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ARTICLE
Diagnostic Value of Flow Cytometry in Kidney Transplant Recipients With Active Pulmonary Tuberculosis

Objectives: Long-term use of immunosuppressant drugs in kidney transplant recipients leads to immuno­suppression. When active pulmonary tuberculosis infection occurs, lymphocyte proliferation and function are impaired, and the clinical symptoms of patients are not typical, which often leads to delay in diagnosis.

Materials and Methods: We collected and analyzed the peripheral blood lymphocytes of hospitalized patients with active pulmonary tuberculosis and other types of pulmonary infection after kidney transplant within 2 years. The proportion and absolute values of lymphocytes were obtained by a flow cytometer.

Results: There were significant differences in the proportion of CD8+ subsets between active pulmonary tuberculosis and bacterial pneumonia in kidney transplant recipients. If the proportion of CD8+ subsets in peripheral blood is over 33.27%, then the active pulmonary tuberculosis diagnosis sensitivity is higher than 88.9% and specificity is higher than 83.3%.

Conclusions: Analysis of peripheral lymphocyte subsets is helpful in the early diagnosis of kidney transplant recipients with active pulmonary tuberculosis. It should be added into routine examinations of kidney transplant recipients who have an ambiguous diagnosis between active pulmonary tuberculosis and bacterial pneumonia.


Key words : Active tuberculosis, Kidney transplantation, Lymphocyte subsets

Introduction

The Global Tuberculosis Report revealed that about 10 million people were newly infected with tuberculosis (TB) in 2018, of which 8.7 million lived in 30 countries with a high prevalence of TB. It was estimated that 1.3 million people die of TB every year.1 Kidney transplant recipients are at high risk of infection with Mycobacterium tuberculosis because of long-term use of immuno­suppressant drugs. The incidence of infection was 4- to 30-fold higher in kidney transplant recipients than that shown in the general population.2 In low-prevalence countries, reactivation of latent TB infection is the most common cause of active pulmonary TB.3 However, in countries with high prevalence, the risk of community exposure after kidney transplant still exists. In previous studies, the mortality rate of kidney transplant recipients with active pulmonary TB ranged from 12% to 24%.4-6 Timely diagnosis of active pulmonary TB after kidney transplant is particularly important. Because of the immuno­suppression caused by M. tuberculosis infection or other types of immunosuppression, interferon-gamma release assay (IGRA) may not be able to confirm active pulmonary TB, nor may it accurately predict the development of TB in kidney transplant recipients.7,8 The results of etiological examinations are often negative, and kidney transplant recipients with active pulmonary TB are often only diagnosed after substantial delay.6

Immunosuppressant drugs inhibit the proliferation and differentiation of lymphocytes. This defect of proliferation and differentiation leads to the decrease of lymphocytes.9 We evaluated the differences among peripheral blood lymphocyte subsets by discri­minating kidney transplant recipients with active pulmonary TB from those with bacterial pneumonia or fungal pneumonia.

Materials and Methods

Study population
Kidney transplant recipients were recruited between January 1, 2018, and January 1, 2020. Thirty-three individuals undergoing the first kidney transplant were divided into TB (n = 9), bacterial (n = 12), and fungal (n = 12) groups according to imaging findings and laboratory examination outcomes. Nineteen individuals undergoing a first kidney transplant matched with the sex, age, and postoperative time of the 3 infection groups were selected as the stable group. All enrolled individuals received tacrolimus in combination with mycophenolate mofetil and prednisolone. All enrolled individuals were negative for HIV. When active pulmonary TB was confirmed, patients were transferred to a special institute for TB control for registration and treatment and followed up at the surgery hospital. Clinical data of the participants are shown in Table 1. No prisoners were used in the study, and participants were neither coerced nor paid. This study was approved by the Organ Procurement Organization and the Institutional Review Board of The Third Xiangya Hospital of Central South University. The study complied with the Helsinki Congress and Istanbul Declaration. All patients signed the informed consent form.

Criteria for diagnosis
Active pulmonary TB was confirmed by etiological evidence of M. tuberculosis (sputum smear, sputum culture, or fiberoptic bronchoscopy), with imaging findings of active pulmonary disease. Bacterial pneumonia was confirmed by etiological evidence of bacteria (sputum smear or sputum culture), with imaging findings of active pulmonary disease, negative IGRA, and negative serum galactomannan antigen test. Fungal pneumonia was confirmed by etiological evidence of fungus (sputum smear or sputum culture), with positive imaging findings of active pulmonary disease, positive serum galacto­mannan antigen test, and negative IGRA. The stable group was defined as those individuals who tested negative at the IGRA screening without any symptom of infection or rejection.

Instruments and materials
A flow cytometer (FACSCanto II, BD Biosciences) was used for examination. Main materials included Multitest 6-color (CD3/CD8/CD45/CD4/­CD16+56/CD19) TBNK reagent (BD Biosciences, Multitest REF 644611), BD Trucount tubes (BD Biosciences), and 10× fluorescence-activated cell sorting hemolysin (BD Biosciences).

Flow cytometric analysis
Peripheral blood samples were collected into anticoagulant tubes and processed within 2 hours. Multitest 6-color TBNK reagent (20 μL) was added to the bottom of BD Trucount tubes, and 50 μL blood was transferred into the tubes. The mixture was incubated in tubes for 20 minutes away from light at room temperature. Diluted hemolysin, 450 μL, was added in each tube and incubated for 20 minutes away from light at room temperature. Samples were then immediately acquired. The proportional and absolute values of lymphocytes were obtained by flow cytometry.

