Chronic pulmonary aspergillosis as a cause of smear negative TB and or anti-TB

treatment failure in Nigerians

Oladele RO1,2, Irurhe NK1, Foden P3, Akanmu AS1, Gbaja-Biamila T4, Nwosu A1, Ekundayo

HA5, Ogunsola FT1, Richardson MD6,7, Denning DW2,7.

1. College of Medicine, University of Lagos

2. Faculty of Biology, Medicine and Health, The University of Manchester,

Manchester Academic Health Science Centre,

3. Medical Statistics Department, University Hospital of South Manchester, UK

4. Nigerian Institute of Medical Research

5. General Hospital, Ilorin. Nigeria

6. Mycology Reference Centre Manchester, University Hospital of South

Manchester, Manchester, UK.

7. National Aspergillosis Centre, University Hospital of South Manchester,

Manchester, UK

Running title: Chronic pulmonary aspergillosis in Nigerians

Keywords: Aspergillosis, Tuberculosis, HIV, Chronic pulmonary aspergillosis,

Aspergillus serology

Corresponding author: Rita Oladele, Department of Medical Microbiology, College of

Medicine, University of Lagos, Lagos. Nigeria

Telephone: +2348171570142

Email: drritaoladele@yahoo.com

Word count: Summary – 202; Main text – 2561; Number of references – 35; Number of

tables – 1; Number of figures – 3

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Abstract

Objective: We evaluated CPA as an alternative diagnosis of smear negative TB and treatment

failure in TB patients in Nigeria.

Methods: We conducted a cross-sectional multicenter survey in HIV positive and negative

adult patients at the end of their TB treatment in clinics in Lagos and Ilorin states. All were

assessed by clinical examination, chest X-ray and Aspergillus IgG serology and some for

sputum fungal culture. CPA was defined as a positive Aspergillus fumigatus IgG titre with

compatible chest X-ray or a positive sputum culture of Aspergillus with a visible fungal ball

on the chest X-ray with symptoms with underlying lung disease.

Findings: 208 patients were recruited between June 2014 and May 2015. 153 (73.6%) were

HIV positive. Mean age was 39.8 years, 124 (59.6%) were female and 39 (18.8%) were

unable to work. Median CD4 count (range) was 169.5 cells/ml (4-593) in the HIV infected

patients with positive Aspergillus IgG. 109 (52.4%) had documented TB and 140 (67.3%) had

productive cough and 50 had haemoptysis. The prevalence of CPA was 8.7%; 10 (6.5%) with

HIV infection and 8 (14.5%) HIV negative patients (Fisher’s exact p=0.092).

Conclusion: CPA is a neglected disease in Nigeria, most matching the WHO diagnostic

criteria for ‘smear-negative’ tuberculosis.

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Introduction

Chronic pulmonary aspergillosis (CPA) is a progressive pulmonary disease, complicating

many other respiratory disorders such as tuberculosis, and affects an estimated three million

people globally.[1] Prior pulmonary disorders are almost universal in CPA patients.[2] The

global prevalence of CPA secondary to TB is estimated to be between 0.8 and 1.37 million

cases with 43 cases per 100,000 in Congo and Nigeria. [3] CPA is associated with significant

morbidity and mortality. Long-term oral antifungal therapy is given because of high

frequency of relapse.[2] Resistance development or intolerable side effects occur in up to 50%

of patients.[4]

Studies of aspergillomas in Taiwan, South Korea, China and India identified tuberculosis as

the aetiological factor in upto 93% of cases of CPA.[5-8] A UK survey found that out of 544

patients who were left with a residual cavity of 2.5 cm one year post TB therapy, 36% had

positive Aspergillus antibodies and 22% had an aspergilloma after 3 years. [9,10] No similar

surveys have been reported since.

