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Diagnostic accuracy and safety of Cy-Tb for detection of tuberculosis infection: A multicentric study from India
#Equal contributions
Present address: $National Institute of Occupational Health, Ahmedabad, Gujarat, India
For correspondence: Dr Manjula Singh, Division of Delivery Research, Indian Council of Medical Research, New Delhi 110 029, India e-mail: drmanjulasb@gmail.com; drmanjulasb@outlook.com
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Received: ,
Accepted: ,
How to cite this article: Singh M, Hussain T, Modi B, Nair S, Kumar S, Alvi Y, et al. Diagnostic accuracy and safety of Cy-Tb for detection of tuberculosis infection: A multicentric study from India. Indian J Med Res. 2026;163:309-18. doi: 10.25259/IJMR_1794_2025
Abstract
Background and objectives
Only 10% of the individuals infected with Mycobacterium tuberculosis (MTB) manifest active tuberculosis. Interferon-γ release assay (IGRA) and tuberculin skin test using purified protein derivative (PPD) are used to identify tuberculosis (TB) infection. We aimed to study the comparative performance of Cy-Tb (the SIILTIBCY skin test) and PPD with IGRA as the reference standard.
Methods
In step-I, 30 TB patients and 32 IGRA-negative health controls; in step-II, 2651 participants aged 1 to 85 yr; and in step- III, 647 household contacts of TB patients aged 1-18 years old were enrolled. Blood for IGRA was drawn immediately prior to intra-dermal administration of Cy-Tb (the SIILTIBCY skin test) or PPD. The size of induration of Cy-Tb ≥5 mm or PPD ≥ 10mm after 48 to 72 h were considered positive. Combined Data from step II and step III were analysed for sensitivity, specificity, agreement, and kappa-coefficient with IGRA as the reference standard using Stata version 14.2.
Results
The step-I results for Cy-Tb showed 90% specificity, 93.8% specificity, and an agreement of 91.9% with IGRA and a Cohen’s kappa coefficient of 0.83. Combined analysis of step-II and step-III data revealed sensitivity of 82.6% (95% CI 79.1-85.8), specificity of 71.1% (95% CI 68.2-73.8), agreement of 74.8%, and kappa of 0.5 for Cy-Tb. PPD showed sensitivity and specificity of 71.0% (95% CI 66.7-75.0) and 71.9% (95% CI 69.0-74·6), respectively with 71.6% agreement and kappa of 0.4 with IGRA.
Interpretation and conclusions
Performance of Cy-Tb was comparable to that of PPD with IGRA as reference standard. Cy-Tb skin test can be used for diagnosis of TB infection.
Keywords
Cy-Tb
IGRA
PPD
TB infection
Tuberculin skin test (TST)
Tuberculosis caused by Mycobacterium tuberculosis (MTB) is diagnosed microbiologically or clinically based on the symptoms and X-rays. Only 10% of the infected individuals progress to active tuberculosis (TB) during their life-time,1 when their immune system is compromised, suggesting the need to detect individuals with TB infection since they are the reservoir of infection.2 Thus, treating the MTB infected individuals with TB preventive therapy (TPT) is important to achieve TB elimination.
Interferon gamma release assay (IGRA) and tuberculin skin test (TST) using purified protein derivative (PPD) are used for detection of TB infection. However, due to shortage of PPD, comparable tests are required to replace PPD. Since Cy-Tb (the SIILTIBCY skin test), has shown to deliver IGRA-like results3,4 we have evaluated its diagnostic accuracy for TB infection in India.
We hypothesised that Cy-Tb skin test is comparable to PPD to detect TB infection. We evaluated whether the sensitivity, specificity, percent agreement and kappa-coefficient of the Cy-Tb skin test was comparable to that of standard TST using PPD with respect to quantiFERON -plus i.e., IGRA as reference standard in general population, house-hold contacts (HHCs) and high-risk population in 6 states of India.
Methods
Study design
This was a cross-sectional, open comparison of the diagnostics performance of Cy-Tb and 2 T.U. tuberculin PPD RT23 SSI with quantiFERON®-TB Gold Plus as reference standard. The trial was designed to address the issue that there is no gold standard that is 100 % sensitive and specific for the diagnosis of TB infection.
