IN VITRO METHOD FOR DISCRIMINATING LATENT FROM ACTIVE TUBERCULOSIS

Abstract

In vitro method for discriminating latent from active TB. The present invention refers to an in vitro method for the differential diagnosis between active TB, preferably early stages of TB, and LTBI.

Claims

1. In vitro method for the differential diagnosis between active TB and LTBI which comprises: a. Determining the level of at least IFN-γ and IP-10 after the stimulation of blood samples obtained from the patient with a M. tuberculosis antigen; b. Determining the level of at least IFN-γ and IP-10 after the stimulation of blood samples obtained from the patient with a mitogen; c. Wherein if the level of IFN-γ after the stimulation with the mitogen is lower as compared with the level of IFN-γ obtained after the stimulation with the antigen, and the level of IP-10 and IFN-γ after stimulation with the antigen and the mitogen are higher as compared with the level of IP-10 and IFN-γ obtained without stimulation, this is an indication that the patient is suffering from active TB; d. Wherein if the level of IFN-γ after the stimulation with a mitogen is higher as compared with the level of IFN-γ obtained after the stimulation with the antigen, and the level of IP-10 and IFN-γ after stimulation with the antigen and the mitogen are higher as compared with the level of IP-10 and IFN-γ obtained without stimulation, this is an indication that the patient is suffering from LTBI.

2. In vitro method, according to the claim 1, wherein if the level of IFN-γ after the stimulation with the mitogen is lower as compared with the level of IFN-γ obtained after the stimulation with the antigen, and the level of IP-10 and IFN-γ after stimulation with the antigen and the mitogen are higher as compared with the level of IP-10 and IFN-γ obtained without stimulation, this is also an indication that the patient is suffering from early stage active TB.

3. In vitro method, according to any of the claim 1 or 2, further comprising the determination of the level of an acute-phase reactant selected from the list comprising: ferritin, c-reactive protein (CRP), procalcitonin (PCT), lactate dehydrogenase (LDH), erythrocyte sedimentation rate (ESR) or lactoferrin, and/or the level of a vitamin D analogue selected from the list comprising: 25-hydroxyvitamin D, paricalcitol, calcitriol, calcidiol, alfacalcidol, tacalcitol, calcipotriol, maxacalcitol, doxercalciferol or falecalcitriol, wherein if the level of the acute-phase reactant and/or the level of the vitamin D analogue is higher as compared with the level obtained in patients suffering from LTBI, this is an indication of active TB, preferably early stage active TB.

4. In vitro method, according to any of the previous claims, wherein the level of IFN-γ and IP-10 is directly determined by ELISA or indirectly determined by counting the number of T cells secreting IFN-γ and IP-10.

5. In vitro method, according to any of the previous claims wherein the mitogen is phytohaemagglutinin and the M. tuberculosis antigen are ESAT-6 and/or CFP-10.

6. In vitro method, according to any of the previous claims wherein the result is confirmed by CT scan, mycobacterial culture, PCR of M. tuberculosis, or by pathological anatomy.

7. In vitro use of at least IFN-γ and IP-10, after the stimulation of blood samples obtained from the patient with a M. tuberculosis antigen and with a mitogen, for the differential diagnosis between active TB and LTBI, or for the diagnosis of early stage active TB.

8. In vitro use, according to claim 7, in combination with the level of an acute-phase reactant selected from the list comprising: ferritin, c-reactive protein (CRP), procalcitonin (PCT), lactate dehydrogenase (LDH), erythrocyte sedimentation rate (ESR) or lactoferrin and/or the level of a vitamin D analogue selected from the list comprising: 25-hydroxyvitamin D, paricalcitol, calcitriol, calcidiol, alfacalcidol, tacalcitol, calcipotriol, maxacalcitol, doxercalciferol or falecalcitriol.

9. Kit for performing the method of claims 1 to 6 which comprises: a. Reagents or media for the determination of the level of IFN-γ and IP-10 after the stimulation of blood samples obtained from the patient with a M. tuberculosis antigen, and b. Reagents or media for the determination of the level of IFN-γ and IP-10 after the stimulation of blood samples obtained from the patient with a mitogen.

10. Kit, according to claim 9, further comprising reagents or media for the detection of the level of an acute-phase reactant selected from the list comprising: ferritin, c-reactive protein (CRP), procalcitonin (PCT), lactate dehydrogenase (LDH), erythrocyte sedimentation rate (ESR) or lactoferrin and/or for the detection of a vitamin D analogue selected from the list comprising: 25-hydroxyvitamin D, paricalcitol, calcitriol, calcidiol, alfacalcidol, tacalcitol, calcipotriol, maxacalcitol, doxercalciferol or falecalcitriol.

Description

DESCRIPTION OF THE FIGURES

[0045] FIG. 1. Flow diagram of enrolment. TB, tuberculosis; LTBI, LTBI; PTB, intrathoracic TB with pulmonary involvement; Mediastinal TB, intrathoracic TB with isolated mediastinal lymphadenopathy in the absence of lung parenchyma involvement.

[0046] FIG. 2. ROC curve of the four markers presented in the model [IFN-γ, IP-10, Ferritin, and 25(OH)D] to discriminate between active TB (N=44) and latent TB infected cases (N=37) with positive QuantiFERON-TB Gold In-Tube result. AUC, Area under ROC curve.

DETAILED DESCRIPTION OF THE INVENTION

Example 1. Materials and Methods

1.1 Study Population

[0047] A prospective case-control study was conducted from August 2015 to December 2016 in the paediatric hospital of Saint Damien in Port-au-Prince (Haiti). Children (0-14 years old) who presented signs and symptoms compatible with active TB and/or documented TB exposure were screened for suspected TB. According to the hospital programme, siblings (0-14 years old) of the children diagnosed with TB were also screened for TB or LTBI. Children (0-14 years old) from a school and a kindergarten were screened as uninfected controls. The exclusion criteria were: children with known immunodeficiency, on current immunosuppressive treatment, with a condition that could potentially compromise the immune system (e.g. children from oncology, rheumatology, nephrology and those who had undergone organ transplantation), children who had been under anti-TB treatment or preventive treatment during the previous year, and children not providing informed consent.

