Improved diagnosis of tuberculosis in HIV-positive ...

Author's personal copy International Journal of Infectious Diseases (2009) 13, 613—622

http://intl.elsevierhealth.com/journals/ijid

Improved diagnosis of tuberculosis in HIV-positive patients using RD1-encoded antigen CFP-10 Parasa V. Ramana Rao a, Madhan Kumar Murthy a, S. Basirudeen a, Pawan Sharma b, Soumya Swaminathan c, Alamelu Raja a,* a

Department of Immunology, Tuberculosis Research Centre (ICMR), Mayor V. R. Ramanathan Road, Chetput, Chennai 600 031, India Immunology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India c HIV/AIDS Division, Tuberculosis Research Centre (ICMR), Chetput, Chennai, India b

Received 17 June 2008; received in revised form 12 September 2008; accepted 27 September 2008 Corresponding Editor: William Cameron, Ottawa, Canada

KEYWORDS RD1; Serodiagnosis; CFP-10; Tuberculosis; HIV—TB; ELISA

Summary Objective: The present study was aimed at determining the serodiagnostic potential of 38-kDa (Rv0934, Mycobacterium tuberculosis complex-specific antigen) and CFP-10 (Rv3874, RD1 antigen) antigens among HIV-positive and HIV-negative patients with pulmonary TB. Methods: The diagnostic potential of native 38-kDa (n38-kDa) and recombinant CFP-10 (rCFP-10) antigens was ascertained in terms of sensitivity and specificity using an indirect ELISA. The study included 508 HIV-seronegative TB patients (TB), 54 HIV-seropositive TB patients (HIV—TB), 30 HIVpositive patients without TB (HIV), and 256 controls. Results: In HIV—TB, the sensitivities for individual antigens ranged from 14.8% to 31.5% and the specificity was >98% for IgG. When IgA results were added to IgG, the sensitivity increased to 25.9% for 38-kDa and 57.4% for CFP-10; specificity changed to 97.5% for 38-kDa and 98.1% for CFP10. The combined results of both the antigens gave 59.3% sensitivity and 95.6% specificity. In TB, the sensitivity was 82.8% when the antigen results were combined. None of the HIV-infected controls showed positivity for IgG to either of the two antigens. Conclusion: Use of CFP-10 enhances the sensitivity of 38-kDa, and therefore the 38-kDa and CFP10 antigen combination can be a diagnostic marker in HIV—TB. # 2008 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Introduction The increasing incidence of tuberculosis (TB) on account of its association with AIDS is unquestionably a cause of global * Corresponding author. Tel.: +91 (044) 2836 9626; fax: +91 (044) 2836 2528. E-mail address: [email protected] (A. Raja).

concern. TB is the leading cause of death among persons with AIDS, killing one of every three people who die of AIDS.1 TB and HIV infections are both intracellular and known to have profound influence on each other’s progression. Moreover, the emergence of multidrug-resistant strains is also a significant threat to TB control.2 In spite of the availability of an adequate treatment regimen for TB, attempts to restrict and eradicate the disease have failed. Delayed diagnosis of

1201-9712/$36.00 # 2008 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijid.2008.09.022

Author's personal copy 614 TB and initiation of appropriate treatment later than 3 weeks after presentation are associated with 45—85% of deaths in HIV-infected patients.3 Therefore accurate and early diagnosis of TB in HIV-infected patients will aid in the better management of this disease and hence improve the survival rate of these patients. The clinical features of TB in HIV-infected persons are often non-specific, hence diagnosis can be difficult. The conventional acid-fast staining method accounts for only 50% case detection in HIV—TB patients.4 By the time the culture results are available, Mycobacterium tuberculosis would have caused clinical deterioration. PCR-based methods are expensive. Decreased tuberculin reactivity, atypical radiographic features, and confusion with other HIV-related infections hinder the diagnosis of TB in HIV-infected patients.5 Therefore, better diagnostic methods are needed for HIV—TB. Alternative methods for the detection of M. tuberculosis based on serologic response to mycobacterial antigens have been reported. There is promise in serodiagnostic tests like ELISA because of their ease of performance, cost-effectiveness, and high specificity if species-specific antigens are used. The importance of purified, well-characterized antigens in ELISA,6—9 as opposed to crude extracts,10,11 is well recognized. The major drawback in such assays is the extensive person-toperson variation in antibody response, leading to poor sensitivity when a single antigen is used. In this scenario, the prospective approach would be to use more than one antigen for the diagnosis of TB in HIV-infected patients. The RD1 gene product, culture filtrate protein-10 (CFP-10, Rv3874) is an early-secreted, M. tuberculosis complex-specific antigen and hence a suitable candidate to be used in diagnosis. Since the RD1 region is absent in Mycobacterium bovis bacille Calmette—Guerin (BCG), prior vaccination with BCG will not give false-positive results. The present study was undertaken in order to evaluate the diagnostic value of CFP-10 in HIV—TB cases. The ability of CFP-10 to enhance the sensitivity when combined with another well-characterized M. tuberculosis complex-specific antigen, 38-kDa (Rv0934), was investigated. Moreover, 38kDa and CFP-10 are known to diagnose advanced cavitary TB and non-cavitary TB, respectively. Hence, their combination may be a potential diagnostic candidate.

