Results

Clinical manifestation:

The disease in this study area was found to affect mainly children below 11 years of age and young adults between age 11 and 30 (Figure 5). There was male predominance (79%). Most of the patients (86%) were migrants of different tribes from western Sudan or Falata from Chad and Nigeria. There were also 5 patients from Eretria. The disease was found to affect mainly those of low socio-economic class (for details see appendix 1).

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

Direct detection of Leishmania parasites in clinical samples: Microscopy:

Overall from the 100 Giemsa-stained thin films, 71 samples were found to be microscopically positive. Twenty two of these were found to be negative when microscopic examination was performed on samples collected from lymph node aspirates. These samples were found to be positive when examination was carried out on samples collected from bone marrow aspirates and one sample from skin smear.

PCR results:

All the 71 selected microscopically positive clinical samples, which were collected from the field work were also positive in nested PCR when ITS1 and ITS2 regions were used as targets for amplification. In the first round of ITS-PCR these samples were also positive, but some showed weak PCR products. Fourteen of the 29 microscopically negative clinical samples were found to be PCR positive, whereas the remaining 15 clinical samples were also PCR negative. All negative controls were negative; i.e. no contamination or inhibition was detected.

Lymph node aspirates: microscopy versus PCR:

Lymph node aspirates were subjected to microscopy and PCR (ITS1 and ITS2 regions). A comparison of the results is given in table (4). PCR detected significantly more Leishmania parasites than did microscopy (P<0.001), in the clinical samples collected from lymph node aspirates. Of the 36 lymph node samples with a positive PCR results but a negative microscopy result, 21 were from patients with confirmed visceral leishmaniasis (VL) on the basis of microscopy of bone marrow aspirates, 15 were from VL suspects (microscopically negative on basis of bone marrow and lymph node aspirates

Microscopy

Total

Positive

Negative

PCR

Positive

49

36

85

Negative

0

15

15

Total

49

51

100

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

P < 0.001
X²=16.9555

but patients had clinical symptoms of leishmaniasis). Of the 15 samples, which were found to be negative by microscopy and PCR, one sample was collected from a lymph node of a post kala-azar dermal leishmaniasis patient (PKDL).

Bone marrow aspirates: microscopy versus PCR:

Bone marrow aspirates were subjected to microscopy and PCR (ITS1 and ITS2 regions). A comparison of the results is given in table (5). PCR detected significantly more Leishmania parasites than did microscopy (P<0.001), in the clinical samples collected from bone marrow aspirates. Of the 15 samples, which were found to be negative by microscopy and PCR one sample was collected from a bone marrow of a PKDL patient.

Microscopy

Total

Positive

Negative

PCR

Positive

21

15

36

Negative

0

15

15

Total

21

30

51

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

P < 0.001
X²= 14.875

Detection of DNA polymorphisms in the ITS sequences:

The ITS sequences were amplified from genomic DNA extracted from the clinicalsamples collected in Sudan but also from 18 cultured strains of L. donovani.

Samples were scored as positive for the presence of Leishmania if a PCR product of approximately 1020 bp was obtained for the entire ITS region. When ITS1 and ITS2 were amplified separately, amplification products of about 320 and 700 bp, respectively, were obtained. When ITS2A and B were amplified separately, PCR products of about 300 and 450bp, respectively, were observed (Figure 6).

To test for sequence heterogeneity which could be detected by RFLP, the entire ITS region was cut with 10 different enzymes, the profiles of all 86 (71 microscopically confirmed cases plus the 15 Sudanese cultured samples) studied strains were, however indistinguishable. Figure (7) showed the restriction pattern obtained by Cfo1 as an example.

Figure. 6:PCR products amplified from 2 different clinical isolates of Leishmania. Lanes 1&2: ITS PCR products (1020bp); Lanes 3&4: ITS1 PCR products (320bp); Lanes 5&6: ITS2 PCR products (700bp); Lanes 7&8: ITS2A PCR products (300bp); Lanes 9&10: ITS2B PCR products (450bp); M: Molecular size (weight) standard marker; Kbp: Kilobase-pairs

When ITS1 PCR products were subjected to SSCP, in total 14 ITS1 polymorphic profiles (Figures 8a & 8b; Appendix 2) could be distinguished among all 89 (71 microscopically confirmed cases plus the 18 {15 from Sudan and 3 from other regions} cultured samples) tested isolates. Two of these patterns namely K and N were demonstrated by the samples of Kenya, India and China.

Figure 7: RFLP banding patterns obtained by digesting the amplified ITS regions of different clinical samples of L. donovani with Cfo1 enzyme. M: molecular size (weight) standard marker; Kbp: Kilobase-pairs.

The Kenyan and Indian samples had the same pattern K, whereas the Chinese isolate exhibited pattern N. The other 12 patterns were demonstrated by the samples from Sudan. The dominant pattern, A, was found in 66 of the 86 VL cases (76.7%) from Sudan, followed by pattern D, C and H, which were observed in 5, 4 and 3 cases, respectively i.e. (5.8%, 4.6% and 3.5%). Each of the other 8 patterns, namely J, E, L, B, F, G, I and M, were obtained from only one VL case each.

