Multiplexed real-time PCRs for the detection and differentiation of Leishmania utilising bisulphite conversion technology

Gow, Ineka C.

Multiplexed real-time PCRs for the detection and differentiation of Leishmania utilising bisulphite conversion technology

Gow, Ineka C.

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Publication Type:: Thesis
Issue Date:: 2023

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Field	Value	Language
dc.contributor.author	Gow, Ineka C.
dc.date.accessioned	2023-07-11T04:55:39Z
dc.date.available	2023-07-11T04:55:39Z
dc.date.issued	2023
dc.identifier.uri	http://hdl.handle.net/10453/171445
dc.description	University of Technology Sydney. Faculty of Science.	en_US.UTF-8
dc.description.abstract	Leishmaniasis, caused by protozoan parasites in the genus Leishmania, is a cause of serious morbidity and mortality around the globe but particularly in the world’s poorest nations. It is for this reason that the disease and its various clinical forms is recognised as a neglected tropical disease (NTD). There are more than 20 species of Leishmania that cause human infection and all are transmitted by the bite of a female Phlebotomus or Lutzomyia sandfly. They can either elicit cutaneous, mucocutaneous or visceral disease; the cutaneous and mucocutaneous forms cause significant morbidity with disfiguring ulcers and resultant scarring, whilst the visceral form is fatal if left untreated. However, many human cases are asymptomatic and both humans and animals act as reservoir hosts, thereby contributing to the spread of the disease. Aside from the devastating impact on impoverished communities, leishmaniasis is also defined as an NTD due to the relative lack of funding for research, interventions, tools, and diagnostics. The wide range of diagnostics currently used includes both traditional microbiological and serological techniques, but also molecular techniques which are not deemed appropriate for resource-constrained regions. The World Health Organisation (WHO) has identified the need for accurate and rapid diagnosis to reach elimination targets in addition to broader public health intervention programs. At the patient level, rapid detection informs dosage and case management and achieves differential diagnosis from circulating diseases with similar clinical presentations. As the gap between the need and availability of apposite Leishmania diagnostics currently stands, it raises the following questions: can modern diagnostics for leishmaniasis be developed commensurate to its context of endemicity; and, can the perceived inappropriateness of these molecular techniques be challenged within these contexts? These questions form the overarching focus of this thesis. The development of a highly sensitive and specific Leishmania molecular diagnostic method, real-time PCR, appropriately solved these research questions. Sensitivity and specificity of 100% were observed for both the detection and the differentiation assays. Moreover, its adaptability in addressing specific priorities of test-of-cure and less invasive sampling was able to be achieved. A detection limit of 100 cells/mL was observed in whole peripheral blood and RNA/DNA ratios were determined in clinical sample nucleic acid. This method was developed on a platform that can easily be deployed as part of routine testing or outbreak response. Thus, it supports the discussion as to whether these methods are appropriate within the regions that need them most.	en_US.UTF-8
dc.format	Thesis (PhD)
dc.language.iso	en_US	en_US.UTF-8
dc.relation	https://opus.lib.uts.edu.au/bitstream/10453/171445/1/thesis.pdf
dc.rights	info:eu-repo/semantics/openAccess
dc.rights	The author owns the copyright in this thesis including all reproduction and reuse rights for the work. The work may not be altered without the permission of the copyright owner. Attribution is essential when quoting or paraphrasing from this thesis.
dc.rights	© 2023 Ineka C. Gow
dc.rights	au.edu.uts.lib/cph
dc.title	Multiplexed real-time PCRs for the detection and differentiation of Leishmania utilising bisulphite conversion technology	en_US.UTF-8
dc.type	Thesis
utslib.copyright.status	open_access	*

Abstract:

Leishmaniasis, caused by protozoan parasites in the genus Leishmania, is a cause of serious morbidity and mortality around the globe but particularly in the world’s poorest nations. It is for this reason that the disease and its various clinical forms is recognised as a neglected tropical disease (NTD). There are more than 20 species of Leishmania that cause human infection and all are transmitted by the bite of a female Phlebotomus or Lutzomyia sandfly. They can either elicit cutaneous, mucocutaneous or visceral disease; the cutaneous and mucocutaneous forms cause significant morbidity with disfiguring ulcers and resultant scarring, whilst the visceral form is fatal if left untreated. However, many human cases are asymptomatic and both humans and animals act as reservoir hosts, thereby contributing to the spread of the disease. Aside from the devastating impact on impoverished communities, leishmaniasis is also defined as an NTD due to the relative lack of funding for research, interventions, tools, and diagnostics. The wide range of diagnostics currently used includes both traditional microbiological and serological techniques, but also molecular techniques which are not deemed appropriate for resource-constrained regions. The World Health Organisation (WHO) has identified the need for accurate and rapid diagnosis to reach elimination targets in addition to broader public health intervention programs. At the patient level, rapid detection informs dosage and case management and achieves differential diagnosis from circulating diseases with similar clinical presentations. As the gap between the need and availability of apposite Leishmania diagnostics currently stands, it raises the following questions: can modern diagnostics for leishmaniasis be developed commensurate to its context of endemicity; and, can the perceived inappropriateness of these molecular techniques be challenged within these contexts? These questions form the overarching focus of this thesis. The development of a highly sensitive and specific Leishmania molecular diagnostic method, real-time PCR, appropriately solved these research questions. Sensitivity and specificity of 100% were observed for both the detection and the differentiation assays. Moreover, its adaptability in addressing specific priorities of test-of-cure and less invasive sampling was able to be achieved. A detection limit of 100 cells/mL was observed in whole peripheral blood and RNA/DNA ratios were determined in clinical sample nucleic acid. This method was developed on a platform that can easily be deployed as part of routine testing or outbreak response. Thus, it supports the discussion as to whether these methods are appropriate within the regions that need them most.

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