158
TRANSACTIONS OFTHEROYAL SOCIETY OFTROPICAL MEDICINE ANDHYGIENE (1992) 86,158-160
Identification probes
of Leishmania
from mucosal leishmaniasis
by recombinant
DNA
H. W. Ghalib’, E. A. Eltoum’, C. C. M. Kroon 3 and A. M. El Hassan’ ’ Department of Microbiology, College of Medicine, University ofJuba, Khartoum Centre, P.O. Box 1270, Khartoum, Sudan; 2Department of Pathology, Faculty of Medicine, University of Khartoum, P.O. Box 102, Khartoum, Sudan; 3Laboratory of Tropical Hygiene, Royal Tropical Institute, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands Abstract Three casesof mucosal leishmaniasisare described. Parasitesisolated from mucosal lesions were identified by Southern blot analysisof their genomic deoxyribonucleic acids (DNAs) using recombinant DNA probe pDK20. Parasitesfrom 2 patients were identified asLeishmania donovani s.1. One of the patients had pure mucosal lesions, while in the secondpatient there was dissemination of the parasite to other organs. The spectrum of the diseasecausedby L. donovani is discussed.The parasite from the third patient was identified asL. major. Introduction Visceral, cutaneousand mucosal leishmaniasisprevail in the Sudan. The presentation, course and outcome of these diseasesare related to the speciesof the parasite and the host-parasiterelationship. Visceral leishmaniasis (VLJ is causedby Leishmania donovani s.l., zymodeme LON-48, and cutaneousleishmaniasis(CL) by L. major zymodeme LON-1. The causativeagent of the uncommon mucosal leishmaniasis(ML) has not beenidentified with certainty. Isoenzyme and serologicalanalysisusing monoclonal antibodies are someof the recent procedures employed for parasiteidentification. The novel recombinant deoxyribonucleic acid (DNA) technology is also now applied to the identification of Leishmania. A recombinant nuclear DNA probe was developed for the identification of ‘Old World’ Leishmania(VANEYSet al. 1989), and the strains within the Leishmania donovani complex (VANEYSet al., 1991). Here we report on the application of these recombinant DNA probes to the identification of the causativeagent of mucosal leishmaniasis in the Sudan.
total and differential white blood cell counts were normal. The patient reactedpositively to leishmanin. Biopsy was obtained from the nasal lesion and processedas described. The patient was treated with 400 mg of ketoconazole (Nizoral@, JanssenPharmaceuticals) once daily for 6 weeks. His condition improved and he was discharged from the hospital but did not report for followup. Patient no. 3 (S4). A 45 year old male from Ginana, western Sudan presented with an 8 year history of de-
Materials and Methods Patients Patient no. I (S2). A 30 year old male from Kosti, cen-
tral Sudan. Dresentedwith a 2 vear historv of nasalswelling, nasal&truction and bleehing. On examination, the nasalmucosawas red, swollen and fissured and the nasal septum was perforated (Fig. 1). There was no primary skin lesion or scar. The patient was afebrile. The spleen and lymph nodes were not palpable. The liver was palpable 4 cm below the costal margin. The haemoglobin was 10.2 eidl. Dackedcell volume (PCV1 36%. ervthrocyte sedim’en&on rate (ESR) 103 km ik the Crst’hour. Total and differential white blood cell counts were normal. The patient reactedpositively to leishmanin. A biopsy was obtained from the nasal lesion. Impression smearswere made and the biopsy was homogenizedand inoculated into a hamster for parasite isolation as detailed below. The patient was successfullytreated with sodium stibogluconate (Pentostamm,Wellcome, UK), 10 mg/kg body weight intravenously for 30 d. Patient no. 2 (S3). A 35 year old male from Nyala, western Sudan, presented with a 6 year history of oral and nasal swelling and anosmia.There was no history of active cutaneouslesion or scar. On examination, the Datient was afebrile; the spleenand liver were not palpable. The sublingual and submandibular lvmuh nodes were enlarged. ‘l?hemucosa covering the cheeks, floor of the mouth, gums and oropharynx was red, swollen and fissured. The mucosaof the noseshowedthe sameappearance. There was no perforation of the nasal septum or palate. He had a cardiomyopathy. The haemoglobinwas 11.7 g/d& PCV 36%, ESR 90 mm in the first hour, and Addressfor correspondence: H. W. Ghalib, P.O. Box 1270, Khartoum,Sudan.
