EP1576371A1

EP1576371A1 - Diagnostic method and assay kit

Info

Publication number: EP1576371A1
Application number: EP03778503A
Authority: EP
Inventors: Simon Barry
Original assignee: Royal Free Hampstead NHS Trust
Current assignee: Royal Free Hampstead NHS Trust
Priority date: 2002-12-18
Filing date: 2003-11-24
Publication date: 2005-09-21
Also published as: AU2003285505A1; GB0229444D0; WO2004055516A1

Abstract

The invention provides a method of diagnosis of infection by a pathogenic organism wherein lymphocytes in or from isolated bronchoalveolar lavage fluid are exposed to an antigen specific to said pathogen and the resulting cytokine production by said lymphocytes is indicative of a positive diagnosis. Also provided is a diagnostic assay kit adapted or assemblable to perform the method.

Description

DIAGNOSTIC METHOD AND ASSAY KIT

Field of the Invention The invention relates to the diagnosis of pathogenic infections practised on isolated bronchoalveolar lavage fluid, and diagnostic assay kits to perform the method of diagnosis.

Review of the Art known to the Applicants) It is estimated that one third of the world's population is infected with Mycobacterium tuberculosis and that there are eight million new cases of TB and nearly three million deaths each year [1]. The most common route of infection is through the inhalation of droplets carrying the mycobacterium. This results in a local lung immune response that generally contains the infection. However, re-infection, or reactivation may occur resulting predominantly in apical lung disease [2].

The gold standard diagnostic test for tuberculosis remains the visualisation of acid-alcohol fast bacilli (AFB) by Ziehl-Neelsen or auramine staining, with confirmatory culture of the organism. This can take up to eight weeks using solid culture medium, although Bactec systems may achieve more rapid culture results [3, 4]. However, only 54% of all cases of TB and 61% of those with pulmonary TB were culture positive in 1999 in the UK [5]. Although the true figures for culture positive TB may be higher due to underreporting, there still remains a large proportion of TB diagnoses that are made on clinical grounds alone. More recently, DNA amplification techniques have been employed as a rapid diagnostic test in suspected TB cases [6-8]. However, the sensitivity of DNA amplification tests may be reduced in those with smear negative disease [9, 10].

An alternative diagnostic strategy is suggested by the discovery that antigen-specific cells can be identified by a variety of different techniques [11-14]. Nevertheless, these studies have so far been almost exclusively directed towards examining responses in the blood.

Summary of the Invention

To overcome the drawback of the available diagnostic methods, we reasoned that in infectious lung diseases antigen-specific responses of lymphocytes recovered from BAL might prove to be clinically more relevant.

To investigate and demonstrate this invention, we used as a case study the diagnosis of Mycobacterium tuberculosis infection performed upon isolated BAL fluid. We used flow cytometry (FCM) to detect CD4 lymphocyte cytokine production in response to PPD (purified protein derivative) in short-term cultures of blood and BAL (bronchoalveolar lavage fluid) in patients with suspected TB (tuberculosis).

The demonstration of high CD4 IFN-γ and TNF-α synthetic responses to PPD in BAL when compared to the low proportions in peripheral blood is powerful evidence for the dominance of lung immune responses and for the active recruitment of TB-specific CD4+ T lymphocytes to the lung during TB infection. Previous human [18-20] and murine [21, 11] studies have also indicated that lung immune responses predominate during TB infection in different experimental systems. In two of these human studies [18, 19] , T cells were separated from the BAL leukocytes and incubated with peripheral blood mononuclear cells (PBMC) and TB antigens, before investigating [3H] thymidine incorporation. These studies used complex experimental techniques due to concerns regarding the possible suppressive effects of alveolar macrophages and their unsuitability as antigen presenting cells (APC). The impressive antigen-specific CD4 lymphocyte cytokine responses demonstrated in our simplified system supports the findings of previous investigators that BAL contains effective APC [23, 24] . The main conclusion of our case study is that the PPD activated CD4+ T cell type-1 cytokine response, measured by IFN-γ or TNF-α synthesis appears to be a useful diagnostic test for active TB in HIN-uninfected individuals. Importantly, high CD4 type-1 cytokine responses were present not only in those with pulmonary disease, but also in three patients with non-pulmonary TB and four patients with disseminated TB in whom extra-pulmonary complication dominated the clinical picture. This finding has particularly significant diagnostic implications as BAL may be both an easier and safer procedure than the biopsy of other tissues in patients investigated for occult TB.

