1  Introduction

• 1.1 Leishmaniasis:

  • 1.1.1 The disease:

  • 1.1.2 Clinial spectrum:

    • 1.1.2.1 Visceral leishmaniasis (VL):

    • 1.1.2.2 Post kala-azar dermal leishmaniasis (PKDL):

    • 1.1.2.3  Cutaneous leishmaniasis (CL):

    • 1.1.2.4 Diffuse cutaneous leishmaniasis (DCL):

    • 1.1.2.5 Mucocutaneous leishmaniasis (MCL):

  • 1.1.3 Treatment:

  • 1.1.4 Parasite and life cycle:

  • 1.1.5 The vector:

  • 1.1.6 The Leishmania genome:

    • 1.1.6.1 Nuclear DNA:

    • 1.1.6.2 Kinetoplast DNA (kDNA):

  • 1.1.7 Classification of Leishmania species:

  • 1.1.8 Diagnosis of visceral leishmaniasis:

  • 1.1.9 Identification and characterization of Leishmania:

    • 1.1.9.1 Phenotypic and immunological methods:

    • 1.1.9.2 Molecular biological methods:

• 1.2 Leishmaniasis in the Sudan:

• 1.3 Selection of genomic targets for the differentiation of species and strains of Leishmania :

  • 1.3.1 Ribosomal internal transcribed spacer (ITS):

  • 1.3.2 gp63 genes:

  • 1.3.3 Anonymous DNA regions:

• 1.4 Objectives of this study:

2 Materials and Methods

• 2.1 Study area:

• 2.2 Samples collection:

• 2.3  DNAextraction:

  • 2.3.1 DNA extraction from clinical samples spotted on filter papers:

  • 2.3.2  DNA extraction from cultured Leishmania:

• 2.4 PCR amplification:

  • 2.4.1 Internal transcribed spacer (ITS):

  • 2.4.2 Major surface protease msp (gp63) gene:

  • 2.4.3 Anonymous DNA markers:

• 2.5  Optimization of PCR protocols:

• 2.6 Single stranded conformation polymorphism (SSCP):

• 2.7 PCR- fingerprinting:

• 2.8 Restriction fragment length analysis (RFLA):

• 2.9  Radioactive cycle sequencing:

  • 2.9.1 Template purification:

  • 2.9.2 Sequencing cycles:

  • 2.9.3 Preparation of the sequencing plates and electrophoresis:

3 Results

• 3.1 Clinical manifestation:

• 3.2  Direct detection of Leishmania parasites in clinical samples:

  • 3.2.1 Microscopy:

  • 3.2.2 PCR results:

  • 3.2.3 Lymph node aspirates: microscopy versus PCR:

  • 3.2.4 Bone marrow aspirates: microscopy versus PCR:

• 3.3 Detection of DNA polymorphisms in the ITS sequences:

• 3.4 Detection of DNA polymorphisms in the gp63 sequences:

• 3.5  Detection of DNA polymorphisms in different anonymous DNA fragments:

• 3.6 PCR fingerprinting:

4  Discussion

• 4.1  Conclusions:

Appendices

Table 1: Specific primer pairs and PCR conditions (per 50 μl) for the amplification of anonymous DNA markers. One U Taq polymerase and 1x PCR buffer (10 mM Tris-HCl, pH 8.0; 50 mM KCl; 1.5 mM MgCl2) were added into all reactions.

Table 2: Conditions for SSCP analysis of different PCR products

Table 3: PCR program for PCR-fingerprinting using the primers T3B, (GTG)5, M13 core and (GACA)4. *ID: Initial denaturation. ** F: Final

Table 4: Comparison of the results obtained by PCR and microscopy with clinical samples collected from lymph node aspirates

Table. 5: Comparison of the results obtained by PCR and microscopy with clinical samples collected from bone marrow aspirates

Appendix 1: Demographic and clinical data for112 visceral leishmaniasis patients from eastern part of Sudan around Gedaref State (April 1997-November 1998).

Appendix 2: Strains of Leishmania donovani analysed in this study and the results of analyses.

Figure 1: Endemic areas of visceral and cutaneous leishmaniasis in Sudan.

Figure 2: A picture of the endemic villages showing the construction of living places and the cracked clay soil.

Figure 3: The position of the internal transcribed spacer (ITS) in the ribosomal operon amplified with leishmania specific primers. Primer sequences are given in the text.

Figure 4: Schematic presentation showing the division of gp63 gene into 3 parts. Primer sequences are given in the text.

Figur. 5: Age and sex distribution of kala-azar patient from eastern Sudan around El Gedaref between April 1997 and November 1998

Figure. 6:PCR products amplified from 2 different clinical isolates of Leishmania.

Figure 7: RFLP banding patterns obtained by digesting the amplified ITS regions of different clinical samples of L. donovani with Cfo1 enzyme.

Figure 8a: SSCP analysis of ITS1 regions amplified from different clinical Leishmania donovani isolates.

Figure 8b: SSCP analysis of ITS1 regions amplifyed from different clinical Leishmania donovani isolates.

Figure 9: SSCP analysis of ITS2 regions amplified from different clinical Leishmania donovani isolates.

Figure 10: The complete dominant ITS sequence amplified from clinical L.donovani sample from eastern Sudan Primers are represented by bold letters. Sequence of the 5.8S rRNA gene is indicated in red.

Figure 11 Alignments of the variable parts of the ITS1 sequences amplified from clinical L. donovani isolates, representing the 14 different SSCP patterns (LdA...LdN).

Figure 12: Alignments of the variable parts of the ITS2 sequences amplified from clinical L. donovani isolates, representing the 5 different SSCP patterns (LdA…LdZ).

Figure 13: PCR products amplified from 3 different clinical isolates of Leishmania donovani.

Figure 14: SSCP analysis of Y regions of gp63 gene amplified from different clinical Leishmania donovani isolates from eastern Sudan.

Figure 15: Alignments of part (Y) of the coding region of the gp63 gene sequences amplified from clinical samples of L.donovani from eastern Sudan representing the 3 different SSCP patterns (A,B,C).

Figure 16: DNA fragments amplified from 2 different clinical isolates of Leishmania donovani.

Figure 17: SSCP analysis of anonymous fragment (114) amplified from different clinical Leishmania donovani isolates from eastern Sudan.

Figure 18: Anonymous DNA loci sequences amplified from clinical L. donovani samples from eastern Sudan.

Figure 19: Results of the PCR fingerprinting using the (GTG)5 primer.

Figure 20: Results of the PCR fingerprinting using the (GACA)4 primer.