Study was conducted after getting necessary approvals from the Central Drugs Standard Control Organization (CDSCO) and the Institutional Human Ethics committees of participating sites. Participants were enrolled after obtaining informed consent/assent following Helsinki Principles, ICMR guidelines, and good clinical practice guidelines.
Step I: For proof of concept, concordance of Cy-Tb results with TB positivity was tested in 30 microbiologically confirmed TB patients and 32 IGRA negative healthy controls with no symptoms or signs suggestive of TB and without a history of contact with a TB patient, from six sites in India.
Step II: Study population and sample size: Participants in the age group of 1 to 85 yr from six states of India, viz. Kerala, Gujarat, Delhi, Odisha, Telangana and Sikkim were included. Participants were recruited from general population at Kerala and Gujarat sites, living in the field practice area of the District TB Centre. This was done utilising routine community outreach by TB health visitors under NTEP. Delhi, Odisha, and Telangana sites, recruited household contacts (HHCs) of TB patients, during routine contact tracing activity. Sikkim site recruited high risk individuals who had no sign and symptoms of TB, but had the risk factors like diabetes mellitus, low body mass index (BMI) (<18.5 kg/m2), elderly (>60 yr), or history of smoking and tribals, selected from routine screening for TB symptoms at the health facilities and within the community catered by the Sikkim site.
With an assumed sensitivity of 80% (null hypothesis) and alternative hypothesis of 90%, and power of >80% sample size was calculated to be 2407 and with a drop-out of 10%, it was 2649, with variable numbers from each site based on the prevalence of TB infection in the target group. A total of 2651 participants were enrolled from six states (Supplementary Table I) and randomly divided in two groups that were followed up between December 2021 to March 2022 (Fig. 1). In the first group, participants received Cy-Tb on one arm and in-vitro QFT-plus test. In the second group participants received PPD on one arm and in-vitro QFT-plus test.
- Study participants flow for recruitment and data availability.
Step III: The study was extended to include additional HHCs of TB patients aged 1 month to 18 years. Sample size was calculated to be 470, assuming estimated prevalence of TB infection in the target population to be 50%, with a margin of error (d) = 0.05, with a sensitivity of 87% and specificity of 85%, and loss to follow up 20%. However, 647 HHCs from five states were enrolled in this step between October 2022 to June 2023 (Fig. 2).
- Flow chart showing study participants recruited for Stage III (children and adolescents aged 1-18 yr and data availability).
Inclusion criteria
Step I: Microbiologically confirmed TB cases, diagnosed within last 3 years were included. IGRA negative participants without history of contact with TB patients and no symptoms or signs suggestive of TB were included as healthy controls.
Step II: Males or non-pregnant females 1 through 85 years of age, both inclusive, were included from general population, high-risk population, and HHCs who were in close contact with microbiologically confirmed pulmonary TB index case for more than 6 h/day for minimum five days (Supplementary Table II). Negative urine pregnancy test and adequate contraception for 28 days prior to ‘Intervention Product’ (IP) administration in female subjects of childbearing potential was assured.
Step III: Additional children and adolescents, 1 month to 18 yr old who were HHCs of microbiologically confirmed pulmonary TB index case for more than 6 hours/day for minimum five days and were willing and likely to comply with the trial procedures.
Exclusion criteria
Participants who were vaccinated with any live vaccine within 6 wk prior to the day of inclusion, had been tested for TST<12 months prior to the day of inclusion, or had active disease affecting the lymphoid organs, and lactating females were excluded. Exclusions also included participant with skin condition/s which interfere with Cy-Tb and PPD reading e.g., tattoos, severe scarring, burns/sunburns, rash/eczema, psoriasis etc. at or near injection site, bleeding or coagulation disorders etc., or were participating in other clinical trials with investigational or non-investigational drugs or devices, or any condition which might interfere with the trial results.
Details of the investigational products (IPs)
Cy-Tb(rESAT-6+rCFP-10), 2T.U. Tuberculin PPD RT23 SSI (PPD) vials were provided by Serum Institute of India, and the reference standard, QuantiFERON®-TB Gold Plus (QFT plus) kits for IGRA were purchased from Qiagen.
Dosage and route of administration of Cy-Tb or PPD
0.1 mL of the Cy-Tb or PPD was administered intradermally by Mantoux technique, into the flexor surface of right/left forearm at junction of the upper one third and lower two-thirds, as indicated by the randomisation code allocated.
