Cutaneous leishmaniasis: An emerging epidemic focus of Leishmania tropica in north Morocco

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TRANSACTIONS OFTHE ROYAL SOCIETY OFTROPICAL MEDICINE AND HYGIENE (1997) 91,660-663

Cutaneous leishmaniasis: an emerging epidemic focus of Leishmania north Morocco

tropica in

N. Guessous-Idrissil, S. Chiheb1y2, A. Hamdanil, M.

Krimech3

Riyad I, M. Bichichil, S. Hamdani3 and A.

‘Laboratoire de Parasitologic-Mycologic, Unite d’Etudes et de Recherche sur les Leishmanioses, Centre Hospi- talier Ibn Rochd et Fact.& de Medecine et Pharmacie, Casablanca, Morocco;2Service de Dermatologie-Venerologie, Hopital Ibn Rochd, Casablanca, Morocco;3Minist&-e de la Sante Publique, Delegation de la Province de Taza, Taza, Morocco

Abstract

Cutaneous leishmaniasis in Morocco occurs mainly in the south and is caused by Leishmania major and L. tropica. In 1995, for the first time, 4 autochthonous cases were confirmed by smear and/or culture from the province ofTaza in north Morocco. An active survey revealed 128 more cases. The number had increased gradually since 1994. Most of the cases (86%) came from the suburbs of the city ofTaza. All cultured and typed parasites were characterized as L. tropica MON-102. A leishmanin skin test survey among a random sample of the exposed population showed an overall positivity rate of 19.9%, with no correlation with age or gender. The spatial distribution of the cases and skin test positivity, their occur- rence in all age groups, the highly variable clinical picture, the severity and large size of lesions in older patients, the slow recovery of some treated patients, and the isoenzymic monomorphism of the parasite, all suggested that cutaneous leishmaniasis caused by L. tropica is an emerging disease inTaza.

Keywords: leishmaniasis, cutaneous leishrnaniasis. Leishmania tropica, Morocco Introduction

Cutaneous leishmaniasis is well known in southern Morocco. During the 198Os, outbreaks of zoonotic cutaneous leishmaniasis due to Leishmania major occurred in the arid and semi-arid areas Tata, Ouarzazate and Er- rachidia (ANONYMOUS, 1992). In 1987, the first case of cutaneous leishmaniasis caused by L. tropica was reported @iARTY et al., 1989) and thereafter widespread sparse hypoendemic rural foci were identified in a sub- arid area ramzing: from Tadla to Aeadir (FRATLONG et Y v

al., 1991). In 1995, 4 indigenous c\taneous leishmaniasis cases originating from the province of Taza, in the north of Morocco, were confirmed by smear and/or culture, and the parasite was characterized as L. tropica. As this province had not previously been known to be an area with cutaneous leishmaniasis, an active survey was started in October 1995. The purpose of this paper is to present and analyse the results obtained.

Material and Methods Study area

The province of Taza is located in the central-north region of Morocco (Fig. 1). It covers 14408 km2 with 708 026 inhabitants (1994 census). The mean altitude is 600 m, with plains and hills but also mountains up to

3250 m. The climate is mainly semi-arid (mean yearly rainfall 600 mm), but some areas have a sub-humid or humid dimate. The capital city of the province is Taza, inhabited by 100678 people (1994 census), located at 600 m altitude and characterized by cold winters and hot summers, with annual temperatures ranging from 5”

to 42°C. The yearly rainfall is about 400 mm but con- siderable variations have been observed during the last 3 years (483 mm, 274 mm and 865 mm, respectively, in 1994. 1995 and 1996). The vrovince of Taza was know& to be a hypoendemic focus of visceral leishmaniasis (L. infanturn) until 1995, with 23 cases recorded from 199 1 to 1995, and no autochthonous case of cutaneous leishmaniasis had been recorded (GUESSOUS- IDRISSI et al., 1996). From April to June 1995, 4 active cutaneous leishmaniasis cases were diagnosed and treated at the University Hospital Ibn Rochd in Casablanca.

All the patients were either living in, or had travelled to, Taza province during the summer of 1994, and none had ever been to the southern leishmaniasis foci or outside Morocco. They were thus confirmed as having been infected in the province in Taza. An active survey was therefore started in October 1995.