Tuberculosis is more common in Africa,[11] and the relationship between tuberculosis and

HIV/AIDS is clear. This raises the possibility that CPA is much more common in Africa. The

first case of CPA in Africa was an aspergilloma in a South African farmer in 1965. [12]

Subsequent cases have documented CPA in Ivory Coast,[13] Senegal,[14] Ethiopia,[15] Nigeria,[16]

and more recently from Uganda[17] and Tanzania.[18] A South African report revealed a 9.9%

rate of aspergillomas in patients with life-threatening haemoptysis in an area of high

tuberculosis incidence.[19]

Currently, many CPA cases are either diagnosed late or misdiagnosed. Here, we evaluated

CPA as a cause of smear-negative TB and or anti-TB treatment failure in HIV-positive and -

negative Nigerians.

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Methodology

Study Population

Patients enrolled in a cross-sectional study conducted in three centers in Nigeria; National

Institute for Medical Research, ART (Antiretroviral therapy) and TB clinic, DOTS (Directly

Observed Therapy, Short course) clinics at Lagos University Teaching Hospital and

University of Ilorin Teaching Hospital. HIV-positive and -negative adults who were in their

last month of TB treatment (smear + GeneXpert® positive) or being treated for ‘smear-

negative TB’ (based on radiological and clinical picture) were recruited. In addition, two

control groups were included: 50 blood donors and 50 HIV positive patients on ART with

CD4 count >350 cells/ml and without a history of TB. Controls underwent venepuncture

only.

Ethical approval was obtained from the institutional Ethics Committee of the study site

hospitals. Informed consent was obtained from each participant.

Study Design

We recruited 208 patients and 100 controls over a 12 months period, from June 2014 to May

2015. 153 HIV-positive patients and 55 HIV-negative patients. All were assessed clinically,

chest x-ray and by Aspergillus IgG serology. Clinical examination including chest

auscultation and blood oxygen level testing. Where patients had a productive cough, sputum

was collected. Sputum was processed for microscopy and fungal culture. 5mls of blood was

obtained from all participants.

Chest X-rays were assessed by two independent consultant radiologists.

Data Collection

A questionnaire was used to collect basic sociodemographic data, clinical parameters and

medical history. Other pertinent data such as drug history, latest CD4 count (within last six

months) and HIV serology-status were obtained.

Laboratory processing

Serum transfered in dry ice to Mycology Reference Centre, Manchester. Aspergillus

fumigatus-specific IgG antibodies were measured using ThermoFisher Scientific

ImmunoCAP® system. An IgG of >40 mg/L was considered as elevated.

Definitions

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CPA was diagnosed based on the criteria proposed by Denning and colleagues.[2][20] It required

the presence of the following: 1 - underlying pulmonary disease, 2 - symptoms, 3 –

radiological findings and 4 - microbiological evidence of aspergillosis, A. fumigatus IgG

antibody positive or culture from sputum of a non-fumigatus species of Aspergillus in patients

with a fungal ball visible on the chest radiograph.

PTB was diagnosed if a patient had a positive sputum smear test for acid and alcohol fast

bacilli or a positive GeneXpert 2110 polymerase chain reaction test.

Anti-TB treatment failure TB was diagnosed in a patient who was sputum smear positive at 5

months after the initiation of anti-TB treatment or in persistently symptomatic patients in the

last one month of anti-TB therapy.

A patient is described as symptomatic if they have at least one of: haemoptysis, cough,

productive cough, and severe fatigue.

Radiological features indicative of CPA were at least one of: cavitation, fungal ball, pleural

thickening, or fibrosis.

An unspecified fungal ball is diagnosed in patients with an apparent aspergilloma on CXR,

but with normal Aspergillus IgG levels.

Data analysis

Analyses were performed using SPSS 22 and a 5% significance level was used. Summary

statistics were presented using frequencies and percentages for binary and categorical

variables, means and standard deviations for normally distributed, continuous variables, and

medians and interquartile ranges (IQR) for non-normally distributed, continuous variables.

Natural logarithm transformations have been used to perform analyses on the non-normally

distributed variables.

