- Email: [email protected]
Identification of Leishmania donovani as a cause of cutaneous leishmaniasis in Sudan
Transactions of the Royal Society of Tropical Medicine and Hygiene (2008) 102, 54—57
available at www.sciencedirect.com
journal homepage: www.elsevierhealth.com/journals/trst
Identification of Leishmania donovani as a cause of cutaneous leishmaniasis in Sudan E.M. Elamin a,b, I. Guizani c, S. Guerbouj c, M. Gramiccia d, A.M. El Hassan a, T. Di Muccio d, M.A. Taha e, M.M. Mukhtar a,∗ a
Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan Faculty of Medical Laboratory Sciences, Al Zaiem Al Azhari University, Khartoum, Sudan c Laboratoire d’Epidemiologie et Ecologie Parasitaire, Institut Pasteur de Tunis, 1002 Tunis Belvedere, Tunisia d Instituto Superiore di Sanita, Viale Regina Elena 229, 00161 Rome, Italy e Department of Dermatology, Alribat Hospital, Khartoum, Sudan b
Received 10 April 2006; received in revised form 5 October 2007; accepted 5 October 2007 Available online 26 November 2007
KEYWORDS Cutaneous leishmaniasis; Leishmania donovani; Diagnosis; PCR; kDNA; Sudan
Summary Eight patients with cutaneous ulcers were referred to the Institute of Endemic Diseases, Khartoum, Sudan, from June 2000 to March 2002 for the diagnosis of suspected cutaneous leishmaniasis (CL). Diagnosis was confirmed parasitologically by both positive Giemsa-stained smears and successful culture of Leishmania promastigotes in NNN medium. The eight parasite isolates were shown to belong to the Leishmania donovani complex by kDNA PCR. Isoenzyme typing of three isolates revealed that they were identical to the L. donovani MON-82 reference strain, and the gp63 PCR—RFLP profile showed similar patterns to a reference strain of MON-82. CL is endemic in most regions of Sudan and has been reported previously as being caused by L. major MON-74. The results of this study suggest that L. donovani is also a cause of CL in Sudan and that further study of isolates from Sudanese patients with cutaneous ulcers is warranted to ascertain whether L. donovani or L. major is the causative agent. © 2007 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction Leishmania parasites cause different clinical forms of leishmaniasis: cutaneous (CL), visceral (VL), mucocutaneous, mucosal and post-kala-azar dermal (PKDL) (El-Hassan and
∗
Corresponding author. Tel.: +249 183 779712; fax: +249 183 779712. E-mail address: [email protected] (M.M. Mukhtar).
Zijlstra, 2001). The cutaneous form in the Old World is caused by L. major, L. tropica and L. aethiopica and is the most common form, constituting 50—75% of all new cases of leishmaniasis in the world (WHO, 2000). CL is endemic in most regions of Sudan and it was reported to be caused by L. major MON-74 only, while VL is endemic in the eastern, western and southern parts of Sudan (El-Hassan and Zijlstra, 2001). CL patients in Sudan usually present with nodular, nodulo-ulcerative and ulcerative lesions (El-Hassan and Zijlstra, 2001).
0035-9203/$ — see front matter © 2007 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2007.10.005
Leishmania donovani as a cause of cutaneous leishmaniasis
55
Leishmania infantum (L. donovani complex) was isolated from cutaneous lesions in Tunisia (Aoun et al., 2000; Gramiccia et al., 1991), Algeria (Harrat et al., 1996), Italy (Gramiccia et al., 1987) and France (Rioux et al., 1980). A few cutaneous ulcers were reported to be caused by L. donovani sensu latu in Kenya (Mebrahtu et al., 1993), Ethiopia (Schonian et al., 2000) and Lebanon (Guerbouj et al., 2001). In Sudan, the L. donovani complex comprises three major zymodemes: MON-18 (L. donovani), MON-30 (L. infantum) and MON-82 (L. archibaldi) (El-Hassan and Zijlstra, 2001). Some authors consider the three zymodemes to be separate species and suggest the endemic focus in Sudan as the origin of leishmaniasis (Rioux et al., 1990). In contrast, the three zymodemes were reported as a genetically homogeneous group causing VL (Jamjoom et al., 2004). Leishmania donovani has not been investigated as a cause of CL in Sudan. We isolated and typed Leishmania parasites from Sudanese patients with cutaneous ulcers to determine whether they were caused by L. donovani.
