Studies in mediterranean leishmaniasis

60 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL. MEDICINE AND HYGIENE. Vol. 69. No. 1. 1975.

STUDIES

IN MEDITERRANEAN

3. THE LEISHMANIN S. PAMPIGLIONE,

LEISHMANIASIS

SKIN TEST IN KALA-AZAR

P. E. C. MANSON-BAHR, M. LA PLACA, AND S. MUSUMECI”t

M. A. BORGATTI

The immune response in human infection with leishmaniae is similar to that in tuberculosis and leprosy. In cutaneous leishmaniasis there is a wide spectrum of immune response varying from the tuberculoid form with good resistance, an epithelioid granulomatous tissue response, few parasites and the development of delayed hypersensitivity to the diffuse cutaneous form with little or no resistance, numerous parasites, little cellular tissue response and no delayed hypersensitivity. Visceral leishmaniasis (kala-azar has always been thought to be unique, in that infection of man with L. donovani resulted in the kala-azar syndrome with little or no immune response, poor cellular tissue response, many parasites and no delayed hypersensitivity. However it was found that delayed hypersensitivity and a positive leishmanin skin test developed 6-8 weeks after treatment and henceforth such cases were immune to reinfection (MANSONBAHR, 1959). The intradermal leishmanin test (MONTENEGRO, 1926) bears a similar relationship to leishmaniasis as does the tuberculin test to tuberculosis, and since it measures delayed hypersensitivity it becomes positive as cell mediated immunity develops and leishmanin positivity correlates well with in vitro lymphocyte transformation in the presence of leishmanial antigen (TREMONTI and WALTON, 1970) The leishmanin test is specific for leishmaniasis but is not species specific (MONTENEGRO, 1926; BORZONE, 1930; PESSOAand PEREIRA BARRETTO, 1948; MANSON-BAHR, 1961a). Leishmanin positivity increases with age in endemic areas of cutaneous leishmaniasis (IMPERATO and DIAKITE, 1969) and in endemic kala-azar areas leishmanin positivity is acquired without any clinical evidence of infection and varies inversely with the incidence of kala-azar (SOUTHGATE and ORIEDO, 1967). indicating population immunity (SOUTHGATE and MANSON-BAHR, 1967). Leishmanin positivity has also been shown to develop without clinical signs of infection in Northern Italy during an outbreak of kalaazar (PAMPIGLIONE, et al., 1974a). This test has been used widely in studies on the epidemiology of kala-azar in Kenya (MANSON-BAHR, 1961) and of mucocutaneous leishmaniasis in South America (PESSOA and PEREIRA BARRFXTO, 1948). In the Mediterranean it has been used only rarely and then in trials using unstandardized antigen of doubtful activity which have given debatable and sometimes conflicting results. The recent (1971-72) outbreak of kala-azar in Emilia-Romagna, Northern Italy (PAMPIGLIONE et al., 1974a) gave a chance to assessthe reliability of this test in the Mediterranean using antigen of proved efficiency. Since the focus in Northern Italy showed features not usually seen in the Mediterranean area (prevalence in adults, acute toxic forms, epithelioid granulomata in the liver) the survey was extended to compare results in the classical endemic area of kala-azar in Eastern Sicily centred on the town of Catania (GABBI, 1909; FELETTI, 1909; LONGO, 1912; ABATE, 1916; ADLER and THEODOR, 1931; PARADISO and PAFUMI, 1938). Material and methods a) Preparation of the antigen The strain “B0/72” of Leishmaniu donovani was isolated during the outbreak of kala-azar observed in Emilia-Romagna region, from a 72 year old male patient with a rather unusual clinical picture, characterized by lesions limited to the tonsils and satellite lymph nodes. *Survey sponsored by a grant from the Italian Ministry of Health, Health Authorities of the EmiliaRomagna region and the London School of Hygiene and Tropical Medicine. tThe authors wish to thank the Italian Health Authorities and the many colleagues whose valuable help made it possible to carry out our investigations. We are especially grateful to Dr. Turci, Regional Deputy for Health in Emilia-Romagna, to Dr. Galia, Provincial Health Officer for Catania, to Dr. Grassi, director of the Hospital of Caltagirone, to the Mayor of Pianoro and the Mayor of Caste1 S. Pietro Terme, to Profs. S. Bonanno, F. Giungi, G. Leocata, G. D’Agata, M. Midulla, A. Nunnari, G. Russo, to Drs. G. Canestri Trotti, S. Falzone, A. Fisher, I. Papale, A. Parenti, M. Samir Yassin, R. Zanetti and to M. Balbo, DVM.