Randomisation
Site-specific simple randomisation was done by statistician to give either Cy-Tb or PPD to be administered in left or right arm of the consecutive participants, using a statistical software. Staff administering the allocated IP as per the randomisation, was aware of the IP and noted it in Case investigation form (CIF). However, the examiner measuring the in duration did not know which test was administered. The trial consisted of three visits:
Visit 1 (Day 0) screening, enrolment and injection
After informed consent/assent was obtained, screening and enrolment was done. Details of medical history and BCG vaccination status was documented and blood for QFT-Plustest (for IGRA) was drawn, immediately prior to intra-dermal administration of Cy-Tb or tuberculin PPDRT23SSI (PPD). Blood was transported to the IGRA labs at 4oC in icebox and incubation started within 6 h, as per manufacturer’s instructions. Cy-Tb and PPD were administered using tuberculin syringe with needle bevel size of 23 g or 24 g by the nurses trained in intradermal injection. Participants were closely monitored for 30 min after intra-dermal administration of Cy-Tb or PPD, for adverse events.
IGRA test was performed as per the manufacturer’s instructions. Briefly, 1 mL of blood was collected in four collection tubes, which included a negative control tube, 2 TB antigen tubes (with recombinant antigens CFP-10 and ESAT-6), and a mitogen tube (positive control). After briefly mixing the blood, tubes were incubated at 37°C for 16 to 24 h, followed by centrifugation, and estimation of IFN-γ (IU/mL) in the supernatants using ELISA. IGRA results with a cut-off at ≥ 0.35 IU/mL (after subtracting their respective negative control values), were considered positive. The positive control value must be ≥ 0.5 IU/mL to ascertain the integrity of the test, such that a positive control values < 0.5 IU/mL, with < 0.35 IU/mL for the TB specific test, would be labelled as indeterminate. However, the same value for TB specific test with ≥ 0.5 IU/mL in the positive control would be negative.
Visit 2 (Day 2-3) follow up
After 48 to 72 h of administration of Cy-Tb or PPD, adverse events were recorded and the size of induration was measured. Margins of the induration were marked using both palpation and ball point method with a marker and readers were trained to measure the induration transversely to the long axis of the fore arm using a scale. Supervisory visits were conducted by the site investigator and readings were verified for inter-reader concordance. Digital images of injection sites were taken along with the scale showing the size of the induration, for the purpose of record and verification (Fig. 3).
- Representative pictures of diameter of induration at the Cy-TB (A, B, C) and PPD (D, E, F) injection site of participants measured transversely to the long axis of the forearm at 2 to 3 days after intra-dermal administration of Cy-TB and PPD according to the cut-off value ≥ 5mm and ≥10 mm Cy-Tb and PPD, respectively in participants showing negative or indeterminate results with IGRA.
Visit 3 (Day 28) follow up
On the 28th day, adverse events were recorded and the size of induration was again recorded along with general medical examination of the participant.
Outcome variable
Outcome variable was to assess the diagnostic accuracy of Cy-Tb with IGRA as reference standard and determine whether the sensitivity, specificity, agreement of Cy-Tb with IGRA was comparable to that of PPD. Induration size of ≥ 5 mm for Cy-Tb and ≥ 10 mm for PPD was considered as positive and less were considered negative. Cut-off size of 5 mm applied in the current study for Cy-Tb, has been validated in earlier trials.5,6
Safety endpoints were the injection site adverse reactions within 2 to 3 days and 28 days after intra-dermal administration of Cy-Tb and PPD.