The other significant development inherent in our assay is that the results are available within 24 hours of acquisition of the BAL sample. It is relevant that in our TB cohort 55% of patients were smear negative and 29% both smear and PCR negative. Thus the immunodiagnostic test avoided delays before waiting for TB culture positivity in these patients.

One case with TB had a low (1.98%) BAL CD4 response to PPD. This patient was asymptomatic and afebrile with normal inflammatory markers, but was referred for further investigation due to an abnormal chest radiograph. The unusual findings of a normal BAL lymphocyte percentage and a low PPD response suggest that this case may have represented a very early reactivation of TB, during the recruitment of antigen- specific lymphocytes to the lung.

A corollary of our observations is that testing the CD4 responses to PPD in blood alone is inadequate to diagnose active TB. The blood lymphocyte responses from TB patients remained indistinguishable from the responses seen in BCG vaccinated control subjects (figure 4). The most likely explanation for the low blood responses seen in the TB patients is that these antigen-specific lymphocytes are actively recruited to the site of infection in the lung resulting in their relative depletion in the blood. Furthermore, it has been demonstrated that BCG vaccinated subjects develop cytokine responses to PPD in an ELISPOT system [25] . Therefore, PPD which shares epitopes with both mycobacterium tuberculosis and BCG is not a discriminating stimulatory antigen when measuring blood lymphocyte responses. Interestingly, BCG vaccination did not provoke a significant BAL cytokine response to PPD as no difference was demonstrated between the BCG vaccinated and unvaccinated control patients (table 1).

One could argue that TB infection and BCG vaccination can be better discriminated by using antigens specific for TB, such as the TB early secretory antigen, ESAT-6.

Overlapping peptides of this antigen have been demonstrated to elicit IFN-γ responses in peripheral blood mononuclear cells (PBMC) in patients with TB, but not in BCG vaccinated subjects when measured by a sensitive ELISPOT technique [13] . However, this test did not distinguish between active and latent disease. Furthermore, the use of ESAT-6 does not solve the problem of impaired cytokine responses in blood from patients with severe TB [26, 27] . These pitfalls are avoided by examining lung immune responses where high cytokine responses by TB antigen-recruited cells in addition to a generalised lymphocytosis are supportive of active TB.

The flow cytometric assay used in our study has a considerable advantage over other methods to determine antigen-specific responses such as the ELISPOT system for three reasons. First, FCM provides a rapid and precise quantification of the total BAL lymphocyte percentage together with the proportions and absolute counts of the responding cell types, CD4 or CD8. Second, different cytokine responses, in our study IFN-γ and TNF-α can be investigated from the same activated lymphocytes. Third, the new generation of cheap red diode laser flow cytometers will render this FCM technology an accessible and affordable option [28] . Nevertheless, fibreoptic bronchoscopy is an expensive diagnostic tool and this sort of study will need to be performed using cheap non-bronchoscopic methods of obtaining a lavage [29] , if it is to be applicable to resource-poor settings.

From an immunological perspective this study highlights several issues. The importance of the type 1 cytokine axis in the control of TB has been documented in both murine experiments [30-32] and in patients with inherited defects in the interleukin-12 and IFN-γ receptors [33-35] . Other investigators using murine TB models have demonstrated that TNF-α is essential for the generation of protective granulomas, without which TB control is insufficient [36, 37] . Our study gives direct support for both IFN-γ and TNF-α in mediating anti-TB responses in the lung. It is intriguing that powerful cytokine responses 004/055516

to PPD in BAL are generated in patients with non-pulmonary TB. These findings are suggestive of a lymphocyte recirculation pathway to the lung, presumably reflecting the fact that even in these patients the origin of post-primary disease was the lung.