Materials and methods Study subjects The study was approved by the institutional ethics committee, and informed consent was obtained from the study subjects. Patients were recruited from the Tuberculosis Research Centre, Chetput, Chennai, India between 2001 and 2005. Eight hundred and forty-eight subjects were included, of whom 508 (59.9%) were HIV-negative pulmonary TB patients, 54 (6.4%) were HIV-infected TB patients, and 286 (33.7%) were controls. Blood was collected from the patients prior to starting treatment. The groups included in the present study are listed below. HIV-seronegative TB patients (TB group; N = 508) The clinical criteria for diagnosis of HIV-seronegative TB patients included the presence of two or more symptoms,

P.V.R. Rao et al. namely cough for more than 2 weeks, fever, weight loss, night sweats, anorexia, and hemoptysis. Of the 508 HIV-negative pulmonary TB patients, 262 were smear- and culture-positive (S+C+), of whom 197 were male and 65 female, with ages ranging from 20 to 87 years, median 42 years. Of the remaining 246 smear-negative cases, 60 (24.4%) were culture-positive (S C+), with a male:female ratio of 3:1 and an age range of 20—80 years, median 44 years. The remaining 186 patients were smear- and culturenegative (S C ), clinically and radiologically diagnosed cases. This group consisted of 139 males and 47 females, with ages ranging from 20 to 80 years, median 40 years. The X-rays were read by two independent readers (and by one more reader in the case of a disagreement between the two). These patients were placed on a regimen of broad-spectrum antibiotics for a period of 3 weeks and the X-rays were repeated. The X-ray abnormality persisted and the patients were placed on anti-tuberculous therapy (Category III Revised National Tuberculosis Control Programme (RNTCP) regimen); they responded to the therapy and had a good clinical outcome, and hence were considered as pulmonary TB patients. Examination of smears and cultures was done according to the methods established at our center.12 Two spot and one overnight sputum specimens were collected from each patient. Staining of acid-fast bacilli (AFB) with auramine— phenol was done for smears and they were examined under a fluorescence microscope. For culture, sputum was concentrated and inoculated onto Lowenstein—Jensen (LJ) medium and incubated for up to 8 weeks at 37 8C in a non-CO2 atmosphere. All patients were HIV-seronegative as tested by two enzyme immunoassays (EIA; Comb Aids-RS, Span Diagnostics, India and HIV TRI-DOT, J. Mitra & Co, India) in serum. There were no co-morbid conditions along with the TB. HIV-seropositive pulmonary TB patients (HIV—TB group; N = 54) There were 41 males and 13 females in this group, and their age ranged from 20 to 67 years, with a median of 35 years. The HIV status was confirmed by two rapid tests (EIA). When a serum was positive in both tests, it was considered HIVpositive. If a serum was positive for only one EIA (which was rare), Western blot was done as a confirmatory test. Pulmonary TB was bacteriologically confirmed by smear and culture positivity (S+C+) in 29 of the patients. Of the remaining 25 smear-negative patients, 14 were negative for smear, but positive for culture (S C+) and 11 were negative for both smear and culture (S C ). These 11 were initially classified as HIV—TB based on clinical suspicion without bacteriological evidence; they recovered on anti-tuberculous therapy, thus justifying their classification. Their CD4 counts ranged from 12 to 1052 103 cells/mm3 (mean CD4 265.24, SD 227.07; median CD4 167). HIV-seropositive patients without TB (HIV group; N = 30) This group consisted of 19 males and 11 females, with age ranging from 20 to 52 years, median 32 years. HIV status was confirmed by two rapid tests as previously described. These patients ranged in their clinical status from asymptomatic cases to those with opportunistic infections

