The soluble urokinase plasminogen activator receptor (suPAR) has been shown to be a strong prognostic biomarker for tuberculosis (TB). In the present study, the profiles of plasma suPAR levels in pulmonary TB patients at high risk for multidrug resistance were analyzed and compared with those in multidrug resistant (MDR)-TB patients. Forty patients were prospectively included, consisting of 10 MDR-TB patients and 30 TB patients at high risk for MDR, underwent clinical assesment. Plasma suPAR levels were measured using ELISA (SUPARnostic, Denmark) and bacterial cultures were performed in addition to drug susceptibility tests. All patients of suspected MDR-TB group demonstrated significantly higher suPAR levels compared with the healthy TB-negative group (1.79 ng/mL). Among the three groups at high risk for MDR-TB, only the relapse group (7.87 ng/mL) demonstrated suPAR levels comparable with those of MDR-TB patients (7.67 ng/mL). suPAR levels in the two-month negative acid-fast bacilli conversion group (9.29 ng/mL) were higher than positive control, whereas levels in the group consisting of therapy failure patients (5.32 ng/mL) were lower. Our results strongly suggest that suPAR levels enable rapid screening of suspected MDR-TB patients, but cannot differentiate between groups.
For decades, tuberculosis (TB) has been one of the leading infectious diseases causing death in Indonesia. Presently, the situation is worsening due to the appearance of multidrug-resistant TB (MDR-TB), which accounts for 4% to 5% of TB cases. MDR-TB is a condition in which
To date, the most common test recommended by the WHO to diagnose multidrug resistance is the drug susceptibility test (DST), which takes two months to complete [
Our previous study showed that suPAR could be used as a biomarker to monitor the progress of therapy in TB-AFB(+) patients [
The sample consisted of 10 MDR-TB patients and 30 TB patients at high risk for multidrug resistance. Ten healthy patients serving as negative controls were recruited from the Dr. Saiful Anwar Hospital, Malang, Indonesia. The patients were categorized into suspected MDR-TB, including negative AFB conversion after two months of intensive-phase therapy, relapse and therapy failure groups. All patients, except the healthy control group, fulfilled the following inclusion criteria: having pulmonary TB, regularly taking medication, male or female between 15 and 50 years of age, having a body mass index (BMI) > 16 kg/m2, agreeing to be a subject of the research, and providing informed consent. TB patients with other diseases, such as severe bacterial pneumonia, HIV-AIDS, heart disease, diabetes mellitus, heart and kidney problems, or extrapulmonary TB, and pregnant patients and patients with psychiatric problems were excluded from the study.
All patients, except TB-AFB(−) conversion, received directly observed, six-month (26-week), short-course anti-TB therapy as recommended by the Indonesian National Tuberculosis Program, which is based on WHO TB guidelines [
For patients in the AFB conversion after two months and therapy failure groups, the drug regimen consisted of a fixed, weight-dependent combination of INH (320–400 mg/day), rifampicin (480–600 mg/day), ethambutol (800–1200 mg/day), and pyrazinamide (1000–1250 mg/day) for the two-month intensive-phase treatment, followed by rifampicin and INH in the four-month continuation phase. In the case of therapy failure groups, the patients showed to be AFB positive at the end of five and six months of therapy.
Several routine clinical and laboratory investigations were performed in accordance with standard procedures conducted in Indonesian hospitals [
To observe the viability of Mtb and to determine resistance to basic anti-TB drugs, sputum samples were collected from patients in each group. Sputa were washed in 0.9% NaCl solution and concentrated, and the sediment was subsequently cultured on Lowenstein-Jensen medium. The DST was performed with at least two anti-TB drugs, rifampicin and INH, using the bacteriological absolute concentration method [
A 3 mL blood sample was obtained from new patients by venipuncture before they initiated second-line anti-TB drug treatment as guided by the WHO [
Serum levels of suPAR were measured in duplicate using commercially available ELISA kits according to the manufacture’s protocol (suPARnostic, ViroGates A/S, Copenhagen, Denmark) [
All statistical analyses were performed using SPSS software. Multivariate analyses were conducted using SPSS version 16 (IBM Corporation, USA). Differences between the groups and controls were analyzed using one-way ANOVA.
The present study was approved by the Ethics Committee of Syaiful Anwar Public Hospital, Brawijaya University, Indonesia. All patients provided written informed consent.
The study initially included 41 patients consisting of 31 pulmonary TB patients at high risk for multidrug resistance and 10 MDR-TB patients. During the course of the study, however, one patient acquired pneumonia and was subsequently excluded. Table
Characteristics of tuberculosis (TB) patients at high risk for multidrug resistance (MDR) and multidrug resistant TB.
