R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c in a T r o p i c a l 2 4 ( 4 ) : 2 3 1 - 2 3 4 , o u t - d e z , 1 9 9 1
P A R A S IT O L O G IC A L D IA G N O S IS O F M U C O C U T A N E O U S
L E IS H M A N IA S IS D U E T O
LEISHMANIA B. BRAZILIENSIS
I N B O L IV IA
L a u r e D im ie r -D a v id , C h r isto p h e D a v id , P ie r r e R a v isse , R e n a to B u st illo s, S u sa n a R e v o llo , P h ilip p e L y è v r e , M a r u sc h k a M u n o z ,
F e r n a n d o V a r ga s a n d J e a n -P ier r e D e d e t
SUMMARY
P a r a s ito lo g ic a l d ia g n o s is , u s in g s ta n e d s m e a r s , c u ltu r e a n d p a th o l o g i c a l e x a m in a tio n o f b io p s y , w a s s tu d i e d in 1 4 6 p a ti e n t s in fe c te d w ith m u c o c u ta n e o u s le is h m a n ia s is , in B o li v ia a n d P e r u . T h e m o s t e ffic ie n t p a r a s i te d e te c tin g te c h n iq u e a p p e a r e d to b e th e s m e a r e x a m in a tio n in c u ta n e o u s le s io n s (3 3 % p o s itiv e ) a n d th e p a th o lo g y in c a s e o f m u c o u s le s io n s (2 8 % p o s i tiv e ). In b o th , c u ta n e o u s a n d m u c o u s le s io n s , th e p a r a s i te s w e r e f o u n d m o s t f r e q u e n t l y in o l d le s io n s . K e y - w o r d s : M u c o c u ta n e o u s le is h m a n ia s is . Leishmania b. braziliensis. D ia g n o s is . P a r a s ite . B o li v ia . P e r u .
M u c o c u ta n e o u s le ish m a n ia sis due to
Leishmania braziliensis braziliensis
Vianna 1911, is endemic in a large part of the American continent8. In Bolivia, it represents a public health problem because o f its individual implications and socio- economical consequences on development of various parts o f the country2A positive diagnosis determines o f parasites patient treatment. It is based on direct evidence in the lesions, which represents certainty o f the disease.
The objective of this document is to present the results o f
L.b. braziliensis
isolations using different techniques, in patients affected with primary cutaneous lesions or with mucosal secondary dissemination.Instituto Boliviano de Biologia de Altura, c/o Embajada de Francia and Servicio de Otorrinolaringologia, Hospital das Clínicas, La Paz, Bolivia. Unité d 'Histopathologie, Institut Pasteur, Paris, France.
A d ress to correspondence: Dr. Jean-Pierre Dedet, Laboratoire d 'Ecologie Médicale et Parasitologie Parasitaire, Annexe de la Faculté de Médecine, 163 rue Auguste Broussonet, 34000 Montpellier France.
Recebido para publicação em 01/07/91.
MATERIAL AND METHODS
The study was realized within a sample o f 146 patients which were seen at the parasitology department of the Instituto Boliviano de Biologia de Altura (La Paz, Bolivia) between 1988 and 1990. They originated from the endemic areas neighbouring La Paz (Yungas, Alto Beni and Beni in Bolivia, Puno and Madre de Dios departments in Peru).
For each patient, the search for parasite was simultaneously conducted using the three following techniques:
1. smears made with material obtained from scratching the cutaneous lesion periphery or by imprints of biopsy section in case o f mucous lesion. The smears were fixed by methanol and Giemsa stained; 2. culture inoculation on biphasic NNN medium containing Schneider medium and antibiotics as the liquid phase, of material obtained from scratching or biopsying the lesion. The isolates obtained were then characterized by isoenzyme electrophoresis6; 3. pathological examination o f biopsy material. The biopsy material was taken with a 4 mm punch biopsy on the edge o f the cutaneous lesion or with a cutting biopsy forceps in the case of mucous lesion, and
D i m i e r - D a v i d L , D a v i d C , R a v i s s e P , B u s t i ll o s R , R e v o l lo S , L y è v r e P , M u n o z M , V a r g a s F , D e d e t J P . P a r a s i t o l o g i c a l d i a g n o s i s o f m u c o c u ta n e o u s l e is h m a n ia s is d u e t o Leishm ania b. brasiliensis in B o li v ia . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c in a T r o p i c a l 2 4 : 2 3 1 - 2 3 4 , o u t- d e z , 1 9 9 1
fixed in B ouin's fluid or 10% buffered formalin. The sections were stained with haemalum-eosin- safranine or Giemsa ' s stain.
