the use of different concentrations of leishmanial antigen in ... - SciELO

Revista da Sociedade Brasileira de M edicina Tropical 18(4): 231-236, Out-Dez, 1985

THE USE OF DIFFERENT CONCENTRATIONS OF LEISHMANIAL ANTIGEN IN SKIN TESTING TO EVALUATE DELAYED HYPERSENSITIVITY IN AMERICAN CUTANEOUS LEISHMANIASIS C ésar A. C uba C u b a 1, Philip D. M a rs d e n 1, A ir C. B a rre tto 1, T h o m as C. J o n e s 2 and F rank R ich ard s 2 Three concentrations o f Leishmania mexicana am azonensis sonicated whole prom astigote antigen (30, 9.6 and 3 ug N in 0.1 m l) wereprepared and 0.1 ml o f each inoculated intraderm ally in topatients who live in one endem ic leishm aniasis region in B razil. P atients were divided into groups with active cutaneous leishm aniasis (AC L), healed cutaneous leishm aniasis (H C L), m ucosal leishm aniasis (M L ), and Controls (C). S kin reactions were recorded by m easuring induration 48 hours after inoculation. S kin tests using 9.6 u g N /0 .1 m lyield e d th e b estd ia g n o stic resu ltssin c e9 7 % o f3 0 patients with active lesions (cutaneous or m ucosal) a n d 8 3 % with H C L showed reactions o f 5 m m orgreater as com pared with 4% Controls. Tests using 30ug N/O. 1 m l causedan unacceptable levei o fsk in reactions with necrosis (1 0 % o fA C L p a tie n ts tested and 17% o f H C L , respectively). Tests using 3 ug N/O. 1 m l were less sensitive since only 87% o f patients with active lesions and 68% with H C L had reactions o f5 m m orgreater. The 3 ug N/O. 1 m l dose was utilized to a sk the questions whether skin delayed hypersensitivity decreased with tim e after the initial lesion a n d whether mucosal involvem ent is associated with enhaced hypersensitivity to leishm anial antigen. Decreased delayed hypersensitivity was noted only in th o sep a tien ts who had an initial lesion more than 30 years ago. The m ean induration o f the reaction in 10 patients with M L was 11.3 m m ± 7.15, in 41 patients with H C L , 9 .27 m m ± 6.78 and in 2 0 patients with A C L 10. 7 m m ± 6.10 mm. The percent o f patients with 5 m m orgreater induration was M L 80% , H C L 71%, A C L 90% . Thus, we could not confirm an association between enhanced delayed hypersensitivity and m ucosal involvem ent in leishmaniasis. Key W ords: Skin test comparison. Antigen concentrations. Clinicai forms. A m erican mucocutaneous leishmaniasis. A delayed hypersensitivity cutaneous reaction to leishmanin is present in m ost forms of cutaneous and mucocutaneous leishmaniasis with the exception of diffuse cutaneous leishmaniasis and some cases o f post kala-azar dermal leishmaniasis^. The skin test was first proposed by W agener in 192328, and was introduced as a diagnostic aid in A m erican cutaneous leishmaniasis in man by M ontenegro in 1926*8. it js the most widely used skin test in parasitic disease and is a valuable ancillary investigation especially in mucosal disease where parasites are often difficult to find. Shaw and Lainson 197527 observed that patients with mucosal leishmaniasis tended to have larger delayed hypersensitivity reactions than those with only cutaneous lesions. This suggested that * This work was supported by N IH G rant A I 16282 and by the Conselho N acional de Pesquisas (C N P q, PID E V 403682/82), the Brazilian M inistry of Health (SU C A M ) and U N D P /W O R L D B A N K /W H O Special programme for research and Training in Tropical D isease (TD R). 1. Faculdade de Ciências da Saúde. N úcleo de M edicina Tropical. Universidade de Brasília. Brasília, Brazil. 2. Com ell University M edicai College, N ew York, U SA . Recebido para publicação em 26/9/84.

mucosal disease could be associated with enhaced hypersensitivity response to leishmanial antigens. This study was designed to compare a) three concentrations of antigen in three forms of American cutaneous leishmaniasis and b) to examine the hypothesis that mucosal disease is associated with more marked cutaneous hypersensitivity than cutaneous lesions. M A T E R IA L S A N D M E T H O D S

1) P atient population - Ninety-nine patients were studied living in the area of Três Braços, Bahia, Brazil, were mucocutaneous leishmaniasis is endemic^. The following groups were established: a) 20 patients with active cutaneous leishmaniasis (A C L) presenting as a skin ulcer. b) 10 patients with mucosal lesions (M L) of the nose or oropharynx as well as a skin scar or active ulcer. c) 41 patients with the old scars of cutaneous leishmaniasis (H C L ) which are very typical in appearance (Figure 2) d) 28 patients living in the same area without signs or a history of leishmaniasis. e) 10 volunteers at the University of Brasília cons231

