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Leishmaniasis in Brazil: X. some observations on intradermal reactions to different trypanosomatid antigens of patients suffering from cutaneous and mucocutaneous leishmaniasis
323 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. 69. No. 3. 1975.
LEISHMANIASIS TO DIFFERENT
IN BRAZIL: X. SOME OBSERVATIONS ON INTRADERMAL REACTIONS TRYPANOSOMATID ANTIGENS OF PATIENTS SUFFERING FROM CUTANEOUS AND MUCOCUTANEOUS LEISHMANIASIS
J. J. SHAW AND R. LAINSON The Wellcome Parasitology Unit, Institute Evandro Chagas, Belbm, Pat-d, Brazil.
In 1923 WAGNER, using an alkaline extract of Leishmania tropica and L. infantum, noted delayed hypersensitivity reactions in guinea pigs that had previously been immunized with the same parasites. Stimulated by these results MONTENEGRO (1926) used the same type of skin test in patients suffering from cutaneous leishmaniasis. He obtained positive results with an alkaline extract of washed promastigotes of a strain of L. braziliensis and with the medium in which they had been grown. Buss (1929) and GOMES (1939) obtained stronger reactions with whole promastigotes suspended in phenolized saline. This antigen is what is generally nowadays called leishmanin and is what most workers use in routine clinical and epidemiological studies. Positive leishmanin tests in bacterial and non-trypanosomatid parasitic diseases are uncommon and seem to occur mainly in casesof tuberculosis with lymph node lesions (CORRGA, 1941). Exceptional positive reactions noted by some authors in certain mycotic infections have been attributed to past leishmanial infection, which on clinical grounds is difficult to exclude. Many species of Leishmania, including species that are not pathogenic to man such as L. enriettii, have given delayed responses in man (MUNIZ and MEDINA, 1948). Apart from whole organism suspensions, sonicated promastigotes (CORR~A and AMATO, 1957) and leishmanial polysaccarides (FURTADO and PELLIGRINO, 1956) have been used successfully as skin test antigens. Trypanosomatids other than Leishmania have given specific delayed responses in patients suffering from cutaneous leishmaniasis (Trypanosoma cruzi-PESSOA and PESTANA, 1940; Crithidia oncopeltiZELDON et al., 1960; T. equiperdum-DEPIEDE et al., 1958; Leptomonas ctenocephali and C. fasciculataRANQUE and DUNAN, 1964; L. pessoai-&RBOsA et al., 1972). Such reactions reflect group antigens, which creates potential difficulties in areas where people may be sensitized by such flagellates as T. cruzi (AMATO et al., 1964) or Leishmania species of lizards (SOUTHGATE,1967). In most of the papers dealing with the reactions caused by heterologous antigens the apparently homologous antigen often gave smaller reactions. This could possibly be due to the difficulty of standardizing antigens used in skin tests. ADLER (1965) produced delayed hypersensitivity reactions in volunteers immune to L. tropica, with antigens secreted by living amastigotes and promastigotes of L. mexicana mexicana. SERGIEV and SHUIKINA (1969) compared the reactions produced by killed promastigotes of L. t. major and the liquid phase of the medium in which they had been cultivated. They concluded that the overlay gave stronger reactions than washed promastigotes. While studying the antigens of T. cruzi TARRANT et al. (1965) noted that the culture forms produced an in vitro exo-antigen (IEX-antigen) that was very specific in complement fixation (CF) tests with human sera. It would seem that all the above antigens and one of those used by MONTENEGRO (1926) are of a similar nature. The present work compares the cutaneous hypersensitivity reactions of patients suffering from cutaneous leishmaniasis to whole organism and IEX-antigen of L. m. amazonensis and T. cruzi. Attempts are made to correlate skin reactions with causative Leishmania species and different clinical conditions.
These studies were conducted under the auspices of the Wellcome Trust, London, Instituto Evandro Chagas of the Funda@o Instituto Oswald0 Cruz of Brazil, Funda@o Serviqo Especial de Sacde Pliblica of Brazil and the World Health Organization, Geneva. We would like to thank Sebastiao Oliveira and Henrique Buna for their able assistance in handling the various Leishmania strains and to Roberto Naiff for his help in isolation of strains and skin testing. We would also like to thank Dr. Barry Bloom for stimulating comments. We are also most grateful to all the volunteers without the co-operation of whom this work would have been impossible.
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J. J. SHAW AND R. LAINSON
325
Materials and methods (i) Parasitological examination. Biopsies were taken from the indurated edges of lesions. Impression smears from this material were rapidly dried, fixed in absolute methanol and stained in double strength Giemsa (2 ml. of stain to 15 ml. of pH 75 buffered distilled water) for 45 minutes. A small piece of tissue was fixed in Carnoy’s fluid. The rest of the material was ground up in a pestle and mortar, containing a small quantity of sterile sand in 1.0-l-5 ml. of sterile saline containing 200 units of penicillin G (crystalline sodium) and 100 pg. streptomycin sulphate per ml. and stored at 4°C. for 4-6 hours. A quantity of O-1 ml. of the ground tissue was inoculated intradermally into the nose and hind foot of 2 hamsters (Mesocricetus auratus). These animals were kept under observation for periods up to 1 year. Smears and cultures were made from any lesions that appeared. The strains isolated in the above manner were grouped according to the classification proposed by LAINSON and SHAW(1972). (ii) Preparation of antkens. The antigens used were prepared from strain Ph 8 of L. m. amazonensis that had been isolated from a naturally infected Lutzomyia jlaviscutellata captured in the Utinga forest Belem (LAINSON and SHAW, 1968) and strain CAN 1 of T. cruzi isolated from an acute case of Chagas’ disease from Beltm (SHAW et al., 1969). The flagellates were grown in dialysis membrane sacs (TOBIE and REES, 1948) according to the method described in detail by FIFE and KENT (1960). We did not, however, use the same culture medium as these authors because during preliminary trials we found that L. m. amazonensis did not grow well in this medium. The other major modification was the use of simple Locke’s solution in the dialysis sac. This was done to avoid contaminating the IEX-antigen with substances likely to provoke non-specific hypersensitivity reactions. The medium outside the sac was a brain/heart broth with 10% (v/v) human blood (group 0, D+) and 6% (v/v) potato extract. The blood was added under sterile conditions to the medium and sterile Locke’s solution was similarly added to the dialysis sac. Although 50 ml. was added to each sac only between 30 to 35 ml. was harvested. This same medium was used with agar and a Locke’s solution overlay: the blood was added, in this case, while the temperature of the medium was 56”C., as was the contents of the dialysis sac. Strains of L. m. amazonensis grew equally well in both types of media but strains of L. braziliensis grew slightly better in the diaphasic medium. Antigen preparations used for skin testing were grown in the broth medium. Promastigotes used to start the dialysis sac cultures were grown in NNN medium prepared from nutrient agar (Oxoid code No. CM3) and 10% (v/v) rabbit blood with an overlay of 0.9% (w/v) saline. The parasites were harvested at 7-10 days whilst in their log phase of growth and washed 3 times in cold Locke’s solution. The flagellates from 4-6 tubes were used to sow the dialysis cultures. Cultures were examined periodically and were harvested while in their log phase of growth: this varied from 16-25 days,
Opposite Page :FIG. 1. Anaphylactic
reaction to L. mexicana amazonensis in vitro exo-antigen in an individual leishmaniasis caused by L. braziliensis guyanensis.
with cutaneous
FIG. 2. Anaphylactic
reaction to L. mexicana amazonensis in vitro exo-antigen in an individual leishmaniasis caused by L. braziliensis bradiensis.
with cutaneous
FIG. 3. Skin test sites to leishmanin (left) and leishmanial in vitro exo-antigen (right) at 30 minutes, in a patient with very strong delayed hypersensitivity reactions to both antigens, showing absence of anaphylactic responses. FIG. 4. A lesion of approximately
3-4 weeks evolution on the elbow (Case 1253) caused by L. mexicana amazonensis associated with a weak leishmanin reaction.
FIG. 5. Extensive
cutaneous lesions caused by L. mexicana amazonensis on the elbow of a 4 year old girl, similar lesions distributed on face, arms and legs, associated with negative delayed responses (diffuse cutaneous or Hansenoid anergid leishmaniasis). FIG. 6. Mucocutaneous leishmaniasis (Case 1287) caused by L. bradiensis braziliensis with extensive destruction of nasal septum and active cutaneous lesions on other parts of the body associated with a strong delayed hypersensitivity reaction.
