INTERNATIONAL JOURNAL OF LEPROSY ^ Volume 65, Number 2 Printed in the U.S.A. (ISSN 0148-916X) Comparison of Bacillary Indexes in Slit-Skin Smears, Skin and Nerve Biopsies; a Study from Malawi' Jorg M. Ponnighaus, Christian Lienhardt, Sebastian Lucas, Paul E. M. Fine, and Jonathan A. C. Sterne The presence of acid-fast bacilli (AFB) in slit-skin smears of patients has long been considered as one of the cardinal signs of leprosy, sufficient on its own to establish the diagnosis (t. 7 ). In 1982, the World Health Organization (WHO) recommended the use of multidrug therapy (MDT) as standard treatment for leprosy and intro- duced an operational classification of lep- rosy distinguishing between paucibacillary (PB) and multibacillary (MB) forms based on the result of the slit-skin smears ("). This definition was revised in 1988 ("). Several authors have expressed concern about the fact that the decision to treat a pa- tient as PB or MB relies solely on slit-skin smear results since this examination is so dependent upon the availability and quality of slit-skin smear services (`. "). If smear services are not reliable, MB cases could be classified wrongly as PB, thus increasing the risk of relapse due to insufficient treat- ment. Concern also has been expressed about the difficulty of comparing the effects of MDT between populations and over time, given the changing criteria for classi- fication ( 3 . 5 ). An increase in case-finding activities within leprosy control programs (LCP) will, in general, lead to an earlier detection ' Received for publication on 16 October 1996. Ac- cepted for publication in revised form on 15 January 1997. = J. NI. Ponnighaus, Dr.Med., D.T.M.&II., D.T.P.H., Lepra, P. 0. Box 46, Chilumba, Malawi. C. Lienhardt, M.D., D.T.M.&H., M.Sc., Medical Research Council, P. 0. Box 273, Fajara, Banjul, The Gambia. S. Lucas, F.R.C.P., Professor, UMDS, Guy's and St. Thomas Medical and Dental School, Department of Histopathology, St. Thomas Hospital, Lambeth Palace Road, London SE I 7EH, U.K. P. E. M. Fine, V.M.D., Ph.D., Professor; J. A. C. Sterne, Ph.D., CDEU, Lon- don School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 71 IT, U.K. Reprint requests to Dr. J. M. Ponnighaus, Haut- klinik, Vogtlandklinikum Plauen, P.F. 39, 08525 Platten, Germany. of leprosy suspects at a stage when clinical signs are still relatively nonspecific and be- fore the onset of typical nerve damage, in- creasing the difficulty of the diagnosis. Skin biopsies can help greatly in the diagnosis and classification of leprosy in such indi- viduals ( 15 ). Taking biopsies is rarely feasi- ble under routine program conditions, how- ever, in particular if the leprosy control ac- tivities are integrated into the general health services. In fact, the vast majority of regis- tered leprosy cases are never biopsied and are diagnosed on clinical examination and on slit-skin smear results alone ( 5 ). Peripheral nerves have long been recog- nized as preferred sites of M ycobacterium leprue. The bacilli enter and then multiply inside the Schwann cells, inducing a greater or lesser tissue response dependent largely on the immune status of the patient (`. ' 3 ). There has been increasing interest over the past 20 years in the pathological findings in nerve biopsies (S. ' 3 ) and the question has arisen whether classifications based on skin and nerve biopsies are comparable (4.21). Several investigators have noted differ- ences in the bacillary load and the histo- pathological aspects in companion skin and nerve lesions ) These discrepan- cies have led some authors to question the usefulness and indication of skin and nerve biopsies for the classification and treatment of leprosy ( 9 ' 2 ). To investigate further the nature, fre- quency and implications of discrepancies between bacteriological findings in skin and nerve lesions, we have compared the results of slit-skin smears, skin biopsies and nerve biopsies in leprosy patients in Karonga Dis- trict, Malawi. MATERIALS AND METHODS Cases for this analysis were ascertained since 1984 within the Lepra Evaluation Project (LEP), a longitudinal study of the 211
Transcript
INTERNATIONAL JOURNAL OF LEPROSY^ Volume 65, Number 2
Printed in the U.S.A.(ISSN 0148-916X)
Comparison of Bacillary Indexes in Slit-Skin Smears,Skin and Nerve Biopsies; a Study from Malawi'
Jorg M. Ponnighaus, Christian Lienhardt, Sebastian Lucas,Paul E. M. Fine, and Jonathan A. C. Sterne
The presence of acid-fast bacilli (AFB) inslit-skin smears of patients has long beenconsidered as one of the cardinal signs ofleprosy, sufficient on its own to establishthe diagnosis (t. 7). In 1982, the WorldHealth Organization (WHO) recommendedthe use of multidrug therapy (MDT) asstandard treatment for leprosy and intro-duced an operational classification of lep-rosy distinguishing between paucibacillary(PB) and multibacillary (MB) forms basedon the result of the slit-skin smears (").This definition was revised in 1988 (").Several authors have expressed concernabout the fact that the decision to treat a pa-tient as PB or MB relies solely on slit-skinsmear results since this examination is sodependent upon the availability and qualityof slit-skin smear services (`. "). If smearservices are not reliable, MB cases could beclassified wrongly as PB, thus increasingthe risk of relapse due to insufficient treat-ment. Concern also has been expressedabout the difficulty of comparing the effectsof MDT between populations and overtime, given the changing criteria for classi-fication ( 3 . 5 ).