Cuba CAC, M arsden PD, Barretto A C , Jones TC, R ichards F. The use o f different concentrations o f leishm anial antigen in skin testing to evaluate delayed hypersensitivity in A m erican cutaneous leishm aniasis. R evista da Sociedade B rasileira de M edicina Tropical 18: 231-236, Out-Dez, 1985

titued a control group without any history of leishmaniasis and living in a nonendemic area.

tigotes in L IT medium (Liver Infusion Tryptose) and was harvested for these studies after eight days. The organisms were washed twice in phosphate O f the thirty patients with active cutaneous or buffered saline (pH 7.2), centrifuged, resuspended in mucosal lesions 23 (76.6% ) had positive indirect distilled water and sonicated (five periods of one fluorescent antibody titers (promastigote antigen) and minute each at 20 kh at 4o C). Specimens of the mixed in 20 patients, parasites were isolated from the lesions suspension were used to determine total nitrogen (Table 1). Eight stocks from these patients were idenconcentration by the micro-Kjeldahl m ethod'0. The tified as L eishm ania braziliensis braziliensis by solution was adjusted to three concentrations of an­ isoenzyme analysis7. tigen namely 3 0 0 ]_lg Nitrogen per ml, 96 yg N /m l and 2) Antigens - A stock L eishm ania m exicana30 ug N /m l using 0.5% phenol in saline. These antigens were kept at - 20° C until use. The antigens am azonensis (Josefa) isolated from a patient in were tested to confirm their activity in hospitalised Brasília was used to prepare the antigens for skin testing. This organism was maintained by serial patients with known leishmanin skin reactions before passage in hamsters. It gave a rich growth of promasuse in this field trial.

Table 1 - Clinicai and parasitological Jindings in 30 active cases o f Leishm aniasis (cutaneous a n d m ucosal diseases) subm itted to skin test.

Patient n.° Clinicai Form 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 * C: ** M: *** m: y: L.b.b.: N.D.: 232

C* C C C C C C C C C C c c c c c c c c c M ** M M M M M M M M M

Tim e o f E volution

N.° o f Lesions Age

2m *** 12y/ 5m 2,5m 8m lm 2m 3m 4m 5m 6m 1.5m 2m 1.5m 3m 5m ly 2.5m 2m 3m 2y 3y 3y 5.5y 4y 1.5y 2y 9y 2y 2y

Cutaneous disease Mucosal disease months years Leishm ania braziliensis braziliensis N ot Done

2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

55 40 42 60 16 19 15 8 18 4 3 18 53 45 50 48 66 65 28 30 35 59 23 35 49 10 24 37 9 34

Serology (IF A T ) 1:40 -

1:80 1:40 -

1:40 1:160 1:160 1:160 1:80 -

1:40 1:40 1:40 1:80 1:40 1:40 1:20 -

1:40 1:20 1:80 1:40 1:640 -

1:160 1:2560 1:160 -

Parasites

Identification ( I = Isoenzym es)

+ +

L.b.b. (I)

-

+ + +

L.b.b. (I) L.b.b. (I) L.b.b. (I)

-

+ + +

L.b.b. (I)

-

+ + + + + +

L.b.b. (I)

L.b.b. (I)

-

+ + -

+ N .D + + N .D +

L.b.b. (I)

Cuba CAC, M arsden PD, Barretto A C , Jones TC, R ichards F. The use o f different concentrations o f leishm anial antigen in skin testing to evaluate delayed hypersensitivity in A m erican cutaneous leishm aniasis. Revista da Sociedade Brasileira de M edicina Tropical 18: 231-236, Out-Dez, 1985

3) The cutaneous te s t- The antigen suspensions were always mixed thoroughly before use. Each concentration was inoculated intradermally into the ventral surface of the forearm in a dose o f 0.1 ml (Fig. 1). A control solution of the same volume of 0.5% phenol in saline was also inoculated at the same time. A t 48 hours the size o f induration at each inoculation site was measured with a millimeter calliper. A n induration of 5 or more millimeters was regarded as a positive reaction but detailed measurements were taken. Figure 2 shows patients with the typical mucosal lesion and a cutaneous scar. The results of skin testing were analysed calculating the mean induration in millimeters and standard deviation for each group.

Figure 1. R esults o f delayed hypersensitivity skin testing using three doses o f antigen a n d control read a t 48 hours. The g raded dose response is seen.