Finally, the data presented here demonstrate the limitations of exclusively measuring CD4 lymphocyte responses. In patients co-infected with HIN, CD4 lymphopenia may limit the applicability of this CD4 IFΝ-γ test. In order to develop a reliable immunodiagnostic TB test in this important group of patients, antigen-specific CD8 responses to TB antigens will need to be explored in the lung. Furthermore, The specificity of this method for identifying active TB will also need to be explored by examining the responses to PPD in BAL from patients with treated disease. In our study one patient (patient 8, table 1) underwent a repeat bronchoscopy immediately after the completion of TB therapy. The proportion of PPD activated IFΝ-γ producing CD4 lymphocytes following therapy was 0.45%, compared with 12.4% at TB diagnosis. Whether antigen-specific responses in BAL always decline to low levels following therapy will need to be confirmed in further patients. In addition, the applicability of this test for determining those with latent, rather than active TB will also need to be explored. Lastly, a more extensive investigation of BAL responses to PPD in patients with non-tuberculous lung disease will be needed before firm conclusions can be drawn as to the specificity of this test.

In conclusion, we have described a novel method for diagnosing TB by measuring intracellular cytokine responses to PPD in BAL by a simple and rapid flow cytometric technique. We have demonstrated large responses in BAL from patients with both pulmonary and non-pulmonary TB, but low responses in patients with non-tuberculous respiratory disease. This test therefore appears to be a promising diagnostic resource, particularly in those with non-pulmonary TB in whom achieving a culture diagnosis may be both difficult and hazardous.

Furthermore, having demonstrated this inventive concept for this particular disease, it is clear that the method and associated apparatus are amenable to perform diagnostic assays for a wide range of diseases that produce lung-specific immunological effects, by routine choice of pathogen-specific antigen. For example, diseases amenable to detection by the 2004/055516

6 method of this invention include infection by Cytomegalovirus (CMN) and Pneumocystis carinii.

In its broadest aspect, the invention provides a method of diagnosis of infection by a pathogenic organism wherein lymphocytes in or from isolated bronchoalveolar lavage fluid are exposed to an antigen specific to said pathogen and the resulting cytokine production by said lymphocytes is indicative of a positive diagnosis. Preferably, the method is characterised by the absence of the use of peripheral blood.

In any aspect of the invention the pathogenic organism is preferably Mycobacterium tuberculosis. More preferably, the infection is nonpulmonary tuberculosis.

Preferably, in any aspect of the invention the lymphocytes are T-lymphocytes.

Preferably also, in any aspect of the invention the lymphocytes are CD4 lymphocytes.

Preferably also, in any aspect of the invention the lymphocytes are CD8 lymphocytes.

Preferably also, in any aspect of the invention the antigen is PPD (purified protein derivative) or an antigenic fraction thereof.

Preferably also, in any aspect of the invention the antigen is ESAT-6 or an antigenic fraction thereof.

Preferably also, in any aspect of the invention the detection of the said cytokine production by said lymphocytes is performed using flow cytometry means.

Preferably also, in any aspect of the invention the said lymphocytes are treated with a protein transport inhibitor to promote retention of said cytokine within the said lymphocytes. More preferably, the said protein transport inhibitor is Befeldin A.

There is also provided a diagnostic assay kit adapted or assemblable to perform any of the methods described above. Description of the Figures The invention will be described by reference to the figures, in which:

Figure 1 shows a flow cytometry dotplot of BAL from a TB patient with a low lymphocyte percentage (a) and with a lymphocytosis (b). The vertical axis of each dotplot distinguishes CD45+ leukocytes in BAL from non-leukocyte debris. The CD45+ pan- leukogate is denoted Rl. Lymphocytes form a homogenous population with low side scatter (SSC, horizontal axis), denoted by gate R2. The lymphocyte percentages are calculated as the proportion of R2 events within Rl.

Figure 2 shows flow cytometry dotplots demonstrating the proportion of CD3+ lymphocytes producing IFN-γ following 16 hour incubation with PPD in BAL (a) and blood (d) from a patient with TB. Dotplot b depicts the TNF-α response to PPD in BAL and dotplot c shows the IFN-γ response in BAL when no antigen is added. The vertical axis in each dotplot describes a population of CD4+ T lymphocytes (upper left quadrant) and CD3+ CD4- (lower left quadrant) CD8 T lymphocytes. The upper right quadrants demonstrate the cytokine response in the CD4+ T cells and the lower right quadrants the cytokine response in the CD8 T lymphocytes. Dotplots a and b demonstrate large cytokine responses in the CD4 cells with limited responses in the CD8 cells. The responses in the BAL control (c) and blood sample (d) are low.

