MICROBIOLOGY - Index of

122A ^ hiternntintrr(l ,Inlu'/ UlI of Lepi'e.1.V ^ 1998
Gelatin minimal (CM) medium :;upplemented with
sodium palmitate was further supplemented with 5% Dextrin,
2% F'ructose, Actinomycin O at 50 ug/ml concentration
and Chis medium was inoculated with 10 1- 106
CFU/ml ol' ira vitro maintained CAN bacteria which was
allowed to grow for 4-6 weeks. Morphology was studied
every •week by ataining with acid fast atais, Oram stain
and Ciemsa stain. Control medium was CM without these
supplements. The resulta showed unequivocal reversion
of is vitro nos-acid fast bacillary, mycelial and
granular forms of CAN bacteria to typical acid fast
bacilli seen ira vivo ia LL leprosa. The rol• of individual
factor by a process of elimination has bern determined
and evaluated.
M149
OXIDATION OF "C-PALMITATE AS AN INDEX OF INFECTIVITI' AND
VIABILITY OF 'FAST AND 'SLOW GROWING \I LEPRAIi ISOLATES
R W Truman. S O Franrblau. R.51. Sandhez. A. listras
G.\V L. lianse i s Discasc Center. /Imo Rouge. Louuuuu. USA
Though there are as cot no recognvcd strams of .1/ hrrur, san.mis
w lnch cshcbn 'for or erossih in U¢ mouse foot pad halo bois desembolo
The factors w h¢h underhe these sanam Imos are nm yet undcnhwd. We
cvmparcd the nmtabrhe acto ny and infetam aí ditTcrent preparamos of 1f.
leprr ncluding knmm Yast' and 'siais' gruo mg isolai.. Inocula were
compare/1 hetwcen Ireshls hanested l/. l.`ru, with assue preparanons and
suspensions prescned at difercnt tempo,. secas and titiles. A mulher of goai
handlmg pracnces are noted 5letabohe acuovy o as tssessed as the ninava
abJm of Id 11 (error m ostdac "C-Pahmuln m xtiddlebrook 71112 IBacTcct
mvbatctl at 33C and nas espressed as a growth rodes (GIL Inf ctntty was
delermined u ing lhe mouse foot pad rechmque Arnuaddlos viera inoculated
with some preparations The metabolic acusas of .11. (Troe 5aned nurkedls lis
ias time of ongut, tirocine of preparation, duraoon of storage. and general
character of the tssuc from schich u vias denc ed. The GI of freshly hanested
preparanons were roughly bunod.dic correlatcd sc uh infecto ny. Prcpamnons
onh low GI teuded to silos favor io no growth UNO rcutoculauon iam animais
w indo Ihose ardi high GI usudn achicvcJ good growth results. Abuse rir bclow
a threslmld, the GI was 1101 proportional to growth rate and nas no[ assonated
with 'Ciar or 'sino' growth charactensucs of different isolares Preparatans held
at 4-33C showed first an uicrease and troar a gradual decline m GI omer a 4 soa
pcnod, but mfcctmny tias lost rapidls and generally climmalcd by 3 ssecks.
Prcpamnons hcld at 1C for leis bati 7 das s retained bati Infectas ny. Metabohc
acheis rias noa deteclable follosing a single freeze-th:nv of assue rir hanllar
suspension and was generally assocmted with a 991' or grcater decline is
infectas Grossth results achionahlc altar inocuiation of amoadillos and mica
were comparable. Oddation of "C-palmnate can be used etTccus elo as a relativa
meosurn of the qualin daí lapso preparammns The l ack of linear correlation
hctwecn metabolic activity and 'fase rir'tios' growth charactenstic suggcsts that
dilTerenual growth rates are minerem and nos be useful markers to help idcntdy
saram sanants.
MI50
CULTURE :\ ND PATI(OGENIC CIIARACTERISTICS OP THE
SI)COH.1CIERIC:V LEPRA!: ISOLATE "TI L 1-53" IN
:ARNIADILLOS (D.impus.VOIGIIC/.VCIU1).
R W Tnrman D Nl. Scollard, R.NI. Sanchez.
G\VL Hansen's Disease Center, Bacon Rouge, Louisiana, USA
Though no venetic variation has bens detected in .t1./Ursa
from difierent patients, regions and animal hosts, some isolares can be
distinguished by their dilierential growth rates the mouse foot pad.
Tltai-53-.1/. /rprae is a weil characterized "fast" growing isolate
commoniy propaeated is athvmic sede erice. To determine ias
pathogenic characteristics is another host ase inoculated 2 groups o)'
10 armadillos with 1.9 X 10' bacilli and compared their time course of
serological, hcmatological and histopathological esents of their
infection with those of olhar animais inoculated with bacilli passaged
from humans to armadillos. Thai-53 showed high metabolic activity
for oxidation of "C-palmitate. Consistent with ias growth
characteristics is the nade mouse, Thai-53 inoculated armadillos
showed more a rapid progression of infection, decelopine fully
dissentinated leprosy within 8-12 months with ali classical signa of the
disease. Serological antibodv protiles for infection with Thai-53 were
low and inconsistent is comparison to that seen with olhar isolates
and the bacilli did noa comntonly disseminara to distant somatic sitos
prior to the animais entering the terminal stages of discase. Necrupsy
exantination showed that armadillos infected with Thai-53 had
extensiva neurological involvement. Intcrspecies transfer of bacilli do
noa ettect their oscrt pathogenicity and similar culture characteristic
can he observed is dilierent hosts.
