- Email: [email protected]
The limitations of polymerase chain reaction in the setting of possible recurrent tuberculosis: 2 instructional cases
Available online at www.sciencedirect.com
Diagnostic Microbiology and Infectious Disease 68 (2010) 293 – 296 www.elsevier.com/locate/diagmicrobio
Case Reports
The limitations of polymerase chain reaction in the setting of possible recurrent tuberculosis: 2 instructional cases Thomas R. Schulz⁎, Alan C. Street, Emma S. McBryde Victorian Infectious Diseases Service, Royal Melbourne Hospital, Victoria 30502, Australia Received 24 April 2010; accepted 30 July 2010
Abstract The interpretation of a positive result for Mycobacterium tuberculosis by nucleic acid amplification such as polymerase chain reaction (PCR) can be challenging. We present 2 cases that illustrate the limitations of tuberculosis PCR on respiratory secretions in previously treated patients, even years after the previous disease episode. Crown Copyright © 2010 Published by Elsevier Inc. All rights reserved. Keywords: Tuberculosis; Nucleic Acid Testing; Polymerase Chain Reaction; False positive
1. Illustrative cases 1.1. Case 1 The patient was a 62-year-old indigenous woman with a history of chronic rheumatoid arthritis treated with weekly methotrexate, who smoked 20 to 30 cigarettes per day. She had previously been treated for pulmonary tuberculosis (TB) on 2 occasions. The first episode was in the 1960s, at which time treatment included 5 months of injections followed by oral medication, and resulted in a good clinical response. (Treatment details are incomplete for this episode.) The second episode was 2 years previously. She was treated for fully sensitive pulmonary TB with rifampicin (R), isoniazid (H), pyrazinamide (Z), and ethambutol (E) for 2 months, followed by HR for a further 7 months. Therapy was successfully completed 14 months before the current episode. On this occasion, she presented with a 4-day history of fever, cough, dyspnea, and left-sided chest pain. A chest radiograph showed left mid and upper zone opacification. Initial routine cultures, including mycobacterial and fungal culture of blood and sputum, were negative. Acidfast bacilli (AFB) smears of 2 sputum specimens and
urinary pneumococcal and Legionella antigen test results were also negative. She remained febrile with increasing opacification on chest radiograph and underwent a bronchoscopy. Bronchial washings were AFB smear and culture negative but positive by TB polymerase chain reaction (PCR). Three subsequent sputum specimens were also PCR positive but AFB smear and culture negative (Table 1). All other microbiologic studies, including PCR for respiratory viruses and Pneumocystis jiroveci pneumonia, were negative. Chest computed tomography (CT) showed 3 areas of peripheral nodular consolidation, one of which showed cavitation and several showed calcified mediastinal nodes and right upper lobe bronchiectasis. A CT-guided biopsy of the pleurally based lesion revealed nonspecific mild interstitial inflammation and fibrosis, with no granulomas or necrosis. The patient had good clinical improvement with 5 days of conventional antibiotics. She was reviewed 5 weeks after discharge and was well, and her chest radiograph showed almost complete resolution of the previously noted acute changes. 1.2. Case 2
⁎ Corresponding author. Tel.: +61-613-9342-7212; fax: +61-613-93427277. E-mail address: [email protected] (T.R. Schulz).
The second patient was an 89-year-old lady living independently and in good health. Her medical history included diabetes, peripheral vascular disease, myocardial
0732-8893/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2010.07.022
294
T.R. Schulz et al. / Diagnostic Microbiology and Infectious Disease 68 (2010) 293–296
Table 1 Summary of investigations for TB during admission in case 1 Day
Sample
Smear Result
2 3 3 4 5 12 13 14 17
Sputum Sputum Blood cultures: 2 sets Sputum Bronchoscopy Sputum Sputum Sputum Lung tissue
No AFB No AFB
a
No AFB No AFB No AFB No AFB No AFB No AFB
PCR resulta
Positive Positive Positive Positive Insufficient sample
Culture result Negative Negative Negative Negative Negative Negative Negative Negative Negative
PCR using TaqMan with IS6110 target (Globan and Fyfe, 2010).
