Mycobacterium tuberculosis causes two million deaths annually and it is estimated that one third of the world's population is latently infected with this pathogen . Despite this enormous and increasing global burden, there is still no reliable test to confirm active M. tuberculosis infection in culture-negative cases, or to detect latent infection in asymptomatic individuals, because the tuberculin skin test (TST) is of limited sensitivity and specificity [2–5]. An increasing proportion of cases of M. tuberculosis infection worldwide occurs in individuals with HIV infection. Active tuberculosis is more difficult to diagnose in this group [6–10], and the sensitivity of the TST for active tuberculosis  is only 40–60% in the presence of asymptomatic HIV infection, declining still further as HIV progresses . Similarly, HIV co-infection lowers the sensitivity of the TST for detecting latent tuberculosis infection (LTBI). In one study in Zambia , only 30% of HIV-positive patients had positive TST compared with 62% of HIV-negative individuals. There is thus a need for an accurate, rapid test for M. tuberculosis infection, which remains effective in HIV co-infected individuals.
RD1 is a recently identified genomic segment that is present in M. tuberculosis complex but absent from all strains of Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccine and most environmental mycobacteria [13,14]. RD1 gene products such as ESAT-6 and CFP-10 [15–19] thus offer the potential to develop more specific diagnostic tests for M. tuberculosis infection, which, unlike the TST, should not be confounded by previous BCG vaccination. Using an ex-vivo enzyme-linked immunospot (ELISPOT) assay for IFN-γ , we recently identified ESAT-6 peptide-specific T cells as an accurate marker of M. tuberculosis infection. Their presence had a diagnostic sensitivity and specificity of over 90% for the detection of M. tuberculosis infection in patients with culture-confirmed tuberculosis in the UK, and was significantly better than the TST . The ESAT-6-based ELISPOT assay was also more accurate than the TST for identifying healthy contacts at high risk of recent infection in London , and its sensitivity for estimating the prevalence of latent infection in a high prevalence region was recently augmented by the incorporation of CFP-10-derived peptides .
The high sensitivity and specificity of the RD1 gene product-based ELISPOT assay, together with its operational speed and simplicity, suggest that it could significantly contribute to global tuberculosis control. However, given the increasing proportion of tuberculosis cases that are HIV-positive, it is essential to evaluate this T cell-based approach in individuals with HIV-associated impairment in T cell immunity. We therefore assessed the assay for the detection of active and latent M. tuberculosis infection in a population with a high burden of tuberculosis and HIV. This study took place in Lusaka, Zambia, where the seroprevalence of HIV infection in adults is 23% , and there are over 500 new cases of active tuberculosis per 100 000 population annually, over 70% of them in HIV-infected individuals . Our aims were twofold: first to investigate whether the RD1-based ELISPOT assay maintains its high sensitivity for detecting active M. tuberculosis infection in HIV-positive individuals; and second, to examine the relationship between the TST and the RD1-based ELISPOT assay in HIV-negative and HIV-positive asymptomatic adults at high risk of LTBI by virtue of their residence in a tuberculosis endemic region. We prospectively recruited 50 Zambian adults with sputum smear-positive pulmonary tuberculosis (39 HIV-positive) and 75 asymptomatic Zambian adults (21 HIV-positive) in whom active tuberculosis was clinically and radiographically excluded; 40 healthy adults resident in the UK represented controls from a country with a low prevalence of tuberculosis.
Ethical approval for the study was granted by the Ethics Committee of the University Teaching Hospital, Lusaka, Zambia. Zambian tuberculosis patients, healthy Zambian adults and UK controls were recruited prospectively in Lusaka and Oxford, and a heparinized blood sample was drawn after obtaining informed consent including for anonymous HIV testing.
Fifty Zambian adults (58% men; mean age 33 years, range 16–68) with clinical and radiological features consistent with active pulmonary tuberculosis and with at least one sputum sample positive for acid alcohol fast bacilli on microscopy were recruited. Patients were either untreated (n = 43), or had received less than one month of antituberculous therapy (n = 7) at the time of recruitment. A total of 78% (39/50) were HIV positive.
