CLINICAL AND VACCINE IMMUNOLOGY, Feb. 2009, p. 260276 1556-6811/09/$08.00 0 doi:10.1128/CVI.00355-08 Copyright 2009, American Society for Microbiology.

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Vol. 16, No. 2

Performance of Puried Antigens for Serodiagnosis of Pulmonary Tuberculosis: a Meta-Analysis

Karen R. Steingart,1* Nandini Dendukuri,2 Megan Henry,3 Ian Schiller,2 Payam Nahid,4 Philip C. Hopewell,1,4 Andrew Ramsay,5 Madhukar Pai,2 and Suman Laal6,7,8
Francis J. Curry National Tuberculosis Center, University of California, San Francisco, California1; Department of Epidemiology, Biostatistics & Occupational Health, McGill University, Montreal, Quebec, Canada2; San Joaquin County Public Health Services, Stockton, California3; Division of Pulmonary and Critical Care Medicine, San Francisco General Hospital, University of California, San Francisco, California4; UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, World Health Organization, Geneva, Switzerland5; Departments of Pathology6 and Microbiology,7 New York University Langone Medical Center, New York, New York; and Veterans Affairs Medical Center, New York, New York8
Received 26 September 2008/Returned for modication 4 November 2008/Accepted 24 November 2008

Serological antibody detection tests for tuberculosis may offer the potential to improve diagnosis. Recent metaanalyses have shown that commercially available tests have variable accuracies and a limited clinical role. We reviewed the immunodiagnostic potential of antigens evaluated in research laboratories (in-house) for the serodiagnosis of pulmonary tuberculosis and conducted a meta-analysis to evaluate the performance of comparable antigens. Selection criteria included the participation of at least 25 pulmonary tuberculosis patients and the use of puried antigens. Studies evaluating 38 kDa, MPT51, malate synthase, culture ltrate protein 10, TbF6, antigen 85B, -crystallin, 2,3-diacyltrehalose, 2,3,6-triacyltrehalose, 2,3,6,6 -tetraacyltrehalose 2 -sulfate, cord factor, and TbF6 plus DPEP (multiple antigen) were included in the meta-analysis. The results demonstrated that (i) in sputum smear-positive patients, sensitivities signicantly >50% were provided for recombinant malate synthase (73%; 95% condence interval [CI], 58 to 85) and TbF6 plus DPEP (75%; 95% CI, 50 to 91); (ii) protein antigens achieved high specicities; (iii) among the lipid antigens, cord factor had the best overall performance (sensitivity, 69% [95% CI, 28 to 94]; specicity, 91% [95% CI, 78 to 97]); (iv) compared with the sensitivities achieved with single antigens (median sensitivity, 53%; range, 2% to 100%), multiple antigens yielded higher sensitivities (median sensitivity, 76%; range, 16% to 96%); (v) in human immunodeciency virus (HIV)-infected patients who are sputum smear positive, antibodies to several single and multiple antigens were detected; and (vi) data on seroreactivity to antigens in sputum smear-negative or pediatric patients were insufcient. Potential candidate antigens for an antibody detection test for pulmonary tuberculosis in HIV-infected and -uninfected patients have been identied, although no antigen achieves sufcient sensitivity to replace sputum smear microscopy. Combinations of select antigens provide higher sensitivities than single antigens. The use of a case-control design with healthy controls for the majority of studies was a limitation of the review. Efforts are needed to improve the methodological quality of tuberculosis diagnostic studies.

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The failure to diagnose tuberculosis (TB) accurately and rapidly is a key challenge in curbing the epidemic (45, 88, 116). Sputum microscopy, currently the sole diagnostic test in most areas where TB is endemic, has several limitations; in particular, the sensitivity compared with that of culture is variable (80, 97, 104, 116), multiple patient visits are required (56, 93, 114), considerable technical training is necessary, and the procedure is labor-intensive (45, 65). Antibody detection tests (serological tests) are used for the diagnosis of many infectious diseases and could potentially improve the means of diagnosis of TB. These tests measure the presence of specic antibodies (most often immunoglobulin G [IgG]) directed against immu* Corresponding author. Mailing address: Francis J. Curry National Tuberculosis Center, University of California, San Francisco, 3180 18th Street, Suite 101, San Francisco, CA 94110-2028. Phone: (415) 502-4600. Fax: (415) 502-4620. E-mail: karenst@u.washington.edu. Supplemental material for this article may be found at http://cvi .asm.org/. Present address: California Department of Public Health, Sacramento, CA. Published ahead of print on 3 December 2008. 260

nodominant antigens of the pathogen in question. Compared with microscopy, antibody detection methods may enable the rapid diagnosis of TB, as these tests have the advantages of speed (results can be available within hours), technological simplicity, and minimal training requirements. In addition, these tests can be adapted to point-of-care formats that can be implemented at lower levels of health services in low- and middle-income countries (21, 22, 57, 65). Efforts to develop antibody detection tests for the diagnosis of TB have been under way for decades, and the performance of these tests has been well described (13, 17, 22, 32, 40, 47, 48, 52, 60, 64, 100, 107). Several systematic reviews of these tests have been published (discussed below) (28, 94, 95). First-generation antibody detection tests were based on crude mixtures of constituents and products of Mycobacterium tuberculosis, for example, culture ltrate proteins and puried protein derivative, the preparation used in the tuberculin skin test. Several of these early tests had low specicities, as the tests contained antigens shared among different bacterial species (1, 22, 48, 57). During the past two decades, an increased understanding of humoral immune responses to M. tuberculosis and the new tools of

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FIG. 1. Flow diagram for selection of subgroups, IgG and/or IgA antibody detection: an example with MPT51. The same sequence of steps was repeated for each antigen. Having at least four studies available was a condition for inclusion in the meta-analysis. , other antibody combinations included IgG and IgM (n 3 studies); IgM and IgA (n 0); IgG, IgA, and IgM (n 7); and not reported (n 10); , other recombinant antigens included 38 kDa (n 13 studies), CFP-10 (n 9), malate synthase (n 8), TbF6 (n 4), -cystallin (n 4), Mtb48 (n 3), Ag85C (n 2), DPEP (n 2), ESAT6 (n 2), and other antigens (n 16) that appeared in only one study each.

genomics and proteomics have led to the discovery of new antigens reported to provide improved sensitivities and specicities for the diagnosis of TB compared with those achieved with the antigens in the rst-generation tests (48). We reviewed the immunodiagnostic potential of different antigens evaluated in research laboratories (in-house) for the serodiagnosis of pulmonary TB and carried out a meta-analysis to evaluate the performance of various antigens singly and in combination. Previous meta-analyses have shown that commercially available serological tests for both pulmonary TB (94) and extrapulmonary TB (95) have variable accuracies and, consequently, a limited clinical role. Another systematic review (searches through 2003) limited studies to the cohort or case series type of design and included only nine studies relating to in-house anti-TB antibody serological tests (28). A recently published expert review (1) did not include a meta-analysis. We are unaware of other systematic reviews on this topic. The current review addresses the following questions. (i) What is the performance of different antigens in the serodiagnosis of pulmonary TB in sputum smear-positive and smearnegative patients? (ii) What is the performance of these antigens in the serodiagnosis of pulmonary TB in patients with human immunodeciency virus (HIV) infection?
MATERIALS AND METHODS Standard guidelines and methods for systematic reviews and meta-analyses of diagnostic tests were followed (25, 31, 61). The following electronic databases

(1990 to November 7, 2007) were queried for primary studies in the English language: PubMed, EMBASE, Biosis, and Web of Science. The search terms included tuberculosis, Mycobacterium tuberculosis, immunological tests, serological tests, antibody detection, antigen detection, ELISA (enzyme-linked immunosorbent assay), Western blot, and sensitivity and specicity. Additional studies were identied by contacting experts and searching the reference lists of primary studies and review articles. The criteria for including studies for the review were as follows. Cross-sectional and case-control study designs were eligible. The sample size had to be at least 25 patients with sputum smear-positive or smear-negative pulmonary TB who provided sera before or within 14 days of receiving antituberculous treatment. For comparison with TB patients, we selected only one group for each study, preferentially, patients in whom pulmonary TB was initially suspected but was later ruled out, as opposed to healthy participants. The index test (serological antibody detection) had to be evaluated in-house with puried antigens; studies that used puried protein derivative, culture ltrates, or sonicated antigens were not included. The reference standard was either the isolation of M. tuberculosis on sputum culture or, for studies conducted in countries where TB is endemic ( 20 cases per 100,000 population in 2005) where cultures are not routinely performed, the presence of acid-fast bacilli detected by sputum smear microscopy (16, 115). For the determination of outcome measures, there had to be sufcient data to construct a two-by-two table for calculations of sensitivity, specicity, and likelihood ratios. The following studies were excluded: (i) studies whose results were published before 1990, for the reason that many studies used crude antigen extracts or obsolete methods; (ii) studies of latent M. tuberculosis infection; (iii) studies of nontuberculous mycobacteria; (iv) studies describing nonimmunologic methods for the detection of antibodies; (v) studies in the basic science literature concerning cloning of new antigens or their immunologic properties (e.g., epitope mapping); and (vi) case reports and reviews. Study selection. Initially, two reviewers independently screened citations retrieved from all sources for relevance. Screening of full-text articles by using prespecied inclusion criteria was carried out by two reviewers, and the articles