[0048] The following information was collected: age, sex, weight, height, previous medical history (including TB history, TB exposure, HIV status, and comorbidities, haemogram), vaccines, and current and previous medication (antibiotics, corticosteroids, antiparasitic drugs).

[0049] The following signs and symptoms were evaluated: cough and/or fever ≥two weeks (with no improvement after at least a seven-day course of amoxicillin), recent unexplained weight loss, and asthenia/fatigue. The TST was performed by a trained laboratory technician. Intradermal injection of 0.1 ml of Tubersol (bioequivalent to 5 tuberculin units; Sanofi Pasteur, Toronto, Canada) was placed into the ventral surface of the lower arm and read after 72 hours. A positive TST result was defined as an induration ≥10 mm in BCG-vaccinated children (those with a BCG scar), and ≥5 mm in non-BCG-vaccinated children or with a known adult TB contact (American Academy of Pediatrics, 2009; Bass et al., 1990). A standardised specific Z— score for detection of nutritional status weight-for-age (WAZ) was determined by WHO Anthro Plus 1.0.4 software (WHO AnthroPlus, 2009). Children with WAZ scores below −2 standard deviation (SD) were defined as underweight, and a WAZ score above 2 SD was defined as overweight.

[0050] A chest radiograph (anterior-posterior image) was performed in all the children screened for TB or LTBI. Chest radiographs were read by two external experts who were blinded to clinical data using a standardized reporting form and a third reader to resolve discordant opinions (Graham et al., 2015). In addition, nasopharyngeal aspirates and induced sputum were collected on three consecutive days for smear examination (auramine stain) by direct fluorescence microscopy in children suspected of having active TB. Histological examination was performed in children with suspicion of extrathoracic TB (ETB) with lymph node adenopathies.

[0051] Of all the children screened for TB or LTBI, only those with a positive TST result and/or microscopic confirmation were invited to participate in the study. Of all the children screened in the schools, only those with a negative TST result were invited to participate in the study as uninfected controls.

[0052] Treatment and preventive treatment were prescribed in all the children diagnosed with TB or LTBI, respectively. During TB treatment and preventive treatment, the patients were followed monthly until the end of treatment. Children with a positive sputum smear were retested at the fifth and sixth month of treatment.

1.2 Ethics Statement.

[0053] This study was approved by the Health Research Ethics Committee of Barcelona University and the Haitian National Ethics Committee (project number IRB00003099). Before participation, written informed consent was obtained from the child's parent or guardian.

1.3 Definitions for Classification of Children Enrolled in the Study.

[0054] Children were classified according to their clinical history, chest radiographs, smear examination, molecular diagnostic test, TST and QFT-GIT results. Active TB cases were defined as confirmed TB—children with relevant signs and symptoms and microbiologic confirmation of M. tuberculosis—, and unconfirmed TB—children without bacteriological confirmation but with relevant signs and symptoms, positive TST and/or QFT-GIT, radiological findings suggestive of TB, known TB contact, and clinical response to anti-TB treatment. Depending on the TB location, active TB cases were classified as ETB or intrathoracic TB. Children with intrathoracic TB were differentiated into those with pulmonary involvement (PTB) and children with isolated mediastinal lymphadenopathy in the absence of lung parenchyma involvement (Mediastinal TB). LTBI cases were defined as children with documented TB exposure, positive TST and/or QFT-GIT, normal chest radiographs, and no clinical signs of TB development in the last six months after diagnosis. Finally, uninfected controls were defined as asymptomatic children with no history of TB exposure, and negative TST and QFT-GIT.

1.4 Laboratory Tests Performed.

1.4.1 Molecular Diagnostic Test.

[0055] Sputum samples were collected from children with microscopy confirmation and/or radiological findings to perform GeneXpert MTB/RIF (Cepheid, USA) according to the manufacturer's instructions.

1.4.2 QuantiFERON-TB Gold In-Tube.

[0056] In all the participants, a 3 ml blood sample was drawn for conventional QFT-GIT; the antigen tube (ESAT-6, CFP-10, and TB-7.7), and positive (phytohemagglutinin, mitogen) and negative (no antigen, nil) controls. Tubes were incubated at 37° C. for 16 to 24 hours and centrifuged according to the manufacturer's instructions. Plasma was harvested and stored at −20° C. until performing the enzyme-linked immunosorbent assay (ELISA). After the screening and recruitment of cases, plasma samples were sent to the laboratories of the Research Institute Germans Trias i Pujol (IGTP, Badalona, Spain) following optimum conservation conditions. Once there, the supernatants were analysed and the results were interpreted according to the manufacturer's instructions.

1.4.3 Ferritin, 25(OH)D, and Toxocara Spp. Detection.

[0057] One ml of blood from all the participants was added to the biochemical tube to perform ferritin, 25(OH)D, and Toxocara canis determination. Ferritin and 25(OH)D concentrations were analysed at the biochemistry laboratory of the Germans Trias i Pujol Hospital (Badalona, Spain) with a Chemiluminescence Immunoassay (CLIA) method using a Liaison instrument (DiaSorin Liaison, Stillwater, Minn.). 25(OH)D is an indirect method to measure vitamin D in the blood. According to the literature, serum 25(OH)D levels equal or above 20 ng/ml were considered normal levels of vitamin D (Michael F. Holick, 2007). To avoid variations during blood sampling, the blood was almost always collected on the same day of the week at approximately the same hour (13-15 h). Seasonal fluctuations did not affect sampling because the weather seasons in Haiti are barely defined. T. Canis Ig-G antibodies were detected at the IGTP laboratory using a commercial ELISA kit (Ridascreen, R-Biopharm AG, Germany). Results with a sample index above 1.1 were considered positive according to the manufacturer's instructions.