An increase in case-finding activitieswithin leprosy control programs (LCP)will, in general, lead to an earlier detection
' Received for publication on 16 October 1996. Ac-cepted for publication in revised form on 15 January1997.
= J. NI. Ponnighaus, Dr.Med., D.T.M.&II., D.T.P.H.,Lepra, P. 0. Box 46, Chilumba, Malawi. C. Lienhardt,M.D., D.T.M.&H., M.Sc., Medical Research Council,P. 0. Box 273, Fajara, Banjul, The Gambia. S. Lucas,F.R.C.P., Professor, UMDS, Guy's and St. ThomasMedical and Dental School, Department ofHistopathology, St. Thomas Hospital, Lambeth PalaceRoad, London SE I 7EH, U.K. P. E. M. Fine, V.M.D.,Ph.D., Professor; J. A. C. Sterne, Ph.D., CDEU, Lon-don School of Hygiene and Tropical Medicine, KeppelStreet, London WC1E 71 IT, U.K.
Reprint requests to Dr. J. M. Ponnighaus, Haut-klinik, Vogtlandklinikum Plauen, P.F. 39, 08525Platten, Germany.
of leprosy suspects at a stage when clinicalsigns are still relatively nonspecific and be-fore the onset of typical nerve damage, in-creasing the difficulty of the diagnosis. Skinbiopsies can help greatly in the diagnosisand classification of leprosy in such indi-viduals ( 15 ). Taking biopsies is rarely feasi-ble under routine program conditions, how-ever, in particular if the leprosy control ac-tivities are integrated into the general healthservices. In fact, the vast majority of regis-tered leprosy cases are never biopsied andare diagnosed on clinical examination andon slit-skin smear results alone ( 5 ).
Peripheral nerves have long been recog-nized as preferred sites of M ycobacteriumleprue. The bacilli enter and then multiplyinside the Schwann cells, inducing a greateror lesser tissue response dependent largelyon the immune status of the patient (`. ' 3 ).There has been increasing interest over thepast 20 years in the pathological findings innerve biopsies (S. ' 3 ) and the question hasarisen whether classifications based on skinand nerve biopsies are comparable (4.21).
Several investigators have noted differ-ences in the bacillary load and the histo-pathological aspects in companion skin andnerve lesions ) These discrepan-cies have led some authors to question theusefulness and indication of skin and nervebiopsies for the classification and treatmentof leprosy ( 9' 2 ).
To investigate further the nature, fre-quency and implications of discrepanciesbetween bacteriological findings in skin andnerve lesions, we have compared the resultsof slit-skin smears, skin biopsies and nervebiopsies in leprosy patients in Karonga Dis-trict, Malawi.