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The sac was removed and the outside carefully washed with cold physiological saline to remove any adherent culture medium, One end of the sac was held in a pair of Spencer Wells forceps, and cut; the cut end was then introduced into the neck of a flask, the pressure of forceps released and the contents of the sac allowed to pass into the flask. The promastigotes were separated from the sac medium by centrifugation at 11,000 G for 30 minutes at 4°C. The supernatant was removed, passed through a millipore filter (pore size 0.22pm.) and dialyzed against 3 changes of 0.9% (w/v) saline during 24 hours at 4°C. It was again passed through a millipore filter and lyophilized in quantities of 05 ml. For use in skin-testing, this was reconstituted with 05 ml. of sterile distilled water and constituted the final leishmanial in vitro exoantigen (LIEX-antigen). Leishmanin was prepared from the promastigote sediment, in saline containing 05% (w/v) phenol, and at a final concentration of 5 x lo6 organisms per ml. Similar antigens were prepared from dialysate culture of T. cruzi (strain CAN 1) to give trypanosomal in vitro exo-antigen (TIEX-antigen), and trypanosomin which was a 5 x lo6 suspension of culture forms in saline containing 0.5% (w/v) phenol. T. cruzi grew more slowly than the Leishmania and was only suitable for harvesting between 30 to 40 days. The IEX-antigen was stored at -20°C. and the leishmanin and trypanosomin were stored at 4°C. Three other antigens were also prepared-a x 5 leishmanin antigen which gave the same number of organisms per ml. as in the original dialysate culture; 1 : 2 dilution of the LIEX-antigen; and a 1 : 2 LIEX-antigen containing 0.5% (w/v) phenol. Uninfected Locke’s solution from a sterile dialysis sac that had been incubated for 25 days was lyophilized and treated in the same way as the supernatant of the dialysate culture. This was used as the control for the IEX-antigens, while 0.9% (w/v) saline containing 0.5% (w/v) phenol was used as the control solution for the leishmanin and trypanosomin. Skin tests. 0.1 ml. amounts of a given antigen and the control solution were inoculated into the inner surface of the forearm of patients with parasitologically proven cutaneous leishmaniasis. In general more tests were performed with the leishmanin; but paired tests were performed with leishmaniu and LIEXantigen and, in chosen cases, with the T. cruzi antigens and control solution. Whenever possible the test . sites were examined at 15,30 and 60 mmutes and then at 8,24,48 and 72 hours. Any erythema or induration was measured and the thickness of paired tests was determined using a pair of calipers. The volume of 4 the skin reaction was calculated according to BRYCESON et al. (1970), in which v = - ic r2t 3 (r = radius of erythema; t = thickness). Both antigen and control solutions were inoculated into a group of normal control volunteers who had no history of leishmaniasis, patients with proven Chagas’ disease, and others with bacterial or mycological skin problems such as tropical ulcer and sporotrichosis. Reactions read at 15 and 30 minutes were considered positive when there was a definite erythema beyond the area of induration. In dark skinned people this was often difficult to judge, and in such casesa clearly oedematous swelling 12 mm. or more was considered as positive. Delayed reactions in which the indurated area measured 5 mm. or more were taken as positive. Results A summary of the hypersensitivity reactions produced by both types of leishmanial antigen are given in Tables I to III and a summary of the different types of reactions that may possibly be provoked by all the antigens is given in Table IV. Examples of some of the different sequences of reactions seen in different infections are given in Figs. 7-12. Not all the skin tests done were recorded, either because the patient failed to return or the infection could not be confirmed and studied. In normal volunteers the leishmanin and trypanosomin produced no hypersensitivity reactions. In two instances false positives of 6 mm. were noted with the leishmanin at 24 hours but these disappeared by 48 hours. The IEX-antigen did not produce false positive reactions in normal control volunteers. The increase in volume of this group varied from 70-420 nun3 at 30 minutes, which by 8 hours had disappeared. Patients with sporotrichosis (6), blastomycosis (2) and Chagas’ disease (5) did not react to any of the antigens. One patient with lepromatous leprosy had a strongly positive leishmanin test at 48 hours (LIEX-antigen was not tested). He had no past history of a possible leishmanial infection and no history of living in an endemic area. Control solutions to the different antigen preparations did not elicit hypersensitivity reactions.
327
J. Je SHAW AND R. LAINSON TAsiE
I. The results of skin reactions read at 30 minutes using an in vitro exo-antigen prepared from Leishmania mexicana amazonensis in patients suffering from cutaneous leishmaniasis. Positive
---
Infecting organism
Type of lesion
Diameter of erythema 15-30 mm. >30 mm.
Negative
Simple cutaneous
7
7
6
Mucocutaneous
3
0
3
L. b. guyanensis
Simple cutaneous
3”
5”
0
L. m. amazonensis
Simple cutaneous
5
0
2
Anergic cutaneous
2
0
0
20
12
11
L. b. braziliensis
---
Total *At 48 hours reactions negative in 3 cases.
TABLE
II. The results of leishmanin tests read at 48 hours using an in vitro exo-antigen prepared from
Leishmania mexicana amazonensis in patients suffering from proved cutaneous or mucocutaneous leishmaniasis.
Infecting organism L. b. braziliensis
L. b. guyanensis
L. m. amazonensis
Type of lesion Simple cutaneous
Positive 5-10 mm. >lO mm.
Negative
17
7
3
Mucocutaneous
5
6
3”
Simple cutaneous
9
1
3
Multiple cutaneous
2
0
3
Simple cutaneous
4
0
2
Anergic cutaneous
0
0
4
37
14
18
Total +Leishmanin positive.
TABLE III. The results of leishmanin tests read at 48 hours using an antigen of phenolized promastigotes of Leishmania mexicana amazonends in patients suffering from proved cutaneous or mucocutaneous leishmaniasis.
---
Infecting organism
L. b. braziliensis
--L. b. guyanensis
L. m. amazonensis
Type of lesion
Positive
Negative
Simple cutaneous
45
8
Mucocutaneous
19
0
Simple cutaneous
15
3
Multiple cutaneous
5
3
Simple cutaneous Anergic cutaneous
5 0
1 4
89
19
Total
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7. Cutaneous hypersensitivity reactions to two types of leishmanial antigen in a cured L. mexicana amazonensis infection (Case 1253) and normal volunteers. FIG.
8. Cutaneous hypersensitivity reactions to two types of Trypanosoma cruzi an&en in a cured L. mexicana ama&nensis infection (Case 1253) and normal volunteers. FIG.
9. Cutaneous hvpersensitivity reactions to - two types of L. mexicana amazonensis antigen in patients with cutaneous lesions caused by L. mexicana u.mazonensis. FIG.
J. J. SHAW AND R. LAINSON
FIG. 10. Cutaneous hypersensitivity reactions to two types of L. mexicana amazonensis antigen in patients with cutaneous lesions caused by L. braziliensis braziliensis.
FIG. 11. Cutaneous hypersensitivity reactions to two types of L. mexicana amazonensis antigen in patients with mucocutaneous lesions caused by L. braziliensis braziliensis.
FIG. 12. Cutaneous hypersensitivity reactions to two types of L. mexicana amazonensis antigen in patients with cutaneous lesions caused by L. braziliensis guyanensis.
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Type of cutaneous hypersensitivity Type of Antigen --Leishmanm Trypanosomin
Immediate anaphylactic
Delayed
-
+
-
+
+
-
-
+
TABLE IV. Summary of skin responsesof individuals with active cutaneous leishmaniasis to different trypanosomatid antigens.