R E SU L T S Table 2 shows the results of the skin test using the three antigen concentrations in patients with active cutaneous lesions (A C L) and mucosal lesions (M L), scars, and Controls from the endemic area. The frequency of positive reactions in the Controls from the

F igure2. A 7y e a ro ld b o y w ith to talabsenceofnasalseptum fro m m ucosal leishm anisasis and a 13 yea r old girl with positive graded skin tests on the left forearm a n d a characteristic scar on the right forearm . Such scars resemble burns being slightlv depressed, hypopigm ented and fibrotic.

endemic region is very low and present only at high antigen concentrations (4% using a dose of 9.6pgN ). In patients with active disease or a scar, skin reactivity increased with the antigen concentration. There was no difference between these two groups in the number of positive skin reactions at the highest concentration. The highest concentration ( 3 0 y g /0 .1 ml) however was unaceptable since it occasionally caused necrosis at

Table 2 -D e la y e d hypersensitivity responses to three concentrations o f leism anin in three p atient groups:

Patient Groups

N um ber o f Patients Studied

N um ber o f Patients with Reaction o f > 5m m Using the Concentrations o f Antigen ( fJg N/O. 1 m l) Given Be low. 3

a - b Active Lesions (A C L and ML) c Scars d Control

9.6

30

30

26 (87% )

29 (97% )

30 * (100% )

41 28

27 (68% ) 0

34 (83% ) 1 (4% )

40 **(98% ) 2 (7% )

* 3(10% ) and ** 7(17% ) of patients with necrotic cutaneous reaction

233

Cuba CAC, M arsden PD, Barretto A C , Jones TC, R ichards F. The use o f different concentrations o f leishm anial anrigen in skin testing to evaluate delayed hypersensitivity in A m erican cutaneous leishm aniasis. R evista da Sociedade B rasileira de M edicina Tropical 18: 231-236, Out-Dez, 1985 10 0

the inoculation site and axillary lymphadenopathy (10% and 17% of patients with active or healed disease, respectively). 9.6yg/0.1 ml was the most satisfactory dose for routine diagnostic work since it was very sensitive but induced a degree of skin reaction which was only of tem porary inconvenience. The 10 volunteers from the University of Brasília were negative to ali three antigen concentrations. Reaction size in relation to antigen dose is expressed diagramatically in F igure 3 for patients with active lesions. This clearly shows the marked difference that concentration of antigen makes in skin test reactivity in this disease. 3 U g/0.1 ml yielded a scatter of skin reaction in patients with active disease, whereas 30 Ug always yielded reaction greater than 10 mm. In Table 3, the three antigen concentrations are related to lesions duration in eight recently acquired cases of L.b.brazilensis infections. Positive reactions to ali three antigens were obtained in one month after onset of the active lesions. The mean skin test reactivity to 3 lJg compared to time after initial lesion was 10 mm for less than 5 years, 9 mm for 6 to 10 years, 14 mm for 11 to 20 years in both groups with healed scars and mucosal disease. The intensity of the skin test could not be related to the age of the scar in the inactive group except that three patients with scars more than 30 years in the past yielded reactions to 3 UgN of 5,4 and 0 mm induration consistent with a slight age related decrease in Montenegro skin test reactivity.

80 -

60 -

40

20 r

ffl ü < jmm

5mm

>5- l 0mm >10iTim

:

>10mm plus necrosis

Figure 3. C om parison o f the m agnitude o f the sk in test reaction to three concentrations o f leishm anial antigen in 3 0 p atients with active lesions.

T he dose of 3 U gN /0.1 ml was selected to examine the relationship between mucosal disease and degree of skin reactivity. Table 4 lists the induration

Table 3 - Results o f the three doses o f skin test antigen in eight patients with proven active L. braziliensis braziliensis infection o f less than 5 m onths in duration. Paiient N um ber

D uration o f D isease (m onths)

1 2 3 4 5 6 7 8

M agnitude o f the Reaction in M illim eters *

1 2 2 2.5 2 3 4 5

3Ug N /0.1 ml

9.6 i-ig N /0.1 ml

30 yg N /0.1 ml

7.5 5.0 5.5 13.1 7.0 20.5 15.0 7.5

11.6 13.2 8.0 18.7 12.0 18.3 14.0 10.8

15.9 14.2 13.0 21.0 19.0 21.7 23.8 10.0

(*) M ean induration size for the three groups are 10, 13.3 and 17.3 mm respectively. Standard deviation. (Sx) 5.50; 3.66; 4.80.