MATERIALS AND METHODSCases for this analysis were ascertained
since 1984 within the Lepra EvaluationProject (LEP), a longitudinal study of the
211
TotalBacterial index (BI)
>20
Slit-skin
smear
Skinbiopsy
Skinbiopsy
Skinbiopsy
Slit-skin
smear
Slit-skin
smear
Slit^Skinskin
biopsysmear
No.skin
lesions
212^ International Journal of Leprosy^ 1997
TABLE 1. Bacterial indexes in slit-skin smears and skin biopsies by numbers of lesionsin 197 newly diagnosed leprosy patients in Karonga District, northern Malawi.
risk factors for leprosy in the Karonga Dis-trict of northern Malawi ("). The entirepopulation was examined at their houses byclosely supervised leprosy control assis-tants (LCA), who had to complete a two-page general examination form for everyindividual seen. All individuals suspectedof having leprosy, and who had no historyof previous antileprosy treatment, were ex-amined by a medical officer (JMP). Slit-skin smears were taken from anyone withskin lesions which were considered as pos-sibly due to MB leprosy. Two smears wereusually taken from the earlobes and twofrom the lesion(s) and examined at projectheadquarters. The highest of the four bacte-rial indexes (BIs) was coded. The percent-ages of solids, fragments and granules werecoded if the BI was 2 or higher. Positivesmears of newly discovered leprosy pa-tients were shown to the medical officer bythe laboratory technicians. Skin biopsieswere obtained from more than 90% of allleprosy suspects newly found in the courseof the LEP, and strenuous efforts weremade to minimize errors in diagnosis ( 1 ').Most biopsies were taken from the most ac-tive part of a lesion using a disposable4-mm punch ( 00). Split-nerve biopsies wereobtained since 1984 a) if the only sign ofleprosy was an enlarged peripheral sensorynerve or b) if a suspect agreed to have biop-sies taken not only from his skin lesion(s)but also from an enlarged sensory nerve.All biopsy specimens were examined byone of us (SL) and the BIs in the skin andnerve specimens were counted in the sameway (oil immersion x1000).
For this analysis two groups of patientshave been formed: 1) new leprosy casesfrom whom a slit-skin smear and a skinbiopsy were taken at the same time. In caseof repeated smears and/or biopsies, wechose the first time that these two investiga-tions were performed together. 2) newly di-agnosed patients who had a skin and anerve biopsy taken at about the same time(within 6 weeks). In all of them split-nervebiopsies were taken from an enlarged sen-sory nerve after the purpose of the investi-gation had been explained to the patient indetail.
RESULTSData were available for the number of
skin lesions, slit-skin smear results and skinbiopsy results for 212 patients. As shown inTable I, among 61 patients with a single le-sion none were slit-skin smear positive andtwo had bacilli detected in skin biopsies. Incontrast. among 119 patients with four ormore lesions 34 (29%) versus 59 (50%) hadbacilli detectable in slit-skin smears or skinbiopsies, respectively. The latter differenceis statistically significant (p <0.01). For the212 patients, the mean BI was higher in theskin biopsies (0.8) than in the smears (0.5).The difference was statistically significant(t = 4.99; p <0.001).
There were 47 patients for whom bothskin and nerve biopsy results were avail-able. Their mean age was 39.6 years (42.4for females; 37.1 for males). Twenty pa-tients had one cutaneous lesion, 5 patients(14%) had two or three lesions and 22(61%) had four or more lesions. The mean
n-6
n-2
n-7
n-26
n-2
65, 2^Ponnighaus et al.: Ms of Smears, Skin and Nerves^913
6
5
4
Mean 3
B
2
0II^TT^BT^BB
^BL^
LL
classification based on skin biopsies
THE FIGURE. Mean bacterial indexes in skin andsplit-nerve biopsies by histopathological classificationbased on the skin biopsies from 44 patients in theKaronga District of northern Malawi. ■ = Skin; E9 =nerve; n = number of patients in each classification; II= indeterminate leprosy.
13I was higher in the nerve biopsies (2.4)than in the skin biopsies (1.1) (t = 5.92; p<0.001). The distribution of the Bls in skinand nerve biopsies is shown in Table 2. In20/47 (43%) patients the Bls were identicalin skin and nerve biopsies, in 26 (55%) theywere higher in nerve biopsies, and in onlyone patient (2C/0) it was higher in the skinbiopsy.
The Figure shows the mean Bls in skinand nerve specimens by the histopathologi-cal classification based on the examinationof the skin biopsies ( 2"). As can be ex-pected, given that high Bls in skin biopsies
will lead to a classification of BB to LL lep-rosy, the difference between nerve and skinbiopsy Bls appears greatest for the PB cases[II (indeterminate), TT and BT leprosy].