Seventy-five healthy Zambian adults (56% men; mean age 32 years, range 17–66) were recruited when they attended the Casualty Department of the University Teaching Hospital in Lusaka with minor injuries, on the basis of the following criteria: (i) no past history of tuberculosis; (ii) no symptoms suggestive of tuberculosis (more than a month's history of cough, fever or breathlessness, or recent weight loss of over 10 kg); and (iii) normal chest radiography. TST were performed in 61 out of 75, and results were available for the 49 individuals who returned for TST reading. BCG scars were present in 57 out of 75; 28% (21/75) were HIV positive.
Forty healthy adults (53% men; mean age 32 years, range 23–49) were recruited in Oxford, UK, on the basis of the following criteria: (i) no past history of tuberculosis; (ii) no history of residence in a tuberculosis-endemic area for more than one month; and (iii) no known tuberculosis contact; 33 out of 40 were BCG vaccinated. HIV testing was not performed in this group, but all donors were from low-risk groups and none had any clinical features of HIV infection.
HIV testing and lymphocyte counts
HIV testing was performed on all Zambian subjects, using a rapid immunochromatographic screening test for the detection of antibodies to HIV1/2 (Fastline, Millenium Biotechnology, Inc., New York, USA). Those who wished to know their HIV status were referred to the voluntary counselling and testing services within Lusaka. Lymphocyte counts were performed in an automated Coulter counter on blood samples from all HIV-positive subjects.
Tuberculin skin testing
All TST were carried out and read by one individual (A.C.), using the Mantoux technique on the volar surface of a forearm, with five tuberculin units (TU) of tuberculin purified protein derivative (PPD) RT23 (Statens Seruminstitut, Copenhagen, Denmark). Tests were read at 48–72 h, and were measured with a ruler as induration diameters along and across the arm. An average diameter of greater than or equal to 10 mm was taken as a positive test.
ESAT-6- and CFP-10-derived peptides
Serial 15-mer peptides, overlapping by 10 amino acid residues, spanning the complete amino acid sequence of each antigen (17 for ESAT-6; 18 for CFP-10) were purchased (Research Genetics, Alabama, USA). Identity was confirmed by mass spectrometry and purity (of over 70%) by high performance liquid chromatography. Sequence homology searches of the SwissProt and translated GenBank databases confirmed that the peptides used during this study are uniquely restricted to the ESAT-6 and CFP-10 proteins of M. tuberculosis complex. A response to one or more of these 35 peptides was scored as indicative of M. tuberculosis infection.
Ex-vivo enzyme-linked immunospot assay for IFN-γ release
Ex-vivo ELISPOT assays using ESAT-6-derived peptides, recombinant ESAT-6 antigen (rESAT-6, a kind gift of A. Whalen and M. Vordermeier, VLA, Addlestone, UK), CFP-10-derived peptides and PPD (RT49, Statens Seruminstitut) were performed for all 165 study participants, except for one Zambian tuberculosis patient, in whom limited cell numbers precluded the testing of CFP-10-derived peptides. Assays were performed as described previously [20,23], with 2.5 × 105 peripheral blood mononuclear cells (PBMC) per well (3 × 105 for UK controls). Single peptides, rESAT-6 protein and PPD were added to individual wells at final concentrations of 10, 10 and 20 μg/ml, respectively. Phytohaemagglutinin (ICN Biomedicals, Aurora, OH, USA) at 5 μg/ml was added to positive control wells, and no peptide was added to duplicate negative control wells.
For wells containing individual peptides, responses were scored as positive if the test well contained at least five IFN-γ spot-forming cells (SFC) more than negative control wells and this number was at least twice that in negative control wells. Responses below this pre-defined threshold for any given peptide were scored as negative for that peptide. This pre-defined cut-off point translates into a detection threshold of 20 peptide-specific T cells per million PBMC, or 1/50 000 PBMC. A higher cut-off of 10 SFC more than negative control wells was used for wells containing rESAT-6, because we have previously noted low level responses (up to five IFN-γ SFC per 2.5 × 105 PBMC) with this preparation of rESAT-6 in tuberculosis-unexposed UK donors, in the absence of any ESAT-6 peptide-specific response.
Although the person performing the assays was not blind to the tuberculosis status of the patients, the read-out in SFC is quantitative, and our criteria for a positive response were stringent and pre-defined. Mean background numbers of SFC in negative control wells were 2.91 (standard deviation 3.11) in tuberculosis patients, and 2.75 (standard deviation 2.54) in asymptomatic Zambian adults.
