1. Chronic pulmonary aspergillosis (CPA) in association with tuberculosis and AIDS
Dr Iain D Page
NIHR Academic Clinical Lecturer in Infectious Diseases
UK National Aspergillosis Centre, University Hospital South Manchester
Manchester Academic Health Sciences Centre (MAHSC)
The University of Manchester 1

3. Chronic Pulmonary Aspergillosis
Now well described in case series from UK, France, Japan and Korea.
Guidance published by ECCMID and IDSA
Diagnostic Criteria
SYMPTOMS - cough or breathlessness or fatigue or haemoptysis for > 3 months
RADIOLOGY - aspergilloma OR progressive cavitation OR paracavitary infiltrates
MICROBIOLOGY -Aspergillus culture in ‘respiratory fluids’ OR anti-Aspergillus antibodies
Exclusion of other active illness to explain symptoms
Denning et al Clinical Infectious Diseases 2003:37:s265-80, Jhun et al, Medical Mycology;51:811-7, Ohba et al, Respiratory Medicine 2012;106:724-9

4. Chronic Cavitary Pulmonary Aspergillosis, with aspergilloma
Case 004 (also published) in case histories section.
Patient JP
July 2001
Denning DW et al, Clin Infect Dis 2003; 37:S265 4

5. Chronic Fibrosing Pulmonary Aspergillosis
Case 004 (also published) in case histories section.
Patient JP
April 2002
Denning DW et al, Clin Infect Dis 2003; 37:S265 5

6. CPA – without aspergilloma
Case 004 (also published) in case histories section.
Only 36% of CPA cases have aspergilloma – Jhun 2013
Denning DW et al, Clin Infect Dis 2003; 37:S265 6

7. CPA – outcome Haemoptysis – 58% (Denning 2003)
5 year mortality - 85% (Ohba 2012)

8. Chronic pulmonary aspergillosis – Treatment outcomes
Oral itraconazole
6 mo mo
35%
41%
Stable
Improved
Standard care
6 mo mo
23% 7%
29%
64%
71%
53%
21%
24%
Deterioration
30% relapse off therapy in 6 months
Natural history with no therapy over 12 months
Agarwal R et al, Mycoses 2013; 56:559.

9. CPA – primary underlying conditions in UK population
Tuberculosis (15.9%)
Atypical mycobacteria (14.3%)
ABPA (11.9%)
COPD / Emphysema (9.5%)
Prior pnuemothorax (9.5%)
Lung Cancer (9.5%)
Sarcoidosis (4.8%)
Smith and Denning. The European Respiratory Journal. 2011;386:865-72

10. Frequency of TB underlying CPA - globally
UK % (Smith and Denning 2011)
Japan - 50% (Ohba et al 2011)
China – 71% (Chen et al 2012 – 256 surgical patients )
Korea – 81% (Jhun et al 2011)
Senegal – 100% (Ba et al 2000 – 24 surgical patients)
Chen et al. Interactive cardiovascular and thoracic surgery. 2012;15:77-80
Ba et al. Dakar Medical. 2000;45:144-6.

11. Sole published measurement of aspergillosis prevalence post TB
Performed in UK between 1968 to 1970
No CT scans!!
Medical Research Council study
544 patients previously treated for TB, with persistent cavities
34% developed precipitating antibodies to Aspergillus fumigatus
63% of patients with Aspergillus antibodies develop aspergilloma within 2 years
42% of those with aspergilloma developed haemoptysis
British Tuberculosis Association Aspergolloma and residual tuberculous cavities - The results of a resurvey. Tubercle 51:227.
British Tuberculosis Association. Tubercle;51:

12. Frequency of Aspergillus antibodies after TB
UK – 34%
Japan – 20%
India – 27%
Brazil – 21%
Iwata et al. Kekkaku. 1989;64:7.13.
Shahid et al. Indian Journal of Medical Microbiology. 2001;19:201-5
Kurhade et al. Indian Journal of Medical Microbiology. 2002;20:141-4.
Ferreira-da-Cruz et al. Memórias do Instituto Oswaldo Cruz.1988;83:357-60

13. Estimated Global Prevalence of CPA secondary to TB
36 million cured of TB worldwide
22% South African patients have cavities post TB
1.1 million cases CPA predicted worldwide
100,000 new cases annually in Africa
Prevalence of 43 per 100,000 in DRC
Denning, Pleuvry and Cole . Bulletins of the World Health Organization 2011;89:

14. Possible reasons for error 1 - Environment

15. Possible reasons for error 2 – HIV co-infection

16. Aspergillosis in AIDS Described in pre-HAART era case series
Mostly seen in neutropaenic pts
Often secondary to zidovudine or ganciclovir
21% documented cases in non-neutropaenics though
Almost always with CD4 <50
Neutrophil function often poor in advanced AIDS even if counts normal
Survival better if patients received antifungals AND antiretrovirals
Adrizzo-Harris et al. Chest 1997;111:612-8
Greenberg et al. Chest 2002;122:886-92
Denning et al. NEJM 1991;324:654-62