Statistical analysis
Sensitivity, specificity, agreement,7 and Cohen’s kappa coefficient of both Cy-Tb and PPD with respect to IGRA as reference standard were determined using Stata Version 14.2 software, to compare the diagnostic accuracy of Cy-Tb with PPD. Since there is no gold standard to diagnose TB Infection, concordance was evaluated between Cy-Tb and QFT and between PPD and QFT assay using proportion agreement, and kappa (k) coefficients, as reported earlier by Dogra et al7 Kappa in the range of 0.21–0.40 was considered ‘fair’ agreement, in the range of 0.41–0.60 was considered ‘moderate’ agreement, in the range of 0.61–0.80 was considered ‘substantial’ agreement, and kappa >0.81 was considered ‘almost perfect’ agreement as proposed by Landis and Koch.8
Results
In step-I,30 TB patients and 32 unexposed healthy controls were recruited to study the sensitivity and specificity of Cy-Tb in known infected and uninfected subjects. Cy-Tb exhibited sensitivity of 90% (95% CI 74.4-96.5), specificity of 93.8% (79.9-98.9), 91.9% agreement with IGRA and Cohen’s kappa co-efficient if 0.83 which is considered almost perfect agreement.8
Supplementary Table II shows the break-up for 2651 participants enrolled in step-II, their gender-wise distribution, number of participants < 18 years and ≥ 18 years old, numbers tested for IGRA, Cy-Tb and PPD. Second day data on induration induced by the IPs, was available for 2631 (99.25%) and IGRA results were available for 2625 (99.8%) participants (Fig. 1, Supplementary Table II). Figure 3 shows the induration induced by the IPs and its diameter. In step III, 647 household contacts aged 1 to 18 yr old were enrolled at five sites (Fig. 2, Supplementary Table III). Combined analysis was done for all participants enrolled in step II and step III.
Induration induced by Cy-Tb ranged between 1-75 mm (Figs. 4A and C) with 17.7±11.5 mm (mean ±SD) in those positive for Cy-Tb. For PPD the induration size ranged between 1-65 mm (Figs. 4B and C) with 19.2±7.93 mm induration size in those positive for PPD.
- Bar graph showing percentages of participants with different sizes of induration (A). after administration of Cy-Tb and (B). after administration of PPD (C). Table showing the numbers and percentages of participants with different sizes of induration after administration of Cy-Tb and PPD in Step II.
Table I shows the sensitivity, specificity, agreement, and Kappa for Cy-Tb and PPD when IGRA was taken as the reference standard. Of 1703 participants for whom both IGRA and Cy-Tb results were available, 523 (30.7%) were IGRA- positive, 1078 (63.3%) were IGRA-negative and 102 (6.0%) were IGRA indeterminate. Sensitivity and specificity of Cy-Tb was 82.6% and 71.1%, respectively with an agreement of 74.8% with IGRA and kappa of 0.5% which is considered moderate agreement. Additionally, 58 (56.9%) of the 102 IGRA indeterminate participants were Cy-Tb positive (Figs. 3A-C).
| IGRA N=1703@ | |||||||
|---|---|---|---|---|---|---|---|
|
Cy-Tb@ N=1707 |
IGRA Positive N=523 (30.7%), n (%) |
IGRA Negative N=1078 (63.3%), n (%) |
IGRA Indeterminate N=102 (6.0%), n(%) |
Sensitivity (95% C.I.) |
Specificity (95% C.I.) |
Agreement | Kappa (95% C.I.) |
|
Cy-Tb+ N=802 |
432 (82.6) | 312 (28.9) | 58 (56.9) |
82.6 (79.1-85.8) |
71.1 (68.2-73.8) |
74.8% |
0.5 (0.4-0.5) |
|
Cy-Tb- N=901 |
91 (17.4) | 766 (71.1) | 44 (43.1) | ||||
| IGRA N=1566* | |||||||
|
PPD* N=1568 |
IGRA Positive N=475 (30.3%) |
IGRA Negative N=995 (63.5%) |
IGRA Indeterminate N=96 (6.1%) |
- | - | - | - |
| n (%) | n (%) | n(%) | |||||
|
PPD+ N=668 |
337 (71.0) | 280 (28.1) | 51 (52.4) |
71.0 (66.6-75.0) |
71.9 (69.0-74.6) |
71.6% |
0.39 (0.35 -0.44) |
|
PPD- N=898 |
138 (29.0) | 715 (71.9) | 45 (47.6) | ||||
@IGRA not done for 4 participants 2 were positive for Cy-Tb and 2 were negative for Cy-Tb
*IGRA not done for 2 participants, one was negative for PPD, and one was positive for PPD
IGRA, interferon-gamma release assay; PPD, purified protein derivative; QFT Plus, QuantiFERON-TB gold plus; C.I., confidence interval
Similarly, of the 1566 participants for whom both IGRA and PPD results were available, 475 (30.3%) participants were IGRA- positive, 995 (63.5%) were IGRA-negative and 96 (6.1%) were IGRA indeterminate (Table I). Sensitivity and specificity of PPD was 71.0% and 71.9% respectively with an agreement of 71.6%, and kappa of 0.39, which is considered fair agreement. Additionally, 51 (52.4%) of the 96 IGRA indeterminate participants were PPD positive (Figs. 3D-F).