MIS1
EFFECT OF UV RADIATION ON .IIFCOHAC/ER/CM LE1'H.-I F.
R 51' Truman T 1' Gillis
G W Lung llanseti s Discas,: Conter, Baton Rouge, La USA
The relatice impunance is Ieprosy transmission of contantinated dust,
druplets or fun dias remain unclear .111csiorr.•rrran l.Trac is as
extremely slow erow ing ohligate intraccllular parasite which is yet to
be cultivated os artificial media. Seseral onvirunmental factors could
limit ias [ransntissibility and survival ira sature including ias Coloras e to
ultraciolet radiation (UV). In the laboratory, UV-C is generally
recognized as a potent sterilizing aid bar ias etfectiveness aeainst ,i/.
lepra,' alvo has sol beco shosn. \Ve exantined tine intluertce of UV os
the growth and metabolic activity of Al. lepra.: Tesnporary static
culturas ira I Ox35mm polystyrene petri dishes with 500 ui 71112 media
containing Ix10' .1/. 1./rrae obtained front foot pads of nade mico
were exposed to varving timed interva)s of U1• radiation generated
from as artificial source Exposure limes ranged from 0-80 secundo
and constitrited doses totaling from 0-1 2_.64x10' ERGS/est i o The
bacilli were irnmediately resuvpended and I x10' oreanisms from each
exposure dose were inoculated isto BacTec 12B vi.ds to assess for
metabolic eticets Another 1x10' bacilli from each exposure acere
inoculated Unto the foot pads of Balb/c coice )NIFP) to assess Ione
ter naeticets
o0 cell disision. The growth index achieved is BacTec
cultores showed an immediate dose response related decline to a
minimum of about 50% of the control activity afiar exposure to
6 3x10' ERGS/cot'. \IFP results assassine cal) eroscth were similar
and showed that doses of 3x10' ERGS/crr resulted ira a 90% killing
and 6 3x10' ERGS/cm' killed 99% Al. urrara shows sensitivity to
UV-C similar to .tí nrh.•rcrrlaats and Iacks mechanisms that might
aliais it to substantially avoid UV injury.
MI52
RAPID DETECTION DF RIFAMPIN RESISTANCE IN MYCOBACTERIAL
PATIIOGENS: EVALUATION OF PCRüIIG-RIF ASSAY
Diana 1 w ' illiartll Lavnette Spring and Thomas P. Grilo
Molecular Biology Research Department, L.abontory Research l(ranch,
G WL Bansrn's Disease Center, School aí 1'etennan Medicine, Louisiana State
Unisersity, P.O. L3oz 25072, !latos Rouge, Louisiana 70894
Rifampm resvstance in Shrahucrmum lepra,. and lhrohrcrcnum
rubrrculons has bern aunbuted to missense rnutations whhin an 81 base pair
fragmant of Toa (Rd regias), encodmg the B'subunit of the DNA-dependent
RNA polymerasse. Using ihrs information, ave have deueloped and characterized
DN:\-based assays using heminested PCR and unis ersal heteroduplcx genenlor
(PCRUIIG-Rd) for the rapid detectam and nfampin-suscepnbih[y of
lhrohocrrnue, ruberculosrs and SAn,hacrermm lograr is clinicai specimens. In
a double-blurd study, 655 spumm speamens were obtained from individuais
suspected of having tuberculosis and analy eed for the presence of .t1 cubrrculons
and nfampinouscepnblity using the PCR UBG-Rif assay. Results of Chis study
demonstrated that the PCRUBG•Rif assay detected ali smear-positive, culturepositive
specimens containing Al. tonere:dos:s. In addition, 50% of smear•
negasse, culture-positive specimens for M. ruhrrculosa sere detected. This assay
also corrrctly iden[itied the rifampin suscepnbility of af.. tuhnculanr from 98% of
[hese specimens. AII specimens containing only nontuberculous mycobaaeria ar
no acid-fast bac illi wele neganve is the PCR UIIG-Rifassay. The presence of
,9f ruberculosrs is culture-negabve specimens from patients with previous culmrepositive
specimens bui receiving ant i -mberculosis therapy when specimens urre
obtained for PCRUIIG-Rif testing was detected by the use of this asssay. In
addition, PCR amplificados primers and conditions were modi fied tu specifically
amplify and desci the rifampin•suscepnbiliry of Sfycohoctermm leprur ia purified
DNA simples and crude dell lysates of skin biopsy specimens from lepromatous
Ieprosy patients. Even though, a small number of rifampin-resistant clinicai
specimens were available for analyses (no. = 4), the I'CRUIIG-Rif assay conectly
identilied the rifampin susceptibility of these specimens. These assays require
approximately sia hours to mo (posa sample preparados) and should prov ide rapid
tools for detection of the rifampin-resistant phenotype of these mycobacterial
pathogens direetly from clinicai :pecimens.
A.
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