Development of a proof of concept immunochromatographic lateral flow assay for point of care diagnosis of Mycobacterium tuberculosis
- Liya Wassie†1Email author,
- Markos Abebe†1,
- Abraham Aseffa1,
- Kidist Bobosha1,
- Martha Zewdie1,
- Menberwork Chanyalew1,
- Lawrence K Yamuah1,
- Arantxa Cortés2,
- Jose R González2,
- Jose M Delgado2,
- Ismail Ceyhan3,
- Ida Rosenkrands4,
- Karin Weldingh4,
- Peter Andersen4 and
- Timothy Mark Doherty4, 5
© Wassie et al.; licensee BioMed Central Ltd. 2013
Received: 5 September 2012
Accepted: 15 May 2013
Published: 21 May 2013
Despite major public health initiatives and the existence of efficacious treatment regimes, tuberculosis (TB) remains a threat, particularly in resource-limited settings. A significant part of the problem is the difficulty of rapidly identifying infected individuals, and as a result, there has been renewed interest in developing better diagnostics for infection or disease caused by Mycobacterium tuberculosis. Many of the existing tools, however, have limitations such as poor sensitivity or specificity, or the need for well-equipped laboratories to function effectively. Serodiagnostic approaches in particular have long drawn attention, due to their potential utility in large field studies, particularly in resource-poor settings. Unfortunately none of the serodiagnostic approaches have so far proven useful under field conditions.
We screened a large panel of antigens with serodiagnostic potential by ELISA and selected a subpanel that was strongly and broadly recognised by TB patients, but not by controls. These antigens were then formulated into a simple immuno-chromatographic lateral flow assay format, suitable for field use, and tested against panels of plasma and blood samples from individuals with different clinical status (confirmed TB patients, household contacts, and apparently healthy community controls), recruited from Ethiopia (a highly TB-endemic country) and Turkey (a TB meso-endemic country). While specificity was good (97-100% in non TB-endemic controls), the sensitivity was not as high as expected (46-54% in pulmonary TB, 25-29% in extra-pulmonary TB).
Though below the level of sensitivity the consortium had set for commercial development, the assay specifically identified M. tuberculosis-infected individuals, and provides a valuable proof of concept.
KeywordsIgG Antibody TB Antigen Serology Diagnosis ELISA
Although a recent report from World Health Organization (WHO) indicated a decline in the absolute incidence of TB cases, globally tuberculosis (TB) remains one of the most serious causes of infectious disease. It is responsible for about 9 million new cases and more than 1 million deaths (among HIV-negative cases of TB) in 2010 . In addition, Mycobacterium tuberculosis is estimated to infect about a third of the world population, and although the vast majority contain the infection without clinical manifestations of the disease [2, 3], they are at risk of later reactivation, and can act as reservoir for future disease . The majority of TB cases in developing countries are still diagnosed using smear microscopy and clinical examination, augmented by chest X-ray. The use of X-ray examination is often restricted to advanced pulmonary TB in resource-limited settings, and the sensitivity of smear microscopy is in the range of 22-80%, or even lower in children and extra-pulmonary TB patients . Newly-developed diagnostic tools such as whole blood interferon gamma (IFN-γ) release assays (IGRAs) and real time polymerase chain reaction (PCR)-based GenXpert [4, 6–8] are unsuitable for routine clinical use in resource poor settings, where the majority of TB cases occur, as many of these techniques require sophisticated laboratory set-ups, equipment and trained personnel. In particular, the lack of tests that can be used at point of care (POC) is a major impediment to effective TB diagnosis.