S.PAMPIGLIONE,P.

E. C. MANSON-BAHR,M.LA

PLACA,M.

A.BORGATTI

AND S.MUSUMEcl

61

The Leishmaniu strain was maintained, by subcultures at 15-20 days intervals, in a diphasic medium with the following composition : So2&phase : trypticase soy agar (BBL) plus 15% of whole rabbit blood; Ziquid phase : one part of brain-heart infusion broth (DIFCO) plus one part of Hanks balanced salt solution, with 200 I.U. of penicillin and 200 pg of streptomycin per ml. As the presence of blood in culture media can be the source of several difficulties in immunological studies, (MANSON-BAHR, 1961; DEDET and LANOTTE, 1969), the growth of promastigotes for the preparation of the antigen has been obtained using a slight modification of the techniques described by CROOK et al., (1969). 500 ml. Erlenmayer’s flasks with a cylindrical neck were filled to about Q with 200 ml. of brain-heart infusion broth and then closed with a rubber stopper with a central hole fitted up with a glass tube of 1 cm. in diameter, protruding for about 3 cm. from both sides. The exterior end of the glass tube was closed with an inverted miniature test tube, while to the interior end was attached a dialysis tube (Visking 20/32) tightly closed at the bottom. The flasks were then sterilized by autoclaving and after cooling, 18 ml. of defibrinated rabbit blood (pooled from at least 5 animals) were introduced aseptically into the dialysis sack through the glass tube. The Leishmuniu promastigotes were inoculated in the culture flask by removing the rubber stopper. The inoculum consisted of about lo* washed promastigotes per ml. of fresh medium and the yield, at the 8th-15th day of incubation at 21°C varied from 1.5 x lo6 to 8.8 x lo6 promastigotes per ml. This technique enabled a pure suspension of promastigotes to be made simply by washing the promastigotes free of the brain-heart infusion broth components and offered the advantage of preventing contamination of the promastigote suspension by bacteria accidentally present in the blood of donor rabbits. At the end of the incubation period, the promastigotes were harvested by centrifugation at 600 g. for 30 min. at 4°C. The pellets were pooled and washed 4 times with 50 volumes of cold sterile saline solution (8.5% NaCl in doubly distilled water). The last sediment was resuspended in saline solution at the concentration of 10 x lo6 promastigotes per ml. The suspension was then processed following, with slight modifications, the technique reported by PESSOA and PEREIRA BARRETTO (1948) : the suspension was kept at 50°C. in a water bath for 3 days with occasional shaking; at the end of this period 0.4% phenol was added and the suspension was exposed to the temperature of 60°C. for 30 min., 3 times at daily intervals (between the exposures at 60°C. the suspension was kept at 4°C.). The suspension was then submitted to routine sterility tests for bacteria and fungi and the lack of viability of the promastigotes was controlled by inoculating 5 ml. out of every 100 ml. of the suspension into 5 tubes of diphasic medium which were incubated at 21°C. for 40 days. The controlled sterile suspension was then distributed into small (2 ml.) glass containers closed with rubber stoppers and stored frozen at -20°C. By the same procedure the antigen from another L. donovani strain, isolated in March 1973 from a dog in Catania and maintained in subculture as the other one, was prepared. Procedure and reading of the reaction The antigen was injected intradermally into the forearm in a dose of O-1 ml. (1 million organisms). A positive reaction showed an area of induration over 5 mm. in diameter when read through a transparent gauge 48-72 hours after injection. A control inoculum (0.5% phenol) was injected into the opposite forearm as a control. The intensity of the reaction was read in 4 grades of induration: 5-6 mm. + ; 6 to 8 mm. + + ; more than 8 mm. + + + ; and with blistering + + + +. Doubtful reactions were read as negative. b)

c)

Subjects tested The leishmanin skin test was performed on the following groups. 1) Kulu-uzur focus in Emiliu-Romugnu region (in 1971-72). 34 subjects who had recovered from kala-azar 3 weeks to 28 months previously. 2 subjects with active kala-azar. 6 subjects considered to have subclinical asymptomatic kala-azar on the basis of serological data and the histological picture at liver biopsy (PAMPIGLIONE et al 1974b).