Table 4 - S k i n testing in the various fo rm s o f Leishmaniasis. M ean diameter o f induration using the 3 Ug N /0 .1 m l Clinicai fo rm o f Leishm aniasis

N um ber o f Patients Studied

Active cutaneous Mucosal Scar

20 10 41

(*) N ot significant (p < 0.05) compared to scars only 234

M ean Induration in M illim eters ± 1 S D 10.7 11.3 9.3

± ± ±

6 .1 0 * 7 .1 5 * 6.78

Cuba CAC, M arsden PD, Barretto A C , Jones TC, R ichards F. The use o f different concentrations o f leishm anial antigen in skin testing to evaluate delayed hypersensitivity in A m erican cutaneous leishm aniasis. R evista da Sociedade Brasileira de M edicina Tropical 18: 231-236, Out-Dez, 1985

diameter in three groups of patients. N o significant difference between mucosal and cutaneous lesions in size of the delayed hypersensitivity reaction was noted. By comparing the percent of patients with reactions at 5 mm or greater, no differences were detected (M L 80% , H C L 71% , A C L 90% ). D IS C U S S IO N O ur study confirms that the M ontenegro skin test is a useful indicator of active or previous cutaneous leishmaniasis, but that the dose of antigen used determines the sensitivity of the test. 9.6 UgN per dose was found to be the minimum concentration which identified m ost patients. W e confirmed that the skin test was always positive by one month after onset of an active lesion, and persisted with the same degree of reactivity for at least 20 years. Thereafter, a slight decrease in skin test reactivity was noted. W e found no correlation between the occurrence of mucosal disease due to leishmaniasis and enhanced skin test reactivity. Skin testing with leishmania antigen to document the prevalence of cutaneous leishmaniasis has been used for many years. It has been considered a test with good s p e c i f i c i t y 19 an observation confirmed in our Brazilian control group, but few careful studies have been d o n e * 3. Furtado* has summarised w hatis known in Brazil regarding cross reactions with other diseases. Patients with leprosy and previous visceral leishmaniasis may react to leishmanin. Both these infections are very rare in our study area. W hen tests have been done in regions endemic for cutaneous leishmaniasis wide variations in skin test reactivity in patients without a history of disease or a scar have been reported. In our area we have found, in other studies^, that 6% of such patients yield a positive test. In the state of São Paulo 25.6% of people with positive skin reactions had no visible sign of leishmaniasis^^ ín M inas G erais 13.4% 12 jn the Xingu N ational Park for Indians in M ato G rosso 6 2 .5 % ^ in A m azônia 32.9% 8 and in Rio de Janeiro state figures vary from 3.7 - 24% depending on the community studied17 26 W hile different conditions of the test may account for some of this variation it is likely that subclinical infections occur without leaving a recongnisable scar. Increased sensitivity of the test by use of high antigen concentration was first reported by R o t b e r g ^ 4 who showed that with a concentration of 1 x 107 promastigotes per milliliter 92% of patients with cutaneous leishmaniasis had positive reactions. M elo et a l,(\ utilizing a concentration of 40 )Jg of protein nitrogen per milliliter had a 96% positivity rate and suggested that an antigen of this strenght was desirable. Our results support their findings but high strength antigens do produce undesirable skin reaction. On the other hand, we found that when the weakest antigen concentration was used 32% of the 41 patients with scars were non reactors. This is important since leishmanin skin test surveys are frequently used to

assess the incidence and prevalence of cutaneous leishmaniasis using similar antigen concentrations. O ur studies also confirm that antigens prepared from a different organism, L. m. am azonensis, induce satisfactory skin reactions in patients with proven lesions due to L. braziliensis braziliensis. This is not surprising since heterologous antigens outside the leishmania genus, such as T. c r u z fl1 22 ancj L eptom onas p esso a fl yield positive reactions in cutaneous leishmaniasis. The time of onset of skin test reactivity and its duration after the initial lesion has b een o f interest. We have noted, as have others^ 21_ that the test may be negative early in infection but, by one month, nearly ali are positive. Recently three groups of workers have studied leishmanin reactivity some years after an initial test and reported a decline in incidence of positive reactors14 15 20. This is a variance with the view that, once established, skin hypersensitivity persists indefinitely. Analysis of our data does not suggest that the time elapsed between the healing and the skin testing influences the intensity of the reaction except when lesions have been healed for more than 2 0 y e a r s *9. T he destructive effects of espundia have been attributed to a hypersensitivity phenomenorP or autoimmune reaction11. It has been suggested that the basic mechanisms of cutaneous and mucosal disease depend on the toxicity and immunogenicity of the parasites25. T o test the hypothesis that mucosal disease is associated with a greater degree of skin hypersensitivity to leishmanin it was necessary to select the results using the lowest concentration of antigen in view of our data in Table 2. W e detected no significant difference in skin test reactivity between mucosal and active cutaneous cases or mucosal and healed cutaneous cases. This is at variance with the observations made by Shaw and Lainson 197527, in a different region of Brazil. O ur data confirm the necessity to standardise the procedure of the M ontenegro both in the antigen used and the methodology of the test reading. Ideally, one carefully standardized antigen should be distributed by an international agency. This standardization should include careful taxonomy of the strain used for antigen production, concentration of promastigotes per milliliter, preparation method for preserving the antigens and total nitrogen determination. Each lot is best standardized biologically in infected man against a stock of antigen of known activity. A n exoantigen has been reported to yield an immediate hypersensitivity reaction in 74.4% of 41 patients27. This observation is of practical importance and should be further investigated since return after two days to read the reaction is often difficult under field conditions. ADDENDUM Recently Castes M, Agnelli A , Verde O & Rondón A J. (Characterization of the cellular immune response in American cutaneous leishmaniasis. Clinicai 235