LIEX TIEX 1 : 2 LIEX Phenolized 1 : 2 LIEX
la. Immediate anaphylactic hypersensitivity. 32 out of 43 cutaneous leishmaniasis cases had typical weal and flare reactions (Figs. 1 and 2) with the LIEX-antigen at 30 minutes, that then subsided. These figures include the 36 cases mentioned in our preliminary note (SHAW and LAINSON, 1974). Table I summarizes these reactions in relation to the different Leishmania. Only two patients reacted similarly to the leishmanin and, in both, the erythematous area elicited by the LIEX-antigen was large, measuring over 50 mm. diameter: that produced by the leishmanin was in the order of 20 mm. diameter. Three individuals with immediate reactions did not have delayed responses to leishmanin or LIEX-antigen. Positive reactions equal in size to that of the standard LIEX-antigen were produced by the 1 : 2 LIEXantigen, but neither by the phenolized 1 : 2 LIEX-antigen norby the x 5 leishmanin. More positive immediate responses were noted in infections of L. b. guyan-ensisthan L. b. braziliensis (P = <0.05). No such differences were seen between infections of these two leishmania and L. m amazonensis. The average volume of the L. b. guyalzensis immediate reactions was 5.13 cm.3 and of the L. b. braziliensis infections 356 cm.3 (Figs. 11 and 12). Appropriate t-tests indicated that these differences were not significant at the 5% level. Two cases of anergid diffuse leishmaniasis due to L. m. amazonensis had weak immediate responses, as did 3 of the mucocutaneous cases. The infections were regrouped according to their estimated time of evolution and it was noted that the larger reactions (I? =
J. J. SHAW AND R. LAINSON
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Individuals with mucocutaneous leishmaniasis (Figs. 4 and 11) had significantly larger (I? =
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LEISHh4AhUSIS IN BRAZIL
antigen composed of at least 5 different types of glycoproteins (NJOGU and HUMPHREYS, 1972). ALLSOPP et al. (1971) concluded that the “exo-antigen” and “4s-antigen” should be replaced by a single term “surface antigen”. In respect to the importance of surface antigens in immunity it is interesting to note that JOHNSON et al. (1963) noted that the surface antigens of T. cruzi blood forms promoted better protection in mice than did vaccine prepared from trypomastigotes. We do not know what, if any, is the relationship of the exo-antigen produced in vitro, by culture forms, to the surface antigens produced by trypanosomes in vivo. Electronmicroscopic studies of Leishmania amastigotes and promastigotes have not indicated the presence of a surface coat. It is possible that the stage of development of a trypanosomatid is the factor that determines the presence or absence of a surface coat. VICKERMAN (1969) noted that the only culture forms with a surface coat in the vector were the metacyclic trypomastigotes. Antigens equivalent to surface antigens may be produced by other forms but, instead of remaining on the surface, they may be directly liberated into the surrounding media. This kind of process could be associated with amastigotes, promastigotes and epimastigotes, either in vivo or in vitro. ADLER (1965) suggested that Leishmania IEX-antigen may be derived from the flagellar vacuole. In leishmaniasis it is not known whether immunity is of the sterile or non-sterile type. Human cases have been recorded as developing long after the persons left an endemic area (CLAY and ROSS, 1956). In South America, mucocutaneous leishmaniasis occurs many years after the initial lesion and is often associated with a debilitated state (WALTON et al., 1973). There is much evidence that leishmanial infections in wild animals may be cryptic (HERRER and TELFORD, 1969; HERRER et al., 1966; HEFXER et al., 1971; LAINSON and SHAW, 1968; 1973). In man it is possible that a successful primary immune response results in the elimination of the parasites: if it is inadequate, however, pathological lesions may be resolved but the parasites remain as an occult infection. The subsequent clinical outcome of such infections may depend on the type of Leishmania. The fact, however, that living organisms liberate antigenic substances means that these could be as or more important in stimulating immune responses of the host than somatic antigens. The latter would presumably only become available on the death of the organisms. TURK (1972) has drawn attention to the differences that exist between delayed hypersensitivity reactions and cellular immunity, and suggested that antigens that cause skin reactions may not necessarily be those that give rise to immunity. Arguing on similar lines it might be said that the antigens that give rise to immunity have not been used as skin test antigens and this could lead to apparently discordant results. In this paper we have shown that, in man, more than one type of antigen can elicit delayed responses and the same may be true of immediate anaphylactic type reactions. In all comparisons of skin-test antigens, however, it is difficult to distinguish between qualitative differences in the absence of suitable methods for antigen standardization. NEAL (pers. comm.) has emphasized this point in his finding that potency of antigen in skin-tests does not appear to be directly related to number of organisms, nor their nitrogen or protein content. Such problems make it even more difficult to carry out quantative studies of skin reactions to different antigens. In the present work this was to some degree compensated for by the use of the same antigen batches for all the comparative studies. The percentage of positive Montenegro reactions in South American cutaneous leishmaniasis varies from 865% (MONTENEGRO, 1926) to 100% (BARBOSA et al., 1972). Such differences may depend upon the method of selection of the patients. Our results are very similar to those of MONTENEGRO (1926) and were chosen, as were his, by simply testing all patients that were available. In comparing the delayed reactions of our parasitologically proven casesof L. braziliensis with similarly diagnosed casesof L. tropica (DOSTROVSKY and SAGHER, 1946), significantly less (I’ = <0*05) patients with L. brazdiensis had a positive leishmanin test. Immediate anaphylactic type responses were less common in L. b. braziliensis than L. b. guyanensis infections. When these infections were grouped according to similar ages the samples were smaller and the differences were no longer significant, although larger reactions occurred significantly more frequently in the infections of 6 months or less. Much of our data failed to indicate significant differences but certain trends were noted, such as the tendency for L. b. bradiensis infections to have larger delayed responses to LIEX-antigen. If the L. b. guyanensis and L. m. amazonensis infections were grouped together, excluding the anergid diffusa cases, the difference was significant (I’ = <0.05). There is therefore evidence suggesting that both time of infection and the type of Leishmania influence the spectrum of hypersensitivity reactions. FURTADO a.nd PELLEGRINO (1956) questioned the specificity of the standard leishmanin and considered that the polysaccaride fraction gave better specificity. Positive leishmanin reactions have been noted in
J. J. SHAW AND R. LAINSON
333
individuals suffering from tuberculous involvement of the lymph nodes (CORR~A, 1941) but not in other types of tuberculosis. In general, cases of leprosy also do not react to leishmanin (ARANTES, 1941) but positive reactions have been noted in the lepromatous form of the disease (FURTADO and PELLEGRINO, 1956, and present work). Such reactions are interesting as they might be regarded as being due to some common antigen. In CF tests the sera of kala-azar patients react strongly with a number of different mycobacterial antigens. The incidence of false positives in apparently healthy individuals varies, with leishmanin, from 15% (SOUTHGATE and ORIEDO, 1967) to 6.3% (DOSTROVSKY and SAGHER, 1946) but such reactions have not been associated with any other infections. To date, skin reactions in Chagas’ patients have been equivocable. MAYER and PIFANO (1941), using culture forms of T. cruzi, obtained positive delayed reactions in 6 patients with Chagas’ disease. Individuals with cutaneous leishmaniasis did not react to the trypanosomal antigen but did react to the leishmanin antigen. PESS~A and PESTANA (1940) found that persons with cutaneous leishmaniasis had equally strong delayed hypersensitivity reactions to both leishmanin and trypanosomin antigen. Subsequently PESSQ and CARDOSA(1942) skin-tested persons with Chagas’ disease and cutaneous leishmaniasis and again found that the individuals with cutaneous leishmaniasis reacted to both antigens, but those with Chagas’ disease reacted to neither. AMATO et al. (1964) performed intradermal tests with lysed amastigotes and trypomastigotes of T. muzi, suspended in distilled water containing 1 : 5,000 merthiolate, in patients with Chagas’ disease and cutaneous leishmaniasis. The test sites were examined at 15 and 30 minutes but there was no evidence of anaphylactic hypersensitivity. Of 12 individuals with Chagas’ disease 8 had weak delayed reactions, while 7 patients with cutaneous leishmaniasis all reacted strongly to this same antigen. In the present work we found, as had PESS~A and CARDOSA (1942), that no hypersensitivity reactions were elicited in Chagas’ disease patients using antigens prepared from the culture forms of Leishmania or T. cruzi, Possibly the type of antigen produced by the culture forms influences the demonstration of delayed hypersensitivity in Chagas’ disease. There is little danger of false positives arising from undiagnosed cases of Chagas’ disease if the IEX leishmanial antigen is used. Diagnostically it would be an advantage to have a skin test that could be read rapidly. The present results with LIEX-antigen indicate that it is potentially useful, especially in early cases when there are specific reactions between 15 to 30 minutes which, in certain instances, are not associated with a delayed response. The LIEX-antigen by comparison with the TIEX-antigen, in delayed responses, would seem to be specific at the generic level, but this is not true of the immediate anaphylactic type responses. Since we have as yet been unable to grow strains of L. braziliensis in dialysate medium we cannot compare