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FIG. 2. Flow of studies through the review process. PTB, pulmonary tuberculosis.

included were checked by a third reviewer. Disagreements were resolved by consensus. Data extraction. A data extraction form was created and pilot tested with a subset of eligible studies and then nalized. Two reviewers (each of whom was responsible for approximately 50% of the studies) extracted data from all included studies with the standardized form. To verify reproducibility, a third reviewer independently performed data extraction on all studies. Differences among reviewers were resolved by consensus. When necessary, authors were contacted for additional information. Assessment of study quality. The quality of studies was appraised by using a subset of criteria from QUADAS, a validated tool for diagnostic studies (see Table S1 in the supplemental material) (110). Antigen classication. Antigens were classied into ve categories according to the type of compound: (i) recombinant proteins, (ii) native proteins, (iii) lipids, (iv) multiple antigens (protein-protein or lipid-lipid additive reactivity), and (v) protein-lipid antigens. Several investigators evaluated antibody responses to multiple antigens in the same patient population to enhance sensitivity. These studies have taken two approaches. In some cases, different antigens (or portions thereof) have been cloned as single protein entities (polyproteins) and tested for their reactivities with sera. In other cases, multiple antigens have been tested as single entities and cumulative results (additive reactivity) were calculated. In the former case, we considered polyproteins to be single antigens; in the latter case, we classied the entities as multiple antigens. Data analysis. Estimates of sensitivity and specicity from individual studies and their exact 95% condence intervals (CIs) were obtained by using MetaDiSc (version 1.4) software (117). Sensitivity refers to the proportion of TB patients with positive test results; specicity refers to the proportion of participants without TB with negative test results. For sensitivity, we included studies that used sputum smear as the reference standard along with studies that used culture. For specicity, we noted the type of comparison group, e.g., healthy

participants or patients with nontuberculous respiratory disease. Likelihood ratio positive was calculated as sensitivity/(1 specicity); likelihood ratio negative was calculated as (1 sensitivity)/specicity. Selection of subgroups for meta-analysis. We recognized that studies were heterogeneous in many respects, particularly concerning antigen characteristics and antibody class. Therefore, in order to address heterogeneity and combine study results, subgroups of comparable antigens were determined (Fig. 1). Initially, studies were grouped by the class of antibody detected by the test: (i) IgG and/or IgA, (ii) IgM, and (iii) other IgM-containing combinations (IgM-IgG, IgM-IgA, and IgM-IgG-IgA). This division was based on the understanding that IgM antibodies are expressed transiently and earlier in infection than other antibodies. Next, studies were stratied by antigen number (single or multiple antigens); the type of compound (protein or lipid); and, for proteins, the source of the compound (recombinant or native). Finally, for each distinct single antigen or multiple-antigen combination, studies were stratied by patient sputum smear status and HIV status. At least four studies were required to be available for inclusion in a subgroup in order to strengthen the results and reduce the possibility of nding a signicant result by chance. In this way, we identied 16 subgroups. To summarize sensitivity and specicity within each subgroup, separate metaanalyses were performed by using the hierarchical summary receiver operating characteristic curve model (72). The advantages of the hierarchical summary receiver operating characteristic are that it jointly models both sensitivity and specicity, weights studies according to the number of participants, and takes into account unmeasured heterogeneity between studies by using random effects (31). The model was estimated by using a Bayesian approach with noninformative prior distributions and was implemented with WinBUGS (version 1.4.1) software program (91). The average sensitivity, specicity, and likelihood ratios from each metaanalysis were estimated. From the posterior distribution of each parameter of

VOL. 16, 2009 TABLE 1. Guide to tables and gures in the review
Category or antigen

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263

TABLE 2. Antigens evaluated for serodiagnosis of pulmonary TB

Name(s) of antigen(s)a Protein Rv designation Reference(s)

Table or Figure

Antigen names ...........................................................................Table 2 Characteristics of study quality ................................................Table 3 Meta-analysis..............................................................................Table 4 Recombinant malate synthase .................................................Table 5 Recombinant CFP-10................................................................Table 6 DAT ............................................................................................Table 7 Specicity estimates by control group.....................................Table 8 Antigen performance by Ig class .............................................Table 9 Recombinant 38 kDa ................................................................Table A1 Recombinant MPT51 ................................................................Table A2 Native 38 kDa ............................................................................Table A3 Native Ag85B .............................................................................Table A4 Questions for quality assessment.............................................Table S1 Recombinant protein antigens.................................................Table S2 Native protein antigens.............................................................Table S3 Lipid antigens.............................................................................Table S4 Multiple antigens .......................................................................Table S5 Protein/lipid antigens ................................................................Table S6 Flow diagram for selection of subgroups ...............................Figure 1 Flow of studies in the review ...................................................Figure 2 SROC curve for recombinant proteins...................................Figure 3A SROC curve for native proteins ..............................................Figure 3B SROC curve for lipid antigens.................................................Figure 3C Sensitivity, TB-HIV coinfection...............................................Figure 4A Specicity, TB-HIV coinfection...............................................Figure 4B

Ag85C, 32.5 kDa, FbpC2, MPT-45 38 kDa, Ag 5, PstS1, PhoS, PhoS1

0129c 0934

Mtb 81/88 kDa, malate synthase, GlcB DPEP, MPT32; 45/47 kDa, Apa, ModD Ag85B 16 kDa; -crystallin; 14 kDa, HspX, Acr 27 kDa; MPT51, FbpC1, MPB 51 CFP-10, MTSA-10, EsxB; Lhp, Mtb11 ESAT-6 Mtb48 DAT TAT SL-I, sulfolipid I Cord factor
a

1837c 1860 1886c 2031c 3803c 3874 3875 3881c

76, 77 3, 8, 15, 18, 20, 27, 33, 35, 37, 55, 59, 69, 76, 77, 81, 102 20, 35, 37, 76, 85, 86, 108 19, 26, 76 23, 67, 77, 103 33, 39, 66, 103 10, 69, 85, 108 27, 33, 59, 118 33, 55 43, 43, 43, 43, 118 44, 83, 105 44 44 44, 101

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Boldface indicates the name of the antigen in common use.

interest, we extracted the mean and the 95% credible interval (the Bayesian equivalent of the classical condence interval) on the basis of the 2.5% and 97.5% quartiles. When feasible, specicity estimates were stratied by type of comparison group. Finally, a summary receiver operating characteristic (SROC) curve from each meta-analysis was obtained. The SROC curve plots sensitivity versus 1 specicity for the range of specicity values observed for each study, as extrapolation beyond this range is not advisable (42). The SROC curve gives an idea of the overall performance of a test across different thresholds (54, 61). The closer that the curve is to the upper-left-hand corner of the plot (sensitivity and specicity are both 100%), the better the performance of the test is (42). The plots were made by using the R (version 2.6.1) software program (70). Descriptive analysis. Descriptive analyses were performed by using SPSS (version 14.0.1.366) software (92). Forest plots were made by using Meta-DiSc (version 1.4) software (117).

RESULTS Description of studies included. The literature searches identied over 5,000 citations, of which 49 publications (254 studies) were included (Fig. 2) (24, 6, 8, 10, 12, 15, 1820, 23, 24, 26, 27, 29, 3337, 39, 43, 44, 55, 58, 59, 6669, 73, 76, 77, 8187, 99, 101103, 105, 108, 109, 118). Mycobacterial culture was used as the reference standard in 199 (78%) studies, sputum smear was used as the reference standard in 29 (11%) studies, and sputum smear and/or culture was used as the reference standard in 26 (10%) studies. Two hundred thirteen (84%) studies involved smear-positive patients, and 41 (16%) involved culture-conrmed smear-negative patients. Four studies involved children younger than 15 years of age, and 30 (12%) studies involved HIV-infected persons. The vast majority (96%) of studies performed antibody tests by ELISA. The median number of participants with TB was 51 (interquartile range, 39 to 105); the median number of participants without TB was 57 (interquartile range, 35 to 83). Two hundred fty-four studies evaluating 51 distinct single antigens (9 native proteins, 27 recombinant proteins, and 15

lipids) and 30 distinct multiple-antigen combinations were identied. Many of these antigens were evaluated in only one study. In order to accommodate the large number of antigens identied in the review, only those antigens appearing in two or more studies are included in Tables S2 though S6 in the supplemental material. A guide to the tables and gures is presented in Table 1. The antigens and their alternative names are listed in Table 2. The most frequently evaluated antigens are described below and in additional tables and gures, as noted. Assessment of study quality. The majority of studies used a case-control study design. Only 65 (26%) studies reported blinded interpretation of index test results. Almost all studies provided sufcient detail describing the execution of the index test (Table 3). Meta-analysis (Table 4 and Fig. 3). (i) Recombinant proteins. (a) Recombinant 38 kDa (Rv0934) (Table A1). 38 kDa, a major protein present in culture ltrates of M. tuberculosis, has been studied extensively (1, 17, 22). Several studies have shown an association between the presence of anti-38 kDa antibodies and advanced cavitary TB (14, 22, 75). In smear-positive patients, recombinant 38 kDa yielded a sensitivity of 47% (95% CI, 39 to 55) and a specicity of 94% (95% CI, 86 to 98) (8, 27, 33, 35, 37, 55, 77, 81). (b) Recombinant malate synthase (Rv1837c) (Table 5). Malate synthase (81 kDa), present in M. tuberculosis culture ltrates, the cell wall, and cytoplasmic subcellular fractions, is an enzyme of the glyoxylate pathway used by M. tuberculosis during intracellular replication in macrophages (90) and has adapted to function as an adhesin that enhances bacterial adherence to host cells (46). In sputum smear-positive patients, malate synthase achieved a sensitivity of 73% (95% CI, 58 to 85) and a specicity of 98% (95% CI, 95 to 100) (35, 37, 85, 86, 108). The likelihood ratio positive (40.78) was considerably higher for malate synthase than for other antigens. Earlier studies have