Author's personal copy Diagnosis of TB in HIV-positive patients using CFP-10 other than TB. The opportunistic infections included oral candidiasis, skin diseases due to fungal infections, diarrhea due to bacterial and viral causes, meningitis other than TB, and carcinomas of the ovary and gastrointestinal tract. Their CD4 counts ranged from 49 to 836 103 cells/mm3 (mean CD4 307.53, SD 169.01; median CD4 286). TB was ruled out by X-ray, smear, and culture. Non-TB controls (N = 96) This category included subjects with symptoms of TB (cough, fever, loss of appetite, etc., but normal X-rays) and individuals with other lung diseases (bronchial asthma, lung carcinoma, pneumonia, etc., with X-ray shadows). The male:female ratio was 2.7:1. Their ages ranged from 20 to 80 years, with a median of 44 years. TB was ruled out in this group by sputum smear and culture. All 96 patients were HIVseronegative as evident by two EIAs. Among patients who presented with other lung diseases, 24 had pneumonia: Streptococcus pneumoniae (n = 10), Klebsiella spp (n = 7), Mycoplasma pneumoniae (n = 4), and Chlamydia spp (n = 3); 52 patients had other lung diseases, including bronchial asthma (n = 25), chronic obstructive pulmonary disease (n = 13), bronchiectasis (n = 7), lung cancer (n = 4), and other miscellaneous conditions (n = 3). The remaining 20 subjects had TB symptoms without any lung disease. Normal healthy subjects (NHS group; N = 160) These subjects were apparently normal without any clinical symptoms. They were laboratory and blood bank volunteers in whom HIV and TB infections were ruled out by blood tests and chest X-ray, respectively. The subjects of this category were negative for smear and culture. One hundred and ten males and 50 females were included. Their age ranged from 20 to 60 years, with a median of 40 years.

Latent TB, BCG status, and the tuberculin skin test The presence of antibodies due to sub-clinical latent TB infection (LTBI) and prior BCG vaccination has often been stressed as a complicating factor for serology of TB in the literature. Since childhood BCG vaccination is a policy in India, the majority of the patients may have been BCG vaccinated. Data on BCG vaccination status were not collected in this study. However, all study subjects were adults (>20 years), and previous studies from our laboratory have established that remote BCG vaccination does not influence the antibody response over and above that of background levels of antibodies found in endemic populations.13 The tuberculin skin test (TST) was not carried out in the study subjects. It is neither specific nor sensitive in this TB endemic area, especially in adults. It has been observed that >70% of females and >80% of males turn positive for purified protein derivative (PPD-S) by 25 years of age due to exposure to M. tuberculosis, as well as other environmental mycobacteria. Thus TST positivity in our population does not mean infection with M. tuberculosis alone, as in the case of nonendemic populations.14,15 Blood samples were collected, and serum was separated and stored sterile at 70 8C until use.

615

Antigens Isolation and purification of native 38-kDa (n38-kDa) The 38-kDa antigen was purified by two-dimensional preparative electrophoresis (employing Rotofor fluid phase isoelectric focusing in the first dimension and Prepcell electrophoresis in the second dimension), from M. tuberculosis H37Rv culture filtrate (CFA).16 Details of the method, data on purity (immunoblotting with a polyclonal antiserum against M. tuberculosis H37Rv culture filtrate), and identity of the antigen to the already reported 38-kDa (Antigen 5, Rv0934; immunoblotting with reference monoclonal antibody MAb IT-23 from the World Health Organization Bank) have been reported previously.16 Isolation and purification of recombinant CFP-10 (rCFP-10) MTSA-10 (M. tuberculosis-specific antigen-10/CFP-10) was prepared in the laboratory of Dr Pawan Sharma, ICGEB, New Delhi, India. In brief, the open reading frame Rv3874 encoding MTSA10 of M. tuberculosis was amplified by PCR from genomic DNA. The PCR product was then directly cloned in an intermediate vector (pGEM-T-Easy). After nucleotide sequence validation, the full-length gene was sub-cloned in the bacterial expression vector pQE-31 for expression as a polyhistidine-tagged recombinant MTSA-10 protein in Escherichia coli. Recombinant MTSA10 was purified by nickel nitrilotriacetic acid (Ni—NTA) metal affinity chromatography. Details of the method, data on purity (immunoblotting with a mouse antipolyhistidine monoclonal antibody), and concentration of lipopolysaccharide in MTSA-10 preparation have been reported previously.17

ELISA ELISA was carried out to estimate the IgG and IgA antibody levels in sera against the n38-kDa and rCFP-10 antigens as described previously.16 The 96-well polystyrene plates (Flatbottom, Nunc-Immuno Maxisorp polystyrene plates, Nunc A/S, Denmark) were coated with an antigen concentration of 1 mg/ ml for n38-kDa and 2 mg/ml for rCFP-10, and incubated overnight at 4 8C. Each antigen was coated individually. Plates were blocked with 1% casein and 10% normal goat serum for 1 h at 37 8C. Sera were used at 1:2000 dilution for IgG measurement and 1:500 for IgA, as fixed by prior standardizing experiments, and then incubated for 1 h at 37 8C. Plates were washed in between every step (3—5) using phosphate-buffered saline containing 0.1% Tween. Goat antihuman IgG/IgA peroxidase conjugates (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were used at 1:10 000 dilution for IgG and 1:5000 for IgA in blocking solution. Plates were washed (7) after incubation for 1 h at 37 8C. Ortho-phenylene diamine (OPD; Sigma Chemical Co., St Louis, USA) was used as the substrate, and the color development was arrested with 8N H2SO4. Finally the plates were read at 490 nm with 650 nm as the correction wavelength using a Spectromax ELISA reader (Molecular Devices, USA).

