R e v is t a d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T r o p ic a l 2 8 ( 2 ) :9 9 - 1 0 3 , a b r - ju n 1 9 9 5 .
A RT1G O S
EV A LUA TIO N O F A D IREC T IM M UN O FLUO RESCEN T
A N TIBO D Y (D IFM A ) TEST USIN G LEISH M A N IA G EN
US-SPEC IFIC M O N O CLO N A L A N TIBO D Y IN TH E RO U TIN E
D IA G N O SIS O F CUTA N EO US LEISHM A N IA SIS
M arth a E. C h ico , R o n ald H . G u d erian , Ph ilip J. C o o p er, R o d rig o A rm ijo s
an d M ax G ro g l
A d i r e c t i m m u n o f l u o r e s c e n t a n t i b o d y ( D IFM A ) t e s t u s in g a L e i s h m a n i a g e n u s -s p e c i f i c m o n o c l o n a l a n t i b o d y w a -s e v a l u a t e d in t h e r o u t i n e d i a g n o -s i -s o f c u t a n e o u -s l e i s h m a n i a s i s ( C L ) i n E c u a d o r . T his t e s t w a s c o m p a r e d w it h t h e s t a n d a r d d i a g n o s t i c t e c h n i q u e s o f s c r a p in g s , c u l t u r e a n d h is t o lo g y . D i a g n o s t ic s a m p l e s w e r e t a k e n f r o m a t o t a l o f 9 0 a c t i v e d e r m a l u lc e r s f r o m p a t i e n t s f r o m a r e a s o f E c u a d o r k n o w n t o b e e n d e m i c f o r c u t a n e o u s l e i s h m a n i a s i s . D IFM A w a s p o s i t i v e i n a l l le s io n s . It w a s s h o w n t o b e s i g n i f i c a n t l y s u p e r i o r t o s t a n d a r d d i a g n o s t i c m e t h o d s e i t h e r a l o n e o r in c o m b i n a t i o n . T h e s e n s it iv it y o f D IFM A d i d n o t d i m i n i s h w it h c h r o n i c i t y o f le s io n s . T his t e s t p r o v e d t o b e e x t r e m e l y u s e f u l i n t h e r o u t i n e d i a g n o s i s o f C L b e c a u s e it is h ig h ly s e n s it iv e , is e a s y t o u s e a n d p r o d u c e s r a p i d r e s u lt s .
K e y - w o r d s : C u t a n e o u s l e i s h m a n i a s i s . D ia g n o s is . M o n o c l o n a l a n t i b o d y .
Cutaneous leishmaniasis is end emic in Ecuador6. Detailed clinical and parasitological stu d ies hav e im p lic ated 5 d ifferen t
sp e c ie s (L e i s h m a n i a b r a z i l i e n s i s , L.
p a n a m e n s i s , L .g u y an e n s is , L. m e x i c a n a a n d L . a m a z o n e n s i s ) as causative agents414. These
sp ecies vary geo grap hically betw een the different tropical and subtropical regions o f the country3.
Diagnosis in developing countries usually relies o n clinical assessment. However, w here facilities permits diagnosis may be made by parasite detection in tissue isolates and/ or by the leishmanin (Montenegro) skin test. The lo w sensitivity o f standard parasite detection metho ds5, the p o o r sp ecificity o f the leishmanin test in endemic areas12, and the need to distinguish cutaneous leishmaniasis (CL) from lesio ns o f different aetiology, have stimulated the search fo r o ther diagnostic assays. To date sérodiagnostic assays have proved disappointing in the diagnosis o f New
N atio n al C e n te r f o r T ro p ic al D ise ase s- Q u ito b ran c h , H o sp ital V o z an d es, In stitu te o f In v estig atio n , Sc h o o l o f M ed ic in e, C en tral U n iv ersity o f Ecu ad o r, Q u ito , Ec u ad o r and W alter R e e d A rm y In stitu te o f R esearc h . W ash ing to n , D C. USA .
A d d r e s s t o : D ra. M artha C h ic o , D ep artm en t o f C linical In v e stig atio n , N atio n al C e n te r f o r T ro p ic al D ise ase s. H o sp ital V o z and es, C asilla 17-17-691, Q u ito , Ecu ad o r, So u th A m eric a. Fax : 593-2-447-263.
R e c e b id o p ara p u b lic aç ão e m 10/ 06/ 94.