MW148). The isolates were mass cultured in 200 ml of RPMI 1640 medium supplemented with 10% fetal calf serum. DNA was extracted from log-phase cultures of the eight isolates using the phenol/chloroform method (Maniatis et al., 1986) and amplified using primers AJS3/DBY for kDNA PCR, which amplify a genomic DNA fragment of 800 bp in the L. donovani complex and 700 bp in the L. major complex (Smyth et al., 1992).
2. Materials and methods 2.1. Patients Eight Sudanese patients with cutaneous ulcers typical of those caused by Leishmania parasites (Figure 1) were referred to the Institute of Endemic Diseases, Khartoum, Sudan, from June 2000 to March 2002 for the diagnosis of suspected CL. All the patients contracted the infection in Khartoum State, central Sudan, and had no history of travelling outside Khartoum State to VL-endemic areas. On examination, all patients looked well and had no fever or enlargement of the liver or spleen. They all gave consent to be included in the study.
2.2. Diagnosis Aspirates were taken from the edges of the ulcers. Half of the aspirate was used for preparation of Giemsastained smears and the other half was inoculated into NNN medium for isolation of the parasite. Inoculated NNN medium was incubated at 24 ◦ C (Ashford et al., 1992). Cultures were examined microscopically for parasite growth. Eight isolates were successfully cultured (MHOM/SD/00/MW3, MHOM/SD/01/MW30, MHOM/SD/02/ MW120, MHOM/SD/00/MW17, MHOM/SD/00/MW79, MHOM/ SD/01/MW144, MHOM/SD/02/MW131 and MHOM/SD/00/
2.2.1. Isoenzyme typing The laboratories in Sudan do not have the facilities to confirm the findings by isoenzyme typing, thus the eight isolates were sent to the Instituto Superiore di Sanita, Rome, Italy, for characterization using the method described by Rioux et al. (1990) based on the isoenzyme profiles of 13 enzymes (representing 15 enzyme loci). Due to lack of funding, three of the eight isolates were randomly selected for isoenzyme typing. 2.2.2. gp63 PCR The gp63 PCR method described by Guerbouj et al. (2001) was used for further characterization of the three isolates that were subjected to isoenzyme typing. Specific primers (SG1 and SG2) that amplify the coding region of gp63 genes were used. Six reference strains were included as controls: L. aethiopica, L100 (MHOM/ET/72/L100); L. tropica, K27 (MHOM/SU/74/K27); L. major, 5ASKH (MHOM/SU/73/5ASKH); L. donovani MON-82, GEBRE1 (MHOM/ET/72/GEBRE1); L. infantum, IPT1 (MHOM/TN/80/IPT1); and L. donovani MON-18, DD8 (MHOM/IN/80/DD8). 2.2.3. gp63 PCR—RFLP The PCR-amplified fragments were analysed using four restriction enzymes, SalI, BalI, HincII (Amersham Pharmacia Biotech, Shanghai, China) and BsiEI (New England BioLabs, Ipswich, MA, USA), as recommended by the manufacturers. The DNA digests were separated by electrophoresis in 3% Small Fragments Agarose (Eurogentec, Liege, Belgium) overnight at 20 V and transferred to a Hybond-N+ nylon membrane using the ‘pocket blotting’ method (Cuny et al., 1991). The gp63 probe (L. chagasi cDNA clone, kindly provided by Dr M. Willson) was labelled with [32 P]dCTP using random primer labelling (Amersham Biosciences, Little Chalfont, UK). Hybridization and high stringency washing were performed at 65 ◦ C according to the manufacturer’s instructions (Amersham Biosciences).
2.3. Treatment of patients The patients were treated with ketoconazole (Janssen, Beerse, Belgium). The drug was administered orally at a dose of 400 mg/d for 4—8 weeks for adults (3 mg/kg/d for children). The drug was given only after liver function tests were found to be normal.
