months, these proportions had almost re-versed, 11 (27%) and 6 (18%) in the treat-ment and placebo arms, respectively. In thelatter group, 75% had recovered sponta-

neously after 12 months. Prednisolone treat-ment of sensory impairment of the ulnar andposterior tibial nerves detectable with themonofilament test, but not with the balloentest, did not improve the long-term outcomein terms of recovery of touch sensibility, nordid it reduce the risk of leprosy reactions ornerve function impairment beyond the initial4-month treatment phase. Two unexpectedmain findings were the strong tendency ofmild sensory impairment to recover sponta-neously and the fact that patients with mildsensory impairment without any other signsor symptoms of reaction or nerve functionimpairment are relatively rare.—T ropicalDisease Bulletin

72, 4 Current Literature, Immunopathology 525

Immunopathology

Antas, P. R., Sales, J. S., Per eira, K. C.,Oliveira, E. B., Cunha, K. S., Sarno, E.N., and Sampaio, E. P. Patterns of intra-cellular cytokines in CD4 and CD8 Tcells from patients with mycobacterial in-fections. Braz. J. Med. Biol. Res. 37(8)(2004) 1119–1129. Epub 2004 Jul 20.

Using a short-term bulk culture protocoldesigned for an intracellular-staining methodbased on a flow cytometry approach to thefrequencies of cytokine-producing cellsfrom tuberculosis and leprosy patients, wefound distinct patterns of T cell subset ex-pression. The method also reveals the pro-file of peak cytokine production and canprovide simultaneous information about thephenotype of cytokine-producing cells, pro-viding a reliable assay for monitoring theimmunity of these patients. The immune re-sponse of Mycobacterium leprae and puri-fied protein derivative (PPD) in vitro to apanel of mycobacteria-infected patientsfrom an endemic area was assessed in pri-mary mononuclear cell cultures. The kinet-ics and source of the cytokine pattern weremeasured at the single-cell level. IFN-gamma-, TNF-alpha-, IL-4- and IL-10-secreting T cells were intracytoplasmic eval-uated in an attempt to identify M. leprae-and PPD-specific cells directly from the pe-ripheral blood. The analysis by this ap-proach indicated that TNF-alpha was thefirst (8 hr) to be produced, followed by IFN-

gamma (16 hr), IL-10 (20 hr) and IL-4 (24hr), and double-staining experiments con-firmed that CD4+ were a greater source ofTNF-alpha than of CD8+ T cells (p <0.05).Both T cell subsets secreted similar amountsof IFN-gamma. We conclude that the proto-col permits rapid evaluation of cytokine pro-duction by different T cell populations. Themethod can also be used to define immunestatus in non-infected and contact individu-als.—Authors’Abstract

Bermudez, L. E., Petrofsky, M., and San-gari, F. Intracellular phenotype of Myco-bacterium avium enters macrophagesprimarily by a macropinocytosis-likemechanism and survives in a compart-ment that differs from that with extracel-lular phenotype. Cell. Biol. Int. 28(5)(2004) 411–419.

Mycobacterium avium uptake by humanmacrophages differs between the phenotypesof bacterium grown in laboratory media (ex-tracellular growth, EG) and bacterium grownwithin macrophages (intracellular growth,IG). Studies in vivo have confirmed that, whenspreading, pathogenic mycobacteria entermacrophages by a complement receptor 3-independent pathway, in contrast to myco-bacteria uptake in vitro. M. avium, grown inmacrophages (IG) for 3 or more days, invadefresh macrophages by a macropinocytosis-

like mechanism, in contrast to bacteriagrown in media (EG), confirmed by the in-hibitory effect of wortmannin, an inhibitor ofphosphoinoside-3-kinase, on the uptake of IG,but not EG, by macrophages. The IG pheno-type was seen in vacuoles with lower pHthan those inhabited by the EG phenotype.Incubation of macrophages with bafilomycinA1, an inhibitor of vacuole acidification, de-creased the viability of intracellular IG, butnot EG, phenotype, suggesting the impor-tance of an acidic environment for the regu-lation of IG genes. In addition, the percent-age of vacuoles that incorporate and retainLAMP-1 is smaller with EG than with IGbacteria. The formation of M. aviummacropinosomes was also shown to be inde-pendent of microtubules. These data suggestthat uptake of extracellular fluid is part of M.avium IG phenotype uptake by macro-phages, and that the IG phenotype inhabitsa slightly dif ferent vacuole than that ofEG.—Authors’Abstract

Cosma, C. L., Humbert, O., and Ra-makrishnan, L. Superinfecting myco-bacteria home to established tuberculousgranulomas. Nat. Immunol. 5(8) (2004)828–835. Epub 2004 Jun 27. Commentin: Nat. Immunol. 5(8) (2004) 778–779.

Acentral paradox of tuberculosis immunityis that reinfection and bacterial persistenceoccur despite vigorous host immune re-sponses concentrated in granulomas, whichare organized structures that form in responseto infection. Prevailing models attribute rein-fection and persistence to bacterial avoidanceof host immunity via establishment of infec-tion outside primary granulomas. Alterna-tively, persistence is attributed to a gradualbacterial adaptation to evolving host immuneresponses. We show here that superinfectingMycobacterium marinum traffic rapidly intopreexisting granulomas, including theircaseous (necrotic) centers, through specificmycobacterium-directed and host cell-mediated processes, yet adapt quickly topersist long term therein. These findingsdemonstrate a failure of established granu-lomas, concentrated foci of activated macro-phages and antigen-specific immune effec-tor cells, to eradicate newly deposited

mycobacteria not previously exposed to hostresponses.—Authors’Abtract

Durai, M., Kim, H. R., and Moudgil, K.D. The regulatory C-terminal determi-nants within mycobacterial heat shockprotein 65 are cryptic and cross-reactivewith the dominant self homologs: impli-cations for the pathogenesis of autoim-mune arthritis. J. Immunol. 173(1) (2004)181–188.

The 65-kDa mycobacterial heat shockprotein (Bhsp65) has been invoked in thepathogenesis of both adjuvant arthritis (AA)in the Lewis rat (R T.1(l)) and humanrheumatoid arthritis. Arthritic Lewis rats inthe late phase of AA show diversification ofthe T cell response to Bhsp65 C-terminal de-terminants (BCTD), and pretreatment ofnaive Lewis rats with a mixture of peptidesrepresenting these neoepitopes affords pro-tection against AA. However, the fine speci-ficity and physiologic significance of theBCTD-directed T cell repertoire, and therole of homologous self (rat) hsp65(Rhsp65), if any, in spreading of the T cellresponse to Bhsp65 have not yet been ex-amined. We observed that T cells primed bypeptides comprising BCTD can adoptivelytransfer protection against AA to the recipi-ent Lewis rats. However, these T cells can beactivated by preprocessed (peptide) form ofBCTD, but not native Bhsp65, showing thatBCTD are cryptic epitopes. The BCTD-reactive T cells can be activated by the nat-urally generated (dominant) C-terminal epi-topes of both exogenous and endogenousRhsp65 and vice versa. Furthermore, certainindividual peptides constituting BCTD andtheir self homologs can also induce protec-tion against AA. These results support amodel for the diversification of T cell re-sponse to Bhsp65 during the course of AAinvolving up-regulation of the display ofcryptic BCTD coupled with spontaneous in-duction of T cell response to the cross-reactive dominant C-terminal epitopes ofRhsp65. The identification of disease-regulating cryptic determinants in Ags im-plicated in arthritis provides a novel ap-proach for immunotherapy of rheumatoidarthritis.—Authors’Abstract


Fischer, K., Scotet, E., Niemeyer, M., Koe-bernick, H., Zerrahn, J., Maillet, S.,Hurwitz, R., Kursar , M., Bonneville,M., Kaufmann, S. H., and Schaible, U.E. Mycobacterial phosphatidylinositolmannoside is a natural antigen for CD1d-restricted T cells. Proc. Natl. Acad. Sci.U.S.A. 101(29) (2004) 10685 –10690.Epub 2004 Jul 08.

A group of T cells recognizes glycolipidspresented by molecules of the CD1 family.The CD1d-restricted natural killer T cells(NKT cells) are primarily considered to beself-reactive. By employing CD1d-bindingand T cell assays, the following structuralparameters for presentation by CD1d weredefined for a number of mycobacterial andmammalian lipids: two acyl chains facili-tated binding, and a polar head group wasessential for T cell recognition. Of the my-cobacterial lipids tested, only a phos-phatidylinositol mannoside (PIM) fulfilledthe requirements for CD1d binding andNKT cell stimulation. This PIM activatedhuman and murine NKT cells via CD1d,thereby triggering antigen-specific IFN-gamma production and cell-mediated cyto-toxicity, and PIM-loaded CD1d tetramersidentified a subpopulation of murine andhuman NKT cells. This phospholipid, there-fore, represents a mycobacterial antigen rec-ognized by T cells in the context ofCD1d.—Authors’Abstract

Kudo, K., Sano, H., Takahashi, H.,Kuronuma, K., Yokota, S., Fujii, N.,Shimada, K., Yano, I., Kumazawa, Y.,Voelker, D. R., Abe, S., and Kuroki, Y.Pulmonary collectins enhance phagocy-tosis of Mycobacterium avium throughincreased activity of mannose receptor. J.Immunol. 172(12) (2004) 7592–7602.

Collectins, including surfactant proteins A(SP-A) and D (SP-D) and mannose bindinglectin (MBL), are the important constituentsof the innate immune system. Mycobacte-rium avium, a facultative intracellular patho-gen, has developed numerous mechanismsfor entering mononuclear phagocytes. In thisstudy, we investigated the interactions of col-lectins with M. avium and the effects of these

lectins on phagocytosis of M. avium bymacrophages. SP-A, SP-D, and MBLexhib-ited a concentration-dependent binding to M.avium. The binding of SP-A to M. aviumwas Ca(2+)-dependent but that of SP-D andMBL was Ca(2+)-independent. SP-A andSP-D but not MBL enhanced the phagocy-tosis of FITC-labeled M. avium by rat alve-olar macrophages and human monocyte-derived macrophages. Excess mannan,zymosan, and lipoarabinomannan derivedfrom the M. avium-intracellular complex, sig-nificantly decreased the collectin-stimulatedphagocytosis of M. avium. Enhanced phago-cytosis was not affected by the presence ofcycloheximide or chelation of Ca(2+). Themutated collectin, SP-A(E195Q, R197D)exhibited decreased binding to M. avium butstimulated phagocytosis to a level compara-ble to wild-type SP-A. Enhanced phagocy-tosis by cells persisted even after preincu-bation and removal of SP-A or SP-D. Ratalveolar macrophages that had been incu-bated with SP-A or SP-D also exhibited en-hanced uptake of (125)I-mannosylated BSA.Analysis by confocal microscopy and flowcytometry revealed that the lung collectinsup-regulated the cell surface expression ofmannose receptor on monocyte-derivedmacrophages. These results provide com-pelling evidence that SP-A and SP-D en-hance mannose receptor-mediated phago-cytosis of M. avium by macrophages.—Authors’Abstract

Luo, Y., Yamada, H., Chen, X., Ryan, A.A., Evanoff, D. P., Triccas, J. A., andO’Donnell, M. A. Recombinant Myco-bacterium bovis bacillus Calmette- Guérin(BCG) expressing mouse IL-18 aug-ments Th1 immunity and macrophage cy-totoxicity. Clin. Exp. Immunol. 137(1)(2004) 24–34.

Interleukin-18 (IL-18) has been demon-strated to synergize with BCG for inductionof a T-helper-type 1 (Th1) immune re-sponse. Since successful treatment of super-ficial bladder cancer with BCG requiresproper induction of Th1 immunity, we havedeveloped a recombinant (r) BCG strain thatfunctionally secretes mouse (m) IL-18. ThisrBCG-mIL-18 strain significantly increased


production of the major Th1 cytokine IFN-gamma in splenocyte cultures, at levelscomparable to that elicited by control BCGplus exogenous rIL-18. IFN-gamma pro-duction by splenocytes was eliminated byaddition of neutralizing anti-IL-18 antibody.Endogenous IL-12 played a favorable rolewhereas IL-10 played an adverse role inrBCG-mIL-18-induced IFN-gamma pro-duction. Enhanced host antimycobacterialimmunity was observed in mice infectedwith rBCG-mIL-18 which showed lesssplenic enlargement and reduced bacterialload compared to control mice infected withBCG. Further , splenocytes from rBCG-mIL-18-infected mice, in response to BCGantigen, displayed increased production ofIFN-gamma and GMCSF , decreased pro-duction of IL-10, elevated cellular prolifer-ation and higher dif ferentiation of IFN-gamma-secreting cells. rBCG-mIL-18 alsoenhanced BCG-induced macrophage cyto-toxicity against bladder cancer MBT-2 cellsin a dose-dependent manner . Neutralizingall endogenous macrophage-derived cy-tokines tested (IL-12, IL-18 and TNF-alpha)as well as IFN-gamma severely diminishedthe rBCG-mIL-18-induced macrophage cy-tolytic activity, indicating a critical role forthese cytokines in this process. Cytokineanalysis for supernatants of macrophage-BCG mixture cultures manifested higherlevels of IFN-gamma and TNF-alpha inrBCG-mIL-18 cultures than in control BCGcultures. Taken together, this rBCG-mIL-18strain augments BCG’s immunostimulatoryproperty and may serve as a better agent forbladder cancer immunotherapy and antimy-cobacterial immunization.—Authors’ Ab-stract

Mendez-Samperio, P., Palma-Barrios, J.,Vazquez-Hernandez, A., and Gar cia-Martinez, E. Secretion of interleukin-8by human-derived cell lines infected withMycobacterium bovis . Mediators In-flamm. 13(1) (2004) 45–49.

BACKGROUND: The variable efficacyof bacillus Calmette-Guérin ( Mycobacte-rium bovis BCG) in protecting humansagainst tuberculosis has prompted a searchfor the mechanisms through which BCG in-duces chemokines. In this study, our exper-

iments were designed to determine the roleof the transcription factor nuclear factor -kappaB (NF-kappaB) and intracellular cal-cium in the production of interleukin (IL)-8,a main chemotactic factor, by human-derivedmonocytic cell line U937 and by a humanepithelial HEp-2 cell line infected with M.bovis BCG. METHODS: The concentra-tions of IL-8 in culture supernatants of U937cells or HEp-2 cells infected with M. bovisBCG were determined by enzyme-linked im-munosorbent assay. We used sulfasalazineand curcumin, which are well-described in-hibitors of NF-kappaB activity, and we usedethylenediamine tetraacetic acid to deplete ex-tracellular Ca2+ or used the cell-permeableagent 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxy-methyl) ester to chelate releasable intracel-lular stores of Ca2+ in order to investigatethe mechanisms through which M. bovisBCG induces IL-8 secretion in our system.RESULTS: The enzyme-linked immunosor-bent assay showed that IL-8 protein secretionwas elevated in M. bovis-infected cell lines.This ef fect was statistically significant (p<0.01). When calcium influx was sup-pressed in M. bovis-infected cell lines, IL-8secretion was inhibited. Notably, specific in-hibitors of NF-kappaB (sulfasalazine andcurcumin) inhibited M. bovis-induced IL-8secretion from U937 cells or HEp-2 cells.CONCLUSIONS: Collectively, these resultsindicate that activation of NF-kappaB is animportant signal transduction pathway in M.bovis-induced IL-8 secretion in monocyticor epithelial cells. Furthermore, the resultsshowed that calcium influx had a directeffect on IL-8 secretion in U937 cells orHEp-2 cells infected with M. bovis.—Au-thors’Abstract

Miyamoto, Y., Mukai, T., Takeshita, F.,Nakata, N., Maeda, Y., Kai, M., andMakino, M. Aggregation of mycobacte-ria caused by disruption of fibronectin-at-tachment protein-encoding gene. FEMSMicrobiol. Lett. 236(2) (2004) 227–234.

The fibronectin-attachment protein (FAP)is conserved among several species of my-cobacteria. Although this protein is associ-ated with attachment and internalization ofbacteria to host cells via fibronectin, the


physiological role of the protein still re-mains unclear. To investigate this point, wegenerated FAP gene disruptant in Mycobac-terium smegmatis. The gene disruption, ver-ified by Southern blot and PCR analysis, in-duced changes on the bacteria, which areassociated with strong aggregation and al-teration of cell surface properties. Increasedhydrophobicity and Congo red accumula-tion was observed in the F AP gene dis-ruptant. In addition, the complementationexperiment demonstrated that the corre-sponding gene restored wild type morphol-ogy in the disruptant. These results indicatethat the FAP affects the cell surface proper-ties, and its deletion lead to enhanced ag-gregation of the M. smegmatis.—Authors’Abstract

Pietersen, R., Thilo, L., and de Chastel-lier, C. Mycobacterium tuberculosis andMycobacterium avium modify the com-position of the phagosomal membrane ininfected macrophages by selective deple-tion of cell surface-derived glycoconju-gates. Eur . J. Cell. Biol. 83(4) (2004)153–158.

The growth of pathogenic mycobacteriain phagosomes of the host cell correlateswith their ability to prevent phagosome mat-uration. The underlying molecular mecha-nism remains elusive. In a previous study ,we have shown that Mycobacterium aviumdepletes the phagosome membrane of cellsurface-derived glycoconjugates (de Chas-tellier and Thilo, Eur . J. Cell Biol. 81,17–25, 2002). We now extended these quan-titative observations to the major humanpathogen, Mycobacterium tuber culosis(H37Rv). At increasing times after infectionof mouse bone marrow-derived macro-phages, cell-surface glycoconjugates werelabelled enzymatically with [3H]galactose.Subsequent endocytic membrane traffic re-sulted in a redistribution of this label fromthe cell surface to endocytic membranes, in-cluding phagosomes. The steady-state dis-tribution was measured by quantitative au-toradiography at the electron microscopelevel. Relative to early endosomes, withwhich phagosomes continued to fuse andrapidly exchange membrane constituents,the phagosome membrane was depleted

about 3-fold, starting during infection and inthe course of 9 days thereafter. These resultswere in quantitative agreement with our pre-vious observations for Mycobacteriumavium. For the latter case, we now showedby cell fractionation that the depletion wasselective, mainly involving glycoproteins inthe 110–210 kDa range. Together, these re-sults indicated that pathogenic mycobacte-ria induced and maintained a bulk change inphagosome membrane composition thatcould be of special relevance for survival ofpathogenic mycobacteria within phago-somes.—Authors’Abstract

Pozos, T. C., and Ramakrishan, L. Newmodels for the study of Mycobacterium-host interactions. Curr. Opin. Immunol.16(4) (2004) 499–505.

The outcome of Mycobacterium infectionis determined by a series of complex inter-actions between the bacteria and host im-munity. Traditionally, mammalian modelsand cultured cells have been used to studythese interactions. Recently , ameba (Dic-tyostelium), fruit flies (Drosophila) and ze-brafish, amenable to forward geneticscreens, have been developed as models formycobacterial pathogenesis. Infection ofthese hosts with mycobacteria has allowedthe dissection of intracellular traf fickingpathways (Dictyostelium) and the roles ofphagocytic versus antimicrobial peptide re-sponses (Drosophila). Real-time visualiza-tion of the optically transparent zebrafishembryo/larva has elucidated mechanisms bywhich Mycobacterium-infected leukocytesmigrate and subsequently aggregate intogranulomas, the hallmark pathological struc-tures of tuberculosis.—Authors’Abstract

Quesniaux, V., Fremond, C., Jacobs, M.,Parida, S., Nicolle, D., Yeremeev, V.,Bihl, F., Erard, F., Botha, T., Drennan,M., Soler, M. N., Le Bert, M., Schny-der, B., and Ryffel, B. Toll-like receptorpathways in the immune responses tomycobacteria. Microbes Infect. 6(10)(2004) 946–959.

The control of Mycobacterium tuberculo-sis infection depends on recognition of the


pathogen and the activation of both the in-nate and adaptive immune responses. Toll-like receptors (TLR) were shown to play acritical role in the recognition of severalpathogens. Mycobacterial antigens recog-nise distinct TLR resulting in rapid activa-tion of cells of the innate immune system.Recent evidence from in vitro and in vivo in-vestigations, summarized in this reviewdemonstrates TLR-dependent activation ofinnate immune response, while the induc-tion of adaptive immunity to mycobacteriamay be TLR independent.—Authors’ Ab-stract

Rambukkana, A. Mycobacterium leprae-induced demyelination: a model for earlynerve degeneration. Curr . Opin. Im-munol. 16(4) (2004) 511–518.

