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Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine

Abstract:

INTRODUCTION

: Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples.

METHODS:

Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested).

RESULTS:

STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary).

CONCLUSIONS:

STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.

Keywords:
Mycobacterium tuberculosis; Molecular diagnostic test; Nested polymerase chain reaction; Blood; Urine

INTRODUCTION

Tuberculosis (TB) is a major cause of morbidity and mortality affecting individuals of various ages and social classes 11. World Health Organization (WHO). Global Tuberculosis Report 2013. Genève: WHO; 2013. 289p. (Acessed 2014 May 15 th) Available at: Available at: http://www.who.int/tb/publications/global_report/en/
http://www.who.int/tb/publications/globa...
. In 2013, 83,310 cases were recorded in Brazil, including 34.7 new cases per 100,000 habitants in the Northeastern region 22. Ministério da Saúde (MS). Sistema de Informação de Agravos de Notificação. DATASUS. Brasília: Ministério da Saúde; 2014. (Acessed 2015 Jul 12 th). Available at: Available at: http://dtr2004.saude.gov.br/sinanweb/tabnet/tabnet?sinannet/tuberculose/bases/tubercbrnet.def.
http://dtr2004.saude.gov.br/sinanweb/tab...
33. World Health Organization (WHO). Global Tuberculosis Report 2014. Genève: WHO;2014. 171p. (Accessed 2015 April 16). Available at: Available at: http://apps.who.int/iris/bitstream/10665/137094/1/9789241564809_eng.pdf?ua=1
http://apps.who.int/iris/bitstream/10665...
. Conventional bacteriological tests for detecting TB do not combine high sensitivity and specificity 44. Tortora GJ, Funke BR, Case CL. Microbiologia. 8th ed. Porto Alegre (RS): Artmed; 2005.. For example, acid-fast bacillus (AFB) smear microscopy is rapid and low cost, but has low sensitivity and it is not specific for Mycobacterium tuberculosis55. Moure R, Muñoz L, Torres M, Santin M, Martín R, Alcaide F. Rapid Detection of Mycobacterium tuberculosis Complex and Rifampin Resistance in Smear-Negative Clinical Samples by Use of an Integrated Real-Time PCR Method. J Clin Microbiol 2011; 49:1137-1139..

