1

Added value of ELISPOT PPD in patients with BCG-infection after intravesical BCG 1

instillations. 2

Karen A. Heemstra1, 2, Ailko W.J. Bossink3, Roan Spermon4, John J. M. Bouwman1, Robert van 3

der Kieft1, Steven F. T. Thijsen1 4

1Diakonessenhuis Utrecht, Medical Microbiology and Immunology, Utrecht, The Netherlands 5

2 University Medical Center Utrecht, Medical Microbiology, Utrecht, The Netherlands 6

3 Diakonessenhuis Utrecht, Pulmonology, Utrecht, The Netherlands 7

4 Diakonessenhuis Utrecht, Urology, Utrecht, The Netherlands 8

9

Short title: ELISPOT PPD in disseminated BCG infection 10

Word count: 50 (abstract) + 3025 11

12

13

14

15

Corresponding author: 16

Karen A. Heemstra 17

Diakonessenhuis, Department of Microbiology & Immunology 18

Bosboomstraat 1, 3582 KE Utrecht, Netherlands 19

Phone:+31 88 2505000 20

Fax: +31 302566695 21

K.A.Heemstra@umcutrecht.nl 22

23

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.05597-11 CVI Accepts, published online ahead of print on 29 March 2012

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Abstract 24

In this case series, we describe four cases in which the use of interferon-γ release assays with 25

purified protein derivate (PPD) as stimulating antigen was able to demonstrate PPD-specific 26

immune activation. This may help to improve the adequate diagnosis of (systemic) BCG 27

infections after intravesical BCG instillations for bladder carcinoma. 28

29

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Introduction 31

Worldwide hundred thousands of people are treated with intravesical Bacillus Calmette Guérin 32

(BCG) instillations for bladder carcinoma each year (1). Less than 5 percent of these patients 33

develop severe complications of this therapy, including BCG pneumonitis and systemic infection 34

(16). One of 15.000 patients develops a septic reaction and needs to be treated with 35

antimycobacterial antibiotics. (15). Unfortunately, it is cumbersome to confirm the diagnosis of 36

BCG pneumonitis or sepsis, because cultures often remain negative and if positive it may take up 37

to 12 weeks until mycobacteria are grown. 38

This hampers to distinguish between (systemic) BCG infections and other causes of infection or 39

other complications of BCG instillations. Hence, in the case of a systemic BCG infection rapid 40

and accurate diagnosis is important because BCG instillations should be ceased and treatment 41

with antimycobacterial medication should be initiated generally for a long period (3-6 months) 42

(14). 43

We hypothesized that in the case of a (systemic) BCG infection as complication after BCG 44

instillations a specific immune response will be initiated, which could be detected using purified 45

protein derivate (PPD) as an antigen. Tuberculin-skin test (TST) detects a cell mediated immune 46

response in the form of a delayed hypersensitivity reaction to PPD (18, 22). This test can be used 47

for diagnosing Mycobacterium tuberculosis infections and has cross-reactivity with other 48

mycobacterial strains, including the BCG strain and non-tuberculous strains. However TST has 49

some limitations, such as interobserver variations, the need to recall people for test-reading, low 50

specificity in people who are vaccinated with BCG and false negative results in patients with 51

immunosuppression (5, 9, 13, 20). 52

A newer immunological method, the interferon-γ release assay (IGRA) overcomes these 53

limitations. Using this method, T cells of patients sensitized to mycobacterial antigens will 54

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produce interferon-γ when stimulated by mycobacterial antigens (18). Two commercial variants 55

of this test exist; QuantiFERON is an enzyme-linked immunosorbent based assay (ELISA) which 56

measures interferon-γ production in patients’ plasma and T-SPOT.TB is an enzyme linked 57

immunospot assay (ELISPOT) which enumerates the number of interferon-γ producing T cells. 58