homologous antigen with the heterologous L. m. amazonensis antigen in patients with L. braziliensis infections. Some workers (SERGIEV and SHUIKINA, 1969a) consider that the most suitable antigen for skin testing is a suspension of unwashed promastigotes at a concentration of 1 x loj promastigotes per ml., in Hank’s solution containing 0.25% phenol. The effect of the phenol on the IEX-antigen, however, would seem to us to be a disadvantage in such an antigen, because immediate reactions would no longer be elicited. The future selection of skin test antigen will depend on careful comparative studies and the development of suitable methods j of standardization of preparations. Cell mediated immunity, dependent on T-lymphocytes, is responsible for delayed hypersensitivity reactions, but antibodies derived from B-cells are important in immediate reactions. PESS~A and PESTANA (1940) and PESS~A (1941) obtained positive Prausnitz-Kiistner reactions with the sera from 3 individuals with cutaneous leishmaniasis, in 5 volunteers. CURBAN (1941) repeated this with sera of 3 patients, in 6 individuals, but obtained negative results. DOSTROVSKY and SAGHER (1946) produced Prausnitz-Kiistner reactions with the sera of 2 patients who had been infected with L. tropica in 4 out of 11 attempts. These would appear to be the first records of a reaction involving reagin (IgE) in the donors. The finding of immediate anaphylactic responses to LIEX and TIEX-antigen in some patients with active cutaneous leishmaniasis is further evidence for the presence of cell-bound antibody. The detection of circulating antibody has been attempted with various serological techniques. Their presence has been reported using indirect fluorescent antibody (IFA) tests but some of the results should be viewed with certain reservations. WALTON et al. (1972) found that promastigotes fluoresced strongly with sera from proven cases but that this type of antigen lacked sensitivity and specificity. These same authors obtained greater specificity using amastigotes grown in tissue culture as antigens. They concluded that antibodies were produced early in the infection but appeared to be unrelated to the extent or duration of the infection. Antibodies have also been detected by the indirect haemaglutination test (BRAY and LAINSON, 1967; ANTUNES et al., 1972), but the lack of specificity makes this test at present unsuitable for detecting low antibody levels. Com-
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LEISHMANIASISIN BRAZIL
plement fixation tests have not been specifically used for the detection of antibodies in cutaneous leishmaniasis but cross reactions have often been noted with certain T. cruzi antigens (TARRANT et al., 1965). In CF tests with sera of mucocutaneous leishmaniasis, we have noted reactions typical of antigen deficiency due to the presence of minor antigens. Such cross reactions did not occur when the TIEX-antigen was used. It would seem, therefore, that both cell-bound and circulating antibodies occur in cutaneous leishmaniasis. The present work indicates that cell-bound antibodies occur early in the infection. There now seems sufficient evidence to indicate that both cell-mediated and humoral responses occur in cutaneous leishmaniasis and that, in part, cutaneous hypersensitivity reactions are reflections of the two systems. It would appear that cell-bound antibodies are formed early in the infection, as shown by the immediate anaphylactic responses, followed by the development of both humoral and cell-mediated responses (TREMONTI and WALTON, 1970). As the infection progresses, humoral antibodies may reach such a level as to block anaphylactic reactions. Subsequently a successful immunological answer is reached whereby the parasite is either eliminated or continues as an occult infection. Epidemiological evidence suggests that many more people contract leishmaniasis in the Amazon region and recover, than seek medical assistance. Some of those that seek medical help presumably had an inadquate initial response. PRESTON (1973), in discussing the disease spectrum of leishmaniasis, suggested that either stimulation or suppression of the immune response could lead to non-healing forms of the disease. Thus the existence of latent infections could lead to an allergic state responsible for such conditions as mucocutaneous leishmaniasis, and suppression, brought about by an initial heavy infection (TURK and BRYCESON, 1971), could lead to diffuse anergid cutaneous leishmaniasis; in both cases the continued presence of parasites would be associated with the liberation of exo-antigen. Such material may be more important in causing immune deviation than somatic amastigote antigen. It may well be that such antigens will be necessary when investigating cell mediated responses of man in vitro. It has recently been shown (MUNRO et al., 1972) that lymphocyte transformation in post-dermal kala-azar dermal leishmanoid was elicited by L. donovani antigen but not by L. tropica. The above mentioned sequence of events is of course hypothetical but seems to fit the available information that we at present have on the immunological responses in cutaneous leishmaniasis in the New World. Summary In spite of the difficulty of antigen standardization, paired intradermal skin tests demonstrated qualitative differences between leishmamn and in vitro exo-antigen. Some of these differences may be due to the destruction of certain antigens by phenol, while others seem to reflect basic antigenic differences. Leishmania mexicana amazonensis in vitro exo-antigen produced immediate anaphylactic responses in 74.4% of the patients with parasitologically proven cutaneous leishmaniasis. Although more individuals responded to leishmanin, 82.4% as compared to 73.9%, the leishmanial in vitro exo-antigen gave significantly larger delayed responses. Trypanosoma cruzi trypamosomin elicited delayed reactions, while trypanosomal in vitro exo-antigen only produced immediate anaphylactic reactions in persons with active cutaneous leishmaniasis. Significantly more immediate anaphylactic responses were seen in L. brasiliensis guyanensis infections, and more marked responses occurred in infections of 6 months or less duration. Patients with mucocutaneous leisbmaniasis had larger delayed responses than individuals with cutaneous infections due to L. b. braziliensis. Individuals with diffuse anergid cutaneous leishmaniasis had negative delayed reactions to all antigens, but two had weak anaphylactic reactions. Arthus-type responses were uncommon; they occurred in 4 people with mucocutaneous leishmaniasis, and in 3 with cutaneous leishmaniasis caused by L. b. guyanensis. No hypersensitivity reactions were seen in patients with Chagas’ disease. REFERENCES ADLER, S. (1965). Israel J. Med. Sci., 1, 9.
B. A., NJOGU, A. R. & HUMPIIREYS, K. G. (1971). Expl Purasit., 29, 271. A. P., TAVARES, C. A. I?. 81 PELLEGRINO, J. (1972). Revta Inst. Med. trop. S. Paulo, 14, 203. AMATO, V., MAGALDI, C. & PESS~~A, S. B. (1964). Revtagoiuna Med., 10, 121. ARANTES, S. C. (1941). Arches Hig. Saiide ptibl., 6 (13), 67. BARBOSA,A., SOUSA,M. C. M., RASSI, D. M., OLIVEIRA, R. L. & MOTA, L. (1972). Revta Pat. trop., 1, 377. BRAY, R. S. & LAINSON, R. (1966). Trans. R. Sot. trop. Med. Hyg., 60, 605. T& (1967). Ibid., 61, 490. ALLSOPP,
ANTUNES, L. J., REIS,
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BRYCESON,A. D. M., BRAY, R. S., WOLSTENCROFT,R. A. & DUMONDE, D. C. (1970). Clin. & .Ex# Immunol., 7, 301. Buss, G. (1929). Arch. Schifls-u. Tropenhyg., 33, 65. CLAY, R. D. & Ross, A. A. (1956). Br. med. J., 1, 1279. CLINTON, B. A., PALCXJK, N. C. & STAUBER, L. A. (1972). J. Immun., 108, 1570. CO&A, C. (1941). Arches Hig. sulide ptibz., 6 (13), 61. COF&A, M. 0. A. & AMATO, V. (1957). Revta Inst. Adolph0 Lutz, 17, 39. CuRBAN, G. V. (1941). Revtu pa&. Med., 19, 174. DEPIEDS, R., COLLOMB, H., MATHORIN, J. & RANQUE, J. (1958). BUZZ. Sot. Path. exot., 51, 501. DOSTROVSKY,A. & SAGHER, F. (1946). Ann. trap. Med. Parasit., 40, 265. FIFE, E. H. & KENT, J. F. (1960). Am. J. trop. Med. Hyg., 9, 512. FURTADO, T. A. & PELLEGRINO, J. (1956). J. invest. Derm., 27, 53. GOMES, L. S. (1939). Bras.-me’d., 53, 1079. JOHNSON, P., NEAL, R. A. & GALL, D. (1963). Nature, Lond., 200, 83. HERRER, A., THATCHER, V. E. & JOHNSON, C. M. (1966). J. Parasit., 52, 954. & TELFORD, S. R. (1969). Science, 164, 1419. & CHRISTENSEN,H. A. (1971). Ann. trop. Med. Purasit., 65, 349. ‘-> LAINSON, R. & SZIAW, J. J. (1968). Trans. R. Sot. trop. Med. Hyg., 62, 385. (1972). Br. med. Bull., 28, 44. -: (1973). Bull. Pan. Am. Hlth Org., 7 (4), 1. MAYER, M. & PIFANO, F. C. (1941). Bras.-mid., 55, 317. MONTENEGRO, J. (1926). Archs Derm. Syph., 13, 187. MUNIZ, J. & MEDINA H. (1948). Hospital, Rio de J., 33, 35. MUNRO, D. D., Du VIVIER, A. & JOPLING, W. H. (1972). Br. J. Derm., 87, 374. NJOGU, A. R. & HUMPHREYS, K. C. (1972). ExpZ Parasit., 31, 178. PESS~A, S. B. (1941). Revtapaul. Med., 19, 236. & CARDOSA,F. A. (1942). Hospital, Rio de J., 21, 187. & PESTANA, B. R. (1940). S PuuZo me’d., 2 (5/6), 133. PRESTON,P. M. (1973). Proc. R. Sot. Med., 66, 776. RANQUE, J. & DUNAN, S. (1964). Ann. Purusit. hum. camp., 39, 117. SCHNUR, L. F., ZUCKERMAN, A. & GREENBLATT, C. L. (1973). J. ProtozooZ., 20, 534. SERGIEV, V. P. & SHUIKINA, E. V. (1969). Medskaya Parazit., 38, 208. -& (1969a). Ibid., 38, 562. SHAW, J. J. & LAINSON, R. (1974). Trans. R. Sot. trop. Med. Hyg., 68, 168. & FRAIHA, H. (1969). Revta S&de Pzibl., 3, 153. -> SOUTHGATE, B. A. (1967). J. trop. Med. Hyg., 70, 33. & ORIEDO, B. V. E. (1967). Ibid., 70, 1. TARRANT, C. J., FIFE, E. H. & ANDERSON, R. I. (1965). J. Parasit., 51, 277. TOBIE, E. J. & REES, C. W. (1948). Ibid., 34, 162. TREMONTI, L. & WALTON, B. C. (1970). Am. J. trop. Med. Hyg., 19, 49. TURK, J. L. (1972). Immunology in Clinical Medicine, p. 56. London: William Heinemann. & BRYCESON,A. D. M. (1971). Adv. ImmunoZ., 13, 209. VICKERMAN, K. (1969). J. CeZZSC;., 5, 163. WAGNER, E. H. (1923). Univ. Calif. Pubis Zool., 20, 477. WALTON, B. C., BROOKS,W. H. & ARJONA, I. (1972). Am. J. trop. Med. Hyg., 21,296. -, CHINEL, L. V. & EGUIA, 0. E. (1973). Ibid., 22, 696. WEITZ, B. G. E. (1960). Nature, Lond., 185, 788. WEISSBERGER,H., SPIRA, D. T. & ZUCKERMAN, A. (1973). 3. Protozool., 20, 534. WILLIAMSON, J. & BROWN, K. N. (1964). ExpZ Par&t., 15, 44. ZELED~N, R., HIDALGO, W. R. & HIDALGO, H. X. (1960). Revta BioZ. trop., 8, 145.