Patient characteristic | MDR |
NC |
TF |
|
Total |
|
---|---|---|---|---|---|---|
BMI (kg/m2) | <18 | 5 (50) | 7 (70) | 4 (40) | 6 (60) | 32 (80) |
18–22 | 3 (30) | 3 (30) | 4 (40) | 4 (40) | 18 (45) | |
>22 | 2 (20) | 0 | 2 (20) | 0 | 4 (10) | |
Lesion width | Normal | 0 (0) | 0 | 1 (10) | 0 | 1 (2.5) |
Minimal | 0 (0) | 1 (10) | 1 (10) | 1 (10) | 3 (7.5) | |
Moderate | 0 (0) | 2 (20) | 3 (30) | 3 (30) | 9 (22.5) | |
Far advanced | 10 (100) | 7 (70) | 5 (50) | 6 (60) | 38 (95) | |
Milier | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 3 (7.5) | |
ESR (mm/h) | 65.1 | 63.5 | 24.6 | 62.1 | ||
Culture and DST | (+) MDR | 10 (100) | 2 (20) | 1 (10) | 2 (20) | 15 (37.5) |
(+) nMDR | 0 (0) | 1 (10) | 4 (40) | 5 (50) | 10 (40) | |
Culture (−) | 0 (0) | 7 (70) | 4 (40) | 2 (20) | 13 (52.5) | |
MOTT | 0 (0) | 0 (0) | 1 (10) | 1 (10) | 2 (5) |
BMI: Body mass index; DST: drug susceptibility test; eSR: erythrocyte sedimentation rate; MOTT:
Examination of chest X-rays showed that lesion areas corresponding to the far advanced category most often occurred in patients in the MDR-TB group (100%). In contrast, the high risk for multidrug resistance group demonstrated variations in lesion width: highly advanced lesions (60% to 70%), moderately advanced lesions (20% to 30%), and minimal lesions (10%).
ESR is often elevated in patients with active TB, although a normal ESR does not rule out TB [
TB patients at high risk for multidrug resistance had significantly higher suPAR levels than those in the healthy purified protein derivative-negative (ie., control) group (1.78 ng/mL) (
Given that each group of suspected MDR patients contained MDR and culture-negative individuals (Table
Measurement of plasma soluble urokinase plasminogen activator receptor (suPAR) levels based on culture growth and drug susceptibility.
In the present study, all TB patients at high risk for multidrug resistance shared characteristics that were also found in MDR-TB patients, including low BMI and high ESR. The fact that BMI values were below normal in all TB groups suggests that BMI is not a characteristic of MDR-TB patients because it was also prevalent in patients in the TB at high risk for multidrug resistance as well as those who were TB-AFB(+) [
The measurement of suPAR levels has been shown to reflect pathological status in pulmonary TB patients, making application in clinical settings relevant to disease progress. We report the profile of suPAR levels from three groups of TB patients at high risk for MDR-TB (ie., two-month negative AFB conversion, therapy failure and relapse). Among the three groups at high risk for MDR-TB, only the relapse group demonstrated suPAR levels comparable with MDR-TB patients (7.67 ng/mL). The other groups (negative AFB conversion after two months and therapy failure) showed either higher or lower levels subsequently (9.29 ng/mL and 5.32 ng/mL, resp.). Given that patients from the relapse group were completely cured of TB for a considerable length of time but then reactivated, results of the present study demonstrate that suPAR levels are quite stably maintained in the plasma of relapse patients. One possible explanation is that a small proportion of Mtb in the macrophages of TB relapse patients survives first-line anti-TB treatment and persists for an uncertain length of time and subsequently reemerges upon host immunosuppression [
The mean suPAR level in patients in the two-month AFB negative conversion group (9.29 ng/mL) was slightly but significantly higher than in patients in the MDR-TB group, indicating that pathological processes continued to proceed, possibly due to the survival of a subpopulation of bacteria. These bacteria persist within latent foci contained in granulomas and in the sputum of large cavities [
The therapy failure group showed the lowest suPAR levels, but these levels were still comparable with those of the cured TB patients from the previous study (5.32 ng/mL versus 5.09 ng/mL) [
To date, culture growth and the DST have been the only methods able to discriminate multidrug resistance from suspected multidrug resistance. Our novel finding that suPAR levels could reflect the immune status of different groups of TB patients at high risk for multidrug resistance adds to the literature. Although suPAR level could not differentiate MDR-TB patients from suspected MDR patients, TB patients with anti-TB drug sensitivity after therapy demonstrated that suPAR levels > 5 ng/mL warrant performance of DSTs.
The results of our study suggest that measurement of suPAR levels could be used to screen TB patients at high risk for multidrug resistance after therapy. However, this marker cannot differentiate between suspected multidrug resistance and MDR-TB patients. Larger studies are expected to confirm the prognostic value for MDR from TB patients at high risk of MDR.
The authors thank the suPARNOSTIC Inc. that gave special price for the kit that enabled them to conduct this paper. This study was funded by the Faculty of Medicine, Brawijaya University. A thank you for Dian, M.D., Rahadi, MD that provided samples and Fitri for technical assistant.