DISCUSSIONThe percentages of slit-skin smears and
skin biopsies with a positive BI increasedwith the number of skin lesions recorded(Table 1). This correlation provides a mea-sure of quality control for slit-skin smearand skin biopsy readings. The histopatholo-gist was not aware of the number of skin le-sions found, neither did the laboratory tech-nicians know the number of lesions. Theprobability of finding bacilli in a patientwas greater in the skin biopsies than in theslit-skin smears. One explanation might bethat, as suggested by Ridley ( 1 '), bacilli inearly PB disease are more common in thedeeper tissue (particularly nerves) than inthe superficial dermal strata, and thus arelikely to be excluded from slit-skin smears.
If either the smear- or biopsy-positive BIwere to be regarded as evidence of MB lep-rosy, then 28/177 (15.8%) patients whowere suspected on clinical grounds to haveMB leprosy would have been "wrongly"classified as PB relying on slit-skin smearsonly.
Several studies have compared bacterio-logical loads in skin and nerve biopsies. Ina study of 50 leprosy patients undergoingreconstructive surgery in Bombay, Antia, etal. found bacilli in 12% of skin biopsies andin 60% of nerve biopsies ('). Among 30randomly selected patients attending anoutpatient clinic in Chengalpattu (India),Srinivasan, et al. found that nerve lesionscontained more bacilli than skin lesions in
TABLE 2. Bacterial indexes in skin and nerve biopsies in 47 newly diagnosed leprosypatients in Karonga District, northern Malawi.
six of them ("). In a study of 42 untreatedleprosy patients in Nepal, Ridley and Rid-ley found that the 13Is (after correction forthe size of granuloma) were generallyhigher in nerves than in skin biopsies (").According to Figure 6 in their publication,differences in the BIs were negligible at thetwo ends of the spectrum but marked forBT to BL leprosy. Negesse, et al. reported astudy in which 220 untreated suspected lep-rosy patients in Ethiopia underwent parallelskin and nerve biopsies. In 46/161 con-firmed cases the bacillary load seems tohave been considerably higher in nervesthan in the skin ("). Similary, in a study of60 consecutive untreated leprosy patientsattending a clinic in Chandigarh (India), theaverage BI was higher in nerve biopsiesthan in skin biopsies of the 28 BL patientsbut not in seven LL leprosy patients (').
Several hypotheses can be formulated asto why the BIs are higher in nerve than inskin biopsies: a) the difference may simplybe due to technical reasons: nerve biopsiesare always taken from the best place (an en-larged stretch near the skin) while skinbiopsies are usually taken from the mostconvenient lesion which may not always bethe site with the highest density of bacilli;b) M. leprae first settle in nerves and thedifferences in the BIs simply reflect thelonger stay of the bacilli in the nerves thanin the skin; c) M. leprae reach nerves andskin at the same time but can survive andmultiply more easily in nerves than in skin,perhaps because within nerves they areshielded from the cell-mediated and/or hu-moral immune system; and d) dead M. lep-rae are removed more easily from the skinthan from the nerves.
It might be possible to refute the first hy-pothesis by comparing nerve biopsies withseveral skin biopsies from the same patient.If the BIs in nerves are higher than in allskin specimens, the first hypothesis wouldbecome unlikely. The last hypothesis couldbe tested by comparing percentages ofsolid-staining (viable) bacilli, which shouldbe higher in the skin than in nerves if deadbacilli are removed more efficiently fromthe skin than from the nerves. On the otherhand, percentages of solids should be simi-lar in the nerves and in the skin if the sec-ond or third hypothesis were true. Both hy-potheses b) and c) would imply that leprosy
does progress from PB to MB withinnerves. If, however, bacilli could be foundin nerve biopsies but not in several skinbiopsies from the same patients, the secondhypothesis, that bacilli initially invadenerves, would be the most likely one.
Given that in 32/47 (68%) of biopsiednerves bacilli could be detected, our find-ings suggest that at least in the absence ofreliable slit-skin smear services a leprosypatient should be treated as a MB patient ifa definitely enlarged peripheral nerve isfound. On the other hand, where slit-skinsmear services exist, relapse rates amongPB patients are already so low, using thepresent definition of PB leprosy, that itwould appear to be unnecessary to usemore stringent criteria for PB leprosy ( 1 ').