Comparisons between numbers of total IFN-γ SFC, and between TST diameters of induration, were performed using the non-parametric Mann–Whitney–Wilcoxon rank sum test. Comparisons between the numbers of donors positive in each assay were performed using Fisher's exact test.
The RD1-based enzyme-linked immunospot assay detects M. tuberculosis infection with a high sensitivity in HIV-negative and -positive Zambian tuberculosis patients
Eleven out of 11 (100%) HIV-negative and 35 out of 39 (90%) HIV-positive Zambian tuberculosis patients responded to one or more ESAT-6- or CFP- 10-derived peptides (Table 1 and Fig. 1). Four HIV-positive patients with active tuberculosis did not respond to any ESAT-6 or CFP-10 peptide in the ELISPOT assay (patients 36, 42, 54 and 60). There was no significant difference in the mean lymphocyte count between responders and non-responders (geometric mean 1.19 × 109/l for responders, 0.93 for non-responders; P = 0.36, t-test). Only one out of three sputum samples from patient 36 was weakly positive on microscopy for acid-fast bacilli (eight bacilli in 100 fields on Ziehl–Neelsen staining). This patient's chest X-ray did not show clear changes consistent with pulmonary tuberculosis, raising the possibility that the single positive sputum result was caused by laboratory contamination. If this were the case, then the sensitivity of the RD1 gene product-based ex-vivo ELISPOT assay among HIV-positive tuberculosis patients would be 92% (35/38). In the remaining three HIV-positive tuberculosis patients with negative results, the diagnosis of active tuberculosis was radiographically and microbiologically secure.
The median numbers of summated ESAT-6 and CFP-10 peptide-specific T cells (totalled for both antigens) in patients who responded were 452 per million PBMC (interquartile range 360–732) in HIV-negative, and 396 per million PBMC (interquartile range 224–560) in HIV-positive patients; this difference was not statistically significant (P = 0.08, Mann–Witney U test).
Fewer patients responded to rESAT-6 antigen than to ESAT-6 peptides (82 versus 100% in HIV-negative patients; 46 versus 87% in HIV-positive patients; Table 1). Responses to ESAT-6 peptides in patients in whom there was no response to whole antigen may have been mediated by HLA class 1-restricted CD8 T cells, as we have previously observed that ESAT-6 peptide-specific CD8 T cells do not respond to rESAT-6 antigen in ex-vivo ELISPOT assays because recombinant protein added exogenously is not processed and presented through the MHC class I antigen processing pathway [26,27]. In addition, HIV-related impairment of antigen presenting cell function may result in the impaired presentation of rESAT-6 through the class II processing pathway. The ability of peptides to elicit higher response rates in individuals with active tuberculosis may prove to be a significant advantage over recombinant antigen. Conversely, one tuberculosis patient responded to rESAT-6 antigen, but not to the peptides, probably because the responding T cells were primed to an epitope situated across two adjacent peptides but not fully contained within either one.
The RD1-based ex-vivo enzyme-linked immunospot assay suggests a high prevalence of latent M. tuberculosis infection in Zambian adults
Thirty-seven out of 54 (69%) HIV-negative asymptomatic Zambian adults had circulating RD1 gene product-specific T cells. Of the 21 HIV-positive asymptomatic Zambian individuals, nine (43%) responded in the RD1 gene product-based ex-vivo ELISPOT assay (Table 1 and Fig. 1). For the HIV-infected group, a comparison of responders and non-responders showed a trend towards lower mean lymphocyte counts in the non-responders (geometric mean 1.55 responders, 1.08 non-responders, P = 0.06). Median numbers of RD1 gene product-specific T cells were significantly lower in HIV-positive responders (92 per million PBMC; interquartile range 68–148) than in the HIV-negative responders (212 per million PBMC; interquartile range 120–364; P = 0.01, Mann–Witney U test). Again fewer asymptomatic Zambian adults responded to rESAT-6 antigen than to ESAT-6 peptides (Table 1).
In contrast to the 69% prevalence of RD1 gene product-specific T cells among healthy HIV-negative Zambian individuals, none of 40 healthy UK residents responded to any of the ESAT-6 or CFP-10 peptides, nor to rESAT-6 protein (Table 1 and Fig. 1).