3. Results Figure 1 Multiple cutaneous lesions due to Leishmania donovani infection on the arm of a Sudanese patient.
The Giemsa-stained smears from all eight patients were positive. In addition, Leishmania parasites were successfully isolated and cultured from the eight patients. Parasite
56 growth was microscopically observed within 5—7 d of incubation at 24 ◦ C. kDNA PCR amplified a DNA fragment of 800 bp from the eight tested isolates, identifying them as members of the L. donovani complex. The isoenzyme profile of the three selected isolates, MHOM/SD/00/MW3, MHOM/SD/01/MW30 and MHOM/SD/02/ MW120, was identical to L. donovani MON-82. PCR amplification of the gp63 coding region of the three tested isolates generated a fragment of 1.2 kb. Hybridization of the gp63 PCR restriction fragments with the gp63 probe showed patterns similar to the L. donovani MON-82 reference strain.
4. Discussion Epidemics of CL were reported in Tuti Island in Khartoum in 1985 (El Safi and Peters, 1991) and in Shendi area, 300 km north of Khartoum (Abdalla and Sherif, 1978). Leishmania major MON-74 has been isolated and characterized from CL ulcers in Sudan and it is known to be transmitted by Phlebotomus papatasi (El-Hassan and Zijlstra, 2001). The study results indicate that L. donovani can cause classical cutaneous ulcers in Sudan. Leishmania donovani is known to be the main cause of VL and PKDL in Sudan and it has been isolated from cutaneous ulcers in Kenya (Mebrahtu et al., 1993), Ethiopia (Schonian et al., 2000) and Lebanon (Guerbouj et al., 2001). In Sudan, L. donovani has been described as the cause of leishmaniomas, which are localized ulcerating swellings at the site of the bite of sandflies in VL-endemic areas in eastern Sudan (El-Hassan and Zijlstra, 2001), where cutaneous ulcers have not been reported previously. Leishmania major isolates were cultured from the other three patients referred to the Institute of Endemic Diseases with cutaneous ulcers during the period of this study. The implication of the possible co-endemicity of L. donovani, causing CL, and L. major in central Sudan on the pathogenesis, clinical outcome, host immune response, interaction with vectors and infectivity of sandflies is not known. The identification of L. donovani MON-82 causing CL in Sudan in this study could be due to the use of more sensitive typing techniques. Further study of isolates from patients with cutaneous ulcers from different endemic areas is needed to ascertain the extent to which L. donovani causes CL in Sudan compared with L. major. Authors’ contributions: MMM co-ordinated the study; EME and MMM contributed to the collection of the samples, culture and maintenance of the parasites, and molecular characterization of the samples; IG and SG contributed to the DNA extraction, and establishment and application of the gp63 PCR and probes; MG and TDM contributed to the isoenzyme characterization of the isolates; AMEH contributed to the clinical diagnosis and management of the patients; MAT contributed to the clinical diagnosis and follow-up of the patients. All authors read and approved the final manuscript. MMM is guarantor of the paper.
E.M. Elamin et al. Acknowledgement: We thank Dr M. Willson for providing the gp63 probe. Funding: UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) grant No. 990559 and grant No. A20743. Conflicts of interest: None declared. Ethical approval: National Ethical Committee, Federal Ministry of Health, Khartoum, Sudan.