Active case detection survey

The neighbourhoods where the 4 index cases lived were visited in October and November 1996. Suspected cases were first identified by visits to schools, markets and houses. They were then invited to a medical consul- tation at their nearest health care centre. Seminars and training schemes were provided for the health workers of the whole province on the diagnosis and treatment of tb.e disease. Data for all suspected cases were recorded on a standardized questionnaire, including name, sex, age, address and number, size, clinical description, and location of the lesions. Family or neighbouring cases were recorded. Special emphasis was given to the travel history and date of appearance of the lesions.

Diagnosis

Whenever possible, the diagnosis was confirmed by examination of a Giemsa-stained smear of the lesion and a sample was taken for culture in NNN medium.

Parasite isolates were transferred to RFMI-1640 medium supplemented with 20% fetal calf serum for bulk culture. Parasites were characterized as follows. (i) Im- munofluorescence using the monoclonal antibodies E5A8, svecific for L. donovani. B8B3. svecific for L. major and iH2A8 and 2B4A11,‘specific for L. tropica (see JAFFX et al., 1990). (ii) Excreted factor analysis using as antigens the reference strains LRC-L47 (L. infanturn Fig. 1. Sketch map showing the study area in Morocco.

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CUTANEOUS LEISHMANIASIS IN MOROCCO 661

serotype B2), LRC-L36 (L. tropica serotype A2) and LRC-L137 CL. maior serotvne Al) and also nolvclonal antisera against L&-L47-&1d LkC-L36 (SCJ%JR et al., 1990). (iii) Isozyme typing on cellulose acetate (IQEUTZER & CHRISTENSEN, 1980), comparing the enzymes nucleotidase 1 and 2, superoxide dismutase, phosphoglucomutase, glucose phosphate isomerase, phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, aspartate aminotransferase, malate dehydrogenase and malic enzyme to those of L.

infuntum, L. tropica and L. major reference strains (respectively MHOM/TN/80/IPTl, MHOM/SU/73//K27 and MHOMISUI74I5ASKI-I) and to south Moroccan L. tropica strains of zymodemes MON-102, 112, 107, 109. 123. 113. and 122 (resnectivelv MHOM/MA/88/

LEtil314, tiHOM/MAiSSiLEM1~51, MHOMIMAI 88/LEM1452, MHOMIMAi89ILEM1591, ISERIMAI 89/LEM1694, MHOM/MA/89/LEM1777 and ISERI MAl89iLEM1828).

Treatment

Glucantime@ (meglumine antimoniate) was given free to all confirmed cases by intralesional injection twice a week for a period of 6 weeks. In cases of bacterial co-infection, local antibiotic treatment was added. Pa- tients with multiple lesions and/or inaccessible lesions, or those with intralesional treatment failure, were given intramuscular Glucantime@ (20-50 mg/kg/d for 15 d).

Leishmanin skin test survey

From the data on the geographical distribution of active cases. 6 nei&bourhoods in Taza citv were identified as having’a highs rate of infection. Fro; a total population of 783 families (4386 inhabitants), a random sample of 173 (1069 people) was asked to submit to a leishmanin skin test in May 1996. Ethical clearance was obtained from the Moroccan Ministry of Health, and the subjects also gave their consent. If a family did not participate, the neighbouring one was tested. Data on sex, age and place of residence were gathered for all par- ticipants. L. infanturn leishmanin, prepared and kindly provided by Zstituto Superiore dz’ Sanitci, Rome, Italy, was used according to the World Health Organization rec- ommendations (WHO, 1984). A Dermojep injector was used to inject 1 mL intradermally in the left forearm and the induration diameter was measured after 48 h by the ‘ball point pen’ method (WHO, 1984). People with positive results (diameter >5 mm) were examined for active lesions or scars and asked about their travel history. The results were analysed using Epi-Info version 6 software.

Entomological survey

Monthly sandfly trapping was carried out from May to November 1996 in the defined areas of high prevalence, using castor oil sticky traps outside and CDC light traps within houses and caves. Live female sandflies were dissected and examined for parasites. Sand- flies caught by the sticky traps were also dissected for species identification using standard methods (KIL LICK-aNDRICK, 1983).

Fig. 2. Age distribution of active cases of cutaneous leishmaniasis in the Taza focus, Morocco.

