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CLINICAL AND VACCINE IMMUNOLOGY, Feb. 2009, p. 260276 Vol. 16, No. 21556-6811/09/$08.000 doi:10.1128/CVI.00355-08Copyright 2009, American Society for Microbiology. All Rights Reserved.

Performance of Purified Antigens for Serodiagnosis of PulmonaryTuberculosis: 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; andVeterans Affairs Medical Center, New York, New York8

Received 26 September 2008/Returned for modification 4 November 2008/Accepted 24 November 2008

Serological antibody detection tests for tuberculosis may offer the potential to improve diagnosis. Recent meta-analyses have shown that commercially available tests have variable accuracies and a limited clinical role. Wereviewed the immunodiagnostic potential of antigens evaluated in research laboratories (in-house) for the serodi-

agnosis of pulmonary tuberculosis and conducted a meta-analysis to evaluate the performance of comparableantigens. Selection criteria included the participation of at least 25 pulmonary tuberculosis patients and the use ofpurified antigens. Studies evaluating 38 kDa, MPT51, malate synthase, culture filtrate protein 10, TbF6, antigen85B, -crystallin, 2,3-diacyltrehalose, 2,3,6-triacyltrehalose, 2,3,6,6-tetraacyltrehalose 2-sulfate, cord factor, andTbF6 plus DPEP (multiple antigen) were included in the meta-analysis. The results demonstrated that (i) in sputumsmear-positive patients, sensitivities significantly>50% were provided for recombinant malate synthase (73%; 95%confidence interval [CI], 58 to 85) and TbF6 plus DPEP (75%; 95% CI, 50 to 91); (ii) protein antigens achieved highspecificities; (iii) among the lipid antigens, cord factor had the best overall performance (sensitivity, 69% [95% CI,28 to 94]; specificity, 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 immunodeficiency 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 insputum smear-negative or pediatric patients were insufficient. Potential candidate antigens for an antibody detec-tion test for pulmonary tuberculosis in HIV-infected and -uninfected patients have been identified, although no

antigen achieves sufficient sensitivity to replace sputum smear microscopy. Combinations of select antigens providehigher sensitivities than single antigens. The use of a case-control design with healthy controls for the majority ofstudies was a limitation of the review. Efforts are needed to improve the methodological quality of tuberculosisdiagnostic studies.

The failure to diagnose tuberculosis (TB) accurately andrapidly is a key challenge in curbing the epidemic (45, 88, 116).Sputum microscopy, currently the sole diagnostic test in mostareas where TB is endemic, has several limitations; in partic-ular, 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 pro-cedure is labor-intensive (45, 65). Antibody detection tests

(serological tests) are used for the diagnosis of many infectiousdiseases and could potentially improve the means of diagnosisof TB. These tests measure the presence of specific antibodies(most often immunoglobulin G [IgG]) directed against immu-

nodominant antigens of the pathogen in question. Comparedwith microscopy, antibody detection methods may enable therapid diagnosis of TB, as these tests have the advantages ofspeed (results can be available within hours), technologicalsimplicity, and minimal training requirements. In addition,these tests can be adapted to point-of-care formats that can beimplemented at lower levels of health services in low- andmiddle-income countries (21, 22, 57, 65).

Efforts to develop antibody detection tests for the diagnosisof TB have been under way for decades, and the performanceof these tests has been well described (13, 17, 22, 32, 40, 47, 48,52, 60, 64, 100, 107). Several systematic reviews of these testshave been published (discussed below) (28, 94, 95).

First-generation antibody detection tests were based on crudemixtures of constituents and products ofMycobacterium tubercu-losis, for example, culture filtrate proteins and purified proteinderivative, the preparation used in the tuberculin skin test. Sev-eral of these early tests had low specificities, as the tests containedantigens shared among different bacterial species (1, 22, 48, 57).During the past two decades, an increased understanding of hu-moral immune responses to M. tuberculosis and the new tools of

* Corresponding author. Mailing address: Francis J. Curry NationalTuberculosis Center, University of California, San Francisco, 318018th Street, Suite 101, San Francisco, CA 94110-2028. Phone: (415)502-4600. Fax: (415) 502-4620. E-mail: [email protected].

Supplemental material for this article may be found at http://cvi.asm.org/.

Present address: California Department of Public Health, Sacra-mento, CA.

Published ahead of print on 3 December 2008.

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genomics and proteomics have led to the discovery of new anti-gens reported to provide improved sensitivities and specificitiesfor the diagnosis of TB compared with those achieved with theantigens in the first-generation tests (48).

We reviewed the immunodiagnostic potential of differentantigens evaluated in research laboratories (in-house) for theserodiagnosis of pulmonary TB and carried out a meta-analysisto evaluate the performance of various antigens singly and incombination. Previous meta-analyses have shown that com-mercially 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 caseseries type of design and included only nine studies relating to

in-house anti-TB antibody serological tests (28). A recentlypublished 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 serodiag-nosis of pulmonary TB in sputum smear-positive and smear-negative patients? (ii) What is the performance of these anti-gens in the serodiagnosis of pulmonary TB in patients withhuman immunodeficiency 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 termsincluded tuberculosis, Mycobacterium tuberculosis, immunological tests,

serological tests, antibody detection, antigen detection, ELISA (en-

zyme-linked immunosorbent assay), Western blot, and sensitivity and speci-

ficity. Additional studies were identified 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-sec-

tional 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 treat-

ment. 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 (serolog-

ical antibody detection) had to be evaluated in-house with purified antigens;

studies that used purified protein derivative, culture filtrates, or sonicated anti-

gens were not included. The reference standard was either the isolation of M.