Fig.1.Patient no.1.Mucosal leishmaniasis ofthenose. Thenose isswollenandthenasal septum isperforated. formity and swelling of the nose, oral swelling, nasalobstruction, tooth ache and loosening of the teeth. He had spontaneouslylost someof his molar teeth. There was no active lesion or scar in the skin. On examination the patient had a low-grade fever (38.5”C), of which he was not aware. The spleen and liver were palpable, 10 cm and 2 cm below the costalmargin respectively. The submandibular, epitrochlear and inguinal lymph nodeswere enlarged. The nasal septum was completely destroyed and
159 the anterior nareswere collapsed. The mucosalining the nosewas red, swollen and fissured. The mucosacovering the gums, cheek, floor of the mouth, soft and hard palates was also red. swollen and fissured. The incisor teeth were loose and there was severerecessionof the gums. Someof his molar teeth were lost. The haemoglob%was 8.8 ddl. PCV 26% and ESR 111 mm in the first hr. Totd white blood cell count was 2200 cells/mm3 with normal differential. Stoolswere negativefor schistosome eggs. The patient was treated with sodium stibogluconate. 10 ma/ka bodv weieht for 30 d. The mucosal lesions healgd ucom&eteli but the spleen remained palpable at 6 months follow-up.
nant probe pDK20. Our findings agreewith the recent report of L. infanturn causing mucocutaneous leishmaniasis from Spain
Isolation of the parasite
Biopsy material from the mucosal lesions was homogenized in RPM1 1640and about 0.5-1.0 ml of the homogenatewas injected intraperitoneally in hamsters. The animals were killed 6 weekslater and infected tissues,including the spleenand liver, were homogenizedin RPM1 1640 under sterile conditions. Aliquots of the homogenate were promptly transferred to RPM1 1640 supolemented with 10% foetal calf serum. incubated at ioom temperature and examineddaily forpromastigotes. The material was transferred to Amsterdam for characterization using DNA probes. Identification of Leishmania strains Leishmania promastigotes were mass cultured in
RPM1 1640 supplemented with 15% foetal calf serum. Parasite nuclear DNA was isolated as described by VAN EYS et aZ. (1989); 8 ug of DNA were digestedwith endonucleasesHi&II and BarnHI, blotted on nitrocellulose paper followed by hybridization with probe pDK20 (VAN EYS et al., 1991). This probe was originally selected from a genomic library of L. major (MHOMISNI OOiDKl) and differentiates between ‘Old World’ Leishmania taxa, in particular within the L. donovani comDlex. Probe DNA was labelled with higoxygenin-1 l-dUTP and the signal was detectedwith a Dig. Detection Kit@ (Boehringer Mannheim). Reference strains used were L. donovani DD8, L. infanturn IPTl and L. major SASKH. Control. Parasites isolated from a Sudanesepatient (Sl) with zoonotic cutaneous leishmaniasis were included in the analysisfor comparison. Results
Leishmania parasiteswere demonstratedin impression smears obtained from the active lesions of the 3 ML casesand the CL patient. The hamsters at post-mortem showed disseminated Leishmania infection. Promastigotes were successfully masscultured and parasite nuclear DNA was isolated. Southern blotting of digested parasite genomic DNAs followed by hybridization showed that parasites cultured from the mucosal leishmaniasis patients no. 1 (S2) and no. 3 (S4) resembledL. donovani DD8, whereas parasitesfrom the mucosal patient no. 2 (S3) and the control CL (Sl) were comparable with L. major SASKH (Fig. 2). Discussion
ML is rather uncommon in the Sudan, only 50 cases having been reported (WHO, 1990). Largely on epidemiologicalevidence,previous reports suggestedL. donovani as the causative agent (SATI, 1963; ABDALLA, 1982). ABDALLA et al. (1975)postulated a multiple aetiology for ML and suggestedthat it might be causedby dermotronic Leishmania parasites in some patients and viscerotropic Leishmania in others. Recombinant DNA orobes were develooed for the prompt identification of’ Leishmania parasites. VAN EYS et al. (1989, 1991) developed probes that identify Leishmania parasites of the ‘Old World’, within the L. donovani complex. Here we were able to isolate parasites from ML patients and characterizethem using recombi-