In recent years, TB serological tests have drawn attention because of their potential to be developed for POC use. Unlike cell-mediated immune responses, the humoral response to M. tuberculosis is often delayed and peaks much later, possibly coinciding with the onset of clinical disease . A specific, sensitive TB serodiagnostic test in a rapid test format could therefore constitute the ideal supplement for current diagnostic approaches in highly TB-endemic countries. Considerable progress has been made in the identification of M. tuberculosis antigens with the help of comparative genomics [10–12], and several promising antigens have been identified for use in serodiagnosis [13, 14]. The main objective of this study was to screen and evaluate a broad panel of M. tuberculosis antigens and identify candidates for construction of an antibody-based immuno-chromatographic lateral flow assay (LFA) in a field-friendly format applicable in point of care screening. The best candidates from the screening panel were then converted to a simple LFA, which could be read without the need for further laboratory equipment, and tested against blood and plasma samples from study cohorts with well-defined clinical status.
Study design and participants
119 HIV-negative TB patients recruited at Danish hospitals,
79 HIV-negative TB patients recruited at Ethiopian hospitals,
99 HIV-negative TB patients recruited at Turkish hospitals,
79 HIV-negative TB patients from Uganda obtained through the WHO/TDR Tuberculosis Specimen bank,
73 close household contacts (HIV-negative) recruited among the families of patients from Ethiopian hospitals,
48 Mantoux-positive, close household contacts (HIV-negative) recruited among the families of patients from Turkish hospitals,
79 non-endemic healthy, HIV-negative controls with no known exposure to TB, recruited from the same communities in Denmark as the TB patients,
58 TB-endemic HIV-negative healthy controls, recruited from the same communities in Ethiopia as the TB patients,
153 TB-endemic HIV-negative healthy controls, recruited from the same communities in Turkey as the TB patients,
8 extra-pulmonary TB patients and 8 symptomatic non-TB community controls, recruited from Turkey and
31 extra-pulmonary TB patients from Ethiopia.
The TB patients were newly diagnosed, bacteriologically confirmed (smear- and/or culture positive, from sputum or needle aspirate) clinical cases with persistent cough for more than 2 weeks, accompanied by signs and symptoms of TB such as productive cough, loss of appetite, weight loss, night sweat or haemoptysis, enlarged lymph nodes or abnormal chest X-ray. Household contacts were defined as individuals who had been living with pulmonary TB (PTB) patients in the same household at least for the past 6 months before recruitment into the study. The majority of these cases were apparently healthy, asymptomatic, and had normal chest X-rays. However, 12 cases had complaints of cough and abnormal chest X-ray (16%), albeit not serious or specific enough for them to be diagnosed as having TB, and were sputum negative. The control groups were apparently healthy volunteers, living in the same community with TB patients. All participants were screened for HIV and were negative. This study received ethical approval from institutional and national ethical review committees of all partners involved: the Statens Serum Institute IRB, København Kommunes Videnskabsetiske Komité in Copenhagen, the Scientific Ethics Committee in Refik Saydam National Hygiene Centre, Ankara and the National Health Science and Technology Council and ethics review committees in Addis Ababa (the AHRI/ALERT Ethics Review Committee and the National Research Ethics Review Committee of Ethiopia).
Antigen selection and plasma sample preparation
Percent antibody positive responders by ELISA in different population groups to candidate antigens
Healthy controls (Denmark, n = 79)
TB patients (Denmark, n = 119)
TB patients (Uganda, n = 77)
Healthy controls (Ethiopia, n = 58)
TB patients (Ethiopia n = 79)
Since the antigens were expressed in an E. coli vector, all plasma samples were adsorbed against E. coli Extract (Promega, San Luis Obispo, CA, USA), to reduce nonspecific responses, according to the manufacturer’s recommendations. Briefly, 20 μl of E. coli extract was added to 200 μl of plasma sample, incubated overnight (O/N) at room temperature (RT), with gentle shaking. The day after, the mix was centrifuged at 10,000 rpm for 10 min and the supernatant transferred into new tubes, where it was treated with 0.05% sodium azide to prevent growth of contaminating bacteria. The samples were then kept at 4°C until use. As a control, a subset of plasma samples were tested against the test antigens before and after adsorption (Additional file 1).
