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INTERNATIONAL. JOURNAL OF LEPROSY^ Volume 64, Number 3Printed in
the U.S.A.
(ISSN 0145-916X)
Microbial Colonizers in Leprosy Skin Ulcers andIntensity of
Inflammation'
A. Willem Sturm, Bushra Jamil, Keith P. W. J. McAdam, Khurram Z.
Khan,Shaheen Parveen, Thomas Chiang, and Rabia Hussain 2
IVIrcobacterium leprae is the causativeagent of leprosy, a
disease in which theclinical presentation varies with the
pa-tient's immune response (' 5 ). Skin lesionsare seen at the
lepromatous as well as thetuberculoid poles of the disease
spectrum.Although the underlying immune pathologyof the lesions is
different across the spec-trum, chronic ulceration can develop
atboth poles of leprosy ("). These ulcers be-come infected with
bacteria other than M.hp/we and show recurrent episodes of
cel-lulitis of the surrounding tissues as well asosteitis of
underlying bone. This, in turn, re-sults in deformities which
disable patientsand contribute to the stigma associated withthe
disease ( 1 ). Early and adequate treat-ment of these episodes of
inflammationcould interrupt this sequence of events.
There are several reports (") whichshow that chronic skin ulcers
in leprosy pa-tients are colonized with gram-positive rodswhich
have not been observed in chronicskin ulcers of different etiology.
However,there are no studies which compare cultureresults of
leprosy-related and other skin ul-cers in the same population.
Also, no at-tempt has been made to relate colonizingspecies with
inflammation.
' Received for publication on 24 October 1995; ac-cepted for
publication in revised form on 12 March1996.
= A. W. Sturm, M.D., Ph.D.; B. Jamil, N1.11.13.S.; K.Z. Khan.
M.Sc., D.Bact.; S. Parveen, B.Sc., NI.Sc.; R.Hussain. B.Sc.
(lions). M.Sc., Ph.D., M.R.C. (Path),Department of Microbiology,
The Aga Kahn Univer-sity, Karachi, Pakistan. K. P. W. J. MacAdam,
M.A.(Nat.Sc.), M.R.C.P., F.R.C.P., London School of Hy-giene and
Tropical Medicine. London, U.K. T. Chiang,M.13.13.S., M.Sc.,
C.T.M., Marie Adelaide LeprosyCenter. Karachi. Pakistan.
Reprint requests to Prof. A. W. Sturm. Departmentof
Microbiology, Medical School, University of Natal,P. 0. Box 17039,
Congella 4013, South Africa.
This study compares organisms presentin leprosy skin ulcers with
those present inskin ulcers of different etiologies, and
cor-relates this with the severity of inflamma-tion. Susceptibility
patterns of these organ-isms are reported and the possible
contribu-tion of antimicrobial treatment towardprevention of
deformity is discussed.
PATIENTS AND METHODSPatients were recruited from the popula-
tion attending Marie Adelaide LeprosyCentre (MALC) in Karachi,
Pakistan. Allpatients with ulcers, from whom informedconsent was
obtained in the period betweenOctober 1991 and October 1992, were
en-rolled. Patients with nonleprosy skin ulcersof varying durations
and not on antimicro-bial treatment for at least 2 weeks were
re-cruited as a control group at the surgeryclinic of Civil
Hospital, Karachi.
Inflammatory scores (IS) were measuredbased on the method of
Ehrenkranz, et al.( 7 ). This included amount and character
ofdrainage, extent of cellulitis, presence offoul odor and presence
of necrosis. Obser-vations were scored as follows: drainage
ondressing = 0; minimal, moderate and co-pious drainage in ulcer
bed = 1, 2 and 4, re-spectively; serous, sanguinous,
sanguino-purulent and purulent drainage = 0, 1, 3 and4,
respectively; widest cellulitis width of< 1 cm, > 1 to 2 cm,
> 2 to < 3 cm, > 3 to 4 cm = 0, I, 2, 3 and 4,
respec-tively; no foul odor = 0; foul odor = 1; nonecrosis = 0;
necrosis in ulcer margin = 1;necrosis in ulcer margin and base = 2.
