Tropical Medicine and International Health

VOLUME I NO. 2 PP 251-254 APRIL 1996

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Leishmania tropicu in Egypt: an undesirable import Emad W. Mohareb, Elen M. Mikhail and Fouad G. Youssef

US Naval Medical Research Unit No. 3

Summary Cutaneous as well as visceral leishmaniasis has been previously reported in Egypt. The former clinical manifestation is attributed to Leishmania major, the latter to L. infantum. In this study, L. tropica was isolated from an Egyptian labourer returning from Saudi Arabia. Amastigotes were detected by both Giemsa staining and indirect immunofluorescence using rabbit anti-gp63. Promastigotes from Schneider’s medium were typed isoenzymatically as L. tropica. In view of the emerging threat of visceralization of L. tropica, the potential risk for its transmission in Egypt is discussed.

keywords Leishmania tropica, transmission, isoenzymes, immunofluorescence

correspondence Research Publications Branch, US Naval Medical Research Unit No. 3 (NAMRU-3), PSC 452 Box 5000, FPO AE 09835-0007.

Materials and methods

Patient

Introduction

Leishmaniasis presents a health problem in several countries. Cutaneous and visceral leishmaniasis have both been reported in Egypt. The cutaneous form has been primarily identified in northern Sinai (Mansour et al. 1987) and was attributed to Leishmania major, according to isoenzyme analysis (Mansour et al. 1989). Visceral leishmaniasis caused by L. infantum has been found near Alexandria, in El Agamy (Awadalla et al. 1987). Leishmania tropica, another causative species for cutaneous leishmaniasis, is widely distributed over the Middle East, especially in countries bordering the Arabian Gulf (Nadim et al. 1979), but not in Egypt. In Saudi Arabia, L. tropica is endemic in the western and south-western part of the Kingdom (Peters et al. 1985). However, the disease seems to affect mostly the non-Saudi expatriates (Al-Gindan et al. 1984). Thousands of Egyptians who are either temporary

A 27-year-old male Egyptian labourer worked in A1 Taif in south-western Saudi Arabia, from October 1992 to October 1993. He visited Makka in May 1992. One month after his return to Egypt 3 small nodule appeared on his nose and was surgically removed. Later an ulcerated lesion measuring 5-6 cm in diameter started to form. Ulcers were surrounded by yellow-brown scaly plaque with an irregular base (Figure I ) . The lesion was clinically diagnosed by a dermatologist as cutaneous leishmaniasis and the patient was given 300 mg rifampin capsules twice a day for one week. The treatment was repeated for 4 months without response. He was then referred to us for conclusive diagnosis.

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workers in the Kingdom or visitors during the ‘El Ha]’ or ‘El Omra’ season return home potentially

Parasitological diagnosis

carrying leishmaniasis. We report on the isolation of L. tropica from an Egyptian who visited Saudi Arabia, and discuss the potential for L. tropica transmission in Egypt and the risk involved.

One hundred microlitres of sterile saline were injected into the outer border of the lesion and then aspirated back. The aspirate was divided into 4 portions: one aliquot each was inoculated into

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Tropical Medicine and International Health VOLUME I N O . 2 PP z 5 ~ - 2 5 4 APRIL '996

E W Mohareb et ol. Leishmania tropica in Egypt

Figure I Ulceration and scaly plaque.

Tanabe's medium and Schneider's Drosophilu medium supplemented with 10% heat inactivated fetal bovine serum and 100 IU penicillin, IOO mg streptomycin and 0.25 pg Fungizone/ml. Another portion was applied onto 2 microscope slides and stained with Giemsa, and the last portion was applied onto a multi-well immunofluorescence slide.

Amastigote detection using the anti-gp63 immunofluorescence assay (IFA)

Lesion aspirate was fixed in acetone at - zo"C for 5 minutes. The antigen was overlaid with a I : IO dilu- tion of rabbit anti-gp63 and incubated for 2 hours in a humid chamber. Slides were washed for I minute in 0.5% phosphate-buffered saline (PBS)-Tween and air dried before adding another 5 pl of a 1:2o dilu- tion of affinity purified goat anti-rabbit IgG labelled with fluorescein isothiocyanate (FITC) (Kirkegaard

and Perry Laboratories, Inc, Gaithersburg, MD) in PBS pH 7.4 for I hour. Counter-staining was carried out with Evans blue at 1:2000 dilution; unbound conjugate was removed by washing the slides in PBS-Tween. Cover slips were mounted in buffered glycerol and slides were examined at x 400 using a Zeiss immunofluorescent Axiskop.