LEISHMANIASIS TO DIFFERENT
IN BRAZIL: X. SOME OBSERVATIONS ON INTRADERMAL REACTIONS TRYPANOSOMATID ANTIGENS OF PATIENTS SUFFERING FROM CUTANEOUS AND MUCOCUTANEOUS LEISHMANIASIS
J. J. SHAW AND R. LAINSON The Wellcome Parasitology Unit, Institute Evandro Chagas, Belbm, Pat-d, Brazil.
In 1923 WAGNER, using an alkaline extract of Leishmania tropica and L. infantum, noted delayed hypersensitivity reactions in guinea pigs that had previously been immunized with the same parasites. Stimulated by these results MONTENEGRO (1926) used the same type of skin test in patients suffering from cutaneous leishmaniasis. He obtained positive results with an alkaline extract of washed promastigotes of a strain of L. braziliensis and with the medium in which they had been grown. Buss (1929) and GOMES (1939) obtained stronger reactions with whole promastigotes suspended in phenolized saline. This antigen is what is generally nowadays called leishmanin and is what most workers use in routine clinical and epidemiological studies. Positive leishmanin tests in bacterial and non-trypanosomatid parasitic diseases are uncommon and seem to occur mainly in casesof tuberculosis with lymph node lesions (CORRGA, 1941). Exceptional positive reactions noted by some authors in certain mycotic infections have been attributed to past leishmanial infection, which on clinical grounds is difficult to exclude. Many species of Leishmania, including species that are not pathogenic to man such as L. enriettii, have given delayed responses in man (MUNIZ and MEDINA, 1948). Apart from whole organism suspensions, sonicated promastigotes (CORR~A and AMATO, 1957) and leishmanial polysaccarides (FURTADO and PELLIGRINO, 1956) have been used successfully as skin test antigens. Trypanosomatids other than Leishmania have given specific delayed responses in patients suffering from cutaneous leishmaniasis (Trypanosoma cruzi-PESSOA and PESTANA, 1940; Crithidia oncopeltiZELDON et al., 1960; T. equiperdum-DEPIEDE et al., 1958; Leptomonas ctenocephali and C. fasciculataRANQUE and DUNAN, 1964; L. pessoai-&RBOsA et al., 1972). Such reactions reflect group antigens, which creates potential difficulties in areas where people may be sensitized by such flagellates as T. cruzi (AMATO et al., 1964) or Leishmania species of lizards (SOUTHGATE,1967). In most of the papers dealing with the reactions caused by heterologous antigens the apparently homologous antigen often gave smaller reactions. This could possibly be due to the difficulty of standardizing antigens used in skin tests. ADLER (1965) produced delayed hypersensitivity reactions in volunteers immune to L. tropica, with antigens secreted by living amastigotes and promastigotes of L. mexicana mexicana. SERGIEV and SHUIKINA (1969) compared the reactions produced by killed promastigotes of L. t. major and the liquid phase of the medium in which they had been cultivated. They concluded that the overlay gave stronger reactions than washed promastigotes. While studying the antigens of T. cruzi TARRANT et al. (1965) noted that the culture forms produced an in vitro exo-antigen (IEX-antigen) that was very specific in complement fixation (CF) tests with human sera. It would seem that all the above antigens and one of those used by MONTENEGRO (1926) are of a similar nature. The present work compares the cutaneous hypersensitivity reactions of patients suffering from cutaneous leishmaniasis to whole organism and IEX-antigen of L. m. amazonensis and T. cruzi. Attempts are made to correlate skin reactions with causative Leishmania species and different clinical conditions.
These studies were conducted under the auspices of the Wellcome Trust, London, Instituto Evandro Chagas of the Funda@o Instituto Oswald0 Cruz of Brazil, Funda@o Serviqo Especial de Sacde Pliblica of Brazil and the World Health Organization, Geneva. We would like to thank Sebastiao Oliveira and Henrique Buna for their able assistance in handling the various Leishmania strains and to Roberto Naiff for his help in isolation of strains and skin testing. We would also like to thank Dr. Barry Bloom for stimulating comments. We are also most grateful to all the volunteers without the co-operation of whom this work would have been impossible.
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J. J. SHAW AND R. LAINSON
325
Materials and methods (i) Parasitological examination. Biopsies were taken from the indurated edges of lesions. Impression smears from this material were rapidly dried, fixed in absolute methanol and stained in double strength Giemsa (2 ml. of stain to 15 ml. of pH 75 buffered distilled water) for 45 minutes. A small piece of tissue was fixed in Carnoy’s fluid. The rest of the material was ground up in a pestle and mortar, containing a small quantity of sterile sand in 1.0-l-5 ml. of sterile saline containing 200 units of penicillin G (crystalline sodium) and 100 pg. streptomycin sulphate per ml. and stored at 4°C. for 4-6 hours. A quantity of O-1 ml. of the ground tissue was inoculated intradermally into the nose and hind foot of 2 hamsters (Mesocricetus auratus). These animals were kept under observation for periods up to 1 year. Smears and cultures were made from any lesions that appeared. The strains isolated in the above manner were grouped according to the classification proposed by LAINSON and SHAW(1972). (ii) Preparation of antkens. The antigens used were prepared from strain Ph 8 of L. m. amazonensis that had been isolated from a naturally infected Lutzomyia jlaviscutellata captured in the Utinga forest Belem (LAINSON and SHAW, 1968) and strain CAN 1 of T. cruzi isolated from an acute case of Chagas’ disease from Beltm (SHAW et al., 1969). The flagellates were grown in dialysis membrane sacs (TOBIE and REES, 1948) according to the method described in detail by FIFE and KENT (1960). We did not, however, use the same culture medium as these authors because during preliminary trials we found that L. m. amazonensis did not grow well in this medium. The other major modification was the use of simple Locke’s solution in the dialysis sac. This was done to avoid contaminating the IEX-antigen with substances likely to provoke non-specific hypersensitivity reactions. The medium outside the sac was a brain/heart broth with 10% (v/v) human blood (group 0, D+) and 6% (v/v) potato extract. The blood was added under sterile conditions to the medium and sterile Locke’s solution was similarly added to the dialysis sac. Although 50 ml. was added to each sac only between 30 to 35 ml. was harvested. This same medium was used with agar and a Locke’s solution overlay: the blood was added, in this case, while the temperature of the medium was 56”C., as was the contents of the dialysis sac. Strains of L. m. amazonensis grew equally well in both types of media but strains of L. braziliensis grew slightly better in the diaphasic medium. Antigen preparations used for skin testing were grown in the broth medium. Promastigotes used to start the dialysis sac cultures were grown in NNN medium prepared from nutrient agar (Oxoid code No. CM3) and 10% (v/v) rabbit blood with an overlay of 0.9% (w/v) saline. The parasites were harvested at 7-10 days whilst in their log phase of growth and washed 3 times in cold Locke’s solution. The flagellates from 4-6 tubes were used to sow the dialysis cultures. Cultures were examined periodically and were harvested while in their log phase of growth: this varied from 16-25 days,
Opposite Page :FIG. 1. Anaphylactic
reaction to L. mexicana amazonensis in vitro exo-antigen in an individual leishmaniasis caused by L. braziliensis guyanensis.
with cutaneous
FIG. 2. Anaphylactic
reaction to L. mexicana amazonensis in vitro exo-antigen in an individual leishmaniasis caused by L. braziliensis bradiensis.
with cutaneous
FIG. 3. Skin test sites to leishmanin (left) and leishmanial in vitro exo-antigen (right) at 30 minutes, in a patient with very strong delayed hypersensitivity reactions to both antigens, showing absence of anaphylactic responses. FIG. 4. A lesion of approximately
3-4 weeks evolution on the elbow (Case 1253) caused by L. mexicana amazonensis associated with a weak leishmanin reaction.
FIG. 5. Extensive
cutaneous lesions caused by L. mexicana amazonensis on the elbow of a 4 year old girl, similar lesions distributed on face, arms and legs, associated with negative delayed responses (diffuse cutaneous or Hansenoid anergid leishmaniasis). FIG. 6. Mucocutaneous leishmaniasis (Case 1287) caused by L. bradiensis braziliensis with extensive destruction of nasal septum and active cutaneous lesions on other parts of the body associated with a strong delayed hypersensitivity reaction.