17. Bombay autopsy series – HIV +ve pts
1717
Bombay autopsy series – HIV +ve pts
Lanjewar & Duggal, HIV Med 2001;2:266

18. Japanese AIDS autopsy series
Katano et al BMC Infectious Diseases 2014, 14:229

19. Italian autopsy series
Antinori et al, Am J Clin Pathol 2009;132:221 19

20. Aspergillosis in ‘smear-negative TB’
‘Smear negative TB’ common in AIDS
WHO diagnostic criteria for ‘smear negative TB’
3 negative sputum smears
Abnormal CXR
Failure to respond to antibiotics
34% mortality at 2 years
Patients NOT routinely tested for any fungal lung diseases
7% of patients with smear negative TB in Kampala grow Aspergillus on BAL culture
Banda et al. Int J of Tuberc Lung Dis. 2000;10:
Yoo et al. Respirology. 2011:16:836-41

21. Research Questions
What is the prevalence of CPA secondary to pulmonary TB?
Does HIV co-infection alter the frequency of CPA
Can CPA be diagnosed with chest X-ray and symptoms alone?
Can CPA be diagnosed with chest X-ray + symptoms + serology?
Can patients at high risk of CPA be identified at the time of active pulmonary TB treatment?

22. CCPA case definition Patients must have ALL of the following:-
Cough or haemoptysis for 3 months or longer.
Raised Aspergillus-specific IgG
or positive precipitins.
Radiological features of CPA – at least one from;
Progressive cavitation on serial CXR.
Paracavitary fibrosis on CT chest.
Aspergilloma on CT scan.
Plus exclusion of active pulmonary TB

23. Measuring anti-Aspergillus antibodies 1 – Precipitins / CIE 2 – ELISA
Time consuming.
Requires highly skilled laboratory staff.
May have poor sensitivity.
Results are subjective.
Can be done in house with antigens from fungal culture.
Very difficult to reproduce at other labs.
Simple automated systems.
Produces objective numerical results.
Antigens produced by the manufacturer at a single site and distributed to labs.
Intra-lab consistency can be assured.
Diagnostic cut-offs for CPA not well defined.
Many brands available – comparative efficacy poorly described.

24. Aspergillus-specific IgG test
Exposure to Aspergillus spores probably ubiquitous
Most adults have some anti-Aspergillus antibodies
Existing published analyses of optimal cut-offs shown below
Test
Suggested cut-off
Type of study
Country
Author
ImmunoCAP
35 mg/L
97.5th centile
Netherlands
van Toorenebergen 2012
70.1 mg/L
Belgium
van Hoeyveld 2006
66.4 mg/L
South Africa
Watkins 2012
35.9 mg/L
ROC (20 mixed cases vs 42 controls)
Siemens Immulite
19.3 mg/L
“Siemens software analysis of healthy controls”
BioRAD
No published data
Serion / Virion
Genesis

25. Study design
242 samples of stored sera from patients with proven CPA at UK National Aaspergillosis Centre tested.
Patients not on antifungals at the time of testing.
100 samples from Ugandan healthy controls tested.
ROC analysis used to define optimal diagnostic cut- offs.
Sensitivity and specificity calculated for those cut-offs.
British Tuberculosis Association. 1970tuberculous cavities - The results of a resurvey. Tubercle 51:227.
British Tuberculosis Association. Tubercle. 1970;51:

26. ROC curves: CPA cases vs. healthy controls

27. levels between COPD and healthy controls for this assay
RESULTS
TEST
CoV
ROC AUC
95% CI
New Proposed cut off
Sensitivity
Specificity in healthy controls
ImmunoCAP -
0.996
20 mg/L
96%
98%
Siemens Immulite
3.4%
0.991
0.982 – 1
10 mg/L
Serion
23.2%
0.973
0.96 – 0.987
35 AU/ml
91%
Dynamiker
12.1%
0.918
0.890 – 0.946
70 U/ml
75%
Genesis
11.1%
0.902
0.871 – 0.933
16 U/ml
78%
97%
CIE
59%
100%
*a new high-specificity COPD cut off was not calculated for Genesis as there was no difference in mean
levels between COPD and healthy controls for this assay

28. Are the cut-offs found in relation to Ugandan controls relevant to Europe?
Environmental exposure to Aspergillus might be different in the two environments
Genetic differences might affect results
The unexpectedly low age of Ugandan controls recruited might bias results
Differences between the current and proposed cut-offs would have significant impacts on test sensitivity and specificity