The molecular events that occur at theearly phase of many demyelinating neu-rodegenerative diseases are unknown. A re-cent demonstration of rapid demyelinationand axonal injury induced by Mycobacte-rium leprae provides a model for elucidat-ing the molecular events of early nerve de-generation which might be common toneurodegenerative diseases of both infec-tious origin and unknown etiology . Theidentification of the M. leprae -targetedSchwann cell receptor, dystroglycan, and itsassociated molecules in myelination, de-myelination and axonal functions suggests arole for these molecules in early nerve de-generation.—Author’s Abstract

Reed, M. B., Domenech, P ., Manca, C.,Su, H., Barczak, A. K., Kreiswirth, B.N., Kaplan, G., and Barry, C. E. 3rd. Aglycolipid of hypervirulent tuberculosisstrains that inhibits the innate immune re-sponse. Nature. 431(7004) (2004) 84–87.

Fifty million new infections with Myco-bacterium tuber culosis occur annually ,claiming 2–3 million lives from tuberculo-sis worldwide. Despite the apparent lack ofsignificant genetic heterogeneity betweenstrains of M. tuberculosis, there is mountingevidence that considerable heterogeneity ex-ists in molecules important in disease patho-genesis. These differences may manifest in

the ability of some isolates to modify thehost cellular immune response, thereby con-tributing to the observed diversity of clini-cal outcomes. Here we describe the identifi-cation and functional relevance of a highlybiologically active lipid species-a polyketidesynthase-derived phenolic glycolipid (PGL)produced by a subset of M. tuberculosis iso-lates belonging to the W-Beijing family thatshow “hyperlethality” in murine diseasemodels. Disruption of PGLsynthesis resultsin loss of this hypervirulent phenotype with-out significantly affecting bacterial load dur-ing disease. Loss of PGL was found to cor-relate with an increase in the release of thepro-inflammatory cytokines tumour-necrosisfactor-alpha and interleukins 6 and 12 invitro. Furthermore, the overproduction ofPGL by M. tuberculosis or the addition ofpurified PGL to monocyte-derived macro-phages was found to inhibit the release ofthese pro-inflammatory mediators in a dose-dependent manner.—Authors’Abstract

Rook, G. A., Hernandez-Pando, R.,Dheda, K., and Teng Seah, G. IL-4 intuberculosis: implications for vaccinedesign. Trends Immunol. 25(9) (2004)483–488.

Current attempts to find a vaccine for tu-berculosis (TB) are based on the assumptionthat it must drive a Th1 response. We reviewthe evidence that progressive disease mightnot be due to absence of Th1, but rather tothe subversive effect of an unusual Th2-likeresponse, involving interleukin-4 (IL-4) andIL-4delta2. This Th2-like response can im-pair bactericidal function and lead to toxic-ity of tumour necrosis factor -alpha (TNF-alpha) and to pulmonary fibrosis. If this isimportant, effective vaccines will need tosuppress pre-existing Th2-like activity. Suchvaccines are feasible and are active thera-peutically in mouse TB.—Authors’Abstract

Sendide, K., Deghmane, A. E., Reyrat, J.M., Talal, A., and Hmama, Z . Myco-bacterium bovis BCG urease attenuatesmajor histocompatibility complex class IItrafficking to the macrophage cell sur-face. Infect. Immun. 72(7) (2004) 4200–4209.


We have previously shown that Myco-bacterium tuberculosis attenuates cell sur-face expression of major histocompatibilitycomplex class II molecules in response togamma interferon (IFN-gamma) by a mech-anism dependent on intracellular sequestra-tion of alpha,beta dimers. In this study weexamined whether intracellular alkaliniza-tion due to mycobacterial urease could ac-count for the defect in intracellular traffick-ing of class II molecules. Phagocytosis ofwild-type Mycobacterium bovis BCG wasassociated with secretion of ammonia intra-cellularly, which increased substantiallyupon addition of exogenous urea to the cul-ture medium. Increased intracellular ammo-nia, due to urea degradation by the bacte-rium, correlated with inhibition of class IIsurface expression. Conversely, no ammo-nia was detected in cells infected with aurease-negative mutant strain of M. bovisBCG, which also displayed a reduced effecton surface expression of class II molecules.A direct cause-effect relationship betweenurease and class II molecule trafficking wasestablished with experiments where cellsingesting beads coated with purified ureaseshowed an increased ammonia level anddecreased surface expression of class II inresponse to IFN-gamma. In contrast toBCG, infection of macrophages with Myco-bacterium smegmatis, which expresses rela-tively greater urease activity in cell-free cul-ture, had a mar ginal ef fect on both theintracellular level of ammonia and class IIexpression. The limited effect of M. smeg-matis was consistent with a failure to resistintracellular killing, suggesting that ureasealone is not sufficient to resist macrophagemicrobicidal mechanisms and that this is re-quired for a more distal effect on cell regu-lation. Our results demonstrate that alkalin-ization of critical intracellular organelles bypathogenic mycobacteria expressing ureasecontributes significantly to the intracellular

retention of class II dimers.—Authors’ Ab-stract

Temmerman, S., Pethe, K., Parra, M.,Alonso, S., Rouanet, C., Pickett, T.,Drowart, A., Debrie, A. S., Delogu, G.,Menozzi, F. D., Sergheraert, C., Bren-nan, M. J., Mascart, F., and Locht, C.Methylation-dependent T cell immunityto Mycobacterium tuberculosis heparin-binding hemagglutinin. Nat. Med. 10(9)(2004) 935–941. Epub 2004 Aug 08.

Although post-translational modificationsof protein antigens may be important com-ponenets of some B cell epitopes, the deter-minants of T cell immunity are generallynonmodified peptides. Here we show thatmethylation of the Mycobacterium tubercu-losis heparin-binding hemagglutinin (HBHA)by the bacterium is essential for effective Tcell immunity to this antigen in infectedhealthy humans and in mice. Me thylatedHBHA provides high levels of protectionagainst M. tuberculosis challenge in mice,whereas nonmethylated HBHA does not.Protective immunity induced by methylatedHBHA is comparable to that af forded byvaccination with bacille Calmette et Guérin,the only available anti-tuberculosis vaccine.Thus, post-translational modifications ofproteins may be crucial for their ability toinduce protective T cell-mediated immunityagainst infectious diseases such as tubercu-losis.—Authors’Abstract

Ziglam, H. M., Daniels, I., and Finch, R.G. Immunomodulating activity of ri-fampicin. J. Chemother . 16(4) (2004)357–361.

See Current Literature, Chemotherapy, p.515.

72, 4 Current Literature, Immunopathology (Leprosy) 531

Immunopathology (Leprosy)

Alves, L., de Mendonca Lima, L., da SilvaMaeda, E., Carvalho, L., Holy , J.,Sarno, E. N., Pessolani, M. C., andBarker, L. P. Mycobacterium leprae in-fection of human Schwann cells dependson selective host kinases and pathogen-

modulated endocytic pathways. FEMSMicrobiol. Lett. 238(2) (2004) 429–437.

Mycobacterium leprae, an obligate intra-cellular pathogen, shows a unique tropismfor Schwann cells (SC). This leads to the pe-

ripheral neuropathy disorder observed inleprosy. In this study, we investigated signaltransduction events and the intracellular fateof M. leprae during the interaction of themicroorganism with SC. First, we demon-strated that the human schwannoma cell lineST88-14 readily phagocytized the bacteriaas observed by time-lapse microscopy, actinstaining and electron microscopy. The effectof specific kinase inhibitors on M. leprae in-ternalization was then investigated showingthat functional protein tyrosine kinase,calcium-dependent protein kinase and phos-phatidylinositol 3-kinase, but not cAMP-dependent protein kinase are essential forphagocytosis of the bacteria. Similar resultswere obtained when irradiated and live bac-teria were compared and when M. lepraewas pre-coated with recombinant histone-like-protein/ laminin binding protein, a bac-terial adhesin. In addition, experiments wereperformed to analyze the bacterial traffick-ing within the endosomal network by label-ing the acidified intracellular compartmentsof M. leprae -infected SC with the L yso-tracker acidotrophic probe. Acidification ofvesicles containing live M. leprae was min-imal in both RAW murine macrophages andSC, although phagosomes containing heat-killed bacteria seem to follow normal endo-cytic maturation. These data indicate that theinvading bacteria interfere with normal en-docytic pathway maturation of bacteria-containing phagosomes within SC.—Au-thors’Abstract

Hagge, D. A., Ray, N. A., Krahenbuhl, J.L., and Adams, L. B. An in vitro modelfor the lepromatous leprosy granuloma:fate of Mycobacterium leprae from targetmacrophages after interaction with nor-mal and activated effector macrophages.J. Immunol. 172(12) (2004) 7771–7779.

The lepromatous leprosy granuloma is adynamic entity requiring a steady influx ofmacrophages (Mphi) for its maintenance.We have developed an in vitr o model tostudy the fate of Mycobacterium leprae in aLL lesion, with and without immunothera-peutic intervention. Target cells, consistingof granuloma Mphi harvested from the foot-pads of M. leprae-infected athymic nu/numice, were cocultured with normal or IFN-

gamma-activated (ACT) effector Mphi. Thebacilli were recovered and assessed for via-bility by radiorespirometry . M. leprae re-covered from tar get Mphi possessed highmetabolic activity, indicating a viable statein this uncultivable organism. M. leprae re-covered from tar get Mphi incubated withnormal effector Mphi exhibited significantlyhigher metabolism. In contrast, bacilli re-covered from target Mphi cocultured withACT effector Mphi displayed a markedlydecreased metabolic activity. Inhibition byACT Mphi required an E:T ratio of at least5:1, a coculture incubation period of 3–5days, and the production of reactive nitrogenintermediates, but not reactive oxygen in-termediates. Neither IFN-gamma nor TNF-alpha were required during the cocultivationperiod. However , cell-to-cell contact be-tween the target and effector Mphi was nec-essary for augmentation of M. leprae me-tabolism by normal effector Mphi as well asfor inhibition of M. leprae by ACT effectorMphi. Conventional fluorescence mi-croscopy and confocal fluorescence mi-croscopy revealed that the bacilli from thetarget Mphi were acquired by the ef fectorMphi. Thus, the state of Mphi infiltrating thegranuloma may markedly affect the viabil-ity of M. leprae residing in Mphi in the lep-romatous lesion.—Authors’Abstract

Kang, T. J., Yeum, C. E., Kim, B. C., You,E. Y., and Chae, G. T. Differential pro-duction of interleukin-10 and interleukin-12 in mononuclear cells from leprosy pa-tients with a Toll-like receptor 2 mutation.Immunology 112(4) (2004) 674–680.

Toll-like receptor 2 (TLR2) is a key me-diator of the immune response to mycobac-terial infections, and mutations in TLR2have been shown to confer susceptibility toinfection with mycobacteria. This studyinvestigated the profiles of cytokines, suchas interferon (IFN)-gamma, interleukin (IL)-10, IL-12 and tumour necrosis factor(TNF)-alpha in response to Mycobacteriumleprae in peripheral blood mononuclearcells (PBMC) with the TLR2 mutationArg677Trp, a recently reported polymor-phism that is associated with lepromatousleprosy. In leprosy patients with the TLR2mutation, production of IL-2, IL-12, IFN-


gamma, and TNF-alpha by M. leprae -stimulated PBMC were significantly de-creased compared with that in groups withwild-type TLR2. However, the cells frompatients with the TLR2 mutation showedsignificantly increased production of IL-10.There was no significant difference in IL-4production between the mutant and wild-type during stimulation. Thus, these resultssuggest that the TLR2 signal pathway playsa critical role in the alteration of cytokineprofiles in PBMC from leprosy patients andthe TLR2 mutation Arg677Trp provides amechanism for the poor cellular immune re-sponse associated with lepromatous lep-rosy.—Authors’Abstract

Kim, B. C., Kang, T. J., Jin, S. H., Kim, S.K., Lee, S. B., and Chae, G. T. Tissue-specific down-regulation of RIPK 2 in My-cobacterium leprae -infected nu/nu mice.Mediators Inflamm. 13(1) (2004) 51–52.

RIPK 2 is adapter molecule in the signalpathway involved in Toll-like receptors.However, there has been no reported asso-ciation between receptor-interacting serine/threonine kinase 2 (RIPK 2) expression andthe infectious diseases involving mycobac-terial infection. This study found that itsexpression was down-regulated in the foot-pads and skin but was up-regulated in theliver of Mycobacterium leprae -infectednu/nu mice compared with those of the M. leprae non-infected nu/nu mice. It wasobserved that the interlukin-12p40 andinterferon-gamma genes involved in the sus-ceptibility of M. leprae were down-regulatedin the skin but were up-regulated in the liver.Overall, this suggests that regulation ofRIPK 2 expression is tissue-specifically as-sociated with M. leprae infection.—Au-thors’Abstract

Kimura, H., Maeda, Y., Takeshita, F .,Takaoka, L. E., Matsuoka, M., andMakino, M. Upregulation of T-cell-stimulating activity of mycobacteria-infected macrophages. Scand. J. Immunol.60(3) (2004) 278–286.

Macrophages are one of the most abundanthost cells to come in contact with mycobacte-

ria. However, the infected macrophages lessefficiently stimulate autologous T cells invitro. We investigated the effect of the induc-tion of phenotypic change of macrophages onthe host cell activities by using Mycobacte-rium leprae as a pathogen. The treatment ofmacrophages with interferon-gamma (IFN-gamma), GM-CSF and interleukin-4 de-prived macrophages of CD14 antigen ex-pression but instead provided them withCD1a, CD83 and enhanced CD86 antigenexpression. These phenotypic features re-sembled those of monocyte-derived den-dritic cells (DC). These macrophage-derivedDC-like cells (MACDC) stimulated autolo-gous CD4+ and CD8+ T cells when infectedwith M. leprae. Further enhancement of theantigen-presenting function and CD1a ex-pression of macrophages was observedwhen treated with IFN-gamma. The M.leprae-infected and -treated macrophagesexpressed bacterial cell membrane-derivedantigens on the surface and were efficientlycytolysed by the cell membrane antigen-specific CD8+ cytotoxic T lymphocytes(CTL). These results suggest that the induc-tion of phenotypic changes in macrophagescan lead to the upregulation of host defenceactivity against M. leprae.—Authors’ Ab-stract

Moraes, M. O., Pacheco, A. G.,Schonkeren, J. J., Vanderborght, P. R.,Nery, J. A., Santos, A. R., Moraes, M.E., Moraes, J. R., Ottenhoff, T. H.,Sampaio, E. P., Huizinga, T. W., andSarno, E. N. Interleukin-10 promotersingle-nucleotide polymorphisms asmarkers for disease susceptibility anddisease severity in leprosy . GenesImmun. Epub 2004 Aug 12.

We have determined IL-10 promotergenotypes of five single-nucleotide poly-morphisms (SNPs): T-3575A, A-2849G, C-2763A, -A-1082G and C-819T. The hap-lotype frequencies were defined in healthysubjects compared to leprosy patients, andanalyzed for their occurrence in multi- (MB)vs paucibacillary (PB) as severe and mildforms of leprosy, respectively. Haplotypesdefined by three SNP positions (–3575,–2849 and –2763) captured significant dif-ferences between controls and patients (p =

72, 4 Current Literature, Immunopathology (Leprosy) 533

0.04). The haplotype carrying –3575A,–2849G and –2763C was associated with re-sistance to leprosy and to the developmentof severe forms of the disease using eithera binomial (controls vs. cases, p = 0.005,OR = 0.35, CI = 0.13–0.91) or ordinal (con-trols vs PB vs MB, p = 0.006, OR = 0.32, CI= 0.12–0.83) model. By contrast, the IL-10haplotype –3575T/–2849A/–2763C wasfound to be associated with susceptibility toleprosy per se (p = 0.027, OR = 2.37, CI =1.04–5.39), but not leprosy type. The datasuggest that the IL-10 locus contributes tothe outcome of leprosy.—Authors’Abstract

Ohyama, H., Kato, N., Takeuchi, K.,Soga, Y., Uemura, Y., Nishimura, F .,and Matsushita, S. Monocytes of dis-tinct clinical types of leprosy are differ-entially activated by cross-linking class IIHLA molecules to secrete IL-12. APMIS112(4–5) (2004) 271–274.

Leprosy is characterized by a wide spec-trum of clinical features depending on theindividual differences in Th1-type immu-nity. The objective of this study was toevaluate whether monocyte activation bystimulus via class II HLAmolecules wouldbe correlated with the differences in cellu-lar immune responses among diverse clin-ical forms of leprosy. IL-1beta and IL-12productivity in monocyte preparations ob-tained from PBMCs was estimated in pa-tients with lepromatous- and tuberculoid-type leprosy . We found that monocytesfrom lepromatous patients produced sig-nificantly higher (about 4-fold higher)amounts of IL-12 as compared to in pa-tients with tuberculoid type of leprosywhen class II HLA molecules were cross-linked with anti-HLA class II antibodies,whereas almost equal amounts of IL-1betawere produced from each monocyte prepa-ration by stimulus via class II HLA mole-cules regardless of the clinical form of lep-rosy. These results suggest that monocyteactivation differs between lepromatous andtuberculoid patients in terms of IL-12 se-cretion, which might be related to indi-vidual differences in the cellular immuneresponses according to the clinical type ofleprosy.—Authors’ Abstract

Reddy, Y. N., Murthy, S. V., Krishna, D.R., and Prabhakar , M. C. Oxidativestress and anti-oxidant status in leprosypatients. Indian J. Lepr . 75(4) (2003)307–316.

Severe oxidative stress has been reportedin leprosy patients because of malnutritionand poor immunity . The purpose of thisstudy was to investigate the serum lipid per-oxidation products, serum LDH and impor-tant free radical scavenging enzymes, i.e.superoxide dismutase (SOD), and catalaseand anti-oxidant glutathione levels and totalanti-oxidant status, in different types of lep-rosy patients. The subjects for this studywere normal human volunteers (NHVs, N =14), paucibacillary leprosy patients (PB, N= 18), untreated MB patients (MB1, N =18), MB patients under treatment (MB2, N= 19), and MB patients released from treat-ment (RFT) (MB3, N = 28). The levels oflipid peroxidation product, malondialdehyde(MDA), and LDH increased significantly (p<0.001) in MB (MB1, MB2, MB3) patients,and both gradually decreased with clinicalimprovement following MDT. The levels ofSOD, catalase and glutathione, and the totalanti-oxidant status decreased significantly inMB (MB1, MB2, MB3) patients (p <0.001),in comparison with NHVs. They graduallyincreased with clinical improvement withMDT. There was no significant variation ofthese parameters in PB leprosy patients incomparison with healthy volunteers. Highfree radical activity and low anti-oxidantlevels observed in MB (MB1, MB2, MB3)leprosy patients indicate that there is an ox-idative stress in MB cases, irrespective ofthe treatment status and suggest a suitableanti-oxidant therapy to prevent possible tis-sue injury.—Authors’Abstract

Walsh, D. S., Lane, J. E., Abalos, R. M.,and Myint, K. S. TUNEL and limitedimmunophenotypic analyses of apoptosisin paucibacillary and multibacillary lep-rosy lesions. FEMS Immunol. Med. Mi-crobiol. 41(3) (2004) 265–269.

Some mycobacterial infections, such astuberculosis, are characterized by apoptosisof infected or by-stander mononuclear im-mune cells. For localized (paucibacillary ,


PB) and disseminated (multibacillary, MB)leprosy, characterized by polarized Th1-likevs. Th2-like immune responses, respec-tively, little is known about lesional apop-tosis. We analyzed sections of paraf fin-embedded, untreated leprosy lesions from21 patients by an indirect immunofluores-cent terminal deoxynucleotide-transferase-mediated dUTP-digoxigenin nick endlabeling (TUNEL) assay . Some TUNEL(+) PB sections were then reacted withphycoerythrin-conjugated (red) antibodiesagainst T cells, monocytes, or antigen-pre-senting (Langerhans) cells. TUNEL (+)bodies were detected in 9 of 16 PB lesions(56%) and in 1 of 5 MB lesions (20%).Some TUNEL (+) bodies in PB diseasewere CD3+ (T cell), as well as CD4+ (T-helper) or CD8+ (T-cytotoxic). Apopto-sis characterizes PB and MB leprosy le-sions and may be more frequent in PB dis-ease. In PB disease, some TUNEL (+)bodies may derive from T cells.—Authors’Abstract

Yamashita, Y., Maeda, Y., Takeshita, F.,Brennan, P. J., and Makino, M.Role ofthe polypeptide region of a 33kDa myco-bacterial lipoprotein for ef ficient IL-12

production. Cell. Immunol. 229(1) (2004)13–20.

