Investigation of genetic and immunological factors that influence the performance of Plasmodium falciparum Histidine-Rich Protein 2 (PfHRP2)- detecting Rapid Diagnostic Tests (RDT) for malaria

Joanne Baker (2011). Investigation of genetic and immunological factors that influence the performance of Plasmodium falciparum Histidine-Rich Protein 2 (PfHRP2)- detecting Rapid Diagnostic Tests (RDT) for malaria PhD Thesis, Population Health, The University of Queensland.

       
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Author Joanne Baker
Thesis Title Investigation of genetic and immunological factors that influence the performance of Plasmodium falciparum Histidine-Rich Protein 2 (PfHRP2)- detecting Rapid Diagnostic Tests (RDT) for malaria
School, Centre or Institute Population Health
Institution The University of Queensland
Publication date 2011-03
Thesis type PhD Thesis
Supervisor Assoc Prof Qin Cheng
Assoc Prof James McCarthy
Dr Michelle Gatton
Total pages 247
Total colour pages 20
Total black and white pages 227
Subjects 11 Medical and Health Sciences
Abstract/Summary Malaria remains, in the 21st Century, a serious infectious disease, with 243 million people developing clinical malaria in 2009, resulting in over 863 000 deaths worldwide. In sub-Saharan Africa, the Asia-Pacific Region and South America, falciparum malaria continues to result in 20% of all childhood deaths, and remains a major impediment to social and economic growth and development in many low-income countries. There remains no vaccine against malaria, and the pace of development of effective treatments must compete with the parasite’s ability to develop resistance, which is proving increasingly difficult as resistance prevalence increases. Case management of malaria relies upon early and accurate diagnosis and effective treatment. Malaria rapid diagnostic tests (RDTs) offer great potential to improve the diagnosis of malaria, particularly in remote areas. Detection of falciparum histidine-rich protein 2 (PfHRP2) is the basis for many of these tests. However, RDT sensitivity in operational use has been reported to be highly variable. There is an urgent need to investigate systematically reasons for performance variation in these tests. In theory, causes for variation in PfHRP2-detecting malaria RDTs could occur in two main areas: problems with the detecting antibody imbedded in the test, and problems with the antigen produced by the parasite which the test detects. Antibody problems relate to the quality of the RDT: production issues, antibody specificity and affinity, heat stability of the antibody, and quality control. Antigen factors include the parasitemia of the infection, genetic diversity, gene deletion, gene transcription and protein expression, and antigen-antibody complex binding in vivo. This thesis concentrated on these antigen factors which could impact on the sensitivity of RDTs. I hypothesized that: • pfhrp2 gene variation in structure • PfHRP2 antigen quantity and expression differences between different strains of the parasite • an overloading of antigens, and • pre-existing antibody to PfHRP2 may all contribute to a variation in sensitivity in the use of PfHRP2-detecting RDTs. To date, no methodical research has been undertaken to investigate the extent of contribution of these factors, to this varied sensitivity. The aims of the research were: • to investigate molecular structural differences in the pfhrp2 gene from a large, geographically diverse sample of Plasmodium falciparum parasite isolates, to fill a knowledge gap in the extent of diversity of this gene; • to examine the transcription of pfhrp2 in different laboratory strains of the parasite, and the expression of PfHRP, and test whether variant strains produced variant amounts of protein, which may be differentially detected on RDTs. This has importance in detection sensitivity particularly at low parasitemia; • to test whether an excess of antigen (prozone phenomenon, or high dose-hook effect), could contribute to the sensitivity of the tests. This has importance in detection sensitivity particularly at high parasitemia and high antigen levels; • to study whether a high level of pre-existing in vivo anti-PfHRP2 antibodies present in the patient could bind strongly with antigen produced by the parasite, preventing the antigen binding to the detection antibody in the PfHRP2-detecting RDT, and produce a so-called “blocking” effect. Key outcomes of the research included: • Characterization of the pfhrp2 gene. Whilst extensive diversity was observed in the sequence of this gene, it was concluded from this research that the pfhrp2 gene structure does not significantly impact upon the sensitivity of the tests at parasitemia > 250 P/µL. The results substantially contributed to the information collated by the World Health Organization in their Round One and Round Two Malaria Rapid Diagnostic Test product testing, 2008 and 2009; • Description of the transcription of pfhrp2 and expression of PfHRP. The research found that differences exist between transcription and expression of this gene and protein in a number of laboratory isolates, and provided a proof of concept that this variation impacts on the sensitivity of a number of RDTs; • Demonstration of a prozone effect. My results showed that no complete negative RDT result could be produced by parasitemia as high as 10%, however a strengthening of the band intensity does occur following dilution of the parasite-infected blood sample; • Demonstration of a “blocking” effect. Incubating a hyperimmune patient serum containing anti-PfHRP2 antibody with soluble parasite antigen and parasitized red blood cells blocked the detection of parasites on a number of PfHRP2-detecting RDTs. This thesis has contributed to scientific knowledge of the genetic variation on a global scale of pfhrp2, and transcription and protein expression level variation of PfHRP2 throughout the intraerythrocytic lifecycle and between different strains. The results suggest that a number of parasite genetic and protein factors may contribute to the sensitivity of the RDTs, findings which are not due to the quality of the tests. The outcomes have been incorporated by WHO into its Malaria Rapid Diagnostic Test Performance program by using a panel of 100 geographically variant parasite isolates with characterized pfhrp2 in its most recent (Round 2, 2009) malaria RDT product testing. This is the first report of an antibody blocking effect impairing the sensitivity of PfHRP2-detecting RDTs for malaria. The work outlined in this thesis has importance in its contribution to public health through identifying factors which can contribute to the variable sensitivity of RDTs for malaria. The work presents challenges for manufacturers and scientists in future work on PfHRP2-detecting RDTs for malaria, to develop better antibodies against less-variable epitopes of PfHRP2 with a minimum level of protein expression, or innovative diagnostic products to detect less-variable antigens. It would also be important to improve the lysis buffer in the RDT kits so antigen-antibody complexes can be interrupted prior to the test completion. As falciparum malaria has the potential to kill very quickly, it is imperative that an RDT be as sensitive as possible to ensure people receive the treatment they need. An RDT also needs to be as specific as possible to ensure negative cases are investigated for other causes of illness. This is particularly important for public health in malarious areas without concurrent microscopy, or microscopy skills that are not optimal, so that patients who are symptomatic and parasitemic will be detected. The outcomes of this thesis will contribute to the work of the WHO, to achieve its Millennium Development Goal of reversing the incidence of malaria and the number of malaria deaths. Through correct diagnosis and treatment of all cases, control and elimination can be achieved with the best use of resources, of particular importance to developing countries.
Keyword Plasmodium falciparum malaria, rapid diagnostic test, histidine-rich protein 2
Additional Notes Colour: 28, 31, 44, 46, 48, 75, 124, 155, 206, 54-55, 99, 100-103, 137, 148, 156, 157, 186-189 Landscape: 54-55, 149-151, 202, 208, 210-212

 
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Created: Mon, 08 Aug 2011, 12:16:19 EST by Mrs Joanne Baker on behalf of Library - Information Access Service