|Year : 2019 | Volume
| Issue : 3 | Page : 262-266
Soluble urokinase-type plasminogen activator receptor as a biomarker of treatment response in childhood tuberculosis
Ery Olivianto1, Sri Sudarwati2, Heda Melinda Nataprawira2
1 Department of Child Health, Division of Pediatric Respirology, Faculty of Medicine, Saiful Anwar General Hospital, Universitas Brawijaya, Malang, Indonesia
2 Department of Child Health, Division of Pediatric Respirology, Faculty of Medicine, Hasan Sadikin General Hospital, Universitas Padjadjaran, Bandung, Indonesia
|Date of Web Publication||12-Sep-2019|
Dr Ery Olivianto
Department of Child Health, Division of Pediatric Respirology, Faculty of Medicine, Saiful Anwar General Hospital, Universitas Brawijaya, Malang
Source of Support: None, Conflict of Interest: None
Background: Childhood tuberculosis (TB) is a major health problem worldwide, especially in developing countries. In 2015, there are estimated 950,000 cases of childhood TB. Since most TB in children is paucibacillary, this gives rise not only to problem in diagnosing but also in monitoring the response to anti-TB treatment as well. Soluble urokinase-type plasminogen activator receptor (suPAR), a glycosylphosphatidylinositol-linked membrane protein of various cells of immune system, is one of the potential biomarkers to be used in the management of TB. The objective of this study is to study the decrease of serum suPAR level after anti-TB treatment in children with TB and its association with patient characteristics. Methods: We conducted a prospective study on children suspected of having TB due to a history of contact with active TB case and symptoms such as coughing, fever, and enlarged lymph nodes. The diagnosis of TB is established by history, physical examination including anthropometric examination. Tuberculin skin test using PPD RT-23 and interferon-gamma releasing assay with Quantiferon TB-Gold Plus was performed. Chest X-rays were read by two independent radiologists. Microbiological examination was performed using microscopic examination and Xpert MTB/RIF. The level of suPAR before and after anti-TB treatment was examined with the Elisa method. Results: There was no significant difference of serum suPAR levels before and after anti-TB treatment (mean 0.71 [standard deviation 0.585] ng/mL; P = 0.072). There was no association between ages (P = 0.112), nutritional status (P = 0.228), diagnosis of pulmonary or extrapulmonary TB (P = 0.956), and radiological feature (P = 0.810) with serum suPAR levels decrease. Conclusion: There is no suPAR serum level decrease 2 months after treatment with anti-TB and there is no association with age, nutritional status, pulmonary or extrapulmonary TB diagnosis, and radiological feature.
Keywords: Childhood tuberculosis, soluble urokinase-type plasminogen activator receptor serum level, treatment response
|How to cite this article:|
Olivianto E, Sudarwati S, Nataprawira HM. Soluble urokinase-type plasminogen activator receptor as a biomarker of treatment response in childhood tuberculosis. Int J Mycobacteriol 2019;8:262-6
|How to cite this URL:|
Olivianto E, Sudarwati S, Nataprawira HM. Soluble urokinase-type plasminogen activator receptor as a biomarker of treatment response in childhood tuberculosis. Int J Mycobacteriol [serial online] 2019 [cited 2020 Jul 3];8:262-6. Available from: http://www.ijmyco.org/text.asp?2019/8/3/262/266494
| Introduction|| |
In 2015, an estimated 950,000 cases of tuberculosis (TB) occurred in children. In Indonesia, 8% of all TB cases are children.,, Besides treatment adherence, one of the problems in the management of childhood TB is to monitor the treatment response. Microbiology examinations have limitations in pediatric patients.,,,,,
The Soluble urokinase-type plasminogen activator receptor (suPAR) is a biomarker that potentially can be used to monitor the response of TB treatment. The inflammatory response increases plasma suPAR levels in various inflammatory diseases.,,,,,,,,,,,,,,,,
This study aimed to study the decrease of suPAR after 2 months of treatment with anti-TB in childhood TB.
| Methods|| |
The study was conducted in Saiful Anwar General Hospital from March to November 2018. Children with presumptive pulmonary or extrapulmonary TB aged <15 years were included. In all participants, tuberculin skin test (TST) and/or interferon-gamma releasing assay (IGRA) was performed; chest X-ray and 5 mL blood sample were obtained. Sputum sample was obtained using the standard procedure of sputum induction and examined both by microscopic examination and rapid molecular test using GeneXpert MTB/RIF.