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STEINGART ET AL. TABLE 3. Characteristics of study quality

Characteristic No. (%) of studies

CLIN. VACCINE IMMUNOL.

Study design Cross-sectional ....................................................................... 39 (15) Case-control............................................................................208 (82) Nested within observational study....................................... 7 (3) Recruitment of participants Consecutive or random......................................................... 20 (8) Convenience or not reported ...............................................234 (92) Selection criteria clearly described..........................................141 (56) Complete verication by use of the reference standard ......107 (42) Execution of test described in sufcient detail ......................253 (100)a Index test results blinded to reference standard? Yes........................................................................................... 65 (26) No ............................................................................................ 1 (0) Not reported...........................................................................188 (74)
a

The description of the test execution was deemed insufcient in one study.

demonstrated that whereas antibodies to the 38-kDa antigen are present in patients with extensive cavitary lesions, antimalate synthase antibodies are elicited earlier during the progression of TB, being present in patients who have not yet developed cavities (74). This is reected in the higher sensitivity of TB diagnosis provided by malate synthase. (c) Recombinant MPT51 (Rv3803c) (Table A2). The 27-kDa protein MPT51, a culture ltrate protein, is closely related to the antigen 85 (Ag85) complex, which comprises Ag85A, Ag85B, and Ag85C. MPT51 is an adhesin (108) reported to be a bronectin-binding protein of M. tuberculosis (113). A sufcient number of studies evaluating the performance of MPT51 were available to stratify the results by HIV infection status. In sputum smear-positive patients, MPT51 provided equivalent

sensitivities in both HIV-negative TB patients (59%; 95% CI, 38 to 76) and HIV-positive TB patients (58%; 95% CI, 30 to 82); the specicities were 94% and 97%, respectively (10, 69, 85, 108). (d) Recombinant CFP-10 (Rv3874) (Table 6). Culture ltrate protein 10 (CFP-10), a culture ltrate and cell wall protein, has been identied as one of the earliest proteins expressed by M. tuberculosis during culture in bacteriological media (9). In sputum smear-positive patients, CFP-10 provided a sensitivity of 48% (95% CI, 29 to 68) and a specicity of 96% (95% CI, 83 to 99) (27, 33, 59, 118). (e) Recombinant TbF6 (see Table S2 in the supplemental material). TbF6 is a single antigen combining four distinct antigens (CFP-10, MTB8, MTB48, and 38 kDa) as a genetically fused polyprotein (37). In sputum-smear positive patients, TbF6 achieved a sensitivity of 70% (95% CI, 37 to 90) and a specicity of 93% (95% CI, 69 to 99) (6, 37). The high sensitivity obtained with TbF6 is likely due to the fact that it comprises immunogenic domains from multiple antigens. (ii) Native proteins. (a) Native 38 kDa (Rv0934) (Table A3). In sputum smear-positive patients, native 38 kDa provided a sensitivity of 49% (95% CI, 37 to 61). In sputum smear-negative patients, the sensitivity reported was lower (31%; 95% CI, 15 to 52). Specicities were 97% in both subgroups (15, 18, 59, 69, 76, 77, 102). (b) Native Ag85B (Rv1886c) (Table A4). Ag85B, present in M. tuberculosis culture ltrates and cell walls, is a major component of the Ag85 complex (112). Like MPT51, Ag85B is a bronectin-binding protein (113). In HIV-negative TB patients, native Ag85B yielded a sensitivity of 53% (95% CI, 20 to 83), and in HIV-positive TB patients, a it yielded a sensitivity of 62% (95% CI, 19 to 92). The specicities were 95% (23, 67, 77, 103). (c) Native -crystallin (2031c) (see Table S3 in the supplemental material). -Crystallin is a 14/16-kDa cell wall protein (106) shown to be induced in bacteria under hypoxia (78). In sputum

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TABLE 4. Overall sensitivities, specicities, and likelihood ratios for antigens evaluated for serodiagnosis of pulmonary TB with assays detecting IgG and/or IgA antibodies
Type of compound Antigen name Rv designation No. of studies Smear status HIV status Sensitivity (%)a Specicity (%)a Likelihood ratio positivea Likelihood ratio negativea

Recombinant

38 kDa Malate synthase MPT51 MPT51 CFP-10 TbF6b TbF6, DPEPc 38 kDa 38 kDa Ag 85B Ag 85B -Crystallin DAT TAT SL-I Cord factor

0934 1837c 3803c 3803c 3874

12 8 5 4 6 4 4 13 7 4 4 6 7 4 4 5

Positive Positive Positive Positive Positive Positive Positive Positive Negative Positive Positive Positive Positive Positive Positive Positive

/ / /

47 (3955) 73 (5885) 59 (3876) 58 (3082) 48 (2968) 70 (3790) 75 (5091) 49 (3761) 31 (1552) 53 (2083) 62 (1992) 54 (3275) 63 (4578) 81 (2199) 80 (5693) 69 (2894)

94 (8698) 98 (95100) 94 (7799) 97 (84100) 96 (8399) 93 (6999) 95 (8699) 97 (9499) 97 (9299) 95 (7899) 97 (8999) 96 (8399) 81 (5096) 44 (2467) 59 (896) 91 (7897)

8.22 (3.4124.85) 40.78 (14.43155.7) 10.50 (2.7069.69) 19.03 (3.73172.3) 12.11 (3.2064.63) 9.61 (2.2353.99) 14.97 (5.4356.66) 15.73 (8.8431.55) 9.13 (3.8824.05) 9.36 (2.5253.81) 17.83 (4.0462.32) 13.23 (3.5266.61) 3.32 (1.3213.35) 1.44 (0.422.31) 1.94 (0.8920.90) 7.03 (2.4420.65)

0.56 (0.480.65) 0.27 (0.160.42) 0.44 (0.260.67) 0.44 (0.190.73) 0.55 (0.350.73) 0.33 (0.130.66) 0.26 (0.100.53) 0.53 (0.410.65) 0.72 (0.510.87) 0.51 (0.200.84) 0.39 (0.080.84) 0.48 (0.280.71) 0.47 (0.300.74) 0.42 (0.031.71) 0.34 (0.142.22) 0.35 (0.060.80)

Native protein

0934 0934 1886c 1886c 2031c

/ / / / /

Lipid

a b c

The data represent the posterior means (95% credible intervals). Polyprotein. Multiple antigen (additive reactivity).