Mycobacterium leprae lipoprotein, LpK,induced IL-12 production from humanmonocytes. To determine the componentsessential for cytokine production and therelative role of lipidation in the activationprocess, we produced lipidated and non-lipidated truncated forms of LpK. While 0.5nM of lipidated LpK-a having N-terminal60 amino acids of LpK produced more than700 pg/ml IL-12 p40, the non-lipidatedLpK-b having the same amino acids as thatof LpK-a required more than 20nM of theprotein to produce an equivalent dose ofcytokine. Truncated protein having the C-terminal 192 amino acids of LpK did notinduce any cytokine production. Fiftynanomolar of the synthetic lipopeptide ofLpK produced only about 200pg/ml IL-12.Among the truncated LpK, only LpK-a andlipopeptide stimulated NF-kB-dependent re-porter activity in TLR-2 transfectant. How-ever, when monocytes were stimulated withlipopeptide in the presence of non-lipidatedprotein, they produced IL-12 synergistically.Therefore, both peptide regions of LpK andlipid residues are necessary for efficient IL-12 production.—Authors’Abstract

72, 4 Current Literature, Immunopathology (Tuberculosis) 535

Immunopathology (Tuberculosis)

Banerjee, S., Nandyala, A., Podili, R., Ka-toch, V. M., Murthy , K. J., and Has-nain, S. E. Mycobacterium tuberculosis(Mtb) isocitrate dehydrogenases showstrong B cell response and distinguishvaccinated controls from TB patients.Proc. Natl. Acad. Sci. U.S.A. 101(34)(2004) 12652–12657. Epub 2004 Aug 16.

Proteins released from Mycobacterium tu-berculosis (Mtb) during late logarithmicgrowth phase are often considered candidatecomponents of immunogenic or autolysismarkers. One such protein is isocitrate de-hydrogenase (ICD), a key regulatory en-zyme in the citric acid cycle. We have eval-uated the immunogenic properties of twoisoforms of Mtb ICD and compared them

with the control antigens heat-shock protein60 and purified protein derivative (PPD).PPD lacks the sensitivity to distinguish be-tween bacillus Calmette-Guérin (BCG)-vac-cinated and tuberculosis (TB)-infectedpopulations, and, therefore, epidemiologicalrelevance of PPD in BCG-vaccinated re-gions is debatable. We show that Mtb ICDselicit a strong B cell response in TB-infectedpopulations and can dif ferentiate betweenhealthy BCG-vaccinated populations andthose with TB. The study population (N =215) was categorized into different groups,namely, patients with fresh infection (N =42), relapsed TB cases (N = 32), patientswith extrapulmonary TB (N = 35), clinicallyhealthy donors (N = 44), nontuberculousmycobacteria patients (N = 30), and non-TB

patients (culture negative for acid-fast bac-teria but carrying other infections, N = 32).The Mtb ICDs showed statistically signifi-cant antigenic distinction between healthyBCG-vaccinated controls and TB patients (p<0.0001) and those with other infections.Although extrapulmonary infections couldnot be discriminated from healthy controlsby heat-shock protein 60 (p = 0.2177), in-terestingly, the Mtb ICDs could significantly(p <0.0001) do so. Our results highlight theimmunodominant, immunosensitive, andimmunospecific nature of Mtb ICDs andpoint to an unusual property of this tricar-boxylic acid ener gy cycle enzyme.—Au-thors’Abstract

Boshoff, H. I., Myers, T. G., Copp, B. R.,McNeil, M. R., Wilson, M. A., Barry,C. E. 3rd. The transcriptional responsesof Mycobacterium tuber culosis to in-hibitors of metabolism: novel insightsinto drug mechanisms of action. J. Biol.Chem. 279(38) (2004) 40174 –40184.Epub 2004 Jul 09.

The differential transcriptional responseof Mycobacterium tuberculosis to drugs andgrowth-inhibitory conditions was monitoredto generate a data set of 430 microarray pro-files. Unbiased grouping of these profiles in-dependently clustered agents of knownmechanism of action accurately and wassuccessful at predicting the mechanism ofaction of several unknown agents. Thesepredictions were validated biochemicallyfor two agents of previously uncategorizedmechanism, pyridoacridones and pheno-thiazines. Analysis of this data set further re-vealed 150 underlying clusters of coordi-nately regulated genes of fering the firstglimpse at the full metabolic potential of thisorganism. A signature subset of these geneclusters was sufficient to classify all knownagents as to mechanism of action. Tran-scriptional profiling of both crude and puri-fied natural products can provide criticalinformation on both mechanism and detox-ification prior to purification that can beused to guide the drug discovery process.Thus, the transcriptional profile generatedby a crude marine natural product recapitu-lated the mechanistic prediction from thepure active component. The underlying gene

clusters further provide fundamental in-sights into the metabolic response of bacte-ria to drug-induced stress and provide a ra-tional basis for the selection of criticalmetabolic tar gets for screening for newagents with improved activity against thisimportant human pathogen.—Authors’ Ab-stract

Briken, V., Porcelli, S. A., Besra, G. S.,and Kremer, L. Mycobacterial lipoara-binomannan and related lipoglycans:from biogenesis to modulation of the im-mune response. Mol. Microbiol. 53(2)(2004) 391–403.

The cell wall component lipoarabino-mannan (ManLAM) from Mycobacteriumtuberculosis is involved in the inhibition ofphagosome maturation, apoptosis and inter-feron (IFN)-gamma signalling in macro-phages and interleukin (IL)-12 cytokine se-cretion of dendritic cells (DC). All theseprocesses are important for the host tomount an efficient immune response. Con-versely, LAM isolated from non-pathogenicmycobacteria (PILAM) have the oppositeeffect, by inducing a potent proinflammatoryresponse in macrophages and DCs. LAMsfrom diverse mycobacterial species differ inthe modification of their terminal arabinoseresidues. The strong proinflammatory re-sponse induced by PILAM correlates withthe presence of phospho-myo-inositol on theterminal arabinose. Interestingly , recentwork indicates that the biosynthetic precur-sor of LAM, lipomannan (LM), which isalso present in the cell wall, displays strongproinflammatory effects, independently ofwhich mycobacterial species it is isolatedfrom. Results from in vitro assays andknock-out mice suggest that LM, likePILAM, mediates its biological activity viaToll-like receptor 2. We hypothesize that theLAM/LM ratio might be a crucial factor indetermining the virulence of a mycobacte-rial species and the outcome of the infection.Recent progress in the identification ofgenes involved in the biosynthesis of LAMis discussed, in particular with respect to thefact that enzymes controlling the LAM/LMbalance might represent targets for new anti-tubercular drugs. In addition, inactivation ofthese genes may lead to attenuated strains of


M. tuberculosis for the development of newvaccine candidates.—Authors’Abstract

Chua, J., and Deretic, V. Mycobacteriumtuberculosis reprograms waves of phos-phatidylinositol 3-phosphate on phago-somal organelles. J. Biol. Chem. 275(35)(2004) 36982–36992. Epub 2004 Jun 21.

The potent human pathogen Mycobacte-rium tuberculosis persists in macrophageswithin a specialized, immature phago-some by interfering with the pathway ofphagolysosome biogenesis. The molecularmechanisms underlying this process remainto be fully elucidated. Here, using four -dimensional microscopy , we detected onmodel phagosomes, which normally matureinto phagolysosomes, the existence of cyclicalwaves of phosphatidylinositol 3-phosphate(PI3P), a membrane trafficking regulatorylipid essential for phagosomal acquisition oflysosomal characteristics. We show that my-cobacteria interfere with the dynamics ofPI3P on phagosomal organelles by alteringthe timing and characteristics of the PI3Pwaves on phagosomes. The default programof cyclical PI3P waves on model phago-somes is composed of an initial stage (phaseI), represented by a strong PI3P burst oc-curring only upon the completion of phago-some formation, and a subsequent stage(phase II) of recurring PI3P waves on ma-turing phagosomes with the average perio-dicity of 20 min. Mycobacteria alter thisprogram in two ways: (i) by inducing, in acholesterol-dependent fashion, a neophaseI* of premature PI3Pproduction, coincidingwith the process of mycobacterial entry intothe macrophage, and (ii) by inhibiting thecalmodulin-dependent phase II responsiblefor the acquisition of lysosomal characteris-tics. We conclude that the default pathwayof phagosomal maturation into the phago-lysosome includes temporally or ganizedcyclical waves of PI3P on phagosomalmembranes and that this process is targetedfor reprogramming by mycobacteria as theyprevent phagolysosome formation.—Au-thors’Abstract

Ciaramella, A., Cavone, A., Santucci, M.B., Garg, S. K., Sanarico, N., Bocchino,

M., Galati, D., Martino, A., Auricchio,G., D’Orazio, M., Stewart, G. R., Ney-rolles, O., Young, D. B., Colizzi, V., andFraziano, M. Induction of apoptosis andrelease of interleukin-1 beta by cell wall-associated 19-kDa lipoprotein during thecourse of mycobacterial infection. J. In-fect. Dis. 190(6) (2004) 1167–1176. Epub2004 Aug. 11.

Mycobacterium tuberculosis induces apop-tosis in human monocyte-derived macro-phages (MDMs) during the early stages ofinfection. We investigated the proapoptoticrole of cell wall-associated mycobacterial 19-kDa lipoprotein and the possible associationbetween 19-kDa lipoprotein signaling andproduction of proinflammatory cytokines. Pu-rified mycobacterial 19-kDa lipoprotein, 19-kDa lipoprotein-expressing M. smegmatis (M.smegmatis 19+), 19-kDa lipoprotein knock-out (KO) M. tuber culosis, and 19-kDalipoprotein KO M. bovis bacille Calmette-Guerin (BCG) strains were analyzed for theirability to induce apoptosis in MDMs. The 19-kDa lipoprotein and infection with M. smeg-matis 19+ induced apoptosis in MDMs. M.tuberculosis and BCG KO strains had signif-icantly decreased abilities to induce apoptosis.The 19-kDa lipoprotein proapoptotic signalwas mediated by Toll-like receptor 2 but notby tumor necrosis factor-alpha. Only the re-lease of interleukin (IL)-1 beta was decreasedafter infection with 19-kDa lipoprotein KOstrains. These findings indicate that the 19-kDa lipoprotein is the main signal required totrigger both apoptosis and the release of IL-1beta during the early stages of mycobacterialinfection.—Authors’Abstract

Cliff, J. M., Andrade, I. N., Mistry , R.,Clayton, C. L., Lennon, M. G., Lewis,A. P., Duncan, K., Lukey , P. T., andDockrell, H. M. Differential gene ex-pression identifies novel markers ofCD4+ and CD8+ T cell activation fol-lowing stimulation by Mycobacterium tu-berculosis. J. Immunol. 173(1) (2004)485–493.

T cell activation in response to antigenicstimulation is a complex process, involvingchanges in the expression level of a lar genumber of genes. We have used cDNAarray


technology to characterize the differences ingene expression between human CD4+ andCD8+ T cells. PBMC from six healthydonors were stimulated with live Mycobac-terium tuberculosis, and the gene expressionprofiles of each donor’s CD4+ and CD8+ Tcells were analyzed separately. ANOVA re-vealed 518 genes that were consistently dif-ferentially expressed between CD4+ andCD8+ T cells. These differentially expressedgenes include a combination of well-known,previously characterized genes with a rangeof biological functions and unknown in sil-ico predicted hypothetical genes. Wherepossible, the novel genes have been charac-terized using bioinformatics, and putativetranscription factors, signaling molecules,transmembrane, and secreted factors havebeen identified. A subset of these differen-tially expressed genes could be exploited asmarkers of CD4+ and CD8+ T cell activa-tion for use in vaccine trials. These observeddifferences in the gene expression profile ofCD4+ and CD8+ T cells following activa-tion by a human pathogen contribute to anincreased understanding of T cell activationand differentiation and the roles these T cellsubsets may play in immunity to infec-tion.—Authors’Abstract

Gehring, A. J., Dobos, K. M., Belisle, J. T.,Harding, C. V., and Boom, W. H. Myco-bacterium tuberculosis LprG (Rv1411c):a novel TLR-2 ligand that inhibits humanmacrophage class II MHC antigen pro-cessing. J. Immunol. 173(4) (2004)2660–2668.

MHC class II (MHC-II)-restricted CD4(+)T cells are essential for control of Myco-bacterium tuberculosis infection. This re-port describes the identification and purifi-cation of LprG (Rv1411c) as an inhibitor ofprimary human macrophage MHC-II Agprocessing. LprG is a 24-kDa lipoproteinfound in the M. tuberculosis cell wall. Pro-longed exposure (>16 hr) of human macro-phages to LprG resulted in marked inhibi-tion of MHC-II Ag processing. Inhibition ofMHC-II Ag processing was dependent onTLR-2. Short-term exposure (<6 hr) toLprG stimulated TLR-2-dependent TNF-alpha production. Thus, LprG can exploitTLR-2 signaling to inhibit MHC-II Ag

processing in human macrophages. Inhibi-tion of MHC-II Ag processing by myco-bacterial lipoproteins may allow M. tuber-culosis, within infected macrophages, toavoid recognition by CD4(+) T cells.—Au-thors’ Abstract

Glatman-Freedman, A., Casadevall, A.,Dai, Z., Jacobs, W. R. Jr., Li, A., Mor-ris, S. L., Navoa, J. A., Piperdi, S., Rob-bins, J. B., Schneerson, R., Schwebach,J. R., and Shapir o, M. Antigenic evi-dence of prevalence and diversity of My-cobacterium tuber culosis arabinoman-nan. J. Clin. Microbiol. 42(7) (2004)3225–3231.

Arabinomannan (AM) is a polysaccharideof the mycobacterial capsule. The capsularpolysaccharides of various microorganismsare diverse, and this diversity is importantfor classification of organisms into serotypesand vaccine development. In the presentstudy we examined the prevalence and di-versity of AM among Mycobacterium tuber-culosis strains using four AM-binding mono-clonal antibodies (MAbs). One of theseMAbs, MAb 9d8, is known to bind to AMspecifically. By whole-cell enzyme-linkedimmunosorbent assay (ELISA), the AM rec-ognized by MAb 9d8 was detected on thesurfaces of 9 of 1 1 strains, while 2 strainsshowed no reactivity with MAb 9d8. How-ever, the AM recognized by MAb 9d8 wasfound in the culture supernatants of all 11 M.tuberculosis strains tested, as demonstratedby capture ELISA. Other AM-binding MAbsreacted both with the surfaces and with theculture supernatants of all 11 strains. Miceimmunized with an experimental AM-recombinant Pseudomonas aeruginosa exo-protein A (rEPA) conjugate vaccine had anincreased antibody response to AM and amoderate reduction in the numbers of CFUin their organs 7 days after challenge. Ourresults indicate that AM was detected in allM. tuberculosis strains tested, with dif fer-ences in epitope distributions of certainstrains. In addition, our results suggest thatan experimental AM-rEPA vaccine has amoderate effect on the numbers of CFU inorgans early after infection.—Authors’Abstract


Guidry, T. V., Olsen, M., Kil, K. S.,Hunter, R. L. Jr ., Geng, Y. J., andActor, J. K. Failure of CD1D–/– mice toelicit hypersensitive granulomas to my-cobacterial cord factor trehalose 6,6 ′-dimycolate. J. Interferon Cytokine Res.24(6) (2004) 362–371.

The present study defines pathologic dif-ferences in acute and hypersensitive responsesto Mycobacterium tuberculosis glycolipid tre-halose-6,6′-dimycolate (TDM, cord factor) innormal BALB/c mice and those deficient ingroup II CD1 molecule CD1d1. Mice immu-nized against TDM demonstrate hypersensi-tive responses, yet the mechanisms for TDMpresentation remain elusive. Mice lackingCD1d (CD1D(–/–)) d emonstrate dysregu-lated granulomatous response to TDM,compared with CD1D(+/–) heterozygouscontrols. Because CD1d-restricted T cellscan regulate macrophage immune functionsat mucosal surfaces, we hypothesized thatCD1D(–/–) mice would show deficientTDM-induced hypersensitive pulmonarygranulomatous response in which T cellsplay a central role. Control CD1D(+/+) micesensitized and subsequently challenged withTDM demonstrated aggressive inflamma-tion defined by monocytic lesions containedby CD3(+) lymphocytic cuffing. CD1D(–/–)mice demonstrated distinctly dif ferentpathologies, with edema present concurrentwith extended, nonfocal mononuclear cell-based granulomatous reactions. Further-more, CD1D(–/–) mice did not demonstratedestructive lesions, and CD3(+) lympho-cytes were only loosely organized in prox-imity to reactive pathology . The CD1d-deficient mice demonstrated rapid increasesin proinflammatory mRNAs, with significantdifferences in interferon-gamma (IFN-gamma) compared to the wild-type group.IFN-gamma, interleukin-6 (IL-6), and IL-12proteins were significantly elevated in theCD1D(–/–) group compared with wild-typemice (p <0.05) 2 days after TDM challenge.However, by 7 days postadministration,similar production for all cytokines andproinflammatory molecules examined waspresent in both groups of mice. These ex-periments provide evidence for a role forCD1d in mediation of pathology during hy-persensitive responses to the mycobacterialglycolipid TDM.—Authors’Abstract

Haydel, S. E., and Clark-Curtiss, J. E.Global expression analysis of two-component system regulator genes duringMycobacterium tuberculosis growth inhuman macrophages. FEMS Microbiol.Lett. 236(2) (2004) 341–347.

In the Mycobacterium tuber culosisH37Rv genome, there are 1 1 paired two-component regulatory system genes, two or-phan histidine kinase genes, and six orphanresponse regulator genes. Expression of the17 response regulator genes and the two or-phan histidine kinase genes during growthof M. tuber culosis in human peripheralblood monocyte-derived macrophages hasbeen analyzed by using cDNAmixtures pre-pared by the selective capture of transcribedsequences (SCOTS) technique. SCOTSprobes were prepared from cDNA obtainedfrom M. tuberculosis grown for 18, 48, and110 hr in human macrophages. Based on ex-pression profiles, the regulatory genes wereassigned to three categories: (i) constitu-tively expressed during growth in macro-phages (three genes); (ii) differentially ex-pressed during growth in macrophages (ninegenes) and (iii) no detectable expressionduring growth in macrophages (sevengenes).—Authors’Abstract

Islam, N., Kanost, A. R., Teixeira, L.,Johnson, J., Hejal, R., Aung, H.,Wilkinson, R. J., Hirsch, C. S., andToossi, Z. Role of cellular activation andtumor necrosis factor-alpha in the earlyexpression of Mycobacterium tuberculo-sis 85B mRNAin human alveolar macro-phages. J. Infect. Dis. 190(2) (2004)341–351. Epub 2004 Jun 18.

BACKGROUND: Infection of alveolarmacrophages (AMs), which constitute thefirst line of defense against Mycobacteriumtuberculosis, initiates an intense interactionbetween the host’s innate immune responseand mycobacteria that may assist in the suc-cessful intracellular parasitism of M. tuber-culosis. METHODS: Expression of tumornecrosis factor (TNF)-alpha and M. tuber-culosis 85B mRNA was studied in M.tuberculosis-infected AMs, to better delin-eate the role of macrophages in the earlyevents in initiation of infection. RESULTS:


Both TNF- alpha mRNA and M. tuberculo-sis 85B were induced in AMs; at 24 hr, thetime point of maximum TNF-alpha induc-tion, the mRNA levels for TNF-alpha andM. tuberculosis 85B correlated with one an-other, and induction of either gene corre-lated strongly with their protein levels. In-hibition of endogenous TNF-alpha bysoluble (s) TNF receptor (R) I and sTNFRIIreduced expression of both TNF-alpha andM. tuber culosis 85B. The activation ofnuclear factor-kappa B was found to under-lie expression of both TNF-alpha and M.tuberculosis 85B. Exogenous TNF-alphawas slightly more potent than interleukin(IL)-6 and granulocyte-macrophage colony-stimulating factor and was significantlystronger than IL-1 in inducing expression ofM. tuberculosis 85B. Interestingly, inhibi-tion of bactericidal mediators, reactive oxy-gen intermediates (ROIs) and reactive ni-trogen intermediates (RNIs), reducedexpression of TNF-alpha and M. tuberculo-sis 85B genes in M. tuberculosis-infectedAMs. CONCLUSION: Activation of AMsby M. tuber culosis initiates a cascade ofevents whereby TNF-alpha, ROI, and RNIenhance the expression of the M. tuberculo-sis 85B gene.—Authors’Abstract

Jang, S., Uematsu, S., Akira, S., and Sal-game, P. IL-6 and IL-10 induction fromdendritic cells in response to Mycobacte-rium tuberculosis is predominantly de-pendent on TLR2-mediated recognition.J. Immunol. 173(5) (2004) 3392–3397.

The initial TLR-mediated interaction be-tween Mycobacterium tuberculosis and den-dritic cells is critical, since the cytokine pro-duction that ensues can greatly influence theclass of adaptive immunity that is generatedto the pathogen. In this study, we thereforedetermined the dependency on TLR2 andTLR4 for M. tuberculosis-induced cytokineproduction by murine dendritic cells. A keynew finding of this study is that productionof IL-6 and IL-10 from dendritic cells in re-sponse to M. tuberculosis is principally de-pendent on TLR2. The study also indicatesthat M. tuberculosis can induce IL-12 pro-duction in the absence of either TLR2 orTLR4, suggesting redundancy or possiblyinvolvement of other receptors in IL-12 pro-

duction. In addition, the data also reveal thatlack of TLR2 or TLR4 does not impact ondendritic cell maturation or on their abilityto influence the polarity of dif ferentiatingnaive T cells. Collectively, data presentedhere provide a mechanistic insight for thecontribution of TLR2 and TLR4 to tubercu-losis disease progression and offer strategiesfor regulating IL-6 and IL-10 production indendritic cell-based vaccine strategies.—Authors’Abstract

Keller, C., Lauber, J., Blumenthal, A., Buer,J., and Ehlers, S. Resistance and sus-ceptibility to tuberculosis analysed at thetranscriptome level: lessons from mousemacrophages. T uberculosis (Edinb).84(3–4) (2004) 144–158.