Chi-squared tests and Fisher’s exact tests were used to compare proportions between groups

and Student’s t-tests were used to compare means. Linear regression was used to assess the

relationship between continuous variables.

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Results

Of the 208 patients recruited into the study, 153 (73.6%) were HIV positive and 55 (26.4%)

were HIV negative. Ninety-five (95/141, 67.4%) were receiving anti-TB therapy for the first

time, while 46 (32.6%) were being retreated having been diagnosed as anti-TB treatment

failure. Thirty-nine (18.8%) were unable to work due to the severity of their illness. There

were 124 (59.6%) females, of whom 52 (41.9%) were 30-39 years old. The mean age of the

participants was 39.8 years (SD 12.3) (range 16-82). Ninety-nine (64.7%) of those HIV-

infected were 30-49 years old and 25 (16.3%) 10-29 years old. The median CD4 count was

212.5 (n=136, IQR 88.5-337.5) in the HIV-infected patients and 169.5 (n=8, range 4-593) in

the HIV-infected patients with positive Aspergillus IgG. One hundred and nine (52.4%) had

TB and 140 (67.3%) had productive cough. The median duration of cough amongst the

studied population was 3 months (n=120, IQR 1-8), a range of 1-96 months. Positive sputum

cultures for Aspergillus spp. were obtained in 28 (20%); A. fumigatus in 15, A. flavus 10 and

A. niger 3. Fifty patients had haemoptysis, nine with moderately severe frank blood

haemoptysis.

Serology results, CPA and risk factors

Seventeen (8.2%) patients had a positive Aspergillus IgG above the 40mg/L cut off. The

median titre was 6.2mg/L (IQR 3.7-14.3) with a range of <2 to 194mg/L. Both CD4+ and

Aspergillus IgG levels were positively skewed, log transformations were performed allowing

a linear regression to be used to determine their statistical relationship; it was not significant.

For 150 patients a smear/GeneXpert result was provided, the remaining 58 patients were

diagnosed radiologically. In the HIV positive subgroup, there was no significant difference in

the proportion of IgG positive patients between patients with confirmed (smear and/or

GeneXpert) TB and smear negative patients (7.4% (2/27) vs 9.6% (7/73); Fisher’s exact

p>0.99). Similarly, in the HIV negative subgroup, there was no significant difference in the

proportion of IgG positive patients with confirmed TB and smear negative patients (5.6%

(1/18) vs 18.8% (6/32); Fisher’s exact p=0.40) (table 1). However, there was a significant

difference in the geometric mean of Aspergillus IgG concentration between the HIV infected

and non-HIV infected patients (6.0 vs 10.6 mg/L; p=0.002). None of the controls had

Aspergillus IgG levels above the threshold.

Using the definition of CPA, 18/208 (8.7%) had CPA, including three patients with

Aspergillus IgG <40mg/L who had fungal balls on chest X-rays and grew A. niger (n=2) and

A. flavus (n=1) in their sputum. A greater proportion of HIV-negative patients had CPA (8/55,

14.5%) compared to HIV-positive patients (10/153, 6.5%); (Fisher’s exact p=0.092). In the

“treatment failure” group, 7 had CPA; of which only 1 was receiving antiTB therapy for the

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first time. In the HIV-positive subgroup, there was no significant difference (p=0.74) in the

CD4 count between those with CPA (n=9, geometric mean=162.5) and those without CPA

(n=127, geometric mean=182.4). A greater proportion of patients with CPA had chest pain

than those without CPA, though the difference was not significant (58.8% (10/17) vs 36.2%

(63/174); p=0.067).

Radiological findings

The proportions of different radiological features are, for the most part, similar for the whole

group and the HIV-positive subgroup (Figures 1-3). 79 (46.5%) patients without CPA had

normal CXR. 84.8% (67/79) of these patients were HIV positive and 52.2% had CD4 count

<250 cells/mm3; none had Aspergillus IgG above 40mg/L. All nine HIV positive patients with

Aspergillus IgG >40mg/L had radiological features suggestive of CPA and in six out of eight

of those with a documented CD4 count result, it was less than 250 cells/mm3.