SUMMARYData analyzed in this paper were col-
lected within the framework of the LepraEvaluation Project, an epidemiologicalstudy of leprosy in Karonga District, north-ern Malawi. For 212 patients informationon the number of skin lesions, slit-skinsmear and skin biopsy results were avail-able. Among 61 patients with a single le-sion none were slit-skin-smear positive andtwo had bacilli detected in skin biopsies. Incontrast, among 119 patients with four ormore lesions 34 (28.6%) versus 59 (49.6%)had bacilli detectable in slit-skin smears orskin biopsies, respectively.
In a further 47 patients skin biopsy re-sults could be compared with split-nervebiopsy results. In 20 of 47 patients the bac-terial indexes (BIs) were identical in skinand nerve biopsies, while in 26 of 47 pa-tients the BIs were higher in nerve than inskin biopsies. This difference, which is con-sistent with several other studies in the lit-erature, provides an insight into the patho-genesis of leprosy.
RESUMENLos datos analizados en este trabajo fueron colecta-
dos dentro del marco del Proyecto de EvaluaciOn de IaLepra, on estudio epidemiolOgico de la lepra en el Dis-trito de Karonga, en el forte de Malawi. Sc pudo con-tar con los datos sobre el nnmero de lesiones en Ia piel,las baciloscopias en linfa cutanea y los resultados delas biopsias en 212 pacientes. Entre los 61 pacientescon una soli lesiOn, ningono tuvo bacilos en linfacuLinea mientras clue dos tuvieron bacilos en las biop-sias de piel. En contrasts, de los 119 pacientes con 4 6
65, 2^Ponnighaus et al.: Bls of Smears, Skin and Nerves^215
alas lesiones, 34 (28.6%) tuvieron bacilos en linfacutzinezt y 59 (49.6%) tuvieron bacilos en la biopsia depiel.
En un grupo adicional de 47 pacientes, los resulta-dos de las biopsias de piel pudieron compararse conlos resultados de las biopsias tornados de los nerviosafectados. En 20 de 47 pacientes, los indices bacteri-anos fueron identicos en las biopsias de piel y denervios, mientras que en 26 de 47 pacientes, los BIsfueron mayores en las biopsias de nervio que en lasbiopsias de piel. Esta diferencia, la cual es consistentecon otros estudios de Ia literatura, es un aspect() queclebe estudiarse a fondo para comprender Ia patogene-sis de la lepra.
RÉSUMÉLes donnees analysees dans cet article ont ete re-
coltees dans le cadre du "Lepra Evaluation Project",une etude epidemiologique de la lepre dans le Districtde Karonga, dans le nord du Malawi. L'informationsur le nombre de lesions cutanees, les resultats desfrottis et des biopsies cutanees etaient disponibles pour212 patients. Aucun des 61 patients porteurs d'une le-sion unique n'etait positif a [examen du frottis cutane,et pour deux on a detecte des bacilles a Ia biopsie cu-tanee. Par contre, parmi les 119 patients avec 4 lesionsou plus, 34 (28.6%) et 59 (49.6%) avaient des bacillesdetectables respectivement dans les froths cutanes et ala biopsie.
Chez 47 patients supplementaires, on a pu comparerles resultats d'une biopsie email& et d'une biopsie denerf. Chez 20 des 47 patients, les indices bacteriens(IB) etaient identiques dans les biopsies de peau et denerfs, tandis que chez 26 des 47 patients, les IB etaientplus eleves dans les biopsies de nerfs que dans lesbiopsies de peau. Cette difference, qui est coherenteavec d'autres etudes de la litterature, foumit certainesinformations quoin a Ia pathogenese de Ia lepre.
Acknowledgment. We thank the leprosy patientsfor their cooperation and the leprosy control assistantsfor their dedication to the project. The following agen-cies provided financial support: LEPRA (The BritishLeprosy Relief Association), ILEP (The InternationalFederation of Anti-Leprosy Organizations) and IMM-LEP (The Immunology of Leprosy component of theWHO/TDR). JMP received a grant from the RobertCochrane Fund for Leprosy to travel to Bamako toprepare this paper together with CL. We thank theMinistry of Health of the Republic of Malawi for theirinterest in the project and permission for publication.
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