Cuba CAC, M arsden PD, Barretto A C , Jones TC, R ichards F. The use o f different concentrations o f leishm anial antigen in skin testing to evaluate delayed hypersensitivity in A m erican cutaneous leishm aniasis. Revista da Sociedade B rasileira de M edicina Tropical 18: 231-236, Out-Dez, 1985

Immunology and Immunopathology 27: 176-186, 1983), have shown that the skin test response (as mean induration in mm.) was significantly greater in mucocutaneous leishmaniasis (M CL, 11 patients) than in 32 patients with localized cutaneous leishmaniasis (LCL). This difference from our results might be explained by the small groups examined in both series and the fact that some of our patients with mucosal disease had secondary malnutrition which would depress skin responses. ACKNOW LEDGEM ENTS W e wish to thank D r. John D avid for the initial suggestions leading to this study. Luiz C. Barreto and M ario Abreu gave valuable practical assistance in the field. R EFEREN CES 1. Ashton D L, Thorley A P. Leishm aniasis in Central Brazil. Results of a M ontenegro skin test survey among Am erindians in the Xingu N ational Park. Transactions of the Royal Society of Tropical Medicine and Hygiene 64: 671, 1970. 2. Azulay RD, Salgado U. Surto epidêmico da leishmaniose tegumentar em pára-quedistas do Exército no Amazonas. M edicina C utânea 1: 347, 1966. 3. Barbosa W . Souza M CM , Rassi D M , O liveira RL, M ota L. Investigação sobre imunologia da leishmaniose te­ gumentar americana. 1. Intraderm orreação concomitante com antígenos de L eptom onas pessoai e L. braziliensis. Revista de Patologia Tropical 1: 377-383, 1972. 4. Barretto A C, C uba C A C , M arsden PD , Vexenat JA , De Belder M. C aracterísticas epidemiológicas de leishma­ niose tegumentar am ericana em uma região endêmica do E stado da Bahia, Brasil. I. Leishmaniose humana. Boletim Oficina Sanitaria Pan-am ericana 90: 415-424 1981. 5. Bittencourt AL, A ndrade ZA . Aspectos immunopatológicos na leishmaniose cutaneomucosa. Hospital 71: 975-984, 1967. 6. Bray RS. Leishmaniasis. In: Houba V (ed)Immunological investigation of Tropical parasitic diseases. Churchill Livingstone, New York. p. 66-74, 1980. 7. Cuba CA. Leishmaniose tegum entar em area endêmica do Estado da Bahia. C aracterização e classificação de L eishm ania. no homem e cão doméstico e aspectos comportamentais de L. braziliensis braziliensis. Tese de Doutoramento. Universidade Federal de M inas G erais. Belo Horizonte-M G. Brasil. 1983. 8. Fonseca JM , Lacaz CS, M achado PA . Inquérito imunoalérgico na Am azônia. Resultados preliminares. Revista do Instituto de M edicina Tropical de São Paulo 15: 409-416, 1973. 9. Furtado TA. Critérios para o diagnóstico da Leishma­ niose Tegum entar A m ericana. Anais Brasileiros de Dermatologia 55: 81-86, 1980. 10. G oa A. A microbiuret method for protein determination of total protein in cerebrospinal íluid. Scandinavian Journal of Clinicai Investigation 5: 218-223. 1953. 11. Heyneman D. Immunology o f Leishmaniasis. Bulletin of the W orld Health Organization 44: 499-514. 1971. 12. Martins AV. Barreto M P, Brener Z, Pellegrino J. O bser­

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