1.4.4 Detection of Soil-Transmitted Parasites Infections.

[0058] Stool samples were collected to detect intestinal parasites in all the participants. Stool samples were processed on the same day of collection at the laboratory of the paediatric hospital of Saint Damien using the Kato-Katz and formalin-gasoline technique (a modification of the formalin-ether sedimentation technique) as described previously (Ahmadi and Damraj, 2009; Katz, N. Chaves, A. Pellegrino, 1972) and were examined with optical microscopy.

1.4.5 Cytokine Measurement.

[0059] Frozen supernatants remaining from the QFT-GIT tubes were used for the measurement of cytokine concentrations using a bead-based multiplex assay (Luminex 11-plex cytokine kit, R&D Systems, UK) and measured by Bioplex manager software (version 5.0, Bio-Rad, USA) according to the manufacturer's instructions. After optimisation experiments, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-gamma (γ), interleukin (IL)-2, IL-5, IL-10, IL-13, IL-22, IL-17, and tumor necrosis factor (TNF)-alpha (a) were analysed in a 1:8-fold dilution, while IL-1RA and induced protein (IP)-10 were analysed in 1:8, 1:80, and 1:160-fold dilutions. In this way, values were within the detection limits marked in the standard curve. IL-32 and vascular endothelial growth factor (VEGF) were measured by DuoSet ELISA (R&D Systems, UK) because of incompatibilities with the human custom multiplex cytokine kit. ELISA was performed according to the manufacturer's instructions.

1.5 Statistical Analysis.

[0060] Qualitative variables were described using frequencies and percentages. Qualitative variables were described using median and interquartile ranges (IQR), or, using mean and standard deviation (SD) in case the of variables were with a normally distribution, using mean and standard deviation. The independent variables analysed were: sex, age, weight by age, BCG, haemoglobin levels, 25(OH)D levels, ferritin levels, the presence of intestinal helminths, IgG T. Canis antibodies, and cytokines responses. The concentration of released cytokines released in response to M. tuberculosis antigens (Ag-TB) and phytohemagglutinin (PHA) was calculated by subtracting the concentration measured in the nil tube (Ag-TB, antigen minus nil; and PHA, mitogen minus nil). For all the variables with a normal distribution, the comparison between several groups was performed using the analysis of variance or Student's t-student test (only two groups). However, for variables non-normally distributed variables, the comparison between groups was performed using Kruskal-Wallis and Mann-Whitney tests. In the case of qualitative variables, comparisons were performed using the Fisher's exact test or chi-squared test. The Tukey method (normal distribution) or Benjamini & Hochberg method (non-normal distribution) was used to correct p-values in multiple comparisons.

[0061] Multivariate logistic regression was performed to detect variables able to classify individuals among the three different study groups (active TB, LTBI, and uninfected). Participants with a positive QFT-GIT result (TB and LTBI) were analysed separately (Binomial) from those with a negative QFT-GIT result (TB, LTBI, and uninfected; Multinomial). The results were expressed using the odds ratios (OR) and their confidence intervals. Sensitivity and specificity were calculated for assessing the value of performing a diagnostic test. The receiving operating characteristic (ROC) curve was used to evaluate diagnostic accuracy. The optimal cut-off value was identified to maximise the difference between true positive and false positives subjects. The level of significance was set at 0.05.

[0062] These analyses were performed using the statistical software IBM SPSS Statistics v. 25 (SPSS, Chicago, US), R package v.3.0.5 (R Foundation for Statistical Computing, Vienna, Austria), and GraphPad PRISM v. 5(GraphPad Software, Inc., San Diego, USA).

Example 2. Results

2.1. Study Subjects.

[0063] A total of 305 children suspected of having LTBI or TB were screened in the hospital, whereas a total of 96 uninfected children were screened in a school and a kindergarten. Among the patients screened at the hospital, 156 were recruited, and 149 were excluded because they did not meet the inclusion criteria (informed consent not obtained, negative TST). Of the 156 participants, 111 (71.2%) were analysed in the study, and 45 (28.8%) were lost to follow-up and were therefore not included in the analysis due to lack of complementary tests necessary for clinical assessment (chest radiographs, biopsies). Among the children screened at school and kindergarten, 55 were recruited and analysed, and 41 were excluded because they did not meet the inclusion criteria (informed consent not obtained, positive TST). Among the 166 children analysed (111 screened at the hospital and 55 screened at school and kindergarten), 74 had active TB (44.6%), 37 had LTBI (22.3%), and 55 were uninfected (33.1%). Of the 74 active TB cases, 8 had ETB, and 66 had intrathoracic TB. Among the intrathoracic TB children, 48 had PTB, and 18 had Mediastinal TB (FIG. 1).

[0064] Table 3 shows the description and demographic and clinical characteristics among study groups according to their QFT-GIT result. Significant differences were observed in age and body weight-for-age z-scores (p=0.001 and p=0.001, respectively) among the study groups with different QFT-GIT results (positive, negative, and indeterminate). Whereas LTBI cases with positive QFT-GIT were older than active TB cases with negative QFT-GIT (p=0.002), active TB cases with positive QFT-GIT had lower weight-for-age z-scores than uninfected controls (p=0.001). However, comparisons among QFT-GIT results within each study group (TB, LTBI, and non-infected) were performed, and no significant differences were observed.

