Entwicklung von Rekombinase-Polymerase-Amplifikations-Verfahren zum schnellen Nachweis von Leishmania spp.
Development of a pan recombinase polymerase amplification assay for detection of leishmania species
by Chiheb Louizi
Date of Examination:2022-03-22
Date of issue:2022-02-24
Advisor:Prof. Dr. Carsten Lüder
Referee:Prof. Dr. Carsten Lüder
Referee:Prof. Dr. Timo Buhl
Referee:Prof. Dr. Ralf Dressel
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EnglishLeishmaniasis is a disease caused by obligate intracellular protozoan and flagellated parasites of the genus Leishmania, belonging to the family Trypanosomatidae, order Kinetoplastidia. The parasite is transmitted to humans via the bite of infected female sandflies of the genus Phlebotomus and Lutzomyia. Leishmaniasis is endemic in nearly 90 countries. They are geographically divided into two groups: "New World" leishmaniasis- typically found in America, and the "Old World" leishmaniasis, which is endemic in several tropical countries of Asia and Africa, and also in the countries of the Mediterranean region. However, the spread of endemic areas owing mostly to the change in sandfly habitats and human mobility are of recent concerns. The main clinical forms of Leishmaniasis are cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), visceral leishmaniasis (VL) and its dermal complication called post Kala-azar dermal leishmaniasis (PKDL). CL is most commonly caused by L. major, L. tropica, L. aethiopica, L. amazonensis, L. mexicana, L. braziliensis, L. panamesis and L. guayanensis depending on the geographical spread of the species in endemic regions. Common pathological consequences include skin ulceration, raised rim and erythematous papules. Mucocutaneous leishmaniasis (MCL) can be caused by L. braziliensis, L. panamensis and L. guyanensis and is developed often, after CL. Visceral leishmaniasis (VL) or "Kala-Azar" caused by the L. donovani complex is the most fatal forms of leishmaniasis if left untreated and is highly endemic in the Indian subcontinent and in East Africa. Parasite infection in viscerotropic organs can lead to splenomegaly, pancytopenia, hepatomegaly, fever, weight loss, and mucosal bleeding. Post Kala-azar Dermal Leishmaniasis (PKDL) is an atypical dermatosis that often develops as a sequel of VL - mostly after treatment. PKDL manifestation is in the form of painless macular or papulo-nodular lesions or a mix of both that harbour parasites. Another less common types is the diffuse cutaneous leishmaniasis (DCL) caused by L. amazonensis and L. aethiopica, which features typical nodular or plaque-shaped solitary lesions. The diagnostic protocol for leishmaniasis involves provisional diagnosis which is based on clinical presentation and symptoms, usually followed by confirmatory test, which is usually by either culture, microscopy or polymerase chain reaction (PCR). Cases of leishmaniasis may pose a diagnostic dilemma as the signs and symptoms are often similar to those of other infectious and/or genetic diseases, particularly in imported cases and for species of Leishmania, which are non-endemic at a particular region. In such cases, parasite detection using the culture and/or microscopy could be limiting as for sensitivity, whereas most PCR methods are mostly targeted for one species or a particular form of leishmaniasis. Antibody detection by the immunofluorescence test (IFT) or by ELISA is only useful for the VL because of the generalized infection. On the other hand, intradermal test, which is possible for CL or MCL is about 95% sensitive and 96-100% specific, however it cannot diagnose VL. Nevertheless, antibody detection tests cannot differentiate between active infection from previous episodes. PCR is known to be more sensitive, specific and robust test for detection of active infection, however, it is limited to laboratories with relatively complex technical equipment. A low cost and easy to establish simple molecular diagnostic method such as recombinase polymerase amplification (RPA) would leverage the setup at the resource-poor settings for the detection of Leishmania spp. RPA runs significantly faster (15-20 minutes) and at a constant temperature of about 37-42°C to produce results that demonstrated sensitivity and specificity comparable to PCR based methods. The unique feature of the RPA amplification lies in the formation of recombinase enzyme-primer complex that upon homology in double-stranded DNA template, performs strand-invasion to insert the primer sequence. In this way, the strand-displacing polymerase can ultimately bind to the primer and synthesize the desired amplicon. For real-time detection, an exo-probe is used and the emitted fluorescence signal can be measured. The lyophilized pellets of the enzyme mix are relatively stable in ambient temperature requiring no cold-chain maintenance, which facilitates its operation in mobile settings in a portable suitcase. Several RPA assays have been developed to date that are either specific for one or a few species of Leishmania. We have previously developed RPA assay kDNA, which was found very sensitive and specific for the detection of L. donovani that typically cause visceral- and post-kala-azar dermal leishmaniasis, and cross-reactive to L. major, L. aethiopica and L. infantum but not the other infectious species. The aim of the study was to develop pan-leishmania RPA assay for the detection of all Leishmania pathotypes. Two RPA assays targeting 18S ribosomal RNA gene- (18S rRNA) and cathepsin L-like cysteine proteinase B (Cpb) gene were developed. Their ability of detecting most identified species was tested and the better performer between the assays was selected for clinical validation.
Keywords: Recombinase polymerase amplification assay; Suitcase laboratory; Leishmania spp.