In short, antigens were coated in duplicate onto 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark) at concentrations ranging from 5 to 0. 1 μg/ml in 100 μl of 0.05 M carbonate buffer (pH 9.6) and then incubated overnight at 4°C. After the plates were coated, they were washed three times with phosphate-buffered saline (PBS; pH 7.2)–0.05% Tween 20 (PBS-T), and serum samples diluted 1:100 in 0.2% PBS-T and 1% bovine serum albumin were applied and incubated for 1 hour at room temperature. The plates were then washed with PBS-T, and then incubated for 1 hour with rabbit anti-human immunoglobulin G antibody conjugated with horseradish peroxidase (D0336; Dako, Hillerød, Denmark) diluted 1:1,000 in PBS-T. The plates were washed with PBS-T and enzyme activity was assayed by incubation for 30 min at room temperature with 100 μl of tetramethylbenzidine peroxidase substrate (Bio-Rad, Hercules, Calif.) per well. To stop the reaction, 100 μl of 4 N sulfuric acid was added and the optical density (OD) was measured at 405 nm. In each experimental plate, known concentration of purified human IgG immunoglobulin (rhIgG) (SIGMA) was serially diluted and run as standard (positive control) along with samples and blank wells as negative controls. OD values were then measured, concentrations extrapolated against known concentration of standard (rhIgG) using GraphPad Prism Software (Version 4.0).
Multi-antigen print immunoassay
Antibody determinations were done by multi-antigen print immunoassay (MAPIA) as described previously . Briefly, the 4 antigens of interest were immobilized on nitrocellulose membrane (Schleicher & Schuell, Keene, NH) at a concentration of 0.05 mg/ml using a semi-automated microsprayer (Linomat IV, Camag Scientific Inc., Wilmington, DE). The membrane was cut perpendicular to the antigen bands into 4 mm wide strips, blocked (1 h) with 1% non-fat skim milk in PBS-T and then incubated for 1 h with serum samples diluted 1:50 in blocking solution. After washing, strips were incubated (1 h) with alkaline phosphatase-conjugated anti-human IgG or anti-human IgM antibody (Sigma Chemical Co., St. Louis, MO) diluted 1:5000 in blocking solution, followed by washing. Bound antibodies were visualized with BCIP/NBT (Kirkegaard & Perry Laboratories, Gaithersburg, MD).
Lateral flow assay (LFA) production
Application of selected antigens to the lateral-flow format
Once the basic parameters were established, the prototype LFAs was assessed for stability and reproducibility in use. Stability was tested at room temperature and at 2-8°C as well as via an accelerated stability study. The stability studies at room temperature (20-25°C) were assessed at 15 days, 1 month, 3 months and then every subsequent three months for 18 months. The performance of the kit at 2-8°C was tested every three months for 18 months. The accelerated stability study was carried out by incubating the LFAs at 37°C and 55°C in a humid atmosphere. Testing was performed at 3, 7, 10, 15, 21, and 30 days at 37°C as well as at 3, 7, 10, and 15 days at 55°C. On each of the indicated days, dilutions of the positive sera pool (from 1:1 to 1:16) were analyzed in duplicate alongside the undiluted negative control pool. For LFAs stored at room temperature, 2-8°C or at 37°C, there was no significant decay of the signal during the testing period, while at 55°C, the intensity of the signal decreased after the seventh day, such that samples at or below 1/4 dilution were clearly weaker in color intensity than the same samples assessed earlier in the study. Finally, reproducibility was tested on three different days, performed with three different sample titrations, and a positive and negative control. The prototype showed excellent reproducibility across different days, samples and operators (Additional file 4). Following these assessments, the LFA test kits were sent to Turkey and Ethiopia for testing under field conditions.
Raw data was measured as OD values and the concentration of IgG in each sample was extrapolated from the standard curve drawn from human rhIgG standard, using GraphPad Prism Software (Version 4.0), with assumption of a non-linear regression model and Sigmoidal-dose response (variable slope) equation. Comparison between groups was done using one-way ANOVA with the assumption of Kruskal-Wallis test statistics. A p-value < 0.05 was taken as indicating statistical significance.