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FIG. 3. SROC curves of antigen performance for serodiagnosis of pulmonary TB. (A) Recombinant proteins; (B) native proteins; (C) lipids.

smear-positive patients, -crystallin provided a sensitivity of 48% (95% CI, 29 to 68) and a specicity of 96% (95% CI, 83 to 99) (66, 103). (iii) Lipids. A variety of lipid-containing antigens are common to mycobacterial species (71). Several lipid moieties have been puried and intensely studied for their serological potential for TB diagnosis (44). Four lipid antigens, all acylated trehaloses (107), were evaluated in smear-positive patients and included in the meta-analysis. (a) DAT (Table 7). 2,3-Diacyltrehalose (DAT), a component of the M. tuberculosis cell wall, has been postulated to play a role in modulating host immune responses (50). DAT yielded a sensitivity of 63% (95% CI, 45 to 78) and a specicity of 81% (95% CI, 50 to 96) (43, 44, 83, 105). (b) TAT (see Table S4 in the supplemental material). 2,3,6Triacyltrehalose (TAT) is an antigenic glycolipid compound found in the M. tuberculosis cell wall (41, 43). TAT provided a sensitivity of 81% (95% CI, 21 to 99) and a specicity of 44% (95 CI, 24 to 67) (43, 44). (c) SL-I (see Table S4 in the supplemental material). 2,3,6,6 Tetraacyltrehalose 2 -sulfate (sulfolipid I [SL-I]), a compound found abundantly in the M. tuberculosis cell wall, may affect the human immune system and play a role in the pathogenesis of TB (51). SL-I yielded a sensitivity of 80% (95% CI, 56 to 93) and a specicity of 59% (95% CI, 8 to 96) (43, 44). (d) Cord factor (see Table S4 in the supplemental material). Cord factor (trehalose 6,6 -dimycolate), a major component of M. tuberculosis cell walls, is named for its central role in aggregating mycobacteria into cord structures (7, 30). Cord factor may contribute to the virulence of M. tuberculosis by facilitating cavity formation (38). Cord factor achieved a sensitivity of 69% (95% CI, 28 to 94) and a specicity of 91% (95% CI, 78 to 97) (43, 44, 101). (e) TbF6 plus DPEP (see Table S5 in the supplemental material). TbF6 polyprotein plus DPEP was the multiple-antigen combination most frequently evaluated; four studies involved HIV-uninfected individuals, and one study involved HIV-infected individuals. As described above, TbF6 is a polyprotein. DPEP, also known as MPT32, is a proline-rich 45/47-kDa antigen suggested to have a role in the cross-linking of molecules produced by or bordering M. tuberculosis (49). Earlier studies with native MPT32 have demonstrated that it is a highly immunogenic protein that provided higher sensitivity than the 38-kDa protein when it was tested in the same patient cohort (74). In HIV-negative TB patients, TbF6 plus DPEP achieved a sensitivity of 75% (95% CI, 50 to 91) and a specicity of 94% (95% CI, 86 to 99) (6, 37). The single study evaluating the serodiagnostic potential of TbF6 plus DPEP in HIV-infected individuals is described below. Assessment of specicity in healthy volunteers compared with assessment of specicity in patients with nontuberculous respiratory diseases (Table 8). Sufcient numbers of studies evaluated ve antigens, four proteins (recombinant 38 kDa, native 38 kDa, malate synthase, and CFP-10) and one lipid (DAT) for comparison of the specicities for healthy and diseased controls. For the four proteins, both subsets showed similar specicity values. For DAT, studies involving patients with nontuberculous respiratory disease yielded a signicantly higher specicity, 57% (95% CI, 30 to 76), than studies with healthy volunteers, 97% (95% CI, 88 to 100).

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TABLE 5. Studies evaluating recombinant malate synthase (Rv1837c) for serodiagnosis of pulmonary TB
Author, yr (reference) Chaudhary et al., 2005 (20) Hendrickson et al., 2000 (35) Hendrickson et al., 2000 (35) Houghton et al., 2002 (37) Houghton et al., 2002 (37) Singh et al., 2003 (86) Singh et al. 2005 (85) Wanchu et al., 2008 (108) Wanchu et al., 2008 (108)
a b

Study design Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control

Reference standard (smear statusa) Smear (SP) Culture (SP) Culture (SP) Culture and/or smear (SP) Culture and/or smear (SP) Smear (SP) Smear (SP) Smear (SP) Smear (SP)

Patient (comparison) country India (India) South Africa and Uganda (China) South Africa and Uganda (China) Sub-Saharan Africa (China) Sub-Saharan Africa (China) India (area of endemicity) India (area of endemicity) India (India) India (United States)

Status of individuals used for comparison Healthy Nontuberculous respiratory disease Nontuberculous respiratory disease Nontuberculous respiratory disease Nontuberculous respiratory disease Healthy Healthy Healthy Healthy

HIV status of patient (comparator) NRd (NR) (NR) (NR) (NR) (NR) (NR) (NR) ( ) ( )

Ig class IgG, IgM IgG IgG IgG IgG IgG IgG, IgA IgG, IgA IgG, IgA

No. of participantsb 44/105 52/31 25/31 59/31 66/31 43/25 40/29 138/38 26/65

Sensitivity (%)c 32 (1948) 60 (4573) 92 (7498) 78 (6588) 58 (4570) 77 (6188) 75 (6782) 73 (5288) 73 (5288)

Specicity (%)c 99 (93100) 97 (83100) 97 (83100) 97 (83100) 97 (83100) 100 (86100) 100 (91100) 100 (91100) 99 (92100)

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SP, smear positive. Number of participants with TB/number of participants without TB. c 95% CIs are given in parentheses. d NR, not reported.

Overall performance of antigens (Fig. 3). Among recombinant proteins, malate synthase and TbF6 plus DPEP (multiple antigen) provided the highest sensitivities for the specicities reported (Fig. 3A). For native proteins, Ag85B in HIV-infected TB patients achieved better performance than other native antigens (Fig. 3B). Among lipid antigens, cord factor had the best performance (Fig. 3C).

Descriptive analysis. (i) Performance of antigens in pulmonary TB patients with HIV infection (Fig. 4). Thirty studies evaluating antigens (all proteins) in HIV-infected TB patients were identied; all studies involved sputum smear-positive patients. Of the total, 23 (77%) studies evaluated assays for the detection of IgG and/or IgA antibodies (23, 35, 37, 69, 103, 108), four studies evaluated assays for the detection of IgM

TABLE 6. Studies evaluating recombinant CFP-10 (Rv3874) for serodiagnosis of pulmonary TB

Author, yr (reference) Dillon et al., 2000 (27) Greenaway et al., 2005 (33) Murthy et al., 2007 (59) Murthy et al., 2007 (59) Murthy et al., 2007 (59) Zhang et al., 2007 (118) Murthy et al., 2007 (59) Murthy et al., 2007 (59) Murthy et al., 2007 (59)
a b c

Study design Case-control Nested within observational study Case-control Case-control Case-control Case-control Case-control Case-control Case-control

Reference standard (smear statusa) Culture and/or smear (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Smear (SP) Culture (SN) Culture (SN) Culture (SN)

Patient (comparison) country Brazil (United States and areas of endemicity) Gambia (Gambia) India (India) India (India) India (India) China (China) India (India) India (India) India (India)

Status of individuals used for comparison Healthy Healthy Nontuberculous respiratory disease Nontuberculous respiratory disease Nontuberculous respiratory disease Healthy Nontuberculous respiratory disease Nontuberculous respiratory disease Nontuberculous respiratory disease

HIV status of patient (comparator) ( ) and and ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( )

Ig class IgG IgG IgG IgA IgG, IgA IgG IgG IgA IgG, IgA

No. of participantsb 250/57 100/100 262/76 262/76 262/76 50/28 60/76 60/76 60/76

Sensitivity (%)c 28 (2234) 63 (5372) 42 (3649) 25 (2031) 57 (5163) 78 (6489) 10 (421) 43 (3157) 50 (3763)

Specicity (%)c 97 (88100) 55 (4565) 99 (93100) 99 (93100) 97 (91100) 96 (82100) 99 (93100) 99 (93100) 97 (91100)

SP, smear positive; SN, smear negative. Number of participants with TB/number of participants without TB. 95% CIs are given in parentheses.

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267

Author, yr (reference)

Study design

Reference standard (smear statusa)

Patient (comparison) country

Status of individuals used for comparison

HIV status patient (comparator)

Ig No. of class participantsb

Sensitivity (%)c

Specicity (%)c

Julian et al., 2002 Cross-sectional Culture (SP) Spain (Spain) Nontuberculous and ( ) IgG (44) respiratory disease Julian et al., 2002 Cross-sectional Culture (SP) Spain (Spain) Nontuberculous and ( ) IgA (44) respiratory disease Julian et al., 2002 Cross-sectional Culture (SP) Spain (Spain) Nontuberculous and ( ) IgM (44) respiratory disease Julian et al., 2004 Cross-sectional Culture (SP) Spain (Spain) Nontuberculous ( ) IgG (43) respiratory disease Julian et al., 2004 Cross-sectional Culture (SP) Spain (Spain) Nontuberculous ( ) IgA (43) respiratory disease Julian et al., 2004 Cross-sectional Culture (SP) Spain (Spain) Nontuberculous ( ) IgM (43) respiratory disease Simonney et al., Cross-sectional Culture (SP) France Healthy and IgG 1997 (83) (France) (NRd) Culture (SP) Mexico Healthy NR (NR) IgG Vera-Cabrera et Case-control (Mexico) al., 1999 (105)e Vera-Cabrera et Case-control Culture (SP) Mexico Healthy NR (NR) IgG al., 1999 (105) (Mexico) Simonney et al., Cross-sectional Culture France Healthy and IgG 1997 (83) (SN) (France) (NR)
a b c

42/48 42/48 42/48 29/35 29/35 29/35 31/50 39/35 39/35 29/50

60 (4374) 79 (6390) 10 (323) 52 (3371) 79 (6092) 7 (123) 32 (1751) 49 (3265) 80 (6491) 21 (840)

58 (4372) 50 (3565) 100 (93100) 57 (3974) 51 (3469) 100 (90100) Downloaded from cvi.asm.org at McGill Univ on January 31, 2009 96 (86100) 97 (85100) 97 (85100) 96 (86100)

