Statistical analysis:



ONLINE REPOSITORY/ DATA SUPPLEMENT

Evaluable quantitative pulmonary T cell responses are useful for the rapid immuno-detection of pulmonary tuberculosis

Keertan Dheda, Richard van Zyl-Smit, Richard Meldau, Surita Meldau, Greg Symons, Hoosain Khalfey, Neville Govender, Andrew Whitelaw, Helen Wainwright, Motasim Badri, Rodney Dawson, Eric Bateman, Alimuddin Zumla

&Lung Infection and Immunity Unit & CTBRI, UCT Lung Institute & Division of Pulmonology, Department of Medicine, University of Cape Town, South Africa

*Centre for Infectious Diseases and International Health, University College Medical School, London;

†Department of Biomedical Sciences, University of Sassari, Sardinia, Italy.

Running title: Rapid immunodiagnosis of tuberculosis

Corresponding author: Keertan Dheda, Lung Infection and Immunity Unit,

Division of Pulmonology, Department of Medicine, University of Cape Town.

Current address: Department of Medicine. J flr. Old Main Bldg. Groote Schuur Hospital.

Observatory, Cape Town, South Africa, 7925.

Tel: +27 21 4066509 , Fax: +27 21 4486815, email: keertan.dheda@uct.ac.za

This work was supported by the South African National Research Foundation (SARChI), the South African Medical Research Council and the UCL-UCT Collaboration Initiative.

METHODS

Alveolar lymphocyte antigen-specific IFN-( responses

The following alveolar antigen-specific IFN-( responses were evaluated:

(i) RD-1 ELISPOT IFN-γ responses to ESAT-6/CFP-10 peptide pools (T-SPOT.®TB; Oxford Immunotec, England) were performed according to manufacturer’s instructions using 2x105 BALMCs per well (cell number selected by performing optimization experiments in 5 patients; see online data supplement for details). BAL fluid was filtered through sterile gauze and washed twice in RPMI.

(ii) RD-1 ELISA-measured IFN-( responses to ESAT-6/ CFP-10 and TB 7.7 antigens (QFT-TB-GIT assay; Cellestis, Victoria) were performed using 1x106 BALMCs re-suspended in 1ml of serum free medium in each tube. The cell number and volume used were derived through optimization experiments using BALMCs from 10 patients (see online data supplement).

(iii) PPD (10(g/ml; Staten Serum Institute, Denmark) IFN-( ELISPOT responses were performed in duplicate using IFN-( pre-coated ELISPOT plates (Mabtech, Sweden) seeded with 2x105 BALMCs per well.

(iv) IFN-( ELISPOT responses to HBHA (concentration of 100(g/ml derived from optimization experiments; see online supplement) were performed in duplicate using IFN-( pre-coated ELISPOT plates. Methylated HBHA antigen was produced in cultures of Mycobacterium smegmatis 3.38 as previously described [19-21].

All antigen-specific ELISPOT assays were enumerated using an ELISPOT reader and using 2x105 BALMCs per well. Results were deemed to be inconclusive if there were insufficient cells to perform the assay, if the positive control failed (indeterminate), there was a high spot count/ level in the negative control well/ tube, or, if there was high background discoloration of the ELISPOT wells precluding meaningful evaluation of the plate. Given the significant frequency of indeterminate results, in the second half of the study, in addition to phytohaemagglutinin (PHA), staphylococcal enterotoxin B (SEB) was used as a positive control.

Peripheral blood-derived antigen-specific IFN-( responses

The following peripheral blood IFN-( responses were evaluated in each patient according to the methods already described:

(i) RD-1 ELISPOT responses using 2.5x105 peripheral blood MCs/ well (T-SPOT.®TB).

(ii) RD-1 ELISA using 1ml of whole blood per tube (QFT-GIT).

(iii) PPD ELISPOT responses using 2.5x105 peripheral blood MCs/ well.

(iv) HBHA ELISPOT responses using 2.5x105 peripheral blood MCs/ well.