Initially, both methods used PPD as stimulating antigen. To avoid the high degree of antigenic 59

cross-reactivity of PPD’s from different mycobacterial species, including the BCG strain and 60

non-tuberculous mycobacterial -strains (12), IGRA’s nowadays use Mycobacterium tuberculosis 61

specific antigens, such as recombinant early secretory antigenic target-6 (ESAT-6) and 62

recombinant culture filtrate protein-10 (CFP-10). IGRA’s for Mycobacterium tuberculosis have a 63

good sensitivity in immunocompromised patients, such as HIV patients (6, 17). Jaffari et al. have 64

shown that IGRA’s could also be used on specimens from the site of infection, such as cells 65

obtained from bronchoalveolar lavage (BAL) (10). 66

We developed an in-house IGRA using PPD as stimulating antigen and hypothesized that this 67

assay can be useful for rapid detection of BCG infections resulting from intravesical BCG 68

therapy. In this case series, we describe 4 cases in which the use of an IGRA with PPD was able 69

to detect PPD-specific immune activation which could be of value for adequately detecting 70

infections due to BCG. 71

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Case 1 73

In May 2010, a 60 year old man was diagnosed with pTaG3 urothelial cell carcinoma of the 74

bladder. He was treated with a trans-urethral resection of the bladder tumour. Hereafter he started 75

with intravesical BCG instillation maintenance therapy. After ten BCG instillations, he presented 76

with chills, fever of 40°C, and vomiting on the emergency department. The patient had no other 77

localizing symptoms. He had a CRP of 104 mg/L and leukocytes of 8.6*109/L. Blood cultures 78

and urine were sampled. Hereafter the patient started with intravenous ciprofloxacin therapy. 79

Blood- and urine cultures stayed negative. After switch to oral ciprofloxacin therapy the patient 80

developed fever again. CT-thorax showed fine nodular lesions with an infiltrate in the lower left 81

lobe, which could fit miliary tuberculosis, sarcoidosis or diffuse metastases. There were no 82

enlarged lymph nodes visible. A BAL was performed. BAL showed negative auramine staining, 83

no pathogenic microbes (including mycobacterial cultures), and a negative PCR on 84

Mycobacterium Tuberculosis complex. ELISPOT TB of peripheral blood mononuclear cells 85

(PBMCs) was negative, while ELISPOT PPD was marginally reactive with 8 spots. ELISPOT 86

PPD on the BAL was strongly reactive (>100 spots), while ELISPOT TB was negative (Figure 87

1). The patient was suspected for systemic BCG infection and treated with ethambutol, isoniazid 88

and rifampicin for 6 months after which the patient recovered. A chest CT 6 months after 89

cessation of the medication showed a decreased number of nodules and disappearance of the 90

infiltrate in the lower left lobe compared with the first chest CT. 91

92

Case 2 93

A 82 year old male patient was treated for high grade non-muscle invasive bladder carcinoma 94

with intravesical BCG instillations. The patient had a trans-urethral resection of the bladder 95

tumour in March 2008. In April 2008 he started with intravesical BCG instillations. After 4 96

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intravesical BCG instillations, he presented with deterioration, fever of 40°C, chills, fatigue and 97

diminished appetite. The patient had a CRP of 173 mg/L, leukocytes of 8.9*109/L and an 98

erythrocyte sedimentation rate of 56 mm/h. CT of the thorax showed some aspecific nodular 99

lesions centrilobular and in the upper left lobe. Urine culture showed an Eschericha coli for 100

which the patient was treated with ceftriaxone. Auramine staining was negative and 101

mycobacterial culture of the urine remained negative. Blood cultures stayed negative as well. 102

ELISPOT TB and ELISPOT PPD of PBMCs were also negative. On May 19th, 2008 CT of the 103

abdomen showed an abscess ventrally of the bladder and prostate, which was drained. 104

Mycobacterium tuberculosis complex PCR of the abscess was positive and Mycobacterium bovis, 105

BCG type Pasteur was cultured after 12 days. The antibiotic susceptibility pattern showed 106

sensitivity to ethambutol, streptomycin, isoniazid and rifampicin and resistance to pyrazinamid. 107

On June 3th 2008, ELISPOT TB of PBMCs remained negative, while ELISPOT PPD was 108

reactive with 7 spots. In July 2008, ELISPOT TB on blood still remained negative, while 109