Diagnoses of pulmonary TB were based on collected data of clinical examination, TST/IGRA results, chest-X-ray, and microbiology examinations. Diagnoses of extrapulmonary TB were supported by appropriate examinations, such as fine-needle biopsy and cerebrospinal fluid analysis.
Those with concomitant bacterial infection such as urinary tract infection, medial otitis, and pneumonia were excluded from the study. Children who had a history of previous anti-TB treatment suffered from concomitant HIV infection, malignancy, and MDR TB were also excluded. Participants who did not adhere to anti-TB treatment, loss to follow-up, or died were not included.
Anti-TB drugs which consisted of isoniazid, rifampicin, pyrazinamid in fixed-dose combination and additional ethambutol (in case of bacteriologically confirmed TB and severe TB) were given at doses recommended by the WHO according to participants body weights.
As much as 1 mL blood sample of those who diagnosed with pulmonary or extrapulmonary TB were obtained 2 months later (i.e., after the completion of intensive phase drugs). Serum suPAR level before the start of anti-TB drugs and after the intensive phase was examined.
Ethical clearance was obtained from Saiful Anwar Hospital Ethics Committee for Health Research. Informed consent was obtained from the participants' parents/guardians.
Tuberculin skin test
We used 0.1 mL of PPD-RT23 2 TU (Staten Serum Institute, Copenhagen, Denmark) to be injected intradermally at the volar area of the lower arm of 32 participants. The induration read 48–72 h after injection was measured and considered positive if the diameter of 10 mm or more was noted.
Interferon-gamma releasing assay
We performed an IGRA examination in all patients using Quantiferon TB-Gold plus (QFT-plus) (Qiagen, Germantown, USA). Four mL of blood specimen obtained was divided into four collection tubes; each of 1 mL. Plasma sample then stored in 2°C to be harvested not to exceed 28 days later. The level of IFN-ɣ (interferon gamma) was measured using ELISA. The results would be interpreted as indeterminate if the concentration of negative control is >8.0 IU/mL or that of the positive control is <0.50 IU/mL. The difference of >0.35 to the negative control and >25% of the negative control was interpreted as positive. As per the study protocol, we did not repeat the examination when the results were indeterminate.
Chest X-ray was performed both in AP/PA and lateral position for all participants. The results were interpreted by two independent radiologists; each is expert in thoracic and pediatric radiology. Chest X-ray expertation would be noted as suggestive TB, normal, or lung diseases than TB.
Soluble urokinase-type plasminogen activator receptor level
suPAR level was examined using human suPAR Elisa kit (Bioassay Technology Laboratory, Shanghai, China). One mL plasma sample was centrifuged for 15 min of 2000 rpm and 40 μL of supernatant was put in the sample well. Ten mL anti-suPAR antibody was added to the well followed by 50 μL streptavidin-HRP, then mixed and incubated at 37°C for 60 min. The plate was washed five times with wash buffer. Fifty μL substrate solution A and then 50 μL substrate solution B was added to each well. The covered plate then was incubated at 37°C for 10 min in the dark. Fifty μL of stop solution was added. Optical density (OD) value was determined using microplate reader set to 450 nm within 10 min after stop solution added. suPAR concentration (ng/L) was determined based on standard curved constructed earlier.
