Baseline characteristics of AFB- IGRA+ TB and PN participants
The characteristics of the AFB− IGRA+ TB and PN participants were shown in Additional file 1: Table S1. No significant age and gender differences were found in the first cohort. Males made up the majority in both two cohorts. Clinical types of TB participants included 162 infiltrative pulmonary tuberculosis, 19 cavitary pulmonary tuberculosis, 8 secondary pulmonary tuberculosis, and 15 tuberculous pleurisy and empyema. Only 44 and 23 participants have received TB-DNA and TB-antibody examiniation, yielding 9.09% (4/44) and 13.04% (3/23) positive rates respectively.
Differential laboratory biomarkers between AFB- IGRA+ TB and PN
To gain more insights into the overall change in correlation structure in laboratory variables between AFB− IGRA+ TB and PN, we used DGCA to visualize all variable pair correlations in both conditions (Fig. 2A). Variables in this heatmap were ordered by their median z-score correlation difference with all of the other variables, without the restriction to positive correlations. By quantified the difference in correlation between all variable pairs using permutation testing[15], we found a lower correlation between most variable pairs in TB than PN (mean difference in z-score (dz) = 0.11, p = 0.04), suggesting that the TB group had a more coordinated perturbation of profile than the PN group.
We further explored the significant variables between the two conditions. Using t-statistics and setting an FDR < 0.2, 13 variables with marked differences were identified (Table 1, Additional file 2: Table S2). Only uric acid (UA) was up-regulated (foldchange > 1.2). Five variables, including aspartate aminotransferase (AST), total bile acid (TBA), triglyceride (TG), alanine aminotransferase (ALT) and glutamyl transpeptidase (GGT), were down-regulated (foldchange < 0.83) in TB compared with PN (Fig. 2B).
Table 1
Differential variables and OR values in AFB− IGRA+ TB compared with PN group.
Variables
|
p-value
|
FDR
|
Foldchange
|
OR
|
p-value of OR
|
2.5% CI
|
97.5% CI
|
UA
|
6.82E-06
|
2.73E-04
|
1.30
|
0.36
|
2.49E-05
|
0.22
|
0.57
|
GLU
|
6.94E-04
|
0.01
|
0.88
|
4.26
|
1.45E-03
|
1.82
|
10.93
|
AST
|
8.54E-04
|
0.01
|
0.82
|
1.78
|
1.47E-03
|
1.26
|
2.56
|
TBA
|
5.90E-03
|
0.06
|
0.66
|
1.59
|
0.01
|
1.14
|
2.30
|
TG
|
1.45E-02
|
0.11
|
0.82
|
2.66
|
0.02
|
1.20
|
6.65
|
RBC
|
0.02
|
0.11
|
1.11
|
0.28
|
0.02
|
0.09
|
0.72
|
ALT
|
0.02
|
0.13
|
0.82
|
1.31
|
0.03
|
1.03
|
1.67
|
Hb
|
0.03
|
0.13
|
1.05
|
0.37
|
0.03
|
0.15
|
0.89
|
GGT
|
0.03
|
0.14
|
0.72
|
1.33
|
0.04
|
1.02
|
1.74
|
ALP
|
0.04
|
0.14
|
0.86
|
1.92
|
0.05
|
1.02
|
3.71
|
LDH
|
0.04
|
0.14
|
0.83
|
1.60
|
0.06
|
1.01
|
2.71
|
ALB
|
0.04
|
0.14
|
1.05
|
0.40
|
0.05
|
0.16
|
0.98
|
WBC
|
0.06
|
0.19
|
0.93
|
1.49
|
0.06
|
0.98
|
2.29
|
FDR, false discovery rate, threshold < 0.2. OR, odds ratio. CI, confidence interval. UA, uric acid. GLU, glucose. AST, aspartate aminotransferase. TBA, total bile acid. TG, triglyceride. RBC, red blood cell. ALT, alanine aminotransferase. Hb, hemoglobin. GGT, glutamyl transpeptidase. ALP, alkaline phosphatase. LDH, lactate dehydrogenase. ALB, albumin. WBC, white blood cell. |
Z-transformed correlation in these 13 variables also revealed differential connectivity between AFB− IGRA+ TB and PN, for example, pair hemoglobin (Hb) and UA had no significant correlation (p-value > 0.05) in TB, while had positive correlation [p-value < 0.05, Corr(ρ) > 0.3] in PN; pair white blood cell counts (WBC) and UA had no significant correlation in TB (p-value > 0.05), while had negative correlation in PN [p-value < 0.05, Corr(ρ) = -0.3, Fig. 2C]. These data revealed variable expression differences between two conditions, and some variables might be useful to distinguish AFB− IGRA+ TB from PN.
In order to find the odds that TB would progress or not given exposure to these laboratory variables, odds ratio (OR) was assessed for each variable by a univariate general linear model. We identified 11 variables significantly associated with TB progression (p-value < 0.05, Table 1). Among them, four variables indicated the protective effect TB progression (OR < 1), including UA, red blood cell (RBC), Hb and albumin (ALB); while seven variables showed as risk factors in TB progression (OR > 1).
Multivariate risk model to predict TB progression probability
Combining with the results above, and using AIC as a stopping rule, we finally selected five laboratory variables (Age, UA, ALB, Hb and WBC) to develop a multivariate risk model with 89 AFB− IGRA+ AFB and 38 PN participants. Figure 2D represented the expression distribution of variables in both groups, showing more unbalance in PN than AFB− IGRA+ TB. The risk model yielded a C-index of 0.7 (95% CI: 0.61, 0.8, Fig. 3A black line), with p-value = 0.01 (Chi-square test). The calibration plot revealed a moderate agreement between the risk model prediction and the actual observation (Fig. 3B, p-value = 0.18, Hosmer-Lemeshow test).
Using the nomogram, we mapped the values for each variable to points on a scale axis ranging from 0 to 10. With a corresponding number of point assigned to given magnitudes of the variables, the risk probability was calculated by corresponding cumulative point score for all the variables[16]. We found that UA had the most protective effect on TB progression, followed by Hb; while Age, WBC and ALB acted as risk factors (Fig. 3C).
The Multivariate risk performed well in an independent validation cohort
Next, we prospectively collected an external validation cohort of 134 participants, of whom 15 were excluded due to missing variables, consisting 77 AFB− IGRA+ TB and 42 PN participants. The total points of each participant in the external cohort were calculated according to the established nomogram, and then used to evaluate the performance of the nomogram. The C-index of nomogram for predicting external cohort was 0.77 (95% CI: 0.68, 0.86, Fig. 3A red line). The calibration plot also showed good agreement between the prediction by nomogram and actual observation (Fig. 3D) with a p-value of 0.13 by Hosmer-Lemeshow test.