tuberculosis on sputum culture or, for studies conducted in countries where TBis 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 sufficient data to construct a two-by-two table for calculations of sensitivity,specificity, 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 con-

cerning 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 re-

trieved from all sources for relevance. Screening of full-text articles by using

prespecified inclusion criteria was carried out by two reviewers, and the articles

FIG. 1. Flow diagram for selection of subgroups, IgG and/or IgA antibody detection: an example with MPT51. The same sequence of steps wasrepeated for each antigen. Having at least four studies available was a condition for inclusion in the meta-analysis. , other antibody combinationsincluded IgG and IgM (n 3 studies); IgM and IgA (n 0); IgG, IgA, and IgM (n 7); and not reported (n 10); , other recombinant antigensincluded 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.

VOL. 16, 2009 META-ANALYSIS OF SERODIAGNOSIS OF PULMONARY TB 261

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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 finalized. Two reviewers (each of whom wasresponsible for approximately 50% of the studies) extracted data from all in-cluded studies with the standardized form. To verify reproducibility, a third

reviewer independently performed data extraction on all studies. Differencesamong 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 classification. Antigens were classified into five 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. Thesestudies have taken two approaches. In some cases, different antigens (or portions

thereof) have been cloned as single protein entities (polyproteins) and tested fortheir reactivities with sera. In other cases, multiple antigens have been tested assingle entities and cumulative results (additive reactivity) were calculated. In the

former case, we considered polyproteins to be single antigens; in the latter case,we classified the entities as multiple antigens.

Data analysis. Estimates of sensitivity and specificity from individual studiesand their exact 95% confidence intervals (CIs) were obtained by using Meta-DiSc (version 1.4) software (117). Sensitivity refers to the proportion of TB

patients with positive test results; specificity refers to the proportion of partici-pants without TB with negative test results. For sensitivity, we included studies

that used sputum smear as the reference standard along with studies that usedculture. For specificity, we noted the type of comparison group, e.g., healthy

participants or patients with nontuberculous respiratory disease. Likelihood ratiopositive was calculated as sensitivity/(1 specificity); likelihood ratio negative

was calculated as (1 sensitivity)/specificity.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 combinestudy results, subgroups of comparable antigens were determined (Fig. 1). Ini-

tially, studies were grouped by the class of antibody detected by the test: (i) IgGand/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 otherantibodies. Next, studies were stratified by antigen number (single or multiple

antigens); the type of compound (protein or lipid); and, for proteins, the sourceof the compound (recombinant or native). Finally, for each distinct single antigenor multiple-antigen combination, studies were stratified by patient sputum smear

status and HIV status. At least four studies were required to be available forinclusion in a subgroup in order to strengthen the results and reduce the possi-

bility of finding a significant result by chance. In this way, we identified 16subgroups.

To summarize sensitivity and specificity within each subgroup, separate meta-

analyses were performed by using the hierarchical summary receiver operatingcharacteristic curve model (72). The advantages of the hierarchical summary

receiver operating characteristic are that it jointly models both sensitivity andspecificity, weights studies according to the number of participants, and takesinto account unmeasured heterogeneity between studies by using random effects

(31). The model was estimated by using a Bayesian approach with noninforma-tive prior distributions and was implemented with WinBUGS (version 1.4.1)

software program (91).The average sensitivity, specificity, and likelihood ratios from each meta-

analysis were estimated. From the posterior distribution of each parameter of

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

262 STEINGART ET AL. CLIN. VACCINE IMMUNOL.

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interest, we extracted the mean and the 95% credible interval (the Bayesianequivalent of the classical confidence interval) on the basis of the 2.5% and

97.5% quartiles. When feasible, specificity estimates were stratified by type ofcomparison group. Finally, a summary receiver operating characteristic (SROC)curve from each meta-analysis was obtained. The SROC curve plots sensitivity

versus 1 specificity for the range of specificity 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 (sensitivityand specificity 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 (ver-

sion 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 searchesidentified over 5,000 citations, of which 49 publications (254studies) 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 culturewas used as the reference standard in 199 (78%) studies, spu-tum smear was used as the reference standard in 29 (11%)studies, and sputum smear and/or culture was used as thereference standard in 26 (10%) studies. Two hundred thirteen(84%) studies involved smear-positive patients, and 41 (16%)involved culture-confirmed smear-negative patients. Fourstudies involved children younger than 15 years of age, and 30(12%) studies involved HIV-infected persons. The vast major-ity (96%) of studies performed antibody tests by ELISA. Themedian number of participants with TB was 51 (interquartilerange, 39 to 105); the median number of participants withoutTB was 57 (interquartile range, 35 to 83).

Two hundred fifty-four studies evaluating 51 distinct singleantigens (9 native proteins, 27 recombinant proteins, and 15

lipids) and 30 distinct multiple-antigen combinations wereidentified. Many of these antigens were evaluated in only onestudy. In order to accommodate the large number of antigensidentified in the review, only those antigens appearing in twoor more studies are included in Tables S2 though S6 in thesupplemental material. A guide to the tables and figures ispresented in Table 1. The antigens and their alternative namesare listed in Table 2. The most frequently evaluated antigens

are described below and in additional tables and figures, asnoted.Assessment of study quality. The majority of studies used a

case-control study design. Only 65 (26%) studies reportedblinded interpretation of index test results. Almost all studiesprovided sufficient detail describing the execution of the indextest (Table 3).

