Hall, Diane Dogcio (2016) Standardisation and commercialisation of a filarial antibody test for use in the global lymphatic filariasis elimination programme. PhD thesis, James Cook University.

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Abstract

This thesis addresses an important research question of how to take a diagnostic test from the research and development laboratory to a commercially-available product that can be used reliably in a global disease control programme. Laboratory procedures, regulatory requirements and administrative steps required to standardise a filarial antibody test are covered. The question is answered by following the progress of a test developed for use in the Global Program to Eliminate Lymphatic Filariasis. The "lessons learnt" in this translational research apply to the development and commercialisation of any similar product.

The need for reliable diagnostic tools for defining end-points in transmission assessment surveys and post-intervention surveillance becomes imperative as the lymphatic filariasis elimination efforts are moving swiftly to achieve the set objectives by 2020. A variety of diagnostic methods are used, including the detection of microfilariae and antigen detection assays. These tests have served very well in the mapping and preventive chemotherapy phases of the elimination programme, but for the final stages of the programme, there is a strong belief that they may not be adequate to confirm that the transmission of infection has ceased. After a few rounds of preventive chemotherapy, these tests no longer have the same sensitivity due to low mf and antigen prevalence.

Exposure to infective larvae can take months or years before an adult worm becomes established, a patent infection develops and filarial antigen and mf can be detected. What is required is a robust test that can detect immunological changes in any individual after the successful implementation of preventive chemotherapy. The absence of filarial antibody is a key point for concluding that transmission has ceased, marking the elimination of lymphatic filariasis.

Assays for filarial antibodies have been available in the 1970s, but they are based upon the use of crude, whole-worm antigens. These consequently cross-reacted with other parasites, resulting with great concern for the lack of specificity. The "researchgrade" methods are varied from one laboratory to the other, making comparisons between institutions virtually impossible. Also, research laboratories do not have the expertise and capability of producing industry-standard commercial kits required for a global programme.

This thesis describes certain specific methods and techniques used in optimisation and standardisation of an ELISA based on a recombinant antigen Bm14. The Bm14 ELISA is specific to brugian and bancroftian filariasis and, therefore, ideal for use in lymphatic filariasis elimination programmes. The Bm14 ELISA aims to address the general agreement that any antibody test for the global LF elimination programme has to meet the following requirements:

• must be based upon recombinant antigens;

• needs to be reactive with all three lymphatic filariasis species;

• must have excellent sensitivity and specificity;

• must be produced as a standardised commercially-produced kit;

• produced under high-quality control standards with on-going quality assurance;

• can be produced in large-scale to satisfy demand, but at an affordable price;

• has excellent transport and storage stability under tropical conditions and;

• preferably produced in an ELISA and rapid test format.

The standardisation process for Bm14 ELISA includes evaluation of raw materials, assay optimisation and validation. The raw materials and reagents for the assay were evaluated for best results by experimentation. Optimisation was achieved by experimenting on the important concepts of ELISA kinetics for each critical assay component. Validation of the final protocols and assay performance of the optimised ELISA was completed to demonstrate that the test will perform as intended. The standardised test has a sensitivity and specificity of 100% and 98% respectively. No cross-reactivity with strongyloidiasis, schistosomiasis, dengue, malaria, Chagas disease and toxoplasmosis was found but it was cross-reactive with onchocerciasis, therefore, cannot be used in other African countries where the disease overlaps. A shelf-life of 12 months was validated. Repeatability and reproducibility calculated by percentage coefficient of variation were 4.2% and 3.3% respectively and both were within the 10% acceptable performance criteria.

To address the need for a convenient field-based test, the ELISA construct was converted to a rapid diagnostic test (RDT) in a dipstick format. Evaluation of the Bm14 dipstick found 100% concordance with ELISA results.

To commercialise the assays, conformity to International Standards of in vitro medical devices manufacturing (ISO13485) (ISO, 2012) is required by the Australian Therapeutic Goods Administration (TGA). A series of documents structured to follow a quality management system (QMS) was prepared. Documents include scientific methods and procedures, kit inserts and performance results to support the design and development of the commercial product. The output aims to provide a set of guidelines on the commercialisation process that can be used as a reference for future developers of innovative diagnostic tests.

Antibody subclass IgG4 is the widely accepted marker for lymphatic filariasis active infection however, studies also show that IgG1 may represent a response to incoming infective larvae exposure. The standardised Bm14 Antibody CELISA was used in an experiment to investigate the role of IgG1 in antibody detection. Serum samples from a high-transmission area of Papua New Guinea were used in an age-profile study. It was found that both subclasses were equally present across the age groups, regardless of antigen status although, levels of IgG4 were higher than IgG1. Follow-up work is needed to ascertain whether a subclass switching occurs much earlier by investigating the antibody response of infants in a low-transmission setting.

The standardisation of the Bm14 Antibody CELISA and information from this study will fill the critical gap in one of the challenges of the lymphatic filariasis elimination programme. There is currently the need for a reliable recombinant-based antibody test for stopping preventive chemotherapy namely:

(1) end-point determination of preventive chemotherapy and;

(2) post-preventive chemotherapy surveillance: for the detection of hot-spots and verification of transmission interruption. This study will provide industry commercialisation information for diagnostic test research, a gap that needs to be addressed to show how to convert a researchgrade test to a standardised, commercially available test that can be utilised for both clinical and research applications. This study provides information for:

(3) industry-based standardisation and optimisation methods for diagnostic tests to ensure product performance meet the intended purpose and;

(4) quality-based development and commercialisation procedures for a diagnostic test that meets international standards ISO13485.

Item ID:	44645
Item Type:	Thesis (PhD)
Keywords:	diagnosis; diagnostic testing; diagnostic tests; elephantiasis; enzyme-linked immunosorbent assay (ELISA); filarial antibody (IgG4); filarial diseases; filarial infections; filarial nematodes; filarial worms; lymphangitis; lymphatic filariasis; philariasis
Date Deposited:	11 Aug 2016 04:37
FoR Codes:	11 MEDICAL AND HEALTH SCIENCES > 1107 Immunology > 110799 Immunology not elsewhere classified @ 33% 11 MEDICAL AND HEALTH SCIENCES > 1108 Medical Microbiology > 110803 Medical Parasitology @ 33% 11 MEDICAL AND HEALTH SCIENCES > 1117 Public Health and Health Services > 111799 Public Health and Health Services not elsewhere classified @ 34%
SEO Codes:	92 HEALTH > 9202 Health and Support Services > 920203 Diagnostic Methods @ 50% 92 HEALTH > 9204 Public Health (excl. Specific Population Health) > 920499 Public Health (excl. Specific Population Health) not elsewhere classified @ 50%
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