4.4-
2.32.0--
Fig. 2. Photographof a Southernblot containingdigestedgenomicDNAs hybridizedto pDK20. StrainsofL. donovaniDD8 (L. don.),L. infanturn I$Tl (L. infl) and L. major5ASKH (L. maj.) are WHO reference strains.Sl is anisolatefrom a Sudanese CL patientwith cutaneous leishmaniasis;S2-S4areisolatesfrompatientswith mucosalleishmaniasis (patientsno. 1,2 and3 respectively). (ALVAR et al., 1990). L. donovani was identified by
isoenzyme analysis from a chronic case of ML in the Sudan(A. M. El Hassanand E. A. Eltoum, unpublished data). In Saudi Arabia L. major was isolated from an infant with mucocutaneousleishmaniasis (AL-GINDAN et al., 1983). The recombinant DNA probe used also confirmed L. major LON-1 as the causative agent of cutaneousleishmaniasisin the Sudan. It is apparent that L. donovani causesa spectrum of manifestations. It causesclassicalVL, characterized by generalized visceralization, cachexia, pancytopenia, immunosuppressionand high mortality in untreated cases (SIDDIG et al.. 1989). The oarasite is also associatedwith the late corn&cations of*VL including post-kala-azar dermal leishmaniasis(PKDL) and post-kala-azaruveitis PKUf (EL-HASSAN. et at.. 1990). In natient no. 1. mucosal leishmaniasiswas a chronic diseasethat was confined to the nasal mucosa. Patient no. 3 had mucosal lesions and parasiteswere found in the lymph nodes, bone marrow and oossiblv in the soleen and liver. He had intermittent low grade fever. Unlike classicalVL, his illness was of long duration and despite this there was no wasting. Furthermore, he was leishmanin positive. We are not certain whether the patient’s illness started with the mucosal diseasethat later disseminated or whether the visceral and mucosal manifestations occurred simultaneously. From patient no. 2, who had oronasal lesions without systemic involvement, L. major was isolated. This showsthat mucosal leishmaniasisin the Sudan may be causedby either L. donovani or L. major, as postulated previously (ABDALLA et aZ., 1975). It is therefore
160 important to type asmany isolatesfrom ML aspossible. ML is associatedwith low mortalitv and is successfullv treated with Pent&tam@ or ketoco&ole. ML patients are immunologically competent to leishmanin sensitization and their peripheral blood mononuclear cells respond vigorously to leishmanial antigens (H. W. Ghalib, unpublished data). Pure ML causedby L. donovani represents the immunologically competent end of the spectrum, whereas VL is at the other end of the spectrum and is usually associated with anergy to leishmanial antigens. There appear to be casesin the middle of the spectrum, as exemplified by patient no. 3. Whether there are severalgenetic variants amongthe isolatesof L. donovani responsible for these different host-parasite manifestations is worth clarifying. This necessitatesthe need for the development of more refined diagnostic DNA probes for use within the genusLeishmnnia and its constituent speciesand complexes.
Rqal Society of Tropical Medicine and Hygiene, 69,443-+49.
Al-Gindan, Y., Omer, A. H. S., Al-Humeidan, Y., Peters, W. & Evans, D. A. (1983). A case of mucocutaneous leishmaniasis in Saudi Arabia causedby Leishmania major and its responseto treatment. Clinical and Expen’mental Dermatology, 8, 185-188. Alvar, J., Balesteros,J. A., Soler, R., Benito, A., van Eys, G. J. J. M., Schoone, G. J. & Cabrer, B. (1990). Mucocutaneous leishmaniasis due to Leishmania (Leishmania) infantum: biochemical characterization. American Journal of Tropical Medicine and Hygiene, 43,614-618.
El Hassan, A. M., Ghalib, H. W., Zijlstra, E., Eltoum, I. A., Ali, M. S. & Ahmed, H. M. A. (1990). Post-kala-azardermal leishmaniasisin the absenceof active visceral leishmaniasis. Lancet, 336,750.