Figure 3 is a graph showing the percentage of CD4 T lymphocytes producing IFN-γ in response following 16 hour incubation with PPD in BAL from immunocompetent patients with TB and controls with non-TB respiratory disease. The IFN-γ responses in the TB patients are demonstrated both in those with pulmonary TB and those with non- pulmonary and disseminated disease. Bars represent the median values.

Figure 4 is a graph showing the percentage of CD4 T lymphocytes producing IFN-g following 16 hour incubation with PPD in the blood of patients with TB compared with the blood from BCG vaccinated normal controls and non-BCG vaccinated controls with respiratory disease other than TB. Bars represent median values Exemplary Embodiment of the Invention: The Diagnosis of Pulmonary and non-Pulmonary Tuberculosis

Patients

The hospital ethics committee approved this study to obtain BAL and blood from patients with suspected or proven TB. Of the 36 patients included in our study, 22 were diagnosed with TB including 15 with pulmonary, four with disseminated and three with non- pulmonary disease (table 1). The diagnosis in all of the TB patients was confirmed by culture, including that from aspirates of bone (patient 1) and lymph node (patients 2 and 3). The patients with disseminated TB had predominantly cerebral disease (patient 4) and lymph node disease (patients 6 and 7). Patient 5 had miliary shadowing of the chest radiograph and culture confirmed TB meningitis. Blood, but not BAL results were available for a further 5 cases with culture positive pulmonary TB. All TB patients tested HIN negative.

The control group comprised of 14 patients with a variety of conditions requiring a diagnostic bronchoscopy in which TB was considered in the differential diagnosis (table 1). Of the two patients with cytomegalovirus (CMN) infection, one had chronic renal failure and the other was a bone marrow recipient. In two patients no diagnosis was determined from the bronchoscopy, but their symptoms resolved. Three controls (patients 29,30 and 31, table 1) were HIN negative and the remainder were not tested. These patients were generally older and felt not to be in a high HIN risk group. Moreover, absolute blood CD4 counts measured from these patients as part of the study methods were all greater than 500 cells/μl.

In addition, blood was taken from 20 BCG-vaccinated control subjects who were nursing, medical and laboratory staff at the Royal Free Hospital. 11 of this latter group were male and the median age was 36 years (range 22-53).

Bronchoalveolar lavage

BAL was undertaken by standard technique with a flexible bronchoscope wedged into a sub-segmental bronchus under intravenous sedation. Aliquots of warmed sterile normal saline were introduced through the bronchoscope and aspirated into a siliconised glass bottle. An area of radiologically affected lung was washed otherwise the right middle lobe was used. In four patients that already had a prior microbiological diagnosis of TB, bronchoscopies were performed within two weeks of commencing TB therapy. In the remaining cases, TB therapy was commenced following bronchoscopy.

Sample preparation

The fresh BAL sample was placed on ice and samples sent to the relevant diagnostic laboratories. The remaining BAL (25-50ml) was centrifuged at 430g for 8 minutes, decanted and filtered through a lOOμm filter (CellTricks, Partec GmbH, Mϋnster, Germany) and then centrifuged again. The pellet was resuspended in 1ml of culture medium (RPMI 1640 with 10% fetal calf serum). Aliquots of BAL in culture medium were then analysed by FCM to assess the BAL lymphocyte percentage, the CD4/CD8 ratio and the absolute CD4 count prior to culturing the sample with PPD.

FCM of BAL and blood lymphocytes

To 25μl of the BAL suspension, CD45-FITC (Becton Dickinson, Oxford, UK) was added for staining at room temperature in the dark for 15 minutes. A second 25μl BAL aliquot was stained with a cocktail of the following monoclonal antibodies: CD4-FITC (Royal Free Hospital), CD8-PE (Royal Free Hospital) and CD3-PE/CY5 (Dako, Ely, UK).

Following staining, phosphate buffered saline (PBS) was added to each tube to a volume of 1ml. 50μl of whole blood collected into lithium heparin tubes from each patient was also stained as described above followed by the addition of lysis buffer (NH4CL) instead of PBS. All antibodies were used in pretitrated optimal concentrations. Fresh BAL and blood samples stained with CD45 and CD4/CD8/CD3 were run on a Cytoron-absolute flow cytometer (Ortho Diagnostics, High Wycombe, UK). A CD45+ pan-leucogate was used to count the absolute number of all leucocytes [15] and lymphocytes were identified by their strong CD45 staining and lymphoid scatter characteristics (figure 1). The numbers of CD4+ cells and the CD4/CD8 ratio in BAL and blood were calculated from the second tube.

