Tuberculosis (TB) is an airborne infectious disease caused by Mycobacterium tuberculosis (MTB) ⁽¹⁾. The bacteria that cause TB are spread from person to person through the inhalation of droplets released into the air through coughing and sneezing ⁽²⁾. The World Health Organization (WHO) TB Report noted that 10 million people fell ill with TB, and 1.5 million people died from TB in 2019 ⁽³⁾. TB incidence fell about 2% per year between 2015 and 2019 ⁽⁴⁾.

Additionally, latent TB infection (LTBI) is defined as a case of infection with MTB with the presence of a small number of living bacteria in the body tissues however not spreading to others and shows normal chest X-ray (CXR) examination results ⁽⁵⁾. Overall, LTBI is defined as an asymptomatic clinical infection with MTB, and 5%–10% of infected people who do not receive treatment for LTBI eventually progress to active TB (ATB) ⁽⁶⁾. The risk of developing ATB from LTBI is significantly increased in people with diabetes, malignancies, and kidney disease, as well as in immunosuppressed patients ⁽⁷⁾.

Therefore, it is important to quickly and accurately distinguish between ATB and LTBI. Currently, the methods for diagnosing LTBI are the tuberculin skin test (TST) and whole blood interferon-gamma (IFN-γ) release assays (IGRAs) ⁽⁸⁾. However, these conventional methods are inadequate for distinguishing between ATB and LTBI ⁽⁹⁾. It is necessary to distinguish between these conditions to discover indicators that can be used for the effective management of TB and for the development of diagnostic methods.

To overcome these limitations, other studies were conducted to distinguish between ATB, LTBI, and healthy populations using TB-related serum proteins ⁽¹⁰⁾. These serum proteins can be used as prognostic and diagnostic biomarkers to identify ATB ⁽¹¹⁾. According to a study conducted in 2017, serum levels of acute phase proteins (APPs) such as procalcitonin (PCT), C-reactive protein (CRP), and α1-acid glycoprotein (AGP) were significantly increased (P < 0.0500) and could be used to distinguish patients with TB. Among serum APP markers, CRP is an APP whose levels are elevated in response to interleukin (IL) 6-mediated purulent infection, such as ATB ^(12,13). Additionally, CRP is a valuable screening marker for increasing the accuracy of TB diagnosis and is the best serum marker for differentiating between ATB, LTBI, and healthy individuals ^(14,15). In the present study, 110 whole blood samples were collected from 28 ATB, 29 LTBI, and 53 healthy patients, and Serum CRP levels were analyzed using quantitative suspension bead array. In the study by Mountjoy et al. ⁽¹⁶⁾, quantitative suspension bead arrays provide cost-effective and time-efficient results compared to ELISA methods. Therefore, we derived quantitative values of serum CRP levels using collected patient samples, the results were compared with those of acid-fast bacilli (AFB) staining, mycobacterial cultures, MTB-polymerase chain reaction (PCR), CXR, complete blood count (CBC), white blood cell (WBC) differential count, and erythrocyte sedimentation rate (ESR) examination.

1. Clinical samples

A total of 110 whole blood samples from 28 ATB patients, 29 LTBI patients, and 53 healthy individuals were obtained from April 2018 to March 2019 at the Department of Laboratory Medicine of Good Samsun Hospital, a secondary general hospital in Busan, Republic of Korea. The present study was approved by the Institutional Review Board (IRB) of the Catholic University of Pusan (IRB Approval No.: CUP IRB-2019-01-010), Busan, Republic of Korea. Participants with acute or chronic diseases, previous history of ATB, or symptoms suggestive of TB were excluded. The study population was divided into three groups: ATB group, LTBI group, and healthy control (HC) group. The ATB group was confirmed positive for Ziehl-Neelsen AFB staining, mycobacterial cultures, MTB-PCR tests using respiratory specimens, and CXR examination. The LTBI group was confirmed to be positive for the whole blood IGRA and QFT-GIT (Qiagen) tests and negative for ATB diagnostic tests; subjects in this group also had no ATB signs and symptoms. The HC group was confirmed negative for the whole blood IGRA test and CXR examination and had no ATB signs and symptoms.

2. Whole blood collection and serum preparation

Whole blood samples were collected using a VACUETTE^® EDTA (Greiner Bio-One) blood collection tube for the CBC, WBC differential count, and ESR. Collected whole blood samples were tested within 4 hours. Serum samples were separated and collected for the suspension bead array to target CRP. A VACUETTE^® Plain (Greiner Bio-One) serum-separating plain blood collection tube containing clot activator was used to collect the serum samples, which were allowed to stand at room temperature for 10 minutes. After that, clotted whole blood samples were centrifuged at 4,000 ×g for 15 minutes. The serum samples were stored at –20°C until use.