TABLE-US-00003 TABLE 3 Description and comparisons of the demographic and clinical characteristics among study groups according to their QFT-GIT result. Study groups classified by QFT-GIT result (n = 166) Uninfected Active TB (n = 74) LTBI (n = 37) (n = 55) Positive Negative Indeterminate Positive Negative Indeterminate Negative p. n = 62 n = 9 n = 3 n = 22 n = 12 n = 3 n = 55 overall Gender 0.548 Female 26 (41.9%) 5 (55.6%) 2 (66.7%) 12 (54.5%) 6 (50.0%) 3 (100%) 25 (45.5%) Male 36 (58.1%) 4 (44.4%) 1 (33.3%) 10 (45.5%) 6 (50.0%) 0 (0%) 30 (54.5%) Age in years 85.3 (44.8) 47.3 (51.6) 59.0 (32.0) .sup.a 114 (45.6) .sup.a 80.6 (44.6) 48.7 (19.5) 76.5 (35.2) 0.001 Range 0.008 ≤5 years 25 (40.3%) 7 (77.8%) 2 (66.7%) 5 (22.7%) 6 (50.0%) 3 (100%) 32 (58.2%)  >5 years 37 (59.7%) 2 (22.2%) 1 (33.3%) 17 (77.3%) 6 (50.0%) 0 (0%) 23 (41.8%) Body weight- for-age Z-score (SD) −1.39 (1.70) .sup.a −1.70 (1.09) −1.78 (1.06) −0.66 (0.98) −1.57 (1.50) −0.92 (1.23) −0.30 (1.00) .sup.a 0.001 BCG scar 0.424 Yes 41 (69.5%) 7 (87.5%) 2 (66.7%) 15 (71.4%) 9 (100%) 1 (50.0%) 39 (72.2%) Hemoglobin (g/dl) 10.4 (2.02) 9.89 (0.97) 9.57 (0.84) 11.2 (1.30) 11.0 (1.37) 11.0 (3.33) 11.5 (0.66) 0.457 Categorical variables expressed described using frequencies (n) and percentages (%), and quantitative variables expressed using median and interquartile ranges (IQR) or standard deviation (SD). Statistical differences between pairwise comparisons .sup.a. QFT-GIT, QuantiFERON-TB Gold In-Tube; TB, tuberculosis; LIBI, latent TB infection; Z-score, default classification system used to present child nutritional status; BCG, Bacillus Calmette-Guérin.
2.2. Characterization of Subjects with a Positive QFT-GIT Test.

[0065] Comparisons between the groups with active TB and positive QFT-GIT were performed (Table 4). The Ag-TB levels of IL-17A, GM-CSF, together with the PHA levels of TNF-α, GM-CSF and, IL-13, and median haemoglobin levels showed significant differences between active TB groups (p=0.009, p=0.027, p=0.027, p=0.040 and p=0.042, p=0.006, respectively). Furthermore, pairwise comparisons showed that Ag-TB levels of IL-17A and GM-CSF, together with the PHA levels of TNF-α and IL-13, were higher in children with Mediastinal TB than in those with PTB (p=0.009 and 0.042, p=0.025 and p=0.050, respectively). Likewise, haemoglobin levels were significantly higher in children with Mediastinal TB than in children with PTB or ETB (p=0.014 and p=0.014, respectively). No biomarker showed significant differences between the PTB and ETB groups. Therefore, the groups of children with PTB and ETB were merged and analysed as a single group (TB, n=44), while children with Mediastinal TB (n=18) were analysed separately.