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LEISHMANIASIS
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The sac was removed and the outside carefully washed with cold physiological saline to remove any adherent culture medium, One end of the sac was held in a pair of Spencer Wells forceps, and cut; the cut end was then introduced into the neck of a flask, the pressure of forceps released and the contents of the sac allowed to pass into the flask. The promastigotes were separated from the sac medium by centrifugation at 11,000 G for 30 minutes at 4°C. The supernatant was removed, passed through a millipore filter (pore size 0.22pm.) and dialyzed against 3 changes of 0.9% (w/v) saline during 24 hours at 4°C. It was again passed through a millipore filter and lyophilized in quantities of 05 ml. For use in skin-testing, this was reconstituted with 05 ml. of sterile distilled water and constituted the final leishmanial in vitro exoantigen (LIEX-antigen). Leishmanin was prepared from the promastigote sediment, in saline containing 05% (w/v) phenol, and at a final concentration of 5 x lo6 organisms per ml. Similar antigens were prepared from dialysate culture of T. cruzi (strain CAN 1) to give trypanosomal in vitro exo-antigen (TIEX-antigen), and trypanosomin which was a 5 x lo6 suspension of culture forms in saline containing 0.5% (w/v) phenol. T. cruzi grew more slowly than the Leishmania and was only suitable for harvesting between 30 to 40 days. The IEX-antigen was stored at -20°C. and the leishmanin and trypanosomin were stored at 4°C. Three other antigens were also prepared-a x 5 leishmanin antigen which gave the same number of organisms per ml. as in the original dialysate culture; 1 : 2 dilution of the LIEX-antigen; and a 1 : 2 LIEX-antigen containing 0.5% (w/v) phenol. Uninfected Locke’s solution from a sterile dialysis sac that had been incubated for 25 days was lyophilized and treated in the same way as the supernatant of the dialysate culture. This was used as the control for the IEX-antigens, while 0.9% (w/v) saline containing 0.5% (w/v) phenol was used as the control solution for the leishmanin and trypanosomin. Skin tests. 0.1 ml. amounts of a given antigen and the control solution were inoculated into the inner surface of the forearm of patients with parasitologically proven cutaneous leishmaniasis. In general more tests were performed with the leishmanin; but paired tests were performed with leishmaniu and LIEXantigen and, in chosen cases, with the T. cruzi antigens and control solution. Whenever possible the test . sites were examined at 15,30 and 60 mmutes and then at 8,24,48 and 72 hours. Any erythema or induration was measured and the thickness of paired tests was determined using a pair of calipers. The volume of 4 the skin reaction was calculated according to BRYCESON et al. (1970), in which v = - ic r2t 3 (r = radius of erythema; t = thickness). Both antigen and control solutions were inoculated into a group of normal control volunteers who had no history of leishmaniasis, patients with proven Chagas’ disease, and others with bacterial or mycological skin problems such as tropical ulcer and sporotrichosis. Reactions read at 15 and 30 minutes were considered positive when there was a definite erythema beyond the area of induration. In dark skinned people this was often difficult to judge, and in such casesa clearly oedematous swelling 12 mm. or more was considered as positive. Delayed reactions in which the indurated area measured 5 mm. or more were taken as positive. Results A summary of the hypersensitivity reactions produced by both types of leishmanial antigen are given in Tables I to III and a summary of the different types of reactions that may possibly be provoked by all the antigens is given in Table IV. Examples of some of the different sequences of reactions seen in different infections are given in Figs. 7-12. Not all the skin tests done were recorded, either because the patient failed to return or the infection could not be confirmed and studied. In normal volunteers the leishmanin and trypanosomin produced no hypersensitivity reactions. In two instances false positives of 6 mm. were noted with the leishmanin at 24 hours but these disappeared by 48 hours. The IEX-antigen did not produce false positive reactions in normal control volunteers. The increase in volume of this group varied from 70-420 nun3 at 30 minutes, which by 8 hours had disappeared. Patients with sporotrichosis (6), blastomycosis (2) and Chagas’ disease (5) did not react to any of the antigens. One patient with lepromatous leprosy had a strongly positive leishmanin test at 48 hours (LIEX-antigen was not tested). He had no past history of a possible leishmanial infection and no history of living in an endemic area. Control solutions to the different antigen preparations did not elicit hypersensitivity reactions.
327
J. Je SHAW AND R. LAINSON TAsiE
I. The results of skin reactions read at 30 minutes using an in vitro exo-antigen prepared from Leishmania mexicana amazonensis in patients suffering from cutaneous leishmaniasis. Positive
---
Infecting organism
Type of lesion
Diameter of erythema 15-30 mm. >30 mm.
Negative
Simple cutaneous
7
7
6
Mucocutaneous
3
0
3
L. b. guyanensis
Simple cutaneous
3”
5”
0
L. m. amazonensis
Simple cutaneous
5
0
2
Anergic cutaneous
2
0
0
20
12
11
L. b. braziliensis
---
Total *At 48 hours reactions negative in 3 cases.
TABLE
II. The results of leishmanin tests read at 48 hours using an in vitro exo-antigen prepared from
Leishmania mexicana amazonensis in patients suffering from proved cutaneous or mucocutaneous leishmaniasis.
Infecting organism L. b. braziliensis
L. b. guyanensis
L. m. amazonensis
Type of lesion Simple cutaneous
Positive 5-10 mm. >lO mm.
Negative
17
7
3
Mucocutaneous
5
6
3”
Simple cutaneous
9
1
3
Multiple cutaneous
2
0
3
Simple cutaneous
4
0
2
Anergic cutaneous
0
0
4
37
14
18
Total +Leishmanin positive.
TABLE III. The results of leishmanin tests read at 48 hours using an antigen of phenolized promastigotes of Leishmania mexicana amazonends in patients suffering from proved cutaneous or mucocutaneous leishmaniasis.
---
Infecting organism
L. b. braziliensis
--L. b. guyanensis
L. m. amazonensis
Type of lesion
Positive
Negative
Simple cutaneous
45
8
Mucocutaneous
19
0
Simple cutaneous
15
3
Multiple cutaneous
5
3
Simple cutaneous Anergic cutaneous
5 0
1 4
89
19
Total
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LEISHMANIASIS
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7. Cutaneous hypersensitivity reactions to two types of leishmanial antigen in a cured L. mexicana amazonensis infection (Case 1253) and normal volunteers. FIG.
8. Cutaneous hypersensitivity reactions to two types of Trypanosoma cruzi an&en in a cured L. mexicana ama&nensis infection (Case 1253) and normal volunteers. FIG.
9. Cutaneous hvpersensitivity reactions to - two types of L. mexicana amazonensis antigen in patients with cutaneous lesions caused by L. mexicana u.mazonensis. FIG.
J. J. SHAW AND R. LAINSON
FIG. 10. Cutaneous hypersensitivity reactions to two types of L. mexicana amazonensis antigen in patients with cutaneous lesions caused by L. braziliensis braziliensis.
FIG. 11. Cutaneous hypersensitivity reactions to two types of L. mexicana amazonensis antigen in patients with mucocutaneous lesions caused by L. braziliensis braziliensis.
FIG. 12. Cutaneous hypersensitivity reactions to two types of L. mexicana amazonensis antigen in patients with cutaneous lesions caused by L. braziliensis guyanensis.
329
330
LEISHMANIASIS
IN BRAZIL
Type of cutaneous hypersensitivity Type of Antigen --Leishmanm Trypanosomin
Immediate anaphylactic
Delayed
-
+
-
+
+
-
-
+
TABLE IV. Summary of skin responsesof individuals with active cutaneous leishmaniasis to different trypanosomatid antigens.