World CL1 19. The availability o f no v el techniques using monoclo nal antibodies or kinetoplast DNA pro bes offers rapid diagnosis w ith high sensitivity and specificity1. The usefulness o f such assays in the routine diagnosis o f CL remains largely untested.
In this study, the direct immunofluorescent mono clo nal antibody (DIFMA) test using a genus-sp ecific m o no clo nal antibo d y7 w as compared w ith standard parasite detection methods.
' MATERIAL AND M ETHODS
S tud y p o p u latio n
Dermatological clinics w ere held in Quito and Santo Domingo de los Colorados in the province o f Pichincha, Ecuador, o ver a 1 year period. Patients w ith active skin lesio ns suggestive o f CL w ere selected foEJfoe study. These patients came fro m the ^Follow ing regio ns o f the co untry, the trb'jijical and subtropical areas o f the Pacific Coast,- the high semi-arid plains o f the A ndes, and the tropical eastern A mazon regions.
Each p atient w as exam ined and
do cumented regarding age, sex, race,
C h ic o M E, G u d e r ia n R H , C o o p e r P J A r m ijo s R , G r o g l M . E v a lu a t io n o f a d ir e c t im m u n o flu o r e s c e n t a n t ib o d y (D IFM A ) t e s t u s in g le i s h m a n ia g e n u s - s p e c ific m o n o c lo n a l a n t ib o d y in t h e r o u t in e d ia g n o s is o f c u t a n e o u s le is h m a n ia s is . R e v is t a d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T r o p ic a l 2 8 :9 9 - 1 0 3 , a b r - ju n , 1 9 9 5 .
S am p le co lle ctio n an d e xam in atio n
Befo re samples w ere taken, necro tic areas w ere d ebrid ed and the lesio ns cleaned thoroughly w ith savlon to avoid seco ndary contamination. From each patient o ne sample o f each o f the follow ing w ere taken: scraping, needle aspirate and punch biopsy. In patients w ith multiple lesio ns additional samples w ere taken.
D IFM A
Impression smears o f the bio psy w ere made o n glass slides and air dried. The touch preparations w ere pro cessed by the DIFMA technique using the g enus-sp ecific anti- leishmanial monoclo nal antibody (Mab) 83- J3D 2. This m etho d has been d escribed elsew here7. The preparations w ere examined under oil im mersion using an ultraviolet m icro sco p e (Z eiss) fo r the p resence o f amastigotes (x43, 100 fields).
S crap in g s
Scrapings w ere taken fro m the active bo rders o f lesio ns using a size 20 scalpel blade. They w ere smeared onto glass slides to make a thin preparation, air dried and fixed in
m ethano l. A fter Giemsa staining, the
preparatio ns w ere exam ined und er oil immersion (xlOO magnification, 100 fields).
C u ltu re
Samples for culture w ere taken by needle aspirate and bio psy punches. Needle aspirates w ere obtained w ith a 3ml syringe and 23 gauge need le. The syringe w as filled w ith 0.1ml sterile phosphate buffered saline (pH 7.4). The needle w as inserted into the outer bo rd er o f the lesio ns, the syringe rotated and the tissue fluid aspirated o n withdrawal.
Full thickness bio psies w ere obtained w ith a 4mm punch from the active bo rd ers o f the ulcers, after anaesthetising w ith 2% lidocaine and adrenaline. The bio psies w ere placed in saline supplemented w ith 100,000 units o f benzylpenicillin fo r 24 hours at 4°C. The bio psies w ere then homo genised in saline befo re culturing.
Needle aspirates and bio psy homogenates w ere each cultured in all the follow ing media: defribinated rabbit blo o d agar-NNN medium, Schneid ers Dro so phila med ium (GIBCO,
Green Island, NY) supplemented w ith 20% heat inactivated fo etal calf serum, and diphasic blo o d agar meduim w ith 0.5ml Dulbeccos phosphate buffered saline as the liquid overlay (DBA -PBS). Cultures w ere maintained at 25°C fo r at least 30 days befo re reporting negative.
H isto lo g y
Punch bio psy fragments w ere obtained as
d escribed abo ve. These w ere fixed
immediately in 10% buffered formaldehyde and later stained w ith haematoxylin and eosin, embedded in paraffin, and sectio ned . At least 100 field s w ere exam ined befo re being reported negative.