infarction, stroke, and pulmonary hypertension. She previously had breast cancer and a retinal vein thrombosis. Twenty-eight months before the current presentation, she had been diagnosed with fully drug-susceptible, smearpositive pulmonary TB. She was treated with 2 months of HRZ followed by 4 months of HR and made a full clinical recovery. Nine months later, she presented with a productive cough. Sputum was AFB smear positive and TB PCR positive, but AFB cultures were negative. A CT scan showed a right lower lobe lung mass with surrounding nodules. Despite the negative sputum cultures, she was retreated for TB with 6 months of HRZE (the pyrazinamide was ceased after 2 months). The right lung lesion was unchanged on a repeat CT scan after completion of therapy. A positron emission tomography scan did not show any increased uptake in the corresponding area of the lung. Seven months later, she presented with an acute upper respiratory illness on the background of ongoing chronic cough. Three sputum specimens were smear positive for AFB, 2 of which were also TB PCR positive (Table 2). Chest CT scan was unchanged from 13 and 7 months previously. A repeat sputum sample was smear and PCR negative for TB.
Table 2 Summary of investigations for TB in case 2 Date
Sample
Smear result
PCR result
TB culture result
7/9/2006 8/9/2006
Sputum Sputum
No AFB Positive
Positive
Negative Positive
6/12/2007 6/12/2007 6/12/2007 15/12/2007 17/12/2007 18/12/2007
Sputum Sputum Sputum Sputum Sputum Sputum
No AFB Positive No AFB
Positive
Negative Negative Negative
29/12/2008 30/12/2008 31/12/2008 13/1/2009 3/2/2009
Sputum Sputum Sputum Sputum Sputum
Positive Positive Positive No AFB No AFB
Positive Positive Positive
Negative Negative Negative Positive Positive Negative
Negative Negative Negative Negative Negative
Although she had already recovered from her respiratory illness, she was commenced on 4-drug antituberculous therapy with HRZ and moxifloxacin. At 2 months of review, all 3 sputum cultures were negative, and so all antituberculous therapy was discontinued. A further 5 months later, she remains well and continues to live independently at home.
2. Discussion TB diagnosis has traditionally been made on the basis of clinical presentation, radiograph findings, microscopy, and culture. Nucleic acid amplification tests (NAAT) are now used widely as an additional diagnostic test. Recently, the US Centers for Disease Control and Prevention (2009) has recommended that PCR testing is performed on at least 1 respiratory specimen from each patient for whom TB is being considered. TB PCR has greater sensitivity and specificity than microscopy but is not as sensitive as culture for the detection of pulmonary TB (Catanzaro et al., 2000; Wobeser et al., 1996). In a meta-analysis of various studies, sensitivity and specificity of TB PCR in smear-negative pulmonary disease are 66% and 98%, respectively, and in smear-positive pulmonary disease, sensitivity is 96% and specificity only 85% (Greco et al., 2006). Organism detection by PCR requires the presence of preserved genetic material, but a positive result does not necessarily indicate that the organism is viable. In patients with untreated disease, a positive PCR can generally be assumed to indicate that viable Mycobacterium tuberculosis is present. After treatment is started, PCR may remain positive in the absence of viable bacteria, as judged by conversion of cultures to negative (Yuen et al., 1993). The length of time that the PCR remains positive is not clearly defined, and the role of PCR in patients being investigated for possible TB recurrence is poorly understood. These 2 patients had both been treated for TB on 2 previous occasions. They then presented with respiratory illnesses, compatible in case 1 with acute communityacquired bacterial pneumonia and in case 2 with an upper respiratory tract infection. In both cases, the subsequent clinical courses were in keeping with these diagnoses, although no laboratory confirmation was achieved. The dilemma that arose on both occasions was how to interpret a repeatedly positive sputum TB PCR results when there was a clear discordance between this finding and the patient's clinical presentation. This dilemma was heightened in case 2, who was also sputum AFB smear positive. In both cases, the final decision was to accept that these PCR results are due to nonviable bacilli. The TB PCR used (Globan and Fyfe, 2010) is specific for the M. tuberculosis complex of organisms; however, because M. tuberculosis had been cultured during prior episodes and because no alternative species was ever cultured, it would be reasonable to assume this was the organism detected by PCR.