SP, smear positive; SN, smear negative. Number of participants with TB/number of participants without TB. 95% CIs are given in parentheses. d NR, not reported. e Dot immunoassay was used; all other studies performed ELISA.

antibodies (69, 103), and three studies evaluated assays for the detection of IgG plus IgA plus IgM antibodies (69, 103). Four studies based on multiple-antigen combinations are described in more detail below (35, 37, 108). Antibodies to all ve single antigens (38 kDa, malate synthase, Ag85B, -crystallin, and recombinant MPT51) evaluated in these studies were detected. As discussed, only MPT51 and Ag85B were investigated in a sufcient number of studies for inclusion in the meta-analysis (Table 4). With assays for the detection of IgG and/or IgA antibodies, the sensitivities reported for 38 kDa (range, 35% to 68%) and -crystallin (range, 15% to 58%) were similar to those provided for MPT51 and Ag85B. Malate synthase achieved higher sensitivities (range, 73% to 92%). Compared with tests for the detec-

TABLE 8. Specicity estimates by type of comparison

Specicity (%)a Antigen name Patients with nontuberculous respiratory disease Healthy subjects

Recombinant 38 kDa Recombinant malate synthase Recombinant CFP-10 Native 38 kDa DAT

97 (9099) (6) 97 (91100) (4) 99 (92100) (3) 96 (9099) (6) 55 (3076) (4)

90 (5799) (6) 99 (81100) (4) 90 (4399) (3) 98 (92100) (4) 97 (88100) (3)

a The data represent the posterior means (95% credible intervals) (number of studies).

tion of only IgG and/or IgA antibodies, tests for the detection of IgM antibodies provided considerably lower sensitivities (range, 4% to 5%). The inclusion of tests for the detection of IgM (IgG plus IgA plus IgM) did not appreciably increase the sensitivity. The specicities provided by all of the above antigens were high (range, 89% to 100%). However, only 6 (20%) studies involved controls with nontuberculous respiratory disease (23, 35, 37), while 14 (47%) studies involved either healthy volunteers or asymptomatic HIV-infected individuals without TB (35, 37, 103, 108). In 10 (33%) studies, the control group involved HIV-infected individuals whose clinical status ranged from asymptomatic to symptomatic with opportunistic infections other than TB (69). (ii) Performance of tests with multiple antigens (see Table S5 in the supplemental material). Assays based on multiple antigens provided higher sensitivities (median, 76%; range, 16% to 96% [57 studies]) than assays based on single antigens (median, 53%; range, 2% to 100% [197 studies]), while they maintained high specicities (median, 96%; range, 79% to 100%) (data not shown). The combination of malate synthase plus MPT51 was evaluated in three studies, two studies involving HIV-negative TB patients (2, 108) and one study involving HIV-infected TB patients (108). The sensitivities provided by malate synthase plus MPT51 were similar with HIV-uninfected and -infected TB patients from India: 80% (95% CI, 73 to 87) and 77% (95% CI, 56 to 91), respectively. The specicities were equivalent (97%) whether the comparison group involved

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Downloaded from cvi.asm.org at McGill Univ on January 31, 2009 FIG. 4. Performance of antigens for the serodiagnosis of pulmonary TB in HIV-infected patients. (A) Sensitivity; (B) specicity. The circles and the lines represent the point estimates and the 95% CIs, respectively. The size of the circle indicates the study size. MS, malate synthase; n, native; r, recombinant; 1, IgG; 2, IgM; 3, IgA; 4, IgG/A; 5, IgG/IgA/IgM.

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TABLE 9. Sensitivity and specicity of in-house antibody detection tests by Ig class

Ig class No. of studies Median (range) % Sensitivity Specicity

IgG IgA IgM IgG and IgA IgG and IgM IgM and IgA IgG, IgA, and IgM Not reported All

151 25 24 34 3 0 7 10 254

61 (8100) 40 (1090) 11 (271) 71 (4397) 36 (3252) 71 (6083) 41 (1674) 58 (2100)

96 (26100) 96 (48100) 98 (89100) 97 (85100) 98 (9899) 93 (9093) 96 (8999) 96 (26100)

HIV-negative healthy volunteers from India or HIV-infected (tuberculin skin test-positive and -negative) asymptomatic individuals from the United States (108). However, this antigen combination yielded a sensitivity of only 55% (95% CI, 36 to 72) with TB patients from the United States (2). The combination of TbF6 plus DPEP plus malate synthase achieved a sensitivity of approximately 85% with both HIVnegative and HIV-infected TB patients (37). The specicities were high (97%; 95% CI, 83 to 100), even when this antigen combination was evaluated with patients with nontuberculous respiratory disease. In studies in which 38 kDa plus malate synthase was evaluated, the sensitivities reported were 71% (95% CI, 57 to 83) for HIV-negative TB patients and 96% (95% CI, 80 to 100) for HIV-infected TB patients; the specicity was 89% (95% CI, 78 to 96) when it was assessed with healthy volunteers (35). With HIV-negative, sputum smearpositive patients, the combination of 38 kDa plus Ag85B and -crystallin achieved a sensitivity of 89% (95% CI, 84 to 93) and, with the addition of MPT51, a sensitivity of 91% (95% CI, 86 to 95) (68). With non-HIV-infected, sputum smear-negative patients, the two combinations provided sensitivities of 73% (95% CI, 57 to 86) and 78% (95% CI, 62 to 89), respectively, and a specicity of 87% (95% CI, 75 to 94) when they were assessed with patients with nontuberculous respiratory diseases (68). Only two studies with multiple lipid antigens were identied (see Table S6 in the supplemental material). (iii) Test performance by Ig class (Table 9). Stratication by Ig class showed that in comparison with the results of studies that detected antibodies to IgG (median sensitivity, 61%; range, 8% to 100%) or IgA (median sensitivity, 40%; range, 10% to 90%), studies that detected antibodies to IgM had considerably lower sensitivities (median, 11%; range, 2% to 71%). The median specicities were similar: 96%, 96%, and 98%, respectively. In addition, compared with the results of tests that detected only anti-IgG or anti-IgA antibodies, tests that detected IgG plus IgA showed higher sensitivities (median, 71%; range, 43% to 97%). The inclusion of IgM (IgG plus IgA plus IgM) did not further enhance the sensitivity (median, 71%; range, 60% to 83%). DISCUSSION Principal ndings. This systematic review yielded 254 studies evaluating 51 distinct single antigens and 30 multiple-antigen combinations. The performance of these antigens was ex-

amined in in-house tests for the serodiagnosis of pulmonary TB. Studies evaluating 13 distinct antigens (recombinant 38 kDa, native 38 kDa, MPT51, malate synthase, CFP-10, TbF6 polyprotein, Ag85B, -crystallin, DAT, TAT, SL-I, cord factor, and TbF6 plus DPEP [multiple antigen]) were included in the meta-analysis. The results demonstrate that (i) in sputum smear-positive patients, only recombinant malate synthase (sensitivity, 73%; 95% CI, 58 to 85) and TbF6 plus DPEP (sensitivity, 75%; 95% CI, 50 to 91) provided sensitivities signicantly 50%; (ii) all protein antigens achieved high specicities; (iii) among the lipid antigens, cord factor had the best overall performance (sensitivity, 69% [95% CI, 28 to 94]; specicity, 91% [95% CI, 78 to 97]); (iv) compared with single antigens (median sensitivity, 53%; range, 2% to 100%), multiple antigens yielded higher sensitivities (median sensitivity, 76%; range, 16% to 96%); (v) in HIV-infected patients who are sputum smear positive, antibodies to several single and multiple antigens were detected; and (vi) data on seroreactivity to specic antigens in sputum smear-negative or pediatric patients were insufcient. These results demonstrate that no single antigen provides a sensitivity that is sufcient for a single antigen to be used to devise a serodiagnostic test for TB and that it is unlikely that a single antigen-based serodiagnostic test can be devised. This is not surprising, since the titers of antibodies to each antigen would differ in individuals and the detection of low titers of antibodies would be occluded due to the formation of immune complexes. It is also interesting that while both DPEP and malate synthase are conserved in the M. tuberculosis complex species and in all clinical isolates of M. tuberculosis whose genomes have been sequenced, antibodies to these antigens are not detected in a vast majority of tuberculin skin test-positive individuals with likely latent infection. Proteins that are approximately 50 to 60% homologous to these antigens are present in some other mycobacteria and whether cross-reactive antibodies exist in nontuberculous mycobacterial diseases remains to be reported. Stratication by Ig class demonstrated that assays for the detection of IgG and/or IgA antibodies provided higher sensitivities than assays for the detection of IgM antibodies. This is not surprising, since IgM antibodies are likely to be expressed early during the onset of infection, with the levels quickly decreasing after this period. By the time that bacteriologically detectable TB manifests, whether it is during primary infection or reactivation, the infection has already progressed for months to years in immunocompetent individuals and weeks to months in immunocompromised patients. Thus, the detection of IgM antibodies may have a role in the identication of early infection, but its value for the serodiagnosis of active TB disease may be limited. Considering that the prole of antigens recognized by antibodies is altered with the progression of M. tuberculosis infection (74), the antigens used in a serodiagnostic test during contact tracing are likely to differ from those used in a test for the diagnosis of clinical TB. To our knowledge, no antigens that can be the basis for an accurate serodiagnostic test for contact tracing have been reported. The discovery, evaluation, and comparison of such tests with gamma interferon release assays need to be considered. This systematic review and meta-analysis had several strengths. Standard protocols for the conduct of the review (61) and assessment of the quality of the studies (110) were