C o n sen t
Info rmed co nsent fo r this study w as obtained from all subjects and procedures w ere explained in the lo cal language. The study w as carried out und er pro to co ls approved by the Ministry o f Public Health o f Ecuador and the Ethical Committee o f Hospital Vozandes, Quito.
S tastistical an aly sis
McNemar’s test for co mparison o f tw o paired samples w as used3.
RESULTS
A total o f 90 active skin ulcers from 88 patients w ere assessed. The results o f the different diagnostic methods are show n in Table 1. DIFMA w as superior to all other methods employed and w as positive in 100% o f lesio ns. Histo lo gy (74.4% ) w as more senstive than either scrapings (51.1% ) o r culture (51.1%).
T a b l e 1 - P o s i t i v e l e s i o n s b y t h e f o u r d i a g n o s t i c m e t h o d s . Diagno stic metho d Postive
N°. %
DIFMA 90 100
Histology 67 74.4
Culture 46 51.1
Scrapings 46 51.1
Thirteen (14.4%) lesions w ere po sitive by DIFMA alone. Positive results in all tests w ere o btained in 29 (32.2% ) lesions. W hen DIFMA is excluded from the analysis no ne o f the o ther d etectio n metho d s either alo ne o r in combination w ere positive in more than 85.6% o f lesio ns (Table 2).
C h ic o M E, G u d e r ia n R H , C o o p e r P J, A r m ijo s R , G r o g lM . E v a lu a t io n o f a d ir e c t im m u n o flu o r e s c e n t a n t ib o d y ( D IF M A ) t e s t u s in g le is h m a n ia g e n u s - s p e c ific m o n o c lo n a l a n t ib o d y in t h e r o u t in e d ia g n o s is o f c u t a n e o u s le is h m a n ia s is . R e v is t a d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T r o p ic a l 2 8 :9 9 - 1 0 3 , a b r - ju n , 1 9 9 5 .
T a b l e 2 - C o m p a r i s o n o f d e t e c t i o n m e t h o d s f o r d i a g n o s i s o f c u t a n e o u s l e i s h m a n i a s i s .
M etho d co mpared N2. % N9. % P DIFMA vs culture 90 100 vs 46 51.1 pcO.OOl DIFMA vs scrapings 90 100 vs 46 51.1 pO.OOl DIFMA vs histo lo gy 90 100 vs 67 74.4 pO.OOl DIFMA vs 3 metho d s 90 100 vs 77 85.6 p<0.005 Histolo gy vs scrapings 67 74.4 vs 46 51.1 PO.OOl Histology vs culture 67 74.4 vs 46 51.1 p<0.005 Cutlure vs scrapings 46 51.1 vs 46 51.1 NS
A direct co mparison o f the 4 methods is show n in Table 2. DIFMA w as far more sensitive than any o f the other detection methods either alo ne o r in combinatio n. Histolo gy w as superior to scrapings and culture. There w as no difference in sensitivity betw een culture and scrapings.
The sensitivity o f standard d etectio n methods decreased w ith duration o f the lesio n (Table 3)- After 6 months duration a small pro po rtio n o f lesio ns w ere p o sitive by histology (40.0% ), culture (30.0% ) or scrapings (30.0% ). On the o ther hand all lesions w ere positive by DIFMA irrespective o f duration.
T a b l e 3 - R a t e s o f p o s i t i v i t y b y t h e f o u r d i a g n o s t i c t e s t s i n r e l a t i o n t o d u r a t i o n o f le s i o n .
Duratio n o f lesio n __________ Diagno stic metho d____________ scrapings culture histo lo gy DIFMA
% % % %
< 3 mo nths 58.1 54.1 67.4 100 (n -43)
3-6 months 32.4 37.6 81.1 100
(n -37)
> 6 mo nths 30.0 30.0 40.0 100 (n -10)
DISCUSSION
O ur results sho w that in the routine diagnosis o f cutaneo us leishmaniasis, the DIFMA method is not only a useful technique w ith high sensitivity, but is greatly superior to standard d iagno stic metho d s. The high sensitivity and specificity o f this monoclonal
antibody (Mab) fo r L e i s h m a n i a has been
demonstrated in o ther studies17. This Mab (83- J3D 2) recognizes a dominant antigen common to promastigotes and amastigotes o f isolates from 3 major species and 5 subspecies o f New World CL1.