3. Measurement of serum C-reactive protein levels

Serum CRP level analysis was performed using a Cardiovascular Disease Magnetic Bead Panel 3 MILLIPLEX^Ⓡ MAP kit (EMD Millipore Corp.) using the manufacturer’s recommendations and a Luminex MAGPIX^Ⓡ System (Luminex Corp.). The results were analyzed using Luminex xPONENT^Ⓡ software (Luminex Corp.).

4. Complete blood count and WBC differential count

The automated hematology analyzer XN-1000^TM (Sysmex Corp.) was used for the CBC analysis containing red blood cell count, hemoglobin (Hb) concentration, and platelet count and the WBC differential count containing neutrophils, lymphocytes, monocytes, eosinophils, and basophils. The white cell nucleated channel was used for the CBC, and the white cell differential channel was used for the WBC differential count. After the analyzer and sample had been checked for ready status, the rack was placed in the sampler pool, and the test was initiated. XN Software v. UR (Sysmex Corp.) was used for data interpretation. The specific thresholds used in the interpretation of the results were analyzed using the manufacturer’s analysis tool.

5. Erythrocyte sedimentation rate analysis

ESR analysis was performed using the TEST-1 BCL (Alifax) automatic ESR analyzer. EDTA-treated whole blood samples were inserted into the rack, and the test was performed. The test principle for the TEST-1 BCL (Alifax) is based on photometric capillary stopped-flow kinetic analysis. The detector measures the amount of infrared light that passes through the capillaries through which blood flows. The number of cells in the sample was measured by a signal. The output was obtained by converting these signals over time to a Westergren ESR value using a regression equation. TEST 1 BCL software v. 8.0A (ALIFAXⓇ) was used for data interpretation. The specific thresholds used in the interpretation of the results were analyzed using the manufacturer's analysis tool (ESR value is a warning if it is over 200, and can be analyzed if it is under 1,000)

6. Statistical analysis

Statistical analysis was performed using GraphPad Prism v. 8.00 (GraphPad). Differences in serum CRP levels between the ATB, LTBI, and healthy groups were statistically analyzed, and 95% confidential intervals were calculated. One-way analysis of variance (ANOVA) was used to compare two or more groups. Additionally, to confirm the clinical usefulness and determine the cut-off value, specificity, and sensitivity, a receiver operator characteristic (ROC) curve analysis was performed on the ATB, LTBI group and the ATB, healthy group (P value < 0.05).

1. Population characteristics

The AFB stain results of the ATB group were +positive (n = 2, 7.1%), ++positive (n = 5, 17.9%), +++positive (n = 3, 10.7%), ++++positive (n = 4, 14.3%), and negative (n = 14, 50.0%). The AFB culture results were positive (n = 18, 64.3%) and negative (n = 10, 35.7%). The MTB-PCR test results were also both positive (n = 26, 92.9%) and negative (n = 2, 7.1%). CXR examination results showed that the ATB group was both positive (n = 22, 78.6%) and negative (n = 6, 21.4%), the LTBI group was positive (n = 3, 10.3%) and negative (n = 26, 89.7%), and the HC group was negative (n = 53, 100.0%). The IGRA results were positive (n = 29, 100.0%) in the LTBI group and negative (n = 53, 100.0%) in the healthy individual group (Table 1).

Table 1

Clinical characteristics of study subject

	Active TB	LTBI	Healthy
AFB stain
+positive	2 (7.1)	NA	NA
++positive	5 (17.9)	NA	NA
+++positive	3 (10.7)	NA	NA
++++positive	4 (14.3)	NA	NA
Negative	14 (50.0)	NA	NA
Mycobacterial culture
Positive	18 (64.3)	NA	NA
Negative	10 (35.7)	NA	NA
MTB-PCR test
Positive	26 (92.9)	NA	NA
Negative	2 (7.1)	NA	NA
Chest X-ray
Positive	22 (78.6)	3 (10.3)	0 (0.0)
Negative	6 (21.4)	26 (89.7)	53 (100.0)

Values are presented as n (%).

TB, tuberculosis; LTBI, latent tuberculosis infection; AFB, acid-fast bacilli; NA, not available (missing value); MTB-PCR, Mycobacterium tuberculosis-polymerase chain reaction.