References Abdalla, R.E., Sherif, H., 1978. Epidemic of cutaneous leishmaniasis in northern Sudan. Ann. Trop. Med. Parasitol. 72, 349—352. Aoun, K., Bouratbine, A., Harrat, Z., Guizani, I., Mokni, M., Bel Hadj Ali, S., Ben Osman, A., Belkaid, M., Dellagi, K., Ben Ismail, R., 2000. Epidemiologic and parasitologic data concerning sporadic cutaneous leishmaniasis in northern Tunisia. Bull. Soc. Pathol. Exot. 93, 101—103 [in French]. Ashford, R.W., Seaman, J., Schorscher, J., Pratlong, F., 1992. Epidemic visceral leishmaniasis in southern Sudan: identity and systematic position of the parasites from patients and vectors. Trans. R. Soc. Trop. Med. Hyg. 86, 379—380. Cuny, G., Veas, F., Roizes, G., 1991. ‘‘Pocket blotting’’: a method for transferring nucleic acids onto nylon membranes. Anal. Biochem. 193, 45—48. El-Hassan, A.M., Zijlstra, E.E., 2001. Leishmaniasis in Sudan. 1. Cutaneous leishmaniasis. Trans. R. Soc. Trop. Med. Hyg. 95 (Suppl. 1), S1/1—S1/17. El Safi, S.H., Peters, W., 1991. Studies on the leishmaniases in the Sudan. 1. Epidemic of cutaneous leishmaniasis in Khartoum. Trans. R. Soc. Trop. Med. Hyg. 85, 44—47. Gramiccia, M., Gradoni, L., Pozio, E., 1987. Leishmania infantum sensu lato as an agent of cutaneous leishmaniasis in Abruzzi region (Italy). Trans. R. Soc. Trop. Med. Hyg. 81, 235— 237. Gramiccia, M., Ben-Ismail, R., Gradoni, L., Ben Rachid, M.S., Ben Said, M.A., 1991. A Leishmania infantum enzymatic variant, causative agent of cutaneous leishmaniasis in north Tunisia. Trans. R. Soc. Trop. Med. Hyg. 85, 370—371. Guerbouj, S., Guizani, I., De Doncker, S., Dujardin, J.C., NuwayriSalti, N., 2001. Identification of Lebanese dermotropic putative Leishmania archibaldi isolates by gp63 PCR—RFLP. Trans. R. Soc. Trop. Med. Hyg. 95, 687—688. Harrat, Z., Pratlong, F., Belazzoug, S., Dereure, J., Deniau, M., Rioux, J.A., Belkaid, M., Dedet, J.P., 1996. Leishmania infantum and L. major in Algeria. Trans. R. Soc. Trop. Med. Hyg. 90, 625—629. Jamjoom, M.B., Ashford, R.W., Bates, P.A., Chance, M.L., Kemp, S.J., Watts, P.C., Noyes, H.A., 2004. Leishmania donovani is the only cause of visceral leishmaniasis in East Africa; previous descriptions of L. infantum and ‘‘L. archibaldi’’ from this region are a consequence of convergent evolution in the isoenzyme data. Parasitology 129, 399—409. Maniatis, T., Fritsch, E.F., Sambrook, K.J., 1986. Purification of nucleic acids, in: Molecular Cloning, thirteenth ed. Cold Spring Harbor Laboratory, New York, pp. 458—459. Mebrahtu, Y.B., Van Eys, G., Guizani, I., Lawyer, P.G., Pamba, H., Koech, D., Roberts, C., Perkins, P.V., Were, J.B., Hendricks, L.D., 1993. Human cutaneous leishmaniasis caused by Leishmania donovani s.l. in Kenya. Trans. R. Soc. Trop. Med. Hyg. 87, 598—601. Rioux, J.A., Lanotte, G., Maazoun, R., Perello, R., Pratlong, F., 1980. Leishmania infantum Nicolle, 1908, the agent of the
Leishmania donovani as a cause of cutaneous leishmaniasis
57
autochthonous oriental sore. Apropos of the biochemical identification of 2 strains isolated in the eastern Pyrenees. C. R. Seances Acad. Sci. D 291, 701—703 [in French]. Rioux, J.A., Lanotte, G., Serres, E., Pratlong, F., Bastien, P., Perieres, J., 1990. Taxonomy of Leishmania. Use of isoenzymes. Suggestions for a new classification. Ann. Parasitol. Hum. Comp. 65, 111—125. Schonian, G., Akuffo, H., Lewin, S., Maasho, K., Nylen, S., Pratlong, F., Eisenberger, C.L., Schnur, L.F., Presber, W., 2000. Genetic variability within the species Leishmania aethiopica does not
correlate with clinical variations of cutaneous leishmaniasis. Mol. Biochem. Parasitol. 106, 239—248. Smyth, A.J., Ghosh, A., Hassan, M.Q., Basu, D., De Bruijn, M.H.L., Adhya, S., Mallik, K.K., Barker, D.C., 1992. Rapid and sensitive detection of Leishmania kinetoplast DNA from spleen and blood samples of kala-azar patients. Parasitology 105, 183— 192. WHO, 2000. The leishmaniases and Leishmania/HIV co-infections. World Health Organization, Geneva, Fact Sheet No. 116, revised May 2000.