The data were analyzed using SPSS 22.0 computer software (SPSS Software - Australia/IBM). Paired t-test was used to compare the suPAR level before and after anti-TB treatment. One-way ANOVA was used to compare the suPAR level between groups. Alternative nonparametric analysis was used for data with skewed distribution. The value of P < 0.05 was considered statistically significant.
| Results|| |
Forty children with pulmonary or extrapulmonary TB were eligible for the study had completed intensive phase treatment during study periods. The median ages of participants were 53 months. [Table 1] shows the demographic characteristics of children, and [Table 2] shows the participants' clinical characteristics.
Median of suPAR level was 0.71 ng/mL before treatment, and the level were not decreased after 2 months of treatment (mean difference 0.17 ng/mL; P = 0.072) [Figure 1]. Twelve participants actually had decrease suPAR level after treatment [Figure 2].
|Figure 1: The level of soluble urokinase-type plasminogen activator receptor before and after treatment. The mean difference was 0.17 (95% CI - 0.02–0.36) ng/L, P = 0.072|
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|Figure 2: Soluble urokinase-type plasminogen activator receptor level of subjects before and after treatment|
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| Discussion|| |
This study is the first to examine the suPAR level as a biomarker to monitor treatment response in childhood TB using the standard diagnostic procedure outlined by the WHO. The previous study had included the subject using the Indonesian pediatric TB scoring system which is criticized due to its specificity and reliability.
In contrast to other studies, the level of suPAR in our participants was not increased before anti-TB treatment. A study by Indumati et al. found increased suPAR level in adults with pulmonary TB. This may be related to the mycobacterium load in their participants, in which all had positive results of microscopy smear examination. In this study, the inflammation in children would be expectedly lower than that in adults. Raras et al. had found increased suPAR level in children with pulmonary TB. However, in contrast to our study which participants' ages ranged widely from under 1 year to 15 years of age, their participants were dominantly aged 7–10 years of age.
We found that suPAR level was not changed after 2 months of treatment with anti-TB in our participants, despite clinical improvement [Table 3]. This result is in contrary to the study in adults participants which found the decrease of suPAR level in 2 months. However, their study design made it less possible to compare the decrease of suPAR level to clinical parameters improvement, because different group were involved in each period of observation.
|Table 3: Soluble urokinase-type plasminogen activator receptor level before and after 2 months anti tuberculosis treatment|
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Raras's et al. study in 25 children with pulmonary TB found that although suPAR level was not decreased after 2 months intensive anti-TB treatment, it was decreased at 4th month and approaching normal level after 6 months. However, the participants recruitment were not standardized as they did not perform microbiology examination while rely solely to pediatric TB scoring system.
In this study, suPAR levels did not appear to be proportional to the participants' age or severity of the disease. Serum suPAR levels were not detected in participants aged 5 years and 6 months with a diagnosis of pulmonary TB confirmed by microbiology, in addition, the tuberculin test and IGRA were positive. Conversely, the upper extreme values are found in patients <1-year-old, with poor nutritional status and with negative IGRA examination results. This indicates that suPAR levels are not in line with cellular immune status of children with TB. Similarly, the suPAR level does not describe the amount of Mycobacterium tuberculosis bacilli that infects TB patients.
Our study reveals that the suPAR level was not affected by the degree of the pulmonary lesion, as it has no association with the presence of pulmonary lesion on chest X-ray. In this study, we have only two positive sputum results for M. tuberculosis, and their suPAR level was not increased before treatment and not decrease after 2 month treatment. This, in part, shows that suPAR level might not be related to the degree of TB disease severity.
Although overall there was no decrease of suPAR level after 2 months of anti-TB treatment, decreased suPAR levels occurred in 12 of 40 patients, nine of which were pulmonary TB, whereas the rest were TB lymphadenitis and tuberculous osteomyelitis. This indicates that the decrease could occur in either pulmonary or extrapulmonary TB, which both have similar pathological features. Therefore, it is understandable that the improvement of the immune response should occur in the same way.