Definitive diagnosis o f CL requires the d em o nstratio n o f amastigo tes in lesio ns. Isolatio n o f amastigotes in tissue samples is fraught w ith difficulties. In cutaneous lesions
success at parasite iso latio n is inversely proportional to the duration o f the lesio ns20. This is also reflected in our findings. However, DIFMA w as positive (100% ) irrespective o f duratio n o f the lesio ns. Furthermore, in
patients receiving antileishmanial
chemotherapy, DIFMA w as sho w n to be useful in quantitatively assessing treatment efficacy (Chico M, Guderian RH, unpublished data).
The findings in this study o f a lo w sensitivity o f culture and scrapings, either alone or in combinatio n, mirrors those o f o ther w orkers5. In this study, histology w as also superior to culture and scrapings, w hich is inco nsistent w ith the findings o f o ther w orkers8 18. The clarity w ith w hich amastigotes can be identified by DIFMA far exceed s that w hich is
seen o n co nv entio nal staining w ith
haem ato xylinn and eo sin. DIFMA also identified a high proportio n o f cases that w ere no t d etected by the o ther m eho d s. The observation that all these lesio ns resolved fo llow ing ad equate co urse o f leishmanial chemotherapy is indirect evidence for a correct diagnosis.
Immunological methods have pro ven o f limited use in the diagnosis o f New World CL. Patients rarely have detectable antibodies. Usually the delayed hypersensitivity response (leishmanin test) has not d eveloped at the time o f clinical p resentatio n19. Similarily such methods cannot distinguish current from past infection. Recent improvements in ELISA have improved the sensitivity o f such assays517, but most assays still suffer from po o r specificity. False positive results have been reported in
patients w ith malaria, to xo p lasm o sis,
amoebiasis15, and Chagas diseases2. Differences betw een lo cations w ith regard to specificity and sensitivity have also been reported20. The
leishmanin test remains a useful
epid emio lo gical to ol, but it’s value as a diagnostic test is limited in end emic areas w here a large pro po rtion o f the po pulatio n may test positive916.
C h ic o M E, G u d e r ia n R H , C o o p e r P J, A r m ijo s R , G r o g l M . E v a lu a t io n o f a d ir e c t im m u n o flu o r e s c e n t a n t ib o d y ( D IF M A ) t e s t u s in g le i s h m a n ia g e n u s - s p e c ific m o n o c lo n a l a n t ib o d y in t h e r o u t in e d ia g n o s is o f c u t a n e o u s le is h m a n ia s is . R e v is t a d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T r o p ic a l 2 8 :9 9 - 1 0 3 , a b r - ju n , 1 9 9 5 .
species causing CL in Ecuador414, diagnosis by species-specific probes w o uld require at least 5 different pro bes befo re a diagosis o f CL could be excluded. Though species-specific Mab pro bes are available13, this w ould clearly be impractical in routine diagnosis. There is, how ever, a need to distinguish more benign
sp ecies fro m L. b r a z i l i e n s i s w hich has ' a
tendency to cause destructive mucocutaneous leishmaniasis. This co uld be achieved by screening suspected lesio ns w ith a genus- specific pro be and then testing all positive
cases w ith a L . b r a z i l i e n s i s sp ecific Mab.
Tho ugh species identification may influence therap eutic o p tio ns, in practice in most developing countries, such cho ice is limited to o ne o r tw o drugs, rend ering spéciatio n irrevelant.
The advantages o f novel diagnostic tests using Mab or kinetoplast DNA (kDNA) probes o ver im m uno lo gical o r parasite iso latio n methods are clear. Such assays are rapid and highly sensitive and specific1711. For example, positive culture may take w eeks, delaying diagnosis, w hile DIFMA can be performed in o ne hour. However, the sensitivity o f Mabs and kDNA pro bes may detect differences so fine as to render their significance impossible to evaluate10. W hile this is not so important in diagnosis in the presence o f characterisitic lesions, their usefulness in epidemiological studies w ould be questio nable.
There remains a need for a highly specific
and sensitiv e d iagno stic test that is
inexpensive, rapid and simple to perform w ithout the need for sophisticated equipment. The DIFMA test em plo yed in this study satisfies most o f these criteria, and is superior to culture, scrapings and histology. DIFMA has a num ber o f ad vantages o ver ind irect
immunofluorescence using Mabs, and in s it u
hybridization using kDNA pro bes, due to its simplicity, rapidity and relatively lo w cost. In addition the availability o f inexp ensiv e ultraviolet systems (FW Kirk Ltd, Cambridge, UK) that can be used w ith any co nventio nal microsco pe makes this techno logy accessible to everyone in developing countries.