The ISvalue is the sum of these scores and rangesfrom 0 to 15.
Ulcers were sampled by slightly modify-ing the
irrigation-aspiration method ( 7 ). Inbrief, at least 1 ml of
sterile 0.85% sodiumchloride without preservatives is
irrigatedunder the margin of the ulcer with a needle-less 5-ml
syringe. Excess fluid is removed
274
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64, 3^.S'turin, et al.: Microbial Flora of Leprosy
Ulcers^275
TABLE I.^Characteristics of skin ulcers in patients with and
without leprosy.
Leprosy patients (N = 55) Control patients (N = 18)
NUE ratio 40/15 15/3Mean (range) duration of ulcer (mos.) 10.3
(0.1-180) 7.5 (0.5-72)Character of ulcer (S/I) ratio)' 28/27
12/6Location and no. of ulcers
/lands 4 3Arms 3 1Feet 46 10 (p = 0.02)Legs 2_ 3Other 0 1
Mean no. of microbial species/sample 3.5 4.2
S/I) ratio = Superficial/deep ulcer ratio.
with a sterile gauze pad. The ulcer marginsare massaged
circumferentially with a ster-ile cotton swab. After this, a second
irriga-tion is performed using I ml of saline and anew syringe.
Without removing the excessfluid, the ulcer margin is massaged
again af-ter which the ulcer base and margins areswabbed with a
sterile cotton-tipped swabwhich is immediately wrung into I ml
ofbrain-heart infusion (BHI) broth for stain-ing and culture.
Smears of broth aspirate suspensionswere stained by means of
Gram as well asKinyoun stain. Cultures were performed ona 7% sheep
blood agar, cysteine lactoseelectrolyte-deficient medium (CLED),
anisovitalex enriched chocolate blood agarand a nalidixic acid (10
mg/I) mecillinam (1mg/I) bilayered 7% sheep blood agar. Allplates
were incubated at 37°C; the latter twoin a CO, incubator. In
addition, a Sabouraudagar with 10 mg/I gentamicin and a Colum-bia
agar with 2% Tween 80 and nalidixicacid 10 mg/1 were inoculated and
incubatedat 30°C. Two sets of Lowenstein-Jensenslants incubated for
12 weeks at 30°C and37°C, respectively, were used for the cul-ture
of mycobacteria. The presence ofanaerobes was evaluated on
microscopy ofGram-stained smears, based on multiplicityof
morphological forms, intracellular loca-tion, pale staining of gram
negatives andsize as well as characteristic appearance.
Identification of organisms was done bymeans of established
methods ("I) andbased on the latest edition of Berger's Man-ual of
Deteminative Bacteriology
Minimal inhibitory concentrations (MIC)for antimicrobial agents
were measured by
means of the agar dilution method on iso-sensitest agar with an
inoculum of 10 1cfu/spot ( 11 ).
Mycobacterial culture media were ob-tained from DIFC0
Laboratories, Detroit,Michigan, U.S.A.; all others were
obtainedfrom Oxoid, Basingstoke, Hampshire, En-gland.
Fisher's exact test was used to determinethe significance of
differences between lep-rosy and control ulcers. Mann-Whitney
andKendall rank order correlation coefficienttests were used for
comparison/correlationbetween groups.
RESULTSFifty-five patients with leprosy skin ul-
cers and 18 control patients were enrolledin the study. Five of
the study patients hadthe tuberculoid form of leprosy, 13 the
lep-romatous and 37 had borderline leprosy.Thirty-nine patients,
from whom a reliablehistory could be taken, had suffered fromthe
disease for many years (mean duration14.2 years). At the time of
enrollment. 30 pa-tients were not on any treatment, 10 were
ondapsone, I was on clofazimine, and 14 wereon the multidrug
therapy (MDT) regimen ofclofazimine, dapsone and rifampin.