Results of the lateral flow test under field conditions
Individuals responding to candidate antigens from clinical cohorts in Turkey and Ethiopia
Results from Turkish clinical cohorts
Number (percentage) responding to antigen
TB patients (n = 99)
Pulmonary (n = 91)
Extra-pulmonary (n = 8)
TB contacts (n = 48)
PPD+, known exposure (n = 48)
Community controls (n = 153)
Symptomatic, non-TB (n = 8)
Healthy, no identified exposure (n = 145)
Results from Ethiopian clinical cohorts
Number (percentage) responding to antigen
TB patients (n = 79)
Pulmonary (n = 48)
Extra-pulmonary (n = 31)
TB contacts (n = 73)
Known exposure (n = 73)
Community controls (n = 60)
Healthy, no identified exposure (n = 60)
In Ethiopia, the results were very similar. Of the 79 index cases, 48% were positive for one or more antigens, compared to 29% of household contacts and 27% of community controls. The Ethiopian TB patient cohort included 48 pulmonary tuberculosis patients and 31 extra-pulmonary tuberculosis patients with needle-aspirate confirmed tuberculosis lymphadenitis, but although the percentage of responders were lower in the latter group, there were no clear differences between the groups in terms of antigen recognition in the LFA. The observation that a significant number of healthy controls were positive for one or more antigens in the LFA is the only point at which the Ethiopian data diverge from the Turkish data set, and this is consistent with earlier studies in this highly TB-endemic community, which have suggested that over 30% of adult Ethiopian community controls may be latently infected . Mantoux results are not available from Ethiopia as this test is not routinely used in the local clinics, but the results suggest that the test does not differentiate latent and acute M. tuberculosis infection. Again, in all cohorts MPT83: Acry was by far the most frequently recognised target, which was unexpected, given the high seropositivity to ESAT-6: CFP10 observed in this population by ELISA . The results (Tables 1 and 2) suggest that the findings seen with the LFA are a result of this particular test formulation’s technical performance, not due to an absence of specific antibody in the participants in the study.
In resource-poor settings, the diagnosis of TB is primarily based on identification of mycobacteria by sputum smear and on clinical evidence. However, bacteriological examination is often time-consuming and relatively insensitive. Better tools are available, but are often impractical due to their cost, technical requirements or the long waiting times required for the diagnostic result. Although the potential for antibody-based diagnostic tools to give a quick answer using a simple blood draw has made them attractive alternatives for TB diagnosis, current test formats have proven ineffective, due to their low sensitivity and specificity  and the fact that many of them still require laboratory infrastructure. Concern over the poor accuracy of available commercial serological tests has been raised by the WHO [23, 24]. The single most important factor for the failure of the current serological tests is the choice of antigens used, the majority of which are shared with other mycobacteria leading to low specificity in individuals living in the developing countries. Many of the antigens are also relatively weakly recognized, accounting for the poor sensitivity of these tests.
We have addressed these problems systematically, by screening a broad panel of antigens and select those which were best recognized [15–18]. We also identified that antigens that had limited specificity for M. tuberculosis infection, by screening with negative controls from both TB-endemic and non-endemic countries. The selected antigens were used to develop an LFA, with the goal of providing a serological test that was inexpensive, simple and suitable for point of care use. Since issues regarding sensitivity and specificity are the major factors that determine the applicability of such test kits , particularly in resource limited settings, validation of such tools in different settings is warranted.
Several important observations have been made in the course of this work. Consistent with prior reports  although a higher percentage of TB patients had a significant antibody response to the specific antigens chosen for the LFA in the ELISA, there were still a significant number of healthy controls from TB-endemic populations who made positive responses to these antigens (Table 1). Given that prior studies have indicated a high rate of latent infection in this population  and the low percentage of responders to these antigens among healthy controls from a non-TB-endemic country, it is likely that this response is indicative of latent TB infection, rather than cross-reactivity. Nonetheless, it poses a problem for using such tests in TB-endemic settings, where the ideal would be to identify active or incipient TB, not just M. tuberculosis infection.