Meta-analysis (Table 4 and Fig. 3). (i) Recombinant pro-

teins. (a) Recombinant 38 kDa (Rv0934) (Table A1). 38 kDa, amajor protein present in culture filtrates ofM. tuberculosis, hasbeen studied extensively (1, 17, 22). Several studies have shownan association between the presence of anti-38 kDa antibodiesand advanced cavitary TB (14, 22, 75). In smear-positive pa-tients, recombinant 38 kDa yielded a sensitivity of 47% (95%CI, 39 to 55) and a specificity of 94% (95% CI, 86 to 98) (8, 27,33, 35, 37, 55, 77, 81).

(b) Recombinant malate synthase (Rv1837c) (Table 5). Malatesynthase (81 kDa), present in M. tuberculosis culture filtrates,the cell wall, and cytoplasmic subcellular fractions, is an en-zyme of the glyoxylate pathway used by M. tuberculosis duringintracellular replication in macrophages (90) and has adaptedto function as an adhesin that enhances bacterial adherence tohost cells (46). In sputum smear-positive patients, malate syn-thase achieved a sensitivity of 73% (95% CI, 58 to 85) and aspecificity of 98% (95% CI, 95 to 100) (35, 37, 85, 86, 108). Thelikelihood ratio positive (40.78) was considerably higher formalate synthase than for other antigens. Earlier studies have

TABLE 1. Guide to tables and figures in the review

Category or antigenTable or

Figure

Antigen names .............. ............... .............. .............. ............... ...Table 2Characteristics of study quality................................................Table 3Meta-analysis..............................................................................Table 4Recombinant malate synthase .................................................Table 5

Recombinant CFP-10................................................................Table 6DAT ............................................................................................Table 7Specificity estimates by control group.....................................Table 8

Antigen performance by Ig class ............. ............... .............. ...Table 9Recombinant 38 kDa ................................................................Table A1Recombinant MPT51................................................................Table A2Native 38 kDa ............................................................................Table A3Native Ag85B.............................................................................Table A4Questions for quality assessment.............................................Table S1Recombinant protein antigens.................................................Table S2Native protein antigens.............................................................Table S3Lipid antigens.............................................................................Table S4Multiple antigens ............. .............. .............. ............... .............. .Table S5Protein/lipid antigens ................................................................Table S6Flow diagram for selection of subgroups ...............................Figure 1Flow of studies in the review...................................................Figure 2

SROC curve for recombinant proteins...................................Figure 3ASROC curve for native proteins..............................................Figure 3BSROC curve for lipid antigens.................................................Figure 3CSensitivity, TB-HIV coinfection...............................................Figure 4ASpecificity, TB-HIV coinfection...............................................Figure 4B

TABLE 2. Antigens evaluated for serodiagnosis of pulmonary TB

Name(s) of antigen(s)aProtein Rvdesignation

Reference(s)

Ag85C, 32.5 kDa, FbpC2, MPT-45 0129c 76, 7738 kDa, Ag 5, PstS1, PhoS, PhoS1 0934 3, 8, 15, 18, 20, 27,

33, 35, 37, 55,59, 69, 76, 77,

81, 102Mtb 81/88 kDa, malate synthase,

GlcB1837c 20, 35, 37, 76, 85,

86, 108DPEP, MPT32; 45/47 kDa, Apa,

ModD1860 19, 26, 76

Ag85B 1886c 23, 67, 77, 10316 kDa; -crystallin; 14 kDa,

HspX, Acr2031c 33, 39, 66, 103

27 kDa; MPT51, FbpC1, MPB 51 3803c 10, 69, 85, 108CFP-10, MTSA-10, EsxB; Lhp,

Mtb113874 27, 33, 59, 118

ESAT-6 3875 33, 118Mtb48 3881c 55DAT 43, 44, 83, 105TAT 43, 44SL-I, sulfolipid I 43, 44

Cord factor 43, 44, 101a Boldface indicates the name of the antigen in common use.


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demonstrated that whereas antibodies to the 38-kDa antigenare present in patients with extensive cavitary lesions, anti-malate synthase antibodies are elicited earlier during the pro-gression of TB, being present in patients who have not yetdeveloped cavities (74). This is reflected in the higher sensitiv-ity of TB diagnosis provided by malate synthase.

(c) Recombinant MPT51 (Rv3803c) (Table A2). The 27-kDaprotein MPT51, a culture filtrate protein, is closely related tothe antigen 85 (Ag85) complex, which comprises Ag85A,Ag85B, and Ag85C. MPT51 is an adhesin (108) reported to bea fibronectin-binding protein of M. tuberculosis (113). A suffi-

cient number of studies evaluating the performance of MPT51were available to stratify the results by HIV infection status. Insputum 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 to82); the specificities were 94% and 97%, respectively (10, 69,85, 108).

(d) Recombinant CFP-10 (Rv3874) (Table 6). Culture filtrateprotein 10 (CFP-10), a culture filtrate and cell wall protein, hasbeen identified as one of the earliest proteins expressed by M.tuberculosis during culture in bacteriological media (9). In spu-tum smear-positive patients, CFP-10 provided a sensitivity of48% (95% CI, 29 to 68) and a specificity of 96% (95% CI, 83to 99) (27, 33, 59, 118).