Onlythree patients had never been on anti leprosy ,medications; all
the others had been treatedpreviously with different regimens.
Ulcer characteristics. Table I com-pares the characteristics of
the skin ulcersof leprosy patients with those of the controlgroup.
In leprosy patients, ulcers were pres-ent predominantly on the feet
comparedwith the nonleprosy patients. A higher per-centage of
leprosy ulcers were deep and of
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276^ International Journal of Leprosy^ 1996
TABLE 2. Microbial species grown from skin ulcers of 55 leprosy
patients and 18 non-leprosy patients.
Leprosy patients (N = 55) Control patients (N = I K)
Staphylococcus aureus 30 6Coagulase-negative staphylococci 19
12Streptococcu.s . pvogenes (1311S group A) 16 3Beta-hemolytic
streptococcus 19 0
Group G (1311S group G)'Other streptococci 10 6Enterococcus
species' K 7Arcanobacterium haemolytictme 10 0Unidentified
branching rods' 7 0Corynebacterium species 40 9Bacillus species' 2
9Proteus mirabilis 7 4Other Etiterobacteriacene 8 5Pseduomonas
aeruginosa 7 2Other Pseudomonas 9 6Acinewbacter calcoaceticus 4
1Other gram-negative rods 5 5Conduit, species 0 2Total 201 72
Present only in leprosy ulcers.h Present in significantly higher
number (p = 0.04) in control ulcers.Present in significantly higher
number (p = 0.03) in control ulcers.
longer duration than the control ulcers.Seven of the 18
nonleprosy patients suf-fered from post-traumatic ulcers, 6 had
un-derlying diabetes mellitus, and 1 ulcer fol-lowed an insect
bite; definite etiology couldnot be confirmed in 4 cases.
Ulcer microflora. When the microbialflora was compared in the
study and controlgroups, no difference was found in the meannumber
of microbial species per sample be-tween the two groups (Table 1).
Table 2shows that leprosy patients had significantlymore
beta-hemolytic streptococci. Group Gstreptococcus was exclusively
present in ul-cers of leprosy patients (9.7% of
isolates).Gram-positive rods accounted for 29.3% ofall isolates
from the ulcers of leprosy pa-tients and 25% in the control
patients. Thedistribution of species from the
generaCorynebacteriutn and Bacillus was differ-ent in the two
patient groups. In the leprosygroup, corynebacteria and branching
rodsaccounted for 97% of gram-positive bacilliand Bacillus species
constituted only 3% ofthe group. In the control patient
group,Bacillus species formed 50% of gram-posi-tive rods; the rest
were corynebacteria (p =0.03). In the leprosy group, one third of
thegram-positive bacilli were branching rods.None of these were
acid fast. Ten strains
were identified as Arcanobacteritan haemo-lytic:um; the
remaining 7 could not he iden-tified with the methods used.
Skin ulcers of patients in the controlgroup grew yeast (two
samples) and highernumbers of species of enterococci (p =0.04) and
Enterobacteriaceae (p = 0.08)compared to the leprosy group.
Moreover,no branching rods were isolated from thecontrol ulcers.
Anaerobes (on microscopy)were found with the same frequency in
thestudy (18%) and in the control (17%)groups. No mycobacteria
could be culturedin either group.
Patients with leprosy had an inflamma-tory score (IS) which
varied between 0 to13 (median of 2; mean score of 3). This
wassignificantly (p = < 0.001) lower than in thecontrol group
which showed a variation inIS between I and 10 (mean 5.5; median
6).No correlations were found between the ISand age of the
patients, duration of ulcers,or type and duration of leprosy. The
fourcontrol group ulcers, which etiology couldnot be ascertained,
had a median IS of 1.5with a mean of 3.25 (range 1-9); this valueis
similar to that in the leprosy group.
In the leprosy group, the number of spe-cies per sample appeared
to be directly re-kited to the IS (Fig. I); no such correlation
-
14
2
8
6
4
2EE5t.1E.