Ten of 18 (55.6%) patients with CPA had cavitation compared to 17.1% (29/170) of the

patients without CPA (Table S1). For fungal ball, 77.8% (14/18) of those with CPA had it

compared to 4.7% (8/170) without CPA. Pleural thickening, 61.1% (11/18) of those with CPA

had it compared to 17.1% (29/170) without CPA. Fibrosis was seen in 12 of 18 (66.7%) of

those with CPA compared to 28.2% (48/170) without CPA. Of the patients with CPA, 90.0%

(9/10) had a fungal ball compared to 5.7% (7/123) of those without CPA; 60.0% (6/10) of the

CPA patients had pleural thickening compared to 14.6% (18/123) of those without CPA

(p=0.002, Fisher exact); and 80.0% (8/10) of the CPA patients had fibrosis compared to

26.0% (32/123) of those without CPA (p=0.001, Fisher exact) (figures S1-S4). No formal

statistical testing was done for these four radiological features since they were part of the

definition of CPA. There were significant differences between the CPA and non-CPA patients

in the proportion with: consolidation (66.7%, 12/18 vs 38.8%, 66/170; p=0.023), opacity

(61.1%, 11/18 vs 30.6%, 52/170; p=0.009), collapse (38.9%, 7/18 vs 8.8%, 15/170; p=0.002),

air crescent/Monod sign (22.2%, 4/18 vs 5.3%, 9/170; p=0.025), pleural effusion (22.2%,

4/18 vs 6.5%, 11/170; p=0.041), calcification (22.2%, 4/18 vs 4.7%, 8/170; p=0.018) and

hilar lymphadenopathy (27.8%, 5/18 vs. 8.2%, 14/170; p=0.023). These features were also

significant in the HIV positive subgroup (supplementary data).

Eight cases of an ‘unspecified’ fungal ball was diagnosed with apparent aspergilloma on

CXR, six of these patients had normal Aspergillus IgG levels and negative sputum cultures

but some symptoms and two patients had Aspergillus IgG levels above the cut-off and no

symptoms.

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Discussion

The African Region accounts for about four out of every five HIV-positive TB cases and TB

deaths among people who were HIV-positive. [21] Nigeria is rated fifth in the 22 high burden

countries of TB24 and CPA is a known complication of TB.[22] Even when treated, CPA has a

20–33% short-term mortality and a 50% mortality over 5 years.[22] The estimated prevalence

of CPA in TB patients was estimated to be 42.9 per 100,000 in Nigeria, but this estimate was

a extrapolation, that required confirmation.[22] Furthermore, there are no studies of CPA in

HIV positive patients. In this multicentre study, we confirmed the presence of CPA amongst

patients being managed for TB in Nigeria with an overall prevalence of 8.7%. However, this

overall figure obscures a proportion nearly twice as high in HIV-negative patients (14.5%),

compared to HIV-positive patients (6.5%). A report from Iran gave a prevalence of 13.7%,

very similar to our findings as they excluded HIV-infected TB patients, [23] and similar to data

(16.7%) from Japan.[24] A possible explanation for the marked difference between HIV and

non-HIV infected patients is that HIV patients might not be able to mount a sufficient

antibody response during Aspergillus infection, especially those with low CD4 counts (as

seen in this study).