T.R. Schulz et al. / Diagnostic Microbiology and Infectious Disease 68 (2010) 293–296
False-positive TB PCR results are generally thought to be due to contamination within the laboratory (Greco et al., 2006; Laraque et al., 2009). It is also recognized that TB PCR can be falsely positive in patients who are on treatment (Greco et al., 2006). NAAT have an expanding role in reducing time to diagnosis and the need for respiratory isolation (Campos et al., 2008). Early correct diagnosis also allows for more directed contact tracing. NAATs are not sensitive enough to replace conventional culture-based diagnostic tests (Ling et al., 2008), and the latter are also necessary for standard drug sensitivity testing. Literature reports of sputum TB PCR remaining positive following adequately treated TB are very limited (Schluger et al., 1994). Carpentier et al. (1995) described that 1 patient was sputum TB PCR positive 2 years after diagnosis and treatment completion. The patient had no symptoms to suggest recurrence. Genotyping was not done to compare pre- and posttreatment specimens. Wobeser et al. (1996) examined 16 patients who were culture negative and PCR positive and found 2 patients PCR positive after 26 weeks of treatment and 1 noncompliant patient with HIV who was positive 130 weeks after treatment commencement. Lim et al. (2000) describe 3 patients with TB, 20, 8, and 5 years previously who were smear negative and PCR positive but culture negative. Yuen et al. (1997) examined the role of PCR for monitoring clinical response and found 30% of patients remained positive after 6 months of treatment. This did not correlate with the clinical response but had a positive correlation with comorbidities, residual radiologic change, and relapse. Although culture is considered to be the gold standard for TB diagnosis, the possibility that the culture was falsely negative must be considered. In neither of the cases, however, was the clinical picture suggestive of ongoing active TB. Most cases that are PCR positive but culture negative are also smear negative. Occasional PCR positive cases are smear positive and culture negative but, to our knowledge, only during therapy (Vlaspolder et al., 1995; Wobeser et al., 1996; Yuen et al., 1993). Case 2 is particularly notable because smears and PCR remained positive after completion of treatment. The mechanism responsible for ongoing detection of TB DNA in the face of negative cultures is unknown. We postulate that in both patients, an unrelated bacterial or viral infection led to disruption of lung architecture and mobilization of previously encased nonviable bacilli. Notably, both of the patients had abnormal underlying lungs due to prior comorbid illness. The phenomenon of a mycobacterial PCR that remains positive even after an organism is no longer viable has been demonstrated in other disease states. Studies of Mycobacterium ulcerans suggest that DNA is persistently detected even in successfully treated lesions (Herbinger et al., 2009).
295
Table 3 Summary of recommendations for use of PCR in suspected pulmonary TB Smear result
Treatment started?
PCR indicated?
Positive
Yes No
Negative
Yes No
Only if planning to stop treatment if negative Yes if high rates of nontuberculous mycobacterium Only if planning to stop treatment if negative Yes
Use caution when interpreting PCR in previously treated patients.