available at www.sciencedirect.com
journal homepage: www.elsevierhealth.com/journals/trst
Identification of Leishmania donovani as a cause of cutaneous leishmaniasis in Sudan E.M. Elamin a,b, I. Guizani c, S. Guerbouj c, M. Gramiccia d, A.M. El Hassan a, T. Di Muccio d, M.A. Taha e, M.M. Mukhtar a,∗ a
Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan Faculty of Medical Laboratory Sciences, Al Zaiem Al Azhari University, Khartoum, Sudan c Laboratoire d’Epidemiologie et Ecologie Parasitaire, Institut Pasteur de Tunis, 1002 Tunis Belvedere, Tunisia d Instituto Superiore di Sanita, Viale Regina Elena 229, 00161 Rome, Italy e Department of Dermatology, Alribat Hospital, Khartoum, Sudan b
Received 10 April 2006; received in revised form 5 October 2007; accepted 5 October 2007 Available online 26 November 2007
KEYWORDS Cutaneous leishmaniasis; Leishmania donovani; Diagnosis; PCR; kDNA; Sudan
Summary Eight patients with cutaneous ulcers were referred to the Institute of Endemic Diseases, Khartoum, Sudan, from June 2000 to March 2002 for the diagnosis of suspected cutaneous leishmaniasis (CL). Diagnosis was confirmed parasitologically by both positive Giemsa-stained smears and successful culture of Leishmania promastigotes in NNN medium. The eight parasite isolates were shown to belong to the Leishmania donovani complex by kDNA PCR. Isoenzyme typing of three isolates revealed that they were identical to the L. donovani MON-82 reference strain, and the gp63 PCR—RFLP profile showed similar patterns to a reference strain of MON-82. CL is endemic in most regions of Sudan and has been reported previously as being caused by L. major MON-74. The results of this study suggest that L. donovani is also a cause of CL in Sudan and that further study of isolates from Sudanese patients with cutaneous ulcers is warranted to ascertain whether L. donovani or L. major is the causative agent. © 2007 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction Leishmania parasites cause different clinical forms of leishmaniasis: cutaneous (CL), visceral (VL), mucocutaneous, mucosal and post-kala-azar dermal (PKDL) (El-Hassan and
∗
Corresponding author. Tel.: +249 183 779712; fax: +249 183 779712. E-mail address: [email protected] (M.M. Mukhtar).
Zijlstra, 2001). The cutaneous form in the Old World is caused by L. major, L. tropica and L. aethiopica and is the most common form, constituting 50—75% of all new cases of leishmaniasis in the world (WHO, 2000). CL is endemic in most regions of Sudan and it was reported to be caused by L. major MON-74 only, while VL is endemic in the eastern, western and southern parts of Sudan (El-Hassan and Zijlstra, 2001). CL patients in Sudan usually present with nodular, nodulo-ulcerative and ulcerative lesions (El-Hassan and Zijlstra, 2001).
0035-9203/$ — see front matter © 2007 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2007.10.005
Leishmania donovani as a cause of cutaneous leishmaniasis
55
Leishmania infantum (L. donovani complex) was isolated from cutaneous lesions in Tunisia (Aoun et al., 2000; Gramiccia et al., 1991), Algeria (Harrat et al., 1996), Italy (Gramiccia et al., 1987) and France (Rioux et al., 1980). A few cutaneous ulcers were reported to be caused by L. donovani sensu latu in Kenya (Mebrahtu et al., 1993), Ethiopia (Schonian et al., 2000) and Lebanon (Guerbouj et al., 2001). In Sudan, the L. donovani complex comprises three major zymodemes: MON-18 (L. donovani), MON-30 (L. infantum) and MON-82 (L. archibaldi) (El-Hassan and Zijlstra, 2001). Some authors consider the three zymodemes to be separate species and suggest the endemic focus in Sudan as the origin of leishmaniasis (Rioux et al., 1990). In contrast, the three zymodemes were reported as a genetically homogeneous group causing VL (Jamjoom et al., 2004). Leishmania donovani has not been investigated as a cause of CL in Sudan. We isolated and typed Leishmania parasites from Sudanese patients with cutaneous ulcers to determine whether they were caused by L. donovani.
