PCR diagnosis of Leishmaniasis in Israel and the West Bank [Elektronische Ressource] : development of a field applicable procedure useful for epidemiological studies / von Gerlind Anders

Gerlind Anders

Sujets

• Biologie

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2003
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Aus dem Institut für Mikrobiologie und Hygiene
Medizinische Fakultät Charité
der Humboldt-Universität zu Berlin

The Kuvin Center for the Study of Infectious and Tropical Diseases
The Hebrew University, Hadassah Medical School
Jerusalem, Israel



DISSERTATION

PCR Diagnosis of Leishmaniasis in Israel
and the West Bank
Development of a field applicable procedure useful for epidemiological studies




zur Erlangung des akademischen Grades
doctor medicinae (Dr. med.)



vorgelegt der Medizinischen Fakultät Charité
der Humboldt-Universität zu Berlin
von
Gerlind Anders


1



















Dekan: Prof. Dr. Joachim W. Dudenhausen


Gutachter: 1. Prof. Dr. med. W. Presber
2. Prof. Dr. med. W. Solbach
3. Prof. Dr. R. Lucius



Datum der Promotion: 05.02.2003









2





This research has been part of a German-Israeli-Palestinian cooperation
project on the epidemiology of leishmaniasis in Israel and the West-Bank
granted by the Deutsche Forschungsgemeinschaft (DFG)


The laboratory work has been carried out at the Kuvin Center
for the Study of Infectious and Tropical Diseases,
Hebrew University, Hadassah Medical School, Jerusalem, Israel




3Travel report from Palestine, 1873
“The men called the illness Jericho fever or malaria. They in fact were two
different diseases, the men probably had both.
Jericho fever accounted for the large sores that took months to heal.
Sergeant Black had been troubled by "a very bad place" on his hands;
Conder complained of an ulcerous sore that kept growing on one hand until
the whole arm was painful; Drake was so plagued by sores on his feet, at
times he could barely walk. Any newcomer would mistakenly think the sore
was a boil, or the result of irritation of some kind, but not a disease. When it
healed it left a large scar. Then you would notice among people in Riha* or
in dealings with the Bedouin, that almostever yone in the valley had on his
hands or face at least one large ugly scar .”
in:
Jericho
Dreams, Ruins, Phantoms
*Riha (Arabic) = Jerichoby Robert Ruby
4Table of Contents:

1. Introduction 11

1.1. General facts about leishmaniasis 11
1.1.1. Epidemiology 11
1.1.2. The parasite 12
1.1.3. Life cycle 13
1.1.4. Clinical forms of leishmaniasis 13
1.1.5. Immunology 15
1.1.5.1. Cutaneous leishmanisis 15
1.1.5.2. Visceral leishmaniasis (kala azar) 16
1.1.5.3. Mucocutaneous (Espundia) 17
1.1.5.4. Leishmaniasis and HIV-coinfection 18
1.1.6. Treatment 18
1.1.7. Transmission 20
1.1.8. Antiepidemic measures 22
1.1.9. Leishmaniasis in the Middle East 23
1.2. Review of the diagnostic methods for leishmaniasis 28
1.2.1. Direct identification by classical routine methods 28
1.2.2. Indirect diagnosis 29
1.2.2.1. Cellular immunity
1.2.2.2. Serological methods
1.2.3. Identification of Leishmania species and subspecies 31
1.2.4. DNA based methods 31
1.2.4.1. Classical methods: hybridization and restriction 31
1.2.4.2. Modern methods based on the polymerase chain reaction (PCR) 32
1.2.4.3. Molecular strain typing of Leishmania 34
1.3. Objectives of the study 35

2. Material and methods 37
2.1. Origin of samples
2.1.1. Reference strains
2.1.2. Patients 38
2.1.3. Animal samples 38
52.1.4. Cultivation of Leishmania promastigotes 39
2.2. Laboratory work 40
2.2.1. Sampling 40
2.2.2. Giemsa staining of smears 40
2.2.3. Collection and cultivation of Leishmania strains 40
2.2.4. DNA extraction 41
2.2.4.1. Choice of extraction methods according to specimens 43
2.2.4.2. Extraction from paraffin embedded biopsies 44
2.2.4.3. Storage conditions 44
2.2.5. PCR 44
2.2.5.1. Oligonucleotides
2.2.5.2. PCR-conditions 48
2.2.6. Agarose gels 52
2.2.7. Avoiding inhibition 52
2.2.8. Avoiding contamination 52
2.2.9. Controls 54
2.2.10. Solutions 54
2.2.11. List of reagents 56