2
0
-2
64, 3^Sturm, et al.: Microbial Flora of Leprosy Ulcers^277
0
0
0
o 00
OO
O 0 0 oO o
Q 0 0 0 0 0 00 0 0 0 0 0 0
O •
-1^0^1^2^3^4^5^6^7^8^9
NO. OF ISOLATES / ULCER
Ito. I. higher inflammatory scores appear to beassociated with
higher numbers of isolates from thesample.
0
00
00O O
0
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278^ International Journal of Leprosy^ 1996
TABLE 3. Susceptibility (MIC in pg/m1) of 20/ bacterial strains
isolated from skinulcers of 55 leprosy patients and seven strains
from skin ulcer.s . of 18 control patients.
Drug' RangeMean MIC MIC,„ MIC
, o,,Leprosy Controls Leprosy Controls Leprosy Controls
P 0.25-64 1.1 1.1 64 >64 >64 >64TRP 0.25-64 34 37.22 32
64 >64 >64OFX 0.001-2 0.3 0.4 0.125 0.25 0.5 IC 0.25-64 19.6
38.4 8 32 64 64
P= Penicillin, A= ampicil lin, CLOX = cloxacillin, CTX =
celotaxime, R = rifampin, F = erythromycin, SMX= sulfamethoxazole,
TMP = trimethoprim, OFX = ofloxacin, C = chloramphenicol.
piing technique used in this study has beenshown to he in
decubitus ulcers as represen-tative for culture as a biopsy ( 7
).
We observed that the microflora of skinulcers in leprosy
patients was different fromthe flora of nonleprosy skin ulcers.
Bothleprosy and control patients were from sim-ilar socioeconomic
backgrounds and envi-ronmental differences are, therefore,
un-likely to be accountable for those differ-ences. The significant
observation was theisolation of gram-positive branching bacilliin
leprosy ulcers, more than half of whichwere identified as A.
haemolyticum. Thisorganism has not been reported previouslyfrom
leprosy ulcers. It has been describedas a case report in relation
to a post-trau-matic skin ulcer and as a causative agent
ofoutbreaks of tonsillo pharyngitis ( 9). GroupG beta-hemolytic
streptococci also were ex-clusively isolated from leprosy ulcers.
Cer-tain species from the genus Corynebac-terium have been
described as unique forleprosy patients ('). The branching rodsfrom
our study patients which remainedunidentified could be similar to
these lep-rosy-derived corynebacteria. Cell-wall com-position and
DNA analysis of these strainsmay provide the answer ( 2 . ").
It has been postulated that the presence ofcertain
grain-positive rods in leprosy ulcersis explained by their
antigenic relatednessto M. leprae "). Tolerance to commonantigens
perhaps allows these organisms tocolonize the leprosy ulcers. This
would im-ply that all of the isolates which are unique
to leprosy ulcers may share some antigenicfeatures with M.
leprae. At least two differ-ent organisms have been reported with
thischaracteristic C• 13 ) hut neither one has beenclassified
officially. From the present studywe may postulate that A.
haemolvticwn,unidentified branching rods, and group Gstreptococci
probably also belong to thisgroup. One would expect differences in
col-onization with these organisms at the polesof the spectrum of
leprosy depending on thepatient's immune status. We could not
es-tablish such a relationship because of thesmall numbers of
patients with tuberculoidand lepromatous leprosy. To resolve the
is-sue of shared antigens, a comparative studybetween these
organisms and M. leprae willbe required.
Isolation of group G streptococci fromleprosy patients only
could reflect cross-infection in the health care facility fromwhich
the patients were recruited. The samemay be true for A.
haemolyticum and otherbranching rods. In such a situation,
therewould be a clustering of cases in time.However, these
organisms were isolatedthroughout the year. Also, if spread
frompatient to patient or from health care workerto patient was a
major factor, one would ex-pect to find the more virulent S.
(litmus andgroup A streptococci. Hence, nosocomialoutbreaks cannot
be held responsible forour observations.