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TABLE A1. Studies evaluating recombinant 38 kDa (Rv0934) for serodiagnosis of pulmonary TB
Author, yr (reference) Study design Reference standard (smear statusa) Culture (SP) Culture (SN) Smear (SP) Smear (SP) Patient (comparison) country Status of HIV status individuals used of patient for comparison (comparator) Ig class IgM IgM IgG IgG, IgM IgG ( ) IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG 52/31 25/31 105/57 40/57 66/57 248/31 51/31 31/31 25/25 33/54 46 (3261) 68 (4785) 57 (4767) 35 (2152) 41 (2954) 48 (4255) 29 (1844) 36 (1955) 97 (83100) 97 (83100) 91 (8197) 91 (8197) 91 (8197) 97 (83100) 97 (83100) 97 (83100) IgG Sensitivity No. of (%)c participantsb 41/30 29/30 50/48 44/105 250/57 100/100 Specicity (%)c

Amicosante et al., Case-control 1995 (3) Amicosante et al., Case-control 1995 (3) Ben Amor et al., 2005 (8) Case-control

Chaudhary et al., Case-control 2005 (20) Dillon et al., 2000 Case-control (27) Greenaway et al., 2005 (33) Hendrickson et al., 2000 (35) Hendrickson et al., 2000 (35) Houghton et al., 2002 (37) Houghton et al., 2002 (37) Houghton et al., 2002 (37) Lodes et al., 2001 (55) Lodes et al., 2001 (55) Lodes et al., 2001 (55) Senthil Kumar et al., 2002 (77) Silva et al., 2003 (81)
a b

Italy and United States Healthy NR (NRd) (Italy and United States) Italy and United States Healthy NR (NR) (Italy and United States) Mexico (Mexico) Nontuberculous ( ) respiratory disease India (India) Healthy NR (NR) Healthy Healthy Nontuberculous respiratory disease Nontuberculous respiratory disease Healthy Healthy Healthy ( ) and and (NR) (NR) (NR) (NR) (NR) ) ) ) (NR)

54 (3769) 100 (88100) 55 (3674) 100 (88100) 56 (4170) 36 (2252) 49 (4355) 49 (3959) 96 (86100) 99 (95100) 91 (8197) 50 (4060)

Nested within observational study Case-control Culture (SP) Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Cross-sectional

Culture and/or Brazil (United States smear (SP) and areas of endemicity) Culture (SP) Gambia (Gambia) South Africa and Uganda (China) South Africa and Uganda (China)

Culture (SP)

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Culture and/or Brazil (United States smear (SP) and areas of endemicity) Culture and/or Philippines (United smear (SP) States and areas of endemicity) Culture and/or Sub-Saharan Africa smear (SP) (United States and areas of endemicity) Culture and/or Brazil (China) smear (SP) Culture and/or South Africa (China) smear (SP) Culture and/or Philippines (China) smear (SP) Culture (SP) Culture (SN) India (India) Canada (Canada)

Nontuberculous ( respiratory disease Nontuberculous ( respiratory disease Nontuberculous ( respiratory disease Healthy NR Healthy

60 (3979) 100 (86100) 39 (2358) 89 (7796)

NR (NR)

SP, smear positive; SN, smear negative. Number of participants with TB/number of participants without TB. c 95% CIs are given in parentheses. d NR, not reported.

followed. The application of a comprehensive search strategy with various overlapping approaches enabled the retrieval of relevant studies published since 1990. Screening and data extraction were performed independently among three reviewers, and authors were contacted to clarify points and obtain missing data. None of the studies in the review used the result from the antibody test as a reference to conrm TB (incorporation bias). When possible, patients with disease were selected, in preference to healthy controls, to evaluate the performance of antigens with persons in whom TB was initially suspected and subsequently ruled out. The meta-analysis was limited by the relatively small number of studies investigating the same antigens or antigen combinations. The small number of comparable antigens made it difcult to relate study quality to antigen performance. However, several important deciencies in study design and quality were noted. Only 20 (7%) studies recruited participants in a random or consecutive manner. Therefore, most studies lacked a sound

probabilistic sampling framework. The majority of studies used a case-control design with healthy controls. This design has been found to overestimate test sensitivity and specicity (53, 111), although for the four protein antigens in this review for which a comparison was feasible, the specicities were found to be similar with healthy and diseased controls. Few (26%) studies reported the use of the blinded interpretation of test results and a reference standard. This was not unexpected, since the primary aims of in-house studies are the discovery of novel antigens, evaluation of their diagnostic potential, and/or comparison of different antigens. Nonetheless, the lack of blinding and the dearth of data from cross-sectional studies are major shortcomings of the currently available literature and may have resulted in an overestimation of antigen performance (53). Both errors in design and deciencies in reporting have been noted as concerns in TB diagnostic studies (62, 89). An additional limitation was the lack of information about

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271

Author, yr (reference) Bethunaickan et al., 2007 (10)d Bethunaickan et al., 2007 (10)d Bethunaickan et al., 2007 (10)d Bethunaickan et al., 2007 (10)d Bethunaickan et al., 2007 (10)d Ramalingam et al., 2003 (69)d Ramalingam et al., 2003 (69)d Ramalingam et al., 2003 (69)d Ramalingam et al., 2003 (69)d Ramalingam et al., 2003 (69)d Singh et al., 2005 (85) Wanchu et al., 2008 (108) Wanchu et al., 2008 (108) Bethunaickan et al., 2007 (10)d Bethunaickan et al., 2007 (10)d Bethunaickan et al., 2007 (10)d
a b

Study design Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control

Reference standard (smear statusa) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Smear (SP) Smear (SP) Smear (SP) Culture (SN) Culture (SN) Culture (SN)

Patient (comparison) country India (India) India (India) India (India) India (India) India (India) India (India) India (India) India (India) India (India) India (India) India (Endemic) India (India) India (United States) India (India) India (India) India (India)

Status of individuals used for comparison Nontuberculous respiratory disease Nontuberculous respiratory disease Healthy Nontuberculous respiratory disease Healthy Mixed disease and asymptomatic Mixed disease and asymptomatic Mixed disease and asymptomatic Mixed disease and asymptomatic Mixed disease and asymptomatic Healthy Healthy Healthy Nontuberculous respiratory disease Nontuberculous respiratory disease Nontuberculous respiratory disease

HIV status of patient (comparator) (NRe) (NR) (NR) (NR) (NR) ( ) ( ) ( ) ( ) ( ) (NR) (NR) ( ) (NR) (NR) (NR)

Ig class IgG IgA IgM IgG, IgA IgG, IgM, IgA IgG IgA IgM IgG, IgA IgG, IgM, IgA IgG, IgA IgG, IgA IgG, IgA IgG IgA IgG, IgA

No. of participantsb 175/60 175/60 175/150 175/60 175/150 55/45 55/45 55/45 55/45 55/45 40/29 138/38 26/65 41/60 41/60 41/60

Sensitivity (%)c 57 (5065) 47 (3955) 13 (818) 71 (6478) 72 (6579) 55 (4168) 40 (2754) 6 (115) 69 (5581) 71 (5782) 60 (4375) 59 (5168) 69 (4886) 34 (2151) 29 (1646) 46 (3163)

Specicity (%)c 93 (8498) 92 (8297) 98 (94100) 87 (7594) 93 (8796) 100 (92100) 93 (8299) 100 (92100) 93 (8299)

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93 (8299) 100 (88100) 97 (86100) 99 (92100) 93 (8498) 92 (8297) 87 (7594)

SP, smear positive; SN, smear negative. Number of participants with TB/number of participants without TB. c 95% CIs are given in parentheses. d Index test blinded to reference standard. e NR, not reported.