62

STUDIES

IN

MEDITERRANEAN

LEISHMANIASIS

75 household contacts and neighbours of kala-azar cases. 269 people (85% of the present population) living along the Zena river in a valley with a high prevalence of illness in 1971-72. 52 people over 65 years of age from Caste1 San Pietro and other villages in the area who were living in an old people’s home attached to the hospital in Caste1 San Pietro to which they had been admitted before the summer of 1971 (the season identified as the period of infection for the cases which occurred the following winter). Since this old people’s home was attached to the Caste1 San Pietro Hospital which had been disinfested and was sandfly free this was considered to be a control group for the situation in the area for many years preceding the outbreak. 280 subjects living in Ferrara and in other centres of Emilia-Romagna where no cases of visceral or cutaneous leishmaniasis had ever been seen in the past were used as a control group. Old endemic focus of kala-azar in Eastern Sicily. 26 cases recovered from kala-azar, 25 from l-10 years and 1, 52 years previously. 2 active cases of kala-azar. 134 household contacts and neighbours of individuals who had had kala-azar in the last 5 years. 933 unselected people living in Catania, in the villages at the foot of Mount Etna (Aci Reale, Belpasso, Biancaville, Bronte, Cibali, Giarre, Misterbianco, Paterno) and in the communes of Avola and Noto (Syracuse province), all these centres being regarded in the past as the main endemic foci of kala-axar in the Mediterranean basin and where new cases still occur every year (ZANGRI,1954, 1962, 1963; PARADISO, 1966; Russo, 1973). 155 mostly poor old people living in old people’s homes in Catania coming from the old well-populated areas of the town or from centres known to have had much kala-azar in the past. 92 old people from Caltagirone (Catania province) 600 metres above sea level where kala-azar is thought not to exist, living in an old people’s home who acted as a control for the previous group. 2)

3)

Other subjects tested

200 people including 12 patients who had recovered from kala-azar from the above 2 endemic areas were tested with leishmanin prepared from 2 different antigens (human strain of L. donovani from EmiliaRomagna and canine from Sicily), in order to assessany differences in reactions from the 2 different strains of L. donovani. Results 1)

Emilia-Romagna focus Kala-azar cases

33 of 34 recovered cases of kala-azar gave positive leishmanin reactions. In one the reaction became positive after 12 months, and in another 21 months following recovery. In the only case with a negative leishmanin reaction there was a slight liver and spleen enlargement with raised gamma globulin, slight elevation of the FAT (16) and CFT (10) suggesting that complete recovery had not taken place. 2 active cases of kala-azar gave negative reactions. The 6 subjects considered asymptomatic were all positive. Household contacts and neighbows

48 out of 75 household contacts and neighbours of kala-azar cases were positive (64%). General population

in Valley Zena.

The population in Valley Zena showed a 44.2% positivity rate (Table, Map, Fig. 1). Of 52 old people living in a home in Caste1 San Pietro, representing the population before 1971-72, only one was positive. All 280 control people were negative. 2)

Eastern Sicily focus Kala-azar cases

All 26 patients who had recovered from kala-azar, including one 52 years previously, gave positive reactions.