SUMMARY
O m é t o d o d e i m u n o f l u o r e s c ê n c i a d i r e t a ( D IF M A ) , c o m a n t i c o r p o s m o n o c l o n a i s g ê n e r o -e s p -e c í f i c o s p a r a L -e i s h m a n i a , f o i a v a l i a d o n a r o t i n a d i a g n o s t i c a d a l e i s h m a n i o s e c u t â n e a n o E q u a d o r .
O m é t o d o f o i c o m p a r a d o c o m t é c n i c a s d i a g n o s t i c a s d e r o t i n a : o e s f r e g a ç o , a c u l t u r a e o e x a m e h is t o p a t o l ô g i c o . A s a m o s t r a s p a r a o d i a g n ó s t i c o f o r a m o b t i d a s d e u m t o t a l d e 9 0 l e s õ e s c u t â n e a s at iv as , d e d o e n t e s d a s ã r e a s d o E q u a d o r , e n d ê m i c a s p a r a l e i s h m a n i o s e c u t â n e a . O D IFM A f o i p o s i t i v o e m t o d a s a s le s õ e s , c o m r e s u l t a d o s s i g n i f i c a t i v a m e n t e s u p e r i o r a o s m é t o d o s d i a g n ó s t i c o s d e r o t i n a , i s o l a d o o u e m c o m b i n a ç ã o . A s e n s i b i l i d a d e d o D FIM A n ã o d i m i n u i e m le s õ e s c r ô n i c a s . O m é t o d o m o s t r a - s e m u it o ú t i l n o d i a g n ó s t i c o d e l e i s h m a n i o s e c u t â n e a , p e l a s u a s e n s i b i l i d a d e , r a p i d e z e f a c i l i d a d e d e
e x e c u ç ã o .
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 c u t â n e a . D ia g n ó s t i c o . A n t i c o r p o s m o n o c l o n a i s .
REFERENCES
1. A ntho n y RL, G rogl M , S acci B, Bailou KW! Rapid
d ete ctio n o f Leishm ania am astigo tes in fluid
asp irates and b iop sies o f h u m an tissues. T h e A m e rican Jo u rn al o f T ro p ical M ed icin e an d
H ygiene 3 7 : 2 7 1 - 2 7 6 , 1 9 8 7 .
2. A ntho n y RL, W illiam s K M , S acci JB , Rub in D C.
S u b cellu lar an d taxo n o m ic sp ecif icity o f
m o n o clo n al antib od ies lo N ew W orld Leishm ania.
T h e A m erican Jo u rn al o f T rop ical M ed icine and
H ygiene 3 4 : 1 0 8 5 - 1 0 9 4 , 1 9 8 5 .
3. A rm itag e P. S tatistical m e th o d s in m ed ical
re s earch . B lack w ell S cien tif ic Pu b licatio n s, O xf ord , 1 9 7 4 .
4. A rm ijos RX , C h ico M E, C ruz M E, G ud erian, RH ,
K reutz er RD , Berm an JD , R o g ers M D , G rogl M .
H um an cu tan eo u s leishm an iasis in Ecu ad o r;
id e n tif icatio n o f p arasites b y en z y m e electro p h o resis .T h e A m erican Jo u rn al o f T rop ical
M ed icine and H ygiene 4 2 : 4 2 4 - 4 2 8 , 1 9 9 0 .
5. Furuy a M , M im ori T, G om ez EA , D e C o ro n el Y K aw ab ata M , H ash ig u ch i Y. Ep id em io lo g ical
su rv ey o f leishm aniasis using skin te st and ELISA in Ecuad o r. Jap an ese Jo u rn al o f T rop ical M ed icine
and H ygiene 1 7 : 3 3 1 - 3 3 8 ,1 9 8 9 .
6 . G rim aldi G ,T esh RB, M cM ahon-Pratt D . A rev iew
o f th e g e o g rap h ical d istrib u tio n and ep id em iolo g y o f leishm aniasis in th e N ew W o rld .