The immunological investigation realized in each patient included: leishmanin skin testing and detection o f circulating antibodies by indirect immunofluorescence.
RESULTS
The patients were divided into 2 groups, according to the type o f lesi on (cutaneous or mucosal) they harboured.
Patients with cutaneous lesions
This group included 30 patients harbouring only cutaneous lesions. O f them, 23 were males and 7 females. The age range was 25.7 + 3.2 years.
Three originated from Peru, the others were Bolivian.
The lesions were ulcerative, w ith marked inflammatory fringe. The lesions were unique in 36.6% of the patients and multiples en 63.3%. They were localized on lower limbs in 53.8 % of the cases and 86.6% o f them had a duration period inferior to one year.
The parasite was found in 15 patients (50%) according to the following identification techniques:
. positivity of smear alone: 2 cases (13.3%) . positivity o f culture alone: 3 cases (20.0%) . positivity o f pathology alone: 2 cases (13.3%) . positivity o f both smear and culture: 2 cases
(13.3%)
. positivity o f both smear and pathology: 5 cases (33.3%)
. positivity o f smear, culture and pathology: lease (6.6% )
Table 1 shows the comparisons o f the three identification techniques.
The six isolates otained in culture were characterized by isoenzyme electrophoresis as
L. b.
braziiiensis6.
According to the duration period of the lesions, the parasite was found in 40 % of the lesions of less
than 1 year of evolution and in 75 % of the lesions o f more than 1 year o f evolution.
The leishmanin skin test was positive in 82.1 % and the IFI result was included between 1/40 and 1/80 in 86.6% of the cases.
Patients with mucosal lesions
The second group o f patients included 116 patients affected with mucosal leishmaniasis. One hundred nine were males and seven females. The age range was 3.7 to 38.5 years. Fifteen o f them originated from Peru, the others were Bolivians. The evolution time of mucous involvement was less than 1 year in 12%, between 1 and 5 years in 58.6%, betw een5 and 10yearsin21.5% , between 10 and 20 years in 6 % and more than 20 years in 1.7% of the cases. The extension o f the mucosal involvement was limited (nose + palate mucosa) in 38.7 %, important (nose + palate mucosa + pharynx or larynx lesion) in 28.4% and very important (all the mucous of the otorhinolaryngeal (ORL) sphere) in 32.7 % of the cases.
The parasite was found in 60 patients (51.7%) according to the following identification techniques:
. positivity of smear alone: 10 cases (8.6% ) . positivity o f culture alone: 15 cases (12.9%) . positivity o f pathology alone: 18 cases (15.5 %) . positivity o f both smear and culture: 2 cases
(1.7%)
. positivity of both smear and pathology: 10 cases (8.6%)
. positivity of smear, culture and pathology: 5 cases (4.3%)
The Table 1 shows the comparison o f the three examination techniques in the two groups o f patients.
The 15 isolates obtained from the 22 positive cultures were characterized as
L. b. braziiiensis
by isoenzyme electrophoresis6.Regarding the evolution time o f the lesions, the parasite was found in 48.7
%
o f the lesions o f less than5 yearsof evolution a n d in 5 8 .8 % ofthe lesions of more than 5 years of evolution.The leishmanin skin test was positive in 75% and the IFI results included between 1/80 and 1/160 in 70.6 % of the cases.