Interestingly, the hierarchy of responses observed in ELISA did not predict the outcome in the LFA. When ranked in terms of the percentage of TB patients who made a significant antibody response, TB patients from TB-endemic regions (Uganda and Ethiopia) responded most frequently in the ELISA to the ESAT-6: CFP10 dimer, with a lower percentage responding to the MPT83: Acry dimer. When testing these same antigens in the LFA, this response was completely reversed, with a greatest number of responders being found to the MPT83: Acry dimer. It is not clear if this is a question of avidity, conformational change when the antigens are bound, or some as yet unidentified interference, but it strongly suggests that screening results using ELISA may not always be predictive for other assay formats, including LFA. In addition, we did not recruit sufficient HIV positive individuals to conduct any meaningful analyses in this study, but given the high risk of TB in HIV positive individuals, this is certainly an area that should be further explored.
The results presented here represent one of the few reports on the testing of an LFA designed to diagnose human TB and provide an important proof of concept. It should be noted that despite the relatively low sensitivity, the test does appear to have a high specificity (over 95%, when assessed against the negative controls from Denmark and Spain, who can be safely assumed to be mostly free of M. tuberculosis infection). Since the molecules most frequently recognised in these studies (ESAT-6: CFP10 and MPT83: Acry) gave a sensitivity of over 90% in an LFA developed for primate use , these results suggest that with further optimisation, a point of care LFA test could be developed for rapid diagnosis of human TB, perhaps using different antigens [27, 28]. It has been suggested that allowing serum antibodies to bind to antigen prior to detecting the antibody-antigen complexes as a second step (as opposed to having antibodies interact with the conjugate prior to antigen binding, as occurs in this assay), can improve the sensitivity of the assay and it may also be possible to improve the signalling conjugate used. Given the ongoing need for a simple, inexpensive point of care test for TB, these options should be further investigated.
Butajira Hospital is acknowledged for facilitating recruitment of study participants. We also would like to extend our appreciation to nurses and physicians who assisted in recruiting the study participants. This study was funded by the European Union through the SERO-TB project (Contract No. 037796), which was a joint consortium project between institutions in Turkey, Spain, Denmark and Ethiopia. The Armauer Hansen Research Institute is supported by core grants from Sida and NORAD.
- Global Tuberculosis Control: Epidemiology, strategy, financing. 2009, Geneva: WHO ReportGoogle Scholar
- Glassroth J: Clinical considerations in designing trials of vaccines for tuberculosis. Clin Infect Dis. 2000, 30 (Suppl 3): 229-232.View ArticleGoogle Scholar
- van Crevel R, Ottenhoff TH, van der Meer JW: Innate immunity to Mycobacterium tuberculosis. Clin Microbiol Rev. 2002, 15: 294-309. 10.1128/CMR.15.2.294-309.2002.PubMedPubMed CentralView ArticleGoogle Scholar
- Kaufmann SH, Parida SK: Tuberculosis in Africa: learning from pathogenesis for biomarker identification. Cell Host Microbe. 2008, 4: 219-228. 10.1016/j.chom.2008.08.002.PubMedView ArticleGoogle Scholar