Gene expression patterns associated withresistance and susceptibility to tuberculosis(TB) were investigated at the macrophagelevel in the well-defined mouse model of in-fection. Oligonucleotide microarrays wereused to analyze the regulation of gene ex-pression in murine bone marrow-derivedmacrophages infected with Mycobacteriumtuberculosis. Four mouse strains, known todiffer in terms of growth permissiveness forM. tuberculosis in infected tissues, in thedevelopment of pulmonary pathology, andin the rate of premature death due to tuber-culosis, were compared: C57BL/6 andBALB/c representing resistant, DBA/2 andCBA/J representing susceptible mousestrains. Genes (55) were regulated more thantwo-fold in macrophages of all strains in-vestigated following M. tuberculosis infec-tion. Importantly, 18 genes were commonlyregulated only in macrophages of the tworesistant strains upon infection, and 102genes were commonly regulated exclu-sively in macrophages of the two suscepti-ble strains. Using this approach, we havetherefore identified more than 100 genespotentially associated with resistance andsusceptibility, respectively , to TB at themacrophage level. A tentative interpretationof our microarray data suggests that macro-phages from susceptible mice predomi-nantly stimulate the recruitment of cells thatcontribute disproportionately to tissue dam-age rather than to microbial elimination. Inconclusion, microarray gene chips are use-


ful tools for generating new hypothesesabout resistance and susceptibility to TB,and the mouse model can now be used tosubject candidate genes identified by this ap-proach to further functional analyses.—Au-thors’Abstract

Manca, C., Reed, M. B., Fr eeman, S.,Mathema, B., Kreiswirth, B., Barry, C.E. 3rd, and Kaplan, G. Differentialmonocyte activation underlies strain-specific Mycobacterium tuber culosispathogenesis. Infect. Immun. 72(9) (2004)5511–5514.

In vitro infection of monocytes with My-cobacterium tuberculosis HN878 and relatedW/Beijing isolates preferentially induced in-terleukin-4 (IL-4) and IL-13, which charac-terize Th2 polarized immunity. In contrast,CDC1551 induced more IL-12 and othermolecules associated with phagocyte activa-tion and Th1 protective immunity. The dif-ferential cytokine-chemokine response wasmediated by extracted lipids, suggesting thatthese molecules regulate host responses toinfection.—Authors’Abstract

Marino, S., Pawar, S., Fuller, C. L., Rein-hart, T. A., Flynn, J. L., and Kirschner,D. E. Dendritic cell trafficking and anti-gen presentation in the human immuneresponse to Mycobacterium tuberculosis.J. Immunol. 173(1) (2004) 494–506.

Mycobacterium tuberculosis (Mtb) is anextraordinarily successful human pathogen,one of the major causes of death by infec-tious disease worldwide. A key issue for thestudy of tuberculosis is to understand whyindividuals infected with Mtb experiencedifferent clinical outcomes. To better under-stand the dynamics of Mtb infection and im-munity, we coupled nonhuman primate ex-periments with a mathematical model wepreviously developed that qualitatively andquantitatively captures important processesof cellular priming and activation. Theseprocesses occur between the lung and thenearest draining lymph node where the keycells mediating this process are the dendriticcells (DC). The nonhuman primate experi-ments consist of bacteria and cell numbers

from tissues of 17 adult cynomolgusmacaques (Macaca fascicularis) that wereinfected with Mtb strain Erdman ( approxi-mately 25 CFU/animal via bronchoscope).The main result of this work is that delays ineither DC migration to the draining lymphnode or T cell trafficking to the site of in-fection can alter the outcome of Mtb infec-tion, defining progression to primary diseaseor latent infection and reactivated tubercu-losis. Our results also support the idea thatthe development of a new generation oftreatment against Mtb should optimallyelicit a fast DC turnover at the site of infec-tion, as well as strong activation of DCs formaximal Ag presentation and production ofkey cytokines. This will induce the mostprotective T cell response.—Authors’ Ab-stract

Mollenkopf, H. J., Kursar, M., and Kauf-mann, S. H. Immune response to post-primary tuberculosis in mice: Mycobac-terium tuberculosis and Mycobacteriumbovis bacille Calmette-Guérin induceequal protection. J. Infect. Dis. 190(3)(2004) 588–597. Epub 2004 Jul 06.

We addressed the question of whetherprotective immunity induced by natural in-fection with Mycobacterium tuber culosisand that induced by vaccination with Myco-bacterium bovis bacille Calmette-Guerin(BCG) differ in the murine model. We in-fected mice with M. tuberculosis Erdman,cured them by chemotherapy , and subse-quently reinfected them with a low dose ofM. tuberculosis H37Rv. The course of tu-berculosis was compared with that in micepreviously vaccinated with BCG Danish1331. Protection against postprimary M. tu-berculosis infection did not dif fer signifi-cantly between the 2 groups. After challengeinfection, numbers of interferon- gamma-positive splenocytes did not differ betweenmice with primary infection and vaccinatedmice. Splenocytes from primary M. tuber-culosis-infected mice conferred marginallyhigher protection than did those from BCG-vaccinated mice. Serum transfer did not pro-tect against reinfection in either group. Ourdata emphasize that natural infection withM. tuberculosis and vaccination with BCGdo not differ in their capacity to induce pro-


tective immunity against tuberculosis andsupport the notions that reinfection con-tributes to the development of active diseaseand that any novel vaccine against tubercu-losis has to perform better than both vacci-nation with BCG and immunity evoked bynatural infection.—Authors’Abstract

Peters, W., Cyster , J. G., Mack, M.,Schlondorff, D., Wolf, A. J., Ernst, J.D., and Char o, I. F . CCR2-dependenttrafficking of F4/80dim macrophages andCD11cdim/intermediate dendritic cells iscrucial for T cell recruitment to lungs in-fected with Mycobacterium tuberculosis.J. Immunol. 172(12) (2004) 7647–7653.

We previously reported that CCR2(–/–)mice are susceptible to Mycobacterium tu-berculosis infection. Susceptibility was as-sociated with an early and sustained macro-phage traf ficking defect, followed bydelayed recruitment of dendritic cells (DCs)and T cells to the lungs. However, the rela-tive importance of the lack of CCR2 ex-pression by macrophages and DCs vs T cellsin susceptibility to infection was unclear. Inthis study, we used mixed bone marrow trans-plantation to create mice in which the geno-type of the T cells was either CCR2(+/+) orCCR2(–/–) while maintaining the genotypeof the myeloid cells as CCR2(+/+). Afterinfection with M. tuberculosis, we foundthat the genotype of the macrophages and/orDCs, but not that of the T cells, was criticalfor both T cell and myeloid cell migration tothe lungs. Further investigation revealed acritical role for CCR2 in the recruitment ofF4/80(dim) macrophages and CD11c (dim/intermediate) DCs to the infected lung.—Authors’Abstract

Sander, P ., Rezwan, M., Walker, B.,Rampini, S. K., Kroppenstedt, R. M.,Ehlers, S., Keller , C., Keeble, J. R.,Hagemeier, M., Colston, M. J.,Springer, B., and Bottger, E. C. Lipo-protein processing is required for viru-lence of Mycobacterium tuber culosis.Mol. Microbiol. 52(6) (2004) 1543–1552.

Lipoproteins are a subgroup of secretedbacterial proteins characterized by a lipi-

dated N-terminus, processing of which ismediated by the consecutive activity of pro-lipoprotein diacylglyceryl transferase (Lgt)and lipoprotein signal peptidase (LspA).The study of LspAfunction has been limitedmainly to non-pathogenic microorganisms.To study a potential role for LspA in thepathogenesis of bacterial infections, wehave disrupted lspA by allelic replacementin Mycobacterium tuberculosis, one of theworld’s most devastating pathogens. Despitethe presence of an impermeable lipid outerlayer, it was found that LspA was dispensa-ble for growth under in vitro culture condi-tions. In contrast, the mutant was markedlyattenuated in virulence models of tubercu-losis. Our findings establish lipoprotein me-tabolism as a major virulence determinant oftuberculosis and define a role for lipoproteinprocessing in bacterial pathogenesis. In ad-dition, these results hint at a promising newtarget for therapeutic intervention, as ahighly specific inhibitor of bacterial lipopro-tein signal peptidases is available.—Au-thors’Abstract

Saunders, B. M., Briscoe, H., and Britton,W. J. T cell-derived tumour necrosis fac-tor is essential, but not sufficient, for pro-tection against Mycobacterium tubercu-losis infection. Clin. Exp. Immunol.137(2) (2004) 279–287.

Tumour necrosis factor (TNF) is criticalfor sustained protective immunity againstMycobacterium tuberculosis infection. Toinvestigate the relative contributions ofmacrophage- and T cell-derived TNF to-wards this immunity T cells from wild-type(WT) or TNF–/– mice were transferred intoRAG–/– or TNF–/– mice which were theninfected with M. tuber culosis. InfectedRAG–/– mice and RAG–/– recipients ofTNF deficient T cells developed over-whelming infection, with extensive pul-monary and hepatic necrosis and succumbedwith a median of only 16 days infection. Bycontrast, RAG–/– recipients of WT T cellsshowed a significant increase in survivalwith a median of 32 days. Although initialbacterial growth was similar in all groups ofRAG–/– mice, the transfer of WT, but notTNF–/–, T cells led to the formation of dis-crete foci of leucocytes and macrophages


and delayed the development of necrotizingpathology. To determine requirements formacrophage-derived TNF, WT or TNF–/– Tcells were transferred into TNF–/– mice atthe time of M. tuberculosis infection. Trans-fer of WT T cells significantly prolongedsurvival and reduced the early tissue necro-sis evident in the TNF–/– mice, however ,these mice eventually succumbed indicatingthat T cell-derived TNF alone is insufficientto control the infection. Therefore, both Tcell- and macrophage-derived TNF play dis-tinct roles in orchestrating the protective in-flammatory response and enhancing sur-vival during M. tuber culosis infection.—Authors’Abstract

Toossi, Z., Mayanja-Kizza, H., Kanost, A.,Edmonds, K., McHugh, M., andHirsch, C. Protective responses in tuber-culosis: induction of genes for interferon-gamma and cytotoxicity by Mycobacte-rium tuber culosis and during humantuberculosis. Scand. J. Immunol. 60(3)(2004) 299–306.

The host effector mechanisms against My-cobacterium tuberculosis infection are notwell understood, and this remains a problemin the development of new vaccines and im-munotherapies in tuberculosis (TB). Here,we studied the expression of genes for in-terferon gamma (IFN-gamma) and mole-cules involved in lymphocyte-mediated cy-totoxicity [granzyme B (grzB), perforin,granulysin and Fas ligand (FasL)] againstM. tuberculosis-infected macrophages. Thekinetics of expression of these moleculeswere first established in peripheral bloodmononuclear cells (PBMC) of healthydonors, and then investigated in TB patientswith and without HIV-1 coinfection and ap-propriate control groups. We found that onlyIFN-gamma and grzB were induced by M.tuberculosis in PBMC from healthy purifiedprotein derivative skin test reactive subjects.However, expression of neither gene norIFN-gamma protein correlated with intra-cellular M. tuberculosis growth containmentby macrophages. Mycobacterium tubercu-losis induction of IFN-gamma, but not grzB,mRNA expression was significantly lower(p <0.03) in TB patients as compared withhealthy subjects.—Authors’Abstract

Vergne, I., Chua, J., Singh, S. B., andDeretic, V. Cell Biology of Mycobacte-rium tuber culosis Phagosome. Annu.Rev. Cell. Dev. Biol. Epub. 2004 Jun. 15[ahead of print].

Phagocytosis and phagolysosome biogen-esis represent fundamental biologicalprocesses essential for proper tissue homeo-stasis, development, elimination of invadingmicroorganisms, and antigen processing andpresentation. Phagosome formation triggersa pre-programmed pathway of maturationinto the phagolysosome, a process con-trolled by Ca2+ and the regulators of or-ganellar traf ficking centered around thesmall GTP-binding proteins Rabs and theirdownstream ef fectors, including lipidkinases, organellar tethering molecules, andmembrane fusion apparatus. Mycobac-terium tuber culosis is a potent humanpathogen parasitizing macrophages. It inter-feres with the Rab-controlled membranetrafficking and arrests the maturing phago-some at a stage where no harm can be doneto the pathogen while the delivery of nutri-ents and membrane to the vacuole continuesharboring the microorganism. This process,referred to as the M. tuberculosis phago-some maturation arrest or inhibition ofphagosome-lysosome fusion, is critical forM. tuberculosis persistence in human popu-lations. It also provides a general model sys-tem for dissecting the phagolysosome bio-genesis pathways. Here we review thefundamental trafficking processes targetedby M. tuberculosis and the mycobacterialproducts that interfere with phagosomalmaturation. Expected online publication datefor the Annual Review of Cell and Devel-opmental Biology Volume 20 is October 6,2004.—Authors’Abstract

Yoseph Haile, and Ryon, J. J. Colorimet-ric microtitre plate hybridization assayfor the detection of Mycobacterium lep-rae 16S rRNA in clinical specimens.Lepr. Rev. 75(1) (2004) 40–49.

We have developed a colorimetric mi-crotiter plate hybridization assay in order tosimplify detection of Mycobacterium lepraein clinical specimens. This system detectsthe products amplified by a sensitive R T-


PCR assay targeting a species-specific se-quence of the bacterial 16S rRNA. Theassay detected as few as 10 bacilli isolatedfrom infected nude mouse lymph nodes orhuman skin biopsies. Sensitivity for diago-sis of clinical specimens was assessed for 58tissue biopsies from untreated leprosy pa-tients seen at the ALERT Hospital in AddisAbaba, Ethiopia [date not given]. The assaydetected M. leprae RT-PCR products in100% of biopsies from patients with multi-bacillary disease and 80% of biopsies frompatients with paucibacillary disease, for an

overall sensitivity of 91.3%. The test washighly specific as no RT-PCR products wereamplified from skin biopsies of normal in-dividuals or patients with skin diseases otherthan leprosy. The colorimetric assay is faster,more sensitive, and simplifies detection ofRT-PCR products compared to Southern blotanalysis. It may be useful for diagnosis ofdifficult cases of leprosy, and, since RNA israpidly degraded after cell death, it may beappropriate for assessing response to ther-apy and for distinguishing relapse from re-action.—Tropical Disease Bulletin


Microbiology

Flint, J. L., Kowalski, J. C., Karnati, P .K., and Derbyshire, K. M. The RD1 vir-ulence locus of Mycobacterium tubercu-losis regulates DNA transfer in Myco-bacterium smegmatis. Proc. Natl. Acad.Sci. U.S.A. 101(34) (2004) 12598–12603.Epub 2004 Aug. 16.

Conjugal DNAtransfer occurs by an atyp-ical mechanism in Mycobacterium smegma-tis. The transfer system is chromosomallyencoded and requires recipient recombina-tion functions for both chromosome andplasmid transfer. Cis-acting sequences havebeen identified that confer mobility on non-transferable plasmids, but these are lar gerand have dif ferent properties to canonicaloriT sites found in bacterial plasmids. Toidentify trans-acting factors required for me-diating DNA transfer, a library of transpo-son insertion mutants was generated in thedonor strain, and individual mutants werescreened for their ef fect on transfer. Fromthis screen, a collection of insertion mutantswas isolated that increased conjugation fre-quencies relative to wild type. Remarkably,the mutations map to a 25-kb region of theM. smegmatis chromosome that is synte-nous with the RD1 region of Mycobacteriumtuberculosis, which is considered to be theprimary attenuating deletion in the relatedvaccine strain Mycobacterium bovis bacillusCalmette-Guérin. The genes of the RD1 re-gion encode a secretory apparatus responsi-ble for exporting Cfp10- and Esat-6, bothpotent antigens and virulence factors. In

crosses using two M. smegmatis donors, weshow that wild-type cells can suppress theelevated transfer phenotype of mutantdonors, which is consistent with the secre-tion of a factor that suppresses conjugation.Most importantly, the RD1 region of M. tu-berculosis complements the conjugationphenotype of the RD1 mutants in M. smeg-matis. Our results indicate that the M. tu-berculosis and M. smegmatis RD1 regionsare functionally equivalent and provide aunique perspective on the role of this criti-cal secretion apparatus.—Authors’Abstract

Lee, R. E., Li, W., Chatterjee, D., and Lee,R. E. Rapid structural characterization ofthe arabinogalactan and lipoarabinoman-nan in live mycobacterial cells using 2Dand 3D HR-MAS NMR. Glycobiology.Epub 2004 Sep. 15 [ahead of print]

Mycobacteria possess a unique, highlyevolved, carbohydrate and lipid-rich cellwall that is believed to be important for theirsurvival in hostile environments. Until now,our understanding of mycobacterial cellwall structure has been based upon destruc-tive isolation and fragmentation of indi-vidual cell wall components. This studydescribes the observation of the major cellwall structures in live, intact mycobacteriausing two-dimensional (2D) and three-dimensional (3D) High-Resolution Magic-Angle Spinning (HR-MAS) NMR. As littleas 20 mg (wet weight) of [(13)C] enriched

cells were required to produce a whole-cellspectra in which discrete cross-peaks corre-sponding to specific cell wall componentscould be identified. The most abundant sig-nals of the arabinogalactan (AG) andlipoarabinomannan (LAM) were assigned inthe HR-MAS NMR spectra by comparingthe 2D and 3D NMR whole-cell spectrawith the spectra of purified cellular compo-nents. This study confirmed that the struc-tures of the AG and LAM moieties in thecell wall of live mycobacteria are consistentwith structural reports in the literature,which were obtained via degradative analy-sis. Most importantly, using intact cells, itwas possible to directly demonstrate the ef-fects of Ethambutol on the mycobacterialcell wall polysaccharides, characterize theeffects of embB gene knockout in the M.smegmatis DeltaembB mutant, and observedifferences in the cell wall structures of twomycobacterial species (M. bovis BCG andM. smegmatis). Herein, we show that HR-MAS NMR is a powerful, rapid, non-destructive technique to monitor changes inthe complex, carbohydrate rich cell wall oflive mycobacterial cells.—Authors’Abstract

Mostowy, S., Onipede, A., Gagneux, S.,Niemann, S., Kremer, K., Desmond, E.P., Kato-Maeda, M., and Behr, M. Ge-nomic analysis distinguishes Mycobacte-rium africanum. Clin. Microbiol. 42(8)(2004) 3594–3599.

See Current Literature, Other Mycobac-terial Diseases, p. 562.

Nigou, J., Gilleron, M., Brando, T., andPuzo, G. Structural analysis of myco-bacterial lipoglycans. Appl. Biochem.Biotechnol. 118(1–3) (2004) 253–267.

Mycobacterium tuberculosis, the causativeagent of tuberculosis, is one of the most ef-fective human pathogens. The mycobacterialcell envelope contains lipoglycans, and ofparticular interest is lipoarabinomannan(LAM), one of the most potent mycobacte-rial immunomodulatory molecules. The im-portance of lipoarabinomannan (LAM) inthe immunopathogenesis of tuberculosis hasincited structural studies on this molecule to

(1) establish a precise structural model of themolecule and (2) decipher the structure/func-tion relationships. In recent years, we havefocused on the two domains essential forLAM biologic activit ies: the mannosyl-phosphatidyl-myo-inositol anchor and thecaps. We review here the recent proceduresdeveloped for the structural analysis of thesedomains.—Authors’Abstract

Pethe, K., Swenson, D. L., Alonso, S., An-derson, J., Wang, C., and Russell, D. G.Isolation of Mycobacterium tuberculosismutants defective in the arrest of phago-some maturation. Proc. Natl. Acad. Sci.U.S.A. 101(37) (2004) 13642–13647.Epub 2004 Aug. 31.