S. PAMPIGLIONE,

P. E. C. MANSON-BAHR,

M. LA PLACA,

M. A. BORGATTI

‘00

-

63

AND S. MUSIJMECI EMILIA-ROMAGNA

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Household contacts and neighbours. 54 of 134 household contacts and neighbours were positive (40.3%). General population in Eastern Sicily Among 933 people of various age groups chosen from Catania and nearby villages known as classical kala-azar foci 151 (16.6%) reacted positively with a higher prevalence in people over 36 years of age (Table, Fig. 2). Almost a half (47.1%) of the old people living in old people’s homes and coming from the poorer districts and surroundings of Catania gave positive reactions. TABLE. Leishmanin tests in 2 foci of Mediterranean kala-azar (VL = visceral leishmaniasis). Focus

Type of subjects

No. Leishrnanin examined negative

1) Emilia Romagna Caste1 S. Pietro etc. Caste1 S. Pietro etc. Caste1 S. Pietro etc. Caste1 S. Pietro etc. Valle Zena Caste1 S. Pietro etc. Ferrara

VL recovered 34 VL active VL asymptomatic : VL households 75 unselected people 269 old people controls 2%

2) Eastern Sicily Catania etc.

VL recovered

Catania etc. Catania, etc. Catania etc. Caltagirone

26

VL households 134 2 active unselected people 933 old people 155 old people (control) 92

Leishmanin positive total 70

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In the commune of Caltagirone where kala-azar has never been endemic 6 of 92 subjects (6.5%) were positive. There was no difference in the reactions to the human or canine antigens either as regards positivity and negativity or the intensity of the reaction.

STUDIES EASTERN

IN

MEDITERRANEAN

LEISHMANIASIS

SICILY

FIG. 2. y0 Leishmanin positivity

according to

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>65

Discussion This survey performed in 2 foci of Mediterranean kala-axar (Emilia-Romagna and Sicily) shows that the leishmanin skin test behaves similarly in Mediterranean as in East African kala-azar and South American leishmaniasis. A new understanding of some of the epidemiological aspects of Mediterranean kala-axar has been obtained. During the course of active kala-axar the skin reaction is negative becoming positive a few weeks to about 2 years after recovery and remains positive for life (52 years) with individual variations. The positive reactions remain evident for a number of days and sometimes a small nodule can be palpated for some months. One recovered patient who gave a negative result can be explained by the possibility that he was not yet cured since he exhibited some criteria of continuing infection The intensity of the reaction was generally higher in patients who were treated with antimony after several months of illness and in the few who recovered without treatment. All 5 of the 6 casesof asymptomatic infection gave strong positive reactions. A particularly significant finding is the high percentage of positivity (64% and 40.3% respectively) of leishmanin positive reactors among the household contacts and neighbours of kala-azar patients in the Emilia-Romagna and Sicily foci (Table). Thus in the population of the Valley Zena a small locality where there were 5 cases of kala-azar in 1971-72 and where a large proportion of the people could be considered as close neighbours 44.2% were positive (Map). This distribution resembles the microfoci described in Kenya by SOUTHGATE and ORIEDO (1967). In contrast in Eastern Sicily only 16.2% of unselected persons were positive ( x2 42.43,1 y0 probability). All 280 controls proved negative. In a previous investigation (PAMPIGLIONE et al., 1974a) it has been observed that among people living together with persons who developed kala-azar (people having more or less the same probability of being exposed to infection by sandflies) serum positive (CFT) subjects were significantly more numerous than among the unselected population of the same area. 6 of these serum positive subjects showed epitbelioid granulomata in the liver (one with LD bodies) a histological picture discussed by PAMPIGLIONE et al., 1974b) which resembles that observed by BRADLEY (1974) in semi-immune mice infected with L. donovani. The presence of leishmanin positive individuals who have never had clinical signs of illness near the