MW148). The isolates were mass cultured in 200 ml of RPMI 1640 medium supplemented with 10% fetal calf serum. DNA was extracted from log-phase cultures of the eight isolates using the phenol/chloroform method (Maniatis et al., 1986) and amplified using primers AJS3/DBY for kDNA PCR, which amplify a genomic DNA fragment of 800 bp in the L. donovani complex and 700 bp in the L. major complex (Smyth et al., 1992).
2. Materials and methods 2.1. Patients Eight Sudanese patients with cutaneous ulcers typical of those caused by Leishmania parasites (Figure 1) were referred to the Institute of Endemic Diseases, Khartoum, Sudan, from June 2000 to March 2002 for the diagnosis of suspected CL. All the patients contracted the infection in Khartoum State, central Sudan, and had no history of travelling outside Khartoum State to VL-endemic areas. On examination, all patients looked well and had no fever or enlargement of the liver or spleen. They all gave consent to be included in the study.
2.2. Diagnosis Aspirates were taken from the edges of the ulcers. Half of the aspirate was used for preparation of Giemsastained smears and the other half was inoculated into NNN medium for isolation of the parasite. Inoculated NNN medium was incubated at 24 ◦ C (Ashford et al., 1992). Cultures were examined microscopically for parasite growth. Eight isolates were successfully cultured (MHOM/SD/00/MW3, MHOM/SD/01/MW30, MHOM/SD/02/ MW120, MHOM/SD/00/MW17, MHOM/SD/00/MW79, MHOM/ SD/01/MW144, MHOM/SD/02/MW131 and MHOM/SD/00/
2.2.1. Isoenzyme typing The laboratories in Sudan do not have the facilities to confirm the findings by isoenzyme typing, thus the eight isolates were sent to the Instituto Superiore di Sanita, Rome, Italy, for characterization using the method described by Rioux et al. (1990) based on the isoenzyme profiles of 13 enzymes (representing 15 enzyme loci). Due to lack of funding, three of the eight isolates were randomly selected for isoenzyme typing. 2.2.2. gp63 PCR The gp63 PCR method described by Guerbouj et al. (2001) was used for further characterization of the three isolates that were subjected to isoenzyme typing. Specific primers (SG1 and SG2) that amplify the coding region of gp63 genes were used. Six reference strains were included as controls: L. aethiopica, L100 (MHOM/ET/72/L100); L. tropica, K27 (MHOM/SU/74/K27); L. major, 5ASKH (MHOM/SU/73/5ASKH); L. donovani MON-82, GEBRE1 (MHOM/ET/72/GEBRE1); L. infantum, IPT1 (MHOM/TN/80/IPT1); and L. donovani MON-18, DD8 (MHOM/IN/80/DD8). 2.2.3. gp63 PCR—RFLP The PCR-amplified fragments were analysed using four restriction enzymes, SalI, BalI, HincII (Amersham Pharmacia Biotech, Shanghai, China) and BsiEI (New England BioLabs, Ipswich, MA, USA), as recommended by the manufacturers. The DNA digests were separated by electrophoresis in 3% Small Fragments Agarose (Eurogentec, Liege, Belgium) overnight at 20 V and transferred to a Hybond-N+ nylon membrane using the ‘pocket blotting’ method (Cuny et al., 1991). The gp63 probe (L. chagasi cDNA clone, kindly provided by Dr M. Willson) was labelled with [32 P]dCTP using random primer labelling (Amersham Biosciences, Little Chalfont, UK). Hybridization and high stringency washing were performed at 65 ◦ C according to the manufacturer’s instructions (Amersham Biosciences).
2.3. Treatment of patients The patients were treated with ketoconazole (Janssen, Beerse, Belgium). The drug was administered orally at a dose of 400 mg/d for 4—8 weeks for adults (3 mg/kg/d for children). The drug was given only after liver function tests were found to be normal.