- Steingart KR, Ng V, Henry M, Hopewell PC, Ramsay A, Cunningham J, Urbanczik R, Perkins MD, Aziz MA, Pai M: Sputum processing methods to improve the sensitivity of smear microscopy for tuberculosis: a systematic review. Lancet Infect Dis. 2006, 6: 664-674. 10.1016/S1473-3099(06)70602-8.PubMedView ArticleGoogle Scholar
- Diel R, Loddenkemper R, Meywald-Walter K, Niemann S, Nienhaus A: Predictive value of a whole blood IFN-gamma assay for the development of active tuberculosis disease after recent infection with Mycobacterium tuberculosis. Am J Respir Crit Care Med. 2008, 177: 1164-1170. 10.1164/rccm.200711-1613OC.PubMedView ArticleGoogle Scholar
- Menzies D, Pai M, Comstock G: Meta-analysis: new tests for the diagnosis of latent tuberculosis infection: areas of uncertainty and recommendations for research. Ann Intern Med. 2007, 146: 340-354. 10.7326/0003-4819-146-5-200703060-00006.PubMedView ArticleGoogle Scholar
- Moure R, Munoz L, Torres M, Santin M, Martin R, Alcaide F: Rapid detection of Mycobacterium tuberculosis complex and rifampin resistance in smear-negative clinical samples by use of an integrated real-time PCR method. J Clin Microbiol. 2011, 49: 1137-1139. 10.1128/JCM.01831-10.PubMedPubMed CentralView ArticleGoogle Scholar
- Steingart KR, Henry M, Laal S, Hopewell PC, Ramsay A, Menzies D, Cunningham J, Weldingh K, Pai M: Commercial serological antibody detection tests for the diagnosis of pulmonary tuberculosis: a systematic review. PLoS Med. 2007, 4: e202-10.1371/journal.pmed.0040202.PubMedPubMed CentralView ArticleGoogle Scholar
- Behr MA, Wilson MA, Gill WP, Salamon H, Schoolnik GK, Rane S, Small PM: Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science. 1999, 284: 1520-1523. 10.1126/science.284.5419.1520.PubMedView ArticleGoogle Scholar
- Brosch R, Gordon SV, Billault A, Garnier T, Eiglmeier K, Soravito C, Barrell BG, Cole ST: Use of a Mycobacterium tuberculosis H37Rv bacterial artificial chromosome library for genome mapping, sequencing, and comparative genomics. Infect Immun. 1998, 66: 2221-2229.PubMedPubMed CentralGoogle Scholar
- Mahairas GG, Sabo PJ, Hickey MJ, Singh DC, Stover CK: Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J Bacteriol. 1996, 178: 1274-1282.PubMedPubMed CentralGoogle Scholar
- Brusasca PN, Colangeli R, Lyashchenko KP, Zhao X, Vogelstein M, Spencer JS, McMurray DN, Gennaro ML: Immunological characterization of antigens encoded by the RD1 region of the Mycobacterium tuberculosis genome. Scand J Immunol. 2001, 54: 448-452. 10.1046/j.1365-3083.2001.00975.x.PubMedView ArticleGoogle Scholar
- Houghton RL, Lodes MJ, Dillon DC, Reynolds LD, Day CH, McNeill PD, Hendrickson RC, Skeiky YA, Sampaio DP, Badaro R, Lyashchenko KP, Reed SG: Use of multiepitope polyproteins in serodiagnosis of active tuberculosis. Clin Diagn Lab Immunol. 2002, 9: 883-891.PubMedPubMed CentralGoogle Scholar
- Hoff ST, Abebe M, Ravn P, Range N, Malenganisho W, Rodriques DS, Kallas EG, Soborg C, Mark Doherty T, Andersen P, Weldingh K: Evaluation of Mycobacterium tuberculosis-specific antibody responses in populations with different levels of exposure from Tanzania, Ethiopia, Brazil, and Denmark. Clin Infect Dis. 2007, 45: 575-582. 10.1086/520662.PubMedView ArticleGoogle Scholar
- Skjot RL, Oettinger T, Rosenkrands I, Ravn P, Brock I, Jacobsen S, Andersen P: Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens. Infect Immun. 2000, 68: 214-220. 10.1128/IAI.68.1.214-220.2000.PubMedPubMed CentralView ArticleGoogle Scholar