LIEX TIEX 1 : 2 LIEX Phenolized 1 : 2 LIEX
la. Immediate anaphylactic hypersensitivity. 32 out of 43 cutaneous leishmaniasis cases had typical weal and flare reactions (Figs. 1 and 2) with the LIEX-antigen at 30 minutes, that then subsided. These figures include the 36 cases mentioned in our preliminary note (SHAW and LAINSON, 1974). Table I summarizes these reactions in relation to the different Leishmania. Only two patients reacted similarly to the leishmanin and, in both, the erythematous area elicited by the LIEX-antigen was large, measuring over 50 mm. diameter: that produced by the leishmanin was in the order of 20 mm. diameter. Three individuals with immediate reactions did not have delayed responses to leishmanin or LIEX-antigen. Positive reactions equal in size to that of the standard LIEX-antigen were produced by the 1 : 2 LIEXantigen, but neither by the phenolized 1 : 2 LIEX-antigen norby the x 5 leishmanin. More positive immediate responses were noted in infections of L. b. guyan-ensisthan L. b. braziliensis (P = <0.05). No such differences were seen between infections of these two leishmania and L. m amazonensis. The average volume of the L. b. guyalzensis immediate reactions was 5.13 cm.3 and of the L. b. braziliensis infections 356 cm.3 (Figs. 11 and 12). Appropriate t-tests indicated that these differences were not significant at the 5% level. Two cases of anergid diffuse leishmaniasis due to L. m. amazonensis had weak immediate responses, as did 3 of the mucocutaneous cases. The infections were regrouped according to their estimated time of evolution and it was noted that the larger reactions (I? =
J. J. SHAW AND R. LAINSON
331
Individuals with mucocutaneous leishmaniasis (Figs. 4 and 11) had significantly larger (I? =
332
LEISHh4AhUSIS IN BRAZIL
antigen composed of at least 5 different types of glycoproteins (NJOGU and HUMPHREYS, 1972). ALLSOPP et al. (1971) concluded that the “exo-antigen” and “4s-antigen” should be replaced by a single term “surface antigen”. In respect to the importance of surface antigens in immunity it is interesting to note that JOHNSON et al. (1963) noted that the surface antigens of T. cruzi blood forms promoted better protection in mice than did vaccine prepared from trypomastigotes. We do not know what, if any, is the relationship of the exo-antigen produced in vitro, by culture forms, to the surface antigens produced by trypanosomes in vivo. Electronmicroscopic studies of Leishmania amastigotes and promastigotes have not indicated the presence of a surface coat. It is possible that the stage of development of a trypanosomatid is the factor that determines the presence or absence of a surface coat. VICKERMAN (1969) noted that the only culture forms with a surface coat in the vector were the metacyclic trypomastigotes. Antigens equivalent to surface antigens may be produced by other forms but, instead of remaining on the surface, they may be directly liberated into the surrounding media. This kind of process could be associated with amastigotes, promastigotes and epimastigotes, either in vivo or in vitro. ADLER (1965) suggested that Leishmania IEX-antigen may be derived from the flagellar vacuole. In leishmaniasis it is not known whether immunity is of the sterile or non-sterile type. Human cases have been recorded as developing long after the persons left an endemic area (CLAY and ROSS, 1956). In South America, mucocutaneous leishmaniasis occurs many years after the initial lesion and is often associated with a debilitated state (WALTON et al., 1973). There is much evidence that leishmanial infections in wild animals may be cryptic (HERRER and TELFORD, 1969; HERRER et al., 1966; HEFXER et al., 1971; LAINSON and SHAW, 1968; 1973). In man it is possible that a successful primary immune response results in the elimination of the parasites: if it is inadequate, however, pathological lesions may be resolved but the parasites remain as an occult infection. The subsequent clinical outcome of such infections may depend on the type of Leishmania. The fact, however, that living organisms liberate antigenic substances means that these could be as or more important in stimulating immune responses of the host than somatic antigens. The latter would presumably only become available on the death of the organisms. TURK (1972) has drawn attention to the differences that exist between delayed hypersensitivity reactions and cellular immunity, and suggested that antigens that cause skin reactions may not necessarily be those that give rise to immunity. Arguing on similar lines it might be said that the antigens that give rise to immunity have not been used as skin test antigens and this could lead to apparently discordant results. In this paper we have shown that, in man, more than one type of antigen can elicit delayed responses and the same may be true of immediate anaphylactic type reactions. In all comparisons of skin-test antigens, however, it is difficult to distinguish between qualitative differences in the absence of suitable methods for antigen standardization. NEAL (pers. comm.) has emphasized this point in his finding that potency of antigen in skin-tests does not appear to be directly related to number of organisms, nor their nitrogen or protein content. Such problems make it even more difficult to carry out quantative studies of skin reactions to different antigens. In the present work this was to some degree compensated for by the use of the same antigen batches for all the comparative studies. The percentage of positive Montenegro reactions in South American cutaneous leishmaniasis varies from 865% (MONTENEGRO, 1926) to 100% (BARBOSA et al., 1972). Such differences may depend upon the method of selection of the patients. Our results are very similar to those of MONTENEGRO (1926) and were chosen, as were his, by simply testing all patients that were available. In comparing the delayed reactions of our parasitologically proven casesof L. braziliensis with similarly diagnosed casesof L. tropica (DOSTROVSKY and SAGHER, 1946), significantly less (I’ = <0*05) patients with L. brazdiensis had a positive leishmanin test. Immediate anaphylactic type responses were less common in L. b. braziliensis than L. b. guyanensis infections. When these infections were grouped according to similar ages the samples were smaller and the differences were no longer significant, although larger reactions occurred significantly more frequently in the infections of 6 months or less. Much of our data failed to indicate significant differences but certain trends were noted, such as the tendency for L. b. bradiensis infections to have larger delayed responses to LIEX-antigen. If the L. b. guyanensis and L. m. amazonensis infections were grouped together, excluding the anergid diffusa cases, the difference was significant (I’ = <0.05). There is therefore evidence suggesting that both time of infection and the type of Leishmania influence the spectrum of hypersensitivity reactions. FURTADO a.nd PELLEGRINO (1956) questioned the specificity of the standard leishmanin and considered that the polysaccaride fraction gave better specificity. Positive leishmanin reactions have been noted in
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individuals suffering from tuberculous involvement of the lymph nodes (CORR~A, 1941) but not in other types of tuberculosis. In general, cases of leprosy also do not react to leishmanin (ARANTES, 1941) but positive reactions have been noted in the lepromatous form of the disease (FURTADO and PELLEGRINO, 1956, and present work). Such reactions are interesting as they might be regarded as being due to some common antigen. In CF tests the sera of kala-azar patients react strongly with a number of different mycobacterial antigens. The incidence of false positives in apparently healthy individuals varies, with leishmanin, from 15% (SOUTHGATE and ORIEDO, 1967) to 6.3% (DOSTROVSKY and SAGHER, 1946) but such reactions have not been associated with any other infections. To date, skin reactions in Chagas’ patients have been equivocable. MAYER and PIFANO (1941), using culture forms of T. cruzi, obtained positive delayed reactions in 6 patients with Chagas’ disease. Individuals with cutaneous leishmaniasis did not react to the trypanosomal antigen but did react to the leishmanin antigen. PESS~A and PESTANA (1940) found that persons with cutaneous leishmaniasis had equally strong delayed hypersensitivity reactions to both leishmanin and trypanosomin antigen. Subsequently PESSQ and CARDOSA(1942) skin-tested persons with Chagas’ disease and cutaneous leishmaniasis and again found that the individuals with cutaneous leishmaniasis reacted to both antigens, but those with Chagas’ disease reacted to neither. AMATO et al. (1964) performed intradermal tests with lysed amastigotes and trypomastigotes of T. muzi, suspended in distilled water containing 1 : 5,000 merthiolate, in patients with Chagas’ disease and cutaneous leishmaniasis. The test sites were examined at 15 and 30 minutes but there was no evidence of anaphylactic hypersensitivity. Of 12 individuals with Chagas’ disease 8 had weak delayed reactions, while 7 patients with cutaneous leishmaniasis all reacted strongly to this same antigen. In the present work we found, as had PESS~A and CARDOSA (1942), that no hypersensitivity reactions were elicited in Chagas’ disease patients using antigens prepared from the culture forms of Leishmania or T. cruzi, Possibly the type of antigen produced by the culture forms influences the demonstration of delayed hypersensitivity in Chagas’ disease. There is little danger of false positives arising from undiagnosed cases of Chagas’ disease if the IEX leishmanial antigen is used. Diagnostically it would be an advantage to have a skin test that could be read rapidly. The present results with LIEX-antigen indicate that it is potentially useful, especially in early cases when there are specific reactions between 15 to 30 minutes which, in certain instances, are not associated with a delayed response. The LIEX-antigen by comparison with the TIEX-antigen, in delayed responses, would seem to be specific at the generic level, but this is not true of the immediate anaphylactic type responses. Since we have as yet been unable to grow strains of L. braziliensis in dialysate medium we cannot compare homologous