Molecular approaches have been used as alternative tools for diagnosing infectious diseases 55. Moure R, Muñoz L, Torres M, Santin M, Martín R, Alcaide F. Rapid Detection of Mycobacterium tuberculosis Complex and Rifampin Resistance in Smear-Negative Clinical Samples by Use of an Integrated Real-Time PCR Method. J Clin Microbiol 2011; 49:1137-1139. 66. Kocagöz T, Yilmaz E, Ozkara S, Kocagöz S, Hayran M, Sachedeva M et al. Detection of Mycobacterium tuberculosis in sputum samples by polymerase chain reaction using a simplified procedure. J Clin Microbiol1993; 31:1435-1438. 77. Lima JFC, Montenegro LML, Montenegro RA, Cabral MML, Lima AS, Abath FGC et al. Performance of nested PCR in the specific detection of Mycobacterium tuberculosis complex in blood samples of pediatric patients. J Pneumol 2009; 35:690-697. 88. Silva MAL, Soares CRP, Medeiros RA, Medeiros Z, Melo FL. Optimization of single-tube nested PCR for the diagnosis of visceral leishmaniasis. Exp Parasitol 2013; 134:206-210. 99. Abath FGC, Werkhauser R, Melo FL. Single-tube nested PCR using immobilized internal primers. Biotechniques 2001; 33:1210-1214.. One of these is polymerase chain reaction (PCR), which has been applied to the detection of M. tuberculosis44. Tortora GJ, Funke BR, Case CL. Microbiologia. 8th ed. Porto Alegre (RS): Artmed; 2005. 55. Moure R, Muñoz L, Torres M, Santin M, Martín R, Alcaide F. Rapid Detection of Mycobacterium tuberculosis Complex and Rifampin Resistance in Smear-Negative Clinical Samples by Use of an Integrated Real-Time PCR Method. J Clin Microbiol 2011; 49:1137-1139. 77. Lima JFC, Montenegro LML, Montenegro RA, Cabral MML, Lima AS, Abath FGC et al. Performance of nested PCR in the specific detection of Mycobacterium tuberculosis complex in blood samples of pediatric patients. J Pneumol 2009; 35:690-697. 88. Silva MAL, Soares CRP, Medeiros RA, Medeiros Z, Melo FL. Optimization of single-tube nested PCR for the diagnosis of visceral leishmaniasis. Exp Parasitol 2013; 134:206-210. 1010. Torrea G, Perre PV, Ouedraogo M, Zougba A, Sawadogo A, Dingtoumda B et al. PCR-based detection of the Mycobacterium tuberculosis complex in urine of HIV-infected and uninfected pulmonary and extrapulmonary tuberculosis patients in Burkina Faso. J Med Microbiol 2005; 54:39-44. 1111. Broccolo F, Scarpellini P, Locatelli G, Zingale A, Brambilla AM, Cichero P et al. Rapid diagnosis of Mycobacterial infections and quantification of Mycobacterium tuberculosis load by two Real-time calibrated PCR assays. J Clin Microbiol2003; 41:4565-4572. 1212. Rebollo MJ, Garrido RSJ, Folgueira D, Palenque E, Díaz-Pedroche C, Lumbreras C et al. Blood and urine samples as useful sources for direct detection of tuberculosis by polymerase chain reaction. Diag Microbiol Infect Dis 2006; 56:141-146. 1313. Cruz HLA, Montenegro RA, Lima JFA, Poroca DR, Lima JFC, Montenegro LML et al. Evaluation of a Nested-PCR for Mycobacterium tuberculosis detection in blood and urine samples. Braz J Microbiol 2011; 42:321-329.at concentrations of < 10 cells/mL in clinical samples 77. Lima JFC, Montenegro LML, Montenegro RA, Cabral MML, Lima AS, Abath FGC et al. Performance of nested PCR in the specific detection of Mycobacterium tuberculosis complex in blood samples of pediatric patients. J Pneumol 2009; 35:690-697. 1414. Lima JFC. Detecção do Mycobacterium tuberculosis em amostras de sangue e urina através da Nested-PCR em único tubo. 2009. 111p. (Master's Dissertation). Centro de Pesquisas Aggeu Magalhães. Fundação Oswaldo Cruz; 2009 Recife. 1515. Butt T, Ahmad RN, Kazmi SY, Afzal RK, Mahmood A. An update on the diagnosis of tuberculosis. J Coll Physicians Surg Pak 2003; 13:728-734.. Nested PCR is a variation of this technique that involves two amplification steps 77. Lima JFC, Montenegro LML, Montenegro RA, Cabral MML, Lima AS, Abath FGC et al. Performance of nested PCR in the specific detection of Mycobacterium tuberculosis complex in blood samples of pediatric patients. J Pneumol 2009; 35:690-697. 88. Silva MAL, Soares CRP, Medeiros RA, Medeiros Z, Melo FL. Optimization of single-tube nested PCR for the diagnosis of visceral leishmaniasis. Exp Parasitol 2013; 134:206-210. 1313. Cruz HLA, Montenegro RA, Lima JFA, Poroca DR, Lima JFC, Montenegro LML et al. Evaluation of a Nested-PCR for Mycobacterium tuberculosis detection in blood and urine samples. Braz J Microbiol 2011; 42:321-329. 1616. Assis NCS, Lopes ML, Cardoso NC, Costa MM, Souza CO, Lima KVB. Diagnóstico molecular da tuberculose pulmonar. J Bras Patol Med Lab 2007; 43:1-7. 1717. Sankar S, Balakrishnan B, Nandagopal B, Thangaraju K, Natarajan S. Comparative Evaluation of Nested PCR and conventional smear methods for the detection of Mycobacterium tuberculosis in sputum samples. Mol Diagn Ther 2010; 14:223-227., yielding greater sensitivity and specificity 88. Silva MAL, Soares CRP, Medeiros RA, Medeiros Z, Melo FL. Optimization of single-tube nested PCR for the diagnosis of visceral leishmaniasis. Exp Parasitol 2013; 134:206-210. 1616. Assis NCS, Lopes ML, Cardoso NC, Costa MM, Souza CO, Lima KVB. Diagnóstico molecular da tuberculose pulmonar. J Bras Patol Med Lab 2007; 43:1-7. 1717. Sankar S, Balakrishnan B, Nandagopal B, Thangaraju K, Natarajan S. Comparative Evaluation of Nested PCR and conventional smear methods for the detection of Mycobacterium tuberculosis in sputum samples. Mol Diagn Ther 2010; 14:223-227.. Single-tube nested polymerase chain reaction (STNPCR) is more rapid than conventional nested PCR 88. Silva MAL, Soares CRP, Medeiros RA, Medeiros Z, Melo FL. Optimization of single-tube nested PCR for the diagnosis of visceral leishmaniasis. Exp Parasitol 2013; 134:206-210. 1414. Lima JFC. Detecção do Mycobacterium tuberculosis em amostras de sangue e urina através da Nested-PCR em único tubo. 2009. 111p. (Master's Dissertation). Centro de Pesquisas Aggeu Magalhães. Fundação Oswaldo Cruz; 2009 Recife.with less probability of cross-contamination and requiring a smaller amount of reagent. In STNPCR, both amplification reactions occur consecutively and the microtubes do not need to be opened or changed for the addition of new reagents 44. Tortora GJ, Funke BR, Case CL. Microbiologia. 8th ed. Porto Alegre (RS): Artmed; 2005. 88. Silva MAL, Soares CRP, Medeiros RA, Medeiros Z, Melo FL. Optimization of single-tube nested PCR for the diagnosis of visceral leishmaniasis. Exp Parasitol 2013; 134:206-210..

There are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB 1818. Ruffino-Netto A Programa de Controle da Tuberculose no Brasil: Situação Atual e Novas Perspectivas. Infor Epidemio SUS (periódico na Internet) 2001; 10:129-138.; this is especially challenging when few bacilli are present, for example in cases of extrapulmonary TB, pulmonary TB with a negative AFB test, childhood TB, and co-infection with TB-human immunodeficiency virus (HIV) 77. Lima JFC, Montenegro LML, Montenegro RA, Cabral MML, Lima AS, Abath FGC et al. Performance of nested PCR in the specific detection of Mycobacterium tuberculosis complex in blood samples of pediatric patients. J Pneumol 2009; 35:690-697. 1010. Torrea G, Perre PV, Ouedraogo M, Zougba A, Sawadogo A, Dingtoumda B et al. PCR-based detection of the Mycobacterium tuberculosis complex in urine of HIV-infected and uninfected pulmonary and extrapulmonary tuberculosis patients in Burkina Faso. J Med Microbiol 2005; 54:39-44. 1212. Rebollo MJ, Garrido RSJ, Folgueira D, Palenque E, Díaz-Pedroche C, Lumbreras C et al. Blood and urine samples as useful sources for direct detection of tuberculosis by polymerase chain reaction. Diag Microbiol Infect Dis 2006; 56:141-146..