Statistical design and analysis The IgG and IgA cut-off value for each antigen was ascertained using receiver—operator characteristic (ROC) curve analysis between normal healthy subjects and S+C+ TB patients. The

Author's personal copy 616 Table 1

P.V.R. Rao et al. Demographic profile of subjects recruited

Group

n (%)

Male (%)

CD4 counts 103 cells/mm3 (median)

Age range in years (median)

TB S+C+ S C+ S C

508 262 60 186

(59.9) (30.9) (7.1) (21.9)

381 197 45 139

(75) (75.2) (75) (74.7)

NA NA NA NA

20—87 20—87 20—80 20—80

(42) (42) (44) (40)

HIV—TB S+C+ S C+ S C

54 29 14 11

(6.4) (3.4) (1.7) (1.3)

41 21 12 9

(75.9) (72.4) (85.7) (81.8)

12—1052 (167) 25—1052 (154) 36—969 (140) 12—513 (202)

20—67 23—54 21—67 20—59

(35) (37) (35) (33)

NHS HIV Non-TB controls

160 (18.9) 30 (3.5) 96 (11.3)

110 (68.8) 19 (63.3) 70 (72.9)

NA 49—836 (286) NA

20—60 (40) 20—52 (32) 20—80 (44)

Total

848 (100)

621 (73.2)

NA

NA

TB, pulmonary tuberculosis patients; HIV—TB, HIV-seropositive tuberculosis patients; NHS, normal healthy subjects; HIV, HIV-infected subjects; NA, not applicable; S+, smear-positive; S , smear-negative; C+, culture-positive; C , culture-negative.

Figure 1 IgG and IgA antibody levels for 38-kDa and CFP-10 antigen. NHS, normal healthy subjects; TB, pulmonary tuberculosis patients; HIV, HIV-seropositive patients without TB; HIV—TB, HIV-seropositive tuberculosis patients; Non-TB, non-TB controls. Each dot represents the optical density (OD) of a single serum specimen. The cut-off point for each antigen was determined using ROC curve analysis. The lowest cut-off value with >95% specificity and good sensitivity was considered optimal and is represented by the horizontal line (0.626 for 38-kDa IgG, 0.237 for CFP-10 IgG, 0.239 for 38-kDa IgA and 0.215 for CFP-10 IgA). Any sample exhibiting absorbance above the cut-off value was classified as positive.

Author's personal copy Diagnosis of TB in HIV-positive patients using CFP-10 lowest cut-off value with >95% specificity and good sensitivity was considered optimal. Any sample exhibiting absorbance above the cut-off value was classified as positive. The individual results for the isotypes IgG and IgA were combined. Pooled TB sera were used as the high optical density positive control and pooled NHS sera as the low optical density positive control, while phosphate-buffered saline containing 0.1% Tween was used as the negative control, in each plate.

Predictive value The predictive values for HIV-positive TB patients were calculated18 based on the prevalence of TB in HIV patients of Tamil Nadu.19,20

CD4 counts The CD4 cell count was estimated in blood samples of HIV and HIV—TB patients by flow cytometry. In brief, CD3+/CD4+ cells from peripheral blood were labeled by the lysed whole blood method, using the IMK lymphocyte kit (BD biosciences, CA, USA). The percentage of CD4 cells among the total lymphocytes was obtained from the FACSort (BD Biosciences, CA, USA) using Cell Quest Software (BD Biosciences, CA, USA), and the absolute CD4 count was calculated by multiplying this with the total lymphocyte count. Pearson’s correlation coefficient was used to measure the correlation between CD4 counts and antibody levels. Significance of correlation was calculated using SPSS version 13.0 (SPSS Inc., Chicago, IL, USA).

Results The demographic profile of the subjects enrolled in the study is presented in Table 1. A scatter plot of IgG and IgA antibody Table 2

617 response to 38-kDa and CFP-10 antigens for the various groups is shown in Figure 1. The cut-off value for each antigen was determined using ROC curve and were identified as 0.626 for 38-kDa IgG, 0.239 for 38-kDa IgA, 0.237 for CFP-10 IgG, and 0.215 for CFP-10 IgA.