Sati, M. H. (1963). Cutaneous and mucocutaneous lesions in kala-azar.Sudan MedicalJournal, 2,88-103. Siddiq, M. A., Gaafar, A., Ghalib, H., Grayson, J., Petersen, E. & Khidir, S. (1989). Kala-azar in the Sudan: clinical and immunological manifestations. Sudan Medical Journal, 27, 26-36. Van Eys, G. J. J. M., Schoone,G. J., Ligthart, G. S., Alvar, J., Evans, D. A. & Terpstra, W. J. (1989). Identification of ‘Old World’ Leishmania by DNA recombinant probes. Molecular
Acknowledgements We thank Dr G. van Eys and Dr S. Meredith (Laboratory of Tropical Hygiene, Amsterdam, The Netherlands) for their support and encouragement, and Dr S. Reed (Seattle Biomedical ResearchInstitute, Seattle, Washington, USA) for kindly supplying the leishmanin. We are indebted to the SudanMRCiUSA NIH (Brigham Young University) Medical Parasitology Project for laboratory facilities.
Van Eys, G. J. J. M., Guizam, I., Ligthart, G. S. & Dellagi, K. (1991). A nuclear DNA probe for the identification of strains within the Leishmania donovanicomplex. Experimental Parasi-
References Abdalla, R. E. (1982). Parasitesin Sudanesecutaneousand mucosal leishmaniasis.Annals of Tropical Medicine and Parasito-
WHO (1990). Control of the Leishmaniases. Report of a WHO Expert Committee. Geneva:World Health Organization, Technical Report Seriesno. 793.
logy, 76,299-309.
Abdalla, R. E., El Hadi, A., Ahmed, M. A. & El Hassan, A. M. (1975). Sudan mucosal leishmaniasis. Transactions of the
1 Announcement
and Biochemical Parasitolog?r, 34, 53-62.
tology, 72,459-463.
Received 24 July 1991; revised 28 August 1991; accepted 29 August 1991
for publication
1 Continuing
Medical Royal
Education
and Training
in Europe: the Future
College of Physicians, London, 1-2 October 1992
UK
The conferenceis designedto examine critically the educationaland political implications for medical training in Europe following
harmonization
in 1992.
Further information can be obtained from: Dr M. W. N. Nicholls or Mrs J. M. Coops, The ConferenceOffice, c/o The Fellowship of PostgraduateMedicine, 6 St Andrew’s Place, London, NW1 4LB, UK. Telephone: (44) (0)71 935 5556; fax: (44) (0)71 224 3219.
TRANSACTIONS OFTHEROYAL SOCIETY OFTROPICAL MEDICINE ANDHYGIENE (1992) 86,158-160
Identification probes
of Leishmania
from mucosal leishmaniasis
by recombinant
DNA
H. W. Ghalib’, E. A. Eltoum’, C. C. M. Kroon 3 and A. M. El Hassan’ ’ Department of Microbiology, College of Medicine, University ofJuba, Khartoum Centre, P.O. Box 1270, Khartoum, Sudan; 2Department of Pathology, Faculty of Medicine, University of Khartoum, P.O. Box 102, Khartoum, Sudan; 3Laboratory of Tropical Hygiene, Royal Tropical Institute, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands Abstract Three casesof mucosal leishmaniasisare described. Parasitesisolated from mucosal lesions were identified by Southern blot analysisof their genomic deoxyribonucleic acids (DNAs) using recombinant DNA probe pDK20. Parasitesfrom 2 patients were identified asLeishmania donovani s.1. One of the patients had pure mucosal lesions, while in the secondpatient there was dissemination of the parasite to other organs. The spectrum of the diseasecausedby L. donovani is discussed.The parasite from the third patient was identified asL. major. Introduction Visceral, cutaneousand mucosal leishmaniasisprevail in the Sudan. The presentation, course and outcome of these diseasesare related to the speciesof the parasite and the host-parasiterelationship. Visceral leishmaniasis (VLJ is causedby Leishmania donovani s.l., zymodeme LON-48, and cutaneousleishmaniasis(CL) by L. major zymodeme LON-1. The causativeagent of the uncommon mucosal leishmaniasis(ML) has not beenidentified with certainty. Isoenzyme and serologicalanalysisusing monoclonal antibodies are someof the recent procedures employed for parasiteidentification. The novel recombinant deoxyribonucleic acid (DNA) technology is also now applied to the identification of Leishmania. A recombinant nuclear DNA probe was developed for the identification of ‘Old World’ Leishmania(VANEYSet al. 1989), and the strains within the Leishmania donovani complex (VANEYSet al., 1991). Here we report on the application of these recombinant DNA probes to the identification of the causativeagent of mucosal leishmaniasis in the Sudan.