Given the difficulties and delays in confirming conventional laboratory results, obtaining a timely diagnosis of TB - especially the paucibacillary and extrapulmonary forms 1010. Torrea G, Perre PV, Ouedraogo M, Zougba A, Sawadogo A, Dingtoumda B et al. PCR-based detection of the Mycobacterium tuberculosis complex in urine of HIV-infected and uninfected pulmonary and extrapulmonary tuberculosis patients in Burkina Faso. J Med Microbiol 2005; 54:39-44.- remains a challenge. STNPCR can achieve a more rapid diagnosis of TB than culture methods and has higher sensitivity than the AFB test using urine and blood 77. Lima JFC, Montenegro LML, Montenegro RA, Cabral MML, Lima AS, Abath FGC et al. Performance of nested PCR in the specific detection of Mycobacterium tuberculosis complex in blood samples of pediatric patients. J Pneumol 2009; 35:690-697. 1313. Cruz HLA, Montenegro RA, Lima JFA, Poroca DR, Lima JFC, Montenegro LML et al. Evaluation of a Nested-PCR for Mycobacterium tuberculosis detection in blood and urine samples. Braz J Microbiol 2011; 42:321-329. 1919. Mehta PK, Raj A, Singh N, Khuller GK. Diagnosis of extrapulmonary tuberculosis by PCR. FEMS Immunol Med Microbiol 2012; 66:20-36. 2020. Piatek AS, Cleef MV, Alexander H, Coggin WL, Rehr M, Kampen SV et al. GeneXpert for TB diagnosis: planned and purposeful implementation. Glob Health Sci Pract 2013; 1:18-23.. However, AFB, culture methods, and the GeneXpert Mycobacterium tuberculosis (MTB)/rifampicin (RIF) test are still considered gold standards for diagnosing pulmonary TB in many regions 2020. Piatek AS, Cleef MV, Alexander H, Coggin WL, Rehr M, Kampen SV et al. GeneXpert for TB diagnosis: planned and purposeful implementation. Glob Health Sci Pract 2013; 1:18-23. 2121. Ministério da Saúde (MS), Departamento de Gestão e Incorporação de Tecnologias em Saúde da Secretaria de Ciência, Tecnologia e Insumos Estratégicos (CONITEC). Proposta de incorporação do Xpert MTB-RIF como teste para diagnóstico de tuberculose e para indicação de resistência à rifampicina. Technical Report Number 49. Brasília (DF): MS, CONITEC; 2013. 2222. Silva Jr JB. Tuberculose: Guia de Vigilância Epidemiológica. J Bras Pneumol (online). 2004; 30:S57-S86. 2323. American Thoracic Society. Diagnostic Standards and Classification of Tuberculosis in adults and children. Am J Respir Crit Care Med 2000; 161:1376-1395..

Various studies have reported the molecular diagnosis of TB using extrapulmonary samples, including in cases of negative AFB results 1212. Rebollo MJ, Garrido RSJ, Folgueira D, Palenque E, Díaz-Pedroche C, Lumbreras C et al. Blood and urine samples as useful sources for direct detection of tuberculosis by polymerase chain reaction. Diag Microbiol Infect Dis 2006; 56:141-146. 1313. Cruz HLA, Montenegro RA, Lima JFA, Poroca DR, Lima JFC, Montenegro LML et al. Evaluation of a Nested-PCR for Mycobacterium tuberculosis detection in blood and urine samples. Braz J Microbiol 2011; 42:321-329. 1919. Mehta PK, Raj A, Singh N, Khuller GK. Diagnosis of extrapulmonary tuberculosis by PCR. FEMS Immunol Med Microbiol 2012; 66:20-36.. The present study evaluated the performance of STNPCR in detecting the presence of M. tuberculosis complex in blood and urine using an in-house DNA extraction method from patients with suspected TB. The results demonstrate that the STNPCR method can improve clinical practice by providing early diagnosis of TB so as to avoid unnecessary invasive procedures and inappropriate treatment.

METHODS

Patients were non-randomly selected from public hospitals located in Recife between July 2006 and August 2009. Patients sought health services spontaneously with diverse simptons and suspected of various diseases, for example, tuberculosis or pneumonia. Sample size was determined by convenience according to the number of patients with suspected TB who sought medical attention at participating hospitals. Control patients showing pulmonary symptoms but without TB were recruited from the same hospitals.

Blood [3-5mL collected in a vacuum tube with anti-coagulant ethylenediaminetetraacetic acid (EDTA)] and urine (10ml/day for 3 consecutive days) samples were obtained from each patient suspected of TB before specific treatment was initiated. The samples were sent to Aggeu Magalhães Research Center, Oswaldo Cruz Foundation [ Centro de Pesquisas Aggeu Magalhães/Fundação Oswaldo Cruz (CPqAM/FIOCRUZ)] within 4h of collection and stored at 4°C-8°C for up to 24h. Urine samples were decontaminated using Petroff's method 2424. Barreto AMW, Campos CED, Martins FM. Manual de Bacteriologia da Tuberculose. 2nd ed. Brasília, DF: Ministério da Saúde; 1994. 2525. Ministério da Saúde (MS). Manual de Bacteriologia da Tuberculose. 3rd ed. Brasília, DF: MS; 2005.. Blood cells were separated from whole blood at room temperature by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden).

Ethical considerations

The study protocol was approved by the Research Ethics Committee of CPqAM/FIOCRUZ (no. 0133.0.095.000-08) with the consent of participating hospitals. All participants provided written, informed consent for collection of biological samples; in cases of minors or those unable to sign or decide for themselves, this was provided by a guardian or legal representative.

Study population

Patients of both genders between the ages of 15 and 69 years and suspected of TB infection after clinical examination were recruited from public hospitals in Recife, the capital of the State of Pernambuco in Northeastern Brazil.

Patients were classified into TB and non-TB groups based on clinical, laboratory, and epidemiological criteria 2222. Silva Jr JB. Tuberculose: Guia de Vigilância Epidemiológica. J Bras Pneumol (online). 2004; 30:S57-S86.by the physician who attended to them at the hospital, without data from molecular tests. Clinical diagnoses were made and STNPCR performed in a double-blinded fashion. Active TB was defined as a positive AFB test or TB culture according to the criteria of the Ministry of Health of Brazil 2222. Silva Jr JB. Tuberculose: Guia de Vigilância Epidemiológica. J Bras Pneumol (online). 2004; 30:S57-S86. 2626. Ministério da Saúde (MS), Secretaria de Políticas de Saúde, Departamento de Assistência Primária. Manual técnico para o controle da tuberculose: cadernos de atenção básica. 6th. ed. Rev Ampl, Brasília, DF: MS; 2002.and American Thoracic Society modified criteria 2323. American Thoracic Society. Diagnostic Standards and Classification of Tuberculosis in adults and children. Am J Respir Crit Care Med 2000; 161:1376-1395. 2727. American Thoracic Society. Diagnostic standards and classification of tuberculosis. Am Rev Respir Dis 1990; 142:725-735.. In cases where conventional tests were negative, inconclusive, or were not conducted, individuals were considered TB-positive if they had a clinical or radiological profile consistent with active TB and if they showed clinical improvement after treatment for 1 month with antibiotics. Latent TB was defined as a positive skin test for tuberculin and negative bacteriological exams (when it was available on patient) and with no clinical, radiological, or bacteriological evidence of active TB 2323. American Thoracic Society. Diagnostic Standards and Classification of Tuberculosis in adults and children. Am J Respir Crit Care Med 2000; 161:1376-1395.. The negative control group consisted of individuals who used health services for reasons other than TB, who had a scar corresponding to the Bacillus Calmette-Guérin (BCG) TB vaccination, negative tuberculin test, had not had contact with a bacillipherous adult, and showed no clinical, epidemiological, or laboratory evidence compatible with TB. Patients who were human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS)-positive, had chronic diseases such as kidney or heart disease, cystic fibrosis, or other chronic lung diseases, or who were using immunosuppressive drugs for more than 15 days were excluded from the study.