(e) Recombinant TbF6 (see Table S2 in the supplemental ma-

terial). TbF6 is a single antigen combining four distinct anti-gens (CFP-10, MTB8, MTB48, and 38 kDa) as a geneticallyfused polyprotein (37). In sputum-smear positive patients,TbF6 achieved a sensitivity of 70% (95% CI, 37 to 90) and aspecificity of 93% (95% CI, 69 to 99) (6, 37). The high sensi-tivity obtained with TbF6 is likely due to the fact that it com-prises immunogenic domains from multiple antigens.

(ii) Native proteins. (a) Native 38 kDa (Rv0934) (Table A3).

In sputum smear-positive patients, native 38 kDa provided asensitivity of 49% (95% CI, 37 to 61). In sputum smear-nega-tive patients, the sensitivity reported was lower (31%; 95% CI,15 to 52). Specificities were 97% in both subgroups (15, 18, 59,69, 76, 77, 102).

(b) Native Ag85B (Rv1886c) (Table A4). Ag85B, present in M.tuberculosis culture filtrates and cell walls, is a major compo-nent of the Ag85 complex (112). Like MPT51, Ag85B is afibronectin-binding protein (113). In HIV-negative TB pa-tients, native Ag85B yielded a sensitivity of 53% (95% CI, 20to 83), and in HIV-positive TB patients, a it yielded a sensi-tivity of 62% (95% CI, 19 to 92). The specificities were 95%(23, 67, 77, 103).

(c) Native -crystallin (2031c) (see Table S3 in the supplemen-tal material). -Crystallin is a 14/16-kDa cell wall protein (106)shown to be induced in bacteria under hypoxia (78). In sputum

TABLE 3. Characteristics of study quality

CharacteristicNo. (%) of

studies

Study designCross-sectional ............. ............... .............. .............. ............... 39 (15)Case-control............................................................................208 (82)Nested within observational study....................................... 7 (3)

Recruitment of participantsConsecutive or random......................................................... 20 (8)Convenience or not reported...............................................234 (92)

Selection criteria clearly described..........................................141 (56)

Complete verification by use of the reference standard ......107 (42)

Execution of test described in sufficient 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 insufficient in one study.

TABLE 4. Overall sensitivities, specificities, and likelihood ratios for antigens evaluated for serodiagnosis of pulmonary TB with assaysdetecting IgG and/or IgA antibodies

Type ofcompound

Antigen nameRv

designationNo. ofstudies

Smearstatus

HIVstatus

Sensitivity(%)a

Specificity(%)a

Likelihood ratiopositivea

Likelihood rationegativea

Recombinant 38 kDa 0934 12 Positive / 47 (3955) 94 (8698) 8.22 (3.4124.85) 0.56 (0.480.65)Malate synthase 1837c 8 Positive / 73 (5885) 98 (95100) 40.78 (14.43155.7) 0.27 (0.160.42)MPT51 3803c 5 Positive 59 (3876) 94 (7799) 10.50 (2.7069.69) 0.44 (0.260.67)MPT51 3803c 4 Positive 58 (3082) 97 (84100) 19.03 (3.73172.3) 0.44 (0.190.73)CFP-10 3874 6 Positive / 48 (2968) 96 (8399) 12.11 (3.2064.63) 0.55 (0.350.73)

TbF6b

4 Positive 70 (3790) 93 (6999) 9.61 (2.2353.99) 0.33 (0.130.66)TbF6, DPEPc 4 Positive 75 (5091) 95 (8699) 14.97 (5.4356.66) 0.26 (0.100.53)

Native protein 38 kDa 0934 13 Positive / 49 (3761) 97 (9499) 15.73 (8.8431.55) 0.53 (0.410.65)38 kDa 0934 7 Negative 31 (1552) 97 (9299) 9.13 (3.8824.05) 0.72 (0.510.87)

Ag 85B 1886c 4 Positive 53 (2083) 95 (7899) 9.36 (2.5253.81) 0.51 (0.200.84)Ag 85B 1886c 4 Positive 62 (1992) 97 (8999) 17.83 (4.0462.32) 0.39 (0.080.84)-Crystallin 2031c 6 Positive / 54 (3275) 96 (8399) 13.23 (3.5266.61) 0.48 (0.280.71)

Lipid DAT 7 Positive / 63 (4578) 81 (5096) 3.32 (1.3213.35) 0.47 (0.300.74)TAT 4 Positive / 81 (2199) 44 (2467) 1.44 (0.422.31) 0.42 (0.031.71)SL-I 4 Positive / 80 (5693) 59 (896) 1.94 (0.8920.90) 0.34 (0.142.22)Cord factor 5 Positive / 69 (2894) 91 (7897) 7.03 (2.4420.65) 0.35 (0.060.80)

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


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smear-positive patients, -crystallin provided a sensitivity of48% (95% CI, 29 to 68) and a specificity of 96% (95% CI, 83to 99) (66, 103).

(iii) Lipids. A variety of lipid-containing antigens are com-mon to mycobacterial species (71). Several lipid moieties havebeen purified and intensely studied for their serological poten-tial for TB diagnosis (44). Four lipid antigens, all acylatedtrehaloses (107), were evaluated in smear-positive patients andincluded in the meta-analysis.