total and differential white blood cell counts were normal. The patient reactedpositively to leishmanin. Biopsy was obtained from the nasal lesion and processedas described. The patient was treated with 400 mg of ketoconazole (Nizoral@, JanssenPharmaceuticals) once daily for 6 weeks. His condition improved and he was discharged from the hospital but did not report for followup. Patient no. 3 (S4). A 45 year old male from Ginana, western Sudan presented with an 8 year history of de-
Materials and Methods Patients Patient no. I (S2). A 30 year old male from Kosti, cen-
tral Sudan. Dresentedwith a 2 vear historv of nasalswelling, nasal&truction and bleehing. On examination, the nasalmucosawas red, swollen and fissured and the nasal septum was perforated (Fig. 1). There was no primary skin lesion or scar. The patient was afebrile. The spleen and lymph nodes were not palpable. The liver was palpable 4 cm below the costal margin. The haemoglobin was 10.2 eidl. Dackedcell volume (PCV1 36%. ervthrocyte sedim’en&on rate (ESR) 103 km ik the Crst’hour. Total and differential white blood cell counts were normal. The patient reactedpositively to leishmanin. A biopsy was obtained from the nasal lesion. Impression smearswere made and the biopsy was homogenizedand inoculated into a hamster for parasite isolation as detailed below. The patient was successfullytreated with sodium stibogluconate (Pentostamm,Wellcome, UK), 10 mg/kg body weight intravenously for 30 d. Patient no. 2 (S3). A 35 year old male from Nyala, western Sudan, presented with a 6 year history of oral and nasal swelling and anosmia.There was no history of active cutaneouslesion or scar. On examination, the Datient was afebrile; the spleenand liver were not palpable. The sublingual and submandibular lvmuh nodes were enlarged. ‘l?hemucosa covering the cheeks, floor of the mouth, gums and oropharynx was red, swollen and fissured. The mucosaof the noseshowedthe sameappearance. There was no perforation of the nasal septum or palate. He had a cardiomyopathy. The haemoglobinwas 11.7 g/d& PCV 36%, ESR 90 mm in the first hour, and Addressfor correspondence: H. W. Ghalib, P.O. Box 1270, Khartoum,Sudan.
Fig.1.Patient no.1.Mucosal leishmaniasis ofthenose. Thenose isswollenandthenasal septum isperforated. formity and swelling of the nose, oral swelling, nasalobstruction, tooth ache and loosening of the teeth. He had spontaneouslylost someof his molar teeth. There was no active lesion or scar in the skin. On examination the patient had a low-grade fever (38.5”C), of which he was not aware. The spleen and liver were palpable, 10 cm and 2 cm below the costalmargin respectively. The submandibular, epitrochlear and inguinal lymph nodeswere enlarged. The nasal septum was completely destroyed and
159 the anterior nareswere collapsed. The mucosalining the nosewas red, swollen and fissured. The mucosacovering the gums, cheek, floor of the mouth, soft and hard palates was also red. swollen and fissured. The incisor teeth were loose and there was severerecessionof the gums. Someof his molar teeth were lost. The haemoglob%was 8.8 ddl. PCV 26% and ESR 111 mm in the first hr. Totd white blood cell count was 2200 cells/mm3 with normal differential. Stoolswere negativefor schistosome eggs. The patient was treated with sodium stibogluconate. 10 ma/ka bodv weieht for 30 d. The mucosal lesions healgd ucom&eteli but the spleen remained palpable at 6 months follow-up.