D i m i e r - D a v i d L , D a v i d C , R a v i s s e P , B u s ti ll o s R , R e v o l l o S , L y è v r e P , M u n o z M , V a r g a s F , D e d e t J P . P a r a s i t o l o g i c a l d i a g n o s i s o f m u c o c u t a n e o u s l e i s h m a n ia s i s d u e t o Leishmania b. brasiliensis in B o l iv i a . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 4 : 2 3 1 - 2 3 4 , o u t - d e z , 1 9 9 1
DISCUSSION
The results o f our study showed that using or techniques the percentage parasite positive patients in cutaneous and mucosal lesions o f L . b . b r a z i l i e n s i s leishm aniasis in B olivia are sim ilar: 50% in cutaneous lesions versus 51.7 % in mucous lesions.
Compared to literature data these percentages appear weak in the case o f cutaneous lesions: Cuba et al1 detected parasites in 71.2% o f cutaneous lesions o f the same mucocutaneous leishmaniais in Três Baços (Bahia, Brasil). Also in Bolivia, Desjeux et al3 obtained 48.6% positive results in parasite detection by culture and hamster inoculation in a sample o f 72 cutaneous lesions biopsied from mucocutaneous leishmaniasis. In a 216 patients sample observed in French Guyana, smear and culture detection o f the parasite were positive en 61.6% o f the cutaneous lesions due to L . b . g u y a n e n s i s (Dedet, unpublished data).
In respect to the mucous lesion, the positive percentage is equivalent to that o f Cuba et al1 who observed 48 % o f positivity in mucosal lesions, but differs largely from the 100 % positivity obtained in a limited sample o f patients in Colombia by Saravia et al1.
According to our results, the most efficient parasite detecting techniques appeared to be the smears in cutaneous lesions (33.3% o f positivity) and the pathology in case o f mucous lesions (28. 4 % ) . F o r logistic reasons, ham ster inoculation was not done although this is an efficient method o f diagnosis. O ur observation stresses the value o f routine p a th o lo g ic a l m e th o d in th e d ia g n o s is o f mucocutaneous leishmaniasis. This technique not only can give direct evidence o f the parasite, as reported here, but can also allow the elim ination of other pathologies as will be discussed in detail elsewhere3.
In our experience, the parasite was found more frequently in the cases o f im portant and very important mucous extension (57.7% positivity) than in the cases o f limited involvem ent (42.2% positivity). M arsden5 observed in the same way that it was “easier to detect parasites in m ultiple mucosal lesions than in single ones” .
Another paradoxical data evidenced by our observation is that in both cutaneous and mucous lesions the parasites w ere found m ost frequently in old lesions (more than one year evolution for cutaneous lesions and m ore than 5 years evolution for mucous lesions) than in recent lesions, to the
D i m i e r - D a v i d L , D a v i d C , R a v i s s e P , B u s tillo s R , R e v o l lo S , L y è v r e P , M u n o z M , V a r g a s F , D e d e t J P . P a r a s i t o l o g i c a l d i a g n o s i s o f m u c o c u t a n e o u s le is h m a n ia s is d u e t o Leishm ania b. brasiliensis in B o li v ia . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c in a T r o p i c a l 2 4 : 2 3 1 - 2 3 4 , o u t- d e z , 1 9 9 1
contrary o f other authors' observations1410. These observation illustrate the importance of further comparative studies, in order to show the variations due to local situations, both in lesion expression and in laboratory conditions.
RESUMO
D ia g n ó s ti c o p a r a s ito lô g ic o , u s a n d o e s fr e g a ç o , c u ltu r a
e exame
a n a t o m o p a to ló g ic o d e b ió p s ia , f o i e s tu d a d o e m 1 4 6 p a c ie n t e s in fe c ta d o s c o m le is h m a n io s e m u c o c u tâ n e a , n a B o lív i a e P e r u . A m a is e fic ie n te té c n ic a d e d e te c ta r p a r a s i t a s p a r e c e s e r o e x a m e d e e s fr e g a ç o p a r a le s õ e s c u tâ n e a s (3 3 % d e p o s i t iv id a d e ) e a n a to m o p a to ló g ic o p a r a l e s õ e s d e m u c o s a s (2 8 % d e p o s itiv id a d e ) . E m a m b a s le s õ e s , c u tâ n e a e m u c o s a , p a r a s i t a s f o r a m m a isfr e q u e n t e m e n t e e n c o n tr a d o s e m v e lh a s le s õ e s . P a l a v r a s - c h a v e s : L e i s h m a n i o s e m u c o c u t â n e a .