3. Results Figure 1 Multiple cutaneous lesions due to Leishmania donovani infection on the arm of a Sudanese patient.
The Giemsa-stained smears from all eight patients were positive. In addition, Leishmania parasites were successfully isolated and cultured from the eight patients. Parasite
56 growth was microscopically observed within 5—7 d of incubation at 24 ◦ C. kDNA PCR amplified a DNA fragment of 800 bp from the eight tested isolates, identifying them as members of the L. donovani complex. The isoenzyme profile of the three selected isolates, MHOM/SD/00/MW3, MHOM/SD/01/MW30 and MHOM/SD/02/ MW120, was identical to L. donovani MON-82. PCR amplification of the gp63 coding region of the three tested isolates generated a fragment of 1.2 kb. Hybridization of the gp63 PCR restriction fragments with the gp63 probe showed patterns similar to the L. donovani MON-82 reference strain.
4. Discussion Epidemics of CL were reported in Tuti Island in Khartoum in 1985 (El Safi and Peters, 1991) and in Shendi area, 300 km north of Khartoum (Abdalla and Sherif, 1978). Leishmania major MON-74 has been isolated and characterized from CL ulcers in Sudan and it is known to be transmitted by Phlebotomus papatasi (El-Hassan and Zijlstra, 2001). The study results indicate that L. donovani can cause classical cutaneous ulcers in Sudan. Leishmania donovani is known to be the main cause of VL and PKDL in Sudan and it has been isolated from cutaneous ulcers in Kenya (Mebrahtu et al., 1993), Ethiopia (Schonian et al., 2000) and Lebanon (Guerbouj et al., 2001). In Sudan, L. donovani has been described as the cause of leishmaniomas, which are localized ulcerating swellings at the site of the bite of sandflies in VL-endemic areas in eastern Sudan (El-Hassan and Zijlstra, 2001), where cutaneous ulcers have not been reported previously. Leishmania major isolates were cultured from the other three patients referred to the Institute of Endemic Diseases with cutaneous ulcers during the period of this study. The implication of the possible co-endemicity of L. donovani, causing CL, and L. major in central Sudan on the pathogenesis, clinical outcome, host immune response, interaction with vectors and infectivity of sandflies is not known. The identification of L. donovani MON-82 causing CL in Sudan in this study could be due to the use of more sensitive typing techniques. Further study of isolates from patients with cutaneous ulcers from different endemic areas is needed to ascertain the extent to which L. donovani causes CL in Sudan compared with L. major. Authors’ contributions: MMM co-ordinated the study; EME and MMM contributed to the collection of the samples, culture and maintenance of the parasites, and molecular characterization of the samples; IG and SG contributed to the DNA extraction, and establishment and application of the gp63 PCR and probes; MG and TDM contributed to the isoenzyme characterization of the isolates; AMEH contributed to the clinical diagnosis and management of the patients; MAT contributed to the clinical diagnosis and follow-up of the patients. All authors read and approved the final manuscript. MMM is guarantor of the paper.
E.M. Elamin et al. Acknowledgement: We thank Dr M. Willson for providing the gp63 probe. Funding: UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) grant No. 990559 and grant No. A20743. Conflicts of interest: None declared. Ethical approval: National Ethical Committee, Federal Ministry of Health, Khartoum, Sudan.