In participants <18 years old (Table II), Cy-Tb showed sensitivity of 88.3%, specificity of 69.7%, agreement of 75.4%, and kappa of 0.5. PPD showed a sensitivity of 74.8%, specificity of 71.0%, agreement of 72.3%, and kappa of 0.42 which is moderate agreement. Additionally, 7 (43.7%) of 16 IGRA indeterminate participants were positive for Cy-Tb and 10 (62.5%) of the 16 IGRA indeterminate participants were positive for PPD.
| N=726 | Age 1 to <18 yr | ||||||
|---|---|---|---|---|---|---|---|
| IGRA N=378 | |||||||
|
Cy-Tb N=378 |
IGRA Positive N=111 (29.37%), n (%) |
IGRA Negative N=251 (66.4%), N (%) |
IGRA Indeterminate N=16 (4.23%), N (%) |
Sensitivity (95% C.I.) |
Specificity (95% C.I.) |
Agreement |
Kappa (95% C.I.) |
|
Cy-Tb+ N=181 |
98 (88.3) | 76 (30.3) | 7 (43.8) |
88.3 (80.8-93.6) |
69.7 (63.6-75.3) |
75.4% |
0.5 (0.4-0.6) |
|
Cy-Tb- N=197 |
13(11.8) | 175 (69.7) | 9 (56.3) | ||||
| IGRA N=348 | |||||||
|
PPD N=348 |
IGRA Positive N=115 (33.0%) |
IGRA Negative N=217 (62.4%) |
IGRA Indeterminate N=16 (4.6%) |
- | - | - | - |
|
PPD+ N=159 |
86 (74.8) | 63 (29.0) | 10 (62.5) |
74.8 (65.8-82.4) |
71.0 (64.4-76.9) |
72.3% |
0.42 (0.33-0.52) |
|
PPD- N=189 |
29 (25.2) | 154 (71.0) | 6 (37.5) | ||||
| N=2549 | Age ≥18 yr old | ||||||
| IGRA N=1325 | |||||||
|
Cy-Tb@ N=1329 |
IGRA Positive N=412 (31.1%) |
IGRA Negative N=827 (62.4%) |
IGRA Indeterminate N=86 (6.5%) |
- | - | - | - |
|
Cy-Tb+ N=621 |
334 (81.1) | 236 (28.5) | 51 (59.3) |
81.1 (76.9-84.7) |
71.5 (88.3-74.5) |
74.7% |
0.47 (0.43-0.52) |
|
Cy-Tb- N=704 |
78 (18.9) | 591 (71.5) | 35 (40.7) | ||||
| IGRA N=1218 | |||||||
|
PPD* N=1220 |
IGRA Positive N=360 (29.6%) |
IGRA Negative N=778 (63.9%) |
IGRA Indeterminate N=80 (6.6%) |
- | - | - | |
|
PPD+ N=509 |
251 (69.7) | 217 (27.9) | 41 (51.3) |
69.7 (64.7-74.4) |
72.1 (68.8-75.2) |
71.4% |
0.38 (0.33-0.44) |
|
PPD- N=709 |
109 (30.3) | 561 (72.1) | 39 (48.8) | ||||
@IGRA not done in 4 participants, 2 Cy-Tb+ and 2 Cy-Tb-
*IGRA not done for 2 participants, 1 PPD+ and 1 PPD-
In participants who were ≥ 18 years old (Table II), Cy-Tb showed the sensitivity of 81.1%, and specificity of 71.5%, agreement of 74.7% and kappa of 0.47. PPD exhibited a sensitivity of 69.7%, specificity of 72.1%, agreement of 71.4% and kappa of 0.38.