Antibody levels to the 38-kDa protein As shown in Table 2, the antibody response to 38-kDa antigenic stimulation in HIV—TB patients was 14.8% (95% CI 6.62— 27.12) for both IgG and IgA. ELISA positivity for both the isotypes was less in HIV—TB patients than in pulmonary TB patients. When individual results were scrutinized, it was found that some sera negative for IgG were positive for IgA. Combining the isotype results (IgG + IgA) gave 25.9% positivity in HIV—TB patients. In the NHS group, the specificity of the assay was 98.8% for IgG, but dropped marginally to 97.5% upon combination of IgG and IgA results. The non-TB control group gave >95% specificity. In the HIV-positive TB-negative group, the specificity was 100% (95% CI 88.43—100.00) for IgG, and there was one false-positive for IgA antibodies (Table 2).

Antibody levels to the CFP-10 protein Antibody positivity to CFP-10 antigen in HIV—TB patients was 31.5% (95% CI 21.09—47.47) for IgG and 35.2% (95% CI 24.29— 51.26) for IgA isotypes (Table 3). ELISA positivity obtained with each of the isotypes was similar in HIV—TB and TB patients. On combining the isotype results, the sensitivity in TB patients was 57.5% and in HIV—TB patients, 57.4%. In the NHS group, the specificity of detection was 98.1% with isotype combination. As for the 38-kDa antigen, the specificity of CFP-10 in the non-TB control group was >95%. In

Antibody positivity for 38-kDa antigen

Groups

IgG No. positive (%) a

IgA No. positive (%) a

IgG + IgA No. positive (%) a

TB (n = 508) HIV—TB (n = 54) NHS (n = 160) HIV (n = 30) Non-TB controls (n = 96)

286 8 2 0 0

153 8 2 1 4

348 14 4 1 4

(56.3) (14.8) (1.2) (0) (0)

(30.1) (14.8) (1.2) (3.3) (4.2)

(68.5) (25.9) (2.5) (3.3) (4.2)

TB, pulmonary tuberculosis patients; HIV—TB, HIV-seropositive tuberculosis patients; NHS, normal healthy subjects; HIV, HIV-infected subjects. a Number of positive sera (percentage of positivity); positivity, OD >0.626 for IgG and >0.239 for IgA.

Table 3

Antibody positivity for CFP-10 antigen

Groups

IgG No. positive (%) a

IgA No. positive (%) a



157 17 1 0 2

203 19 2 5 1

292 31 3 5 3

(30.9) (31.5) (0.6) (0) (2.1)

(40.0) (35.2) (1.2) (16.7) (1.0)

(57.5) (57.4) (1.9) (16.7) (3.1)

TB, pulmonary tuberculosis patients; HIV—TB, HIV-seropositive tuberculosis patients; NHS, normal healthy subjects; HIV, HIV-infected subjects. a Number of positive sera (percentage of positivity); positivity, OD >0.237 for IgG and >0.215 for IgA.

Author's personal copy 618

P.V.R. Rao et al.

the HIV-positive TB-negative group, the specificity was 100% (95% CI 88.43—100.00) for IgG, but five of 30 subjects were positive for IgA antibodies (Table 3).

Table 4

Combination of antigen results (38-kDa + CFP-10)

Groups

Combination of antigens The results of 38-kDa and CFP-10 antigens were combined and analyzed. In HIV—TB subjects, 38-kDa gave a positivity of 25.9% (Table 2), CFP-10 gave a positivity of 57.4% (Table 3), and on combining the antigen results (38-kDa and CFP-10) the sensitivity was 59.3% (Table 4). The specificity was marginally reduced.

Antibody levels according to smear and culture status The overall results were split based on smear and culture positivity and then analyzed. Of the 54 HIV—TB cases, 29 presented with smear- and culture-positive TB. Seven of these (24.1%) were positive for 38-kDa and 18 (62.1%) were positive for CFP-10. Of the 14 S C+ HIV—TB patients, four (28.6%) were positive for 38-kDa and six (42.9%) for CFP-10. Among 11 S C HIV—TB patients, three (27.3%) were positive for 38-kDa, while seven (63.6%) were positive for CFP-10. When 38-kDa and CFP-10 results were combined and analyzed, 18 of 29 (62.1%) S+C+, six of 14 (42.9%) S C+, and eight of 11 (72.7%) S C HIV—TB cases were diagnosed (Table 5). In 508 HIV-negative TB patients, 182 of 262 (69.5%) S+C+ patients were positive for 38-kDa, 174 (66.4%) were positive for CFP-10, and on combining the antigen results 234 (89.3%) showed positivity. Of the 60 S C+ TB patients, 31 (51.7%) were positive for 38-kDa, 39 (65%) for CFP-10, and 46 (76.7%) were positive for the combined antigens. Among 186 S C TB patients, 135 (72.6%) were positive for 38-kDa and 79 (42.5%) for CFP-10, which increased to 141 positives (75.8%) on combining the antigen results (Table 6).