Optimisation data

(i) RD-1 ELISPOT assay (T-SPOT.®TB): serial data were obtained for 8 patients at different cell concentrations per well (2.5 x 104, 5 x 104, 1 x 105, 2 x 105, 4 x 105). At the time of sample acquisition the disease status of the participants was unknown. Three patients had positive result (> 6 SFU/ 106 BALMCs in the antigen-containing wells). In these three subjects serial spot counts are shown in Table E1. In summary, responses (cells/ well) were absent at 5 x 104, were recordable at 1 x 105 and increased at 2 x 105 cells/well. Responses were not significantly better at 4 x 105 cells/well.

(ii) RD-1 ELISA assay (QFT-GIT): data obtained from 13 participants at different cell counts are summarized in Figure E1. At the time of sample acquisition the disease status of the participants was unknown and three of the assays were inconclusive. Changing the volume of supernatant did not impact on the result (figure 2E; n=4). Thus, 1 x 106 cells per tube suspended in 1000ul of serum free medium was used (Figure E2).

(iii) HBHA ELISPOT assay: the antigen concentration of M. smegmatis-derived HBHA is unknown. Because of limited cell numbers HBHA optimization data were derived using PBMCs (Figure E3). Thus, 100 (g/ml of HBHA per well was used.

Statistical analysis:

Categorical variables were compared using the χ2 test or Fisher exact test and continuous variables were compared using t-student test, whenever appropriate. Non-parametric tests (Mann-Whitney) were used for non-normally distributed variables. Concordance between tests was measured using the kappa co-efficient. Diagnostic accuracy, including 95% confidence intervals, was assessed using sensitivity, specificity, predictive values and area under the ROC in the TB and non-TB sub-groups; comparative analysis was also done by combining the definite and probable TB groups. The study satisfies the Standards for Reporting of Diagnostic Accuracy (STARD initiative) checklist (19).

RESULTS

Causes of pulmonary infiltrates in the non-TB group

The most common causes of pulmonary infiltrates in the non-TB group were: 9 lung primary or metastatic carcinoma; 18 bacterial, pneumocystis or fungal pneumonia; 6 idiopathic interstitial pneumonias including non-specific interstitial pneumonitis, respiratory bronchiolitis interstitial lung disease, and usual interstitial pneumonitis; 3 sarcoidosis; 2 lymphocytic interstitial pneumonitis and several others with a variety of different pathologies.

Positive controls used in RD-1-driven ELISPOT assay

In 28 ELISPOT assays paired positive controls were used (PHA and SEB); of these significantly more PHA-driven than SEB-driven positive controls failed [1/28 (3%) vs. 7/28 (25%); p= 0. 02; Chi square; Table E2).

RD1-driven ELISA and ELISPOT assays

Performance outcome values for BAL and blood when the definite and probable TB groups were combined are shown in Table E3.

BALMC/ PBMC IFN- γ ratio

In an attempt to improve the discriminatory value of the ELISPOT assay we compared the BALMC/ PBMC IFN- γ ratio in the TB and non-TB groups. Whilst this ratio had a high diagnostic accuracy (AUC = 0.87 at a cut-point of > 0.092; 93% sensitivity and 71% specificity; Figure E4) it was no better that the BAL ELISPOT alone.

HIV effect on RD-1 responses

T-SPOT.®TB and QFT-GIT in the definite, definite + probable TB, and non-TB groups stratified by HIV status is shown in Figure E5.

PPD-driven ELISPOT assays

Performance outcome values for BAL and blood when the definite and probable TB groups were combined are shown in Table E4.

HBHA-driven ELISA and ELISPOT assays

Performance outcome values for BAL and blood when the definite and probable TB groups were combined are shown in Table E4.

Inconclusive results for RD1-driven ELISA and ELISPOT assays

Inconclusive results occurred in many patients for several reasons (failure of positive or negative controls, inadequate cells etc.) Details of the reason for inconclusive results are contained in Table E8.