Analysis of high-risk group, i.e., HHCs of TB patients and vulnerable population (Table III) revealed sensitivity of 88.7% and specificity of 64.3% for Cy-Tb with 73.5% agreement and kappa of 0.48. PPD showed 78% sensitivity, 63.2% specificity, 69.1% agreement and kappa of 0.39. The sensitivity and specificity of Cy-Tb in general population was 77.5% and 74.9%, respectively with an agreement of 75.7% and kappa of 0.47. The sensitivity and specificity of PPD in general population was 63.0% and 77.12%, respectively with an agreement with IGRA of 73.3% and kappa of 0.37.
| High Risk group, N=1310 | |||||||
|---|---|---|---|---|---|---|---|
| IGRA (N=656) | |||||||
|
Cy-Tb N=660@ |
IGRA Positive N=238 (36.3%), n (%) |
IGRA Negative N=389 (59.3%), n (%) |
IGRA Indeterminate N=29 (4.4%), n (%) |
Sensitivity (95% C.I.) |
Specificity (95% C.I.) |
Agreement |
Kappa (95% C.I.) |
|
Cy-Tb+ N=363 |
211 (88.7) | 139 (35.7) | 13(44.8) |
88.7 (83.9-92·4) |
64.3 (59.3-69.0) |
73.5% |
0.48 (0.42-0.54) |
|
Cy-Tb- N=293 |
27 (11.3) | 250 (64.3) | 16 (55.2) | ||||
| IGRA (N=648) | |||||||
|
PPD* N=650 |
IGRA Positive N=245 (37.8%) |
IGRA Negative N=370 (57.1%) |
IGRA Indeterminate N=33 (5.1%) |
- | - | - | - |
|
PPD+ N=348 |
191 (78.0) | 136 (36.8) | 21 (63.6) |
78.0 (72.2-83.0) |
63.2 (58.1-68.2) |
69.1 |
0.39 (0.32-0.46) |
|
PPD- N=300 |
54 (22.0) | 234 (63.2) | 12 (36.4) | ||||
| General Population, N=1965 | |||||||
| IGRA (N=1047) | |||||||
|
Cy-Tb N=1047 |
IGRA Positive N=285 (27.2%) |
IGRA Negative N=689 (65.9%) |
IGRA Indeterminate N=73 (7.0%) |
- | - | - | - |
|
Cy-Tb+ N=439 |
221 (77.5) | 173 (25.1) | 45 (61.6) |
77.5 (72.2-82.3) |
74.9 (71.5-78.1) |
75.7% |
0.47 (0.41-0.52) |
|
Cy-Tb- N=608 |
64 (22.5) | 516 (74.9) | 28 (38.46) | ||||
| IGRA (N=918) | |||||||
|
PPD N=918 |
IGRA Positive N=230 (25.1%) |
IGRA Negative N=625 (68.1%) |
IGRA Indeterminate N=63 (6.9%) |
- | - | - | - |
|
PPD+ N=318 |
145 (63.0) | 143 (22.9) | 30 (47·6) |
63.0 (56.5-69.3) |
77.1 (73.6-80.4) |
73.3% |
0.37 (0.3-0.43) |
|
PPD- N=600 |
85 (37.0) | 482 (77.1) | 33 (52.4) | ||||
@IGRA not done in 4 participants, 2 Cy-Tb+ and 2 Cy-Tb-
*IGRA not done for 2 participants, 1 PPD+ and 1 PPD-
Solicited adverse events
Solicited adverse events noted after 48-72 h of Cy-Tb and PPD administration included pain, pruritus, swelling, erythema, and rashes as the most common adverse events observed, which were self-resolving and reduced substantially by day 28 (Supplementary Tables IV and V). Small percentage of participants had any two or three of these events observed simultaneously in the same individuals at 48-72 h which was negligible by day 28. In children and adolescents <18 yr old, pain and pruritus induced by PPD was observed in significantly higher number of subjects compared to Cy-Tb (P=0.02, OR=1.6, 95% CI 1.07 to 2.44 for pain and P=0.05, OR=1.7, 95% CI 1.02 to 2.86 for pruritus) in participants enrolled for step III.