antigen with the heterologous L. m. amazonensis antigen in patients with L. braziliensis infections. Some workers (SERGIEV and SHUIKINA, 1969a) consider that the most suitable antigen for skin testing is a suspension of unwashed promastigotes at a concentration of 1 x loj promastigotes per ml., in Hank’s solution containing 0.25% phenol. The effect of the phenol on the IEX-antigen, however, would seem to us to be a disadvantage in such an antigen, because immediate reactions would no longer be elicited. The future selection of skin test antigen will depend on careful comparative studies and the development of suitable methods j of standardization of preparations. Cell mediated immunity, dependent on T-lymphocytes, is responsible for delayed hypersensitivity reactions, but antibodies derived from B-cells are important in immediate reactions. PESS~A and PESTANA (1940) and PESS~A (1941) obtained positive Prausnitz-Kiistner reactions with the sera from 3 individuals with cutaneous leishmaniasis, in 5 volunteers. CURBAN (1941) repeated this with sera of 3 patients, in 6 individuals, but obtained negative results. DOSTROVSKY and SAGHER (1946) produced Prausnitz-Kiistner reactions with the sera of 2 patients who had been infected with L. tropica in 4 out of 11 attempts. These would appear to be the first records of a reaction involving reagin (IgE) in the donors. The finding of immediate anaphylactic responses to LIEX and TIEX-antigen in some patients with active cutaneous leishmaniasis is further evidence for the presence of cell-bound antibody. The detection of circulating antibody has been attempted with various serological techniques. Their presence has been reported using indirect fluorescent antibody (IFA) tests but some of the results should be viewed with certain reservations. WALTON et al. (1972) found that promastigotes fluoresced strongly with sera from proven cases but that this type of antigen lacked sensitivity and specificity. These same authors obtained greater specificity using amastigotes grown in tissue culture as antigens. They concluded that antibodies were produced early in the infection but appeared to be unrelated to the extent or duration of the infection. Antibodies have also been detected by the indirect haemaglutination test (BRAY and LAINSON, 1967; ANTUNES et al., 1972), but the lack of specificity makes this test at present unsuitable for detecting low antibody levels. Com-
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plement fixation tests have not been specifically used for the detection of antibodies in cutaneous leishmaniasis but cross reactions have often been noted with certain T. cruzi antigens (TARRANT et al., 1965). In CF tests with sera of mucocutaneous leishmaniasis, we have noted reactions typical of antigen deficiency due to the presence of minor antigens. Such cross reactions did not occur when the TIEX-antigen was used. It would seem, therefore, that both cell-bound and circulating antibodies occur in cutaneous leishmaniasis. The present work indicates that cell-bound antibodies occur early in the infection. There now seems sufficient evidence to indicate that both cell-mediated and humoral responses occur in cutaneous leishmaniasis and that, in part, cutaneous hypersensitivity reactions are reflections of the two systems. It would appear that cell-bound antibodies are formed early in the infection, as shown by the immediate anaphylactic responses, followed by the development of both humoral and cell-mediated responses (TREMONTI and WALTON, 1970). As the infection progresses, humoral antibodies may reach such a level as to block anaphylactic reactions. Subsequently a successful immunological answer is reached whereby the parasite is either eliminated or continues as an occult infection. Epidemiological evidence suggests that many more people contract leishmaniasis in the Amazon region and recover, than seek medical assistance. Some of those that seek medical help presumably had an inadquate initial response. PRESTON (1973), in discussing the disease spectrum of leishmaniasis, suggested that either stimulation or suppression of the immune response could lead to non-healing forms of the disease. Thus the existence of latent infections could lead to an allergic state responsible for such conditions as mucocutaneous leishmaniasis, and suppression, brought about by an initial heavy infection (TURK and BRYCESON, 1971), could lead to diffuse anergid cutaneous leishmaniasis; in both cases the continued presence of parasites would be associated with the liberation of exo-antigen. Such material may be more important in causing immune deviation than somatic amastigote antigen. It may well be that such antigens will be necessary when investigating cell mediated responses of man in vitro. It has recently been shown (MUNRO et al., 1972) that lymphocyte transformation in post-dermal kala-azar dermal leishmanoid was elicited by L. donovani antigen but not by L. tropica. The above mentioned sequence of events is of course hypothetical but seems to fit the available information that we at present have on the immunological responses in cutaneous leishmaniasis in the New World. Summary In spite of the difficulty of antigen standardization, paired intradermal skin tests demonstrated qualitative differences between leishmamn and in vitro exo-antigen. Some of these differences may be due to the destruction of certain antigens by phenol, while others seem to reflect basic antigenic differences. Leishmania mexicana amazonensis in vitro exo-antigen produced immediate anaphylactic responses in 74.4% of the patients with parasitologically proven cutaneous leishmaniasis. Although more individuals responded to leishmanin, 82.4% as compared to 73.9%, the leishmanial in vitro exo-antigen gave significantly larger delayed responses. Trypanosoma cruzi trypamosomin elicited delayed reactions, while trypanosomal in vitro exo-antigen only produced immediate anaphylactic reactions in persons with active cutaneous leishmaniasis. Significantly more immediate anaphylactic responses were seen in L. brasiliensis guyanensis infections, and more marked responses occurred in infections of 6 months or less duration. Patients with mucocutaneous leisbmaniasis had larger delayed responses than individuals with cutaneous infections due to L. b. braziliensis. Individuals with diffuse anergid cutaneous leishmaniasis had negative delayed reactions to all antigens, but two had weak anaphylactic reactions. Arthus-type responses were uncommon; they occurred in 4 people with mucocutaneous leishmaniasis, and in 3 with cutaneous leishmaniasis caused by L. b. guyanensis. No hypersensitivity reactions were seen in patients with Chagas’ disease. REFERENCES ADLER, S. (1965). Israel J. Med. Sci., 1, 9.
B. A., NJOGU, A. R. & HUMPIIREYS, K. G. (1971). Expl Purasit., 29, 271. A. P., TAVARES, C. A. I?. 81 PELLEGRINO, J. (1972). Revta Inst. Med. trop. S. Paulo, 14, 203. AMATO, V., MAGALDI, C. & PESS~~A, S. B. (1964). Revtagoiuna Med., 10, 121. ARANTES, S. C. (1941). Arches Hig. Saiide ptibl., 6 (13), 67. BARBOSA,A., SOUSA,M. C. M., RASSI, D. M., OLIVEIRA, R. L. & MOTA, L. (1972). Revta Pat. trop., 1, 377. BRAY, R. S. & LAINSON, R. (1966). Trans. R. Sot. trop. Med. Hyg., 60, 605. T& (1967). Ibid., 61, 490. ALLSOPP,
ANTUNES, L. J., REIS,
J. J. SHAW AND R. LAINSON
335
BRYCESON,A. D. M., BRAY, R. S., WOLSTENCROFT,R. A. & DUMONDE, D. C. (1970). Clin. & .Ex# Immunol., 7, 301. Buss, G. (1929). Arch. Schifls-u. Tropenhyg., 33, 65. CLAY, R. D. & Ross, A. A. (1956). Br. med. J., 1, 1279. CLINTON, B. A., PALCXJK, N. C. & STAUBER, L. A. (1972). J. Immun., 108, 1570. CO&A, C. (1941). Arches Hig. sulide ptibz., 6 (13), 61. COF&A, M. 0. A. & AMATO, V. (1957). Revta Inst. Adolph0 Lutz, 17, 39. CuRBAN, G. V. (1941). Revtu pa&. Med., 19, 174. DEPIEDS, R., COLLOMB, H., MATHORIN, J. & RANQUE, J. (1958). BUZZ. Sot. Path. exot., 51, 501. DOSTROVSKY,A. & SAGHER, F. (1946). Ann. trap. Med. Parasit., 40, 265. FIFE, E. H. & KENT, J. F. (1960). Am. J. trop. Med. Hyg., 9, 512. FURTADO, T. A. & PELLEGRINO, J. (1956). J. invest. Derm., 27, 53. GOMES, L. S. (1939). Bras.-me’d., 53, 1079. JOHNSON, P., NEAL, R. A. & GALL, D. (1963). Nature, Lond., 200, 83. HERRER, A., THATCHER, V. E. & JOHNSON, C. M. (1966). J. Parasit., 52, 954. & TELFORD, S. R. (1969). Science, 164, 1419. & CHRISTENSEN,H. A. (1971). Ann. trop. Med. Purasit., 65, 349. ‘-> LAINSON, R. & SZIAW, J. J. (1968). Trans. R. Sot. trop. Med. Hyg., 62, 385. (1972). Br. med. Bull., 28, 44. -: (1973). Bull. Pan. Am. Hlth Org., 7 (4), 1. MAYER, M. & PIFANO, F. C. (1941). Bras.-mid., 55, 317. MONTENEGRO, J. (1926). Archs Derm. Syph., 13, 187. MUNIZ, J. & MEDINA H. (1948). Hospital, Rio de J., 33, 35. MUNRO, D. D., Du VIVIER, A. & JOPLING, W. H. (1972). Br. J. Derm., 87, 374. NJOGU, A. R. & HUMPHREYS, K. C. (1972). ExpZ Parasit., 31, 178. PESS~A, S. B. (1941). Revtapaul. Med., 19, 236. & CARDOSA,F. A. (1942). Hospital, Rio de J., 21, 187. & PESTANA, B. R. (1940). S PuuZo me’d., 2 (5/6), 133. PRESTON,P. M. (1973). Proc. R. Sot. Med., 66, 776. RANQUE, J. & DUNAN, S. (1964). Ann. Purusit. hum. camp., 39, 117. SCHNUR, L. F., ZUCKERMAN, A. & GREENBLATT, C. L. (1973). J. ProtozooZ., 20, 534. SERGIEV, V. P. & SHUIKINA, E. V. (1969). Medskaya Parazit., 38, 208. -& (1969a). Ibid., 38, 562. SHAW, J. J. & LAINSON, R. (1974). Trans. R. Sot. trop. Med. Hyg., 68, 168. & FRAIHA, H. (1969). Revta S&de Pzibl., 3, 153. -> SOUTHGATE, B. A. (1967). J. trop. Med. Hyg., 70, 33. & ORIEDO, B. V. E. (1967). Ibid., 70, 1. TARRANT, C. J., FIFE, E. H. & ANDERSON, R. I. (1965). J. Parasit., 51, 277. TOBIE, E. J. & REES, C. W. (1948). Ibid., 34, 162. TREMONTI, L. & WALTON, B. C. (1970). Am. J. trop. Med. Hyg., 19, 49. TURK, J. L. (1972). Immunology in Clinical Medicine, p. 56. London: William Heinemann. & BRYCESON,A. D. M. (1971). Adv. ImmunoZ., 13, 209. VICKERMAN, K. (1969). J. CeZZSC;., 5, 163. WAGNER, E. H. (1923). Univ. Calif. Pubis Zool., 20, 477. WALTON, B. C., BROOKS,W. H. & ARJONA, I. (1972). Am. J. trop. Med. Hyg., 21,296. -, CHINEL, L. V. & EGUIA, 0. E. (1973). Ibid., 22, 696. WEITZ, B. G. E. (1960). Nature, Lond., 185, 788. WEISSBERGER,H., SPIRA, D. T. & ZUCKERMAN, A. (1973). 3. Protozool., 20, 534. WILLIAMSON, J. & BROWN, K. N. (1964). ExpZ Par&t., 15, 44. ZELED~N, R., HIDALGO, W. R. & HIDALGO, H. X. (1960). Revta BioZ. trop., 8, 145.