T h e A m erican Jo u rn al o f T rop ical M ed icine and
H ygiene 4 1 : 6 8 7 7 2 5 , 1 9 8 9
-7. G rogl M , M ilhous W K , M artin RK , K yle D E. K its for
th e diagnosis o f cu tan eo u s leishm aniasis in field lab o ratories. Pro ceed in g s o f th e S o ciety o f A rm ed
Fo rces Lab o rato ry S cien ce 1 8 : 2 2 ,1 9 8 9
-8. H end rick s L, W rig h t N . D iagnosis o f cu tan eo u s leishm aniasis b y in v itro cu ltiv ation o f saline
C h ic o M E, G u d e n a n R H , C o o p e r P J, A r m ijo s R , G r o g l M . E v alu at io n o f a d ir e c t im m u n o flu o r e s c e n t a n t ib o d y (D IFM A ) test u s in g le is h m a n ia g e n u s - s p e c ific m o n o c lo n a l a n t ib o d y in t h e r o u t in e d ia g n o s is o f c u t a n e o u s le is h m a n ia s is . R e v is t a d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T r o p ic a l 2 8 :9 9 - 1 0 3 , ab r - ju n , 1 9 9 5 .
aspirates in Schneiders Drosophila medium. The A merican Jo urnal o f Tro pical M ed icine and Hygiene 28:962-964,1979.
9. Kerdell-Vegas F. A merican leishmaniasis.
International Journal o f Dermatology 21:291-303, 1982.
10. Lainson R, Shaw JJ. Evolutjgg, classification and geographical distribution. M PgfgfS W, Killicfc.
Kendrick R (eds) T h e leishmaniasis in biology
and m ed icine, 1st ed ition, A esdpmjg Press, London p » l-120,1987.
11. Lynch NR, Malave C, Ifante RB, Modlin R, Convit J. In situ d etection o f amastigotes in american cutaneo us leishmaniasis using m o no clo nal antibsdieg. f^BSSetio ns o f the Royal Socipty g f T rop ical M g d ieist g s ä M ygtese § 8 ;# # , w k
-12
. Manson-Bahr PC, Biagiie§i§;M
Pilg fiM
JÖUiek- Kendrick R (ed ) The leishmaniasis in biology and medicine. A cademic Press, London p .703-729, 1987.13. McMahon-Pratt D, David JR. A pgliggtißfß g f
monoclonal antibodies §gggj§g Ltishmania
species, In : Proceedings o f a w orkshop held at
the Pan American Health Organization, 9-11
December, 1980. UNQF.Wwld BiffifeW SQ Iggeial
Programme fo r Research m Trapieal
Diseases, Geneva, Sw itzerland p .247-257,1982. 14. Mimori T, Grimaldi G, Kreutzer RD, Gomez EA,
McMahon--Pratt Q, TegH S i , HgshigueW Y, identification, using enyi?mg tl§etrep hertg ii and monoclonal antibodies, Of leishmania isolated from human and w ild animals in Ecuador. The A merican Jo urnal o f Tro pical M ed icine and Hygiene 40:154-158,1989.
15. Pappas MG, M cGreevy PB, Hajkow ski R, Hendricks LD, O ster CN, Ho ckmeyer WT. Evaluation o f pro mastigo te and amastigote antigens in the ind irect fluo rescent antibody test fo r am erican cutaneo us leishmaniasis. The A merican Jo urnal o f Tro pical M ed icine and Hygiene 32:1260-1267,1983.
I# , Restrepo M. La reaccio n de Montenegro en al epidemiologia de la leishmaniasis sudamericana. Bo letin de O ficina Sanitaria Panamericana 89:130-136,1980.
17. Scott JE, ShrefflerWG, Ghalib HW, El Asad A, Siddig M, Badro R, Reed SG. A rapid and simple digga@§tie test for active visceral leishmaniasis. The A merican Journal o f Tropical Medicine and Hygiene 44:272-277,1991.
18. Weigle KA, De Davalos M, Heredia P, Molineros R, Saravia NG, Alessandro A. Diagnosis o f cutaneous and muco cutaneous leishmaniasis in Colombia: A comp&fi§@n o f seven methods. The A merican Journal o fTropical Medicine and Hygiene 36.489-
4 9 6 ,1987.
19- World Health Organization. Repo rt o f a training seminar on epidemiological methods fo r the leishmaniasis, UNDP/ World Bank/ WHO Special Programme for Research and Training in Tropical Diseases,TDR/ LEISH-SEM/ 80.3 p 23-24,1980. 20. World Health Organization. Repo rt o f the fourth