ELISPOT PPD rose further to 80 spots. This finding in combination with the systemic symptoms 110

made us to conclude that the patient suffered from a systemic BCG infection and was treated with 111

with ethambutol, isoniazid and rifampicin for 9 months and he recovered. 112

113

Case 3 114

A 74 year old male was treated for intermediate grade non-muscle invasive bladder carcinoma 115

with intravesical BCG instillations. In November 2005, the patient was treated with a trans-116

urethral resection of the bladder tumour. In December 2006, he was treated with a second trans-117

urethral resection of the bladder tumour and intravesical mitomycin instillitations for recurrence 118

of disease. In September 2007, he was treated with a trans-urethral resection of the bladder 119

tumour once more for recurrence, after which he started with intravesical BCG instillations. In 120

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November 2007, one day after the second BCG instillation, the patient developed fever, nausea, 121

vomiting and erytrocyturia. The general practitioner treated him with 122

trimethoprim/sulfamethoxazole suspecting cystitis. Because the patient did not recover, 123

antibiotics were switched to ciprofloxacin. Twelve days after onset of the symptoms the patient 124

started coughing and became dyspnoeic. At the same time, he developed night sweats. The 125

patient was referred to the pulmonary outpatient clinic on suspicion of a systemic BCG infection. 126

On physical examination normal breath sounds with diffuse crackles were heard. The patient had 127

a CRP of 84 mg/L, leucocytes of 4.6*109/L and BSE of 40 mm/h. Chest X-ray showed fine 128

disseminated nodules in all lung fields with hilar and mediastinal lymphadenopathy. Urine, BAL, 129

bone marrow aspiration, a lung biopsy and blood cultures were sampled. Auramine staining of 130

urine, BAL, and lung biopsy were negative. Mycobacterium tuberculosis complex PCR and –131

culture of the urine, BAL, lung biopsy and bone marrow were also negative. No bacteria were 132

cultured from the lung biopsy or urine. Cultivation of the BAL showed haemolytic Streptococci 133

group G and Candida albicans. Blood cultures remained negative. In blood ELISPOT TB was 134

negative and ELISPOT PPD was marginally reactive with 4 spots. In this patient no ELISPOT 135

analysis on BAL fluid was performed. Because the patient was suspected for disseminated BCG 136

sepsis he was treated with ethambutol, isoniazid and rifampicin and he responded well on this. In 137

March 2008, ELISPOT TB of PBMCs was still negative, while ELISPOT PPD of PBMCs rose to 138

more than 100 spots. The patients had no clinical sign at that moment and medication was 139

stopped. In December 2008, ELISPOT PPD of PBMCs decreased to 25 spots, while ELISPOT 140

TB was still negative. A CT of the chest showed a decreased number and size of the nodules with 141

no pathologic lymphadenopathy. 142

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Case 4 145

A 74 year old male was treated adjuvant with intravesical BCG instillations after transurethral 146

resection of a high grade non-muscle invasive bladder cancer In September 2007, the patient had 147

a trans-urethral resection of the bladder tumour after which he started with intravesical BCG 148

instillations. After the tenth BCG instillation the patient developed dyspnoea, night sweats, chills 149

and fatigue. Physical examination was unremarkable. A chest CT showed small nodules in all 150

lungfields comparable with miliary tuberculosis. The patient had a CRP of 11 mg/L and 151

leukocytes of 5.8*109/L. Auramine staining of urine, BAL and bone marrow were negative. 152

Mycobacterium tuberculosis complex PCR of the bone marrow and BAL were negative. Culture 153

of urine, bone marrow and BAL were negative. ELISPOT TB of BAL was negative, while 154

ELISPOT PPD on the same BAL fluid showed more than 100 spots. ELISPOT TB on blood was 155

negative, while ELISPOT PPD was marginally reactive with 6 spots. The patient was treated for 156

a suspected BCG-infection with isoniazid, rifampicin, ethambutol. After 3 months of treatment 157

the symptoms were resolved and the chest CT was almost normalized. ELISPOT PPD of PBMCs 158

showed 1 spot, whereas ELISPOT TB was negative. After 6 months the medication was stopped 159

and ELISPOT PPD of PBMCs was now 12 spots, while ELISPOT TB remained negative. A 160

chest CT 3 months after cessation of the medication showed a decreased number of nodules 161

compared with the first chest CT. ELISPOT PPD on PBMCs revealed 10 spots, whereas 162