TABLE-US-00004 TABLE 4 Description and comparisons of demographic, clinical, and cytokine responses among active TB cases with positive QFT-GIT. Study subjects with Active TB and a positive QFT-GIT assay classified by disease location (n = 62) PTB ETB Mediastinal TB p. n = 36 n = 8 n = 18 overall Gender 0.935 Male 20 (55.6%) 5 (62.5%) 11 (61.1%) Age in years 76.0 [34.5; 115] 89.5 [55.5; 100] 99.0 [70.2; 130] 0.187 Range 0.406 <5 years 17 (47.2%) 3 (37.5%) 5 (27.8%) >5 years 19 (52.8%) 5 (62.5%) 13 (72.2%) Body weight-for-age Z-score −1.24 [−2.74; 0.08] −2.07 [−2.79; −1.13] −0.98 [−2.37; −0.21] 0.334 BCG scar 0.854 Yes 23 (69.7%) 5 (62.5%) 13 (72.2%) Hemoglobin (g/dl) 10.3 [8.90; 11.6] .sup.a 9.00 [8.15; 10.9] .sup.b 12.0 [10.8; 12.1] .sup.a b 0.006 Intestinal parasites Protozoa (cyst) 0 (0%) 0 (0%) 2 (33.3%) 0.308 Helminths (ova) 0 (0%) 1 (33.3%) 0 (0%) 0.120 Serology Toxocara spp. 0.099 Negative 31 (86.1%) 5 (62.5%) 11 (64.7%) Positive 5 (13.9%) 3 (37.5%) 6 (35.3%) Ferritin (ng/ml) 98.4 [33.0; 201] 138 [78.5; 218] 64.4 [47.4; 117] 0.276 25 (OH) D (ng/ml) 26.6 [20.6; 34.5] 30.2 [26.3; 34.2] 28.6 [21.8; 31.2] 0.735 Range 1.000 Normal levels 27 (75.0%) 6 (75.0%) 14 (77.8%) Deficiency 9 (25.0%) 2 (25.0%) 4 (22.2%) Cytokine responses (pg/ml) Ag-TB TNF-α 105 [−46.08; 399] 53.1 [36.3; 248] 136 [66.2; 386] 0.400 IP-10 31153 [12825; 47686] 37425 [10304; 57206] 33127 [29369; 48717] 0.778 IL-10 39.5 [23.9; 59.3] 36.4 [16.9; 56.7] 52.4 [47.2; 62.0] 0.060 IFN-γ 3922 [1150; 10449] 3855 [2957; 6969] 7020 [4866; 13847] 0.200 IL-1RA 27796 [12141; 42882] 25015 [13848; 66213] 23673 [15354; 51940] 0.928 IL-17A 7.58 [1.51; 12.6] .sup.a 5.67 [0.00; 19.2] 23.1 [9.13; 35.9] .sup.a 0.009 GM-CSF 41.4 [8.04; 97.1] .sup.a 28.9 [11.5; 54.8] .sup.b 83.6 [39.6; 201] .sup.a b 0.027 IL-13 716 [315; 1350] 1342 [534; 1876] 1326 [984; 1755] 0.071 IL-5 3.32 [0.00; 7.05] 0.00 [0.00; 13.5] 8.59 [3.53; 25.7] 0.204 IL-32 30.1 [−122.86; 64.1] 59.4 [−25.44; 396] 77.1 [−10.03; 360] 0.222 VEGF −381.77 [−784.77; −120.49] −903.69 [−1128.04; −499.36] −468.37 [−931.27; −173.86] 0.150 PHA TNF-α 4961 [2833; 8837] .sup.a 4850 [3446; 7646] 9762 [4829; 12489] .sup.a 0.027 IP-10 4718 [2862; 7972] 5335 [2623; 16865] 6952 [814; 19877] 0.840 IL-10 −2.79 [−11.05; 0.00] 0.00 [−0.30; 0.91] 0.00 [−1.99; 0.92] 0.071 IFN-γ 1102 [632; 3913] 1029 [876; 1878] 1828 [1131; 8723] 0.272 IL-1RA 46679 [27310; 80379] 39813 [17196; 46881] 82727 [29720; 142991] 0.311 IL-17A 39.8 [13.4; 129] 9.07 [6.54; 13.1] 19.8 [8.22; 81.8] 0.057 GM-CSF 65.2 [34.1; 118] 26.7 [9.59; 46.5] .sup.b 74.7 [54.8; 112] .sup.b 0.040 IL-13 1211 [664; 1970] .sup.a 1536 [1132; 2023] 2150 [1423; 2608] .sup.a 0.042 IL-5 4.28 [0.00; 7.38] 0.00 [0.00; 0.00] 2.98 [0.52; 4.76] 0.064 IL-32 43.9 [−74.11; 243] 7.99 [−98.12; 686] 243 [69.0; 739] 0.109 VEGF −62.75 [−368.65; 228] −434.83 [−559.52; −156.10] −73.02 [−503.76; 517] 0.297 Nil TNF-α 151 [40.3; 250] 33.6 [17.3; 178] 106 [55.3; 209] 0.388 IP-10 673 [412; 1220] 745 [529; 1082] 973 [616; 1201] 0.573 IL-10 8.52 [1.62; 16.3] 0.88 [0.00; 5.10] 5.45 [3.49; 9.86] 0.168 IFN-γ 197 [114; 509] 310 [106; 454] 200 [171; 451] 0.872 IL-1RA 14867 [9160; 22252] 11778 [6783; 27489] 12406 [8385; 20155] 0.765 IL-17A 0.00 [0.00; 11.3] 0.00 [0.00; 0.00] 3.33 [0.00; 19.7] 0.239 GM-CSF 1.92 [0.00; 16.2] 0.00 [0.00; 2.41] 2.67 [0.85; 5.27] 0.414 IL-13 311 [147; 622] 257 [0.00; 423] 341 [292; 444] 0.604 IL-5 0.00 [0.00; 5.88] 0.00 [0.00; 8.95] 3.26 [0.00; 5.52] 0.937 IL-32 1096 [508; 2241] 1606 [699; 2579] 1558 [1101; 2840] 0.376 VEGF 702 [175; 1801] 935 [726; 1128] 693 [368; 1323] 0.830 Categorical variables expressed described using frequencies (n) and percentages (%), and quantitative variables expressed using median and interquartile ranges (IQR) or standard deviation (SD). Statistical differences between pairwise comparisons (a, b). QFT-GIT, QuantiFERON-TB Gold In-Tube; TB, tuberculosis; PTB, Intrathoracic TB with pulmonary involvement; ETB, Extrathoracic TB; Mediastinal TB, Intrathoracic TB with isolated mediastinal lymphadenopathy in the absence of lung parenchyma involvement; Z-score, default classification system used to present child nutritional status; BCG, Bacillus Calmette-Guérin; 25(OH)D, 25-hydroxyvitamin D; Ag-TB, antigen-dependent response; PHA, mitogen-induced response.

[0066] Comparisons between the TB group (n=44) and LTBI cases with a positive QFT-GIT (n=22) were performed (Table 5). The Ag-TB levels of IP-10 along with ferritin levels were significantly higher in children with TB than in children with LTBI (p=0.005 and p=0.019, respectively). However, the PHA levels of IFN-γ were significantly lower in children with TB than in those with LTBI (p<0.001). Although nil levels of IL-5 showed significant differences between groups (p=0.007), this cytokine was not considered in the analysis because almost all the results of the LTBI cases (20/22) and more than half of those of the children who belonged to the TB group (24/44) were below the standard curve. Regarding the association of T. canis with IL-5, no significant differences were found between Ag-TB, PHA and nil IL-5 levels and T canis in the groups with positive QFT-GIT (p-values of 0.202, 0.508, and 0.053, respectively). Moreover, the eosinophil count was determined, but because only 25% of the children had an eosinophil count, this variable was not included in the analysis of the study. Despite the few results, no significant differences were found between Ag-TB, PHA and nil IL-5 levels and eosinophil count in the groups with a positive QFT-GIT (p-values of 0.848, 0.923, and 0.768, respectively).