Leishmania b. braziliensis. D ia g n ó s tic o . P a r a s ita . B o lív ia . P e r u .
ACKNOWLEDGEMENTS
We thank Pr. Fernando Cardenas and Pr. Luis Valda of the department o f Dermatology, Hospital de Clinicas of La Paz, were the patients were hospitalised. The Instituto Boliviano deBiologia de Altura receives a support from the M inistère des Affaires Etrangères (Paris, France), the Ministerio de Prevision Social y Salud Publica and the Universidad Mayor de San Andrés (La Paz, Bolivia).
REFERENCES
1. C u b a C C , L lanos-C uentas E A , B arreto A C, M agalhas AV, Lago E L , Reed SG, M arsden PD. Human mucocutaneous leishmaniasis in Três Baços, Bahia - Brazil. An area o f Leishmania braziliensis braziliensis transm ission. I. Laboratory diagnosis. Revistada SociedadeBrasileira deM edicina Tropical 17:161-167, 1984.
2. D esjeux P, M ollinedo S, Le Pont F , Paredes A, Ugarte G. Cutaneous leishmaniasis in Bolivia. A study o f 185 human cases from Alto Beni (La Paz d e p a r tm e n t) . Is o la tio n a n d iso e n z y m e ch aracterizatio n o f 26 strains o f L eishm ania braziliensis braziliensis. Transactions o f the Royal Society o f Tropical M edicine and Hygiene 81:742- 746, 1987.
3. Dimier-David L , Ravisse P , Bustillos R , David C , L y e v re P, M allea F , V ald a L , D ed et JP . H isto p ath o lo g ie d e la leish m an io se cutaneo- m uqueuse à Leishm ania braziliensis braziliensis. Bulletin d e la Societé d e Pathologie Exotique (submitted).
4. FurtadoTA . Diagnóstico laboratorial de leishmaniose te g u m e n ta r a m erican a. A nais B rasileiro s de D erm atologia 47:211-228, 1972.
5. M arsden PD . M ucosal leishm aniasis ( “espundia” E scom el, 1911). T ransactions o fth e R o y al Society o f T ropical M edicine and H ygiene 80:859-876,
1986.
6. Revollo S, Dimier-David L , David C, L yevre P, Camacho C, Dedet JP. Isoenzymatic characterization o f Leishm ania braziliensis braziliensis isolates obtained from Bolivian and Peruvian patients. Transactions o f the Royal Society o f Tropical M edicine and Hygiene 86:388-391, 1992. 7. Saravia N G , Holguin A F, M cM ahon-Pratt D,
D 'A lessandro A. M ucocutaneous leishmaniasis in Colom bia: L eishm ania braziliensis subspecies diversity. The American Journal ofTropical Medicine and Hygiene 34:714-720, 1985.
8. Shaw JJ, Lainson R. Ecology and Epidemiology: New W orld. In : The leishmaniasis in Biology and M edicine. Peters W , K illick-K endrick R (ed). Academic Press, London 1 p .291-363, 1987. 9. T orres Espejo M , L e Pont F , M ouchet J , D esjeux P,
Richard A. E pidem iologie de la leishm aniose tégum entaire en Bolivie. 1. Description des zones d 'é tu d e et fréquence d e la maladie. Annales de la Société Belge de M édecine Tropicale 69:297-306, 1989.
10. Urgel R, D e M uynck A, Recacoechea M , Azogue E. El diagnôstico de la leishmaniasis cutanea con particular referen d a a los hallazgos parasitolôgicos. B oletin Inform ativo del C entro N acio n al d e Enfermedades Tropicales 6:31-37, 1980.