2. Serum C-reactive protein levels measured using a quantitative suspension bead array in ATB, LTBI, and healthy individual groups

Based on the serum CRP analysis, the mean level of serum CRP was found to be 83.5 mg/L for the ATB group, 1.8 mg/L for the LTBI group, and 2.8 mg/L for the healthy group. Compared to the LTBI and healthy groups, the mean serum CRP level in the ATB group was significantly higher (P = 0.0004 and P < 0.0001, respectively). The ATB, LTBI, and healthy groups were statistically significant, with a P-value of less than 0.0001 (Fig. 1).

Fig. 1. Comparison of serum CRP levels analyzed by quantitative suspension bead array in active TB, LTBI, and healthy individual groups. CRP, C-reactive protein; TB, tuberculosis; LTBI, latent TB infection patients. Data are presented ad mean ± standard error of the mean. *P < 0.05, **P <0.01, ***P < 0.001.

3. Serum C-reactive protein levels compared to the results of other tuberculosis diagnostic assays

The mean CRP level of the AFB stain was 19.0 mg/L for negative, 368.5 mg/L for +positive, 689.0 mg/L for ++positive, 107.0 mg/L for +++positive, and 178.0 mg/L for ++++positive. The mean CRP level of the AFB culture was 1.2 mg/L for negative and 165.0 mg/L for positive. The mean CRP level of MTB-PCR was 54.1 mg/L for negative and 90.0 mg/L for positive. The mean level of CXR was 5.1 mg/L for negative and 132.0 mg/L for positive. Comparing serum CRP levels of the AFB stain, AFB culture, MTB-PCR, and CXR examination, serum CRP levels in patients positive for AFB culture, MTB-PCR, and CXR examination were higher than in individuals who were negative for these examinations (Fig. 2).

Fig. 2. Serum CRP levels compared to the result of active tuberculosis diagnostic assays. (A) (A) Comparison of CRP levels according to AFB satining results. (B) Comparison of CRP levels according to AFB culture results. (C) Comparison of CRP levels according to MTB-PCR results. (D) Comparison of CRP levels according to chest X-ray results. Data are presented ad mean ± standard error of the mean. CRP, C-reactive protein; AFB, acid-fast bacilli; MTB-PCR, Mycobacterium tuberculosis-polymerase chain reaction. *P < 0.05, **P <0.01, ***P < 0.001.

4. Correlation coefficients between serum CRP levels and CBC, WBC differential count, and ESR results

The correlation coefficients between serum CRP levels and CBC, WBC differential count, and ESR test results (Fig. 3) were obtained. In the CBC analysis, total WBC was found to be positively correlated with serum CRP levels (P < 0.0001) and Hb was negatively correlated with serum CRP levels (P = 0.0003). In the WBC differential count analysis, neutrophils (P < 0.0001) and lymphocytes (P < 0.0001) were positively correlated with serum CRP levels. Monocytes were negatively correlated with serum CRP levels (P < 0.0001). Additionally, ESR values were positively correlated with serum CRP levels (P < 0.0001). The statistical significance of the correlations is insufficient due to the low R² values, but a trend can be identified.

Fig. 3. Correlation coefficient between serum CRP levels and complete blood count, WBC differential count, ESR. (A) Linear regression of CRP levels and total WBC, (B) Linear regression of CRP levels and ESR, (C) Linear regression of CRP levels and Neutrophil, (D) Linear regression of CRP levels and monocyte, (E) Linear regression of CRP levels and lymphocyte, (F) Linear regression of CRP levels and hemoglobin. CRP, C-reactive protein; WBC, white blood cell; ESR, erythrocyte sedimentation rate. *P < 0.05, **P < 0.01, ***P < 0.001.

5. Receiver operating characteristic curve analysis of serum CRP levels between ATB and LTBI groups and between LTBI and healthy individual groups

The ROC curve analysis of serum CRP protein levels was performed to ensure that the results were clinically applicable. The ROC curve analysis of serum CRP levels among the ATB, LTBI, and healthy groups are shown in Fig. 4. The area of the ROC curve represents the accuracy of the diagnostic test, and this value ranges from 0 to 1. A larger area indicates a higher discriminatory ability of the diagnostic test. The P-value of serum CRP levels for the ATB and LTBI groups was 0.0003, and the area under the curve (AUC) was approximately 0.7820. The P-value of serum CRP levels for the ATB and healthy groups was less than 0.0001, and the AUC was approximately 0.7790 (Fig. 4).