The limitation of this study is that it is difficult to get positive microbiological results so that there is no reliable standard to compare with changes in suPAR levels. The initial diagnosis of TB is based more on the clinical presence of clinical symptoms of TB, contact with adult TB patients, supported by a positive tuberculin or IGRA test and suggestive TB images of TB., Although sputum samples are taken by sputum induction and examined using Xpert MTB/RIF devices that are more sensitive than microscopic examination, positive results are only obtained in two participants. This may be due to the paucibacillary nature of pediatric TB.,
Moreover, the period to monitor suPAR levels only taken once during the treatment, after the intensive phase, could not describe changes in suPAR levels during overall TB treatment.
| Conclusions|| |
We conclude that suPAR may not useful as biomarker to monitor treatment response after 2-month intensive phase anti-TB. However, further study is required to explore the biomarker for treatment monitor during maintenance phase and completion of anti-TB therapy.
We would like to thank UBC Medical Indonesia, Ltd. for supporting the research by providing reagens and equipment for QFT-plus examination.
Financial support and sponsorship
UBC Medical Indonesia, Ltd., has supported the research for QFT-plus reagens and equipment.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Mayers MM. Tuberculosis. Pediatr Rev 2008;29:140-2.
Direktorat Jenderal Pengendalian Penyakit dan Penyehatan Lingkungan Kementerian Kesehatan Republik Indonesia. Petunjuk Teknis Manajemen TB Anak. Jakarta: Kementerian Kesehatan RI; 2016.
World Health Organization. Global Tuberculosis Report 2017. Geneva: World Health Organization; 2017.
Nataprawira HM, Wonoputri N. Obstacles facing tuberculosis treatment in children from a developing country: A hospital-based study. Am J Epidemiol Infect Dis 2014;2:8-12.
TB CARE I. International Standards for Tuberculosis Care, Edisi ke-3. The Hague: TB CARE I; 2014.
Ruiz Jiménez M, Guillén Martín S, Prieto Tato LM, Cacho Calvo JB, Álvarez García A, Soto Sánchez B, et al.
Induced sputum versus gastric lavage for the diagnosis of pulmonary tuberculosis in children. BMC Infect Dis 2013;13:222.
World Health Organization. Guidance for National Tuberculosis Programmes on the Management of Tuberculosis in Children. 2nd
ed. Geneva: World Health Organization; 2013.
World Health Organization. Automated Real-time Nucleic Acid Amplification Technology for Rapid and Simultaneous Detection of Tuberculosis and Rifampicin Resistance: Xpert MTB/RIF System for the Diagnosis of Pulmonary and Extrapulmonary TB in Adults and Children: Policy Update. Geneva: World Health Organization; 2013. Available from: http://www.apps.who.int/iris/handle/10665/112472
. [Last accessed on 2017 Mar 28].
Roya-Pabon CL, Perez-Velez CM. Tuberculosis exposure, infection and disease in children: A systematic diagnostic approach. Pneumonia (Nathan) 2016;8:23.
Hertting O, Shingadia D. Childhood TB: When to think of it and what to do when you do. J Infect 2014;68 Suppl 1:S151-4.
Can U. The role of soluble urokinase-type plasminogen activator receptor (suPAR) in multiple respiratory diseases. Recept Clin Invest 2015;2:e473.
Del Rosso M, Margheri F, Serratì S, Chillá A, Laurenzana A, Fibbi G. The urokinase receptor system, a key regulator at the intersection between inflammation, immunity, and coagulation. Curr Pharm Des 2011;17:1924-43.
El-Mekkawy MS, Saleh NY, Sonbol AA. Soluble urokinase plasminogen activator receptor: A new biomarker in the pediatric intensive care unit. Indian J Pediatr 2016;83:661-9.
Erkut N, Menteşe A, Özbaş HM, Ermantaş N, Sümer A, Örem A, et al.