S.PAMLIGLIONE,P.E.C.MANSON-BAHR,M.LAPLACA,M.A.BORGATTIAND

S.MUSUMECI

65

kala-azar cases provides additional evidence that the spread of kala-azar follows the same pattern as other infectious diseases. This confirms the views of other observers in the past (LEISHMAN, 1909; JEMNA and DI CRISTINA, 1910) and of others (see PAMPIGLIONE et al., 1974b), that clinically obvious cases of kala-azar represent the centre of a microfocus or the tip of an iceberg where many people are infected, some have no symptoms at all, others have few symptoms and recover spontaneously. The clinical cases therefore represent a minority of infected people who do not develop an immune response, allow the parasite to multiply and who will die unless treated Leishmanin positivity denotes an immune state which prevents further infection provided no immunodepressive factors intervene (YOUNG, 1924; NAPIER and KRISHNAN, 1931; SEN GUPTA, 1947; CORKILL 1948, 1949) and this immunity has been demonstrated in Africa by SOUTHGATE and MANSON-BAHR(1967). In old endemic foci where there are frequent contacts with infected sandflies only new generations of children are likely to contact the infection since the older generations are already immune. This phenomenon is aided by the fact that foci often remain constant, strictly limited to the same district or to the same group of houses or even the same house for years (ADLER et al., 1938). In Catania when comparing the old distribution map of the disease (ABATE, 1916) we identified new cases in the same streets and in the same districts and populated outskirts of the town where cases had occurred in the past. The higher incidence of kala-azar in very young children in the classical Mediterranean foci can be explained on these grounds. Leishmanin sensitivity in the old endemic focus of Eastern Sicily supports this view and interference with transmission in the last two decades by adverse influences on the sandfly population (antimalaria campaigns, reduction of gardens etc.) is suggested by the marked increase in sensitivity shown by those of 36 years and above. As seen in Fig. 2 adults and old people show a high positivity rate which is lower in young people and virtually absent in children. A high positivity rate (47*1%) is seen in old people from a geriatric home in Catania who were alive when the optimum conditions for transmission were found in the past in an old focus in the town. This compares with the results found in old people from a home in Caltagirone a none endemic area (6~5%) positive and is statistically significant ( x2 41.84; 1% probability). On comparison leishmanin skin testing in the recent focus in Emilia-Romagna gives a strikingly different picture (Fig. 1). All age groups are strongly affected. The situation previous to the outbreak is shown by the results found in the old people in a home in Caste1 San Pietro (Table) where only one resident was positive. This individual was an 87 year old woman who had lived in the country as a neighbour of kalaazar patients during the summer of 1971 (transmission season) and only entered the home later. This suggests that the population of the Valley Zena had not been previously exposed to infection but had developed immunity in 1971, when all age groups were attacked and in fact clinical caseswere found among all age groups. Since leishmanin positivity appears early as might be expected in asymptomatic cases i.e. contemporaneously with clinical signs in those individuals with evident disease, this could be used as an indirect diagnostic reaction (Indirect LST). If someone is suspected of having kala-azar and the leishmanin skin test is positive in one or more members of the household, this observation along with a negative reaction in the patient can suggest the diagnosis of kala-azar. The percentage of leishmanin positivity according to age can be compared in 3 areas, Emilia-Romagna (Fig. l), Eastern Sicily (Fig. 2), and Kenya (Fig. 3). In the endemic area of Kenya there is a steady increase in positivity with age to a high level suggesting a high level of transmission for a long time. In the classical endemic area of Eastern Sicily there is an increasing positivity with age, low at first but increasing fairly suddenly about the age of 36, suggesting a sudden change from a high to a low level of transmission 20-30 years ago (coinciding with the appearance of insecticides and malaria eradication in the Mediterranean). In Emilia-Romagna all age groups show a similar percentage of positivity suggesting the sudden appearance of transmission at one time in the past so that all age groups of a non-immune population were infected at the same time. It is interesting to note that all over the world, the prevalence in males is greater than in females (Fig. 4). These observations suggest that the leishmanin skin test is a valuable tool to measure&e past present and possiblv future incidence of kala-azar in all areas of the world.

66

STUDIES

IN

MEDITERRANEAN

LSIEHMANIASIS

100 F

KENYA

(Southgate

and O&do

1967)

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FIG.

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--/ %I/ 1906 3387 Summary

Leishmanin skin testing was carried out in the Emilia-Romagna region of Northern Italy, the site of an outbreak of kala-azar in 1971-72, and in Catania, Eastern Sicily an old endemic focus of Mediterranean kala-azar. Nearly all the people who had recovered from kala-azar in the past gave positive skin tests. Active cases of kala-azar gave negative tests. There was a higher proportion of positive reactors amongst the household contacts and neighbours of cases of kala-azar than among the general population. Age specific leishmanin rates showed an increasing positive rate with age in Catania, comparable to those found in endemic areas in Kenya, but in the Emilia-Romagna area all age groups showed a high positivity rate suggesting a simultaneous exposure to infection. The age specific rates from Catania suggest an interruptiotin transmission 20-30 years ago. The leishmanin skin test is a useful tool for the study of the epidemiology of Mediterranean kala-azar.