DNA extraction

Deoxyribonucleic acid (DNA) was extracted using a modified version of a previously published in-house protocol 2828. Rossetti MLR, Jardim SB, Rodrigues VFS, Moura AR, Oliveira H, Zaha A. Improvement of Mycobacterium tuberculosis detection in clinical samples using DNA purified by glass matrix. J Microbiol Methods 1997; 28:139-146.. After DNA was bound to the Sephaglas BandPrep kit resin (Amersham Pharmacia Biotech, Little Chalfont, UK), 200μl of sodium iodide (0.9g/mL) was added to each microtube to increase bond strength. A tube without DNA served as a negative control.

Single-tube nested polymerase chain reaction

The assay was based on a previously described STNPCR method 99. Abath FGC, Werkhauser R, Melo FL. Single-tube nested PCR using immobilized internal primers. Biotechniques 2001; 33:1210-1214.and was optimized for human blood and urine. The IS6110 insert sequence (GenBank accession no. X52471) was the target for amplification. The outer primers TJ5 (5'-CCG CAA AGT GTG GCT AAC-3') and TJ3 (5'-ATC CCC TAT CCG TAT GGT G-3') amplified a 409-bp fragment, and the inner primers OLI5 (5'-AAC GGC TGA TGA CCA AAC-3') and STAN3 (5'-GTC GAG TAC GCC TTC TTG TT-3') amplified a 316-bp fragment 2929. Ritis K, Tzoanopoulos D, Speletas M, Papadopoulos E, Arvanitidis K, Kartali S et al. Amplification of IS6110 sequence for detection of Mycobacterium tuberculosis complex in HIV-negative patients with fever of unknown origin (FUO) and evidence of extrapulmonary disease. J Intern Med 2000; 248:415-424.. The primers have been previously tested in blood and urine samples using a conventional nested PCR system 1313. Cruz HLA, Montenegro RA, Lima JFA, Poroca DR, Lima JFC, Montenegro LML et al. Evaluation of a Nested-PCR for Mycobacterium tuberculosis detection in blood and urine samples. Braz J Microbiol 2011; 42:321-329..

Amplification was carried out on an automatic Eppendorf Gradient thermal cycler (Hamburg, Germany). The first stage of PCR consisted of 15 cycles (94°C for 1min, 57°C for 1 min, and 72°C for 1 min) and the second of 45 cycles (94°C for 1 min, 60°C for 1 min, and 72°C for 1 min). For the first 15 cycles, each tube contained 0.5pmoles of outer primers in a final volume of 50μl containing 200mM Tris-HCl (pH 8.4), 500mM KCl (10× buffer), 2.5mM MgCl 2, 2mM dNTP (5μl), and 5U Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). The inner primers were diluted with equal volumes of water and bromophenol blue (2μg/ml) to a final concentration of 50 pmoles of each primer. This mixture was placed on the inner surface of the microtube cap followed by incubation at 37°C for 30 min, and then eluted from the cap surface into the reaction mixture by briefly interrupting the PCR after the 15 thcycle and repeatedly inverting the tubes 99. Abath FGC, Werkhauser R, Melo FL. Single-tube nested PCR using immobilized internal primers. Biotechniques 2001; 33:1210-1214. 3030. Abath FGC, Werkhauser RP, Melo FL. Método, kit e iniciadores para a identificação de sequências específicas de nucleotídeos através da reação em cadeia da polimerase tipo Nested em um único tubo de reação. Número de Registro: BR n. PI 015740-5, 29 nov 2001.. A 10-μl volume of Tris-EDTA served as a negative control and a 2-μl volume of M. tuberculosis strain H37Rv DNA with 8μl Milli-Q-purified water served as a positive control for each set of reactions. PCR was repeated for all samples yielding negative results as confirmation.

Electrophoresis

Amplicons generated by STNPCR were resolved by 2% agarose gel electrophoresis. The gel was stained with ethidium bromide (20μl per liter of 1× Tris-EDTA buffer) and visualized under ultraviolet light. The Low Mass Ladder (Invitrogen) was used as a marker.

Statistical analysis

A database was created using Statistical Package for the Social Sciences (SPSS) v.10.0 software (SPSS Inc., Chicago, IL, USA) to store clinical, epidemiological, and laboratory data for all patients. PCR sensitivity and specificity and positive and negative predictive values were analyzed in parallel in leukocytes, plasma and urine 3131. Medronho RA. Epidemiologia. 2nd ed. São Paulo: Ed. Atheneu; 2002.for each clinical sample along with their 95% confidence intervals. Note that denominated blood sample means leukocytes and plasma analyzed in parallel. The χ 2test was used to evaluate the results, along with Fisher's Exact test when necessary. OpenEpi v.2.3.1 (http://www.openepi.com/oe2.3/DiagnosticTest/DiagnosticTest.htm) and SPSS v.10.0 software were used for data analysis. P < 0.05 was considered statistically significant.