Mycobacterium tuberculosis resides withinthe phagocytes of its host. It ensures its con-tinued survival through arresting the normalmaturation of its phagosome, which isretained within the early endosomal system ofthe macrophage. Although individual bacte-rial components have been shown to modulatephagosome biogenesis, the mechanism(s) ac-tive in live, intact bacteria remain elusive. Wehave developed a genetic screen that facili-tates the isolation of mutants defective in ar-resting the maturation of their phagosomes.Macrophages were incubated with iron-dextran that was chased into lysosomes. Thecells were subsequently infected with M. tu-berculosis from a library of transposon-mutagenized bacteria. After four rounds of en-richment, the majority of mutants isolatedwere unable to prevent acidification of theirphagosomes and were attenuated for intracel-lular survival. The genes af fected range infunction from those with no known homo-logues to putative transporters and lipid syn-thesis enzymes. Further characterization ofthese bacteria is needed. In addition to clari-fying the processes active in modulation ofphagosome biogenesis by M. tuberculosis,this screen may be applicable to otherpathogens that restrict the maturation of theirphagosome.—Authors’Abstract

Sarkola, A., Makinen, J., Marjamaki, M.,Marttila, H. J., Viljanen, M. K., andSoini, H. Prospective evaluation of theGenoType assay for routine identification

72, 4 Current Literature, Microbiology 545

of mycobacteria. Eur. J. Clin. Microbiol.Infect. Dis. 23(8) (2004) 642–645. Epub2004 Jul. 28.

In order to evaluate the proficiency of theGenoType Mycobacteria strip hybridizationassay (Hain Lifescience, Nehren, Germany)for the routine identification of mycobacte-ria, the assay was used to identify 178 clin-ical isolates during a 6-month prospectivestudy. The GenoType results were comparedto the identification results obtained withAccuProbe (GenProbe, San Diego, CA,USA) or 16S rDNA sequencing, and anoverall agreement of 89.3% between Geno-Type and the two reference methods wasreached. The GenoType assay is, thus, arapid and reliable method for the identifica-tion of clinically important mycobacteria,and it is well suited for use in a routine lab-oratory.—Authors’Abstract

Semret, M., Zhai, G., Mostowy, S., Cleto,C., Alexander, D., Cangelosi, G.,Cousins, D., Collins, D. M., van Soolin-gen, D., and Behr, M. A. Extensive ge-nomic polymorphism within Mycobacte-rium avium. J. Bacteriol. 186(18) (2004)6332–6334.

We have initiated comparative genomicanalysis of Mycobacterium avium subspeciesby DNAmicroarray, uncovering 14 large se-quence polymorphisms (LSPs) comprisingover 700 kb that distinguish M. avium subsp.avium from M. avium subsp. paratuberculo-sis. Genes predicted to encode metabolicpathways were overrepresented in the LSPs,and analysis revealed a polymorphismwithin the mycobactin biosynthesis operonthat potentially explains the in vitr o my-cobactin dependence of M. avium subsp.paratuberculosis.—Authors’Abstract

Torrelles, J. B., Khoo, K. H., Sieling, P. A.,Modlin, R. L., Zhang, N., Marques, A.M., Treumann, A., Rithner , C. D.,Brennan, P . J., and Chatterjee, D.Truncated structural variants of lipoara-binomannan in Mycobacterium lepraeand an ethambutol-resistant strain of My-cobacterium tuberculosis. J. Biol. Chem.

279(39) (2004) 41227– 41239. Epub2004 Jul. 19.

Current knowledge on the structure oflipoarabinomannan (LAM) has resulted pri-marily from detailed studies on a few se-lected laboratory strains of Mycobacteriumtuberculosis, Mycobacterium bovis BCG,and Mycobacterium smegmatis. Our previ-ous work was the first to report on the salientstructural features of M. tuberculosis clini-cal isolates and demonstrated significantstructural variations. A prime effort is to cor-relate a particular structural characteristicwith observed differences in eliciting an im-munobiological response, especially in thecontext of CD1-restricted presentation ofLAM to T cells. T cell clones derived fromthe cutaneous lesions of leprosy patientshave been shown to recognize specificallyLAM from Mycobacterium leprae and notfrom M. tuber culosis Erdman or H37Rv .Herein we provide further fine structuraldata on LAM from M. leprae (LepLAM)and a tuberculosis clinical isolate, CSU20(CSU20LAM), which was unexpectedlyrecognized by the supposedly LepLAM-specific CD1-restricted T cell clones. Incomparison with the de facto laboratoryLAM standard from M. tuberculosis H37Rv(RvLAM), LepLAM derived from in vivogrown M. leprae is apparently simpler in itsarabinan architecture with a high degree ofexposed, non-mannose-capped termini. Onthe other hand, CSU20, an ethambutol-resistant clinical isolate, makes a vastly het-erogeneous population of L AM rangingfrom rather small and non-mannose-cappedto full-length and fully capped variants.LepLAM and CSU20LAM contain a higherlevel of succinylation than RvLAM, which,in the context of truncated or less elaboratedarabinan, may contribute to selective recog-nition by T cells. LAM from all speciescould be resolved into discrete forms by iso-electric focusing based apparently on theirarabinan heterogeneity. In the light of ourcurrent and more recent findings, we reasonthat all immunobiological data should becautiously interpreted and that the actualLAM variants that may be present in vivoduring infection and pathogenesis need to betaken into consideration.—Authors’ Ab-stract


Marques, M. A., Espinosa, B. J., XavierdaSilveira, E. K., Pessolani, M. C., Cha-peaurouge, A., Perales, J., Dobos, K. M.,Belisle, J. T., Spencer, J. S., and Br en-nan, P. J. Continued proteomic analysis ofMycobacterium leprae subcellular frac-tions. Proteomics. 4(10) (2004) 2942–2953

Recently the sequence of the Mycobacte-rium leprae chromosome, the only knownobligate intracellular mycobacterium, wascompleted. It has a dramatic reduction infunctional genes, with a coding capacity ofonly 49.5%, the lowest one so far observedamong bacterial genomes. The leprosybacillus seems to preserve a minimal set ofgenes that allows its survival in the host. Theidentification of genes that are actually ex-pressed by the bacterium is of high signifi-

cance in the context of mycobacterial patho-genesis. In this current study , a proteomicapproach was undertaken to identify theproteins present in the soluble/cytosol andmembrane subcellular fractions obtainedfrom armadillo derived M. leprae. Proteinsfrom each fraction were separated by two-dimensional gel electrophoresis (2-DE) andidentified by mass spectrometry. A total of147 protein spots were identified from 2-DEpatterns and shown to comprise products of44 different genes, twenty eight of them cor-responding to new proteins. Additionally,two highly basic proteins (with p I >10.0)were isolated by heparin affinity chromatog-raphy and identified by N-terminal sequencing.This study constitutes the first application ofproteomics to a host-derived Mycobacte-rium.—Authors’Abstract

72, 4 Current Literature, Microbiology (Leprosy) 547

Microbiology (Leprosy)

Microbiology (Tuberculosis)

We investigated how Mycobacterium tu-berculosis responded to a reduced oxygentension in terms of its pathogenicity andgene expression by growing cells under ei-ther aerobic or low-oxygen conditions inchemostat culture. The chemostat enabledus to control and vary the oxygen tension in-dependently of other environmental param-eters, so that true cause-and-effect relation-ships of reduced oxygen availability couldbe established. Cells grown under low oxy-gen were more pathogenic for guinea pigsthan those grown aerobically. The effect ofreduced oxygen on global gene expressionwas determined using DNA microarray.Spearman rank correlation confirmed thatmicroarray expression profiles were highlyreproducible between repeat cultures. Usingmicroarray analysis we have identifiedgenes that respond to a low-oxygen envi-ronment without the influence of other pa-rameters such as nutrient depletion. Some ofthese genes appear to be involved in thebiosynthesis of cell wall precursors and theirinduction may have contributed to increased

Ahmed, N., Alam, M., Rao, K. R., Kauser,F., Kumar, N. A., Qazi, N. N., Sangal,V., Sharma, V. D., Das, R., Katoch, V.M., Murthy , K. J., Suneetha, S.,Sharma, S. K., Sechi, L. A., Gilman, R.H., and Hasnain, S. E. Molecular geno-typing of a large, multicentric collectionof tubercle bacilli indicates geographicalpartitioning of strain variation and has im-plications for global epidemiology of My-cobacterium tuberculosis. J. Clin. Micro-biol. 42(7) (2004) 3240–3247.

See Curr ent Literatur e, Molecular andGenetic Studies, p. 565.

Bacon, J., James, B. W., Wernisch, L.,Williams, A., Morley, K. A., Hatch, G.J., Mangan, J. A., Hinds, J., Stoker, N.G., Butcher, P. D., and Marsh, P. D. Theinfluence of reduced oxygen availabilityon pathogenicity and gene expression inMycobacterium tuberculosis. Tuberculosis(Edinb.) 84(3–4) (2004) 205–217.

infectivity in the guinea pig. This study hasshown that a combination of chemostat cul-ture and microarray presents a biologicallyrobust and statistically reliable experimen-tal approach for studying the ef fect of rele-vant and specific environmental stimuli onmycobacterial virulence and gene expres-sion.—Authors’Abstract

Bahk, Y. Y., Kim, S. A., Kim, J. S., Euh,H. J., Bai, G. H., Cho, S. N., and Kim,Y. S. Antigens secreted from Mycobacte-rium tuberculosis: Identification by pro-teomics approach and test for diagnosticmarker. Proteomics. Epub 2004 Sep. 20[ahead of print]

Tuberculosis caused by mycobacteria,mainly Mycobacterium tuber culosis, is amajor infectious disease of the respiratorysystem. An early diagnosis followed bychemotherapy is the major control strategy.In an effort to identify the antigens suitablefor immunodiagnosis and vaccines, the pro-teins secreted in a culture medium from theM. tuberculosis K-strain, which is the mostprevalent among the clinical isolates inKorea and belongs to the Beijing family ,were analyzed by two-dimensional poly-acrylamide gel electrophoresis (2-D PAGE)and compared with those from the M. tu-berculosis H37Rv and CDC1551 strains.Eight proteins, Rv0652, Rv1636, Rv2818c,Rv3369, Rv3865, Rv0566c, MT3304, andRv3160, were identified by matrix-assistedlaser desorption/ionization-time of flight-massspectrometry (MALDI-TOF-MS) or liquidchromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and foundto be relatively abundant in the culturemedium from the M. tuberculosis K-strainbut less so from the CDC1551 or H37Rvstrains. In addition, Rv3874 (CFP-10), Rv-0560c and Rv3648c, which were ex-pressed increasingly in the K and CDC1551strains, were also identified using the sameproteomics technology. All proteins wereprepared by molecular cloning, expressionin Escherichia coli followed by affinity pu-rification. Among them, three proteins,rRv3369, rRv0566c, and rRv3874, were se-lected by prescreening and examined fortheir potential as serodiagnostic antigensusing an enzyme-linked immunosorbent

assay. When 100 sera from tuberculosis pa-tients and 100 sera from the healthy controlswere analyzed, rR V3369, rRv3874, andrRv0566c showed a sensitivity of 60% ,74%, and 43%, and a specificity of 96%,97%, and 84%, respectively. These resultssuggest that the rRv3369 and rRv3874 pro-teins, which were expressed more abun-dantly in the more recently obtained clinicalisolates of M. tuberculosis than in the labo-ratory-adapted H37Rv strain, are promisingfor use in the serodiagnosis of tuberculo-sis.—Authors’Abstract

Choudhary, R. K., Pullakhandam, R.,Ehtesham, N. Z., and Hasnain, S. E.Expression and characterization ofRv2430c, a novel immunodominant anti-gen of Mycobacterium tuberculosis. Pro-tein Expr. Purif. 36(2) (2004) 249–253.

About 10% of the coding sequence of My-cobacterium tuber culosis corresponds tohitherto unknown members of the PE andPPE protein families which display sig-nificant sequence and length variation attheir C-terminal region. It has been sug-gested that this could possibly represent arich source of antigenic variation within thepathogen. We describe the purification andbiophysical characterization of the recombi-nant PPE protein coded by hypotheticalORF Rv2430c, a member of the PPE genefamily that was earlier shown to induce astrong B cell response. Expression of the re-combinant PPE protein in Escherichia coliled to its localization in inclusion bodies andsubsequent refolding using dialysis after itsextraction from the same resulted in exten-sive precipitation. Therefore, an on-columnrefolding strategy was used, after which theprotein was found to be in the soluble form.CD spectrum of the recombinant proteindisplayed predominantly alpha helicalcontent (81%) which matched significantlywith in silico and web-based secondary struc-ture predictions. Furthermore, fluorescenceemission spectra revealed that aromaticamino acids are buried inside the protein,which are exposed to aqueous environmentunder 8M urea. These results, for the firsttime, provide evidence on the structural fea-tures of PPE family protein which, viewedwith its reported immunodominant charac-


teristics, have implications for other proteinsof the PE/PPE family.—Authors’Abstract

Hampshire, T., Soneji, S., Bacon, J.,James, B. W., Hinds, J., Laing, K., Sta-bler, R. A., Marsh, P. D., and Butcher,P. D. Stationary phase gene expression ofMycobacterium tuberculosis following aprogressive nutrient depletion: a modelfor persistent or ganisms? Tuberculosis(Edinb.) 84(3–4) (2004) 228–238.

The majority of individuals infected withTB develop a latent infection, in which or-ganisms survive within the body whileevading the host immune system. Such per-sistent bacilli are capable of surviving sev-eral months of combinatorial antibiotictreatment. Evidence suggests that stationaryphase bacteria adapt to increase their toler-ance to environmental stresses. We have de-veloped a unique in vitr o model of dor-mancy based on the characterization of asingle, large volume fermenter culture ofM.tuberculosis, as it adapts to stationary phase.Cells are maintained in controlled and de-fined aerobic conditions (50% dissolvedoxygen tension), using probes that measuredissolved oxygen tension, temperature, andpH. Microarray analysis has been used inconjunction with viability and nutrient de-pletion assays to dissect differential gene ex-pression. Following exponential phasegrowth the gradual depletion of glucose/glycerol resulted in a small population ofsurvivors that were characterized for periodsin excess of 100 days. Bacilli adapting tonutrient depletion displayed characteristicsassociated with persistence in vivo, includ-ing entry into a non-replicative state and theup-regulation of genes involved in beta-oxidation of fatty acids and virulence. A re-duced population of non-replicating bacilliwent on to adapt suf ficiently to re-initiatecellular division.—Authors’Abstract

Hisert, K. B., Kirksey, M. A., Gomez, J. E.,Sousa, A. O., Cox, J. S., Jacobs, W. R. Jr.,Nathan, C. F., and McKinney, J. D.Iden-tification of Mycobacterium tuberculosiscounterimmune (cim) mutants in immun-odeficient mice by dif ferential screening.Infect. Immun. 72(9) (2004) 5315–5321.

Tuberculosis (TB) is characterized bylifetime persistence of Mycobacterium tu-berculosis. Despite the induction of a vigor-ous host immune response that curtails dis-ease progression in the majority of cases, theorganism is not eliminated. Subsequent im-munosuppression can lead to reactivationafter a prolonged period of clinical latency.Thus, while it is clear that protective im-mune mechanisms are engaged during M.tuberculosis infection, it also appears thatthe pathogen has evolved effective counter-mechanisms. Genetic studies with animalinfection models and with patients have re-vealed a key role for the cytokine gamma in-terferon (IFN-gamma) in resistance to TB.IFN-gamma activates a large number of an-timicrobial pathways. Three of these IFN-gamma-dependent mechanisms have beenimplicated in defense against M. tuberculo-sis: inducible nitric oxide synthase (iNOS),phagosome oxidase (phox), and the phago-some-associated GTPase LRG-47. In orderto identify bacterial genes that provide pro-tection against specific host immune path-ways, we have developed the strategy ofdifferential signature-tagged transposon mu-tagenesis. Using this approach we haveidentified three M. tuberculosis genes thatare essential for progressive M. tuberculosisgrowth and rapid lethality in iNOS-deficientmice but not in IFN-gamma-deficient mice.We propose that these genes are involved inpathways that allow M. tuber culosis tocounter IFN-gamma-dependent immunemechanisms other than iNOS.—Authors’Abstract

Karakousis, P . C., Yoshimatsu, T.,Lamichhane, G., Woolwine, S. C.,Nuermberger, E. L., Gr osset, J., andBishai, W. R. Dormancy phenotype dis-played by extracellular Mycobacteriumtuberculosis within artificial granulomasin mice. J. Exp. Med. 200(5) (2004)647–657.

Mycobacterium tuber culosis residingwithin pulmonary granulomas and cavitiesrepresents an important reservoir of persis-tent organisms during human latent tuber-culosis infection. We present a novel in vivomodel of tuberculosis involving the encap-sulation of bacilli in semidiffusible hollow

72, 4 Current Literature, Microbiology (Tuberculosis) 549

fibers that are implanted subcutaneously intomice. Granulomatous lesions developaround these hollow fibers, and in this mi-croenvironment, the organisms demonstratean altered physiologic state characterized bystationary-state colony-forming unit countsand decreased metabolic activity. Moreover,these organisms show an antimicrobial sus-ceptibility pattern similar to persistentbacilli in current models of tuberculosischemotherapy in that they are more suscep-tible to the sterilizing drug, rifampin, than tothe bactericidal drug isoniazid. We used thismodel of extracellular persistence withinhost granulomas to study both gene expres-sion patterns and mutant survival patterns.Our results demonstrate induction of dosR(Rv3133c) and 20 other members of theDosR regulon believed to mediate the tran-sition into dormancy, and that rel(Mtb) is re-quired for Mycobacterium tuberculosis sur-vival during extracellular persistence withinhost granulomas. Interestingly , the dor-mancy phenotype of extracellular M. tuber-culosis within host granulomas appears tobe immune mediated and interferon-gammadependent.—Authors’Abstract

Lightbody, K. L., Renshaw, P. S., Collins,M. L., Wright, R. L., Hunt, D. M., Gor-don, S. V., Hewinson, R. G., Buxton, R.S., Williamson, R. A., and Carr, M. D.Characterisation of complex formation be-tween members of the Mycobacterium tu-berculosis complex CFP-10/ESAT-6 pro-tein family: towards an understanding ofthe rules governing complex formationand thereby functional flexibility. FEMSMicrobiol. Lett. 238(1) (2004) 255–262.

We have previously shown that the se-creted M. tuber culosis complex proteinsCFP-10 and ESAT-6 form a tight, 1:1 com-plex, which may represent their functionalform. In the work reported here a combina-tion of yeast two-hybrid and biochemicalanalysis has been used to characterise com-plex formation between two other pairs ofCFP-10/ESAT-6 family proteins (Rv0287/Rv0288 and Rv3019c/Rv3020c) and to de-termine whether complexes can be formedbetween non-genome paired members of thefamily. The results clearly demonstrate thatRv0287/Rv0288 and Rv3019c/3020c form

tight complexes, as initially observed forCFP-10/ESAT-6. The closely related Rv0287/Rv0288 and Rv3019c/Rv3020c proteins arealso able to form non-genome paired com-plexes (Rv0287/Rv3019c and Rv0288/Rv3020c), but are not capable of binding tothe more distantly related CFP-10/ESAT-6proteins.—Authors’Abstract

Liu, X., Tiwari, R. K., Geliebter, J., Wu, J.M., and Godfrey, H. P. Interaction of aMycobacterium tuber culosis repetitiveDNA sequence with eukaryotic proteins.Biochem. Biophys. Res. Commun. 320(3)(2004) 966–972.

Mycobacterium tuberculosis infects one-third of the world’s population and causestwo million deaths annually. Its intracellularresidence raises the possibility that bacterialnucleic acids might interact with key hostproteins and contribute to the pathophysiol-ogy of infection. To test this hypothesis, wesearched for motifs closely resembling eu-karyotic transcription factor binding sites inthe M. tuber culosis H37Rv genome andfound activator protein-2 and zinc fingerprotein-5 binding motifs in a 36-nucleotiderepetitive mycobacterial DNA sequence(RPT1). RPT1 bound specifically to nuclearextract proteins from U937, A549, andHeLa cells in electrophoretic mobility shiftassays but not to activator protein-2. Severalnuclear and cytosolic proteins showing atleast partial binding specificity for RPT1were isolated from U937 and A549 cells bypull-down assays, including Ku70 (DNA-dependent protein kinase subunit) andpoly(ADP-ribose) polymerase-1. RPT1 alsospecifically activated DNA-dependent pro-tein phosphorylation. These results suggestthat mycobacterial nucleic acid fragmentsmay interact specifically with eukaryoticregulatory proteins which might contributeto bacterial life-cycle maintenance.—Au-thors’Abstract

Mostowy, S., Cleto, C., Sherman, D. R.,and Behr, M. A. The Mycobacterium tu-berculosis complex transcriptome of at-tenuation. Tuberculosis (Edinb). 84(3–4)(2004) 197–204.