Statistical analyses
Statistical analyses were performed using SPSS 23.0 software (SPSS: An IBM Company, version 17.0, IBM Corporation, Armonk, NY, USA). The measurement data were tested by normal distribution test and variance homogeneity test. The normal distribution data are expressed as means ± standard deviation. Variance analysis was used for pairwise comparison between 2 groups. We analyzed the diagnostic

value of lymphocyte subsets by using the receiver operating characteristic (ROC) curve. Area under the curve (AUC) values ranging from 0.5 to 0.7 indicated low diagnostic value. An AUC value > 0.8 indicated high diagnostic value. Statistical significance was set at P < .05.

Results

Renal function
Blood urea nitrogen and serum creatinine levels of individuals in the stable group, TB group, bacteria group, and fungus group are shown in Table 1. Compared with the stable group, blood urea nitrogen and serum creatinine in the TB group, bacteria group, and fungus group increased significantly (P = .015, .010, .013; .030, .022, and .030, respectively).

Predictive effect of lymphocyte subset analysis in kidney transplant recipients
Lymphocyte subsets were compared between the stable group and infection group. There were no significant differences in the proportion of CD3+, CD8+, CD4+, NK, and CD19+ subsets between the stable group and the infection group (P > .05, for all). Compared with the stable group, the absolute values of CD3+, CD8+, CD4+, NK, and CD19+ subsets decreased significantly in the infection group (P < .001, for all).

Predictive effect of lymphocyte subset analysis in different pathogens
The proportional and absolute values of lymphocyte subsets in the TB group, bacteria group, and fungus group are shown in Tables 2 and 3. The proportion of CD8+ subsets in the TB group was significantly higher than that in the bacteria group (P = .033). There was no significant difference in the proportion of CD8+ subsets between the TB group and fungus group (P = .976). The absolute value of CD4+ subsets in the bacteria group was significantly higher than that in the fungus group (P = .025).

The ROC curves of the proportion of CD8+ subsets for discriminating TB and bacteria were plotted (Figure 1). The AUC was 0.880 (95% CI, 0.73-1.00), the standard error was 0.008, and P = .004. The critical value of proportion of CD8+ subsets for diagnosis of active pulmonary TB was 33.27%, with a sensitivity of 88.9% and a specificity of 83.3% (Table 4).

Discussion

Specific symptoms of active pulmonary TB, for example, fever, night sweat, cough, and weight loss, were found in most patients, but many patients also had nonspecific symptoms. High-resolution com­puted tomography was the first choice for imaging examination. Imaging findings of kidney transplant recipients with active pulmonary TB typically included pleural effusion, cavity formation, cavity necrosis, tree-bud sign, interstitial thickening, diffuse reticular nodule shadow, and enlarged and necrotic lymph nodes.9 However, the specificity of high-resolution computed tomography in identifying the pathogens of pulmonary infection is limited.10

In this study, the proportion of lymphocyte subsets presented no significant difference between the stable group and infection group. Absolute values of lymphocyte subsets decreased significantly in the infection group. Absolute values of lymphocyte subsets directly reflected the state of immunosuppression.

In our ROC curve analysis of the proportion of CD8+ subsets for discriminating TB and bacteria, the AUC was 0.880 (95% CI, 0.73-1.00) and P was less than 0.05. This revealed that the proportion of CD8+ subsets has good predictive ability and may help clinicians to discriminate kidney transplant recipients with active pulmonary TB from those with bacterial pneumonia in the early stage. CD8+ lymphocytes dissolve the infected target cells via cytotoxicity to expose and clear M. tuberculosis infection. Furthermore, TB-specific CD8+ lymphocytes play important roles in long-term immune response of M. tuberculosis.11 Active TB infection induces pro­liferation of CD8+ subsets for limiting M. tuberculosis infection and TB pathology.12-14 In addition, the proliferation and metabolism of lymphocytes are suppressed by immunosuppressant drugs.15,16 Impaired proliferation and function of CD8+ subsets may promote latent TB infection to progress to active pulmonary TB.17 The defect of number and function of lymphocytes may accelerate reactivation of latent TB infection.

Fungal pneumonia remains a severe problem in kidney transplant recipients with pulmonary infection. In this study, lymphocyte subset analysis showed a poor ability to discriminate active pulmonary TB from fungal pneumonia. The serum galactomannan antigen test was more effective to diagnose fungal pneumonia in clinical work. To evaluate the immune status of kidney transplant recipients with fungal pneumonia, further study of the dynamic changes of lymphocyte subsets is needed.

There were several limitations to this single-center research study. First, the sample size was small due to difficulty of diagnosis. Second, rapid progress of the disease may have resulted in a rapid change of lymphocyte subsets, which may have affected the accuracy of the statistical data.

Conclusions

The results of this study suggest that flow cytometric analysis is an effective method for discriminating kidney transplant recipients with active pulmonary TB from those with bacterial pneumonia. If the proportion of CD8+ subsets in peripheral blood is over 33.27%, then the active pulmonary TB diagnosis sensitivity is higher than 88.9% and specificity is higher than 83.3%. Further studies are required to expand the sample size and analyze the dynamic changes of lymphocyte subsets during the anti-TB treatments.


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DOI : 10.6002/ect.2020.0104


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From the Department of Transplant Surgery, the Third Xiangya Hospital of Central South University, Changsha, China
Acknowledgements: The authors have not received any funding or grants in support of the presented research or for the preparation of this work from funding agencies in the public, commercial, or not-for-profit sectors and have no potential declarations of interest.
The authors certify that each author participated sufficiently in the study conception or design, data analysis or interpretation, and drafting or revision of the manuscript, so that each author takes responsibility for the validity and objectivity of the entire study. Each author has approved the final version of the manuscript.
Corresponding author: Ke Cheng, Department of Transplant Surgery, the Third Xiangya Hospital of Central South University, Tongzipo Road, 410013, Changsha, China
Phone: +86 731 8861 8312
E-mail: chke1972@163.com