sputum microscopy procedures. Smear status may be determined through the examination of unprocessed sputum (direct smear microscopy) or through the more sensitive examination of sputum after its digestion and concentration prior to the inoculation of cultures (concentrated smear microscopy). The former procedure is more common in low- and middle-income countries, where cultures are rarely done, while the latter procedure is more common in high-income countries. Furthermore, uorescence microscopy, which is commonly used in high-income countries, is associated with a sensitivity higher than that achieved by conventional light microscopy, which is commonly used in low-income countries (96). Conversely, it is widely appreciated that prociency in sputum smear microscopy requires regular exposure to positive smears, and this experience is more likely to be gained in low- and middleincome countries where TB is endemic. Thus, the site where smear microscopy was performed may have had an undeterminable impact on the assessment of antigen performance distinct from the nature of the patient population. Another limitation may be that the patient population differed between richer and poorer countries, with persons in the latter having more advanced disease and perhaps being more likely to be

infected with HIV. In this review, approximately 75% of the studies involved patients who resided in low-income countries. The maximum benet of improved diagnosis through serology would manifest in these countries. However, the ndings of this review suggest that the assays evaluated could not replace sputum microscopy in these countries. In TB diagnostic trials, culture is considered the gold standard. A majority (78%) of studies used culture as the reference standard. Along with studies that used culture, we also included studies from countries where TB is endemic that used sputum smear microscopy, a test with modest sensitivity. The use of an insensitive reference standard may have led to biased estimates of antigen performance (111). Our choice of reference standard (culture and/or smear) may have limited the inclusion of studies involving children (four studies). Pediatric TB is difcult to diagnose on a bacteriological basis because of the paucibacillary nature of the disease (79). Although statistical tests and graphical methods for the detection of potential publication bias in meta-analyses of randomized control trials are available, to our knowledge such techniques have not been adequately evaluated for diagnostic data (98). It is therefore difcult to rule out publication bias in our review. In addition,

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TABLE A3. Studies evaluating native 38 kDa (Rv0934) for serodiagnosis of pulmonary TB
Author, yr (reference) Bothamley and Rudd, 1994 (15)b Bothamley and Rudd, 1994 (15)b Chan et al., 1990 (18) Chan et al., 1990 (18) Murthy et al., 2007 (59) Murthy et al., 2007 (59) Murthy et al., 2007 (59) Murthy et al., 2007 (59) Murthy et al., 2007 (59) Murthy et al., 2007 (59) Ramalingam et al., 2003 (69)b Ramalingam et al., 2003 (69)b Ramalingam et al., 2003 (69)b Ramalingam et al., 2003 (69)b Ramalingam et al., 2003 (69)b Study design Cross-sectional Cross-sectional Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Status of HIV status of Reference Patient (comparison) individuals used patient standard country a for comparison (comparator) (smear status ) Culture (SP) Culture (SN) Culture (SP) Culture (SN) Culture (SP) Culture (SP) Culture (SP) Culture (SN) Culture (SN) Culture (SN) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SN) Culture (SN) Culture (SN) Ig class No. of participantsc 25/82 27/82 88/140 37/140 262/76 262/76 262/76 60/76 60/76 60/76 55/45 55/45 55/45 55/45 55/45 42/83 25/25 175/60 175/60 175/60 175/60 175/60 41/60 41/60 41/60 Sensitivity (%)d 72 (5188) 56 (3575) 45 (3556) 16 (632) 48 (4254) 23 (1828) 62 (5668) 25 (1538) 22 (1234) 43 (3157) 40 (2754) 40 (2754) 5 (115) 60 (4673) 62 (4875) 38 (2454) 52 (3172) 61 (5368) 30 (2337) 10 (616) 68 (6175) 71 (6478) 54 (3769) 15 (629) 5 (117) Specicity (%)e 96 (9099) 96 (9099) 98 (94100) 98 (94100) 100 (95100) 96 (8999) 96 (8999) 100 (95100) 96 (8999) 96 (8999) 100 (92100) 93 (8299) 100 (92100) 93 (8299) 93 (8299) 100 (96100) 100 (86100) 87 (7594) 97 (88100) 97 (88100) 96 (9199) 90 (8494) 87 (7594) 97 (88100) 97 (88100)

Samanich et al., Case-control 2000 (76) Senthil Kumar et Case-control al., 2002 (77) Uma Devi et al., Case-control 2001 (102) Uma Devi et al., 2001 (102) Uma Devi et al., 2001 (102) Uma Devi et al., 2001 (102) Uma Devi et al., 2001 (102) Uma Devi et al., 2001 (102) Uma Devi et al., 2001 (102) Uma Devi et al., 2001 (102)
a b

United Kingdom Nontuberculous ( ) IgG (United Kingdom) respiratory disease United Kingdom Nontuberculous ( ) IgG (United Kingdom) respiratory disease Hong Kong (Hong Healthy NRd (NR) IgG Kong) Hong Kong (Hong Healthy NR (NR) IgG Kong) India (India) Nontuberculous ( ) IgG respiratory disease India (India) Nontuberculous ( ) IgA respiratory disease India (India) Nontuberculous ( ) IgG, IgA respiratory disease India (India) Nontuberculous ( ) IgG respiratory disease India (India) Nontuberculous ( ) IgA respiratory disease India (India) Nontuberculous ( ) IgG,/A respiratory disease India (India) Mixed disease ( ) IgG and asymptomatic India (India) Mixed disease ( ) IgA and asymptomatic India (India) Mixed disease ( ) IgM and asymptomatic India (India) Mixed disease ( ) IgG, IgA and asymptomatic India (India) Mixed disease ( ) IgG, IgM, and IgA asymptomatic United States Healthy ( and ) IgG (United States) India (India) Healthy NR (NR) IgG India (India) India (India) India (India) India (India) India (India) India (India) India (India) India (India) Nontuberculous respiratory disease Nontuberculous respiratory disease Nontuberculous respiratory disease Healthy Healthy Nontuberculous respiratory disease Nontuberculous respiratory disease Nontuberculous respiratory disease ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) IgG IgA IgM IgG, IgA IgG, IgM, IgA IgG IgA IgM

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Case-control Case-control Case-control Case-control Case-control Case-control Case-control

SP, smear positive; SN, smear negative. Index test blinded to reference standard. c Number of participants with TB/number of participants without TB. d 95% CIs are given in parentheses. e NR, not reported.

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273

Author, yr (reference) Daniel et al., 1994 (23)b Daniel et al., 1994 (23)b Raja et al., 2004 (67)b Raja et al., 2004 (67)b Raja et al., 2004 (67)b Raja et al., 2004 (67)b Raja et al., 2004 (67)b Senthil Kumar et al., 2002 (77) Senthil Kumar et al., 2002 (77) Uma Devi et al., 2003 (103)b Uma Devi et al., 2003 (103)b Uma Devi et al., 2003 (103)b Uma Devi et al., 2003 (103)b Raja et al., et al., 2004 (67)b Raja et al., 2004 (67)b Raja et al., 2004 (67)b
a b

Study design Cross-sectional Cross-sectional Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control Case-control

Reference standard (smear statusa) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SP) Culture (SN) Culture (SN) Culture (SN)

Patient (comparison) country Uganda (Uganda) Uganda (Uganda) India (India) India (India) India (India) India (India) India (India) India (India) India (India) India (India) India (India) India (India) India (India) India (India) India (India) India (India)

Status of individuals used for comparison Nontuberculous respiratory disease Nontuberculous respiratory disease Nontuberculous respiratory disease Nontuberculous respiratory disease Nontuberculous respiratory disease Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Nontuberculous respiratory disease Nontuberculous respiratory disease Nontuberculous respiratory disease

HIV status of patient (comparator) ( ( and and ) )

Ig class IgG IgG IgG IgA IgM IgG, IgA IgG, IgM, IgA IgG IgG IgG IgA IgM IgG, IgA IgG IgA IgM

No. of participantsc 68/35 141/35 175/60 175/60 175/60 175/150 175/150 25/25 25/25 55/150 55/150 55/150 55/150 41/60 41/60 41/60

Sensitivity (%)d 62 (4973) 28 (2136) 67 (6074) 15 (1021) 14 (1020) 71 (6478) 73 (6680) 40 (2161) 60 (3979) 67 (5379) 67 (5379) 4 (013) 84 (7192) 44 (2960) 10 (323) 12 (426)

Specicity (%)e 89 (7397) 89 (7397) 80 (6889) 100 (94100) 93 (8498) 97 (9299) 92 (8696) 96 (80100)

( ) ( ) ( ) ( ) ( ) NR (NR) NR (NR) ( ) ( ) ( ) ( ) ( ) ( ) (NR)

d

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100 (86100) 99 (95100) 97 (9299) 96 (9299) 97 (9299) 80 (6889) 100 (94100) 93 (8498)

SP, smear positive; SN smear negative. Index test blinded to reference standard. c Number of participants with TB/number of participants without TB. d 95% CIs are given in parentheses. e NR, not reported.