Another possible reason for the differ-ences in colonization of
skin ulcers of lep-rosy and control patients could be the
selec-
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64, 3^Sturm, et al.: Microbial Flora of Leprosy Ulcers^279
tion of organisms resistant to the drugs usedfor the treatment
of leprosy. Therefore, dap-sone (sulfonamide) and rifampin
resistancewas examined. All branching rods andgroup G streptococci
were resistant to sul-fonamides hut so were all of the other
or-ganisms in the leprosy as well as the controlgroup. Resistance
to rifampin showed amore complicated picture. There was a
sig-nificantly higher level of resistance amongdifferent groups of
bacterial isolates fromleprosy patients, including group G
strepto-cocci. This difference is, however, based ona broad range
of MIC values and not onhigher MICs for all isolates. The
branchingrods were fully susceptible as were 15 outof the 19 group
G streptococci; the other 4showed high-level resistance. These
fourwere responsible for the high mean MICand highMIC )) values.
Susceptibility testsfor clofazimine were not performed. Al-though
resistance to the antileprosy drugswas found with higher frequency
in the lep-rosy group, it does not provide an explana-tion for the
observed difference in coloniz-ing species. The presence of
drug-suscepti-ble strains (branching rods) cannot havebeen due to
the selective pressure of the an-tibiotics used.
Leprosy ulcers showed a low mean in-flammatory score (IS) as
compared to thecontrol group, the IS of the latter being
inaccordance with the findings of others ( 7 ).The presence of
pyogenic streptococci to-gether with S. aureus resulted in a lower
ISthan either of these separately, while thepresence of A.
haemolyticum or unidenti-fied branching rods together with
faculta-tive gram-negative bacilli enhanced inflam-mation. The
mechanism of these bacterialinteractions is unclear and warrants
furtherstudy. If a high IS is related to the develop-ment of
cellulitis and osteitis of adjacenttissue, leading to systemic
disease and de-formity, regular IS evaluation with
timelyintervention could prevent this. Based onobservations of this
study, the aim shouldhe continuous ulcer care to reduce the num-ber
of colonizing bacteria and antimicrobialtreatment when there is a
rise in the inflam-matory reaction. The number of
colonizingorganisms also can be kept low by applyingdisinfecting
dressings, while the choice ofantimicrobial drugs should focus on
entero-bacteria as well as pyogenic cocci. A com-
bination of a fluorinated quinolone withpenicillin would be
adequate. However, theefficacy of such an approach has yet to
beestablished.
SUMMARYThe microflora of 55 patients with lep-
rosy skin ulcers was studied and related to aweighted
inflammatory score (IS). The con-trol group consisted of 18 ulcers
with dif-ferent underlying pathology. Leprosy ulcerswere
characterized by the exclusive pres-ence of two types of branching
gram-posi-tive rods; a particular interesting proposal isthat
Mycobacterium leprae share commonantigens with these unusual
"leprosy ulcerassociated" organisms and group G beta-hemolytic
streptococci. In the leprosygroup, corynebacteria and branching
rodsaccounted for 97% of gram-positive bacilliand Bacillus species
constituted only 3%.In the control group, B. species formed 50%of
gram-positive rods; the rest werecorynebacteria (p = 0.03). In the
leprosygroup, one third of the gram-positive bacte-ria were
branching rods; none of them wasacid fast. Ten of them were
identified as Ar-canobacterium haemolyticum, and the re-maining 7
could not be identified. The IS ofleprosy patients was lower than
in the con-trol group. The presence of more than twospecies of
facultative or aerobic gram-neg-ative rods or single species of
pyogenicgram-positive cocci correlated with a highIS. The presence
of two or more differentpyogenic cocci resulted in a lower IS.
Fur-ther studies into the nature of leprosy-unique organisms as
well as the inflamma-tion inhibition factors in mixed infectionsare
warranted. It is recommended that man-agement of ulcers should
consist of the ap-plication of local disinfection and
earlytreatment of episodes of inflammation witha combination of
fluoroquinolone and peni-cillin.
