Development of serology diagnosis of chronic aspergillosis and histoplasmosis in Indonesia

Lead Research Organisation: University of Manchester


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.

Technical Summary

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

Planned Impact

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.


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Ashraf N (2020) Re-drawing the Maps for Endemic Mycoses. in Mycopathologia

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Bongomin F (2019) Essential in vitro diagnostics for advanced HIV and serious fungal diseases: international experts' consensus recommendations. in European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology

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Denning DW (2021) Diagnosing pulmonary aspergillosis is much easier than it used to be: a new diagnostic landscape. in The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease

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Kwizera R (2020) Algorithm-aided diagnosis of chronic pulmonary aspergillosis in low- and middle-income countries by use of a lateral flow device. in European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology

Description New blood test detecting Aspergillus antibody paves the way for faster and earlier diagnosis of chronic pulmonary aspergillosis (CPA). With over 7 million people suffering TB in the lungs each year and 5-10% of them later getting the fungal infection aspergillosis in the lungs, this simple cost-effective test for aspergillosis provides a win-win for early diagnosis and reducing lung scarring and death. The test, available commercially from LDBio diagnostics, is very simple and needs no power source or equipment and therefore is likely to be very useful in resource poor settings.
Testing on blood samples from 154 CPA patients identified at the National Aspergillosis Centre in Manchester and 150 healthy volunteers found an improved sensitivity (91.6%) and specificity (98.0%) compared with existing tests.
This work has been repeated in Indonesia, with slightly lower sensitivity and specificity, but the first such work in a LMIC. A project is ongoing in Uganda to also examine the suitability of lateral flow testing in a low income, high TB and high HIV setting, and a separate trial in India found similar results to Indonesia.
Exploitation Route We anticipate that this assay will become the workhorse screening tool for patients with possible TB, in whom the diagnosis is excluded. Discussions are ongoing with the WHO and others.
Sectors Healthcare

Description The inclusion of Aspergillus antibody testing in the WHO 3rd Essential Diagnostic List was partly attributable to the work conducted during this award. Impacting on country policy towards improving the accuracy of the diagnosis of TB
First Year Of Impact 2022
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Policy & public services