Table 5


38-kDa + CFP-10 IgG No. positive (%) a


341 22 3 0 2

421 32 7 6 6

(67.1) (40.7) (1.9) (0) (2.1)

(82.9) (59.3) (4.4) (20) (6.3)

TB, pulmonary tuberculosis patients; HIV—TB, HIV-seropositive tuberculosis patients; NHS, normal healthy subjects; HIV, HIVinfected subjects. a Number of positive sera (percentage of positive sera); positivity of 38-kDa, OD >0.626 for IgG and >0.239 for IgA; positivity of CFP-10, OD >0.237 for IgG and >0.215 for IgA.

Predictive values A positive predictive value (PPV) of 0.78 for 38-kDa and 0.91 for CFP-10 was found in the HIV—TB group. Negative predictive values (NPV) were 0.80 for 38-kDa and 0.87 for CFP10. Combining the 38-kDa and CFP-10 antigens gave a PPV of 0.82 and an NPV of 0.87 (Table 7).

Antibody response of HIV—TB based on CD4 counts The antibody levels and CD4 counts of HIV-infected TB patients were analyzed for a correlation (Figure 2). CFP-10 antibody levels for IgG had a trend to increase with CD4 count (r = 0.108) but the converse was true for the IgA isotype (r = 0.167). The trend was not significant for either of

ELISA positivity among HIV—TB patients based on smear and culture status

Isotype

S+C+ HIV—TB (n = 29) No. positive (%) a

S C+ HIV—TB (n = 14) No. positive (%) a

S C HIV—TB (n = 11) No. positive (%) a

38-kDa IgG IgA IgG + IgA

3 (10.3) 5 (17.2) 7 (24.1)

2 (14.3) 3 (21.4) 4 (28.6)

3 (27.3) 0 (0) 3 (27.3)

CFP-10 IgG IgA IgG + IgA

13 (44.8) 9 (31.0) 18 (62.1)

4 (28.6) 3 (21.4) 6 (42.9)

0 (0) 7 (63.6) 7 (63.6)

38-kDa + CFP-10 IgG IgA IgG + IgA

14 (48.3) 10 (34.5) 18 (62.1)

5 (35.7) 4 (28.6) 6 (42.9)

3 (27.3) 7 (63.6) 8 (72.7)

S+C+ HIV—TB, smear-positive and culture-positive HIV—TB patients; S C+ HIV—TB, smear-negative and culture-positive HIV—TB patients; S C HIV—TB, smear-negative and culture-negative HIV—TB patients. a Number of positive sera (percentage of positive sera); positivity of 38-kDa, OD >0.626 for IgG and >0.239 for IgA; positivity of CFP-10, OD >0.237 for IgG and >0.215 for IgA.

Author's personal copy Diagnosis of TB in HIV-positive patients using CFP-10 Table 6

619

ELISA positivity among TB patients based on smear and culture status

Isotype

S+C+ TB (n = 262) No. positive (%) a

S C+ TB (n = 60) No. positive (%) a

S C TB (n = 186) No. positive (%) a

38-kDa IgG IgA IgG + IgA

148 (56.5) 70 (26.7) 182 (69.5)

19 (31.7) 16 (26.7) 31 (51.7)

119 (64.0) 67 (36.0) 135 (72.6)

CFP-10 IgG IgA IgG + IgA

125 (47.7) 97 (37.0) 174 (66.4)

11 (18.3) 34 (56.7) 39 (65)

21 (11.3) 72 (38.7) 79 (42.5)

38-kDa + CFP-10 IgG IgA IgG + IgA

200 (76.3) 123 (46.9) 234 (89.3)

21 (35) 36 (60) 46 (76.7)

120 (64.5) 86 (46.2) 141 (75.8)

S+C+ TB, smear-positive and culture-positive TB patients; S C+ TB, smear-negative and culture-positive TB patients; S C TB, smearnegative and culture-negative TB patients. a Number of positive sera (percentage of positive sera); positivity of 38-kDa, OD >0.626 for IgG and >0.239 for IgA; positivity of CFP-10, OD >0.237 for IgG and >0.215 for IgA.

Table 7

Test characteristics for 38-kDa and CFP-10 antigens

Antigens

38-kDa CFP-10 38-kDa + CFP-10

IgG + IgA Sensitivity %

Specificity %

PPV

NPV

25.9 57.4 59.3

97.5 98.1 95.6

0.78 0.91 0.82

0.80 0.87 0.87

PPV, positive predictive value; NPV, negative predictive value. Positivity of 38-kDa, OD >0.626 for IgG and >0.239 for IgA; positivity of CFP-10, OD >0.237 for IgG and >0.215 for IgA.

the isotypes ( p > 0.5). Similar results were observed with the 38-kDa antigen (data not shown).