S. PAMPIGLIONE,

P. E. C. MANSON-BAHR,

M. LA PLACA,

M. A. BORGATTI

AND S. MUSUMBCI

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STUDIES IN MEDITERRANEAN LEISHMANIASIS

REFERENCES ABATE, A. (1916). Leishmaniosi interna infantile. Catania, Di Mattei, ADLER, S. & THEODOR, 0. (1931). Proc. R. Sot. B. 108, 447. & WITENBERG, G. (1938). Ibid., 125, 491.

G&, R. (1930). Reun. Sot. urgent. Putol. reg. N., 5, 600. BRADLEY, D. J. (1974). Unpublished. CORKILL, N. L. (1948). Ann. trop. Med. Purusit., 42, 224. (1949). Ibid., 43, 261. CROOK, G. H., SCOTT, L. V. & PATNADA, R. A. (1969). J. Purusit., 55, 977. DEDET, J. P. & LANOTTE, G. (1969). Archs. Inst. Pasteur, Tunis. 46, 69. FELETTI, R. (1909). Rtf. med., 25, 1373. GABBI, U. (1909). Policlinico (Med.), 16, 1. IMMPERATO, P. J. & DIAKITE, S. (1969). Trans. R. Sot. trop. Med. Hyg., 63,236. JEMMA, R. & DI CRISTINA, G. (1910). Annuli Clin. med. Puletmo., 1, 467. LEISHMAN, W. B. (1906). Hundbuch der Tropenkrunkheiten, ed. C. Mense, 2. Aufl. IV. Leipzig: A. Barth. LONGO, A. (1912) cit. by PARADISO (1934) in Pediutriu Med. Prut., 9, 474. MANSON-BAHR, P. E. C. (1959). Truns. R. Sot. trap. Med. Hyg., 53, 123. (1961a). E. Afr. med. J., 38, 165. (1961b). Truns. R. Sac. trap. Med. Hyg., 55, 550. & SOUTHGATE, B. A. (1964). J. trop. Med. Hyg., 67, 79. MONTENRGRO, J. (1926). Arch. Derm. Syph., 13, 187. NAPIER, L. E. & KRISHNAN, K. V. (1931). Indian med. Guz., 66, 603. PAMPIGLIONE, S., LA PLACA, M. & SCHLICK, G. (1974a). Trans. R. Sot. trop. Med. Hyg., 68, 349, -, MANSON-BAHR, P. E. C., GIUNGI, F., GIUNTI, G., PARENTI, A. & CANESTRI TROTTI, G. (1974b). Ibid., 68, 447. PARADISO, F. (1934). Pediutrtu Med. Prat., 9, 474. (1966). Riv. pediut. sicil., 8, 3. & PAFUMI, A. (1938). Pediutriu Med. Prut., 13, 676. PESSOA, S. B. & PEREIRA BARRETTO, M. (1948). Leishmuniose tegumentur americana. Ministerio da Educacao e Saude, Rio de Janeiro: Imprensa National. RUSSO, G. (1973). Personal communication. SEN GUPTA, P. C. (1947). Indian med. Guz., 82, 726. SOUTHGATE, B. A. & MANSON-BAHR, P. E. C. (1967). J. trap. Med. Hyg., 70,29. & ORIEDO, B. V. (1967). Ibid., 70, 1. TRRMONTI, L. & WALTON, B. C. (1970). Am. J. trop. Med. Hyg., 19, 49. YOUNG, T. C. & MCCOMBIE, W. (1924). RuEa uzur in Assam. London: Lewis & Co. Ltd. ZANGRI, G. (1954). Riv. pediut., sicil., 9, 375. (1960). Ibid., 15, 1.