Although the deletion of RD1 is likely cor-related to attenuation from virulence formembers of the Mycobacterium tuberculosis(MTB) complex, the reasons for this pheno-type remain to be fully explained. As ge-nomic variation is responsible for at least acomponent of variability in gene expression,we looked to the in vitro global expressionprofile of the RD1 artificial knockout from M.tuberculosis H37Rv (H37Rv:deltaRD1) forclues to elucidate its phenotypic shift towardsattenuation. By comparing the transcriptomeof H37Rv:deltaRD1 to that of virulentH37Rv, 15 regulated genes located in ninedifferent regions outside of RD1 have beenidentified, capturing an effect of RD1’s dele-tion on the rest of the genome. To assesswhether these regulations are characteristic ofattenuated MTB in general, expression pro-files of natural RD1 mutants (BCG Russia,BCG Pasteur, and M. microti) as well as the“avirulent” M. tuberculosis H37Ra, whoseRD1 region is genomically intact, were ob-tained. Results indicate that attenuated strainslack the expression of RD1 genes includingcfp10 and esat6, whether through deletion orreduced expression. Furthermore, compara-tive transcriptomics reveals the concurrentdown-regulation of several gene neighbor-hoods beyond RD1. The potential relevanceof these other expression changes towardsMTB virulence is discussed.—Authors’ Ab-stract

Waddell, S. J., Stabler, R. A., Laing, K.,Kremer, L., Reynolds, R. C., and Besra,G. S. The use of microarray analysis todetermine the gene expression profiles ofMycobacterium tuberculosis in responseto anti-bacterial compounds. Tuberculosis(Edinb.) 84(3–4) (2004) 263–274.

The response of Mycobacterium tubercu-losis to six anti-microbial agents was deter-mined by microarray analysis in an attemptto define mechanisms of innate resistance inM. tuberculosis. The gene expression pro-files of M. tuberculosis after treatment at theminimal inhibitory concentration (MIC) for4 hr with isoniazid, isoxyl, tetrahydrolip-statin, SRI#221, SR1#967 and SR1#9190were compared to untreated M. tuberculosis.A common response to drug exposure wasdefined, and this expression profile over-lapped with a number of other mycobacte-rial stress responses recently identified bymicroarray analysis. Compound-specific re-sponses were also distinguished including anumber of putative transcriptional regulatorsand translocation-related genes. These genesmay contribute to the intrinsic resistance of M. tuberculosis to anti-microbial com-pounds. Further investigation into thesemechanisms may elucidate novel pathwayscontributing to mycobacterial drug resis-tance and influence anti-mycobacterial drugdevelopment strategies.—Authors’Abstract

72, 4 Current Literature, Experimental Infections 551

Experimental Infections

Algood, H. M., and Flynn, J. L. CCR5-deficient mice control Mycobacterium tu-berculosis infection despite increasedpulmonary lymphocytic infiltration. J Im-munol. 173(5) (2004) 3287–3296.

The control of Mycobacterium tuberculo-sis infection is dependent on the develop-ment of an adaptive immune response,which is mediated by granulomas. The gran-uloma is a dynamic structure that forms inthe lung and consists primarily of macro-phages and lymphocytes. For this structureto be effective in containment of the bacil-lus, it must develop in an or ganized andtimely manner. The formation of the granu-

loma is dependent on recruitment of acti-vated cells through adhesion molecules andchemokines. M. tuberculosis infection causesan increase in the expression of beta-chemokines CCL3, CCL4, and CCL5, andtheir receptor CCR5, in the lungs. In thisstudy, we demonstrate that CCR5-transgenicknockout mice were capable of recruitingimmune cells to the lung to form granulo-mas. CCR5(–/–) mice successfully induceda Th1 response and controlled infection. Un-expectedly, M. tuber culosis infection inthese mice resulted in greater numbers oflymphocytes migrating to the lung andhigher levels of many inflammatory cy-tokines, compared with wild-type mice,

without apparent long-term detrimental ef-fects. In the absence of CCR5, there weremore dendritic cells in the lung-draininglymph nodes and more primed T lympho-cytes in these mice. Bacterial numbers in thelymph nodes were also higher in CCR5(–/–)mice. Therefore, CCR5 may play a role inthe migration of dendritic cells to and fromthe lymph nodes during M. tuberculosis in-fection.—Authors’Abstract

Brennan, M. J., Morris, S. L., and Size-more, C. F. Tuberculosis vaccine devel-opment: research, regulatory and clinicalstrategies. Expert. Opin. Biol. Ther. 4(9)(2004) 1493–1504.

In the past decade, while the global tu-berculosis (TB) epidemic has continued todevastate mankind, considerable progresshas nevertheless been made in the develop-ment of new and improved vaccines for thisancient disease. Recombinant bacillusCalmette-Guerin strains, DNA-based vac-cines, live attenuated Mycobacterium tuber-culosis vaccines and subunit vaccines for-mulated with novel adjuvants have shownpromise in preclinical animal challengemodels. Three of these vaccines are beingevaluated at present in human clinical stud-ies, and several other vaccine preparationsare being targeted for clinical trials in thenear future. Although the preclinical char-acterisation and testing of new TB vaccineshas clearly led to exciting new findings,complex regulatory and clinical trial designissues remain as a challenge to TB vaccinedevelopment. This report reviews some ofthe exciting advances in TB research thathave led to the development of new TB vac-cines, and addresses the unique regulatoryand clinical issues associated with the test-ing of novel anti-TB preparations in humanpopulations.—Authors’Abstract

Brodin, P ., Majlessi, L., Br osch, R.,Smith, D., Bancr oft, G., Clark, S.,Williams, A., Leclerc, C., and Cole, S.T. J . Enhanced protection against tuber-culosis by vaccination with recombinantMycobacterium microti vaccine that in-duces T cell immunity against region of

difference 1 antigens. Infect. Dis. 190(1)(2004) 115–122. Epub 2004 Jun. 04.

Mycobacterium microti, the vole bacillus,which was used as a live vaccine against tu-berculosis until the 1970s, confers the sameprotection in humans as does Mycobacte-rium bovis bacille Calmette-Guerin (BCG).However, because the efficacy of the BCGvaccine varies considerably, we have tried todevelop a better vaccine by reintroducinginto M. microti the complete region of dif-ference 1 (RD1), which is required for se-cretion of the potent T cell antigens early se-creted antigen target (ESAT)-6 and culturefiltrate protein (CFP)-10. The resultant re-combinant strain, M. microti OV254::RD1-2F9, induced specific ESAT-6 and CFP-10immune responses in mice with CD8(+) Tlymphocytes that had strong expression ofthe CD44(hi) activation marker. This vac-cine also displayed better ef ficacy againstdisseminated disease in the mouse and theguinea pig models of tuberculosis than wasseen in animals vaccinated with M. microtialone or with BCG. The M. micr otiOV254::RD1-2F9 vaccine was less virulentand persistent in mice and than wasBCG::RD1-2F9 may represent a safer alter-native to BCG::RD1-2F9.—Authors’ Ab-stract

Gagliardi, M. C., Teloni, R., Mariotti, S.,Iona, E., Pardini, M., Fattorini, L.,Orefici, G., and Nisini, R. BacillusCalmette-Guérin shares with virulentMy-cobacterium tuberculosis the capacity tosubvert monocyte dif ferentiation intodendritic cell: implication for its efficacyas a vaccine preventing tuberculosis. Vac-cine. 22(29–30) (2004) 3848–3857.

The only available vaccine against tuber-culosis (TB) is Bacillus Calmette-Guérin(BCG) whose ef ficacy in preventing pul-monary tuberculosis is however controver-sial. Here, we show that BCG infection ofmonocytes causes their differentiation intomature dendritic cells (DCs) lacking CD1molecules expression, coupled with subop-timal up-regulation of HLA class II, CD80and CD40 molecules and a marked unre-sponsiveness to lipopolysaccharide stimula-


tion. In addition, alloreactive naïve T lym-phocytes primed by these subverted DCs didnot undergo defined functional polarization, aswitnessed by their inability to produce IFN-gamma. Since efficient antigen presentationand IFN-gamma production by mycobacterial-specific T lymphocytes are required for pro-tection against Mycobacterium tuberculosis,our data might provide additional explana-tion for the low ef ficacy of BCG vaccina-tion.—Authors’Abstract

Harth, G., Maslesa-Galic, S., and Hor-witz, M. A. A two-plasmid system forstable, selective-pressure-independentexpression of multiple extracellular pro-teins in mycobacteria. Microbiology150(Pt. 7) (2004) 2143–2151.

Recombinant mycobacteria expressingMycobacterium tuber culosis extracellularproteins are leading candidates for new vac-cines against tuberculosis and other myco-bacterial diseases, and important tools bothin antimycobacterial drug development andbasic research in mycobacterial pathogene-sis. Recombinant mycobacteria that stablyoverexpress and secrete major extracellularproteins of M. tuberculosis in native form onplasmids pSMT3 and pNBV1 were previ-ously constructed by the authors. To en-hance the versatility of this plasmid-basedapproach for mycobacterial protein expres-sion, the Escherichia coli /mycobacteriashuttle plasmid pGB9 was modified to ac-commodate mycobacterial genes expressedfrom their endogenous promoters. Previousstudies showed that the modified plasmid,designated pGB9.2, derived from the cryp-tic Mycobacterium fortuitum plasmidpMF1, was present at a low copy number inboth E. coli and mycobacteria, and expres-sion of recombinant M. tuberculosis proteinswas found to be at levels paralleling its copynumber, that is, approximating their en-dogenous levels. Plasmid pGB9.2 was com-patible with the shuttle vectors pSMT3 andpNBV1 and in combination with them itsimultaneously expressed the M. tuberculo-sis 30 kDa extracellular protein FbpB. Plas-mid pGB9.2 was stably maintained in theabsence of selective pressure in three myco-bacterial species: Mycobacterium bovis

BCG, M. tuber culosis and M. smegmatis .Plasmid pGB9.2 was found to be self-transmissible between both fast- and slow-growing mycobacteria, but not from myco-bacteria to E. coli or between E. coli strains.The combination of two compatible plas-mids in one BCG strain allows expression ofrecombinant mycobacterial proteins at dif-ferent levels, a potentially important factorin optimizing vaccine potency.—Authors’Abstract

Mark Doherty, T. New vaccines against tu-berculosis. Trop. Med. Int. Health. 9(7)(2004) 818–826

In September 2000, recognizing the effectof communicable diseases as obstacles todevelopment in poorer countries, the Euro-pean Commission assembled a specialround table on “accelerated action targetedat major communicable diseases within thecontext of poverty reduction.” The threemajor communicable diseases discussedwere tuberculosis (TB), malaria and HIV .One outcome of this discussion was a work-shop examining issues related to the fightagainst TB in Africa, which took place inGorée, Sénégal, in May 2001. The timingwas propitious, as new vaccines for TB (re-combinant MVA and BCG, and adjuvanatedrecombinant fusion proteins or peptide con-structs), are just beginning to enter humanclinical trials. All but the last of these haveshown promise in animal models, up to andincluding non-human primates, and all arestrongly immunogenic and apparently safe.Humans trials for safety and ef ficacy arethus the logical next step. This review sum-marizes recent advances in tuberculosis vac-cine development, with a special emphasison issues raised at the Gorée meeting abouttesting and deploying new generation vac-cines in TB-endemic areas such as Africa.—Authors’Abstract

Mollenkopf, H. J., Dietrich, G., Fensterle,J., Grode, L., Diehl, K. D., Knapp, B.,Singh, M., O’Hagan, D. T., Ulmer, J.B., and Kaufmann, S. H.Enhanced pro-tective efficacy of a tuberculosis DNAvaccine by adsorption onto cationic PLG

72, 4 Current Literature, Experimental Infections 553

microparticles. Vaccine 22(21–22) (2004)2690–2695.

Immunization with plasmid DNAvectorsrepresents a promising new approach to vac-cination. It has been shown to elicit humoraland cellular immunity and protection in var-ious infection models. Here, we assessed theimmunogenicity and protective efficacy of aDNA vaccine vector encoding the antigen85A(Ag85A) of Mycobacterium tuberculosis.Since intramuscular (i.m.) immunization withnaked DNArequires considerable amounts ofDNA in order to be effective, we evaluateda strategy to reduce the amount of DNAneeded. To this end, we used Ag85A DNAadsorbed onto cationic poly(DL-lactide-co-glycolide) (PLG) microparticles and ob-served similar levels of protection againstaerosol challenge in mice using doses of PLG-DNA two orders of magnitude lower thanwith naked DNA itself.—Authors’Abstract

Sharma, N., and Agrewala, J. N. Potentrole of vaccines prepared from macro-phages infected with live bacteria in pro-tection against Mycobacterium tubercu-losis and Salmonella typhimuriuminfections. J. Infect Dis. 190(1) (2004)107–114. Epub 2004 Jun. 08.

The present study describes a novel andsimple vaccination strategy that involves theculturing of live Mycobacterium tuberculo-sis and Salmonella typhimurium in syn-geneic, allogeneic, and xenogeneic macro-phages, followed by drug treatment andgamma irradiation, to kill the bacteria. No-table observations were that the lymphocytesobtained from the vaccinated mice prolifer-ated and secreted mainly interferon- gammaand IgG2a, but not interleukin-4 and IgG1.The enumeration of viability of M. tubercu-losis indicated a significant level of protec-tion in the vaccinated mice after challengewith live M. tuberculosis. This vaccinationstrategy worked successfully for tuberculo-sis but also showed a significant decrease inmortality of mice challenged with live S. ty-phimurium.—Authors’Abstract

Skeiky, Y. A., Alderson, M. R., Ovendale,P. J., Guderian, J. A., Brandt, L., Dil-lon, D. C., Campos-Neto, A., Lobet, Y.,

Dalemans, W., Orme, I. M., and Reed,S. G. Differential immune responses andprotective ef ficacy induced by compo-nents of a tuberculosis polyprotein vac-cine, Mtb72F, delivered as naked DNAorrecombinant protein. J. Immunol. 172(12)(2004) 7618–7628.

Key Ags of Mycobacterium tuberculosisinitially identified in the context of hostresponses in healthy purified proteinderivative-positive donors and infectedC57BL/6 mice were prioritized for the de-velopment of a subunit vaccine against tu-berculosis. Our lead construct, Mtb72F ,codes for a 72-kDa polyprotein geneticallylinked in tandem in the linear orderMtb32(C)-Mtb39-Mtb32(N). Immunizationof C57BL/6 mice with Mtb72F DNA re-sulted in the generation of IFN-gamma re-sponses directed against the first two com-ponents of the polyprotein and a strongCD8(+) T cell response directed exclusivelyagainst Mtb32(C). In contrast, immuniza-tion of mice with Mtb72F protein formu-lated in the adjuvant AS02A resulted in theelicitation of a moderate IFN-gamma re-sponse and a weak CD8(+) T cell responseto Mtb32c. However, immunization with aformulation of Mtb72F protein in AS01Badjuvant generated a comprehensive and ro-bust immune response, resulting in the elic-itation of strong IFN-gamma and Ab re-sponses encompassing all three componentsof the polyprotein vaccine and a strongCD8(+) response directed against the sameMtb32(C) epitope identified by DNA im-munization. All three forms of Mtb72Fimmunization resulted in the protection ofC57BL/6 mice against aerosol challengewith a virulent strain of M. tuberculosis.Most importantly, immunization of guineapigs with Mtb72F, delivered either as DNAor as a rAg-based vaccine, resulted inprolonged survival (>1 year) after aerosolchallenge with virulent M. tuber culosiscomparable to bacillus Calmette-Guérin im-munization. Mtb72F in AS02A formulationis currently in phase I clinical trial, makingit the first recombinant tuberculosis vaccineto be tested in humans.—Authors’Abstract

Wang, Q. M., Sun, S. H., Hu, Z. L., Zhou,F. J., Yin, M., Xiao, C. J., and Zhang, J.


C. Epitope DNA vaccines against tuber-culosis: spacers and ubiquitin modulatescellular immune responses elicited byepitope DNA vaccine. Scand. J. Immu-nol. 60(3) (2004) 219–225.

Cell-mediated immune responses are cru-cial in the protection against tuberculosis. Inthis study, we constructed epitope DNAvac-cines (p3-M-38) encoding cytotoxic T lym-phocyte (CTL) epitopes of MPT64 and 38kDa proteins of Mycobacterium tuberculo-sis. In order to observe the influence ofspacer sequence (Ala-Ala-Tyr) or ubiquitin(UbGR) on the efficacy of the two CTLepi-topes, we also constructed DNA vaccines,p3-M-S(spacer)-38, p3-Ub (UbGR)-M-S-38and p3-Ub-M-38. The immune responseselicited by the four DNA vaccines were

tested in C57BL/6 (H-2b) mice. The cyto-toxicity of T cells was detected by LDH-release method and by enzyme-linked im-munospot assay for epitope-specific cellssecreting interferon-gamma. The resultsshowed that DNAimmunization with p3-M-38 vaccine could induce epitope-specificCD8+ CTL response and that the spacer se-quence (AAY) only enhanced M epitopepresentation. The protein-targeting sequence(UbGR) enhanced the immunogenicity ofthe two epitopes. The finding that definedspacer sequences at C-terminus and protein-targeting degradation modulated the im-mune response of epitope string DNA vac-cines will be of importance for the furtherdevelopment of multi-epitope DNA vac-cines against tuberculosis.—Authors’ Ab-stract

72, 4 Current Literature, Epidemiology and Prevention 555

Epidemiology and Prevention

Bakker, M. I., Hatta, M., Kwenang, A.,Faber, W. R., Van Beers, S. M., Klatser,P. R., and Oskam, L. Population surveyto determine risk factors for Mycobacte-rium leprae transmission and infection.Int. J. Epidemiol. EPub 2004 Jul. 15.

BACKGROUND: Not every leprosy pa-tient is equally effective in transmitting My-cobacterium leprae. We studied the spatialdistribution of infection (using seropositiv-ity as a marker) in the population to identifywhich disease characteristics of leprosy pa-tients are important in transmission. METH-ODS: Clinical data and blood samples foranti-M. leprae ELISA were collected duringa cross-sectional survey on five Indonesianislands highly endemic for leprosy. A geo-graphic information system (GIS) was usedto define contacts of patients. We investi-gated spatial clustering of patients andseropositive people and used logistic regres-sion to determine risk factors for seroposi-tivity. RESULTS: Of the 3986 people ex-amined for leprosy , 3271 gave blood.Seroprevalence varied between islands(1.7–8.7%) and correlated significantly withleprosy prevalence. Five clusters of patientsand two clusters of seropositives were de-tected. In multivariate analysis, seropositiv-ity significantly differed by leprosy status,

age, sex, and island. Serological status ofpatients appeared to be the best discrimina-tor of contact groups with higher seropreva-lence: contacts of seropositive patients hadan adjusted odds ratio (aOR) of 1.75 (95%CI 0.92–3.31). This increased seropreva-lence was strongest for contact groups liv-ing ≤75 m of two seropositive patients (aOR= 3.07; 95% CI 1.74 –5.42). CONCLU-SIONS: In this highly endemic area for lep-rosy, not only household contacts ofseropositive patients, but also people livingin the vicinity of a seropositive patient weremore likely to harbour antibodies against M.leprae. Through measuring the serologicalstatus of patients and using a broader defi-nition of contacts, higher risk groups can bemore specifically identified.—Authors’Ab-stract

Deps, P. D., Faria, L. V., Gonçalves, V. C.,Silva, D. A., Ventura, C. G., and Zan-donade, E. Epidemiological features ofthe leprosy transmission in relation to ar-madillo exposure. Hansen. Int. 28(2)(2003) 138–144. [Article in Portuguese].

Introduction and purpose: Some authorsdemonstrated the possibility of the armadil-los, Dasypus novemcinctus, being an envi-

ronmental source of Mycobacterium leprae.An epidemiologic survey was done to checkthe correlation between the human contactwith armadillos and the incidence of lep-rosy. It discusses some features that could beinvolved in the dynamic process of the lep-rosy development. The objective of this re-search is to check the frequency of cases ofleprosy contacts with armadillos and alsothe interhuman contact before their diagno-sis to establish the possibility of the M.leprae transmission to the human beingthrough the contact with armadillos. Meth-ods: One hundred and seven leprosy patientswere surveyed (leprosy patients that had fin-ished the MTD treatment) who lived in thePedro Fontes Colony-Hospital, in Cariacica,Espirito Santo State, Brazil, 29 leprosy pa-tients and 173 non leprosy patients from adermatology service of the city of Vitória,Brazil. The survey included data about thearmadillo meat consumption before leprosydiagnosis, the existence of known casesand/or familial leprosy cases. The data wereanalyzed by Qui-square test, correlation andExact Fischer Test. Results: 90.4% of theleprosy patients or cured leprosy patientshad once eaten armadillo meat before theirleprosy diagnosis, while 15% of the non lep-rosy patients had already eaten armadillomeat. In a group without leprosy contact be-fore the diagnosis, 96,1% ate armadillomeat, and 3,9% didn’t eat. This study sup-poses a possible source of the M. leprae bythe armadillo meat consumption, mainly, ina leprosy patients without previous leprosycontact.—Hansenologia Internationalis

Douglas, J. T., Cellona, R. V., Fajardo, T.T. Jr., Abalos, R. M., Balagon, M. V.,and Klatser, P. R. Prospective study ofserological conversion as a risk factor fordevelopment of leprosy among house-hold contacts. Clin. Diagn. Lab. Im-munol. 11(5) (2004) 897–900.