References Abdalla, R.E., Sherif, H., 1978. Epidemic of cutaneous leishmaniasis in northern Sudan. Ann. Trop. Med. Parasitol. 72, 349—352. Aoun, K., Bouratbine, A., Harrat, Z., Guizani, I., Mokni, M., Bel Hadj Ali, S., Ben Osman, A., Belkaid, M., Dellagi, K., Ben Ismail, R., 2000. Epidemiologic and parasitologic data concerning sporadic cutaneous leishmaniasis in northern Tunisia. Bull. Soc. Pathol. Exot. 93, 101—103 [in French]. Ashford, R.W., Seaman, J., Schorscher, J., Pratlong, F., 1992. Epidemic visceral leishmaniasis in southern Sudan: identity and systematic position of the parasites from patients and vectors. Trans. R. Soc. Trop. Med. Hyg. 86, 379—380. Cuny, G., Veas, F., Roizes, G., 1991. ‘‘Pocket blotting’’: a method for transferring nucleic acids onto nylon membranes. Anal. Biochem. 193, 45—48. El-Hassan, A.M., Zijlstra, E.E., 2001. Leishmaniasis in Sudan. 1. Cutaneous leishmaniasis. Trans. R. Soc. Trop. Med. Hyg. 95 (Suppl. 1), S1/1—S1/17. El Safi, S.H., Peters, W., 1991. Studies on the leishmaniases in the Sudan. 1. Epidemic of cutaneous leishmaniasis in Khartoum. Trans. R. Soc. Trop. Med. Hyg. 85, 44—47. Gramiccia, M., Gradoni, L., Pozio, E., 1987. Leishmania infantum sensu lato as an agent of cutaneous leishmaniasis in Abruzzi region (Italy). Trans. R. Soc. Trop. Med. Hyg. 81, 235— 237. Gramiccia, M., Ben-Ismail, R., Gradoni, L., Ben Rachid, M.S., Ben Said, M.A., 1991. A Leishmania infantum enzymatic variant, causative agent of cutaneous leishmaniasis in north Tunisia. Trans. R. Soc. Trop. Med. Hyg. 85, 370—371. Guerbouj, S., Guizani, I., De Doncker, S., Dujardin, J.C., NuwayriSalti, N., 2001. Identification of Lebanese dermotropic putative Leishmania archibaldi isolates by gp63 PCR—RFLP. Trans. R. Soc. Trop. Med. Hyg. 95, 687—688. Harrat, Z., Pratlong, F., Belazzoug, S., Dereure, J., Deniau, M., Rioux, J.A., Belkaid, M., Dedet, J.P., 1996. Leishmania infantum and L. major in Algeria. Trans. R. Soc. Trop. Med. Hyg. 90, 625—629. Jamjoom, M.B., Ashford, R.W., Bates, P.A., Chance, M.L., Kemp, S.J., Watts, P.C., Noyes, H.A., 2004. Leishmania donovani is the only cause of visceral leishmaniasis in East Africa; previous descriptions of L. infantum and ‘‘L. archibaldi’’ from this region are a consequence of convergent evolution in the isoenzyme data. Parasitology 129, 399—409. Maniatis, T., Fritsch, E.F., Sambrook, K.J., 1986. Purification of nucleic acids, in: Molecular Cloning, thirteenth ed. Cold Spring Harbor Laboratory, New York, pp. 458—459. Mebrahtu, Y.B., Van Eys, G., Guizani, I., Lawyer, P.G., Pamba, H., Koech, D., Roberts, C., Perkins, P.V., Were, J.B., Hendricks, L.D., 1993. Human cutaneous leishmaniasis caused by Leishmania donovani s.l. in Kenya. Trans. R. Soc. Trop. Med. Hyg. 87, 598—601. Rioux, J.A., Lanotte, G., Maazoun, R., Perello, R., Pratlong, F., 1980. Leishmania infantum Nicolle, 1908, the agent of the
Leishmania donovani as a cause of cutaneous leishmaniasis
57
autochthonous oriental sore. Apropos of the biochemical identification of 2 strains isolated in the eastern Pyrenees. C. R. Seances Acad. Sci. D 291, 701—703 [in French]. Rioux, J.A., Lanotte, G., Serres, E., Pratlong, F., Bastien, P., Perieres, J., 1990. Taxonomy of Leishmania. Use of isoenzymes. Suggestions for a new classification. Ann. Parasitol. Hum. Comp. 65, 111—125. Schonian, G., Akuffo, H., Lewin, S., Maasho, K., Nylen, S., Pratlong, F., Eisenberger, C.L., Schnur, L.F., Presber, W., 2000. Genetic variability within the species Leishmania aethiopica does not
correlate with clinical variations of cutaneous leishmaniasis. Mol. Biochem. Parasitol. 106, 239—248. Smyth, A.J., Ghosh, A., Hassan, M.Q., Basu, D., De Bruijn, M.H.L., Adhya, S., Mallik, K.K., Barker, D.C., 1992. Rapid and sensitive detection of Leishmania kinetoplast DNA from spleen and blood samples of kala-azar patients. Parasitology 105, 183— 192. WHO, 2000. The leishmaniases and Leishmania/HIV co-infections. World Health Organization, Geneva, Fact Sheet No. 116, revised May 2000.