Direct comparison of RD1-based ex-vivo enzyme-linked immunospot assay and tuberculin skin test in healthy Zambian adults
TST results were available for 49 asymptomatic Zambian adults, of whom 36 (74%) were BCG vaccinated and 14 (29%) were HIV-positive (Table 2). For the 35 HIV-negative individuals, 28 (80%) had positive TST and 24 (69%) responded in the RD1 gene product-based ex-vivo ELISPOT assay. Of the 28 individuals with positive TST, the median diameter of TST induration in the 19 individuals with positive RD1-based ELISPOT assays (median 17 mm; range 10–23 mm) was significantly higher than the median induration in the nine individuals with negative ELISPOT assays (median 13 mm; range 10–17 mm) (P = 0.004, Mann–Witney–Wilcoxon rank sum) (Fig. 2). The weaker TST responses in this latter group may reflect BCG vaccination rather than latent M. tuberculosis infection; indeed, eight out of these nine subjects had BCG vaccination scars. Conversely, of the five individuals with negative TST and positive ELISPOT assay results, only two had BCG scars.
For the 14 HIV-positive Zambian individuals, five (36%) had positive TST, whereas six (43%) responded in the ELISPOT assay (Table 2). Meaningful comparison of the sizes of the positive TST responses associated with positive (n = 3) and negative (n = 2) RD1-based ELISPOT assays is not feasible, because the numbers of individuals are too low. Three out of 14 HIV-positive individuals had negative TST but positive RD1-based ELISPOT assays, suggesting that the ELISPOT assay may be able to detect latent M. tuberculosis infection in the context of HIV-induced cutaneous anergy. Six HIV-positive individuals were negative in both tests (four out of the six were BCG-vaccinated). Owing to the lack of a gold standard test for latent M. tuberculosis infection, it is not clear what proportion of these may have had latent infection, and false negative TST and RD1-based ELISPOT assay results associated with HIV-induced immunosuppression, although all responded to the positive control in the ELISPOT assay.
Purified protein derivative-based ex-vivo enzyme-linked immunospot assay does not distinguish M. tuberculosis infection from Bacillus Calmette-Guérin vaccination
One-hundred per cent (11/11) and 72% (28/39) of HIV-negative and -positive tuberculosis patients, respectively, responded to PPD in the ex-vivo ELISPOT assay. Among asymptomatic Zambian adults, 83% (45/54) of HIV-negative individuals and 29% (six out of 21) of HIV-positive individuals responded (Table 1 and Fig. 1). By comparison, of 40 healthy unexposed UK donors (33 of whom were BCG vaccinated), 33 (83%) responded to PPD in the ELISPOT assay, whereas none responded to any of the ESAT-6 or CFP-10 peptides, or to whole rESAT-6 protein.
Effect of co-existent HIV infection on the sensitivity of the RD1-based enzyme-linked immunospot assay, purified protein derivative-based enzyme-linked immunospot assay and tuberculin skin test
Table 1 shows the impact of HIV infection on the sensitivity of the RD1 gene product-based ELISPOT assay, the PPD-based ELISPOT assay and the TST. In patients with active tuberculosis, the RD1-based ex-vivo ELISPOT assay showed a trend towards being less sensitive in HIV-positive patients, sensitivity falling from 100 to 90% (P = 0.52, Fisher's exact test). In contrast, the proportion of tuberculosis patients responding to PPD by ex-vivo ELISPOT fell markedly from 100 to 72% (P = 0.09). Among asymptomatic Zambian individuals without active tuberculosis, the proportion with positive TST results fell significantly from 80 to 36% (P = 0.006), in line with previous studies in Lusaka , and HIV infection appeared to have an even larger impact on response rates in the PPD-based ELISPOT assay in this group (83% in HIV-negative versus 29% in HIV-positive individuals, P ≤ 1 × 105). The prevalence of positive responses in the RD1 gene product-based ELISPOT assay also declined from 69 to 43% in HIV-infected individuals, but this fall was less marked than with the other tests and was not statistically significant (P = 0.064).