3. Results 57
3.1. Efficiency of the tested DNA extraction methods 57
3.1.1. Suitability of different specimens for PCR diagnosis 58
3.1.2. Experiments with crude samples 59
3.2. Amplification of whole minicircle DNA with primers Uni21/Lmj4 60
3.2.1. Results with DNA purified from cultured reference strains 60
3.2.2. Results from lysed cultured isolates wi 61
3.2.3. Results with primers Uni21/Lmj4 with dermal scrapings 63
3.2.4. Selected patient cases diagnosed with primers Uni21/Lmj4 65
3.3. Genus specific amplification with kDNA primers 13A/13B 66
3.4. Studies on reservoir animals 66
3.5. Specific detection of Leishmania braziliensis (MPH3/MPL1) 69
3.6. Amplification of the ITS-1 region and RFLP analysis 73
3.7. A new focus of L.tropica in Wadi Albethan, West Bank 75
3.8. The aims of the study have been reached 77
64. Discusion 85
4.1. Sampling and preservation 85
4.1.1. Collection of dermal scrapings 85
4.1.2. Specimens other than dermal scrapings for direct PCR-diagnosis 86
4.1.3. Preservation on filter paper 86
4.1.4. Classical preservation methods 87
4.2. Discussion of DNA extraction methods 87
4.2.1. Phenol-chloroform-extraction 87
4.2.2. Guanidine-extraction 88
4.2.3. Chelex-extraction 88
4.2.4. Crude extraction methods 89
4.2.5. Extraction methods related to different types of specimens 89
4.2.6. Storage of extracted samples 90
4.3. Reliability of the employed PCR-methods 90
4.3.1. Sensitivity 90
4.3.2. False negatives/ inhibition 91
4.3.3. positives/ contamination 92
4.3.4. Reproducibility of PCR-results 93
4.4. Discussion of PCR results 93
4.4.1. Results with primers Uni21/Lmj4
4.4.2. Results with the genus specific kDNA primers 13A/13B 97
4.4.3. ith L.braziliensis specific primers MP3H/MPL1 97
4.4.4. Results with ITS-1 primers and RFLP-analysis 99
4.5. Wadi Albethan, a newly identified focus of L.tropica in (West Bank) 99
4.6. Discussion of PCR results from animals 100
4.7. Choice of treatment on the basis of PCR-results 101
4.8. PCR diagnosis in VL 102
4.9. Guidelines for future applications 103
4.9.1 Sampling, preservation and extraction 103
4.9.2. Recommended approach for PCR diagnosis in the future 104
4.10. Leishmaniasis in non-endemic areas (Germany) 105
4.11. Concluding remarks 106


75. Summary/Zusammenfassung 107/109
6. Bibliography 113
7. Acknowledgements 126
8. CV 128
9. List of publications 129

Tables

Table 1 Species of Leishmania 19
Table 2 Reference strains 37
Table 3PCR methods 49
Table 4Cycling 51
Table 5: PCR results with Uni21/Lmj4 from lysed cultures 62
Table 6: Results with primers Uni21/Lmj4 from dermal scrapings 64
Table 7: Results from animal samples 69
Table 8: Results of patients from the Tel Hashomer Hospital 72
Table 9 BsuI restriction fragments of ITS-1 amplificates 74
Table 10: Results from patients from Wadi Albethan 76


Figures

Figure 1 Amastigotes in Giemsa stained smears 12 2 Promastigotes sandfly gut 13
Figure 3 Life cycle of Leishmania 14
Figure 4 Map of CL in Israel and the West Bank 26
Figure 5 Map of VL in Israel and the West Bank 27
Figure 6 Section of the L.major minicircle 46
Figure 7 kDNA minicircle with primers Uni21/Lmj4 and 13A/13B 46
Figure 8 Internal transcribed spacer of the ribosomal operon 47
Figure 9 Experiment with additives against inhibition 59
Figure 10 PCR with primers Uni21/Lmj4 on different Kinetoplastidae 60
Figure 11 Comparison of sensitivity with Gel star stain and ethidium bromide 61
Figure 12 PCR with primers Uni21/Lmj4 on lysed cultures 63


8Figure 13 PCR with primers Uni21/Lmj4 on crudely extracted
filter paper samples 64
Figure 14 PCR with primers Uni21/Lmj4 on filter paper extractions 65
Figure 15 Screening of desert rodents with primers 13A/13B 67
Figure 16 Psammomys ear scrapings with primers Uni 21/ Lmj4 68
Figure 17 PCR with ITS-1 primers and restriction with BsuI 68
Figure 18 L.braziliensis primers MP3H/MPL1 71
Figure 19 genus specific PCR with primers 13A/13B 71
Figure 20 i 71
Figure 21 PCR wiith