RESULTS

We collected 167 blood and urine samples from 98 individuals (mostly male) with a mean age of 33.4 ± 18.3. A total of 83 patients were diagnosed with active or latent TB ( Table 1) that were of two clinical forms, pulmonary and extrapulmonary. Clinical forms of extrapulmonary TB are described on Table 2. The performance and accuracy of STPCR were analyzed using blood and urine samples according to the clinical form of TB ( Table 3). When previously tested using M. tuberculosis strain H37Rv genomic DNA, the STNPCR system showed a detection limit of 1ag (0.001fg) (results not shown) 1414. Lima JFC. Detecção do Mycobacterium tuberculosis em amostras de sangue e urina através da Nested-PCR em único tubo. 2009. 111p. (Master's Dissertation). Centro de Pesquisas Aggeu Magalhães. Fundação Oswaldo Cruz; 2009 Recife..

Table 1:
Clinical and epidemiological characteristics of study participants.
Table 2:
Frequency of clinical forms of extrapulmonary TB.
Table 3:
Determination of clinical forms of TB (pulmonary or extrapulmonary) by STNPCR analysis of blood and urine samples from individuals with or without TB.

The sensitivity of STNPCR for detecting DNA in leukocytes, plasma and urine samples from patients with or without TB was 50%, 34% and 36.5%, respectively, while the specificity was 100% relative to negative samples ( Table 4). The p value for STNPCR on leukocytes was <0.001 and on plasma p = 0.007. The sensitivity and specificity in blood samples (leukocytes and plasma analyzed as one sample) were also determined 3131. Medronho RA. Epidemiologia. 2nd ed. São Paulo: Ed. Atheneu; 2002., where a result was positive when at least one sample was positive by PCR. The sensitivity and specificity in blood were 61.5% and 100%, respectively ( Table 4), as compared to 65.4% and 100%, respectively, for blood and urine samples together ( Table 4). None of patients was diagnosed with micobacteremia and only one was diagnosed as renal TB, but none had a simple genito-urinary infection.

Table 4:
Accuracy of STNPCR for clinical samples from TB patients and healthy individuals.

DISCUSSION

A posteriori hematogenous lymphatic dissemination of Koch bacilli follows the establishment of Ghon's complex in infected patients 3232. Campos HS. Tuberculosis: etiopathogenesis and clinical presentations. Pulmão RJ 2006; 15:29-35., and previous studies have detected circulating bacilli in blood and urine by PCR 77. Lima JFC, Montenegro LML, Montenegro RA, Cabral MML, Lima AS, Abath FGC et al. Performance of nested PCR in the specific detection of Mycobacterium tuberculosis complex in blood samples of pediatric patients. J Pneumol 2009; 35:690-697. 1212. Rebollo MJ, Garrido RSJ, Folgueira D, Palenque E, Díaz-Pedroche C, Lumbreras C et al. Blood and urine samples as useful sources for direct detection of tuberculosis by polymerase chain reaction. Diag Microbiol Infect Dis 2006; 56:141-146. 1313. Cruz HLA, Montenegro RA, Lima JFA, Poroca DR, Lima JFC, Montenegro LML et al. Evaluation of a Nested-PCR for Mycobacterium tuberculosis detection in blood and urine samples. Braz J Microbiol 2011; 42:321-329.. In the present study, we evaluated the efficiency of detecting the M. tuberculosis complex in clinical samples by STNPCR. This method has the advantage of using samples that are easily collected with minimal invasiveness from patients with suspected pulmonary or extrapulmonary TB. This is especially useful for children. The gold standard of gastric lavage cultures requires hospitalization of the patient and is extremely invasive, and should be reserved for patients with negative AFB and extrapulmonary TB 1010. Torrea G, Perre PV, Ouedraogo M, Zougba A, Sawadogo A, Dingtoumda B et al. PCR-based detection of the Mycobacterium tuberculosis complex in urine of HIV-infected and uninfected pulmonary and extrapulmonary tuberculosis patients in Burkina Faso. J Med Microbiol 2005; 54:39-44. 1212. Rebollo MJ, Garrido RSJ, Folgueira D, Palenque E, Díaz-Pedroche C, Lumbreras C et al. Blood and urine samples as useful sources for direct detection of tuberculosis by polymerase chain reaction. Diag Microbiol Infect Dis 2006; 56:141-146. 1313. Cruz HLA, Montenegro RA, Lima JFA, Poroca DR, Lima JFC, Montenegro LML et al. Evaluation of a Nested-PCR for Mycobacterium tuberculosis detection in blood and urine samples. Braz J Microbiol 2011; 42:321-329. 1414. Lima JFC. Detecção do Mycobacterium tuberculosis em amostras de sangue e urina através da Nested-PCR em único tubo. 2009. 111p. (Master's Dissertation). Centro de Pesquisas Aggeu Magalhães. Fundação Oswaldo Cruz; 2009 Recife. 3333. Petera J, Greenc CD, Hoelschere MF, Mwaba P, Zumlac AD, Dheda K. Urine for the diagnosis of tuberculosis: current approaches, clinical applicability, and new developments. Curr Opin Pulm Med 2010; 16:262-270..

Although the STNPCR showed high sensitivity for detecting M. tuberculosis in blood and urine samples, is unclear why circulating bacilli were found in these samples in patients that do not have a bacteremia nor genitourinary TB 3434. Banada PP, Koshy R, Alland D. Detection of Mycobacterium tuberculosis in Blood by Use of the Xpert MTB/RIF Assay. J Clin Microbiol2013; 51:2317-2322.; the presence of M. tuberculosis in the blood and urine of active TB cases is typically very low, since these are paucibacillary samples. It is possible that patients with active infection have mycobacterial DNA in their macrophages and in other immune cells 3535. Mirza S, Restrepo BI, McCormick JB, Fisher-Hoch SP. Diagnosis of tuberculosis lymphadenitis using polymerase reaction on peripheral blood mononuclear cells. Am J Trop Med Hyg 2003; 69:461-465..