In total 5 ITS2 polymorphic SSCP profiles could be distinguished among all 89 (71 microscopically confirmed cases plus 18 cultured samples) tested isolates (Figure 9; appendix 2). Concerning the 86 Sudanese isolates, all of them had the same pattern A with exception of one sample, which had the pattern J. This sample had the pattern J in ITS1 SSCP analysis. Samples from Kenya, India and China had the patterns X, Y and Z, respectively.

Figure 8a: SSCP analysis of ITS1 regions amplified from different clinical Leishmania donovani isolates. Each SSCP pattern is designated by a capital letter. Patterns A, C and D were very similar but the size of the upper fragment in pattern D was slightly larger than in pattern A. The upper fragment in pattern C had two very close bands. Lane 1: L. donovani MHOM/SD/62/LRC-L61 (positive control). Lane 2-12: strains of L. donovani from eastern Sudan.

ITS-PCR products of Leishmania donovani samples representing different SSCP banding patterns were sequenced in both directions to determine the underlying nucleotide polymorphisms.

The complete dominant ITS sequence (A) is shown in figure (10). The alignments of the variable parts of the ITS1 and ITS2 sequences, which showed variation from the dominant ITS sequence (A) are shown in figures (11&12), respectively. All the variations in the ITS1 SSCP banding patterns were due to deletion of AT pairs or adenine bases from stretches in the first part of the ITS1 (first 192 nucleotides) with exception of the Chinese isolate which has in addition 2 substitutions at positions 27 and 89 (sequence LdN, Figure 11). In the patterns E, H and L sequences between the nucleotides 73 to 110 were not readable because different nucleotides were found in the same position (2 or 3 and sometimes all 4). These findings were confirmed by

repeating sequencing using forward and reverse primers.

Figure 8b: SSCP analysis of ITS1 regions amplifyed from different clinical Leishmania donovani isolates. Each SSCP pattern is designated by a capital letter. Lane 1: L. donovani MHOM/SD/62/LRC-L61 (positive control). Lanes 2&3: L. donovani strains from eastern Sudan; Lane 4: L.donovani MHOM/KE/85/NLB323 (Kenya); Lane 5: L.donovani MHOM/IN/71/LRC-L51a (India); Lane 6: l.donovani MHOM/CN/??/Wangjiel (China).

No sequence variation was observed in the ITS2A region (positions 320-602) in all isolates. The Sudanese isolates had the same ITS2B sequence (positions 603-1042) with the exception of one sample, which had a deletion of one guanine base from a G stretch, at position 831 (LdJ sequence).

Isolates from Kenya and India (LdX and LdY sequences, respectively) differed in a single nucleotide, at position 762 in their ITS2B sequence, but showed many nucleotide differences when compared with the Sudanese isolates (Sequences, LdA, J and D) and to the Chinese isolate (sequence LdZ).

Figure 9: SSCP analysis of ITS2 regions amplified from different clinical Leishmania donovani isolates. Each SSCP pattern is designated by a capital letter. Lane1: L.donovani MHOM/SD/62/LRC-L61 (positive control); Lanes 2&3: L.donovani strains from eastern Sudan; Lane 4: L.donovani MHOM/KE/85/NLB323 (Kenya); Lane 5: L.donovani MHOM/IN/71/LRC-L51a (India); Lane 6: l.donovani MHOM/CN/??/Wangjiel (China).

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). Dots represent the absence of nucleotids; bold underlined letters represent point mutations (substitutions);X: A/C/G/T_

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). Dots represent absence of nucleotides; Bold underlined letters represent point mutations (substitutions).

Detection of DNA polymorphisms in the gp63 sequences:

When part of the coding region of gp63 (XYZ) was amplified from leishmania clinical samples, PCR products of about 1350bp were obtained. When X, Y and Z were amplified separately, PCR products of about 400bp, 350bp and 600bp respectively, were obtained ( Figure 13; Appendix2).

Figure 13: PCR products amplified from 3 different clinical isolates of Leishmania donovani. Lanes 1,2&3: XYZ gp63 PCR products (1350); Lanes 4,5&6: X gp63 PCR products (450bp); Lanes 7,8&9: Y gp63 PCR products (350bp); Lanes 10,11&12: Z gp63 products (600bp); M: Molecular size (weight) standard marker; Kbp: Kilobase-pairs.

In total 31 clinical Leishmania samples spotted on filter papers were investigated. When X and Z regions were used as a target for SSCP analysis, no differences were observed, all the samples had the same SSCP profiles. When Y region was used as a target, 3 polymorphic patterns were observed (Figure 14; Appendix 2). The dominating pattern A was represented by 27 samples. The other patterns B and C were represented by 1 and 3 samples, respectively.

Figure 14: SSCP analysis of Y regions of gp63 gene amplified from different clinical Leishmania donovani isolates from eastern Sudan. Each SSCP pattern is designated by a capital letter. Patterns A and B were very similar but the size of the upper fragment in pattern A was slightly larger than in pattern B. Also the size of the lower fragment of pattern A is slightly smaller than in pattern B and there is very faint additional band. Lane 1: L. donovani MHOM/SD/62/LRC-L61 (positive control). The position of the bands are indicated by arrows.

