DISSERTATION Titel der Dissertation Prevalence and phylogeny of Plasmodium ovale and P. malariae in Bangladesh. Subtitle: A novel PCR technique in comparison with standard PCR methods for the preva- lence screening of Plasmodium sp. in Bangladesh, with a main focus on the distri- bution and phylogeny of P. ovale , P. malariae , and P. knowlesi , as well as the ex- tended short term culture of P. vivax under field conditions. Verfasser Mag.rer.nat. Hans-Peter Führer angestrebter akademischer Grad Doktor der Naturwissenschaften (Dr.rer.nat.) Wien, 2012 Studienkennzahl lt. Studienblatt: A 091 439 Dissertationsgebiet lt. Studienblatt: Zoologie Betreuerin / Betreuer: Univ.-Prof. Dr. Christa Frank 1 Declarations “I declare in lieu of an oath that I have written this doctoral thesis myself and that I have not used any sources or resources other than stated for its preparation. I further declare that I have clearly indicated all direct and indirect quotations. This doctoral thesis has not been submitted elsewhere for examination purposes.” 2 Table of Contents 1 Declarations................................................................................................................... 2 2 Table of Contents .......................................................................................................... 3 3 Abbrevations.................................................................................................................. 6 4 Introduction ................................................................................................................... 8 4.1 Malaria................................................................................................................... 8 4.2 History of malaria.................................................................................................. 9 4.3 Life cycle of Plasmodium sp. .............................................................................. 11 4.3.1 Asexual life cycle ........................................................................................ 11 4.3.2 Sexual reproduction (Sporogony)................................................................ 12 4.4 Plasmodium falciparum (Welch, 1897)............................................................... 13 4.4.1 Morphological features................................................................................ 14 4.4.2 Pathogenesis ................................................................................................ 15 4.5 Plasmodium vivax (Grassi and Feletti, 1890)...................................................... 17 4.5.1 Morphological Features............................................................................... 18 4.5.2 Pathogenesis ................................................................................................ 19 4.6 Plasmodium ovale (Stephens, 1922) ................................................................... 20 4.6.1 History......................................................................................................... 20 4.6.2 Distribution and Epidemiology ................................................................... 21 4.6.3 Life Cycle.................................................................................................... 23 4.6.4 Pathogenesis ................................................................................................ 24 4.7 Plasmodium malariae (Grassi and Feletti 1890)................................................. 25 4.7.1 Morphological features................................................................................ 26 4.7.2 Pathogenesis ................................................................................................ 27 4.8 Plasmodium knowlesi Sinton and Mulligan, 1932 .............................................. 28 4.8.1 Morphology................................................................................................. 29 4.8.2 Pathogenesis ................................................................................................ 30 4.8.3 Epidemiology .............................................................................................. 32 4.9 Diagnosis of malaria............................................................................................ 33 4.9.1 Microscopy.................................................................................................. 33 4.9.2 Rapid Diagnostic Tests (RDTs)................................................................... 33 4.9.3 Molecular tools for the diagnosis of Plasmodium sp. ................................. 34 4.9.3.1 Diagnosis of Plasmodium sp. by nested PCR of the SSU rRNA gene.... 35 3 4.9.3.1.1 NP-1993 .............................................................................................35 4.9.3.1.2 NP-2002 – The problem in the diagnosis of P. ovale becomes obvious 36 4.9.3.1.3 NP-2005 – The problem with P. ovale proceeds ...............................37 4.9.3.1.4 P. knowlesi – Problems in the diagnosis of this newly recognized malaria parasite in human ....................................................................................38 4.10 Molecular phylogeny of Plasmodium sp. using the mitochondrial genome ........40 4.11 Malaria treatment, drug resistances and drug susceptibility ................................43 4.12 Bangladesh ...........................................................................................................45 4.12.1 The Chittagong Hill Tracts and the District of Bandarban ......................... 45 4.13 Malaria Situation in Bangladesh ..........................................................................48 5 Aims .............................................................................................................................50 6 Studies..........................................................................................................................52 6.1 Status of Peer Reviewed Publications..................................................................52 6.2 Indigenous Plasmodium ovale malaria in Bangladesh.........................................53 6.2.1 Abstract ....................................................................................................... 54 6.2.2 Introduction................................................................................................. 55 6.2.3 Materials and Methods................................................................................ 56 6.2.4 Results......................................................................................................... 57 6.2.5 Discussion ................................................................................................... 58 6.3 A novel nested Direct PCR technique for malaria diagnosis...............................67 from filter paper samples..................................................................................................67 6.3.1 Abstract ....................................................................................................... 68 6.3.2 Introduction................................................................................................. 69 6.3.3 Methods....................................................................................................... 69 6.3.4 Results and Discussion................................................................................ 71 6.4 Plasmodium ovale in Bangladesh: genetic diversity and the first evidence of the sympatric distribution of P. ovale curtisi and P. ovale wallikeri in South Asia ..............77 6.4.1 Abstract ....................................................................................................... 79 6.4.2 Background ................................................................................................. 80 6.4.3 Methods....................................................................................................... 81 6.4.4 Results......................................................................................................... 83 6.4.5 Discussion ................................................................................................... 85 4 6.5 Molecular epidemiology of Plasmodium malariae and P. knowlesi in Bangladesh. ................................................................................................................... 103 6.5.1 Introduction ............................................................................................... 103 6.5.2 Materials and Methods .............................................................................. 104 6.5.3 Results ....................................................................................................... 104 6.5.4 Discussion.................................................................................................. 105 6.6 A highly sensitive in vitro-assay for the detection and quantification of Plasmodium vivax parasite biomass after the usage of different cultivation methods.. 108 6.6.1 Introduction ............................................................................................... 108 6.6.2 Materials and Methods .............................................................................. 109 6.6.3 Results and Conclusions............................................................................ 111 7 Conclusion................................................................................................................. 114 8 Abstract.....................................................................................................................