Development of serology diagnosis of chronic aspergillosis and histoplasmosis in Indonesia

Chronic pulmonary aspergillosis mimics pulmonary tuberculosis, and can follow it. An estimated 1.2 million are affected worldwide, and ~85,000 in Indonesia. Histoplasmosis is endemic in Indonesia but no burden estimate is possible as the only tools for diagnosis are culture and histopathology, which are slow and insensitive. The burden of histoplasmosis is not known in the world or in Indonesia, partly because of poor diagnostic tools. Chronic pulmonary histoplasmosis complicates other lung disease, notably COPD, and mimics tuberculosis. Both fungal infections are misdiagnosed as 'smear negative TB'. These patients are often investigated for MDR TB, and treated inappropriately with anti-tuberculous medication. The 5 year mortality of chronic pulmonary aspergillosis is 75-80%, but reduced to ~40% with antifungal therapy.

In this grant we will:
1. Develop and evaluate in the UK and Indonesia the world's first point of care Aspergillus antibody screening assay to enable the diagnosis of chronic pulmonary aspergillosis.
2. Develop and evaluate in Indonesia a new Histoplasma antibody assay to enable the diagnosis of acute and chronic pulmonary histoplasmosis.
3. Identify for the first time 'hot spots' for histoplasmosis in Indonesia, so as to develop a sera bank for evaluating the antibody test, to support subsequent commercialisation.

We have access to highly purified recombinant Aspergillus fumigatus proteins, that have been used in an ELISA format to detect Aspergillus IgG antibody with a 97% sensitivity and a 90% specificity. In Manchester at the Mycology Reference Centre laboratories and National Aspergillosis Centre, we will use these proteins to develop a lateral flow device utilising our huge sera bank from patients with chronic pulmonary aspergillosis and other respiratory disorders to optimize performance against a gold standard assay (ImmunCap). We will then utilize the assay in field trials in the UK as a prospective screen for Aspergillus antibody in the laboratory, and in Jakarta in a cross-sectional study (n=600 patients) and a longitudinal study (n= 200 patients) both in patients with PTB. In the UK, the gold standard assay will be the ImmunoCap and in Jakarta the Siemens assay. During the field trials we will open up commercialization discussions.

In Jakarta, we will identify additional immunodominant proteins in Histoplasma capsulatum yeast phase. We will standardize growth and purification conditions for supernatant production to optimize sensitivity and specificity of a new ELISA Histoplasma antibody test using sera from non-immunocompromised patients with histoplasmosisand controls. We will further validate this assay on additional sera (see Aim 3), to optimize performance. We will prepare the materials for subsequent production of recombinant proteins for a second generation antibody test.

In parallel with this laboratory activity, we will undertake several skin testing surveys with histoplasmin in Jakarta and its surrounding cities (Bogor, Depok, Tanggerang and Bekasi - Jabodetabek), Bandung, Semarang, Surabaya, Bali, and Manado. This will be done by setting up a new Histoplasmosis network in Indonesia. We need to identify more sera for test validation and cutoffs, and the simplest way to do this in Indonesia, is to pinpoint areas of high endemicity and then actively seek out patients in those areas with chronic pulmonary histoplasmosis, through existing TB clinics, focusing on those with 'smear negative' TB. So in areas of >10% local skin test positivity, we will ask the local participating clinicians to identify possible patients and submit fungal diagnostic samples. Fungal culture of sputum or bronchoscopy fluid will be the gold standard for diagnosis.

Timelines
Year 1
Aim 1 - development of lateral flow device (LFD) to prototype stage and initiation of Manchester extended validation
Aim 2 - development of histoplama antibody assay: western blotting with small number of sera complete. Partial identification of key antigenic bands. Large scale batch cultures for supernatant completed and different purification, concentration and periodate oxidation methods compared for optimisation.
Aim 3 - Ethical approval for histoplasmin skin testing gained. Shipping of histoplasmin from Mexico. 30% of skin testing completed.