RESUMENSe estudió la microOora de las Meeras de piel de 55
pacientes con lepra y se relacion6 con el registro delpeso
inflamatorio (1)1). Li grupo control cstuvo confor-mad() por I8
pacientes con tileeras de patologias diver-sas no leprosas. Las
Mceras de la lepra se caraeteri-zaron por la exclusiva presencia dc
2 tipos de baci losramiticantes Gram-positivos. Se propone que
My-
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280^ hitemational Journal of Leprosy^ 1996
co/mete/1/mi /eprae comparte antigenos commies conestos
organismos asociados con las dIceras de la lepray con estroptococos
beta-hemoliticos del grupo G. Enel grupo de lepra las
corinebacterias y los bacilos ram-incomes constituyeron el 97% de
los bacilos Gram-positi vos y las especies del genero Bacillus sólo
el 3%.En el grupo control, las especics de Bacillus fOrmaronel 50%
de los bacilos Gram-positivos, el resto fueroncorinebacterias (p =
0.03). l'.11 el grupo de lepra, tm ter-cio de las hacterias
Gram-positivas fueron bacteriasramificantes; ninguna de ellas fue
acido-resistente.Diez de ellas fueron identificadas como
Arcanobac-terium haemolyticum y los 7 restantes no
pudieronidentificarse. El PI en los pacientes con lepra Inc
Indsbajo que el PI en el grupo control. La presencia de Indsde 2
especies de hacilos facultativios o acrobicosGrain-ncgativos o do
especies tinicas de corns pió-genus Gram-positivos correlaciona con
un P1 elevado.Se requieren nuis estudios sobre la naturaleza de
losorganismos asociados a la lepra y sobre los factores
in-hibitorios de inflamación en las infecciones mixtas.
Serecomienda que el manejo de las Ulceras incluya Iadesinfección
local y el tratamiento temprano de losepisodios de inflamación con
una combinación de fiu-oroquinolona y penicilina.
RÉSUMÉ
On a étudie la microflorc de 55 patients avec des ul-ceres
cutanes lépreux, en relation avec un score in-flammatoire (SI)
pondéré. Le groupe temoin se coin-posait de 18 ulceres avec
inverses maladies sous-ja-centes. Les ulcéres lépreux se
caractérisaient par hipresence exclusive de deux types de baguettes
nutle-t -lees gram-positives; une proposition
particuliérementinteressante est que Mycobacterium leprae portage
desantigénes communs avec ces organismes inhahituels"associes aux
ulcéres lépreux" et des streptocoquesbeta hémolytiques du groupe G.
Dans le groupe lepre,les corynebacteries et les baguettes ramétiées
comp-taient pour 97% des hacilles gram positifs et Bacillusspecies
seulement pour 3%. Dans le groupe temoin, B.species formait 50% des
baguettes grain positives; lereste emit des coryne bacteries (p =
0.03). Dans legroupe lepre, un-tiers des bactéries grain positives
emitdes gahuettes raménées; aucune d'entre elles ne rCsis-tait a
l'acide. Dix &entre elles ont été identitiées entant que
Anyozobacteriton haemolvticutm et les 7autres Wont pu étre
identifiées. Le SI des patientslépreux était plus foible que cent'
du groupe temoin.La presence de plus de deux especes de
baguettesgram negatives facultatives ou aérobiques on dunesettle
espéce de coques gram positifis pyogénes emitassocie 3 un SI élevé.
La presence de deux coques pyo-geniques differents ou plus
resultait dans tin SI plusfoible. Des etudes ulterieures se
rapportant a la naturede ces organismes uniques it Ia lepre ainsi
que stir lesfacteurs d'inhibition de [inflammation dans les
infec-tions mixtes vont titre féalisées. On recommande que
le traitement des ulceres CoIlsiste en l'applicationdune
desinfection locale ainsi que le traitment precocedes episodes d'
[illumination par une combinaison defluoroquinolone et de
penicilline.
Acknowledgment. 'this study was financiallysupported by LEPRA,
the British Leprosy Relief As-sociation, through a grant to
AWS.
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