ELISPOT TB remained negative. 163

To date, all four cases do not have any signs of recurrence of infection. 164

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Discussion 166

In this report, we describe four cases with systemic BCG infections after intravesical BCG 167

instillations for bladder carcinoma. All patients were treated for systemic BCG infections and 168

survived, only in 1 case culture turned positive for BCG. This case series shows that IGRA’s 169

using PPD as stimulating antigen are able to detect immune activation both in blood and BAL 170

fluid which could be valuable in diagnosing infections with BCG strains in patients treated with 171

intravesical BCG instillations. 172

Yearly 357.000 patients are diagnosed with bladder carcinoma worldwide (19), of which 70-75% 173

are non-muscle-invasive (25). Intravesical BCG instillations are recommended as adjuvant 174

therapy for patients with intermediate-risk and high-risk non-muscle-invasive bladder carcinoma 175

(1). After instillation, BCG induces a complex immune response in the bladder with an increase 176

in cytokines and chemokines. Repeated instillations lead to a local type 1 cellular immune 177

response, which lasts for 6 months. The exact antitumour mechanism is not known (25). The 178

success of BCG immunotherapy relies on the intravesical administration of live BCG strains and 179

the generation of a localised immune response in the bladder. Since BCG contains viable bacteria 180

it has the potential to produce (systemic) adverse events. Major adverse reactions, such as 181

pneumonitis, prostatitis or sepsis occur in less then 5% of patients with intravesical BCG 182

instillations (16). Therefore, all patients with fever after intravesical BCG instillations are 183

suspected for BCG sepsis. BCG sepsis might be the result of systemic absorbtion (26) which may 184

induce a hypersensitivity reaction in which elevated levels of cytokines are released in the 185

bloodstream (16). Historically, poor technique of intravesical instillations and non-recognition of 186

BCG related adverse events have led to serious morbidity and, in some cases, to mortality. Risk 187

factors for BCG sepsis include traumatic catheterisation and absorption through the inflamed 188

bladder wall in patients in which BCG was given soon after the trans-urethral resection of the 189

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bladder tumour (14, 25). Cultures generally stay negative and treatment is started based on 190

clinical suspicion (14). Patients with a BCG infection are treated for 3-6 months with 191

antimycobacterial medication and intravesical BCG instillations are ceased until the patient is 192

recovered. To distinguish between BCG infection and other causes of infection or other 193

complications of intravesical BCG instillation, IGRA’s using PPD as stimulating antigen could 194

be valuable in diagnosing disseminated BCG infection. Both ESAT-6 and CFP-10 are not 195

presented by any commercially used BCG strain, so IGRA’s using ESAT-6 or CFP-10 are not 196

able to detect BCG sepsis (7). Therefore IGRA’s using PPD should be used to diagnose 197

disseminated BCG infection. 198

Silverman et al. studied the value of TST and IGRA’s using ESAT-6 and CFP-10 as antigens in 199

patients receiving intravesical BCG instillations for bladder carcinoma who were exposed to 200

tuberculosis. They concluded that these patients should be assessed for latent tuberculosis 201

infection with IGRA’s rather than with TST (21). To our knowledge no other studies have been 202

performed using IGRA’s in patients treated with intravesical BCG instillations for bladder 203

carcinoma. 204

In case 1 and 4, ELISPOT PPD in BAL fluid is strongly reactive, while ELISPOT PPD in blood 205

is weakly reactive. During active infection T cells are clonally increased and recruited to the site 206

of infection (2, 8). In patients with active Mycobacterium tuberculosis infection some studies 207

have been performed on this issue. Wilkinson et al. showed a much higher concentration of 208

ESAT-6 specific T-cells in pleural effusions compared to peripheral blood in patients with pleural 209

tuberculosis (24). Jafari et al. showed that, in sputum acid fast bacilli smear–negative 210

tuberculosis, IGRA’s on BAL mononuclear cells are superior to IGRA’s on peripheral blood 211

mononuclear cells in diagnosing pulmonary tuberculosis (11). Thus performing IGRA’s on cells 212

obtained at the actual site of infection could be more sensitive than on peripheral blood (10), 213

