Molecular epidemiology, clinical molecular diagnosis and genetic diversity of cutaneous Leishmaniasi in Jericho, Palestine [Elektronische Ressource] / von Amer Al-Jawabreh

Benutzer1

Sujets

• Gesundheit

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2005
• Nombre de lectures: 16
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Aus dem Institut für Mikrobiologie und Hygiene
der Medizinischen Fakultät der Charité – Universitätsmedizin Berlin



DISSERTATION


Molecular Epidemiology, Clinical Molecular Diagnosis and
Genetic Diversity of Cutaneous Leishmaniasis
in Jericho, Palestine


zur Erlangung des akademischen Grades
Doctor rerum medicarum
(Dr. rer. medic)


vorgelegt der Medizinischen Fakultät Charité
der Universitätsmedizin- Charité

Von
Amer Al-Jawabreh
Aus Palästina
Dissertation Examination Committee (Gutachter):
1. Prof. Dr. med. W. Presber
2. Prof. Dr. W. Solbach
3. Prof. Dr. Ch. Jaffe

thDatum der Promotion: 25 November, 2005

























iiThis research has been part of the scholarship granted to A. Al-Jawabreh by
Deutsche Akademische austausch Dienst (DAAD).
The molecular work has been carried out at the laboratory of the Institut für
Mikrobiologie und Hygiene- Universitätsmedizin- Charité, Berlin Germany under
close supervision of Dr. G. Schoenian and the isolation and the diagnosis at ICS-
Jericho Medical laboratory in Jericho, Palestine