In smear-positive patients, TB PCR clearly has a role when nontuberculous mycobacterial species are a significant possibility. A negative PCR allows the cessation of treatment and avoids the need for unnecessary patient isolation and tracing of contacts (Laraque et al., 2009). In smear-negative patients, physician surveys have indicated that a positive PCR would prompt treatment if this had not already been commenced, whereas a negative PCR following commencement of treatment would not lead to treatment cessation. This would suggest that if treatment has already started on the basis of a clinician's judgment as to the likelihood of TB, a PCR is of little value, because it will not change management (Conaty et al., 2005; Guerra et al., 2007). TB PCR has been shown to be highly specific in extrapulmonary TB but with poor sensitivity (Pai et al., 2003, 2004), and similarly, results did not significantly affect clinical decisions (Weiner et al., 2005). The use of reverse transcription PCR to quantify TB messenger RNA has been used as a marker of bacterial replication and is more closely correlated with progression to culture negativity. In a small study, this was a good marker of early bactericidal activity, but this was at the expense of sensitivity. One patient who had initially developed a negative result at 30 days later relapsed (Desjardin et al., 1999). In our 2 cases, a positive result may have been a useful marker of the presence of live bacilli, but a negative outcome would not have excluded pulmonary TB. In summary, TB PCR is very useful in previously untreated smear-negative patients. When the sputum smear is positive and infection with a nontuberculous mycobacterial species is considered a possibility, TB PCR has a definite role. For nonrespiratory samples, caution should be used when interpreting negative results because sensitivity is poor. The TB PCR is not of value if the level of clinical suspicion means the result will not change management. Our 2 cases illustrate that positive results should be interpreted in light of the clinical context, in patients who have commenced or been previously treated for TB. Our recommendations are summarized in Table 3. Acknowledgments The authors thank Drs David Leslie and Maria Globan (Victorian Infectious Diseases Reference Laboratory).
296
T.R. Schulz et al. / Diagnostic Microbiology and Infectious Disease 68 (2010) 293–296
References Campos M, Quartin A, Mendes E, Abreu A, Gurevich S, Echarte L, Ferreira T, Cleary T, Hollender E, Ashkin D, Campos M, Quartin A, Mendes E, Abreu A, Gurevich S, Echarte L, Ferreira T, Cleary T, Hollender E, Ashkin D (2008) Feasibility of shortening respiratory isolation with a single sputum nucleic acid amplification test. Am J Respir Crit Care Med 178:300–305. Carpentier E, Drouillard B, Dailloux M, Moinard D, Vallee E, Dutilh B (1995) Diagnosis of tuberculosis by Amplicor Mycobacterium tuberculosis test; a multicentre study. J Clin Microbiol 33:3106–3110. Catanzaro A, Perry S, Clarridge J (2000) The role of clinical suspicion in evaluating a new diagnostic test for active tuberculosis: results of a multicentre prospective trial. JAMA 283:639–645. Centers for Disease Control and Prevention (2009) Updated guidelines for the use of nucleic acid amplification tests in the diagnosis of tuberculosis. MMWR - Morb Mortal Wkly Rep 58:7–10. Conaty S, Claxton A, Enoch D, Hayward A, Lipman M, Gillespie S (2005) The interpretation of nucleic acid amplification tests for tuberculosis: do rapid tests change treatment decisions? J Infect 50:187–192. Desjardin L, Perkins M, Wolski