(a) DAT (Table 7). 2,3-Diacyltrehalose (DAT), a componentof the M. tuberculosis cell wall, has been postulated to play arole in modulating host immune responses (50). DAT yieldeda sensitivity of 63% (95% CI, 45 to 78) and a specificity of 81%(95% CI, 50 to 96) (43, 44, 83, 105).

(b) TAT (see Table S4 in the supplemental material). 2,3,6-Triacyltrehalose (TAT) is an antigenic glycolipid compoundfound in the M. tuberculosis cell wall (41, 43). TAT provided asensitivity of 81% (95% CI, 21 to 99) and a specificity 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 compoundfound abundantly in the M. tuberculosis cell wall, may affect thehuman immune system and play a role in the pathogenesis ofTB (51). SL-I yielded a sensitivity of 80% (95% CI, 56 to 93)and a specificity 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 ofM. tuberculosis cell walls, is named for its central role in ag-gregating mycobacteria into cord structures (7, 30). Cord fac-tor may contribute to the virulence ofM. tuberculosis by facil-itating cavity formation (38). Cord factor achieved a sensitivityof 69% (95% CI, 28 to 94) and a specificity of 91% (95% CI,78 to 97) (43, 44, 101).

(e) TbF6 plus DPEP (see Table S5 in the supplemental mate-rial). TbF6 polyprotein plus DPEP was the multiple-antigencombination most frequently evaluated; four studies involvedHIV-uninfected individuals, and one study involved HIV-in-fected individuals. As described above, TbF6 is a polyprotein.DPEP, also known as MPT32, is a proline-rich 45/47-kDaantigen suggested to have a role in the cross-linking of mole-cules produced by or bordering M. tuberculosis (49). Earlierstudies with native MPT32 have demonstrated that it is ahighly immunogenic protein that provided higher sensitivitythan the 38-kDa protein when it was tested in the same patientcohort (74). In HIV-negative TB patients, TbF6 plus DPEPachieved a sensitivity of 75% (95% CI, 50 to 91) and a speci-ficity of 94% (95% CI, 86 to 99) (6, 37). The single studyevaluating the serodiagnostic potential of TbF6 plus DPEP inHIV-infected individuals is described below.

Assessment of specificity in healthy volunteers compared

with assessment of specificity in patients with nontuberculous

respiratory diseases (Table 8). Sufficient numbers of studiesevaluated five antigens, four proteins (recombinant 38 kDa,native 38 kDa, malate synthase, and CFP-10) and one lipid(DAT) for comparison of the specificities for healthy and dis-eased controls. For the four proteins, both subsets showedsimilar specificity values. For DAT, studies involving patientswith nontuberculous respiratory disease yielded a significantlyhigher specificity, 57% (95% CI, 30 to 76), than studies withhealthy volunteers, 97% (95% CI, 88 to 100).

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


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Overall performance of antigens (Fig. 3). Among recombi-nant proteins, malate synthase and TbF6 plus DPEP (multipleantigen) provided the highest sensitivities for the specificitiesreported (Fig. 3A). For native proteins, Ag85B in HIV-in-fected TB patients achieved better performance than othernative antigens (Fig. 3B). Among lipid antigens, cord factorhad the best performance (Fig. 3C).

Descriptive analysis. (i) Performance of antigens in pulmo-

nary TB patients with HIV infection (Fig. 4). Thirty studiesevaluating antigens (all proteins) in HIV-infected TB patientswere identified; all studies involved sputum smear-positive pa-tients. Of the total, 23 (77%) studies evaluated assays for thedetection of IgG and/or IgA antibodies (23, 35, 37, 69, 103,108), four studies evaluated assays for the detection of IgM

TABLE 5. Studies evaluating recombinant malate synthase (Rv1837c) for serodiagnosis of pulmonary TB

Author, yr(reference)

Study designReferencestandard

(smear statusa)

Patient(comparison)

country

Status ofindividuals usedfor comparison

HIV status ofpatient

(comparator)Ig class

No. ofparticipantsb

Sensitivity(%)c

Specificity(%)c

Chaudhary et al.,2005 (20)

Case-control Smear (SP) India (India) Healthy NRd (NR) IgG, IgM 44/105 32 (1948) 99 (93100)

Hendrickson etal., 2000 (35)

Case-control Culture (SP) South Africa andUganda(China)

Nontuberculousrespiratorydisease

(NR) IgG 52/31 60 (4573) 97 (83100)


Case-control Culture (SP) South Africa andUganda(China)


(NR) IgG 25/31 92 (7498) 97 (83100)

Houghton et al.,2002 (37)

Case-control Culture and/orsmear (SP)

Sub-SaharanAfrica (China)


(NR) IgG 59/31 78 (6588) 97 (83100)



Sub-SaharanAfrica (China)


(NR) IgG 66/31 58 (4570) 97 (83100)

Singh et al., 2003(86)

Case-control Smear (SP) India (area ofendemicity)

Healthy (NR) IgG 43/25 77 (6188) 100 (86100)

Singh et al. 2005(85)

Case-control Smear (SP) India (area ofendemicity)

Healthy (NR) IgG, IgA 40/29 75 (6782) 100 (91100)

Wanchu et al.,2008 (108)

Case-control Smear (SP) India (India) Healthy () IgG, IgA 138/38 73 (5288) 100 (91100)