Although the prevalence of leprosy hasdeclined over the years, there is no evidencethat incidence rates are falling. A method ofearly detection of those people prone to de-velop the most infectious form of leprosywould contribute to breaking the chain oftransmission. Prophylactic treatment of sero-logically identified high-risk contacts of inci-

dent patients should be an operationally fea-sible approach for routine control programs.In addition, classification of high-risk house-hold contacts will allow control programre-sources to be more focused. In this prospec-tive study , we examined the ability ofserology used for the detection of antibodiesto phenolic glycolipid I of Mycobacteriumleprae to identify those household contactsof multibacillary leprosy patients who hadthe highest risk of developing leprosy. Afterthe start of multidrug therapy for the indexcase, a new case of leprosy developed in onein seven of the 178 households studied. Inhouseholds where new cases appeared, theseropositivity rates were significantly higher(p <0.001) than those in households withoutnew cases. Seropositive household contactshad a significantly higher risk of developingleprosy (relative hazard adjusted for age andsex [aRH], 7.2), notably multibacillary lep-rosy (aRH = 24), than seronegative con-tacts.—Authors’Abstract

Mak, D. B., Platt, E. M., and Heath, C. H.Leprosy transmission in the Kimberley ,Western Australia. Med. J. Austral. 179(8)(2003) 452.

Between 1986 and 2002, a total of 28 newleprosy cases were notified to the KimberlyPublic Health Unit in Western Australia,Australia. At diagnosis, the patients wereaged 8–63 years. In several recent cases, di-agnosis was delayed despite multiple pre-sentations to primary health care staf f andmedical specialists. Eleven patients (39%)had multibacillary disease, and can transmitthe disease. The need for the proper man-agement of leprosy patients and to controlthe increasing population movement in andout of leprosy-endemic areas to prevent lep-rosy transmission to other parts of Australiais discussed.—Tropical Diseases Bulletin

Meima, A., Smith, W. C., van Oort-marssen, G. J., Richardus, J. H., andHabbema, J. D. The future incidence ofleprosy: a scenario analysis. Bull. WorldHealth Organ. 82(5) (2004) 373–380.

See Current Literature, General and His-torical, p. 511.


Deepak, S. Answering the rehabilitationneeds of leprosy-affected persons in inte-grated setting through primary healthcare services and community-based reha-bilitation. Indian J. Lepr . 75(2) (2003)127–142.

This article aims to discuss the strategiesfor answering the rehabilitation needs ofpersons with leprosy-related disabilities inintegrated settings through primary healthcare (PHC) services and community-basedrehabilitation (CBR). While the provisionof rehabilitation services through the PHCsystem remains problematic in most devel-oping countries, the article concludes thatCBR programs have the potential for reha-bilitation of leprosy-affected persons in in-tegrated settings. However, the limited cov-erage of CBR programs may pose anobstacle to such an approach. The authorsuggests the use of existing specific reha-bilitation infrastructures meant only forleprosy-affected persons for initiating, sus-taining and extending the CBR coverage tothe surrounding communities. At the sametime, the author asks for support andstrengthening of organizations of leprosy-affected persons, promoting their active in-volvement in all rehabilitation processes.—Authors’ Abstract

Kaur, H., and Gandhi, A. People’s percep-tion of leprosy—a study in Delhi. IndianJ. Lepr. 75(1) (2003) 37–46.

Leprosy is considered to cause more so-cial than medical problems. The presentstudy focussed on this aspect in order to in-vestigate the level of awareness amongpeople—about their attitude towards the dis-ease and the afflicted. The results are basedon interviews with 104 persons in Delhi.The sample data revealed that the level ofknowledge of leprosy was inadequate. Thecause of the disease was known to 44.2% ofthose interviewed, while 31.7% were com-

pletely ignorant; 6.7% believed it to be theconsequence of an individual’ s past mis-deeds, and 1.9% believed it to have beencaused by divine curse. 63.1% were awarethat the disease is curable. 73.1% of the per-sons interviewed sympathised with leprosy-afflicted beggars. 61.5% favored leprosypatients to stay with their families andwithin their communities. 67.3% felt that thecured could marry, while 25% felt that theleprosy-afflicted should stay in leprosycolonies away from the society. 54.8% werereluctant to employ the leprosy-afflicted asdomestic help, and 31.7% were reluctant toestablish matrimonial relationship with afamily having a leprosy-afflicted person. Thedata call for intensification ofpublic aware-ness regarding the aetiology of leprosy. Pos-itive and scientific information should bedisseminated to minimize the social preju-dices associated with the disease.—Authors’Abstract

Qian, J. G., Yan, L. B., Li, W. Z., andZhang, G. C. Posterior tibialis transferfor foot-drop due to leprosy: a case with40 years follow-up. Br . J. Plast. Sur g.57(5) (2004) 450–452.

A 56-year-old male was transferred to ourcentre because of a relapse of leprosy neuri-tis in the hands. We found that the patienthad received a posterior tibialis tendontransfer for correction of his left droppedfoot 40 years previously . On examinationactive dorsiflexion of the left ankle joint wasclose to 0 degrees with grade 4 power ofdorsiflexion, and the plantar flexion wasabout 35 degrees. Walking gait was almostnormal. There were some scars on the plan-tar surface of the left metatarsal area; butwith the continuous use of a soft dressingpad under the middle part of the sole, plan-tar ulceration has been avoided for manyyears even with active daily activities of thepatient. The patient is very satisfied with theoperative results.—Authors’Abstract

72, 4 Current Literature, Rehabilitation and Social Concerns 557

Rehabilitation and Social Concerns

Arend, S. M., Cerda de Palou, E., deHaas, P., Janssen, R., Hoeve, M. A.,Verhard, E. M., Ottenhoff, T. H., vanSoolingen, D., and van Dissel, J. T.Pneumonia caused by Mycobacteriumkansasii in a series of patients withoutrecognised immune defect. Clin. Micro-biol. Infect. 10(8) (2004) 738–748.

The clinical and epidemiological charac-teristics of 17 patients diagnosed with My-cobacterium kansasii pneumonia within alimited geographical region over a period of10 years are described. An in-depth evalua-tion of the innate and adaptive immune sys-tems was performed for five available pa-tients. A comparison was made of thegenetic fingerprint patterns of the isolatesobtained by restriction fragment lengthpolymorphism (RFLP) analysis, with themajor polymorphic tandem repeat (MPTR)as a probe. Predisposing factors consisted ofsmoking, airway abnormalities, substanceabuse, diabetes or poor general condition,but in two patients no risk factor was identi-fied. In the five patients tested, no abnor-malities or deficiencies were detected in theinnate or adaptive type-1 immunity. All M.kansasii isolates had identical MPTR RFLPpatterns, although no epidemiological con-nection could be established, and these wereidentical to those of clinical isolates fromAustralian patients. These data do not sup-port the theory that defects in the innate oradaptive type-1 immunity have a role in thepathogenesis of invasive M. kansasii infec-tions. The identical fingerprint patterns ofthe isolates suggested the existence of a vir-ulent strain of M. kansasii.—Authors’ Ab-stract

Debacker, M., Aguiar, J., Steunou, S. Z.,Meyers, W. M., Guédénon, Scott, J. T.,Dramaix, M., and Portaels, F . Myco-bacterium ulcerans disease (BuruliUlcer) in rural hospital, Southern Benin,1997–2001. Emer g. Infect. Dis. 10(8)(2004) 1391–1398.

Data from 1700 patients living in south-ern Benin were collected at the Centre San-

itaire et Nutritionnel Gbemoten, Zagnanado,Benin, from 1997 through 2001. In the Zouregion in 1999, Buruli ulcer (BU) had ahigher detection rate (2.15/100,000) thanleprosy (13.4/100,000) and tuberculosis(20.0/100,000). More than 13% of the pa-tients had osteomyelitis. Delay in seekingtreatment declined from 4 months in 1989 to1 month in 2001, and median hospitalizationtime decreased from 9 months in 1989 to 1month in 2001. This reduction is attributed,in part, to implementing an international co-operation program, creating a national BUprogram, and making advances in patientcare.—Emerging Infectious Diseases

Desmond, E., Ahmed, A. T., Probert, W.S., Ely, J., Jang, Y., Sanders, C. A., Lin,S. Y., and Flood, J. Mycobacteriumafricanum cases, California. Emerg. In-fect. Dis. 10(5) (2004) 921–923.

Five Mycobacterium tuberculosis com-plex isolates in California were identified asM. africanum by spoligotyping, single nu-cleotide polymorphisms, a deletion muta-tion, and phenotypic traits, confirming it asa cause of tuberculosis in the United States.Three of the five patients from whom M.africanum was isolated had lived in Africa.—Authors’Abstract

Drummond, C., and Butler, J. R. Myco-bacterium ulcerans treatment costs, Aus-tralia. Emerg. Infect. Dis. 10(6) (2004)1038–1043.

Mycobacterium ulcerans gives rise to se-vere skin ulceration that can be associatedwith considerable illness. The cost of diag-nosis, treatment, and lost income has neverbeen assessed in Australia. A survey of 26confirmed cases of the disease in Victoriawas undertaken. Data were collected ondemographic details, diagnostic tests, treat-ment, time of f work, and travel to obtaintreatment. All costs are reported in Aus-tralian dollars in 1997–1998 prices. The costvaries considerably with disease severity .For mild cases, the average direct cost is


Other Mycobacterial Diseases

6803 Australian dollars, and for severe cases27,681 Australian dollars. Hospitalizationaccounts for 61% to 90% of costs, and indi-rect costs amount to 24% of the total percase. M. ulcerans can be an expensive dis-ease to diagnose and treat. Costs can be re-duced by early diagnosis and definitivetreatment. Research is needed to find cost-effective therapies for this disease.—Au-thors’Abstract

Eddyani, M., Ofori-Adjei, D., Teugels, G.,De Weirdt, D., Boakye, D., Meyers, W.M., and Portaels, F . Potential role forfish in transmission of Mycobacterium ul-cerans disease (Buruli ulcer): an envi-ronmental study. Appl. Environ. Micro-biol. 70(9) (2004) 5679–5681.

This study reports a potential role that fishmay play in the transmission of Mycobac-terium ulcerans disease (Buruli ulcer). Fishfound positive for M. ulcerans DNA all ap-pear to feed on insects or plankton and arebelieved to concentrate M. ulcerans fromthis usual food source. These observationsprovide additional data supporting our pre-vious hypothesis on sources of M. ulceransand modes of transmission.—Authors’ Ab-stract

Field, S. K., Fisher, D., and Cowie, R. L.Mycobacterium avium complex pul-monary disease in patients without HIVinfection. Chest 126(2) (2004) 566–581.

Mycobacterium avium complex (MAC) isubiquitous. It is found in various freshwa-ter and saltwater sources around the world,including hot water pipes. Although the or-ganism was identified in the 1890s, its po-tential to cause human disease was only rec-ognized 50 years later. Only a minority ofpeople exposed to the organism will acquireMAC lung disease, usually those with un-derlying lung disease or immunosuppres-sion. MAC may, however, cause progressiveparenchymal lung disease and bronchiecta-sis in patients without underlying lung dis-ease, particularly in middle-aged and elderlywomen. Preliminary data suggest that theinterferon-gamma pathways may be defi-cient in elderly women with MAC lung dis-

ease. Other groups of patients who are morelikely to harbor MAC in their lungs includepatients with a cystic fibrosis or an abnormalalpha(1)-antiproteinase gene and patientswith certain chest wall abnormalities. Treat-ment results continue to be disappointing,and the mortality of patients with MAC lungdisease remains high. A PubMed searchidentified 38 reports of the treatment ofMAC lung disease. Apart from the BritishThoracic Society study, the only publishedcontrolled investigation, the studies pub-lished since 1994 have included a macro-lide, either clarithromycin or azithromycin,usually in combination with ethambutol anda rifamycin. If success is defined as eradica-tion of the organism without relapse over aperiod of several years after treatment hasbeen discontinued, the reported treatmentsuccess rate with the macrolide containingregimens is approximately 55%. The pro-longed treatment period, side ef fects, andpossibly reinfection rather than relapse areresponsible for the high failure rate.—Au-thors’Abstract

Fujita, J., Kishimoto, T., Ohtsuki, Y.,Shigeto, E., Ohnishi, T., Shiode, M., Ya-maji, Y., Suemitsu, I., Yamadori, I.,Bandoh, S., Nishimura, K., Mat-sushima, T., and Ishida, T. Clinical fea-tures of eleven cases of Mycobacteriumavium-intracellulare complex pulmonarydisease associated with pneumoconiosis.Respir. Med. 98(8) (2004) 721–725.

The relationship between silicosis and tu-berculosis is well known. Also other myco-bacteria such as Mycobacterium kansasiioften occur in association with pneumoco-niosis. However, there are few reports de-scribing an association of M. avium -intracellulare complex (MAC) lung diseaseand pneumoconiosis. The purpose of thepresent study is to describe clinical featuresof MAC respiratory infection associatedwith pneumoconiosis. Eleven patients withMAC respiratory infection associated withpneumoconiosis (all men, 6 with silicosisand 5 with welders’ pneumoconiosis) werecollected. A determination of whether or notMAC caused pulmonary disease was madeusing the 1997 criteria required by theAmerican Thoracic Society. Radiologically,

72, 4 Current Literature, Other Mycobacterial Diseases 559

cavity formation as well as upper lung fieldpredominance of MAC disease were ob-served in 8 of 11 cases (72.7%). Two of 11patients died of respiratory failure. Our pres-ent study clearly demonstrates that clinicalfeatures of MAC respiratory infection asso-ciated with pneumoconiosis were differentfrom MAC without underlying diseases.—Authors’Abstract

Garcia-Pelayo, M. C., Caimi, K. C., In-wald, J. K., Hinds, J., Bigi, F., Romano,M. I., van Soolingen, D., Hewinson, R.G., Cataldi, A., and Gordon, S. V. Mi-croarray analysis of Mycobacterium mi-croti reveals deletion of genes encodingPE-PPE proteins and ESAT-6 family anti-gens. T uberculosis (Edinb.) 84(3–4)(2004) 159–166.

Mycobacterium microti is the agent of tu-berculosis in wild voles and has been usedas a live vaccine against tuberculosis in manand cattle. To explore the M. microti genomein greater detail, we used a M. tuberculosisH37Rv genomic DNA microarray to detectgene deletions among M. microti isolates. Anumber of deletions were identified that cor-related with those described previously (In-fect. Immun. 70 (2002) 5568) but a novel M.microti deletion was also found (MiD4)which removes 5 genes that code for ESAT-6family antigens and PE-PPE proteins.Southern blot experiments showed that thisregion was also deleted from M. pinnipedii,a mycobacterium isolated from seals that isclosely related to M. microti. Genes encod-ing ESAT-6 antigens and PE-PPE proteinsappear to be frequently deleted from M. mi-croti, and the implications of this are dis-cussed.—Authors’Abstract

Haverkamp, M. H., Arend, S. M., Linde-boom, J. A., Hartwig, N. G., and vanDissel, J. T. Nontuberculous mycobacte-rial infection in children: a 2-yearprospective surveillance study in theNetherlands. Clin. Infect. Dis. 39(4)(2004) 450–456. Epub 2004 Jul. 23.

We performed a prospective, 2-year na-tionwide study to assess incidence and dis-ease characteristics of suspected infections

with nontuberculous mycobacteria (NTM)in children, via the Netherlands PediatricSurveillance Unit. Data for 61 children werereported (median age, 31 months; interquar-tile range, 22–50 months; female sex, 37subjects); 2 subjects had an underlying dis-ease. Most children (53 [87%] of 61) hadcervical lymph node enlargement, with ab-scess in 25 (47%) and fistula in 1 1 (21%).The estimated annual incidence of NTM in-fection was 77 cases per 100,000 children.In 16 children, the diagnosis was basedsolely on the results of skin tests with my-cobacterial antigens. Cultures were per-formed in 36 cases and yielded mycobacte-ria in 27 (75%); Mycobacterium avium wasisolated from 18 cultures. Children with aculture positive for mycobacteria did notdiffer in presentation, complications, ortreatment from those whose cultures showedno growth. Thirty children underwentsurgery, and chemotherapy was the singletreatment in 24 (39%) of the cases. Thetreatment of localized NTM infection in im-munocompetent children by antimycobacte-rial drugs should be evaluated further.—Au-thors’Abstract

Heckman, G. A., Hawkins, C., Morris, A.,Burrows, L. L., and Berger on, C.Rapidly progressive dementia due toMycobacterium neoaurum meningo-encephalitis. Emer g. Infect. Dis. 10(5)(2004) 924–927.

Dementia developed in a patient withwidespread neurologic manifestations; shedied within 5 months. Pathologic findingsshowed granulomatous inflammation withcaseation necrosis, foreign body-type giantcells, and proliferative endarteritis with vas-cular occlusions. Broad-range polymerasechain reaction identified Mycobacteriumneoaurum as the possible pathogen. Centralnervous system infection by M. neoaurummay result in rapidly progressive demen-tia.—Authors’Abstract

Hyon, J. Y., Joo, M. J., Hose, S., Sinha, D.,Dick, J. D., and O’Brien, T. P. Com-parative efficacy of topical gatifloxacinwith ciprofloxacin, amikacin, and clar-ithromycin in the treatment of experi-


mental Mycobacterium chelonae kerati-tis. Arch. Ophthalmol. 122(8) (2004)1166–1169.

OBJECTIVE: To determine the compara-tive ef ficacy of topical gatifloxacin withciprofloxacin, fortified amikacin, and clar-ithromycin against Mycobacterium che-lonae keratitis in an animal model. METH-ODS: Experimental M. chelonae keratitiswas induced via intrastromal inoculation ina rabbit model. Thirty-five rabbits were ran-domly divided into 5 groups and each groupwas treated hourly for 12 hours with topical0.9% balanced salt solution, 0.3% gati-floxacin, 0.3% ciprofloxacin hydrochloride,a combination of topical fortified amikacinsulfate (50 mg/mL) and clarithromycin (10mg/mL), or a triple combination of topical0.3% gatifloxacin, fortified amikacin sulfate(50 mg/mL), and clarithromycin (10 mg/mL). Antibacterial efficacy of each regimenwas determined by quantitative bacterio-logic analysis. RESULTS: Treatment with0.3% gatifloxacin or the triple combinationof 0.3% gatifloxacin, topical fortifiedamikacin sulfate (50 mg/mL), and clar-ithromycin (10 mg/mL) reduced the numberof mycobacterial or ganisms more signifi-cantly than the controls that were treatedwith a topical balanced salt solution (both p<.001). Therapy with 0.3% gatifloxacin wasmore ef fective than 0.3% ciprofloxacinalone (p <.001) and demonstrated synergyby enhancing the efficacy of the combina-tion of fortified amikacin (50 mg/mL) andclarithromycin (10 mg/mL) (p <.001). Nei-ther 0.3% ciprofloxacin nor the combinationof fortified amikacin (50 mg/mL) and clar-ithromycin (10 mg/mL) demonstrated a sig-nificant difference in activity against myco-bacteria compared with the topical balancedsalt solution. CONCLUSION: These resultssuggest that topical 0.3% gatifloxacin oph-thalmic solution can be a new initial treat-ment agent against M. chelonae kera-titis.Clinical Relevance Topical gatifloxacin0.3% may provide an initial alternative intherapy of M. chelonae keratitis.—Authors’Abstract

Kapur, N., Schuster , H., Parker , N.,Kelsey, M. C., and Goldsmith, P. C. Se-vere sporotrichoid infection with Myco-

bacterium szulgai. Clin. Exp. Dermatol.29(4) (2004) 377–379.

Mycobacterium szulgai is a nontubercu-lous, acid-fast bacillus or atypical mycobac-teria, which prior to 1972 was not thought ofas a pathogen. Since then most cases re-ported in the literature have been of pul-monary disease with only a few case reportsof cutaneous disease. Our patient, who hadan underlying, uncategorized, immunosup-pressive condition, presented with multiplesevere ulcers spreading proximally up thearms in a sporotrichoid pattern with morescatttered lesions on his legs. He made a fullrecovery with appropriate antimicrobialtreatment.—Authors’Abstract

Lewis, C. G., Wells, M. K., and Jennings,W. C . Mycobacterium fortuitum breastinfection following nipple-piercing, mim-icking carcinoma. Breast J. 10(4) (2004)363–365.