nant probe pDK20. Our findings agreewith the recent report of L. infanturn causing mucocutaneous leishmaniasis from Spain
Isolation of the parasite
Biopsy material from the mucosal lesions was homogenized in RPM1 1640and about 0.5-1.0 ml of the homogenatewas injected intraperitoneally in hamsters. The animals were killed 6 weekslater and infected tissues,including the spleenand liver, were homogenizedin RPM1 1640 under sterile conditions. Aliquots of the homogenate were promptly transferred to RPM1 1640 supolemented with 10% foetal calf serum. incubated at ioom temperature and examineddaily forpromastigotes. The material was transferred to Amsterdam for characterization using DNA probes. Identification of Leishmania strains Leishmania promastigotes were mass cultured in
RPM1 1640 supplemented with 15% foetal calf serum. Parasite nuclear DNA was isolated as described by VAN EYS et aZ. (1989); 8 ug of DNA were digestedwith endonucleasesHi&II and BarnHI, blotted on nitrocellulose paper followed by hybridization with probe pDK20 (VAN EYS et al., 1991). This probe was originally selected from a genomic library of L. major (MHOMISNI OOiDKl) and differentiates between ‘Old World’ Leishmania taxa, in particular within the L. donovani comDlex. Probe DNA was labelled with higoxygenin-1 l-dUTP and the signal was detectedwith a Dig. Detection Kit@ (Boehringer Mannheim). Reference strains used were L. donovani DD8, L. infanturn IPTl and L. major SASKH. Control. Parasites isolated from a Sudanesepatient (Sl) with zoonotic cutaneous leishmaniasis were included in the analysisfor comparison. Results
Leishmania parasiteswere demonstratedin impression smears obtained from the active lesions of the 3 ML casesand the CL patient. The hamsters at post-mortem showed disseminated Leishmania infection. Promastigotes were successfully masscultured and parasite nuclear DNA was isolated. Southern blotting of digested parasite genomic DNAs followed by hybridization showed that parasites cultured from the mucosal leishmaniasis patients no. 1 (S2) and no. 3 (S4) resembledL. donovani DD8, whereas parasitesfrom the mucosal patient no. 2 (S3) and the control CL (Sl) were comparable with L. major SASKH (Fig. 2). Discussion
ML is rather uncommon in the Sudan, only 50 cases having been reported (WHO, 1990). Largely on epidemiologicalevidence,previous reports suggestedL. donovani as the causative agent (SATI, 1963; ABDALLA, 1982). ABDALLA et al. (1975)postulated a multiple aetiology for ML and suggestedthat it might be causedby dermotronic Leishmania parasites in some patients and viscerotropic Leishmania in others. Recombinant DNA orobes were develooed for the prompt identification of’ Leishmania parasites. VAN EYS et al. (1989, 1991) developed probes that identify Leishmania parasites of the ‘Old World’, within the L. donovani complex. Here we were able to isolate parasites from ML patients and characterizethem using recombi-
4.4-
2.32.0--
Fig. 2. Photographof a Southernblot containingdigestedgenomicDNAs hybridizedto pDK20. StrainsofL. donovaniDD8 (L. don.),L. infanturn I$Tl (L. infl) and L. major5ASKH (L. maj.) are WHO reference strains.Sl is anisolatefrom a Sudanese CL patientwith cutaneous leishmaniasis;S2-S4areisolatesfrompatientswith mucosalleishmaniasis (patientsno. 1,2 and3 respectively). (ALVAR et al., 1990). L. donovani was identified by
isoenzyme analysis from a chronic case of ML in the Sudan(A. M. El Hassanand E. A. Eltoum, unpublished data). In Saudi Arabia L. major was isolated from an infant with mucocutaneousleishmaniasis (AL-GINDAN et al., 1983). The recombinant DNA probe used also confirmed L. major LON-1 as the causative agent of cutaneousleishmaniasisin the Sudan. It is apparent that L. donovani causesa spectrum of manifestations. It causesclassicalVL, characterized by generalized visceralization, cachexia, pancytopenia, immunosuppressionand high mortality in untreated cases (SIDDIG et al.. 1989). The oarasite is also associatedwith the late corn&cations of*VL including post-kala-azar dermal leishmaniasis(PKDL) and post-kala-azaruveitis PKUf (EL-HASSAN. et at.. 1990). In natient no. 1. mucosal leishmaniasiswas a chronic diseasethat was confined to the nasal mucosa. Patient no. 3 had mucosal lesions and parasiteswere found in the lymph nodes, bone marrow and oossiblv in the soleen and liver. He had intermittent low grade fever. Unlike classicalVL, his illness was of long duration and despite this there was no wasting. Furthermore, he was leishmanin positive. We are not certain whether the patient’s illness started with the mucosal diseasethat later disseminated or whether the visceral and mucosal manifestations occurred simultaneously. From patient no. 2, who had oronasal lesions without systemic involvement, L. major was isolated. This showsthat mucosal leishmaniasisin the Sudan may be causedby either L. donovani or L. major, as postulated previously (ABDALLA et aZ., 1975). It is therefore
160 important to type asmany isolatesfrom ML aspossible. ML is associatedwith low mortalitv and is successfullv treated with Pent&tam@ or ketoco&ole. ML patients are immunologically competent to leishmanin sensitization and their peripheral blood mononuclear cells respond vigorously to leishmanial antigens (H. W. Ghalib, unpublished data). Pure ML causedby L. donovani represents the immunologically competent end of the spectrum, whereas VL is at the other end of the spectrum and is usually associated with anergy to leishmanial antigens. There appear to be casesin the middle of the spectrum, as exemplified by patient no. 3. Whether there are severalgenetic variants amongthe isolatesof L. donovani responsible for these different host-parasite manifestations is worth clarifying. This necessitatesthe need for the development of more refined diagnostic DNA probes for use within the genusLeishmnnia and its constituent speciesand complexes.