In 2014 in Nigeria, pulmonary TB was reported in 100,000 patients with HIV infection and

470,000 without.[21] However, only 22,000 with HIV infection survived, compared to 300,000

who were HIV-infected. Applying the rates of CPA we found, we would anticipate an annual

incidence of ~1,430 HIV positive CPA cases and 43,500 HIV negative CPA cases. Assuming

a 15% annual mortality or lobectomy (cure) rate, we would anticipate a 5 year period

prevalence of 141,619 CPA cases, substantially greater than the first estimate of 60,383, [22]

but close to our more recent estimate of 120,753.[25] While the HIV patients were not selected

for any particular characteristic, we did explicitly study a number of smear-negative HIV

negative patients with continuing symptoms. Over 50% of patients met the criteria of anti-TB

treatment failure (67 were in their last month of treatment and were still symptomatic and 46

were being retreated for TB). Therefore, extrapolation to the whole HIV-negative population

may over-estimate numbers but conversely we omitted the two patients with probable simple

aspergillomas from this calculation.

We measured A. fumigatus antibodies because A. fumigatus is the most commonly implicated

Aspergillus spp. accounting for over 90% of aspergillosis in Europe. [26,27] However, a recent

report from South–Western Nigeria using PCR revealed that A. fumigatus only accounted for

57.1% of clinical and environmental isolates, followed by A. niger 28.6% and A. flavus 7.4%.[28] Earlier studies from Eastern Nigeria showed A. fumigatus 51.9%, A. niger 33.3%, A. flavus

14.8%,[29] and from Northern Nigeria, Kwanashie and colleagues found A. fumigatus

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accounting for 52.4%, A. flavus 21.9%, A. niger 11.4%.[30] A. fumigatus antibody assays may

have a poor sensitivity for infection with other Aspergillus species.[31] This might account for

the three patients (who ended up being classified with CPA) with Aspergillus IgG <40mg/L

but with positive sputum cultures for A. niger (n=2) and A. flavus (n=1). However culture has

low sensitivity also, so this is likely an underrepresentation of the true estimate of the disease

in an area where up to 40% of clinical Aspergillus species are non-fumigatus.

Conventional chest radiography is the imaging modality for initial evaluation of patients with

pulmonary complications in LMICs. However, it has limited sensitivity and specificity

compared to CT-scan especially in immunocompromised patients.[32] We found 43.8% of the

HIV-positive patients to have a normal chest X-ray, despite being managed for tuberculosis –

consistent with others’ data (20-50% of bacteriological confirmed cases). [7,33] Possibly all

abnormal features returned to normal or they were normal previously. Presently, chest X-ray

often cannot fully distinguish between pulmonary TB and CPA because cavities and fibrosis

are seen in both.[34,35] There are no published data documenting the post-therapy CT

appearances of pulmonary TB in HIV-positive patients, whereas in HIV-negative patients

cavitation has been seen in 21-23% of Africans.[22] There is a need for a predictive model that

will help to increase the index of suspicion of CPA amongst clinicians in LMICs.

We found 22 patients with fungal balls, including 14 (63.6%) who had CPA. A further eight

patients had unspecified fungal ball without either symptoms or positive Aspergillus IgG.

This could be due to over-reading of compound shadows in the lung apex, which would be

resolved with CT scanning. Haemoptysis is a known presentation of pulmonary aspergillomas

with associated morbidity and mortality. [19] Frank haemoptysis was observed in nine patients,

similar to the frequency found in another study amongst TB patients with aspergillomas. [19]

Haemoptysis from aspergillomas in Nigeria was associated with a high mortality (37.5%)

despite surgical intervention.[16] This life-threatening condition could be pre-empted by earlier

diagnosis.

Limitations of this study included the lack of follow up of the study population so serial chest

X-rays were not done to document progression or later development of disease. Also only one

centre was performing geneXpert testing at the time of the study and we did not recruit

patients on second line anti-TB therapy that were being managed for multidrug resistant TB

to exclude comorbidity.

Acknowledgements:

Rebecca Cottage, Department of Immunology, Manchester Royal Infirmary.

Authors’ contribution

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ROO, DWD, NKI, ASA and MDR were involved in 1) substantial contributions to

conception and design, acquisition of data, 2) drafting the article or revising it critically for

important intellectual content; and 3) final approval of the version to be published.

PF; 1) analysis and interpretation of data; 2) drafting the article or revising it critically for

important intellectual content; and 2) final approval of the version to be published.