PPD stimulation and FCM analysis Aliquots of the BAL suspension containing 1X105 CD4+ lymphocytes in 1ml of culture medium were placed into two sterile 5ml polypropylene tubes (Thermo Life Sciences, UK). In addition, 1ml of peripheral blood from the same patient collected into lithium heparin tubes was also placed into two polypropylene tubes. To one of the BAL and blood samples, lOμg of PPD (Statens Serum Institute, Copenhagen, Denmark) was added. The other tubes were unstimulated control samples. In five cases, 0.08 IU of tetanus toxoid (Pasteur Merieux, Lyon, France) was added to a third BAL sample as a control antigen. The samples were incubated for two hours at 37°C and 5% CO2, after which time 5μg of Brefeldin A (Epicentre Technologies, Cambridge, UK) was added and the samples incubated for a further 14 hours.

Following incubation, the samples were vortexed vigorously to detach cells from the walls of the tube. First, lymphocyte surface markers were stained using CD4-FITC (Royal Free Hospital) and CD3-PerCP (Becton Dickinson) as described above and the samples were washed. Fixation and permeabilisation of the cells was performed as previously described using Fix-and-Perm (An-Der-Grub, Kaumberg, Austria) [16] Following this, IFN-γ-PE (Caltag Laboratories, Towcester, UK) and TNF-α-APC (Becton Dickinson) were added and the samples stained at 4°C for 30 minutes, followed by a final wash step. Analysis of the stained preparations was performed on FACSCalibur (Becton Dickinson). 40,000 CD4+ events were acquired and the proportion of IFN-γ and TNF-α staining cells within the total lymphocyte and CD4+ T cell populations were analysed using WINMDI software (Version 4.2a; M Trotter) in both the PPD activated and control cultures. The responses attributable to specific PPD effects were calculated by subtracting the responses in the control tubes from those in the PPD activated tubes (figure 2).

Statistics

Median values and absolute ranges were recorded in the text and non-parametric statistical analysis was performed with the Maim- Whitney test to determine the statistical difference between data sets.

RESULTS BAL lymphocyte percentages and CD4/CD8 ratios

BAL from most of the TB patients demonstrated a significant lymphocytosis (table 1, median 28.3%; range 0.7-70.4%). In three cases (patients 19,20 and 21), low lymphocyte percentages were present in BAL. In two of these individuals (patients 20 and 21), washings were taken from apical cavities where pus was aspirated and therefore the vast majority of cells were neutrophils.

It was interesting that a BAL lymphocytosis was present both in those with pulmonary (median 20.6%) and non-pulmonary TB (median 32.0%). The BAL CD4/CD8 ratios (table 1) in the TB group were highly variable (range 0.4 to 11.7), although the median ratio (2.6) was within the range for healthy control subjects [17] .

When the non-TB patients were investigated, the lymphocyte percentages and CD4/CD8 ratios in BAL were also highly variable as expected in such a heterogenous group. The median lymphocyte percentage in this group as a whole was 21.2%, ranging from 2.0- 81.7%. The highest lymphocyte numbers were seen in three patients with sarcoidosis (median 76.0%), in agreement with the documented abnormalities in this disease. Similarly, the CD4/CD8 ratio in the control group was also variable (median 1.7; range 0.5-43.4).

Comparison of IFN-γ and TNF-a responses to PPD in BAL between TB-infected and uninfected individuals

The median percentage of BAL CD4 lymphocytes producing IFN-γ in response to PPD in patients with TB was high (24.0%; range 1.98-37.2%). Even higher BAL TNF-α CD4 responses were noted in these patients (median response 31.4%; range 3.81 -47.4%). FCM analysis determined that both cytokines were mostly produced by the same activated CD4 lymphocyte population. High CD4 IFN-γ responses to PPD in BAL were demonstrated both in TB patients with pulmonary (median IFN-γ 21.8%) and non- pulmonary/disseminated (median IFN-γ 24.4%) disease. BAL TNF-α responses were also similar between these two groups. By contrast, the BAL CD4 IFN-γ production in patients with non-TB respiratory disease was low (median 0.33%; range 0.01-1.12%; figure 3). These results were highly statistically significantly (p<0.0001) when compared to the BAL CD4 IFN-γ production from the TB patients. CD4 TNF-α responses to PPD in BAL were also low in the non-TB control patients (median 0.78%). The control values were low irrespective of whether the patients had been BCG-vaccinated (n=5) or not (n=9).