29. How to identify optimal European cut-off?
Challenges
Solution
At least 100 cases and 100 controls needed for analysis
UK NAC alone has > 100 CPA cases, but does not have access to healthy control sera
No funding available
Collaboration!!
All authors of existing publications on the topic were contacted
All agreed to share their data from testing of healthy controls
ROC analyses performed comparing UK NAC CPA cases to controls from other European centers

30. Samples available for testing
241 sera from CPA cases at UK NAC tested by ImmunoCAP, Siemens Immulite and Serion.
118 separate sera from CPA cases at UK NAC tested by Bio-Rad
Controls
Test
Control Group
Country
Number of controls
Median age of controls
ImmunoCAP
Immulite
Serion
Blood donors from existing study
Uganda
100
19 years
Laboratory technicians
Belgium
114 -
Blood donors
Dutch
152
Bio-RAD
Pregnant women
France
222

31. ROC AUC results and cut-off comparison
Test
ROC AUC
Ugandan controls cut-off
Existing manufacturer’s recommended cut-off
European controls cut-off
Sensitivity (%)
Specificity (%)
ImmunoCAP
0.956
20 mg/L
40 mg/L*
50 mg/L 84 96
Siemens Immulite
0.948
10 mg/L
21.4 mg/L
25 mg/L 93 99
Serion / Virion
0.944
35 U/ml
Variable with batch
50 U/mL 91
Bio-Rad
0.955 -
10 AU/ml
1.5 AU/ml 98
Performance of existing manufacturer’s cut offs
Test
Sensitivity (%)
Specificity (%)
ImmunoCAP 87 90
Immulite 93 99
Bio-Rad 85
100

32. Potential issues with original study
Significantly lower than the manufacturer’s upper reference range of 19.3 mg/L
Based on analysis of results in 152 healthy Dutch controls
ImmunoCAP cut of was 20mg/L
Significantly lower than 40mg/L currently used in UK labs
Large number of adolescents in Ugandan control cohort
Controls were blood donors
Blood donation drive underway in “colleges” at the time
Median age = 19 years
Age range = 17 – 39 years

33. Research question Methods
Are the lower levels of Aspergillus-specific IgG levels found in the previous Ugandan healthy control cohort due to
The young age of those controls?
Genuine difference due to environmental or genetic factors?
Methods
We repeated ROC analyses and optimal cut-off calculations comparing Immulite Aspergillus-specific IgG levels in CPA cases to the following control groups
100 older Ugandan healthy controls
88 older Ugandan control with treated pulmonary tuberculosis, but asymptomatic with no radiological evidence of CPA
152 Dutch healthy controls

34. Research Questions
What is the prevalence of CPA secondary to pulmonary TB?
Does HIV co-infection alter the frequency of CPA
Can CPA be diagnosed with chest X-ray and symptoms alone?
Can CPA be diagnosed with chest X-ray + symptoms + serology?
Can patients at high risk of CPA be identified at the time of active pulmonary TB treatment?

35. Gulu, Northern Uganda

38. Unpublished cross-sectional survey of CPA prevalence in Uganda
CCPA diagnosed in patients with ALL
of the following:-
Cough or haemoptysis > 1 month
Positive Aspergillus – specific IgG
Siemens Immulite
Cut off 20 mg/L
Radiological features (any of three)
Progressive cavitation on serial Chest X-ray
Paracavitary fibrosis on CT thorax
Aspergilloma on CT thorax
All reporting done by 2 consultant radiologists blinded to clinical and serological results
Deciding report by senior radiologist at UK National Aspergillosis Centre
400 unselected patients with previous pulmonary TB
Community based survey
Completed TB treatment in last 7 years
50% HIV positive
All had CXR and Aspergillus IgG
re-surveyed 18 months later
Repeat CXR
CT scan in those with positive IgG test or suspicion of aspergilloma on CXR

41. Could anything be done about it?
Treatment with Itraconazole prevents CPA progression (ref below)
RCT in India without drug level monitoring
Stability or improvement in 76% treated pts vs 35% untreated (p=0.02)
Voriconazole and Posaconazole also effective
Expensive now, but will come off patent
Anti-retroviral drugs distributed in Africa despite patents
IV Amphotericin for sub-acute invasive disease in AIDS
Already used in Africa for cryptococcal meningitis
Amphotericin B can be made cheaply
Surgery
Case series in Senegal shows it can be done in Africa
Agarawal et al. Mycoses. 2013;56:

42. Conclusion
CPA complicating TB is likely to be a major under-recognised Global public health issue
Treatment is plausible, even in resource poor setting
Further studies now need to be done to confirm prevalence in areas of high TB prevalence
Diagnostic tests suitable for use in low-resource settings needed
Access to anti-fungal treatments needs to be improved