Discussion The routine diagnostic methods for TB (smear and X-ray) are not ideal for use in HIV—TB, due to lower sensitivity and nonspecificity. Serologic diagnostic tests using crude mycobacterial antigens such as culture filtrate antigen (CFA), PPD, and antigen A60,10,11 as well as purified lipid antigens21—24 and protein antigens,6—9 have been tried with varying results. Among the available purified protein antigens, 38-kDa and its epitopes have been well studied due to their specificity. In the present study, using the n38-kDa antigen, a reduced sensitivity of 14.8% (IgG, 95% CI 6.62—27.12) was obtained in HIV—TB patients, compared with 56.3% (95% CI 52.46—61.24) for TB patients. These results are comparable with those of earlier reports.8,25 In our previous study,26 a sensitivity of 38% was obtained (IgG) for HIV—TB patients, compared with 61% for TB patients on using this antigen. In contrast, using r38kDa antigen, better sensitivities in HIV—TB (67—70%) have been obtained compared to TB (40—58%).7,9 The response to 38-kDa is associated with advanced cavitary TB.27 In the present study, the patients were not categorized as cavitary or non-cavitary, based on X-ray. However,

there is evidence in the literature to show that HIV—TB is associated with a reduced ability to develop cavitary lesions.28 In order to improve the sensitivity in HIV—TB coinfection, we included an early-secreted antigen, CFP-10, apart from the 38-kDa antigen, in the present study. Unlike 38-kDa, CFP-10 is a less reported M. tuberculosis complex-specific RD1 antigen in serodiagnosis. The RD1 region is absent in attenuated strains of M. bovis BCG, Mycobacterium avium, and other non-tuberculous mycobacteria (exceptions are Mycobacterium kansasii, Mycobacterium szulgai, and Mycobacterium marinum).29—33 Since the RD1 region is absent in M. bovis BCG, prior vaccination with BCG will not give false-positive results. In the present study, CFP-10 had a sensitivity of 57.4% in HIV—TB co-infection and 57.5% in HIV-seronegative TB patients, with a specificity of 98.1%. When the 38-kDa antibody response was combined with that of CFP-10, sensitivity improved to 59.3% in HIV—TB patients and 82.9% in TB patients. The improved sensitivity on combining the results of both antigens may be attributed to the fact that they recognize different sets of patients, i.e., 38-kDa reacting with advanced cavitary cases and CFP-10 reacting with noncavitary cases. Many of the earlier serodiagnostic studies in HIV—TB considered patients as a single group, but did not classify


P.V.R. Rao et al.

Figure 2 Correlation of antibody response (CFP-10) with CD4 levels of HIV infected TB patients. OD indicates optical density. Each dot in the graph represents an individual and the straight line represents the trend of correlation between antibody response and CD4 counts of HIV—TB patients. r represents Pearson’s correlation coefficient and p represents significance of correlation.

them based on smear status. Smear-negative HIV—TB is an important group where diagnostic tests are urgently needed, because failure to diagnose TB in this group may lead to a higher mortality rate. In our study, 38-kDa antigen gave 28% (7/25) sensitivity in smear-negative HIV—TB cases. The sensitivity improved to 56% (14/25) when CFP-10 antigen was also included. A similar increase to 62.1% was seen in smearpositive cases. Isotypes other than IgG were measured in bronchoalveolar lavage fluid and serum in our earlier studies.16,34 Measuring isotypes other than IgG is useful, because some samples that were negative for IgG were still positive for IgA antibodies. Our previous study in HIV—TB patients from South India reported that when IgA results were added to IgG, MPT51 antigen sensitivity was augmented to 63% from 50%.26 Another study in HIV—TB patients showed improved sensitivity from 65% to 84% for 30-kDa, and from 15% to 56% for 16kDa antigen upon combining the isotype results.35 However, the significance of the IgA response is unclear. Some reports state that the production of antibody of a particular class is determined by the concentration of cytokines secreted by CD4+ T-cells. Interleukin (IL)-2, IL-4, IL-6, and interferon (IFN)-g have been found to contribute to the formation of IgG-producing clones, while IL-3, IL-5, and transforming growth factor (TGF)-b have been found to contribute to the formation of IgA-producing plasma cells.36 Since our population is heterogeneous, interpreting the cytokine profile with respect to antibody isotype is difficult, and therefore it was not included in this study. It is interesting to note that IgG showed 100% specificity while IgA showed only 83.3% for CFP-10 antigen in HIVinfected patients. The increased false-positivity in IgA is not due to non-specificity, since in the NHS group IgA showed 98.8% specificity for this antigen. One possible reason for such an enhanced positivity might be the ability of CFP-10 to diagnose subjects who are progressing to active TB. Longterm follow-up studies are needed to confirm this speculation, which is beyond the scope of this paper. In studies using different M. tuberculosis protein antigens for HIV—TB serodiagnosis, 30-kDa has yielded a sensitivity of 0—65%35,37,38 and 27-kDa has given 50% sensitivity,26 whereas 16-kDa has shown low sensitivity.25,35 The only protein anti-