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which could explain the discrepant ELISPOT PPD results between the BAL and the peripheral 214

blood. where the reactive T cells have been recruited to the site of infection and are therefore not 215

available in circulating blood 216

Figure 1 shows the results of the ELISPOT of case 1. The negative control of the BAL shows 217

more background signals probably due to impurity of the material and the presence of 218

macrophages in the BAL. These background signals are on the same level as the samples 219

stimulated with the M. tuberculosis specific antigens ESAT-6 and CFP-10. PPD stimulated cells 220

give rise to a comparable signal as the positive control indicating the presence of large amounts 221

of PPD specific T-cells. In the lower figure it seems that the positive control of the blood is not 222

strongly positive. However the wells are measured by an automatic reader, which is more 223

objective way to enumerate the activated T cells and gave a clear positive result. Cells stimulated 224

with M. tuberculosis specific antigens ESAT-6 and CFP-10 give no signal whereas stimulation 225

with PPD antigen give rise to clearly visible spots indicating the presence op PPD specific T-cells 226

in peripheral blood although at a much lower level as compared to the BAL . 227

As direct specimens are sometimes difficult to obtain and therefore not always available, it is 228

useful to repeat ELISPOT PPD on PBMCs as they can become more reactive over time. In case 3 229

the initial ELISPOT PPD on PBMCs is negative, while ELISPOT PPD on blood became reactive 230

a few months after the start of the BCG sepsis. In case 2 ELISPOT PPD is more reactive one 231

month after start of the infection. This could be explained by the fact that during active infection 232

T cells are clonally increased and recruited to the site of infection. Consequently ELISPOT of a 233

direct specimen is positive early in the infection, while ELISPOT of PBMCs becomes positive 234

later during the infection. 235

Patients who are BCG vaccinated or are exposed to non-tuberculous mycobacteria (NTM) are at 236

risk for false positive results in the ELISPOT PPD (4, 5, 23). Brock et al. showed that 47% of 237

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patients who are BCG vaccinated respond to PPD, while 10% respond to the Mycobacterium 238

tuberculosis specific antigens ESAT-6 and CFP-10 (5). However, in the Netherlands BCG 239

vaccination is rare and not commonly used. In addition, the prevalence of NTM in the 240

Netherlands is low. Not much is known about the specificity of ELISPOT PPD in this group of 241

patients. In a non-published study of our department 70 % and 86.4 % of 103 patients with 242

interstitial lung diseases had less then 4 spots or less then 14 spots on ELISPOT PPD of PBMCs 243

respectively (Thijsen et al. non-published data). 244

For intradermal PPD, reactivity is expected 12 weeks after exposure to BCG or mycobacteria. 245

However, this timeframe is arbitrarily and conservative; in other words after 12 weeks most 246

exposed are responsive to intradermal PPD. Nevertheless, a local immune response to PPD can 247

be expected earlier. Patients who are treated with intravesical BCG instillations receive much 248

higher doses antigen (i.e.BCG) as compared to intradermal BCG vaccinates. In BCG vaccination 249

0.8-1.2*106 CFU Mycobacterium bovis is given percutaneously, while in intravesical BCG 250

instillations 2-8*108 CFU Mycobacterium bovis is given. Bilen et al. showed that 68% of patients 251

became positive for intradermal PPD reactivity one week after they were treated for the first time 252

with intravesical BCG instillations (3). So intravesical BCG instillations may provoke a T-cell 253

mediated immune response to PPD which can be expected earlier than the 3 months period for 254

TST which is used in TB contact tracing. 255

Our cases were treated for 4-9 months with antimycobacterial medication. Pyrazinamid was not 256

given, because Mycobacterium bovis is usually resistant for pyrazimid. This is confirmed in case 257

2 in which Mycobacterium bovis was cultured. In our department treatment for 9 months is given 258

to patients with a positive culture for Mycobacterium bovis. The patients in whom there was no 259

positive culture were treated with with antimycobacterial medication until clinical recovery. 260

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In conclusion, this case series shows that IGRA’s using PPD as stimulating antigen are 261

potentially valuable in diagnosing infection with BCG especially when fluids of affected organs 262

such as BAL fluids can be tested. BCG instillations are widely used in the treatment for non-263

muscle-invasive bladder carcinoma IGRA’s using PPD as stimulating antigen are a promising 264

new tool in diagnosing and/or monitoring complications of this treatment and should be evaluated 265

in greater series. 266

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