Further characterisation of the cytokine responses to PPD in BAL from TB patients

In the short incubation period of our assay, PPD was demonstrated to mainly activate CD4 lymphocytes and not CD8 T lymphocytes (figure 2). The PPD-activated CD4 populations in the lung remained high in patients who had been on anti-tuberculous therapy for up to two weeks (median eight days) prior to BAL (patients 1,2,4,10 and 16, table 1). In addition, steroid therapy did not significantly attenuate the response in patient 4 who was treated with corticosteroids for ten days before BAL.

To investigate whether the induction of cytokines was really antigen specific, tetanus toxoid was added as a control antigen to BAL samples from five TB patients. The responses were very low and similar to those in the control tube to which no antigen was added, thus confirming PPD antigen-specificity.

Our results demonstrate that high cytokine responses to PPD are present throughout the lung and not just localised to radiologically abnormal areas in patients with TB. In patients with non-pulmonary TB, BAL were performed from the radiologically unaffected right middle lobe and in each case high PPD responses were noted (table 1). In addition, comparative washings from areas of radiologically affected and unaffected lung were performed on selected patients with pulmonary TB, demonstrating high responses from both areas (data not shown).

Comparison of IFN-γ and TNF- responses in the blood of TB patients with BCG- vaccinated controls

We next investigated whether there were differences between the CD4 PPD responses in the blood of TB patients when compared to a group of healthy BCG-vaccinated control subjects (figure 4). The median frequency of IFN-γ producing CD4+ T lymphocytes in response to PPD in the TB patients was 0.07% (range 0.01-1.43%). This value was lower than in the BCG vaccinated controls (median 0.14%; range 0.01-0.91%), and there was no statistically significant difference between the two groups (p=0.80). Similarly, the blood CD4 TNF-α responses in the TB patients were low (median 0.18%) as were those from the BCG vaccinated controls (median 0.32%).

Table 1

Demographic and diagnostic results in patients with TB and non-TB respiratory conditions.

Patient Sex Ethnicity Diagnosis BAL TB diagnosis³ BAL Lymph BAL BAL Blood and And AFB PCR Culture %⁴ CD4/CD8 CD4 CD4 age¹ BCG² IFN-γ⁵ IFN-γ⁶