Nucleotide polymorphisms, obtained by sequencing of part (Y) of the coding region of gp63, and representing the 3 different SSCP banding patterns were due to single base exchanges (one transition and 2 transversions) as shown in figure (15).

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). Dots: represent absence of nucleotides. Bold underlined letters represent point mutations (substitutions). Primers are represented by bold letters.

Detection of DNA polymorphisms in different anonymous DNA fragments:

When anonymous DNA fragments, namely L0110, L510, L413, LK413, L0308 and L0114 were amplified from leishmanial genomic DNA, PCR products that were about 580, 250, 300, 350,120 and 230 bp respectively, were obtained ( Figure 16). Unfortunately we succeeded to get good PCR products only for DNA extracted from the 8 successful cultures. Only for the fragment 110 we were able to get good PCR products from 31 samples spotted on filter papers as well. For the other anonymous fragments only, weak, faint and sometimes no PCR products could be amplified directly from clinical samples spotted on filter papers.

Figure 16: DNA fragments amplified from 2 different clinical isolates of Leishmania donovani. Lanes 1&2: PCR products obtained for anonymous fragment (110); Lanes 3&4: PCR products obtained for anonymous fragment (510); Lanes 5&6: PCR products obtained for anonymous fragment (413); Lanes 7&8: PCR products obtained for anonymous fragment (K413); Lanes 9&10: PCR products obtained for anonymous fragment (308); Lanes 11&12; PCR products obtained for anonymous fragment (114); M: Molecular size (weight) standard marker; Kbp: Kilobase-pair.

SSCP was performed for the 6 anonymous DNA fragments namely, L0110, L510, L413, LK413, L0308 and L0114. Concerning the region L0110, 31 samples were investigated and no differences in SSCP patterns were observed. For the other 5 fragments the 8 cultured samples were investigated and also no differences in SSCP patterns were observed with exception of 114 region, which showed two patterns. Pattern A was represented by 7 samples. The other pattern B was represented by one sample (Figure 17; Appendix 2). When SSCP was applied, for the anonymous DNA fragments obtained from faint PCR products amplified from samples spotted on filter papers, no reliable results were obtained.

Figure 17: SSCP analysis of anonymous fragment (114) amplified from different clinical Leishmania donovani isolates from eastern Sudan. Each SSCP pattern is designated by a capital letter. Lane 1: L. donovani MHOM/SD/62/LRC-L61 (positive control).

Figure 18: Anonymous DNA loci sequences amplified from clinical L. donovani samples from eastern Sudan. Primers are represented by bold letters. Polymorphism in locus 114 is represented by bold underlined letters.

The sequences of the 6 amplified anonymous loci in the L.donovani DNA were shown in figure (18). Only locus 114 sequence showed 2 polymorphic patterns A&B which differed by one A/C exchange in position 127 and a small insertion/deletion in position 111.

PCR fingerprinting:

PCR fingerprinting technique using M13 and T3B primers revealed no differences in profiles of all 26 studid L. donovani strains (8 successful cultures from eastern Sudan plus the 12 obtained from the collection of the Royal Tropical Institute, Amsterdam and the 6 WHO reference strains).

Figure 19: Results of the PCR fingerprinting using the (GTG)₅ primer. Lane 1-3: Sudanese strains of L. donovani MHOM/SD/62/LRC-L61, MHOM/SD/98/GCI, and Don 13. Lane 4: L. donovani MHOM/KE/85/NLB323. Lane 5: L. donovani MHOM/IN/71/LRC-L51a. Lane6: L. donovani MHOM/CN/??/Wangjie1. M; molecular size (weight) standard marker. Kbp: Kilobase-pairs.

When the (GTG)5 primer was used, L. donovani from India showed a unique profile (Fig.19, lane 5 ); the fingerprint of the Chinese strain (Fig.19, Lane 6) was very similar to those of the Sudanese strains; however, the high molecular weight bands appeared very faint, which might be due to partial degradation or

Figure 20: Results of the PCR fingerprinting using the (GACA)₄ primer. Lane 1-3: Sudanese strains of L.donovani MHOM/SD/62/LRC-L61, MHOM/SD/98/GCI, and Don 13. Lane 4: L.donovani MHOM/KE/85/NLB323. Lane 5: L. donovani MHOM/IN/71/LRC-L51a. Lane6: L.donovani MHOM/CN/??/Wangjie1. M; molecular size (weight) standard marker. Kbp: Kilobase-pairs.

lower concentration of DNA in this sample. With the primer (GACA) ₄identical

fingerprinting patterns were obtained for all isolates from Sudan with the exception of the one ITS2 pattern J isolate (Fig. 20, Lane 3). This Isolate differed slightly from the other Sudanese isolates as did the isolate from China (Fig.20, Lane 6). The Kenyan and the Indian isolates (Fig.20, lanes 4&5, respectively) showed the same profiles with this primer.