Results

Active case survey

Between October 1995 and November 1996, 128 cutaneous leishmaniasis cases were confirmed either by direct smear and/or culture or clinically, making, with the 4 index cases, a total of 132; the female/male ratio was 75/57 and the age at diagnosis ranged from 8 months to 85 years (mean=24*6 years, ~~=21.5) (Fig. 2). Twelve of the 132 cases were estimated to have been infected in

1994,57 in 1995 and 53 cases between January and July 1996. The mean duration of the disease was 5 months (range 15 d to 2.5 years). Eighty-five patients (64%) had single lesions, mostly on the face and arms, but up to 6 lesions were recorded. The papulo-nodular form was observed in 43 cases (39%) and ulcerated lesions were seen in 67 cases (61%). The diameter of the lesions ranged from 0.5 to 8 cm. Clustering was observed with 41 cases (31%), with up to 3 cases per family. Severe large, disfiguring and erythematous lesions were observed in 11 patients (age range 60-85 years in 7 patients, and 8 months to 6 years in 4 patients). Diagnosis was confirmed by direct smear examination in 63 of 126 cases (50.0%) and by culture in 28 of 67 (41.8%).

Twenty of the 28 isolates have been characterized to date, all as L. tropica, and the isoenzyme profile was indistinguishable from that of zymodeme MON-102; typing of the remaining isolates is continuing. Intralesional administration of Glucantime@ was followed by im- provement of the lesions in 125 cases; the other 7 received intramuscular treatment. One hundred and sixteen of the 132 cases originated from the city of Taza, and 100 of them lived on its margins. The remaining 16 cases originated from 6 rural communities surrounding the city of Taza within a radius of 15-90 km.

Leishmanin skin test survey

Six hundred and thirty-two people were skin-tested (compliance rate 59%). There was no statistical difference in terms of age or place of residence between the compliant and the randomly chosen populations (x2=14*71, P=O*O65, and ~,~=8.98, P=O.ll, ._- respectively) but women were more compliant than males (&S-99. P--0*0027>. One hundred and twentv-six people (19.9%) gave ‘positive results. No statistical difference was found between age groups k2=10.96, eO.14) (Fig. 3) or gender k2=0.31, -0.58).

Sandfly fauna

Between June and November, 1996, 3483 sandflies were caught. Phlebotomus sergenti was the predominant species and the most endophilic, and the trapped population of that species contained the highest proportion of females (Table). The population of l? sergenti showed 2 seasonal peaks, one in August and the second in October (Fig. 4).

Fig. 3. Age distribution of positive skin tests for Leishmania tropica infection in the Taza focus, Morocco.

Discussion

An emerging focus of cutaneous leishmaniasis that reached epidemic proportions in 1995 among the inhabitants of a well defined area of the city of Taza was found to be due to L. tropica. While in the southern Mo- roccan foci most of the 68 cases documented originally had single small, dry lupoid lesions (ANONYMOUS,

1992), in the new focus of Taza, discussed here, some patients exhibited severe, large, multiple lesions, which could be related to the virulence of the parasite having

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662 N. GUESSOUS-IDRISSI &-AL.

Table. Sandfly fauna in the Taza city focus of cutaneous leishmaniasis in Morocco

Species of Phlebotomus l? sergenti I? longicuspis l? perniciosus l? ariasi I? chabaudi l? alexandri l?papatasi l? langeroni Total

Percentages No. Endophilic Female

1503(43%) 40 37.5

935 (27%)

:z

19.5

792 (23%) 8.7

166 (5%) 12 26.5

58 (2%) 0 31.0

12(<1%) 0 8.3

16(<1%) 81 33.3

1 (Cl%) 0 -

3483(100%) - -

Jul. Aug. Sep.

+

act. NOV.

Fig. 4. Seasonal density (numbers caught on 300 sticky traps) of Phlebotomus sergenti in the Taza focus of cutaneous leishmaniasis in Morocco, 1995.

been enhanced by the lack of immunity of the people in a new focus. The leishmanin skin test survey showed no significant difference of infection rates between age groups, confirming that the disease had been newly iti- traduced. or reintroduced. in the citv of Taza. The province ‘of Taza was pre$ously a stable hypoendemic focus of visceral leishmaniasis. In 1993, 3 cutaneous leishmaniasis cases were noted and, since no survey was carried out to determine the area of transmission, they were considered to have been imported from the southern endemic foci. Our results indicated that an outbreak had started after the sandfly season of 1993, and increased thereafter. Most cases appeared during 2 peri- ods of each year, August-November and January-April, correlated with the observed seasonal prevalence of the probable vector, i.e. I? sergenti (Fig. 4). However, the studied area was unusually windy with marked temper- ature variations during the summer of 1996, which could have influenced the sandfly distribution and sea- sonality.