1 F 16 BA + Spinal TB⁷ - 42.8 2.7 23.5 0.05

2 28 BA + Lymph node TB⁷ - - - 32.0 1.2 22.7 0.04

3 F 42 BA + Lymph node TB - - - 29.0 2.9 17.7 0.56

4 M 32 BA + Disseminated TB⁷ - + + 43.6 7.3 34.0 0.02

5 M 21 BA + Disseminated TB _ + + 44.6 2.2 25.5 0.01

6 F 32 o + Disseminated TB ND⁸ + 16.7 2.7 25.2 0.07

7 F 41 BUK+ Disseminated TB - + + 27.6 4.1 24.4 0.06

8 F 24 C + Pulmonary TB + + + 33.0 2.5 12.4 0.03 θ M 24 A + Pulmonary TB + + + 62.0 0.4 11.3 1.43

10 F 21 BA + Pulmonary TB⁷ + + + 20.8 11.7 17.0 0.13

11 M 18 BA + Pulmonary TB _ + + 33.5 2.0 37.2 0.39

12 M 24 A + Pulmonary TB _ - + 70.4 2.6 17.2 0.02

13 M 27 C - Pulmonary TB _ _ + 15.7 2.4 31.7 0.15

14 M 37 A + Pulmonary TB + + + 32.0 1.7 24.9 0.23

15 M 35 C + Pulmonary TB + + + 4.60 1.2 25.6 0.06

16 M 31 C - Pulmonary TB⁷ + + + 15.2 1.1 31.3 0.21

17 F 26 BA + Pulmonary TB + + + 46.8 5.9 21.8 0.02 18 F27 BA + Pulmonary TB - - + 20.3 1.3 5.71 0.92

19 M28 A + Pulmonary TB - + + 7.0 1.3 1.98 0.08

20 31 C + Pulmonary TB + + + 3.2 3.0 31.6 0.07

21 M50 BUK- Pulmonary TB + + + 0.7 2.9 3.01 0.03

22 M19 BA + Pulmonary TB + + + 18.0 4.3 28.4 0.26

23 F71 A- Sarcoid ND - 81.7 2.5 0.19 0.03

24 F41 C + Sarcoid ND - 53.3 9.5 0.05 0.02

25 32 C + Sarcoid - - - 76.0 43.4 0.14 0.07

26 F71 o- Carcinoma ND - 74.5 6.2 0.0 0.01

27 66 A- Carcinoma ND 17.5 1.8 0.23 0.01

28 F56 C- Carcinoma - 48.1 0.7 0.55 0.01

29 M26 A + Cytomegalovirus - - - 21.0 0.7 1.12 0.15

30 19 o- Cytomegalovirus - - - 13.0 0.5 0.35 0.06

31 F38 BUK + Pneumonia ND - 24.2 2.7 0.57 0.06

32 M66 0- Lymphoma. ND - 21.4 1.7 0.06 0.02

33 F50 C + Bronchiectasis - - - 4.1 0.4 0.31 0.06

34 F78 C- Lung fibrosis - - - 8.4 1.0 0.45 0.0

35 F66 A- No diagnosis - - - 17.9 1.7 0.16 0.02

36 F68 C- No diagnosis ND 2.0 1.0 0.48 0.06

Footnotes to Table 1

1. Age in years.

2. Ethnicity: BA=black African, BUK=black UK born, C=Caucasian, A= Asian, O=other. BCG scar present (+) and absent (-).

3. Diagnosis of TB from the BAL specimen by AFB smear, PCR (polymerase chain reaction) and culture. Note patients 1,2 and 3 with non-pulmonary TB were diagnosed from tissue biopsies. These were all smear and PCR negative, but culture positive. 4. Percentage of lymphocytes in the total BAL leukocyte population.

5. Percentage of CD4 lymphocytes producing IFN-γ following incubation with PPD in BAL.

6. Percentage of CD4 lymphocytes producing IFN-γ following incubation with PPD in blood.

7. Patients with TB who had started anti-tuberculous therapy prior to BAL and PPD testing. The number of days following the start of TB therapy was 8 for patient 1, 3 for patient 2, 14 for patient 4, 8 for patient 8 and 7 for patient 14. For all other patients with TB, anti-tuberculous therapy was started following BAL. 8. ND =Not done.

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Claims

1. A method of diagnosis of infection by a pathogenic organism wherein lymphocytes in or from isolated bronchoalveolar lavage fluid are exposed to an antigen specific to said pathogen and the resulting cytokine production by said lymphocytes is indicative of a positive diagnosis.

2. The method of Claim 1 characterised by the absence of the use of peripheral blood.

3. The method of either of the preceding claims wherein the pathogenic organism is Mycobacterium tuberculosis.

4. The method of Claim 3 wherein the infection is nonpulmonary tuberculosis.

5. The method of any of the preceding claims wherein the lymphocytes are T-lymphocytes.

6. The method of any of the preceding claims wherein the lymphocytes are CD4 lymphocytes.

1. The method of any of the preceding claims wherein the lymphocytes are CD 8 lymphocytes.

8. The method of any of the preceding claims wherein the antigen is PPD (purified protein derivative) or an antigenic fraction thereof.

9. The method of any of the preceding claims wherein the antigen is ESAT-6 or an antigenic fraction thereof.

10. The method of any of the preceding claims wherein the detection of the said cytokine production by said lymphocytes is performed using flow cytometry means.

11. The method of any of the preceding claims wherein the said lymphocytes are treated with a protein transport inhibitor to promote retention of said cytokine within the said lymphocytes.

12. The method of Claim 11 wherein the said protein transport inhibitor is Brefeldin A.

13. A diagnostic assay kit adapted or assemblable to perform the method of any of the above claims.