ESAT-6 and CFP-10 epitope maps in Zambian tuberculosis patients and healthy adults
Epitope maps for ESAT-6 and CFP-10 are shown in Fig. 3. Every peptide from both ESAT-6 and CFP-10 was recognized by T cells from at least one Zambian tuberculosis patient and asymptomatic adult. ESAT-6 contained several widely recognized epitopes at the amino terminus and towards the carboxy terminus (Fig. 3a), as previously described . The two most immunodominant epitopes, however, were in the middle of the molecule, peptides ESAT-631–45 and ESAT-651–65, with over 50% of healthy adults responding to ESAT-631–45, a region that has not been found to be immunodominant in other populations. Epitopes in CFP-10 were distributed across the protein, particularly in the central portion (Fig. 3b). The two strikingly immunodominant domains identified in Indians, CFP-1051–70 and CFP-1071–90 , were also widely recognized among Zambian individuals, but in addition a very high proportion of Zambian individuals responded to peptides CFP-1046–60 and CFP-1081–95.
This is the first study to characterize the T cell response to M. tuberculosis RD1 gene products in HIV-infected individuals. The RD1 gene product-based ELISPOT assay showed a sensitivity of 100% for the detection of M. tuberculosis infection in Zambian HIV-negative tuberculosis patients, and this sensitivity was maintained at 90% in HIV-positive tuberculosis patients. This compares favourably with the 72% sensitivity of the PPD-based ex-vivo ELISPOT assay in this series of HIV-positive tuberculosis patients and the 40–60% sensitivity of the TST documented in HIV-positive tuberculosis patients with even a mild degree of immunosuppression [2,11]. Of the four out of 39 HIV-positive tuberculosis patients who did not respond to any of the peptides in the RD1 gene product-based ex-vivo ELISPOT assay, one may not have had tuberculosis (sputum microscopy and chest radiography were both equivocal), and another responded to rESAT-6 antigen. The sensitivity of this assay could thus be as high as 95% (36/38) in HIV-infected tuberculosis patients, if responses to rESAT-6 as well as peptides are included.
The prevalence of LTBI in a population is a crucial parameter for the design and monitoring of tuberculosis control measures, yet TST prevalence surveys are bedevilled by the poor intrinsic specificity of the TST, its susceptibility to HIV-induced cutaneous anergy and operational drawbacks. The RD1-based ELISPOT assay may offer a more accurate approach [21,28]. In the present study, 37 out of 54 healthy HIV-negative adult Zambian individuals with no clinical or radiographic evidence of active tuberculosis had circulating T cells specific for one or more RD1 gene product-derived peptides, suggesting a 69% prevalence of latent infection. In contrast, of 40 healthy adults resident in the UK (33 of whom were BCG vaccinated) none responded to any of the RD1 gene product-derived peptides, highlighting the specificity of this approach. By comparison, 83% (45/54) of healthy HIV-negative Zambian individuals responded in the PPD-based ELISPOT assay, along with 83% (33/40) of the healthy UK residents.
Comparison of RD1-based ELISPOT and TST results was possible in 35 healthy HIV-negative Zambian adults (Table 2). Twenty-one individuals were concordant (19 were positive in both tests; two were negative in both tests). Of the 14 discordant results, the nine individuals that were TST-positive and RD1 ELISPOT-negative had a lower median induration of TST response than the 19 individuals whose positive TST responses were associated with a positive RD1 ELISPOT result (Fig. 2). In general, the stronger the TST response, the greater the likelihood of M. tuberculosis infection , and thus the RD1 ELISPOT may be identifying the subgroup of TST-positive individuals who actually have M. tuberculosis infection. Conversely, the weaker TST in individuals with negative RD1-based ELISPOT assays may reflect BCG vaccination rather than M. tuberculosis infection, and eight out of nine of these individuals did, in fact, have BCG vaccination scars.
The response rates of the RD1 gene product-based ELISPOT assay, the PPD-based ELISPOT assay and the TST were all reduced in healthy HIV-positive compared with HIV-negative Zambian individuals, but the reduction for the RD1 gene product-based ELISPOT assay was less marked than for the other tests (Table 1). Therefore, RD1-specific T cells, as enumerated by ELISPOT assay, appear to be less affected by HIV than are PPD-specific T cell responses, as quantified directly by PPD-based ELISPOT assay, or indirectly by the TST. In tuberculosis patients and asymptomatic Zambian individuals who did respond in the RD1 gene product-based ELISPOT assay, those who were HIV co-infected had a lower median frequency of summated ESAT-6 and CFP-10 peptide-specific T cells than their HIV-negative counterparts. As the majority of peptide-specific T cells enumerated in the RD1 gene product-based ELISPOT assay are CD4 T cells , the lower response rate among HIV-infected individuals, and the reduced frequency of RD1 peptide-specific T cells in those who do respond, probably result from an HIV-associated loss of CD4 T cells.