K, Haun S, Teixeira L, Chen Y (1999) Measurement of sputum Mycobacterium tuberculosis messenger RNA as a surrogate for response to chemotherapy. Am J Respir Crit Care Med 160:203–210. Globan M, Fyfe J (2010) Mycobacterium tuberculosis complex. In: PCR for clinical microbiology: an Australian and international perspective. Schuller M, Sloots TP, James GS, Halliday CL, Carter IWJ, Eds. Dordrecht, Heidelberg, London, New York: Springer, pp. 165–170. Greco S, Girardi E, Navarra A, Saltini C (2006) Current evidence on diagnostic accuracy of commercially based nucleic acid amplification tests for the diagnosis of pulmonary tuberculosis. Thorax 61:783–790. Guerra RL, Hooper NM, Baker JF, Alborz R, Armstrong DT, Maltas G, Kiehlbauch JA, Dorman SE, Guerra RL, Hooper NM, Baker JF, Alborz R, Armstrong DT, Maltas G, Kiehlbauch JA, Dorman SE (2007) Use of the amplified mycobacterium tuberculosis direct test in a public health laboratory: test performance and impact on clinical care. Chest 132: 946–951. Herbinger KH, Adjei O, Awua-Boateng NY, Nienhuis WA, Kunaa L, Siegmund V, Nitschke J, Thompson W, Klutse E, Agbenorku P, Schipf A, Reu S, Racz P, Fleischer B, Beissner M, Fleischmann E, Helfrich K,
van der Werf TS, Löscher T, Bretzel G (2009) Comparative study of the sensitivity of different diagnostic methods for the laboratory diagnosis of Buruli ulcer disease. Clin Infect Dis 48:1055–1064. Laraque F, Griggs A, Slopen M, Munsiff SS (2009) Performance of nucleic acid amplification tests for diagnosis of tuberculosis in a large urban setting. Clin Infect Dis 49:46–54. Lim TK, Gough A, Chin NK, Kumarasinghe G (2000) Relationship between estimated pretest probability and accuracy of automated Mycobacterium tuberculosis assay in smear-negative pulmonary tuberculosis. Chest 118:641–647 [see comment]. Ling DI, Flores LL, Riley LW, Pai M, Ling DI, Flores LL, Riley LW, Pai M (2008) Commercial nucleic-acid amplification tests for diagnosis of pulmonary tuberculosis in respiratory specimens: meta-analysis and meta-regression. PLoS ONE [Electronic Resource] 3:e1536. Pai M, Flores LL, Pai N, Hubbard A, Riley LW, Colford Jr JM (2003) Diagnostic accuracy of nucleic acid amplification tests for tuberculous meningitis: a systematic review and meta-analysis. Lancet Infect Dis 3: 633–643 [see comment]. Pai M, Flores LL, Hubbard A, Riley LW, Colford Jr JM, Pai M, Flores LL, Hubbard A, Riley LW, Colford Jr JM (2004) Nucleic acid amplification tests in the diagnosis of tuberculous pleuritis: a systematic review and meta-analysis. BMC Infect Dis 4:6. Schluger NW, Kinney D, Harkin TJ, Rom WN (1994) Clinical utility of the polymerase chain reaction in the diagnosis of infections due to Mycobacterium tuberculosis. Chest 105:1116–1121. Vlaspolder F, Singer P, Roggeveen C (1995) Diagnostic value of an amplification method (Gen-Probe) compared with that of culture for diagnosis of tuberculosis. J Clin Microbiol 33:2699–2703. Weiner R, Della-Latta P, Schluger N (2005) Effect of nucleic acid amplification for Mycobacterium tuberculosis on clinical decision making in suspected extrapulmonary tuberculosis. Chest 128:102–107. Wobeser W, Krajden M, Conly J, Simpson H, Yim B, D'Costa M (1996) Evaluation of Roche Amplicor PCR assay for Mycobacterium tuberculosis. J Clin Microbiol 34:134–139. Yuen K, Chan K, Chan C, Ho B, Dai L, Chau P (1993) Use of PCR in routine diagnosis of treated and untreated pulmonary tuberculosis. J Clin Pathol 46:318–322. Yuen K, Chan K, Chan C, Ho P, Ng M (1997) Monitoring the therapy of pulmonary tuberculosis by nested polymerase chain reaction assay. J Infect 34:29–33.