Discussion
We demonstrated here that Cy-Tb is comparable to PPD to diagnose TB infection in children, adolescents, and adults. Cy-Tb was found to be safe with local solicited adverse events like PPD which were self-resolving before day 28. In step-I, Cy-Tb missed three TB positive subjects (10%) and showed positive results in 2 (6.3%) healthy controls, in spite of Cohen’s kappa coefficient of 0.83, suggesting almost perfect agreement.8 Hoff et al,4 have concluded that since TB is an immunosuppressive condition, the sensitivity estimates of active TB may not be similar to that of TB infection. IGRA too has its limitations, 6% of the participants tested for Cy-Tb and 6.1% of the participants tested for PPD were indeterminate for IGRA. However, 56.9% and 52.4% of the IGRA indeterminate participants were positive for Cy-Tb and PPD respectively, suggesting that these may actually be M.Tb infected participants.5 Interestingly, 318 (28.9%) of IGRA negative participants were positive for Cy-Tb, while both Cy-Tb and IGRA are based on the same antigens ESAT-6 and CFP-10. This could be due to limitations of IGRA9 which has been shown to lack the required sensitivity resulting in negative or indeterminate results.6,10 Indeterminate results have been attributed to delay in incubation of the tubes from 6 to 12 h.10 Also, difference in the incubation time of the two assays i.e., overnight for IGRA vs. 2–3 days for Cy-Tb could also result in better sensitivity of Cy-Tb.9 In the absence of any other gold standard to diagnose latent TB, IGRA has been used in the current study as the reference standard.9
IGRA and TST are used for detection of MTB infection. However, due to shortage of PPD, safety and efficacy of Cy-Tb has been established earlier.6,11 Prior clinical trials have reported Cy-Tb to be safe and well tolerated in healthy subjects and TB patients.6 A sensitivity similar to TST and IGRA has been reported for Cy-Tb both in children and HIV co-infected persons having active TB in a phase III trial.6 Cy-Tbis very specific to MTB antigens CFP-10 and ESAT-6 and does not recognise BCG, however, PPD comprises of more than 100 different antigens3 along with MTB antigens. PPD also recognises exposure of the participants to BCG vaccination or a previous infection that has been cleared, resulting in false positive results.
This was a large study conducted in both general population and highly vulnerable household contacts of TB patients including children, adolescents, and adults. Study was conducted on different individuals who got either Cy-Tb or PPD, resulting in systemic immune response specific to the IP administered. However, it has limitations too, like the follow up period was too short to analyse the predictive value of Cy-Tb since the incubation of symptom-free, TB disease can be quite long. Diagnostic accuracy of Cy-Tb was determined using IGRA as the reference standard, which itself is not a perfect test and may miss latent infections. Studies in a larger sample of microbiologically confirmed TB patients will strengthen the estimates of sensitivity of Cy-Tb.
Since recombinant antigens are used in Cy-Tb, it can easily be produced in large scale, and can be used to detect M. tuberculosis infection with a single, universal cut-off.4 To conclude, diagnostic accuracy study showed that Cy-Tb is safe and comparable to PPD when IGRA was taken as reference standard in terms of sensitivity, specificity, percent agreement and kappa co-efficient with moderate concordance with IGRA. Based on these results and due to shortage of standardised PPD, ICMR expert committee recommended Cy-Tb to National TB elimination programme (NTEP) and has been adopted by the programme for the detection of latent TB infection.
Acknowledgment
Authors acknowledge the contributions of the NTEP and respective State and District TB cells in supporting the study despite Covid-19 pandemic.
Author contributions
MS: Conceptualised the study and was involved in study design, overall coordination, data curation and data analysis, manuscript writing; AMK, KR, SM, RPJ, RR and MSC: Study design, oversaw the study conduct; TH and SP: Data collection and coordination and communication with sites; TH, BM, SN, SK, YA, KDB, SJ, PP, JO, RG, HS, CPK, SD, AK, RB, AP, SP, VV: Data collection, participant recruitment, examination and follow up of TB patients and their contacts at their respective sites and documentation of participant profile, sample collection, laboratory investigations; MS and RR: Compilation and analysis of the results, preparation of report, manuscript writing; SP: Facilitated the study throughout by providing all necessary support; PKH, STO, Odisha: Facilitated the study. All authors have read and approved the final printed version of the manuscript.
Financial support and sponsorship
The study received funding support from the India TB Research Consortium (ITRC), Indian Council of Medical Research (Grant number- 5/8/5/54/ITRC/2020/ECD-1).
Conflicts of Interest
Indian Council of Medical Research is the sponsor of the study and was involved in conceptualisation, study design, coordination, monitoring of the study to ensure compliance to protocol and timely completion, however, had no role in participant enrolment and data acquisition.
Use of Artificial Intelligence (AI)-Assisted Technology for manuscript preparation
The authors confirm that there was no use of AI-assisted technology for assisting in the writing of the manuscript and no images were manipulated using AI.
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