Fig. 4. ROC curve analysis of serum CRP levels among the ATB patients, LTBI, and healthy individual groups. the area of the ROC curve is 0.5 < AUC ≤ 0.7, it is interpreted as low accuracy (less), when 0.7 < AUC ≤ 0.9, it is interpreted as moderate accuracy, and when 0.9 < AUC < 1.0, it is interpreted as high accuracy. ROC, receiver operator characteristic; CRP, C-reactive protein; ATB, active tuberculosis; LTBI, latent tuberculosi infection; AUC, area under the curve; CI, confidence interval. *P < 0.05, **P < 0.01, ***P < 0.001.

The WHO reported that TB is one of the top 10 causes of death worldwide and is the leading cause of death from a single infectious disease agent ⁽¹⁷⁾. In addition, most people are latently infected with MTB and have no symptoms of the disease ⁽¹⁸⁾. Therefore, it is important to quickly and accurately distinguish between ATB and LTBI. The rapid differentiation of LTBI from ATB is important for TB management and appropriate anti-TB treatment.

Currently, the two available methods for the diagnosis of LTBI are TST and IGRAs. However, the ability to distinguish between ATB and LTBI is still limited due to the individual’s differentiated immune system and various inflammatory conditions ^(19,20). To overcome these limitations, another study was conducted using serum protein markers to distinguish among ATB patients, LTBI, and healthy individual groups ⁽²¹⁾. TB-related proteins can be used as prognostic and diagnostic biomarkers to identify ATB ^(22,23). The results showed that the levels of serum APPs, such as PCT, CRP, and AGP, were significantly increased (P < 0.05) in ATB patients. CRP is also known to be a useful biomarker with a high secretion level for applying rapid immunodiagnostic techniques.

The present study assessed the usefulness of these potential indicators for differential diagnosis and provided basic data for effective diagnosis and treatment by comparing the levels of biomarkers in the whole blood of ATB, LTBI, and healthy groups. The results showed that serum CRP levels in ATB patients were significantly increased compared to LTBI and healthy individual groups. Serum CRP levels of patients who tested positive in mycobacterial culture, MTB-PCR tests, and CXR examinations were higher than those of patients who tested negative. Moreover, CRP, ESR, total WBC, and neutrophil and monocyte levels were positively correlated with serum CRP levels. In contrast, Hb and lymphocyte levels were negatively correlated with serum CRP levels. CRP, an APP, is a general inflammatory marker. Although CRP is not specific to a single disease process ⁽²⁴⁾, it is commonly associated with disease activity, severity, and prognosis at the screening stage. In particular, in TB, CRP levels may indicate the presence of residual MTB ⁽²⁵⁾, Therefore, monitoring CRP levels is expected to play a key role in TB management. These findings are valuable for the rapid and accurate differentiation of ATB patients from LTBI patients. The present study assessed the usefulness of these potential indicators for differential diagnosis and provided basic data for effective diagnosis and treatment by comparing the levels of biomarkers in the whole blood of ATB, LTBI, and healthy groups.

This study has several limitations. First, the sample size was limited to validate CRP as a screening tool for potential TB diagnosis. CRP is commonly used as an inflammatory marker ⁽²⁶⁾. The diagnostic potential of CRP in the correlation between CRP levels and various diagnostic markers in this study requires additional investigation in a larger cohort. Secondly, although CRP can be used as a monitoring tool for TB diagnosis, it has limitations as a single useful biomarker. It can show false positives due to other infections or inflammatory conditions other than TB.

In conclusion, IGRA results combined with total WBC, neutrophil, lymphocyte, monocyte, and CRP levels, and ESR were statistically significant indicators for the differentiation of ATB, LTBI, and healthy groups. Comparing the expression levels of these biomarkers in the whole blood of ATB, LTBI, and healthy groups is useful for differential diagnosis and provides basic data for effective TB diagnosis and treatment.

None.

No potential conflict of interest relevant to this article was reported.

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1C1B108888 and 2020R1C1007169) and the Brain Busan 21 Plus Project.

Conceptualization: YRL, HP. Data curation: Junseong K, YJK. Formal analysis: SBP, HP. Funding acquisition: Jungho K, SK. Investigation: YRL, YJK. Methodology: HP, Junseong K. Project administration: SK. Software: HP. Supervision: Jungho K, SK. Validation: SBP. Visualization: HP. Writing – original draft: YRL, HP. Writing – review and editing: HP, SK.