TABLE-US-00005 TABLE 5 Description and comparisons of demographic, clinical, and cytokine responses between groups with a positive QFT-GIT. Subjects with a positive QFT-GIT assay (n = 66) Active TB* LTBI p. n = 44 n = 22 overall Body weight-for-age Z-score −1.32 [−2.74; −0.17] −0.64 [−1.58; 0.08] 0.061 Intestinal parasites Protozoa (cyst) 0 (0%) 1 (50.0%) 0.200 Helminths (ova) 1 (5.88%) 1 (16.7%) 0.462 Toxocara spp. 0.324 Negative 36 (81.8%) 13 (68.4%) Positive 8 (18.2%) 6 (31.6%) Ferritin (ng/ml) 109 [38.2; 201] 52.2 [32.5; 70.9] 0.019 25 (OH) D (ng/ml) 27.6 [20.8; 34.5] 24.7 [21.2; 30.3] 0.563 Range 0.755 Normal levels 33 (75.0%) 18 (81.8%) Deficiency 11 (25.0%) 4 (18.2%) Cytokine responses (pg/ml) Ag-TB TNF-α 71.0 [−40.32; 364] 47.5 [−26.19; 151] 0.654 IP-10 31545 [12093; 48379] 7762 [3676; 26629] 0.005 IL-10 39.3 [22.1; 59.3] 22.9 [12.1; 44.2] 0.121 IFN-γ 3922 [1201; 10427] 1832 [672; 9569] 0.221 IL-1RA 27796 [12141; 48396] 13918 [4821; 33546] 0.138 IL-17A 7.58 [0.40; 14.9] 10.5 [0.88; 60.2] 0.173 GM-CSF 34.5 [8.04; 97.1] 30.2 [18.4; 60.3] 0.828 IL-13 728 [315; 1468] 427 [171; 1229] 0.118 IL-5 2.90 [0.00; 7.05] 0.00 [0.00; 8.99] 0.579 IL-32 34.7 [−99.73; 72.6] −63.80 [−176.88; 59.4] 0.206 VEGF −442.15 [−913.46; −138.11] −357.76 [−1013.21; −81.31] 0.775 PHA TNF-α 4850 [2833; 8837] 6290 [4103; 9904] 0.226 IP-10 4718 [2680; 9102] 4392 [1800; 8974] 0.644 IL-10 −0.47 [−9.25; 0.00] −5.14 [−8.32; −1.05] 0.307 IFN-γ 1102 [717; 2984] 4420 [2137; 11309] <0.001 IL-1RA 42206 [27310; 69517] 45310 [27013; 112972] 0.812 IL-17A 26.0 [9.06; 112] 99.5 [18.9; 229] 0.053 GM-CSF 55.1 [24.4; 103] 79.4 [47.3; 135] 0.121 IL-13 1232 [683; 1973] 1180 [796; 1812] 0.946 IL-5 2.92 [0.00; 6.98] 0.91 [0.00; 10.1] 0.967 IL-32 32.9 [−74.11; 304] −1.83 [−57.38; 75.4] 0.391 VEGF −108.91 [−462.16; 181] −315.24 [−871.29; −16.37] 0.135 Nil TNF-α 139 [35.0; 250] 90.0 [38.0; 185] 0.654 IP-10 680 [423; 1189] 435 [208; 1208] 0.161 IL-10 7.63 [0.19; 14.9] 9.40 [5.28; 13.0] 0.526 IFN-γ 226 [111; 508] 241 [179; 388] 0.523 IL-1RA 14041 [8927; 23001] 11090 [6497; 17128] 0.106 IL-17A 0.00 [0.00; 10.4] 0.00 [0.00; 2.46] 0.764 GM-CSF 0.89 [0.00; 12.3] 0.83 [0.00; 5.30] 0.429 IL-13 302 [114; 595] 256 [146; 499] 0.683 IL-5 0.00 [0.00; 6.96] 0.00 [0.00; 0.00] 0.007 IL-32 1112 [556; 2241] 1726 [1033; 2590] 0.334 VEGF 809 [209; 1610] 688 [317; 1381] 0.870 Categorical variables expressed described using frequencies (n) and percentages (%), and quantitative variables expressed using median and interquartile ranges (IQR) or standard deviation (SD). QFT-GIT, QuantiFERON-TB Gold In-Tube; TB, tuberculosis; LTBI, latent TB infection; Z-score, default classification system used to present child nutritional status; BCG, Bacillus Calmette-Guérin; 25(OH)D, 25-hydroxyvitamin D; Ag-TB, antigen-dependent response; PHA, mitogen-induced response. *In this table, children with Mediastinal TB were not included into the active TB cases.
2.2. Characterization of Subjects with a Negative QFT-GIT Test.

[0067] Comparisons among active TB cases (n=9), LTBI cases (n=12) and uninfected controls (n=55) with a negative QFT-GIT (n=22) were performed (Table 6). The PHA levels of IL-10, IL-13, and IL-32, together with the nil levels of TNF-α and IL-10, the underweight levels, and 25(OH)D levels showed significant differences (p<0.001, p=0.007, and p=0.004, p=0.001 and p=0.044, p=0.001, and p=0.037, respectively). Furthermore, pairwise comparisons showed that PHA levels of IL-10 were significantly higher in TB and LTBI cases than in uninfected controls (p=0.020 and p=0.001, respectively), whereas the PHA levels of IL-32 were significantly higher in LTBI cases than in TB cases, and uninfected controls (p=0.034 and p=0.004, respectively), and the PHA levels of IL-13 were significantly higher in LTBI cases compared to uninfected controls (p=0.006). Otherwise, the nil TNF-α levels were significantly higher in uninfected controls compared to LTBI cases (p<0.001). Regarding levels of malnourishment, the weight-for-age z-scores were significantly lower in TB and LTBI cases than in uninfected controls (p=0.006 and p=0.006, respectively). On the contrary, the median 25(OH)D levels were significantly higher in LTBI cases compared to uninfected controls (p=0.022).