Isoenzyme characterization

Stationary phase promastigotes were harvested, lysed and identified by enzyme electrophoresis according to the methods of Kreutzer et ul. (1983) against ref- erence strains (L . major MTAT/Ke/??/NL Bo91A and M H O M / E G / ~ I / S ~ - ~ ~ ; L. tropica MHOM/IQ/G$ L75; MCAN/SA/~I/WR 1091, MHOM/IQ/~I/WRI 1095; and MHOM/SA/qr/WR 1063. The latter z isolates were from patients with viscerotropic leishmaniasis; L. donovani MHOM/IN/8o/DD8).

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Tropical Medicine and International Health VOLUME I NO. 2 PI' 25L-254 APRIL 1996

E W Mohareb et ol. Leishmania tropico in Egypt

G6PD

1 2 3 4 5 6 1 8 1 2 3 4 5 6 7 8

Figure 2 Results of zymographic analysis.

Results

Giemsa staining revealed amastigotes in one of the two slides, while anti-gp63 IFA demonstrated bright- green fluorescing amastigotes with no background interference. Both media types showed promastigotes 4 days post-inoculation.

From the zymographic analysis (Figure 2 ) of the isoenzyme characterized it is clearly evident that the test isolate displayed enzyme phenotypes identical to those of the L. tropica zymodeme family. Glucose-6- phosphate dehydrogenase (G6PD I . I . I . ~ ~ ) , rnalate dehydrogenase ( M D H I . I . I . . ~ ~ ) , glucose phosphate isomerase (GPI 5.3.1.9), 6-phosphogluconate dehy- drogenase (6PGD 1.1.1.44). malic enzyme (ME 1.1.1.40) and phosphoglucomutase (PGM 2.7.5.1) differentiated between the test isolate and the L. major Si 28 isolate, a representative of the Leishmania spp. known to cause cutaneous leishmaniasis in Egypt.

Discussion

In Egypt, cutaneous leishmaniasis (CL) has always been attributed to L. major (Mansour et al. 1987). The incidence of the disease has been low, however, with sporadic cases in different governorates. Abdel Wahab et al. (1986) clinically diagnosed 36 CL patients from different Arab countries. These patients presented with 5 clinical types of lesions, some non-healing and forming satellite lesions. How- ever, the causative agent in their study was not identified. Recently, different Leishmania spp. have been isolated in Saudi Arabia from natives of the Kingdom as well as from expatriates (Morsy et al. 1993). Due to the continuous influx of temporary labourers and visitors from endemic areas, the inci- dence of leishmaniasis in Egypt may be underesti- mated. In the present communication, we describe a case of CL in a male who worked for I year in A1 Taif and visited Makka, Saudi Arabia, during that period. Lesion aspirate revealed Leishmania amastig- otes by both Giemsa staining and IFA, and iso- enzyme analysis of the promastigote forms characterized it as L. tropica. Infection of humans with L. tropica in Saudi Arabia has been reported previously, especially in the west and south-western part of the Kingdom (Peters eta! . 1985). Phle- botomus sergenti, the known vector for L. tropica transmission, has also been found in A1 Taif (Nadim et al. 1979), where our patient worked. The belief that certain clinical profiles are assigned to specific Leishmania species has recently been challenged. Leishmania tropica, commonly accepted as causing CL, has been isolated from patients with visceral leishmaniasis in Kenya (Mebrahtu et al. 1989) as well as in Saudi Arabia from US marines during Operation Desert ShieldlStorm (Magi11 et a!. 1993). These isolates were typically characterized by iso- enzyme analysis as belonging to the L. tropica polymorphic family, which is commonly reported to cause CL (Kreutzer et a/. 1993). Whether this atypical presentation for L. tropica is patient-related or an inherent quality of certain strains of the organisms or both, is not yet known. It was demonstrated earlier that some Leishmania spp. are not exclusively restricted to the skin or the viscera and that a given parasite may on occasion cause cutaneous or visceral disease, possibly affected by

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Tropical Medicine and International Health VOLUME I NO. 2 PP 251-254 APRIL 1996

E W Mohareb et ol. Leishmania tropica in Egypt

the host’s immunological status (Schnur et al. 1981). This hypothesis is well supported by the fact that some of the recently isolated visceralizing L. tropica were from either malnourished (Mebrahtu et al. I 989) or irnmunocompromised individuals (Magill et al. 1993). On the other hand, based on the clinicopathologic presentation of L. tropica from Lebanese expatriates in Saudi Arabia, it was speculated that different strains of the parasite can cause different pathologies (Kibbi et a/ . 1987). It is interesting to note that our test isolate shared a similar zymodeme pattern for 8 enzymes with one or both of the viscerotropic reference strains. Although our patient had no sign of splenic involvement and his lesion was self-limiting, the potential for this parasite to visceralize must be acknowledged.