Wanchu et al.,2008 (108)

Case-control Smear (SP) India (UnitedStates)

Healthy () IgG, IgA 26/65 73 (5288) 99 (92100)

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

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



(smear statusa)

Patient (comparison)country


HIV statusof patient


No. ofparticipantsb

Sensitivity(%)c

Specificity(%)c

Dillon et al.,2000 (27)


Brazil (United Statesand areas ofendemicity)

Healthy () IgG 250/57 28 (2234) 97 (88100)

Greenaway etal., 2005(33)

Nested withinobservationalstudy

Culture (SP) Gambia (Gambia) Healthy and (and )

IgG 100/100 63 (5372) 55 (4565)

Murthy et al.,2007 (59)

Case-control Culture (SP) India (India) Nontuberculousrespiratorydisease

() IgG 262/76 42 (3649) 99 (93100)


Case-control Culture (SP) India (India) Nontuberculousrespiratory

disease

() IgA 262/76 25 (2031) 99 (93100)


Case-control Culture (SP) India (India) Nontuberculousrespiratorydisease

() IgG, IgA 262/76 57 (5163) 97 (91100)

Zhang et al.,2007 (118)

Case-control Smear (SP) China (China) Healthy () IgG 50/28 78 (6489) 96 (82100)


Case-control Culture (SN) India (India) Nontuberculousrespiratorydisease

() IgG 60/76 10 (421) 99 (93100)



() IgA 60/76 43 (3157) 99 (93100)



() IgG, IgA 60/76 50 (3763) 97 (91100)

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


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antibodies (69, 103), and three studies evaluated assays for the

detection of IgG plus IgA plus IgM antibodies (69, 103). Fourstudies based on multiple-antigen combinations are describedin more detail below (35, 37, 108).

Antibodies to all five single antigens (38 kDa, malate syn-thase, Ag85B, -crystallin, and recombinant MPT51) evalu-ated in these studies were detected. As discussed, only MPT51and Ag85B were investigated in a sufficient number of studiesfor inclusion in the meta-analysis (Table 4). With assays for thedetection of IgG and/or IgA antibodies, the sensitivities re-ported for 38 kDa (range, 35% to 68%) and -crystallin(range, 15% to 58%) were similar to those provided forMPT51 and Ag85B. Malate synthase achieved higher sensitiv-ities (range, 73% to 92%). Compared with tests for the detec-

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 ofIgM (IgG plus IgA plus IgM) did not appreciably increase thesensitivity. The specificities provided by all of the above anti-gens were high (range, 89% to 100%). However, only 6 (20%)studies involved controls with nontuberculous respiratory dis-ease (23, 35, 37), while 14 (47%) studies involved eitherhealthy volunteers or asymptomatic HIV-infected individualswithout TB (35, 37, 103, 108). In 10 (33%) studies, the controlgroup involved HIV-infected individuals whose clinical statusranged from asymptomatic to symptomatic with opportunisticinfections other than TB (69).

(ii) Performance of tests with multiple antigens (see Table

S5 in the supplemental material). Assays based on multipleantigens 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 theymaintained high specificities (median, 96%; range, 79% to100%) (data not shown). The combination of malate synthaseplus MPT51 was evaluated in three studies, two studies involv-ing HIV-negative TB patients (2, 108) and one study involvingHIV-infected TB patients (108). The sensitivities provided bymalate synthase plus MPT51 were similar with HIV-uninfectedand -infected TB patients from India: 80% (95% CI, 73 to 87)and 77% (95% CI, 56 to 91), respectively. The specificitieswere equivalent (97%) whether the comparison group involved

TABLE 7. Studies evaluating DAT for serodiagnosis of pulmonary TB



(smear statusa)

Patient(comparison)

country


HIV statuspatient

(comparator)

Igclass

No. ofparticipantsb

Sensitivity(%)c

Specificity(%)c

Julian et al., 2002(44)

Cross-sectional Culture (SP) Spain (Spain) Nontuberculousrespiratorydisease

and () IgG 42/48 60 (4374) 58 (4372)



and () IgA 42/48 79 (6390) 50 (3565)



and () IgM 42/48 10 (323) 100 (93100)



() IgG 29/35 52 (3371) 57 (3974)



() IgA 29/35 79 (6092) 51 (3469)



() IgM 29/35 7 (123) 100 (90100)

Simonney et al.,

1997 (83)

Cross-sectional Culture (SP) France

(France)

Healthy and

(NRd

)

IgG 31/50 32 (1751) 96 (86100)

Vera-Cabrera etal., 1999 (105)e

Case-control Culture (SP) Mexico(Mexico)

Healthy NR (NR) IgG 39/35 49 (3265) 97 (85100)

Vera-Cabrera etal., 1999 (105)

Case-control Culture (SP) Mexico(Mexico)

Healthy NR (NR) IgG 39/35 80 (6491) 97 (85100)

Simonney et al.,1997 (83)

Cross-sectional Culture(SN)

France(France)

Healthy and (NR)

IgG 29/50 21 (840) 96 (86100)

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

TABLE 8. Specificity estimates by type of comparison

Antigen name

Specificity (%)a

Patients withnontuberculous

respiratory diseaseHealthy subjects

Recombinant 38 kDa 97 (9099) (6) 90 (5799) (6)Recombinant malate synthase 97 (91100) (4) 99 (81100) (4)Recombinant CFP-10 99 (92100) (3) 90 (4399) (3)Native 38 kDa 96 (9099) (6) 98 (92100) (4)DAT 55 (3076) (4) 97 (88100) (3)

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


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FIG. 4. Performance of antigens for the serodiagnosis of pulmonary TB in HIV-infected patients. (A) Sensitivity; (B) specificity. The circles andthe 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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HIV-negative healthy volunteers from India or HIV-infected(tuberculin skin test-positive and -negative) asymptomatic in-dividuals from the United States (108). However, this antigencombination yielded a sensitivity of only 55% (95% CI, 36 to72) with TB patients from the United States (2).