Year 2
Aim 1 - LFD validation on prospective samples in Indonesia complete (1000+ samples). Manufacture methodology defined precisely. Commercialisation discussions ongoing. Paper submitted for publication.
Aim 2 - Development of ELISA assay complete. Partial validation done, with test and control sera. Second generation recombinant assay antigens decided.
Aim 3 - Histoplasmin skin testing completed and paper written up. Possible chronic cases of histoplasmosis identified and sera collected for 100 cases.

Skin testing
Skin tests are performed by intradermally injecting 0.1mL of histoplasmin antigen in a 0.1ug protein/0.1mL into the inside of the left forearm of each volunteer using disposable tuberculin-type syringes . The same investigator will perform the intradermal tests and readings. Tests are read at 24 and 48h after injection. Induration over 8 mm in transverse diameter after 48 or 72 hours are considered to be histoplasmin positive

This proposal has the potential to transform several aspects of the management of patients thought to have pulmonary TB, in Indonesia and other countries. The primary beneficiaries will be patients who actually have histoplasmosis (unknown number) - those misdiagnosed and inappropriately treated, and those who develop a well recognized complication of prior TB - chronic pulmonary aspergillosis. With a 75-80% mortality over 5 years without therapy, the new diagnostic tests we will develop are important on several fronts:
- Earlier diagnosis of chronic pulmonary aspergillosis or histoplasmosis,
- Reducing patient morbidity, allowing them to return to work
- Lengthening patients' lives
- Reducing unnecessary antituberculous therapy, and so contributing to the antimicrobial resistance (AMR) reduction agenda.
- Provide greater confidence in TB programs, because alternative diagnoses can be reached, in the same consultation, enhancing patient confidence and facilitating earlier referral of other patients with similar symptoms, many of whom with have transmissible TB.
- Provide economic benefit directly by sale of tests and indirectly by improving health,
- Public Health in Indonesia will gain because some of the TB patients can be removed from the annual statistics, and so TB control efforts will look better.
- Tests for resistant (MDR or XDR) TB will be reduced, as alternative diagnoses can be reached first.
- Some data on high areas of endemicity for histoplasmosis will be provided, allowing certain laboratories to be equipped better for diagnosing all forms of histoplasmosis, including class 3 containment culture facilities, if required.

The researchers will gain from doing the research, and the university will increase its rank due to publication in the international journals. The scientific and research community will gain from the experiences and be able to replicate this work in other countries and regions.

This work will go some way to developing a Mycology Reference Laboratory in Indonesia, which is sorely needed. Specialized mycology diagnostic services are required for HIV patients, those with cancer, severe asthma, liver failure, critical care and COPD patients admitted to hospital, as well as large numbers of patients with cutaneous problems, such as mycetoma, chromoblastomycosis and sporotrichosis or patients with disseminated mycoses to the skin. In addition, even though organ transplantation in Indonesia at present is small scale, the knowledge of how to handle fungal infection will be beneficial in the future. Provision of timely, accurate and innovative diagnostic tools for the detection of serious fungal infections will show the path for other laboratories still focusing on conventional (victorian) culture technologies.

The information collected through this project will open the opportunity to develop more research in the mycology field, including determining the social and economic impact of these infections in Indonesia; aspects related to length of survival related to adequate and timely provision of health care services, as well as the study of disease patterns such as, geographical delineatation of endemic areas, potential vectors, and other factors associated with morbidity.
A major deficiency internationally in healthcare is the absence of public health mycology. Apart from the Centres for Disease Control and Prevention, the Pasteur Institute and the collection of Candida bloodstream isolate numbers and resistance in the UK, there are no national programs assessing burden of fungal disease, antifungal resistance or impact. This program will contribute to public health mycology, probably through integration of testing for chronic pulmonary aspergillosis and histoplasmosis in TB programs and improve the general health of 100,000s of patients.