The active cases were concentrated in a newly settled area on the margins of Taza, where the people were living close to likely sandflies breeding sites in garbage, etc.

The high rate of l? sergenti, previously demonstrated to be the vector of L. tropica in the south (GUILVAFCD et aZ.,

1991), and its high rate of endophilic females, support this assumption.

Contrasting with the well known genetic polymor- phism of L. tropica (see KREUTZER et al., 1993), isoenzyme analysis of the 20 characterized L. tropica stocks (16 urban and 4 rural) has so far shown a monomorphic pattern indistinguishable from zymodeme MON-1 02 be- longing to the L. tropica subgroup IIc, previously de- scribed in the south of Morocco together with 6 others (MON-107,109, 112,113, 122 and 123) (PRATLONGet

al., 199 1). Zymodeme MON-102 could have been introduced into Taza from the south by human travel and this zymodeme monomorphism could be considered as one more argument for an epidemic situation, in which one virulent strain becomes predominant. However, considering that 16 other cases were found in sparse and rural areas around Taza, there are 2 other possibilities.

(i) The parasite could have been present in rural locali- ties in Taza province and, when introduced into the city of Taza, it found conditions suitable for an outbreak, including perhaps environmental changes associated with rapid and unplanned development, increased human and sandfly density, and decreased population immunity. (ii) New settlements were established close to many suitable sources of food for potential animal res- ervoirs such as dogs and rodents, so that a zoonotic situation should not be excluded, perhaps co-existing with the anthroponotic transmission usually associated with cutaneous leishmaniasis due to L. tropica. The fact that, in 1995, we reported one case of visceral leishmaniasis caused by L. tropica in a dog from Taounate province (GLJESSOUS-IDR~SSI et al., 1997) again raises the hypothesis of zoonotic transmission of this species;

all the previously reported infections in dogs were strict- ly cutaneous (DEREURE et al., 1991), and therefore un- likely to constitute a reservoir of infection. Finally, it has appeared during the last few years that L. tropica infections may result in a wide spectrum of clinical pictures, ranging from benign cutaneous lesions to self-regressing lymphadenopathy (ARDEHALI et al., 1995) or mild to complete visceralization (MBBRAHTU et al., 1989;

OREN et al., 1991; MAGILL et al., 1993; SACKS et al., 1995). The mechanisms of this pathogenicity and tissue tropism could involve both parasites and patients (KRWTZER et aZ., 1993). In the case of Taza, the disease was demonstrated to be newly introduced, which could explain the observed severe lesions and slow recovery, especially in elderly and young patients. This highlights the necessity of caution and awareness in cases of unusual or visceral leishmaniasis in the future.

The current ioint effort of researchers and the Ministrv of Health authorities in Morocco must be reinforced 6 order to delineate the distribution of the disease, by cutaneous and visceral leishmaniasis case finding and treatment, parasite isolation, and vector and reservoir surveys in the adjacent areas.

Acknowledgements

This work was supported by the US Agency for Intemation- al Development and the National Institutes of Health, through

the MERC DroPram (contract NOl-A0451851.

We are&gra;eful ;o Professors C. Jaffe ani L. Schnur from the Hadassah Medical School, Jerusalem, Israel, who kindly provided the monoclonal antibddies, excreted factor antibodies and reference antigens; Dr M. Gramiccia from Zstituto Su- p&ore di Sanit& Rome, Italy, who kindly provided the L.

infanturn leishmanin; Professor J.-P. Dedet and Dr F. Pratlong from the World Health Organization Reference Laboratory for Leishmaniasis in Montpellier, France, who kindly provided the reference south Moroccan stocks of Leishmania; Dr P. Melby, of the University of Texas Health Science Center, San Anto- nio, Texas, USA, for his comments; the Moroccan Ministry of Health (Drs Mahjour and Laamrani-Idrissi) and the local authorities of the province of Taza (Dr Rihani), for their logistical support; and Drs Zidouh (Ministry of Health) and El Kadioui- El Idrissi (University Hospital Ibn Rochd, Casablanca), for their contribution to the design and analysis of the leishmanin skin test survey.

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Received 12 March 1997; revised 9 June 1997; accepted for publication 10 June 1997

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