Recently, two automated tests for diagnosing TB directly from clinical samples have been evaluated 3636. Friedrich SO, Venter A, Kayigire XA, Dawson R, Donald PR, Diacon AH. Suitability of Xpert MTB/RIF and Genotype MTBDR plus for patient selection for a Tuberculosis Clinical Trial. J Clin Microbiol2011; 49:2827-2831., i.e., GeneXpert MTB/RIF for pulmonary TB and GenoType MTBDRplus (Hain Lifescience GmbH, Nehren, Germany). Both methods can detect TB in clinical samples as well as resistance to RIF and/or isoniazid 3636. Friedrich SO, Venter A, Kayigire XA, Dawson R, Donald PR, Diacon AH. Suitability of Xpert MTB/RIF and Genotype MTBDR plus for patient selection for a Tuberculosis Clinical Trial. J Clin Microbiol2011; 49:2827-2831. 3737. Lacoma A, Garcia-Sierra N, Prat C, Ruiz-Manzano J, Haba L, Roses S, et al. GenoType MTBDRplus Assay for Molecular Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Strains and Clinical Samples. J Clin Microbiol2008; 46:3660-3667. 3838. Piatek AS, Cleef MV, Alexander H, Coggin WL, Rehr M, Kampen SV et al. GeneXpert for TB diagnosis: planned and purposeful implementation. Glob Health Sci Pract2013; 1:18-23.. Despite these advantages, the tests are only indicated for use with sputum. Therefore, a subset of TB patients with no sputum or with the extrapulmonary form of the disease cannot be diagnosed by these methods according to World Health Organization recommendations 11. World Health Organization (WHO). Global Tuberculosis Report 2013. Genève: WHO; 2013. 289p. (Acessed 2014 May 15 th) Available at: Available at: http://www.who.int/tb/publications/global_report/en/
http://www.who.int/tb/publications/globa...
.

The nested PCR approach increases accuracy of detection 88. Silva MAL, Soares CRP, Medeiros RA, Medeiros Z, Melo FL. Optimization of single-tube nested PCR for the diagnosis of visceral leishmaniasis. Exp Parasitol 2013; 134:206-210. 1414. Lima JFC. Detecção do Mycobacterium tuberculosis em amostras de sangue e urina através da Nested-PCR em único tubo. 2009. 111p. (Master's Dissertation). Centro de Pesquisas Aggeu Magalhães. Fundação Oswaldo Cruz; 2009 Recife.. In STNPCR, reactions take place in one tube for each sample, and the tubes do not need to be opened between the first and nested cycles. This decreases the risk of cross contamination between the two steps 88. Silva MAL, Soares CRP, Medeiros RA, Medeiros Z, Melo FL. Optimization of single-tube nested PCR for the diagnosis of visceral leishmaniasis. Exp Parasitol 2013; 134:206-210.and thereby increases sensitivity and specificity relative to the conventional nested PCR 1313. Cruz HLA, Montenegro RA, Lima JFA, Poroca DR, Lima JFC, Montenegro LML et al. Evaluation of a Nested-PCR for Mycobacterium tuberculosis detection in blood and urine samples. Braz J Microbiol 2011; 42:321-329.method.

STNPCR can amplify a smaller quantity of genomic DNA than is present in a single bacillus (5fg DNA/bacillus) 1414. Lima JFC. Detecção do Mycobacterium tuberculosis em amostras de sangue e urina através da Nested-PCR em único tubo. 2009. 111p. (Master's Dissertation). Centro de Pesquisas Aggeu Magalhães. Fundação Oswaldo Cruz; 2009 Recife. 3939. Kox LFF, Rhienthong D, Miranda AM, Udomsantisuk N, Ellis K, van-Leeuwen J et al. A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples. J Clin Microbiol1999; 32:672-678. 4040. Portillo-Gómez L, Morris SL, Panduro A. Rapid and efficient detection of extrapulmonary Mycobacterium tuberculosis by PCR analysis. Int J Tuberc Lung Dis 2000; 4:361-370., implying that STNPCR can detect M. tuberculosis in samples containing only a single or even fragments of a cell 1414. Lima JFC. Detecção do Mycobacterium tuberculosis em amostras de sangue e urina através da Nested-PCR em único tubo. 2009. 111p. (Master's Dissertation). Centro de Pesquisas Aggeu Magalhães. Fundação Oswaldo Cruz; 2009 Recife.. It may therefore be useful in cases with no bacteriological confirmation, which would eliminate the possibility of patients receiving nonspecific treatments. Around 30% of suspected TB cases are treated in an empirical manner without bacteriological confirmation based on a set of clinical, epidemiological, and laboratory criteria or on radiological evaluation 4141. Mello FCQ, Bastos LGV, Soares SLM, Rezende VM, Conde MB, Chaisson RE et al. Predicting smear negative pulmonary tuberculosis with classification trees and logistic regression: a cross-sectional study. BMC Public Health 2006; 6:1-8..

The main limitation of this study was the reference test that was used; it was neither the gold standard culture method, which has high sensitivity and specificity (10-100 bacilli/ml is considered as positive, but the test requires 3 to 8 weeks to obtain a final result 2222. Silva Jr JB. Tuberculose: Guia de Vigilância Epidemiológica. J Bras Pneumol (online). 2004; 30:S57-S86. 2323. American Thoracic Society. Diagnostic Standards and Classification of Tuberculosis in adults and children. Am J Respir Crit Care Med 2000; 161:1376-1395., nor the AFB test, which is rapid and low-cost but less sensitive (> 5,000 bacilli/mL is considered as positive 55. Moure R, Muñoz L, Torres M, Santin M, Martín R, Alcaide F. Rapid Detection of Mycobacterium tuberculosis Complex and Rifampin Resistance in Smear-Negative Clinical Samples by Use of an Integrated Real-Time PCR Method. J Clin Microbiol 2011; 49:1137-1139. 4242. Khan MA, Mirza SH, Abbasi SA, Butt T, Anwar M. Peripheral blood-based Polymerase Chain Reaction in diagnosis of pulmonary tuberculosis. J Ayub Med Coll Abbottabad 2001; 18:25-28.. Given that patients were paucibacillary, the reference test was based on an set of criteria and not one test 4343. Lin LI. Assay Validation Using the Concordance Correlation Coefficient. Biometrics 1992; 48:599-604.; using this approach, the sensitivity of detecting M. tuberculosis IS6110 in blood was greater than that of gold standard methods 1212. Rebollo MJ, Garrido RSJ, Folgueira D, Palenque E, Díaz-Pedroche C, Lumbreras C et al. Blood and urine samples as useful sources for direct detection of tuberculosis by polymerase chain reaction. Diag Microbiol Infect Dis 2006; 56:141-146. 3939. Kox LFF, Rhienthong D, Miranda AM, Udomsantisuk N, Ellis K, van-Leeuwen J et al. A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples. J Clin Microbiol1999; 32:672-678., suggesting that it is reliable for confirming the presence of TB.