We reviewed a rare breast infectionoccurring 4 months after nipple piercing.Clinical examination suggested carcinoma.Mycobacterium fortuitum was eventuallyisolated after surgical biopsy and debride-ment. Antibiotic therapy was initiated intra-venously using two drugs and oral therapywas continued for 6 months. A contralateralmycobacterial lesion emerged and was ex-cised along with a residual fibrotic nodule atthe original biopsy site. When adequatesampling of a complex and suspicious breastmass is benign and initial bacterial culturesare sterile, mycobacterial infection shouldbe considered, particularly when there is ahistory of previous nipple piercing proce-dures.—Authors’Abstract

Lopez-Calleja, A. I., Lezcano, M. A.,Samper, S., de Juan, F., and Revillo, M.J. Mycobacterium malmoense lym-phadenitis in Spain: first two cases in im-munocompetent patients. Eur. J. Clin. Mi-crobiol. Infect. Dis. 23(7) (2004) 567–569.Epub 2004 Jun. 26.

Reported here are two cases of Mycobac-terium malmoense lymphadenitis that oc-curred in two immunocompetent children in


Spain. To the best of our knowledge, theseare the first documented cases of extrapul-monary infection by M. malmoense inSpain. This report serves to draw attentionto this emerging nontuberculous mycobac-terium that is gaining increasing recognitionas a pulmonary and extrapulmonary patho-gen in dif ferent countries.—Authors’ Ab-stract

Majoor, C. J., Schreurs, A. J., and Weers-Pothoff, G. Mycobacterium xenopi in-fection in an immunosuppressed patientwith Crohn’ s disease. Thorax. 59(7)(2004) 631–632.

A 48 year old patient with active Crohn’sdisease presented with bilateral nodules overhis lungs resembling malignant metastasis.Bronchoscopic and pathological examina-tion of the airways and sputum did not showany malignancy. After 6 weeks Mycobacte-rium xenopi was cultured from his bronchialwashings while all other cultures remainednegative. Treatment was started with ri-fampicin, ethambutol, and clarithromycinand, after 9 months of treatment, there wasan almost complete resolution of his chestradiograph.—Authors’Abstract

Marras, T. K., and Daley, C. L. ASystem-atic Review of the Clinical Significanceof Pulmonary Mycobacterium kansasiiIsolates in HIV Infection. J. Acquir. Im-mune Defic. Syndr. 36(4) (2004) 883–889.

SUMMARY: Guidelines recommendtreating HIV -infected patients with pul-monary Mycobacterium kansasii infectiononly in the presence of multiple positive cul-tures and clinical and radiographic abnor-malities. Some authors suggest a single pos-itive culture warrants treatment. Asystematic literature review was done to de-termine whether HIV-infected patients whohad M. kansasii isolated from respiratoryspecimens may have an indolent infection(often referred to as colonization) not re-quiring treatment and to determine the util-ity of diagnostic criteria in distinguishingdisease from indolent infection. Sixteenstudies were included, with at least 573 pa-tients: mean age 44 years; 91% male; 64%

men who had sex with men; 35% injectiondrug users; and median CD4 lymphocytecount of 2–381 cells/microL. The medianrate of indolent infection was 8%. Of thefew patients who did not satisfy diagnosticcriteria and were left untreated, outcomeswere generally favorable. Overall, survivalwas longer in treated patients (mean 12 vs.4 months). Indolent pulmonary infectionwith M. kansasii may exist in the setting ofHIV, but published data do not provide ad-equate information to identify such patients.It is unclear whether unfulfilled diagnosticcriteria necessarily imply the absence of dis-ease in this context.—Authors’Abstract

Mostowy, S., Onipede, A., Gagneux, S.,Niemann, S., Kremer, K., Desmond, E.P., Kato-Maeda, M., and Behr, M. Ge-nomic analysis distinguishes Mycobacte-rium africanum. Clin. Microbiol. 42(8)(2004) 3594–3599.

Mycobacterium africanum is thought tocomprise a unique species within the Myco-bacterium tuberculosis complex. M. afri-canum has traditionally been identified byphenotypic criteria, occupying an interme-diate position between M. tuberculosis andM. bovis according to biochemical charac-teristics. Although M. africanum isolatespresent near-identical sequence homology toother species of the M. tuberculosis com-plex, several studies have uncovered largegenomic regions variably deleted from cer-tain M. africanum isolates. To further inves-tigate the genomic characteristics of organ-isms characterized as M. africanum , theDNA content of 12 isolates was interrogatedby using Affymetrix GeneChip. Analysis re-vealed genomic regions of M. tuberculosisdeleted from all isolates of putative diag-nostic and biological consequence. The dis-tribution of deleted sequences suggests thatM. africanum subtype II isolates are situatedamong strains of “modern” M. tuberculosis.In contrast, other M. africanum isolates(subtype I) constitute two distinct evolu-tionary branches within the M. tuberculosiscomplex. To test for an association betweendeleted sequences and biochemical attri-butes used for speciation, a phenotypicallydiverse panel of “M. africanum-like” isolatesfrom Guinea-Bissau was tested for these


deletions. These isolates clustered togetherwithin one of the M. africanum subtype Ibranches, irrespective of phenotype. Theseresults indicate that convergent biochemicalprofiles can be independently obtained forM. tuber culosis complex members, chal-lenging the traditional approach to M. tu-berculosis complex speciation. Furthermore,the genomic results suggest a rational frame-work for defining M. africanum and providetools to accurately assess its prevalence inclinical specimens.—Authors’Abstract

Phillips, R., Adjei, O., Lucas, S., Benjamin,N., and Wansbrough-Jones, M. Pilotrandomized double-blind trial of treatmentof Mycobacterium ulcerans disease (Bu-ruli ulcer) with topical nitrogen oxides.Antimicrob. Agents Chemother. 48(8)(2004) 2866–2870.

Mycobacterium ulcerans disease (Buruliulcer) is a serious ulcerating skin diseasewhich is common in many tropical coun-tries. Standard treatment, by extensive exci-sion and skin grafting, is not available inrural communities where the disease is com-mon. We evaluated the efficacy and safety oftreatment with topical nitrogen oxides.Thirty-seven patients with a clinical diag-nosis of Buruli ulcer caused by M. ulceransdisease were randomly assigned to one oftwo groups. In one group, two creams con-taining sodium nitrite (6%, wt/wt) or citricacid monohydrate (9%, wt/wt) were applieddaily for 6 weeks, while the other group re-ceived a placebo. In the second 6 weeks,both groups received the nitrogen oxide-generating combination of creams. Treat-ment was continued for another 4 weeks forpatients whose ulcers were not healed after12 weeks. The ulcer surface area was mon-itored by weekly tracings made by assessorsblinded to the treatment. In the first 6 weeks,patients on sodium nitrite and citric acidmonohydrate (group I, active treatment)showed a rapid decrease in ulcer size from28.6 ± 5.6 cm2 (mean ± standard error) to12.6 ± 3.2 cm 2, a decrease significantlygreater than that in group II (from 15.3 ±3.1to 11.7 ± 3.7 cm2; p = 0.03). Five ulcers inthe placebo group enlarged during this pe-riod, compared with one in the active group.In the second 6 weeks (both groups on ac-

tive treatment), the rates of healing weresimilar for the two groups and there was asignificant reduction in ulcer size in group II(previously on placebo) compared to thefirst 6 weeks. Yellow pigmentation of theskin, which disappeared 3 days after treat-ment was stopped, was the only side ef fectto date. We conclude that creams releasingnitrogen oxides increase the healing rate ofulcers caused by M. ulcerans infection withminimal adverse events. This is the first con-trolled trial of any form of therapy whichdemonstrates efficacy in treating this dis-ease.—Authors’Abstract

Phillips, R., Kuijper , S., Benjamin, N.,Wansbrough-Jones, M., Wilks, M., andKolk, A. H. In vitro killing of Mycobac-terium ulcerans by acidified nitrite. An-timicrob. Agents Chemother. 48(8)(2004) 3130–3132.

Mycobacterium ulcerans , which causesBuruli ulcer, was exposed to acidified nitriteor to acid alone for 10 or 20 min. Killingwas rapid, and viable counts were reducedbelow detectable limits within 10 min of ex-posure to 40 mM acidified nitrite. M. ulcer-ans is highly susceptible to acidified nitritein vitro.—Authors’Abstract

Piersimoni, C., Goteri, G., Nista, D., Mar-iottini, A., Mazzarelli, G., and Borni-gia, S. Mycobacterium lentiflavum as anemerging causative agent of cervicallymphadenitis. J. Clin. Microbiol. 42(8)(2004) 3894–3897.

A lymph node excision was performed ona 45-year -old woman with left cervicalswelling. The disorder which developedafter the patient had undergone oral surgeryfor a severe periodontal disease failed to re-spond to antimicrobial chemotherapy. A my-cobacterial strain subsequently identified byhigh-performance liquid chromatographyanalysis of cell wall mycolic acids as Myco-bacterium lentiflavum grew from the ex-cised specimen. This case and previouslypublished reports highlight the relevance ofM. lentiflavum as an emer ging causativeagent of mycobacterial cervical lymph-adenitis.—Authors’Abstract


Reddy, V. M., Suleman, F. G., and Hay-worth, D. A. Mycobacterium aviumbinds to mouse intestinal mucus aldolase.Tuberculosis (Edinb.) 84(5) (2004) 303–-310.

SETTING: Mycobacterium avium com-plex (MAC) is known to colonize the gas-trointestinal tract of human immunodefi-ciency virus (HIV) infected patients beforecausing bacteremia and disseminated dis-ease. However , the mechanism involved in the gastrointestinal colonization is notknown. OBJECTIVE: To identify putativeintestinal mucus receptors which serve asanchor for MAC colonization. DESIGN:C57BL/6 mouse intestinal mucus was sub-jected to single and two-dimensional elec-trophoresis and blotted on nitrocellulosemembranes. MAC specific mucus proteinswere identified by probing the mucus west-ern blots with biotinylated proteins derivedfrom M.avium strain 101 (MAC101). RE-SULTS: Biotinylated MAC 101 proteinsrecognized a 39 kDa intestinal mucus gly-coprotein. The protein displaying an iso-electric point (pI) of 9.0, was found to be pe-riodate sensitive but resistant to sialidase,heparinase I and chondroitinase ABC. Theinternal amino acid sequence of the 39 kDaprotein displayed homology with fructose-1-6-bisphosphate aldolase B (aldolase). Theproclivity between MAC adhesins and al-dolase was confirmed by probing rabbitmuscle aldolase with MAC proteins. Fur-thermore, both 25 and 31 kDa MAC ad-hesins, superoxide dismutase and heparinbinding protein, respectively, were found tobind to aldolase. CONCLUSIONS: MACbinds to intestinal mucus aldolase, conceiv-ably facilitating intestinal colonization ofthe organism.—Authors’Abstract

Schulzke, S., Adler, H., Bar , G., Hein-inger, U., and Hammer, J. Mycobacte-rium bohemicum—a cause of paediatriccervical lymphadenitis. Swiss. Med.Wkly. 134(15–16) (2004) 221–222.

We report on two toddlers suffering fromMycobacterium bohemicum lymphadenitis.Acid-fast bacilli were cultured from sub-mandibular lymph nodes and identified bymolecular methods as Mycobacterium bo-

hemicum. Surgical treatment was successfuland complemented by oral treatment withclarithromycin and rifampicin.—Authors’Abstract

Sedlacek, L., Rifai, M., Feldmann, K.,and Bange, F. C. LightCycler-based dif-ferentiation of Mycobacterium abscessusand Mycobacterium chelonae . J. Clin.Microbiol. 42(7) (2004) 3284–3287.

In this study we introduce a rapid proce-dure to identify Mycobacterium abscessus(types I and II) and M. chelonae usingLightCycler-based analysis of the hsp65gene. Results from 36 clinical strains werecompared with hsp65 gene restriction analy-sis and biochemical profiles of bacilli. As allthree methods yielded identical results foreach isolate, this procedure offers an excel-lent alternative to previously established nu-cleic acid amplification-based techniques forthe diagnosis of mycobacterial diseases.—Authors’Abstract

Tan, H. H., Tan, A., Theng, C., and Ng, S.K. Cutaneous Mycobacterium haemo-philum infections in immunocompro-mised patients in a dermatology clinic inSingapore. Ann. Acad. Med. Singapore.33(4) (2004) 532–536.

INTRODUCTION: Mycobacterium hae-mophilum, a nontuberculous mycobacte-rium (NTM) that was first described in1978, is a pathogen that can cause an arrayof symptoms in immunocompromised pa-tients, predominantly cutaneous. CLINI-CAL PICTURE: We report our hospital’ sexperience with the first 3 patients diag-nosed with this infection from 1994 to 2002.All were women; one had systemic lupuserythematosus (SLE), one had mycosis fun-goides and the last had Sjogren’s syndromewith recurrent bacterial infections, althoughthe specific nature of her immunocompro-mised state has not been defined. All wereHIV negative. All 3 women presented withcutaneous lesions—the first with recurrenterythematous plaques on the limbs andback, the second with tender nodules andabscesses on the knees, and the third withpapular eruptions on the cheek. TREAT-


MENT/OUTCOME: All responded to acombination of antibiotics and are presentlystill undergoing treatment and follow-up.CONCLUSION: Infections caused by M.haemophilum occur mainly in immunocom-promised patients. They can present with avariety of cutaneous manifestations, whichrequire a high index of suspicion and coor-dination between the treating physician andthe laboratory for diagnosis. Combinationantibiotic treatment is recommended, andpatients should be followed up after treat-ment to survey for possible relapse.—Au-thors’Abstract

Tortoli, E., Rindi, L., Gar cia, M. J.,Chiaradonna, P., Dei, R., Garzelli, C.,Kroppenstedt, R. M., Lari, N., Mattei,R., Mariottini, A., Mazzarelli, G., Mur-cia, M. I., Nanetti, A., Piccoli, P., andScarparo, C. Proposal to elevate the ge-netic variant MAC-A, included in theMycobacterium avium complex, to speciesrank as Mycobacterium chimaera sp. nov.Int. J. Syst. Evol. Microbiol. 54(Pt. 4)(2004) 1277–1285.

The possibility that the strains includedwithin the Mycobacterium avium complex(MAC), but not belonging either to M.avium or to Mycobacterium intracellulare,may be members of undescribed taxa, hasalready been questioned by several taxono-mists. A very homogeneous cluster of 12strains characterized by identical nucleotidesequences both in the 16S rDNA and in the16S–23S internal transcribed spacer was in-vestigated. Similar strains, previously re-ported in the literature, had been assigned ei-ther to the species M. intracellulare on thebasis of the 16S rDNA similarity or to thegroup of MAC intermediates. However, sev-eral phenotypical and epidemiological char-acteristics seem to distinguish these strains

from all other MAC organisms. The uniquemycolic acid pattern obtained by HPLC isstriking as it is characterized by two clustersof peaks, instead of the three presented byall other MAC organisms. All of the strainshave been isolated from humans and all butone came from the respiratory tract of el-derly people. The clinical significance ofthese strains, ascertained for seven patients,seems to suggest an unusually high viru-lence. The characteristics of all the strainsreported in the literature, genotypically iden-tical to the ones described here, seem to con-firm our data, without reports of isolationsfrom animals or the environment or, amonghumans, from AIDS patients. Therefore, anelevation of the MAC variant was proposedand characterized here, with the nameMycobacterium chimaera sp. nov .; thisincreases the number of species included inthe M. avium complex. The type strain is FI-01069T (=CIP 107892T=DSM 44623T).—Authors’Abstract

van Hest, R., van derZanden, A., Boeree,M., Kremer, K., Dessens, M., Weste-nend, P., Maraha, B., van Uffelen, R.,Schutte, R., and de Lange, W. Myco-bacterium heckeshornense infection in animmunocompetent patient and identifica-tion by 16S rRNA sequence analysis ofculture material and a histopathology tis-sue specimen. J. Clin. Microbiol. 42(9)(2004) 4386–4389.

Mycobacterium heckeshornense is a rareisolate in clinical specimens. We per-formed simultaneous 16S rRNA sequenceanalysis of a mycobacterium culture and ahistopathology specimen to determine therelevance of M. heckeshornense infectionin an immunocompetent patient initiallypresenting with pneumothorax.—Authors’Abstract

72, 4 Current Literature, Molecular and Genetic Studies 565

Molecular and Genetic Studies

Ahmed, N., Alam, M., Rao, K. R., Kauser,F., Kumar, N. A., Qazi, N. N., Sangal,V., Sharma, V. D., Das, R., Katoch, V.M., Murthy , K. J., Suneetha, S.,

Sharma, S. K., Sechi, L. A., Gilman, R.H., and Hasnain, S. E. Molecular geno-typing of a large, multicentric collectionof tubercle bacilli indicates geographical

partitioning of strain variation and hasimplications for global epidemiology ofMycobacterium tuberculosis. J. Clin. Mi-crobiol. 42(7) (2004) 3240–3247.

Tuberculosis continues to be a majorkiller disease, despite an all-out ef fortlaunched against it in the postgenomic era.We describe here the population structure ofMycobacterium tuberculosis strains, as re-vealed by a chromosome-wide scan of fluo-rescent amplified fragment length polymor-phisms (F AFLPs), for more than 1 100independent isolates from 11 different coun-tries. The bacterial strains were genotypedbased on a total of 136 ± 1 different FAFLPmarkers at the genome sequence interface,with details on IS6110 profiles, drug resist-ance status, clinicopathological observa-tions, and host status integrated into theanalysis process. The strains were found tocluster with possible geographic affinities,including the parameters of host speciestype, IS6110 profile, and drug susceptibilitystatus. Of the five most commonly amplifiedfragment sets (or amplitypes), type A pre-dominated in strains of mixed origin, de-posited in The Netherlands; type B was ex-clusively observed for Indian isolates; typeC was found mainly in strains from Peru andAustralia; and types D and E predominatedin European strains from France and Italy.The amplitypes were independent of certainlarge sequence polymorphisms representingtwo important deletions, TbD1 and Rd9. Itappears that M. tuberculosis has a high ge-nomic diversity with a possible geographicevolution. This may have occurred due tospecific genomic deletions and synonymoussubstitutions selected rigorously against hostdefenses and environmental stresses on anevolutionary timescale. The genotypic datareported here are additionally significant forgenotype-phenotype correlations and for de-termining whether pathogen diversity is areflection of the host population diversity.—Authors’Abstract

Butcher, P. D. Microarrays for Mycobacte-rium tuberculosis. Tuberculosis (Edinb).84(3–4) (2004) 131–137.

This special microarray issue of Tubercu-losis recognises the important contributions

of M. tuberculosis whole genome DNA mi-croarrays to tuberculosis research by bring-ing together a range of papers that addressM. tuberculosis physiology, host-pathogeninteractions, mechanisms of drug action, invitro and in vivo gene expression, host re-sponses, comparative genomics and func-tional analysis of particular genes. A numberof complete datasets of M. tuber culosismRNA expression levels are provided to fa-cilitate multiple cross-condition compari-son. Microarrays represent one of the newfunctional genomics technologies exploitinggenome sequence information that willbring us closer to realising the scientific andmoral imperatives of better vaccines, diag-nostics and new drugs for the control of tu-berculosis throughout the world.—Author’sAbstract

Fabre, M., Koeck, J. L., Le Fleche, P .,Simon, F., Herve, V., Vergnaud, G., andPourcel, C. High genetic diversity re-vealed by variable-number tandem repeatgenotyping and analysis of hsp65 genepolymorphism in a lar ge collection of“Mycobacterium canettii” strains indicatesthat the M. tuberculosis complex is a re-cently emerged clone of “M. canettii.” J.Clin. Microbiol. 42(7) (2004) 3248–3255.

We have analyzed, using complementarymolecular methods, the diversity of 43strains of “Mycobacterium canettii” origi-nating from the Republic of Djibouti, on theHorn of Africa, from 1998 to 2003. Geno-typing by multiple-locus variable-numbertandem repeat analysis shows that all thestrains belong to a single but very distantgroup when compared to strains of the My-cobacterium tuberculosis complex (MTBC).Thirty-one strains cluster into one lar gegroup with little variability and five strainsform another group, whereas the other sevenare more diverged. In total, 14 genotypes areobserved. The DR locus analysis revealsadditional variability, some strains being de-void of a direct repeat locus and others hav-ing unique spacers. The hsp65 gene poly-morphism was investigated by restrictionenzyme analysis and sequencing of PCRamplicons. Four new single nucleotide poly-morphisms were discovered. One strain wascharacterized by three nucleotide changes in


441 bp, creating new restriction enzymepolymorphisms. As no sequence variabilitywas found for hsp65 in the whole MTBC,and as a single point mutation separates M.tuberculosis from the closest “M. canettii”

strains, this diversity within “ M. canettii”subspecies strongly suggests that it is themost probable source species of the MTBCrather than just another branch of theMTBC.—Authors’Abstract

72, 4 Current Literature, Molecular and Genetic Studies 567

Date post:	17-Jan-2020
Category:	Documents
Upload:	others
View:	4 times
Download:	0 times

INTERNATIONAL JOURNALOF LEPROSY Volume 72, Number 4 ...ila.ilsl.br/pdfs/v72n4a17.pdf · Eating...

Documents