- Weldingh K, Hansen A, Jacobsen S, Andersen P: High resolution electroelution of polyacrylamide gels for the purification of single proteins from Mycobacterium tuberculosis culture filtrate. Scand J Immunol. 2000, 51: 79-86. 10.1046/j.1365-3083.2000.00655.x.PubMedView ArticleGoogle Scholar
- Weldingh K, Rosenkrands I, Okkels LM, Doherty TM, Andersen P: Assessing the serodiagnostic potential of 35 Mycobacterium tuberculosis proteins and identification of four novel serological antigens. J Clin Microbiol. 2005, 43: 57-65. 10.1128/JCM.43.1.57-65.2005.PubMedPubMed CentralView ArticleGoogle Scholar
- Rosenkrands I, Aagaard C, Weldingh K, Brock I, Dziegiel MH, Singh M, Hoff S, Ravn P, Andersen P: Identification of Rv0222 from RD4 as a novel serodiagnostic target for tuberculosis. Tuberculosis (Edinb). 2008, 88: 335-343. 10.1016/j.tube.2007.12.001.View ArticleGoogle Scholar
- Lyashchenko KP, Singh M, Colangeli R, Gennaro ML: A multi-antigen print immunoassay for the development of serological diagnosis of infectious diseases. J Immunol Methods. 2000, 242: 91-100. 10.1016/S0022-1759(00)00241-6.PubMedView ArticleGoogle Scholar
- Piefer AJ, Jonsson CB: A comparative study of the human T-cell leukemia virus type 2 integrase expressed in and purified from Escherichia coli and Pichia pastoris. Protein Expr Purif. 2002, 25: 291-299. 10.1016/S1046-5928(02)00011-6.PubMedView ArticleGoogle Scholar
- Demissie A, Leyten EM, Abebe M, Wassie L, Aseffa A, Abate G, Fletcher H, Owiafe P, Hill PC, Brookes R, alRook G, Zumla A, Arend SM, Klein M, Ottenhoff TH, Andersen P, Doherty TM, VACSEL Study Group: Recognition of stage-specific mycobacterial antigens differentiates between acute and latent infections with Mycobacterium tuberculosis. Clin Vaccine Immunol. 2006, 13: 179-186. 10.1128/CVI.13.2.179-186.2006.PubMedPubMed CentralView ArticleGoogle Scholar
- Steingart KR, Ramsay A, Pai M: Commercial serological tests for the diagnosis of tuberculosis: do they work?. Future Microbiol. 2007, 2: 355-359. 10.2217/174609126.96.36.1995.PubMedView ArticleGoogle Scholar
- Morris K: WHO recommends against inaccurate tuberculosis tests. Lancet. 2011, 377: 113-114. 10.1016/S0140-6736(11)60005-6.PubMedView ArticleGoogle Scholar
- Keeler E, Perkins MD, Small P, Hanson C, Reed S, Cunningham J, Aledort JE, Hillborne L, Rafael ME, Girosi F, Dye C: Reducing the global burden of tuberculosis: the contribution of improved diagnostics. Nature. 2006, 444 (Suppl 1): 49-57.PubMedView ArticleGoogle Scholar
- Lyashchenko KP, Greenwald R, Esfandiari J, Meylan M, Burri IH, Zanolari P: Antibody responses in new world camelids with tuberculosis caused by Mycobacterium microti. Vet Microbiol. 2007, 125: 265-273. 10.1016/j.vetmic.2007.05.026.PubMedView ArticleGoogle Scholar
- Ireton GC, Greenwald R, Liang H, Esfandiari J, Lyashchenko KP, Reed SG: Identification of Mycobacterium tuberculosis antigens of high serodiagnostic value. Clin Vaccine Immunol. 2010, 17: 1539-1547. 10.1128/CVI.00198-10.PubMedPubMed CentralView ArticleGoogle Scholar
- Khan N, Alam K, Nair S, Valluri VL, Murthy KJ, Mukhopadhyay S: Association of strong immune responses to PPE protein Rv1168c with active tuberculosis. Clin Vaccine Immunol. 2008, 15: 974-980. 10.1128/CVI.00485-07.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.