The combination of TbF6 plus DPEP plus malate synthaseachieved a sensitivity of approximately 85% with both HIV-negative and HIV-infected TB patients (37). The specificitieswere high (97%; 95% CI, 83 to 100), even when this antigencombination was evaluated with patients with nontuberculousrespiratory disease. In studies in which 38 kDa plus malatesynthase 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 speci-ficity was 89% (95% CI, 78 to 96) when it was assessed withhealthy volunteers (35). With HIV-negative, sputum smear-positive 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-negativepatients, the two combinations provided sensitivities of 73%(95% CI, 57 to 86) and 78% (95% CI, 62 to 89), respectively,and a specificity of 87% (95% CI, 75 to 94) when they wereassessed with patients with nontuberculous respiratory diseases(68). Only two studies with multiple lipid antigens were iden-tified (see Table S6 in the supplemental material).

(iii) Test performance by Ig class (Table 9). Stratification byIg class showed that in comparison with the results of studiesthat 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 hadconsiderably lower sensitivities (median, 11%; range, 2% to71%). The median specificities were similar: 96%, 96%, and98%, respectively. In addition, compared with the results oftests that detected only anti-IgG or anti-IgA antibodies, teststhat detected IgG plus IgA showed higher sensitivities (me-dian, 71%; range, 43% to 97%). The inclusion of IgM (IgGplus IgA plus IgM) did not further enhance the sensitivity(median, 71%; range, 60% to 83%).

DISCUSSION

Principal findings. This systematic review yielded 254 stud-ies evaluating 51 distinct single antigens and 30 multiple-anti-gen combinations. The performance of these antigens was ex-

amined in in-house tests for the serodiagnosis of pulmonaryTB. Studies evaluating 13 distinct antigens (recombinant 38kDa, native 38 kDa, MPT51, malate synthase, CFP-10, TbF6polyprotein, Ag85B, -crystallin, DAT, TAT, SL-I, cord factor,and TbF6 plus DPEP [multiple antigen]) were included in themeta-analysis. The results demonstrate that (i) in sputumsmear-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 sig-nificantly 50%; (ii) all protein antigens achieved high speci-ficities; (iii) among the lipid antigens, cord factor had the bestoverall performance (sensitivity, 69% [95% CI, 28 to 94]; spec-ificity, 91% [95% CI, 78 to 97]); (iv) compared with singleantigens (median sensitivity, 53%; range, 2% to 100%), mul-tiple antigens yielded higher sensitivities (median sensitivity,76%; range, 16% to 96%); (v) in HIV-infected patients whoare sputum smear positive, antibodies to several single andmultiple antigens were detected; and (vi) data on seroreactivityto specific antigens in sputum smear-negative or pediatric pa-tients were insufficient. These results demonstrate that no sin-

gle antigen provides a sensitivity that is sufficient for a singleantigen to be used to devise a serodiagnostic test for TB andthat it is unlikely that a single antigen-based serodiagnostic testcan be devised. This is not surprising, since the titers of anti-bodies to each antigen would differ in individuals and thedetection of low titers of antibodies would be occluded due tothe formation of immune complexes. It is also interesting thatwhile 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, antibodiesto these antigens are not detected in a vast majority of tuber-culin 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 andwhether cross-reactive antibodies exist in nontuberculous my-cobacterial diseases remains to be reported.

Stratification by Ig class demonstrated that assays for thedetection of IgG and/or IgA antibodies provided higher sensi-tivities than assays for the detection of IgM antibodies. This isnot surprising, since IgM antibodies are likely to be expressedearly during the onset of infection, with the levels quicklydecreasing after this period. By the time that bacteriologicallydetectable TB manifests, whether it is during primary infectionor reactivation, the infection has already progressed formonths to years in immunocompetent individuals and weeks tomonths in immunocompromised patients. Thus, the detectionof IgM antibodies may have a role in the identification of earlyinfection, but its value for the serodiagnosis of active TB dis-ease may be limited. Considering that the profile of antigensrecognized by antibodies is altered with the progression of M.tuberculosis infection (74), the antigens used in a serodiagnos-tic test during contact tracing are likely to differ from thoseused in a test for the diagnosis of clinical TB. To our knowl-edge, no antigens that can be the basis for an accurate serodi-agnostic test for contact tracing have been reported. The dis-covery, evaluation, and comparison of such tests with gammainterferon release assays need to be considered.