The prognostic significance of soluble urokinase plasminogen activator receptor in acute myeloid leukemia. Turk J Haematol 2016;33:135-40.
Georgescu AM, Szederjesi J, Voidăzan S, Dobreanu M, Copotoiu SM, Huţanu A, et al
. Soluble urokinase-type plasminogen activator receptor (suPAR) – A possible biomarker for bacteremia in sepsis. Rev Romana Med Lab 2015;23:5-73.
Indumati V, Vijay V, Krishnaswamy D, Rajeshwari V, Ramesh A, Shantala D, et al.
Serum levels of soluble urokinase plasminogen activator receptor (suPAR) as a marker of tuberculosis treatment efficacy. Indian J Tuberc 2017;64:206-11.
Okulu E, Arsan S, Akin IM, Ates C, Alan S, Kilic A, et al.
Serum levels of soluble urokinase plasminogen activator receptor in infants with late-onset sepsis. J Clin Lab Anal 2015;29:347-52.
Rabna P, Andersen A, Wejse C, Oliveira I, Gomes VF, Haaland MB, et al.
Utility of the plasma level of suPAR in monitoring risk of mortality during TB treatment. PLoS One 2012;7:e43933.
Mardining Raras TY, Noor Chozin I. The soluble plasminogen activator receptor as a biomarker on monitoring the therapy progress of pulmonary TB-AFB(+) patients. Tuberc Res Treat 2010;2010:406346.
Raras TY, Hamid MA, Chandra Kusuma MS, Olivianto E. Soluble urokinase plasminogen activator receptor (suPAR) is prospective biomarker to evaluate treatment progress in children with pulmonary tuberculosis during maintenance phase. Edorium J Pediatr 2017;1:6-11.
Rudolf F, Wagner AJ, Back FM, Gomes VF, Aaby P, Østergaard L, et al.
Tuberculosis case finding and mortality prediction: Added value of the clinical TBscore and biomarker suPAR. Int J Tuberc Lung Dis 2017;21:67-72.
Siahanidou T, Margeli A, Tsirogianni C, Charoni S, Giannaki M, Vavourakis E, et al.
Clinical value of plasma soluble urokinase-type plasminogen activator receptor levels in term neonates with infection or sepsis: A prospective study. Mediators Inflamm 2014;2014:375702.
Smith HW, Marshall CJ. Regulation of cell signalling by uPAR. Nat Rev Mol Cell Biol 2010;11:23-36.
Thunø M, Macho B, Eugen-Olsen J. SuPAR: The molecular crystal ball. Dis Markers 2009;27:157-72.
Vasarhelyi B, Toldi G, Balog A. The clinical value of soluble urokinase plasminogen activator receptor (suPAR) levels in autoimmune connective tissue disorders. J Int Fed Clin Chem Lab Med 2017;27:122-9.
Wardhani SO, Burhan N, Ismanoe G, Raras TY. Correlation between soluble urokinase plasminogen activator receptor with CD4 T lymphocyte and WHO clinical staging of HIV infection. Indones J Trop Infect Dis 2010;1:32-5.
Zhang Y, Xiao W, Jiang Y, Wang H, Xu X, Ma D, et al.
Levels of components of the urokinase-type plasminogen activator system are related to chronic obstructive pulmonary disease parenchymal destruction and airway remodelling. J Int Med Res 2012;40:976-85.
Marais BJ, Schaaf HS. Tuberculosis in children. Cold Spring Harb Perspect Med 2014;4:a017855.
Marais BJ, Graham SM, Cotton MF, Beyers N. Diagnostic and management challenges for childhood tuberculosis in the era of HIV. J Infect Dis 2007;196 Suppl 1:S76-85.
Caulfield AJ, Wengenack NL. Diagnosis of active tuberculosis disease: From microscopy to molecular techniques. J Clin Tuberc Other Mycobact Dis 2016;4:33-43.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]