TABLE-US-00006 TABLE 6 Description and comparisons of demographic, clinical, and cytokine responses between groups with a negative QFT-GIT. Subjects with a Negative QFT-GIT assay (n = 76) Active TB LTBI Uninfected p. n = 9 n = 12 n = 55 overall Body weight-for-age Z-score (SD) −1.44 [−2.45; −1.00] .sup.a −1.55 [−2.03; −0.58] .sup.b −0.59 [−1.08; 0.32] .sup.a b 0.001 Intestinal parasites Protozoa (cyst) 0 (0%) 3 (50.0%) 0 (0%) 0.464 Helminths (ova) 0 (0%) 0 (0%) 2 (4.55%) 1.000 Toxocara spp. 0.314 Negative 6 (66.7%) 7 (58.3%) 42 (77.8%) Positive 3 (33.3%) 5 (41.7%) 12 (22.2%) Ferritin (ng/ml) 68.5 [51.0; 84.6] 44.0 [28.4; 77.7] 37.1 [20.3; 55.4] 0.075 25 (OH) D (ng/ml) 29.6 [24.1; 30.1] 30.4 [27.7; 34.4] .sup.b 26.5 [24.6; 30.0] .sup.b 0.037 Range Deficiency 0 (0%) 0 (0%) 2 (3.84%) Cytokine response (pg/ml) Ag-TB TNF-α −26.62 [−59.20; 4.27] −7.89 [−37.82; 0.51] −57.31 [−321.36; 72.9] 0.385 IP-10 544 [−34.59; 795] 94.6 [49.9; 1332] 219 [85.6; 594] 0.894 IL-10 1.44 [−2.71; 9.21] 3.77 [0.85; 6.40] 0.85 [−1.64; 9.29] 0.918 IFN-γ 88.4 [0.00; 196] 54.4 [−5.32; 239] 23.3 [−2.05; 82.1] 0.525 IL-1RA 4361 [1455; 9833] 3204 [1280; 7608] 3812 [1853; 9435] 0.886 IL-17A 0.00 [0.00; 0.00] 0.00 [−22.20; 6.61] 0.00 [0.00; 1.12] 0.751 GM-CSF 1.45 [−1.10; 5.08] 0.00 [0.00; 1.45] 0.04 [−5.15; 1.52] 0.670 IL-13 361 [67.3; 591] 81.5 [23.5; 565] 56.0 [0.00; 200] 0.240 IL-5 0.00 [−0.46; 0.00] 0.00 [0.00; 0.49] 0.00 [0.00; 1.88] 0.171 IL-32 5.89 [−145.98; 222] 106 [−143.37; 256] −52.81 [−218.53; −6.17] 0.197 VEGF −769.46 [−1673.90; 0.00] −57.79 [−1369.04; 19.9] −243.45 [−466.44; −22.59] 0.295 PHA TNF-α 7244 [2691; 8401] 4609 [2837; 6360] 6932 [5104; 9965] 0.261 IP-10 7177 [4600; 11211] 6415 [5166; 9139] 6524 [3733; 10553] 0.819 IL-10 −8.92 [−13.01; −4.87] .sup.a −9.43 [−11.82; −7.98] .sup.b −0.43 [−6.29; 0.00] .sup.a b <0.001 IFN-γ 3092 [2179; 4970] 4808 [3212; 9358] 3998 [1897; 7064] 0.536 IL-1RA 72506 [42906; 111675] 148877 [70665; 201214] 68339 [30968; 172755] 0.184 IL-17A 105 [8.75; 180] 131 [80.7; 224] 80.0 [30.4; 216] 0.390 GM-CSF 39.3 [33.8; 99.2] 64.1 [46.6; 93.7] 71.5 [42.6; 143] 0.186 IL-13 1948 [1467; 2146] 2400 [2021; 2978] .sup.b 1563 [789; 2236] .sup.b 0.007 IL-5 5.24 [0.91; 5.69] 2.99 [0.00; 11.4] 3.42 [1.15; 6.68] 0.789 IL-32 −198.07 [−315.19; −9.88] .sup.c 280 [67.2; 367] .sup.b c −24.34 [−160.79; 66.4] .sup.b 0.004 VEGF 0.00 [−853.06; 782] 0.00 [−780.01; 374] −27.36 [−358.31; 138] 0.703 Nil TNF-α 228 [71.7; 300] 69.5 [40.4; 99.2] .sup.b 312 [155; 809] .sup.b 0.001 IP-10 938 [301; 1667] 561 [413; 2085] 487 [317; 770] 0.291 IL-10 12.3 [8.92; 27.5] 14.0 [10.1; 16.5] 7.75 [3.59; 16.0] 0.044 IFN-γ 321 [0.00; 862] 255 [67.9; 440] 199 [89.0; 434] 0.911 IL-1RA 11598 [7467; 18694] 10372 [8682; 16165] 13409 [9432; 22543] 0.304 IL-17A 0.00 [0.00; 44.3] 0.00 [0.00; 26.5] 0.00 [0.00; 0.71] 0.220 GM-CSF 4.98 [0.00; 9.43] 0.00 [0.00; 3.76] 1.30 [0.00; 14.6] 0.258 IL-13 1166 [478; 1519] 376 [324; 727] 373 [206; 947] 0.069 IL-5 0.46 [0.00; 4.96] 0.00 [0.00; 2.49] 3.07 [0.00; 5.52] 0.122 IL-32 1242 [1006; 1740] 2020 [1384; 4215] 1532 [657; 3754] 0.332 VEGF 1029 [626; 1674] 1546 [491; 2629] 484 [222; 1189] 0.113 Categorical variables expressed described using frequencies (n) and percentages (%), and quantitative variables expressed using median and interquartile ranges (IQR) or standard deviation (SD). Statistical differences between pairwise comparisons corrections (a, b, c). QFT-GIT, QuantiFERON-TB Gold In-Tube; TB, tuberculosis; LTBI, latent TB infection; Z-score, default classification system used to present child nutritional status; BCG, Bacillus Calmette-Guérin; 25(OH)D, 25-hydroxyvitamin D; Ag-TB, antigen-dependent response; PHA, mitogen-induced response.
2.3. Discriminative Biomarker Profiles in Subjects with Positive QFT-GIT.

[0068] The adjusted logistic model showed a set of 4 biomarkers able to discriminate between active TB and LTBI: ferritin, 25(OH)D, IP-10, and IFN-γ (Table 1).