Rqal Society of Tropical Medicine and Hygiene, 69,443-+49.
Al-Gindan, Y., Omer, A. H. S., Al-Humeidan, Y., Peters, W. & Evans, D. A. (1983). A case of mucocutaneous leishmaniasis in Saudi Arabia causedby Leishmania major and its responseto treatment. Clinical and Expen’mental Dermatology, 8, 185-188. Alvar, J., Balesteros,J. A., Soler, R., Benito, A., van Eys, G. J. J. M., Schoone, G. J. & Cabrer, B. (1990). Mucocutaneous leishmaniasis due to Leishmania (Leishmania) infantum: biochemical characterization. American Journal of Tropical Medicine and Hygiene, 43,614-618.
El Hassan, A. M., Ghalib, H. W., Zijlstra, E., Eltoum, I. A., Ali, M. S. & Ahmed, H. M. A. (1990). Post-kala-azardermal leishmaniasisin the absenceof active visceral leishmaniasis. Lancet, 336,750.
Sati, M. H. (1963). Cutaneous and mucocutaneous lesions in kala-azar.Sudan MedicalJournal, 2,88-103. Siddiq, M. A., Gaafar, A., Ghalib, H., Grayson, J., Petersen, E. & Khidir, S. (1989). Kala-azar in the Sudan: clinical and immunological manifestations. Sudan Medical Journal, 27, 26-36. Van Eys, G. J. J. M., Schoone,G. J., Ligthart, G. S., Alvar, J., Evans, D. A. & Terpstra, W. J. (1989). Identification of ‘Old World’ Leishmania by DNA recombinant probes. Molecular
Acknowledgements We thank Dr G. van Eys and Dr S. Meredith (Laboratory of Tropical Hygiene, Amsterdam, The Netherlands) for their support and encouragement, and Dr S. Reed (Seattle Biomedical ResearchInstitute, Seattle, Washington, USA) for kindly supplying the leishmanin. We are indebted to the SudanMRCiUSA NIH (Brigham Young University) Medical Parasitology Project for laboratory facilities.
Van Eys, G. J. J. M., Guizam, I., Ligthart, G. S. & Dellagi, K. (1991). A nuclear DNA probe for the identification of strains within the Leishmania donovanicomplex. Experimental Parasi-
References Abdalla, R. E. (1982). Parasitesin Sudanesecutaneousand mucosal leishmaniasis.Annals of Tropical Medicine and Parasito-
WHO (1990). Control of the Leishmaniases. Report of a WHO Expert Committee. Geneva:World Health Organization, Technical Report Seriesno. 793.
logy, 76,299-309.
Abdalla, R. E., El Hadi, A., Ahmed, M. A. & El Hassan, A. M. (1975). Sudan mucosal leishmaniasis. Transactions of the
1 Announcement
and Biochemical Parasitolog?r, 34, 53-62.
tology, 72,459-463.
Received 24 July 1991; revised 28 August 1991; accepted 29 August 1991
for publication
1 Continuing
Medical Royal
Education
and Training
in Europe: the Future
College of Physicians, London, 1-2 October 1992
UK
The conferenceis designedto examine critically the educationaland political implications for medical training in Europe following
harmonization
in 1992.
Further information can be obtained from: Dr M. W. N. Nicholls or Mrs J. M. Coops, The ConferenceOffice, c/o The Fellowship of PostgraduateMedicine, 6 St Andrew’s Place, London, NW1 4LB, UK. Telephone: (44) (0)71 935 5556; fax: (44) (0)71 224 3219.