Description Application to the WHO for Aspergillus antibody testing to be listed as an 'Essential Diagnostic'
Geographic Reach Multiple continents/international 
Policy Influence Type Implementation circular/rapid advice/letter to e.g. Ministry of Health
Impact Serological detection of Aspergillus antibody is critical to the diagnosis of CPA, especially to differentiate CPA from similarly presenting conditions including pulmonary tuberculosis (TB) and COPD. The burden of TB is particularly high in low- and middle- income countries and CPA both complicates and mimics treated TB, with high 5-year mortality. Chronic pulmonary aspergillosis (CPA) is usually a progressive fungal disease, most often complicating other respiratory disorders. There are an estimated 1.2 million cases following pulmonary TB (Denning, 2011) and overall ~3 million CPA cases worldwide (GAFFI Roadmap, 2015), and with an annual mortality of ~15%, 400,000 deaths (Brown, 2012). CPA is associated with severe morbidity and mortality, but outcomes are improved with long-term antifungal therapy or surgery. While CPA is regarded as a rare disease in high-income countries, its burden in low-income and middle-income countries with high incidences of pulmonary tuberculosis (TB) is considerable. An estimate for India put the 5-year prevalence at 290,147 cases, or 24 per 100,000 (Agarwal et al. 2014), and for Pakistan at 72,438 cases, or 39 per 100,000 population (Jabeen et al. 2017). Put another way, a prospective study in Uganda in both human immunodeficiency virus (HIV)-infected and uninfected patients found an annual rate of CPA development of 6.5% in those with a residual cavity at the end of TB treatment, typically found in 22% to 35% of cases (Page et al. 2019). It is likely that substantial numbers of patients are incorrectly diagnosed as having pulmonary TB rather than CPA, as was found in 19% of the members of a group of HIV-negative, GeneXpert-negative, and smear-negative patients in Nigeria (Oladele et al. 2017). About 45% of the global population of patients with pulmonary TB, representing around 2.5 million cases, are currently not confirmed microbiologically (WHO 2015).
Description Case Definition of Chronic Pulmonary Aspergillosis in Resource-Constrained Settings
Geographic Reach Multiple continents/international 
Policy Influence Type Membership of a guideline committee
Impact Chronic pulmonary aspergillosis (CPA) is a recognized complication of pulmonary tuberculosis (TB). In 2015, the World Health Organization reported 2.2 million new cases of non-bacteriologically confirmed pulmonary TB and many probably do not have TB. The proposed definition will facilitate advancements in research, practice, and policy in lower- and middle-income countries as well as in resource-constrained settings and directly influence the practice of TB access the world.
Description UK-Indonesia: Joint Partnership Call on Infectious Diseases
Amount £393,000 (GBP)
Funding ID MR/S019898/1 
Organisation University of Manchester 
Sector Academic/University
Country United Kingdom
Start 02/2019 
End 02/2021
Title Improved means of estimating burden of chronic pulmonary aspergillosis 
Description We have published an improved mathematical model of CPA incidence and prevalence, based on data generated during the study, which will inform all countries estimates. 
Type Of Material Data analysis technique 
Year Produced 2021 
Provided To Others? Yes  
Impact CPA was previously seen only as a con=mplication (sequela) of pulmonary TB. Our work has shown that this in only part of the story, and it is occurs during TB and can be lethal. Our new modelling reflects this and in Indonesia pushed the cases up from 83,000 to 378,700 cases (which carries a 20% year mortality and 7.5% subsequent year mortality). 
Description Diagnosing histoplasmosis in Cameroon 
Organisation University of Buea
Country Cameroon 
Sector Academic/University 
PI Contribution Our grant has enabled the skin test prevalence of histoplasmosis in Cameroon (a highly endemic country for histoplasmosis) to be ascertained.
Collaborator Contribution On the ground patient and volunteer testing
Impact Masters thesis.
Start Year 2019
Description Histoplasmin supply and skin testing 
Organisation National Autonomous University of Mexico
Country Mexico 
Sector Academic/University 
PI Contribution We have established a strong research relationship with Dr Conchita Toriello of Facultad de Medicina, Universidad Nacional Auto ´noma de Me ´xico, Mexico City, Mexico and this work is allowing detailed new maps to be drawn on the geographical distribution of Histoplasma spp.
Collaborator Contribution Dr Toriello provides a very valuable reagent for our global health work.
Impact Prior outputs and others to emerge as the work we ahve done with the grant gets analysed.
Start Year 2017
Description Study of chronic pulmonary aspergillosis in Sierra Leone 
Organisation University of Sierra Leone
Country Sierra Leone 
Sector Academic/University 
PI Contribution Study design and provision of rapid Aspergillus antibody tests
Collaborator Contribution recruitment of 197 patients with possible TB or aspergillosis. Fifty-two (26.4%) patients were HIV positive, 41 (20.8%) were seropositive for Aspergillus and 23 (11.6%) had chronic pulmonary aspergillosis.
Impact paper of results in draft form
Start Year 2021
Description Advances Against Aspergillosis and Mucormycosis 2022 International Conference 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Alternate year international conference (heald this time fully online) with 14 speakers, 91 scientific posters and ~350 participants from 54 countries. I chaired the meeting and gave an introduced talk.
Year(s) Of Engagement Activity 2022
Description Advances Against Aspergillosis and Mucormycosis international conference 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Advances Against Aspergillosis and Mucromycosis (9th) has now clearly established itself as the premier forum for detailed and dedicated discussion of all aspects of Aspergillus and Mucorales infection and research, and previously published proceedings have been very well-received. Prior meetings have yielded 267 papers = published in 9 Supplements, comprising 2,026 pages of full papers, as well as the 1,429 abstracts from these meetings in the programs. The 9th meeting attracted 325 attendees from >35 countries and 165 abstracts.