Tuberculosis is endemic in the Recife region 4444. Ministério da Saúde (MS). Controle do Brasil Bol Epidemiol Ministério da Saúde 2014; 44:1-13., and without bacteriological confirmation the status of excluded TB cases is unclear. Patients classified as non-TB were diagnosed by a physician based solely on clinical suspicion 11. World Health Organization (WHO). Global Tuberculosis Report 2013. Genève: WHO; 2013. 289p. (Acessed 2014 May 15 th) Available at: Available at: http://www.who.int/tb/publications/global_report/en/
http://www.who.int/tb/publications/globa...
; TB was excluded if the patient did not respond to the treatment that was administered 11. World Health Organization (WHO). Global Tuberculosis Report 2013. Genève: WHO; 2013. 289p. (Acessed 2014 May 15 th) Available at: Available at: http://www.who.int/tb/publications/global_report/en/
http://www.who.int/tb/publications/globa...
. It is therefore possible that some excluded TB cases were falsely diagnosed as negative. On the other hand, in some cases the STNPCR was positive but these were classified as falsely positive, although some studies indicate that PCR can be considered the gold standard for infectious diseases owing to the high specificity 4545. Centers for Disease Control and Prevention (CDC). Guidance for Clinicians on the Use of Rapid Influenza Diagnostic Tests. v. unique. 13p. CDC; 2011. (Acessed 2014 March 13). Available at: Available at: http://www.cdc.gov/flu/pdf/professionals/diagnosis/clinician_guidance_ridt.pdf.
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4646. Centers for disease control and prevention (CDC). CDC Researchers Verify 'Nested' PCR Assay as Gold Standard for Malaria Dx. Abril 2010. (Acessed 2014 March 13). Available at: Available at: http://www.genomeweb.com/pcrsample-prep/cdc-researchers-verify-nested-pcr-assay-gold-standard-malaria-dx
http://www.genomeweb.com/pcrsample-prep/...
.

The heme group of hemoglobin in whole blood acts as an inhibitor of PCR. When leukocytes and plasma were analyzed separately, this factor was eliminated 1414. Lima JFC. Detecção do Mycobacterium tuberculosis em amostras de sangue e urina através da Nested-PCR em único tubo. 2009. 111p. (Master's Dissertation). Centro de Pesquisas Aggeu Magalhães. Fundação Oswaldo Cruz; 2009 Recife. 4747. An SF, Fleming KA. Removal of inhibitors of the polymerase chain reaction from formalin fixed, paraffin-wax embedded tissues. J Clin Pathol 1991; 44:924-927. 4848. Barea JA, Pardini MIMC, Gushiken T. Extração de DNA de materiais de arquivo e fontes escassas para utilização em reação de polimerização em cadeia (PCR). Rev Bras Hematol Hemoter 2004; 26:274-281.. In these samples, STNPCR had higher sensitivity than conventional nested PCR using whole blood 77. Lima JFC, Montenegro LML, Montenegro RA, Cabral MML, Lima AS, Abath FGC et al. Performance of nested PCR in the specific detection of Mycobacterium tuberculosis complex in blood samples of pediatric patients. J Pneumol 2009; 35:690-697. 1313. Cruz HLA, Montenegro RA, Lima JFA, Poroca DR, Lima JFC, Montenegro LML et al. Evaluation of a Nested-PCR for Mycobacterium tuberculosis detection in blood and urine samples. Braz J Microbiol 2011; 42:321-329. 3939. Kox LFF, Rhienthong D, Miranda AM, Udomsantisuk N, Ellis K, van-Leeuwen J et al. A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples. J Clin Microbiol1999; 32:672-678.. The separation of blood into leukocytes and plasma as well as different primer sequences 2929. Ritis K, Tzoanopoulos D, Speletas M, Papadopoulos E, Arvanitidis K, Kartali S et al. Amplification of IS6110 sequence for detection of Mycobacterium tuberculosis complex in HIV-negative patients with fever of unknown origin (FUO) and evidence of extrapulmonary disease. J Intern Med 2000; 248:415-424.may improve the results of STNPCR 77. Lima JFC, Montenegro LML, Montenegro RA, Cabral MML, Lima AS, Abath FGC et al. Performance of nested PCR in the specific detection of Mycobacterium tuberculosis complex in blood samples of pediatric patients. J Pneumol 2009; 35:690-697. 88. Silva MAL, Soares CRP, Medeiros RA, Medeiros Z, Melo FL. Optimization of single-tube nested PCR for the diagnosis of visceral leishmaniasis. Exp Parasitol 2013; 134:206-210. 1313. Cruz HLA, Montenegro RA, Lima JFA, Poroca DR, Lima JFC, Montenegro LML et al. Evaluation of a Nested-PCR for Mycobacterium tuberculosis detection in blood and urine samples. Braz J Microbiol 2011; 42:321-329..

We analyzed the sensitivity and specificity of STNPCR for each sample regardless of the clinical form of TB ( Table 4). We concluded that the sensitivity of STNPCR was higher for detection of M. tuberculosis in plasma and leukocytes as compared to plasma alone. The sensitivity in blood samples in the present study was higher than the previously reported value 1313. Cruz HLA, Montenegro RA, Lima JFA, Poroca DR, Lima JFC, Montenegro LML et al. Evaluation of a Nested-PCR for Mycobacterium tuberculosis detection in blood and urine samples. Braz J Microbiol 2011; 42:321-329., indicating that it is important to analyze patient plasma instead of leukocytes only.