our search strategy may have missed some relevant studies by excluding non-English-language publications (28% of the citations initially identied). Conclusion. In summary, despite the limitations discussed above, this systematic review and meta-analysis has identied potential candidate antigens for inclusion in an antibody detection-based diagnostic test for pulmonary TB in HIV-infected and -uninfected individuals. However, none of the antigens achieves sufcient sensitivity to replace sputum smear microscopy. Combinations of select antigens provide higher sensitivities than single antigens. Our ndings should be interpreted in the context of the availability and the variability in the design and the quality of studies of antigen performance. Clearly, unpublished studies did not feature in this review, and a number of published studies did not meet the eligibility criteria for inclusion. Recently, the number of systematic reviews of TB diagnostic tests has been growing (63). The current review adds to this expanding evidence base and may offer guidance to research-

ers, test developers, and grant-awarding bodies on directions for future research. Activities leading to the evaluation of combinations of existing potential candidate antigens, as well as the discovery of additional antigens with diagnostic potential that complement current antigens, need to be intensied to devise assays that can improve upon microscopy. A focus on antigen discovery for the serodiagnosis of TB in smear-negative and pediatric patients should be encouraged. Biobanks (such as the TB Specimen Bank of the Special Programme for Research and Training in Tropical Diseases [TDR]) may be useful for the rapid evaluation of new antigen combinations prior to the consideration of the performance of eld studies. Finally, research is also urgently needed to determine whether combinations of microscopy and antibody detection with multiple antigens could improve case nding in high-prevalence countries and whether antibody detection tests can be delivered in an appropriate format. Efforts are needed to improve both the methodological quality and reporting of TB diagnostic studies. TDR has developed

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11. Bossuyt, P. M., J. B. Reitsma, D. E. Bruns, C. A. Gatsonis, P. P. Glasziou, L. M. Irwig, J. G. Lijmer, D. Moher, D. Rennie, and H. C. de Vet. 2003. Towards complete and accurate reporting of studies of diagnostic accuracy: the STARD initiative. Ann. Intern. Med. 138:4044. 12. Bothamley, G., H. Batra, V. Ramesh, A. Chandramui, and J. Ivanyi. 1992. Serodiagnostic value of the 19 kilodalton antigen of Mycobacterium tuberculosis in Indian patients. Eur. J. Clin. Microbiol. Infect. Dis. 11:912915. 13. Bothamley, G. H. 1995. Serological diagnosis of tuberculosis. Eur. Respir. J. Suppl. 20:676s688s. 14. Bothamley, G. H., R. Rudd, F. Festenstein, and J. Ivanyi. 1992. Clinical value of the measurement of Mycobacterium tuberculosis specic antibody in pulmonary tuberculosis. Thorax 47:270275. 15. Bothamley, G. H., and R. M. Rudd. 1994. Clinical evaluation of a serological assay using a monoclonal antibody (TB72) to the 38 kDa antigen of Mycobacterium tuberculosis. Eur. Respir. J. 7:240246. 16. Broekmans, J. F., G. B. Migliori, H. L. Rieder, J. Lees, P. Ruutu, R. Loddenkemper, and M. C. Raviglione. 2002. European framework for tuberculosis control and elimination in countries with a low incidence. Recommendations of the World Health Organization (WHO), International Union Against Tuberculosis and Lung Disease (IUATLD) and Royal Netherlands Tuberculosis Association (KNCV) Working Group. Eur. Respir. J. 19:765775. 17. Chan, E. D., L. Heifets, and M. D. Iseman. 2000. Immunologic diagnosis of tuberculosis: a review. Tuber. Lung Dis. 80:131140. 18. Chan, S. L., Z. Reggiardo, T. M. Daniel, D. J. Girling, and D. A. Mitchison. 1990. Serodiagnosis of tuberculosis using an ELISA with antigen 5 and a hemagglutination assay with glycolipid antigens. Results in patients with newly diagnosed pulmonary tuberculosis ranging in extent of disease from minimal to extensive. Am. Rev. Respir. Dis. 142:385389. 19. Chanteau, S., V. Rasolofo, T. Rasolonavalona, H. Ramarokoto, C. Horn, G. Auregan, and G. Marchal. 2000. 45/47 kilodalton (APA) antigen capture and antibody detection assays for the diagnosis of tuberculosis. Int. J. Tuberc. Lung Dis. 4:377383. 20. Chaudhary, V. K., A. Kulshreshta, G. Gupta, N. Verma, S. Kumari, S. K. Sharma, A. Gupta, and A. K. Tyagi. 2005. Expression and purication of recombinant 38-kDa and Mtb81 antigens of Mycobacterium tuberculosis for application in serodiagnosis. Protein Expr. Purif. 40:169176. 21. Daniel, T. M. 1988. Antibody and antigen detection for the immunodiagnosis of tuberculosis: why not? What more is needed? Where do we stand today? J. Infect. Dis. 158:678680. 22. Daniel, T. M., and S. M. Debanne. 1987. The serodiagnosis of tuberculosis and other mycobacterial diseases by enzyme-linked immunosorbent assay. Am. Rev. Respir. Dis. 135:11371151. 23. Daniel, T. M., A. A. Sippola, A. Okwera, S. Kabengera, E. Hatanga, T. Aisu, S. Nyole, F. Byekwaso, M. Vjecha, L. E. Ferguson, et al. 1994. Reduced sensitivity of tuberculosis serodiagnosis in patients with AIDS in Uganda. Tuber. Lung Dis. 75:3337. 24. David, H. L., F. Papa, P. Cruaud, H. C. Berlie, M. De Fatima Maroja, J. I. Salem, and M. F. Costa. 1992. Relationships between titers of antibodies immunoreacting against glycolipid antigens from Mycobacterium leprae and M. tuberculosis, the Mitsuda and Mantoux reactions, and bacteriological loads: implications in the pathogenesis, epidemiology and serodiagnosis of leprosy and tuberculosis. Int. J. Lepr. 60:208224. 25. Deville, W. L., F. Buntinx, L. M. Bouter, V. M. Montori, H. C. de Vet., D. A. van der Windt, and P. D. Bezemer. 2002. Conducting systematic reviews of diagnostic studies: didactic guidelines. BMC Med. Res. Methodol. 2:9. 26. Diagbouga, S., F. Fumoux, A. Zoubga, P. T. Sanou, and G. Marchal. 1997. Immunoblot analysis for serodiagnosis of tuberculosis using a 45/47-kilodalton antigen complex of Mycobacterium tuberculosis. Clin. Diagn. Lab. Immunol. 4:334338. 27. Dillon, D. C., M. R. Alderson, C. H. Day, T. Bement, A. Campos-Neto, Y. A. Skeiky, T. Vedvick, R. Badaro, S. G. Reed, and R. Houghton. 2000. Molecular and immunological characterization of Mycobacterium tuberculosis CFP-10, an immunodiagnostic antigen missing in Mycobacterium bovis BCG. J. Clin. Microbiol. 38:32853290. 28. Dinnes, J., J. Deeks, H. Kunst, A. Gibson, E. Cummins, N. Waugh, F. Drobniewski, and A. Lalvani. 2007. A systematic review of rapid diagnostic tests for the detection of tuberculosis infection. Health Technol. Assess. 11:1196. 29. Fujita, Y., T. Doi, K. Sato, and I. Yano. 2005. Diverse humoral immune responses and changes in IgG antibody levels against mycobacterial lipid antigens in active tuberculosis. Microbiology 151:20652074. 30. Gao, Q., K. Kripke, Z. Arinc, M. Voskuil, and P. Small. 2004. Comparative expression studies of a complex phenotype: cord formation in Mycobacterium tuberculosis. Tuberculosis (Edinburgh) 84:188196. 31. Gatsonis, C., and P. Paliwal. 2006. Meta-analysis of diagnostic and screening test accuracy evaluations: methodologic primer. Am. J. Roentgenol. 187:271281. 32. Gennaro, M. L. 2000. Immunologic diagnosis of tuberculosis. Clin. Infect. Dis. 30(Suppl. 3):S243S246. 33. Greenaway, C., C. Lienhardt, R. Adegbola, P. Brusasca, K. McAdam, and D. Menzies. 2005. Humoral response to Mycobacterium tuberculosis anti-

guidelines (Diagnostics Evaluation Expert Panel) that researchers can use to assess the performance and operational characteristics of diagnostics for infectious diseases (5). Use of the Standards for Reporting of Diagnostic Accuracy may also lead to improvements in the quality of studies (11). Future studies should evaluate outcomes that go beyond the conventional diagnostic accuracy of sensitivity and specicity. These outcomes include the accuracy of diagnostic algorithms (rather than single tests) and their relative contributions to the health care system, the incremental or added value of new tests, the impacts of new tests on clinical decision making and therapeutic choices, the cost-effectiveness of new tests in routine programmatic settings, and the impacts of new tests on patientcentered outcomes (63). APPENDIX Tables A1 to A4 provide additional details about studies that have evaluated the use of recombinant 38 kDa (Rv0934), recombinant MPT51 (Rv3803c), native 38 kDa (Rv0934), and native Ag85B (Rv1886c) for the serodiagnosis of pulmonary TB, respectively.
ACKNOWLEDGMENTS We thank Gloria Won of the University of California, San Francisco, and Madelyn Hall of Southwest Washington Medical Center, Vancouver, WA, for help with the search strategy and article retrieval. We are grateful to Maya, Bhat, Vancouver, WA, and Donna Hammar and Anna Meddaugh, Portland, OR, for technical assistance. In addition, we thank Deb Grantz of the University of California, San Francisco, and Izabela Suder-Dayao and Melissa Anthony Vega of WHO/TDR, Geneva, Switzerland, for administrative assistance.
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