Diagnostic Microbiology and Infectious Disease 68 (2010) 293 – 296 www.elsevier.com/locate/diagmicrobio
Case Reports
The limitations of polymerase chain reaction in the setting of possible recurrent tuberculosis: 2 instructional cases Thomas R. Schulz⁎, Alan C. Street, Emma S. McBryde Victorian Infectious Diseases Service, Royal Melbourne Hospital, Victoria 30502, Australia Received 24 April 2010; accepted 30 July 2010
Abstract The interpretation of a positive result for Mycobacterium tuberculosis by nucleic acid amplification such as polymerase chain reaction (PCR) can be challenging. We present 2 cases that illustrate the limitations of tuberculosis PCR on respiratory secretions in previously treated patients, even years after the previous disease episode. Crown Copyright © 2010 Published by Elsevier Inc. All rights reserved. Keywords: Tuberculosis; Nucleic Acid Testing; Polymerase Chain Reaction; False positive
1. Illustrative cases 1.1. Case 1 The patient was a 62-year-old indigenous woman with a history of chronic rheumatoid arthritis treated with weekly methotrexate, who smoked 20 to 30 cigarettes per day. She had previously been treated for pulmonary tuberculosis (TB) on 2 occasions. The first episode was in the 1960s, at which time treatment included 5 months of injections followed by oral medication, and resulted in a good clinical response. (Treatment details are incomplete for this episode.) The second episode was 2 years previously. She was treated for fully sensitive pulmonary TB with rifampicin (R), isoniazid (H), pyrazinamide (Z), and ethambutol (E) for 2 months, followed by HR for a further 7 months. Therapy was successfully completed 14 months before the current episode. On this occasion, she presented with a 4-day history of fever, cough, dyspnea, and left-sided chest pain. A chest radiograph showed left mid and upper zone opacification. Initial routine cultures, including mycobacterial and fungal culture of blood and sputum, were negative. Acidfast bacilli (AFB) smears of 2 sputum specimens and
urinary pneumococcal and Legionella antigen test results were also negative. She remained febrile with increasing opacification on chest radiograph and underwent a bronchoscopy. Bronchial washings were AFB smear and culture negative but positive by TB polymerase chain reaction (PCR). Three subsequent sputum specimens were also PCR positive but AFB smear and culture negative (Table 1). All other microbiologic studies, including PCR for respiratory viruses and Pneumocystis jiroveci pneumonia, were negative. Chest computed tomography (CT) showed 3 areas of peripheral nodular consolidation, one of which showed cavitation and several showed calcified mediastinal nodes and right upper lobe bronchiectasis. A CT-guided biopsy of the pleurally based lesion revealed nonspecific mild interstitial inflammation and fibrosis, with no granulomas or necrosis. The patient had good clinical improvement with 5 days of conventional antibiotics. She was reviewed 5 weeks after discharge and was well, and her chest radiograph showed almost complete resolution of the previously noted acute changes. 1.2. Case 2
⁎ Corresponding author. Tel.: +61-613-9342-7212; fax: +61-613-93427277. E-mail address: [email protected] (T.R. Schulz).
The second patient was an 89-year-old lady living independently and in good health. Her medical history included diabetes, peripheral vascular disease, myocardial
0732-8893/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2010.07.022
294
T.R. Schulz et al. / Diagnostic Microbiology and Infectious Disease 68 (2010) 293–296
Table 1 Summary of investigations for TB during admission in case 1 Day
Sample
Smear Result
2 3 3 4 5 12 13 14 17
Sputum Sputum Blood cultures: 2 sets Sputum Bronchoscopy Sputum Sputum Sputum Lung tissue
No AFB No AFB
a
No AFB No AFB No AFB No AFB No AFB No AFB
PCR resulta
Positive Positive Positive Positive Insufficient sample
Culture result Negative Negative Negative Negative Negative Negative Negative Negative Negative
PCR using TaqMan with IS6110 target (Globan and Fyfe, 2010).
infarction, stroke, and pulmonary hypertension. She previously had breast cancer and a retinal vein thrombosis. Twenty-eight months before the current presentation, she had been diagnosed with fully drug-susceptible, smearpositive pulmonary TB. She was treated with 2 months of HRZ followed by 4 months of HR and made a full clinical recovery. Nine months later, she presented with a productive cough. Sputum was AFB smear positive and TB PCR positive, but AFB cultures were negative. A CT scan showed a right lower lobe lung mass with surrounding nodules. Despite the negative sputum cultures, she was retreated for TB with 6 months of HRZE (the pyrazinamide was ceased after 2 months). The right lung lesion was unchanged on a repeat CT scan after completion of therapy. A positron emission tomography scan did not show any increased uptake in the corresponding area of the lung. Seven months later, she presented with an acute upper respiratory illness on the background of ongoing chronic cough. Three sputum specimens were smear positive for AFB, 2 of which were also TB PCR positive (Table 2). Chest CT scan was unchanged from 13 and 7 months previously. A repeat sputum sample was smear and PCR negative for TB.