TABLE-US-00007 TABLE 1 Percentages of classification (%) and area under the ROC curve of the study groups. Patient classification Model Signature: active TB* vs LTBI % TB % LTBI AUC 1 Ag (IFN-γ) 100 0 0.59 2 Ag (IFN-γ + IP-10) 88.6 27.3 0.72 3 Ag (IFN-γ + IP-10) + Mit 86.4 59.1 0.84 (IFN-γ + IP-10) 4 Ag (IFN-γ + IP-10) + Mit 90.9 70.0 0.91 (IFN-γ + IP-10) + Ferritin 5 Ag (IFN-γ + IP-10) + Mit 90.9 63.6 0.87 (IFN-γ + IP-10) + 25(OH)D 6 Ag (IFN-γ + IP-10) + Mit 90.0 93.2 0.95 (IFN-γ + IP-10) + Ferritin + 25 (OH) D TB, tuberculosis; LTBI, latent TB infection; 25(OH)D, 25-hydroxyvitamin D; Ag, lymphocytes stimulated with Ag-TB (QFT). Mit, lymphocytes stimulated with mitogen (PHA). *In this table, patients with mediastinal TB were not included into the active TB cases since they are analyzed separately in Table 2

[0069] The best model included 6 variables. The coefficients of this model were showed in Table 7. For each increased unit of IFN-γ (in Ag-TB responses), IP-10 (in Ag-TB and PHA responses), ferritin, and 25(OH)D, the odds of being classified as TB case will increase by a factor of 1.08, 1.80, 1.13, 1.02, and 1.22, respectively. However, for each increased unit of IFN-γ (in PHA responses), the odds of being classified as a TB case will decrease by a factor of 0.46. Therefore, the profile to classify a subject with TB instead of LTBI will be that with low levels of IFN-γ (in PHA responses) but high levels of IP-10 (in Ag-TB and PHA responses), 25(OH)D, and ferritin.

TABLE-US-00008 TABLE 7 Classification table of the model for the study groups with positive QFT-GIT results and variables in the equation. Classification table of the Active TB* and LTBI cases with positive QFT-GIT results and variables in the equation B P-value Odds Ratio [95% CI] 25 (OH) D (ng/ml) 0.20 0.012 1.22 [1.05; 1.43] Ferritin (ng/ml) 0.02 0.010 1.02 [1.01; 1.03] Ag-TB (pg/ml) .sup.† IP-10 0.08 0.017 1.08 [1.01; 1.15] INF-γ 0.12 0.064 1.13 [0.99; 1.29] PHA (pg/ml) .sup.† IP-10 0.59 0.007 1.80 [1.18; 2.75] INF-γ −0.77 0.008 0.46 [0.26; 0.82] Constant −8.67 0.004 Binomial Logistic Regression. B, regression coefficient; OR, Odds Ratio; CI, Confidence Interval. QFT-GIT, QuantiFERON-TB Gold In-Tube; TB, tuberculosis; LTBI, LTBI; 25(OH)D, 25-hydroxyvitamin D; Ag-TB, antigen-dependent response; PHA, mitogen-induced response. *In this table, children with Mediastinal TB were not included into the active TB cases. .sup.† Cytokine levels were multiplied x10.

[0070] The model based on the four host-markers mentioned above was able to correctly classify 93.2% of children with TB, and 90.0% of children with LTBI. In addition, the area under the ROC curve of this model was 0.955 (CI 95%: 0.91 to 1.00), and the positive and negative likelihood ratio were 9.32 and 0.08, respectively (FIG. 2). Furthermore, when this model was applied to Mediastinal TB (i.e. early stages of TB) to compare with LTBI, the model classified correctly 76.5%, and the area under the ROC curve was 0.83 (CI95%; 0.7 to 0.96).

TABLE-US-00009 TABLE 2 Percentages of classification (%) and area under the ROC curve of the study groups. Patient classification Signature: Mediastinal TB % Early % Model vs LTBI stages of TB LTBI AUC 1 Ag (IFN-γ)  100% 0.0% 0.5 2 Ag (IFN-γ + IP-10) 88.9% 27.3% 0.58 3 Ag (IFN-γ + IP-10) + Mit 83.3% 59.1% 0.71 (IFN-γ + IP-10) 4 Ag (IFN-γ + IP-10) + Mit 88.2% 70.0% 0.79 (IFN-γ + IP-10) + Ferritin 5 Ag (IFN-γ + IP-10) + Mit 88.9% 63.6% 0.76 (IFN-γ + IP-10) + 25(OH)D 6 Ag (IFN-γ + IP-10) + Mit 76.5% 93.2% 0.83 (IFN-γ + IP-10) + Ferritin + 25(OH)D TB, tuberculosis; Ag, lymphocytes stimulated with Ag-TB (QFT). Mit, lymphocytes stimulated with mitogen (PHA); 25(OH)D, 2 5-hydroxyvitamin D.
2.4. Discriminative Biomarker Profiles in Subjects with Negative QFT-GIT.

[0071] The adjusted logistic model showed a set of 3 biomarkers, namely, IL-10, IL-13, and IL-32, able to discriminate among active TB, LTBI, and uninfected controls (Table 8). For each increased unit of IL-13 and IL-32 (in PHA responses), the odds of being classified as LTBI case will increase by a factor of 1.003 and 1.003, respectively. However, for each increased unit of IL-10 (in PHA responses), the odds of being classified as uninfected control will decrease by a factor of 0.93 and 0.01, respectively.

[0072] The combination of PHA levels of IL-10, IL-13, and IL-32 detected 11.1% of active TB cases, 50.0% of LTBI cases, and 96.4% of uninfected controls.

TABLE-US-00010 TABLE 8 Classification table for the study groups with negative QFT-GIT results and variables in the equation. Classification table of the study groups with negative QFT-GIT results and variables in the equation Model .sup.a B P-value Odds Ratio [95% CI] Active TB PHA (pg/ml) Constant −4.67  IL-10 −0.071 0.036 0.93 [0.87; 0.99] IL-13 1.25 × 10.sup.−3 0.073 1.01 [1.00; 1.01] IL-32 3.42 × 10.sup.−4 0.751 1.00 [0.99. 1.01] LTBI PHA (pg/ml) Constant −8.873 IL-10 −0.095 0.009 0.910 [0.85; 0.98] IL-13 3.06 × 10.sup.−3 0.001 1.003 [1.001; 1.005] IL-32 2.60 × 10.sup.−3 0.007 1.003 [1.001; 1.005] .sup.a The reference category is the uninfected control group. Multivariate Logistic Regression. B, regression coefficient; OR, Odds Ratio; CI, Confidence Interval. QFT-GIT, QuantiFERON-TB Gold In-Tube; TB, tuberculosis; LTBI, LTBI; PHA, mitogen-induced response.