iiiTable of contents
ABBREVIATIONS
ABSTRACT……………………………………………………………………………………………. 1
CHAPTER 1: INTRODUCTION………………………………………………….............................. 2
Forward: Molecular epidemiology………………………………………………............... 2
1.1 Historical background……………………………………………………………………...2
1.2 Clinical symptoms of leishmaniasis…………………………………….. ……………….. 3
1.3 Epidemiology of leishmaniasis…………………………………………………………….4
1.3.1 Global view………………………………………………………………………………...4
1.3.2 Local view………………………………………………………………………………… 4
1. 4 Leishmaniasis: Public Health Surveillance……………………………………………….. 5
1.5 Clinical diagnosis and identification……………………………………............................ 6
1.6 Multilocus enzyme electrophoresis (MLEE)……………………………………………… 7
1. 7 Microsatellites…………………………………………………………………………….. 8
1. 7. 1 Mutation mechanism…………………………………………………………………........ 9
1. 7. 2 Functions of microsatellites……………………………………………………………….. 10
1. 7. 3 Application of microsatellites for Leishmania strain typing……………………………… 11
1. 8 Two models for microsatellite evolution……..………………………………………........ 11
1. 9 Genetic distance measures used in microsatellite analyses……………………………….. 12
1. 10 L. major and L. tropica……………………………………………………………………. 13
1. 10. 1. Distribution……………………………………………….……………………………….. 13
1. 10.2 Clinical features…………………………………………………………………………… 16
1. 10.3 Clinical diagnosis and characterization…………………………………………………… 17
1.10. 4 Vector …………………………………………………………………………………….. 18
1.10.5 Reservoir ………………………………………………………………………………….. 19
1.10.6 Control…………………………………………………………………………………….. 20
1.10.7 Treatment………………………………………………………………………………….. 21
1.11 Population genetics of Leishmania parasites ……………………………………………... 21
1.12 Genetic diversity and bottleneck theory…………………………………………………... 23
1. 18 Objectives…………………………………………………………………………………. 24
CHAPTER 2: MATERIALS AND METHODS …………………………………………………….. 26 2.1 Clinical molecular diagnosis and local molecular epidemiology…………………………. 26
2.1.1 Patients and study area……………………………………………………………………. 34
2.2 Sample collection…………………………………………………………………………. 27
2.2.1. Patient data sheets………………………………………………………………………….27
2.2.2. Collection and Giemsa staining of skin scrapings……………………………………........ 28
2.2.3. Sampling using filter papers and unstained smears ...……………………………………. 28
2.2.4. Cultivation of parasites from dermal tissue aspirate…………………………………......... 28
2.3. DNA extraction…………………………………………………………………………….29
2.3.1 DNA extraction of cultured parasites……………………………………………………... 29
2.3.2 DNA extraction of Clinical samples………………………………………………………. 30
2.4 PCR amplification: Internal transcribed spacer (ITS1) …………………………….…….. 30
2.5 Panel of controls used in diagnostic PCR…………………………………………………. 32
2.5.1 DNA extraction control………………………………………………………………........ 32
2.5.2 Positive and negative controls…………………………………………………………….. 33
2.5.3 Inhibition control………………………………………………………………………….. 33
2.6 RFLP analysis of ITS1 amplicon….………………………………………………............ 33
2.7 Evaluation studies……………………………………………………………………......... 34
2.7.1 Graded microscopy vs ITS1-PCR……………………………………………………........ 34
2.7.1.1 Patients and study area……………………………………………………………………. 34
2.7.1.2 Sample collection and preparation……………………………………………………….. 34
2.7.1.3 Standardized graded microscopy………………………………………………………….. 35
2.7.2 Filter Paper vs Unstained smears and conventional methods vs PCR ……………………. 36
2.8 Genetic microsatelite variation and global molecular epidemiology……………............... 36
2.8.1 Study area……………………………………………………………………………......... 36
2.9 Microsatellite markers…………………………………………………………………….. 37
2.9.1 Amplification of microsatellite markers by PCR……………………………………......... 38
2.9.2 Polyacrylamide gel electrophoresis (PAGE) ……………………………………………... 39
2.9.3 Silver staining……………………………………………………………………………... 39
2.9.4 Capillary electrophoresis (CE) using CEQTM 8000 Beckman coulter…………………… 40
2.10 Data analysis: clustering methods and presentation of genetic data……………………..... 40
2.10.1 Calculating a distance matrix using MICROSAT…………………………………….......... 40
2.10.2 Drawing of NJ and UPGMA consensus trees using PHYLIP/ PAUP ……………………. 41
v2.10.3 Structuring populations with Structure 2.0………………………………...…………....... 41
2.10.4 F-Statistic by F-STAT and GENEPOP………………………………………………........ 41
2.10.5 Descriptive statistics for for markers by GDA………………………………………......... 42
2.11 Epidemiological data banking and analysis: Epi Info™………………………………….. 42
2.12 Geographical clustering and public health surveillance…………………………………... 42
2. 12. 1 Spatial scan statistics ……………………………………………………………………... 43
2. 12. 2 Space-time scan statistics …………………………………………………………….. … 43
2. 12.3 Adjustment for season relative risk ………………………………………………………. 44
2. 12. 4 Adjusting for covariates…………………………………………………………………… 44
2. 13 Shewhart’s Chart………………………………………………………………………….. 44
CHAPTER 3. RESULTS………………………………………………………..…………………….. 46
3. 1. Method comparison……………………………………………………………………….. 46
3.1.1 46 Graded microscopy and ITS1-PCR………………………………………………………..
3.1.1.1 Positivity rates and sensitivities of ITS1-PCR and graded microscopy…………………... 46
3.1.1.2 Statistical comparison of sensitivities ……………………………………………………. 46
3.1.1.3 Diagnostic relevance of ITS1-PCR ………………………………………………………. 47
3.1.2 Clinical diagnosis of cutaneous leishmaniasis: filter paper and unstained smears as
potential sampling methods for ITS1-PCR …………………………………………........ 50
3.2 Molecular epidemiology………………………………………………………………….. 54
3.2.1 L. major vs. L. tropica in Jericho ……………………………………………………........ 54
3.2.2 Sex-age group distribution………………………………………………………………… 57
3.2.3 Annual rain fall (ARF) and CL……………………………………………………………. 59
3.2.4 Seasonality ofCL…...…………………………………………………………………….. 60
3.3 Public health surveillance and cluster analysis……………………………………………. 62
3.3.1 Descriptive data of CL cases…………………………………………………………........ 62
3.3.2 Purely spatial analysis, adjusted for season……………………………………………….. 63
3.3.3 Space-time analysis, adjusted for season………………………………………………….. 64
3.4 Shewhart’s Plot: Early warning system.………………………………………………….. 66
3.5 Genetic variability within L. major as revealed by Multilocus Microsatellite Analysis
(MLMT)………………………………………………………………………………....... 67
3.5.1 Description of the microsatellite markers used in this study……………………………… 67
3.5.2 Assignment of multilocus microsatellite profiles to the strains of L. major under study 70
vi
3.5.3 Population structure of L. major using structure………………………………………….. 76
3.5.3.1 Estimation of population structure using non-predefined populations……………………. 76
3.5.3.2 Estimation of population structure using predefined population…………………………. 79
3.5.4 Analysis of L. major population structure using distance-based methods………………... 80
3.5.5 Genetic isolation of the L. major populations identified in this study……………………. 85
3.5.6 Estimation of allele numbers in geographical groups of strains …………………………. 86
CHAPTER 4: DISCUSSION…………………………………………………………………………. 88
4. 1. Method comparison ………………………………………………………………………. 46
4.1.1 Graded microscopy and ITS1-PCR……………………………………………………….. 88
4.1.2 Clinical diagnosis of cutaneous leishmaniasis ……………………………………………. 91
4.2 Molecular epidemiology………………………………………………………………….. 96
4.2.2 Seasonality of CL…………………………………………………………………………. 98
4.3 Public health surveillance and cluster analysis……………………………………………. 99
4.4 Multilocus microsatellite analysis and population structure…………………………........ 103
4.4.1 Optimal number of markers and isolates…………………………………………………. 103
4.4.2 Distance and model-based methods: congruence and contradiction…………………….. 105
4.4.3 Origin of L. major and bottleneck theory…………………………………………………. 106
4.5 Recommendations………………………………………………………………………… 107