Might the high prevalence of RD1 gene product-specific T cells in asymptomatic Zambian adults result from exposure to a cross-reactive environmental antigen unrelated to M. tuberculosis that is present in Zambia but not in the UK? We consider this to be unlikely as the peptide sequences in this study are uniquely restricted to ESAT-6 and CFP-10 of the M. tuberculosis complex. However, the esat-6 gene (and therefore very likely the gene for CFP-10, which is within the same operon as esat-6 ) is present in four atypical mycobacteria: Mycobacteria kansasii, szulgai, flavescens and marinum . It is therefore theoretically possible that exposure to one of these organisms may have contributed to the high prevalence of ESAT-6- and CFP-10-specific T cells in asymptomatic Zambian individuals. However, unlike M. tuberculosis, none of these organisms has a geographical distribution consistent with the observed pattern of RD1 ELISPOT assay positivity [31–33]. An ESAT-6 homologue has recently been identified in M. leprae and T cells from some M. tuberculosis-infected individuals showed cross-reactivity with this antigen . However, since the prevalence of leprosy in Lusaka is very low, the T cell responses in our study population are most unlikely to result from M. leprae exposure. We conclude, therefore, that a positive RD1-based ELISPOT assay signifies previous infection with M. tuberculosis. If, after infection with M. tuberculosis, viable bacilli persist in a dormant state for many years, as is believed to be the case, then the presence of circulating RD1 gene product-specific T cells is consistent with ongoing latent infection with M. tuberculosis. This is supported by the progressive decline in the frequency of ESAT-6 peptide-specific IFN-γ-secreting T cells in tuberculosis patients during anti-tuberculosis chemotherapy [23,29].
This study has enabled us to map the immunodominant regions in ESAT-6 and CFP-10 in Zambian individuals. Differences in these ‘epitope maps’ between this population and others we have studied almost certainly reflect genetic differences in HLA backgrounds. However, certain general conclusions, with implications for the future role of these antigens as diagnostic tools or vaccine candidates, can be drawn. First, both ESAT-6 and CFP-10 contain multiple CD4 epitopes across their entire lengths; second, several immunodominant peptides are recognized by T cells from a very high percentage of donors, and are therefore probably permissively restricted by several different HLA class II molecules; third, many M. tuberculosis-infected individuals respond to several peptides from the same antigen.
This study has characterized the impact of HIV infection on M. tuberculosis-specific T cell populations in an area with a high prevalence of both pathogens, and has shed light on the potential clinical and epidemiological utility of the RD1-based ELISPOT assay in HIV-infected individuals. Clearly, the precise role of the assay will depend upon the prevalence of latent M. tuberculosis infection in a given population. The RD1-based ELISPOT maintains a sufficiently high sensitivity in HIV-positive patients to facilitate the rapid presumptive diagnosis of tuberculosis in patients with clinically suspected active disease in low prevalence populations, allowing the early initiation of therapy. In tuberculosis-endemic regions, the high proportion of latently infected individuals would preclude the application of this assay for the presumptive diagnosis of active tuberculosis in adults, but it could nonetheless be useful for diagnosis in children and epidemiologically to estimate the prevalence of latent M. tuberculosis infection. Our findings suggest that this application may be impaired in HIV-positive individuals, but to a lesser extent than the TST. If this is confirmed in larger studies, then this new T cell-based approach could contribute to tuberculosis control by improving the targeting of isoniazid preventative therapy to HIV-positive individuals with latent tuberculosis infection.
The authors would like to thank all patients and healthy donors who participated in this study, and also the medical and nursing staff of the Department of Medicine, University Teaching Hospital, Lusaka, Zambia, for their cooperation. They would also like to thank David Warrell, Chris Conlon and Geoffrey Pasvol for valuable help with the facilitation of this project, and they are also grateful to Paul Fine for helpful discussions.
Sponsorship: This work was supported by the Wellcome Trust.
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