gen reported in the literature to have >80% sensitivity in HIV—TB is the malate synthase recombinant antigen (MTB81).7 This study reported 92% sensitivity in a small number of patients (N = 27), the majority of them from Uganda. The same article also reported a sensitivity of 70% for the same antigen in another cohort of 37 patients from South Africa. Earlier investigators have repeatedly observed that single antigens will never suffice for highly sensitive diagnosis, even for smear-positive cases, and that the sensitivities that could be achieved are around 75% at best,39—41 since there is qualitative and quantitative heterogeneity in antibody response. This heterogeneous recognition of antigens in TB probably arises due to multiple factors like the difference in immunogenetic background of the infected host, stage of their disease, bacillary load, etc. Therefore, an important implication is that serodiagnostic assays for TB must be based on a rational design of antigen combinations to achieve high diagnostic accuracy. The 38-kDa protein is the antigen that has been most studied for the serodiagnosis of TB. Because of its species specificity, 38-kDa offers a test of >95% specificity. But since this antigen is associated with advanced, sputum smear-positive TB alone, sensitivity is limited to 60—70%. Therefore the rational design for a diagnostic test is one with multiple specific antigens, with 38-kDa definitely being one of them. The other antigens may add to the test positivity in cases not diagnosed by 38-kDa. It is the basis of the majority of commercial tests for the serodiagnosis of TB. In this way, the 38-kDa antigen has previously been combined with other proteins to increase the sensitivity of the test. A test sensitivity of 56.3% was achieved when the 38-kDa antigen was combined with MTB48 (a gain of 12.9%).42 When the 38-kDa antigen was mixed with MTB81, the sera from 68% of the TB patients analyzed reacted,7 and when the 38-kDa antigen was added to recombinant CFP-10, the rates of antibody detection increased from 49% to 58% for smearpositive TB patients and from 21% to 40% for smear-negative patients.43 When 38-kDa results were combined with MPT51, sensitivity was enhanced from 62% to 82%.26 Our control population included non-TB controls as well as normal healthy subjects. The inclusion of this population (which many studies do not include or include in insufficient

Author's personal copy Diagnosis of TB in HIV-positive patients using CFP-10 numbers) is crucial for clinical evaluation of diagnostic tests, since they are likely to be confused with TB patients. Non-TB controls might include LTBI too, since our area is endemic for TB. Besides non-TB controls, the NHS group might also contain LTBI. In the present study TST or the interferongamma release assay (IGRA) were not done to identify latent infection in our study population, since it is known that >70% of females and >80% of males turn positive for PPD-S by 25 years of age due to exposure to M. tuberculosis as well as other environmental mycobacteria.14,15 Studies have reported 40% LTBI in healthy community controls recruited from India.44 We observed only up to 6.3% false-positivity in non-TB controls and 4.4% in NHS, which implies that the serodiagnostic assay did not identify latent infection, but only active disease. In the present study, no significant correlation between CD4 cell counts and antibody levels in HIV—TB patients was observed, as demonstrated in other studies.26,45 In a study by Ratanasuwan et al.,46 a difference in antibody response based on the CD4 counts was observed with lipoarabinomannan antigen. The sensitivity among HIV—TB patients who had a CD4 count 200 103 cells/mm3 was significantly higher than for patients with a CD4 count < 200 103 cells/mm3. In the present study, routine sputum smear examination diagnosed 29 out of 54 (53.7%) HIV—TB cases, while ELISA identified 32/54 (59.3%). Forty-three of 54 (79.6%) HIV-positive TB cases were positive for either of the tests. Thus, it can be concluded that ELISA, used along with sputum smear can be a good diagnostic test for HIV—TB co-infection. It is evident that the use of CFP-10 supplements the sensitivity of 38-kDa antigen, and hence CFP-10 antigen has diagnostic utility in HIV—TB as well as in HIV-negative TB irrespective of the sputum smear status. Furthermore, measurement of IgA antibodies along with IgG results in better test outcomes.

Acknowledgements We acknowledge the Indian Council of Medical Research, New Delhi, India for providing Junior Research Fellowship to Mr Ramana Rao and Mr Basirudeen. Mr Madhan Kumar is the recipient of Junior Research Fellowship from the Council of Scientific and Industrial Research, New Delhi, India. The help rendered by our colleague Mr D. Anbarasu in manuscript preparation is kindly acknowledged. Conflict of interest: No conflict of interest to declare.

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