The Aspergillus and Mucorales field continues in a state of rapid advancement, including the publication of numerous post-genomic papers and substantial advances in translational, immunologic, epidemiologic and diagnostic research. The launch of several antifungals in the last few years and anticipated clinical trials of newer compounds with novel mechanisms of action is an exciting time for mycology. Pan-azole, and echinocandin resistance has emerged and requires unique approaches, and combination therapy remains an important area of interest. Greatly increased awareness of allergic aspergillosis has opened new opportunities for both antifungal agents and immunotherapies. New molecular strategies for diagnosis continue to make progress, and recent guidelines offer increased diagnostic insight. There is a continuing high death toll from invasive aspergillosis and mucormycosis, including patient groups not usually associated with these opportunistic infections. This meeting is another chance to gather the world's aspergillosis and mucormycosis experts in one venue. A fundamental tenet of this colloquium, dedicated by the founding Co-Organizers before the first meeting, continues to be to engender collaborative relationships amongst clinicians, scientists, and industry to further advance the field.
Year(s) Of Engagement Activity 2020
Description Inclusion of Aspergillus antibody testing on the WHO Essential Diagnostic List 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Application to the WHO for Aspergillus antibody to be included - and accepted as one of 175 test categories included in the 3rd EDL. Aspergillus antibody is critical for the diagnosis of chronic pulmonary aspergillosis which both mimics and complicates TB of the lungs. About 10% of TB patients have aspergillosis, not TB. Many patients with fungal asthma and Aspergillus sinusitis have positive Aspergillus antibodies too. Aspergillus antibody is 80-92% sensitive depending on the test used, again much more sensitive than culture.
Year(s) Of Engagement Activity 2020,2021
Description The Aspergillus Website 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The Aspergillus Website is the most comprehensive source of information about Aspergillus and the diseases it causes available on the internet. There are extensive sections for clinicians, scientists and laypeople (patients) including comprehensive collection of treatment protocols covering 43 distinct therapeutic areas and all approved antifungal drug SPC's & PIL/VIPIL's. The Aspergillus Website also provides information on evidence supporting (or otherwise) some herbal and alternative remedies. New sections introduced over the last 24 months include those on biodeterioration, prehistoric fungi, antifungal stewardship, and marijuana use and its medical consequences.

The Aspergillus Website provides simple, free access to >74,000 pages all indexed in Google, ready to be searched, over 13,300 scientific articles (including a unique collection of 831 historical articles) and ~15,900 conference abstracts from 1974 onwards.

The Aspergillus Website is listed at number 1, 1, 2 and 3 in, number 1, 1, 2 and 3 in, number 1, 1, 4, 3 in Bing and Yahoo! for 'aspergillus', 'ABPA' 'aspergillosis' and 'aspergilloma'respectively. If 'aspergillus' is searched in Google, there are 11.5 million results. We intend to build a searchable section on resistance in Aspergillus with a good graphical display in 2020.

Current monthly figures show that 100,000 - 130,000 individual computers accessed The Aspergillus Website and Patients' website alone per month, 5,000 to 8,000 people daily. This equates to 8.5 million 'requests for content'. About 65% of visits are using smaller devices. Over 54% of users access the Aspergillus Website using iPhone of iPad, 10% use an Android phone and only 36% use a laptop or personal computer. Twitter posts are put out almost every day (@AspergillusWeb - 1,350 followers), Blogs twice a week.

Mapping of (see figure below for 2019 figures, darker shading = more users) shows that The Website reaches people in over 147 countries. USA is the country from which we get most visits with UK and India in the next 2 places, France and China in 4th & 5th place respectively. We would like to boost usage from other parts of the world.
The Patients Website ( has had a complete refresh in 2019 which includes moving it to a new domain name - is more easily found via Google if a patient was to search for more information. This was important as our communities have expanded enormously since the old website was built in 2012.

Since the changeover the domain name does not register in the top page of Google searches so as yet is not being as intensively used as the old website. The old website had declined in use over the last 5 years as most activity switched to Facebook. Despite these limitations use is gradually increasing and in June 2019 attracted 1800 unique visitors who looked at 4300 pages. The top countries utilising the Patients' Website by origin are: 1. UK, 2. USA, 3. Australia, 4. India, 5. Russia. There are >400 active links to this website.
Year(s) Of Engagement Activity 2018,2019,2020
Description World Aspergillosis Day 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Clinicians need better awareness to be able to 'think fungus' when repeated attempts to treat infections fail. Serious fungal infections are underdiagnosed worldwide
People with one of the multiple forms of aspergillosis need much better support and information as aspergillosis is a rare infection that isolates people.
People who live with and care for people with aspergillosis often need to better understand what the patient is going through and themselves need understanding & support
Last but certainly not least those who fund our health services need to be more aware and to better provide diagnostics and treatments worldwide.

Between each talk today you will see an impact statement from a person living with aspergillosis. These statements have come from people who use the online support group hosted by the National Aspergillosis Centre at Wythenshawe. We hope that these statements will bring the patient voice and their lived experiences to today. Please remember that behind the statistics are real people living with this debilitating condition. They are counting on us to deliver better diagnostics, better treatments and better understanding. Use the hashtag worldaspergillosisday and show your support.

Visit the website and download email signatures, social media header images, we have a twitter frame and a FaceBook frame. Links to all of this can be found on the aspergillosisday website. Please take a look and show your support.
Year(s) Of Engagement Activity 2020