The risk of introducing a new form of Leishmania in Egypt is highlighted by the presence of its known transmitting vector, P. sergenti, in different governo- rates of Egypt (Morsy et a/ . 1990). Experiments are under way to determine whether local sandfly species are capable of transmitting L. tropica.

Acknowledgement

This work was supported by the US Naval Medical Research and Development Command, Bethesda, MD, work unit no. oo1o1.EPX.3411. The opinions and assertions contained herein are those of the authors and not to be construed as official o r reflect- ing the view of the Department of the Navy, Depart- ment of Defense, or the US Government.

References

Abdel Wahab RM, Morsy TA, El Garem AA, Bahgat A & Essa M H (1986) Introduced cases of cutaneous leishmaniasis in Egypt. Journal of the Egyptian Society of

Parasitology 16, 9-16. Al-Gindan Y, Abdul Aziz 0 & Kubba R (1984) Cutaneous

leishmaniasis in Al-Hassa, Saudi Arabia. International Journal of Dermatology 23, 194-197.

Awadalla HN, Mansour NS & Mohareb EW (1987) Further characterization of Leishmania isolates from children with visceral infection in Alexandria area, Egypt. Transactions of the Royal Society of Tropical Medicine and Hygiene 81, 9’5-917.

Kibbi A, Kararn PG & Kurban AK (1987) Sporotrichoid leishmaniasis in patients from Saudi Arabia: Clinical and

histologic features. Journal of the American Academy of Dermatology 17, 759-764.

Kreutzer RD, Semko M, Hendricks LD & Wright N (1983) Identification of Leishmania spp. by multiple isoenzyme analysis. American Journal of Tropical Medicine and Hygiene 32, 703-715.

Kreutzer RD, Grogl M, Neva FA, Fryauff DJ, Magill AJ & Aleman-Munoz M M (1993) Identification and genetic comparison of Leishmania parasites causing viscerotropic and cutaneous disease in soldiers returning from Operation Desert Storm. American Journal of Tropical Medicine and Hygiene 49,

3 57-363.

(1993) Visceral infection caused by Leishmania tropica in veterans of Operation Desert Storm. N e w England Journal of Medicine 328, 1383-1387.

Mansour NS, Youssef FG, Mohareb EW, Dees W H & Karuru ER (1987) Cutaneous leishmaniasis in North Sinai. Transactions of the Royal Society of Tropical Medicine and Hygiene 81, 747.

Mansour NS, Youssef FG, Mohareb EW, Dees W H & Karuru ER (1989) Cutaneous leishmaniasis in peace keeping force in East Sinai. Journal of the Egyptian Society of Parasitology 19, 725-732.

Mebrahtu Y, Lawyer P, Githure J et al. (1989) Visceral leishmania unresponsive t o Pentostam caused by Leishmania tropica in Kenya. American Journal of

Tropical Medicine and Hygiene 41, 289-294. Morsy TA, El-Missiry AG, Kamal AM, Fayad ME &

El-Sharkawy IM (1990) Distribution of Phleboromus species in the Nile delta, Egypt. Journal of the Egyptian Society of Parasitology 20, 589-598.

Morsy TA, Sarwat MA, A1 Shaiby AZ, Fawzy AF & Arafa MA (1993) Isoenzyme characterization of Leishmania isolates from patients in El Nour specialized hospital Saudi Arabia. Journal of the Egyptian Society of Parasitology 23, 871-876.

leishrnaniasis in Saudi Arabia: An overview. Bulletin de la Socidti de Pathologie Exotique et de ses Filiales 72, 7-33,

Peters W, Elbihari S, Ching L et al. (1985) Leishmania infecting man and wild animals in Saudi Arabia. Part I General survey. Transactions of the Royal Society of Tropical Medicine and Hygiene 79, 831-839.

Schnur LF, Chance ML, Ebert F, Thomas SC & Peters W (1981) The biochemical and serological taxonomy of visceralizing Leishmania. Annals of Tropical Medicine and Parasitology 75, 131-144.

Magill AJ, Grogl M, Gasser RA, Sun W & Oster CN

Nadim A, Rashti MA& Ashi J (1979) Cutaneous

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