Despite the low sensitivity of STNPCR in urine samples, when these were analyzed in parallel with blood samples, sensitivity in the latter was increased by 8%. This is consistent with another study that found that analysis of urine samples increased detection sensitivity in blood samples by 10% 1212. Rebollo MJ, Garrido RSJ, Folgueira D, Palenque E, Díaz-Pedroche C, Lumbreras C et al. Blood and urine samples as useful sources for direct detection of tuberculosis by polymerase chain reaction. Diag Microbiol Infect Dis 2006; 56:141-146.. For this reason, the use of urine samples in PCR is recommended for TB diagnosis, especially for extrapulmonary forms or when using extrapulmonary samples 3939. Kox LFF, Rhienthong D, Miranda AM, Udomsantisuk N, Ellis K, van-Leeuwen J et al. A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples. J Clin Microbiol1999; 32:672-678. 4949. Myeong-Hee K, Yang HY, Suh JT, Lee HJ. Comparison of In-house PCR with conventional techniques and Cobas Amplicor M. tuberculosis TM kit for detection of Mycobacterium tuberculosis . Yonsei Med J 2008; 49:537-544.. The use of more than one clinical sample from the same patient enhances sensitivity 1212. Rebollo MJ, Garrido RSJ, Folgueira D, Palenque E, Díaz-Pedroche C, Lumbreras C et al. Blood and urine samples as useful sources for direct detection of tuberculosis by polymerase chain reaction. Diag Microbiol Infect Dis 2006; 56:141-146.since it increases the probability of detecting DNA circulating in body fluids 1010. Torrea G, Perre PV, Ouedraogo M, Zougba A, Sawadogo A, Dingtoumda B et al. PCR-based detection of the Mycobacterium tuberculosis complex in urine of HIV-infected and uninfected pulmonary and extrapulmonary tuberculosis patients in Burkina Faso. J Med Microbiol 2005; 54:39-44.. Another advantage of the STNPCR system is that it can be applied to blood and urine regardless of the infection site and clinical form of TB.

As for AFB and culture methods, the main difficulty in analyzing extrapulmonary TB samples by STNPCR is that they are paucibacillary 3939. Kox LFF, Rhienthong D, Miranda AM, Udomsantisuk N, Ellis K, van-Leeuwen J et al. A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples. J Clin Microbiol1999; 32:672-678.. PCR has been proposed as the method of choice for diagnosing TB when AFB results are negative and infection or disease is strongly suspected 4040. Portillo-Gómez L, Morris SL, Panduro A. Rapid and efficient detection of extrapulmonary Mycobacterium tuberculosis by PCR analysis. Int J Tuberc Lung Dis 2000; 4:361-370.. However, GeneXpert MTB/RIF has been suggested as being more accurate in detecting TB in smear-negative cases 5050. Theron G, Peter J, Meldau R, Khalfey H, Gina P, Matinyena B et al. Accuracy and impact of Xpert MTB/RIF for the diagnosis of smear-negative or sputum-scarce tuberculosis using bronchoalveolar lavage fluid. Thorax 2013; 68:1043-1051.. For positive AFB cases, PCR may still be useful in determining whether or not bacilli of the M. tuberculosis complex are present 3838. Piatek AS, Cleef MV, Alexander H, Coggin WL, Rehr M, Kampen SV et al. GeneXpert for TB diagnosis: planned and purposeful implementation. Glob Health Sci Pract2013; 1:18-23.. Nonetheless, STNPCR should only be used as an auxiliary tool for diagnosing TB since it cannot distinguish between active and latent forms of the disease 3939. Kox LFF, Rhienthong D, Miranda AM, Udomsantisuk N, Ellis K, van-Leeuwen J et al. A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples. J Clin Microbiol1999; 32:672-678..

In almost all cases where AFB was negative or spontaneous expectoration was not tested, STNPCR using blood and urine samples helped to establish a diagnosis of TB by detecting M. tuberculosis . As such, STNPCR can be useful for confirming the disease in cases that would not otherwise be diagnosed or that would be submitted to therapeutic trial. This system also has the advantages of rapidity and high specificity and sensitivity as compared to AFB and culture methods 1414. Lima JFC. Detecção do Mycobacterium tuberculosis em amostras de sangue e urina através da Nested-PCR em único tubo. 2009. 111p. (Master's Dissertation). Centro de Pesquisas Aggeu Magalhães. Fundação Oswaldo Cruz; 2009 Recife. 5151. Abadco DL, Steiner P. Gastric lavage is better than bronchoalveolar lavage for isolation of Mycobacterium tuberculosis in childhood pulmonary tuberculosis. Pediatr Infect Dis J 1992; 11:735-738..

ACKNOWLEDGMENTS

The authors thank to Bruno César Silva and Marta Maciel Lyra for clinical assistance; Carlos Luna for statistical support; and Regina Bressan for input in the writing of the manuscript

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  • This work was supported by the International Clinical, Operational, and Health Services Research and Training Award Program (NIH #U2RTW006885 and #5U2RTW006883-02 AI066994); Ministério da Ciência e Tecnologia/CNPq 15/2007
  • Centro de Pesquisas Aggeu Magalhães/Fundação Oswaldo Cruz ; Programa de Desenvolvimento Tecnológico em Insumos para Saúde/ FIOCRUZ (RID-08); Projetco Universal and Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco /PP-SUS

Data availability

Data citations

Ministério da Saúde (MS). Sistema de Informação de Agravos de Notificação. DATASUS. Brasília: Ministério da Saúde; 2014. (Acessed 2015 Jul 12 th). Available at: Available at: http://dtr2004.saude.gov.br/sinanweb/tabnet/tabnet?sinannet/tuberculose/bases/tubercbrnet.def.

Publication Dates

  • Publication in this collection
    Dec 2015

History

  • Received
    17 Aug 2015
  • Accepted
    10 Nov 2015
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