This systematic review and meta-analysis had severalstrengths. Standard protocols for the conduct of the review(61) and assessment of the quality of the studies (110) were

TABLE 9. Sensitivity and specificity of in-house antibody detectiontests by Ig class

Ig classNo. ofstudies

Median (range) %

Sensitivity Specificity

IgG 151 61 (8100) 96 (26100)IgA 25 40 (1090) 96 (48100)

IgM 24 11 (271) 98 (89100)IgG and IgA 34 71 (4397) 97 (85100)IgG and IgM 3 36 (3252) 98 (9899)IgM and IgA 0IgG, IgA, and IgM 7 71 (6083) 93 (9093)Not reported 10 41 (1674) 96 (8999)

All 254 58 (2100) 96 (26100)


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followed. The application of a comprehensive search strategywith various overlapping approaches enabled the retrieval ofrelevant studies published since 1990. Screening and data ex-traction were performed independently among three review-ers, and authors were contacted to clarify points and obtainmissing data. None of the studies in the review used the resultfrom the antibody test as a reference to confirm TB (incorpo-ration bias). When possible, patients with disease were se-lected, in preference to healthy controls, to evaluate the per-formance of antigens with persons in whom TB was initiallysuspected and subsequently ruled out.

The meta-analysis was limited by the relatively small numberof studies investigating the same antigens or antigen combina-tions. The small number of comparable antigens made it dif-ficult to relate study quality to antigen performance. However,several important deficiencies in study design and quality werenoted. Only 20 (7%) studies recruited participants in a randomor consecutive manner. Therefore, most studies lacked a sound

probabilistic sampling framework. The majority of studies useda case-control design with healthy controls. This design hasbeen found to overestimate test sensitivity and specificity (53,111), although for the four protein antigens in this review forwhich a comparison was feasible, the specificities were foundto be similar with healthy and diseased controls. Few (26%)studies reported the use of the blinded interpretation of testresults and a reference standard. This was not unexpected,since the primary aims of in-house studies are the discovery ofnovel antigens, evaluation of their diagnostic potential, and/orcomparison of different antigens. Nonetheless, the lack ofblinding and the dearth of data from cross-sectional studiesare major shortcomings of the currently available literatureand may have resulted in an overestimation of antigen per-formance (53). Both errors in design and deficiencies inreporting have been noted as concerns in TB diagnosticstudies (62, 89).

An additional limitation was the lack of information about

TABLE A1. Studies evaluating recombinant 38 kDa (Rv0934) for serodiagnosis of pulmonary TB



(smear statusa)

Patient (comparison)country


HIV statusof patient


No. ofparticipantsb

Sensitivity(%)c

Specificity(%)c

Amicosante et al.,1995 (3)

Case-control Culture (SP) Italy and United States(Italy and UnitedStates)

Healthy NR (NRd) IgM 41/30 54 (3769) 100 (88100)

Amicosante et al.,1995 (3)

Case-control Culture (SN) Italy and United States(Italy and UnitedStates)

Healthy NR (NR) IgM 29/30 55 (3674) 100 (88100)

Ben Amor et al.,2005 (8)

Case-control Smear (SP) Mexico (Mexico) Nontuberculousrespiratorydisease

() IgG 50/48 56 (4170) 96 (86100)

Chaudhary et al.,2005 (20)

Case-control Smear (SP) India (India) Healthy NR (NR) IgG, IgM 44/105 36 (2252) 99 (95100)

Dillon et al., 2000(27)



Healthy () IgG 250/57 49 (4355) 91 (8197)

Greenaway et al.,2005 (33)

Nested withinobservationalstudy

Culture (SP) Gambia (Gambia) Healthy and (and )

IgG 100/100 49 (3959) 50 (4060)


Case-control Culture (SP) South Africa andUganda (China)


(NR) IgG 52/31 46 (3261) 97 (83100)


Case-control Culture (SP) South Africa andUganda (China)

Nontuberculousrespiratory

disease

(NR) IgG 25/31 68 (4785) 97 (83100)




Healthy (NR) IgG 105/57 57 (4767) 91 (8197)



Philippines (UnitedStates and areas ofendemicity)

Healthy (NR) IgG 40/57 35 (2152) 9 1 (8197)



Sub-Saharan Africa(United States andareas of endemicity)

Healthy (NR) IgG 66/57 41 (2954) 9 1 (8197)

Lodes et al., 2001(55)


Brazil (China) Nontuberculousrespiratorydisease

() IgG 248/31 48 (4255) 97 (83100)



South Africa (China) Nontuberculousrespiratorydisease

() IgG 51/31 29 (1844) 97 (83100)



Philippines (China) Nontuberculousrespiratorydisease

() IgG 31/31 36 (1955) 97 (83100)

Senthil Kumar etal., 2002 (77)

Case-control Culture (SP) India (India) Healthy NR (NR) IgG 25/25 60 (3979) 100 (86100)

Silva et al., 2003(81)

Cross-sectional C ulture (SN) Canada (Canada) Healthy NR (NR) IgG 33/54 39 (2358) 89 (7796)

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


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sputum microscopy procedures. Smear status may be deter-mined through the examination of unprocessed sputum (directsmear microscopy) or through the more sensitive examinationof sputum after its digestion and concentration prior to theinoculation of cultures (concentrated smear microscopy). Theformer procedure is more common in low- and middle-incomecountries, where cultures are rarely done, while the latter pro-cedure is more common in high-income countries. Further-more, fluorescence microscopy, which is commonly used inhigh-income countries, is associated with a sensitivity higherthan that achieved by conventional light microscopy, which iscommonly used in low-income countries (96). Conv

413 pub art sorologiametanalisetb-steingart cvi 2009

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