FTO, AN, TG, AHE were involved in; 1) acquisition of data, 2) drafting the article or revising

it critically for important intellectual content; and 3) final approval of the version to be

published.

Potential conflict of interest:

Dr Denning and family hold Founder shares in F2G Ltd, a University of Manchester spin-out

antifungal discovery company, in Novocyt which markets the Myconostica real-time

molecular assays. He acts or has recently acted as a consultant to Astellas, Sigma Tau,

Basilea, Scynexis, Cidara, Biosergen, Quintilles, Pulmatrix and Pulmocide. In the last 3 years,

he has been paid for talks on behalf of Astellas, Dynamiker, Gilead, Merck and Pfizer. He is a

longstanding member of the Infectious Disease Society of America Aspergillosis Guidelines

group, the European Society for Clinical Microbiology and Infectious Diseases Aspergillosis

Guidelines group and the British Society for Medical Mycology Standards of Care committee.

Dr Oladele In the last 3 years, he has been paid for talks on behalf of AstraZeneca and Pfizer

Dr Richardson acts a consultant for Gilead Science eEurope, Astellas, MSD, Pfizer and

Basilea. He is a member of the European Society for Clinical Microbiology and Infectious

Diseases Aspergillosis Guidelines writing groups.

Dr Irufhe, Mr Foden, Dr Aakanmu, Dr Gbaja-Biamila, Dr Nwosa, Dr Ekundayo and Dr

Ogunsola have nothing to disclose.

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439440441

442

443

444

445

446

447

448

449

Table1: Relationship between smear status and clinical features in HIV positive and negative

patients separately

HIV positive (153)

p-

value

HIV negative (55)

p-

valueSmear/GeneXpert

With data: 100 With data: 50

Positive

27

Negative

73

Positive

18

Negative

32

Aspergillus IgG

positive2 (7%) 7 (10%) >0.99 1 (6%) 6 (19%) 0.40

Aspergillus IgG

titre

geometric mean

(range)

6.0

(1.1-135.0)

6.8

(1.0-194.0)0.66

9.3

(1.0-159.0)

11.9

(1.0-186.0)0.52

Age

mean (sd)40.6 (11.6) 39.6 (10.4) 0.70 42.7 (17.6) 42.5 (13.8) 0.98

Symptoms

Marked

haemoptysis0 (0%) 1/70 (1%) >0.99 2 (11%) 5/23 (22%) 0.44

Fatigue 11 (41%)33/72

(46%)0.65 12 (67%)

11/20

(55%)0.46

Productive cough 12 (44%)48/72

(67%)0.044 16 (89%)

19/25

(76%)0.43

Chest pain 8/26 (31%) 20 (27%) 0.74 7 (39%)15/20

(75%)0.024

450

451

452

453

454

455

456

457

Figure 1: A graph showing the percentage with radiological features in IgG positive and negative patients.

458

459

460

461

462

463

464465

466

467

468

CPAWithout CPAWith CPA

Percentage

100

90

80

70

60

50

40

30

20

10

0

CollapseOpacityConsolidationFibrosisPleural thickeningFungal ballCavitation

Radiological features

469

470

471

472

473

474

475

476

477

478

479

480

481

482

483

484

485

486

Figure 2: A graph showing the percentage with radiological features in those with and

without CPA, including only HIV positive patients.

CPAWithout CPAWith CPA

Percentage

100

90

80

70

60

50

40

30

20

10

0

CollapseOpacityConsolidationFibrosisPleural thickeningFungal ballCavitation

Radiological features

487

488489

490

491

Figure 3: A graph showing the percentage with radiological features in those with and

without CPA, including only HIV negative patients.

CPAWithout CPAWith CPA

Percentage

100

90

80

70

60

50

40

30

20

10

0

CollapseOpacityConsolidationFibrosisPleural thickeningFungal ballCavitation

Radiological features

492

493

494

495

496

497

498