Table E1. Serial responses at different BALMC counts in 3 out of 6 participants that had a positive result (> 6 SFCs; unadjusted spot count)

| |2.5 x 104 |5 x 104 |1 x 105 |2 x 105 |4 x 105 |

|Patient 1 |0 |0 |6 |25 |34 |

|Patient 2 |0 |5 |10 |22 |30 |

|Patient 3 |ND |50 |100 |120 |200 |

Table E2. Concordance between phytohaemaglutinnin and staphylococcal enterotoxin-B-driven positive controls in 35 paired RD-1 ELISPOT assays

| |PHA positive |PHA negative |PHA indeterminate |TOTAL |

|SEB positive |21 |6 |6 |33 |

|SEB negative |0 |1 |0 |1 |

|SEB indeterminate |0 |0 |1 |1 |

|TOTAL |21 |7 |7 |35 |

Table E3. Performance outcomes (95% CI) of IFN-( release assays (T-SPOT®.TB, QFT-TB GIT) using alveolar lavage cells and whole blood, at different cut-points, in pulmonary TB suspects [definite TB + probable TB were combined and compared to the non-TB group for the analysis; 55 and 46 T-SPOT®.TB (SFCs per 106 alveolar mononuclear cells) and QFT-TB GIT (iu/ l) assays were performed, respectively].

| |Cut-point |Sens |

|T-SPOT.®TB |≥ 30 SFC* |65% |

|(combined RD-1 antigen) | |(45;81) |

| | | |

| |≥ 50 SFC** |61% (41;78) |

| | | |

| | |78% (58;90) |

| |≥ 20 SFC# | |

|T-SPOT.®TB |> 30 SFC* |83% (65;92) |45% (31;60) |51% (37;65) |79% (60;91) |

| | | | | | |

|BAL T-SPOT.®TB (highest spot |Positive (43) |Positive |Indeterminate (n/a) |Negative |Negative |

|count)* | |(10) | |(0) |(1) |

|BAL QFT GIT |Negative (0.00) |Indeterminate (n/a) |Positive (0.98) |Positive (4.09) |Positive (16.29) |

|(IFN-( iu/ml) | | | | | |

|Alternative diagnosis |Carcinoma+ |Pneumonia |LIP* |Carcinoma+ |LIP* |

|Method of establishing diagnosis|Cytology |Culture & histology |Histology |Cytology |Histology |

|Age |64 |22 |35 |51 |32 |

|HIV status |Unknown# |Negative |Positive |Unknown# |Positive |

|Previous TB |No |Unknown |Yes |Unknown |yes |

|Blood T-SPOT®.TB |Negative |Negative |Positive |Positive |Negative |

|Blood QFT |Indeterminate |Positive |Indeterminate |Positive |Indeterminate |

|BAL bacterial and fungal culture|Negative |Klebsiella |Negative |Proteus |Candida |

|result | | | | | |

+ Small cell carcinoma, * Lymphocytic interstitial pneumonitis, # Subject refused testing.

In all patients mycobacterial cultures were negative and follow-up did not reveal any evidence of clinical tuberculosis. None of the patients received anti-TB treatment.

*Actual values of the test results are given below the categorical result in rows 1 and 2.

Table E5. Clinical details and laboratory results of all definite and probable TB patients with a negative BAL T-SPOT®.TB or QFT-GIT assay result.

|Subject |BAL QFT |BAL TSPOT |Final Diagnosis|age |HIV status |Blood QFT |Blood TSPOT |

|Antigen-specific IGRAs using alveolar lavage cells | | |

|HBHA |≥ 3 SFC* |50% (27;73) |56% (33;77) |50% (27;73) |56% (33;77) |53% (36;70) |0.57 |

| | | | | | | | |

| | |21% (8;48) |94% (72;99) |75% (30;95) |58% (39;75) |60% (42;75) | |

| |≥ 68 SFC** | | | | | | |

|PPD |≥ 2 SFC* |56% (34;75) |56% (39;72) |42% (25;62) |69% (50;84) |56% (42;69) |0.60 |

| | | | | | | | |

| | |33% (16;56) |94% (80;98) |75% (41;93) |71% (56;83) |72% (58;83) | |

| |≥ 68 SFC** | | | | | | |

|Antigen-specific IGRAs using peripheral whole blood | | |

|HBHA | ................
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