Table 2 Summary of investigations for TB in case 2 Date
Sample
Smear result
PCR result
TB culture result
7/9/2006 8/9/2006
Sputum Sputum
No AFB Positive
Positive
Negative Positive
6/12/2007 6/12/2007 6/12/2007 15/12/2007 17/12/2007 18/12/2007
Sputum Sputum Sputum Sputum Sputum Sputum
No AFB Positive No AFB
Positive
Negative Negative Negative
29/12/2008 30/12/2008 31/12/2008 13/1/2009 3/2/2009
Sputum Sputum Sputum Sputum Sputum
Positive Positive Positive No AFB No AFB
Positive Positive Positive
Negative Negative Negative Positive Positive Negative
Negative Negative Negative Negative Negative
Although she had already recovered from her respiratory illness, she was commenced on 4-drug antituberculous therapy with HRZ and moxifloxacin. At 2 months of review, all 3 sputum cultures were negative, and so all antituberculous therapy was discontinued. A further 5 months later, she remains well and continues to live independently at home.
2. Discussion TB diagnosis has traditionally been made on the basis of clinical presentation, radiograph findings, microscopy, and culture. Nucleic acid amplification tests (NAAT) are now used widely as an additional diagnostic test. Recently, the US Centers for Disease Control and Prevention (2009) has recommended that PCR testing is performed on at least 1 respiratory specimen from each patient for whom TB is being considered. TB PCR has greater sensitivity and specificity than microscopy but is not as sensitive as culture for the detection of pulmonary TB (Catanzaro et al., 2000; Wobeser et al., 1996). In a meta-analysis of various studies, sensitivity and specificity of TB PCR in smear-negative pulmonary disease are 66% and 98%, respectively, and in smear-positive pulmonary disease, sensitivity is 96% and specificity only 85% (Greco et al., 2006). Organism detection by PCR requires the presence of preserved genetic material, but a positive result does not necessarily indicate that the organism is viable. In patients with untreated disease, a positive PCR can generally be assumed to indicate that viable Mycobacterium tuberculosis is present. After treatment is started, PCR may remain positive in the absence of viable bacteria, as judged by conversion of cultures to negative (Yuen et al., 1993). The length of time that the PCR remains positive is not clearly defined, and the role of PCR in patients being investigated for possible TB recurrence is poorly understood. These 2 patients had both been treated for TB on 2 previous occasions. They then presented with respiratory illnesses, compatible in case 1 with acute communityacquired bacterial pneumonia and in case 2 with an upper respiratory tract infection. In both cases, the subsequent clinical courses were in keeping with these diagnoses, although no laboratory confirmation was achieved. The dilemma that arose on both occasions was how to interpret a repeatedly positive sputum TB PCR results when there was a clear discordance between this finding and the patient's clinical presentation. This dilemma was heightened in case 2, who was also sputum AFB smear positive. In both cases, the final decision was to accept that these PCR results are due to nonviable bacilli. The TB PCR used (Globan and Fyfe, 2010) is specific for the M. tuberculosis complex of organisms; however, because M. tuberculosis had been cultured during prior episodes and because no alternative species was ever cultured, it would be reasonable to assume this was the organism detected by PCR.
T.R. Schulz et al. / Diagnostic Microbiology and Infectious Disease 68 (2010) 293–296
False-positive TB PCR results are generally thought to be due to contamination within the laboratory (Greco et al., 2006; Laraque et al., 2009). It is also recognized that TB PCR can be falsely positive in patients who are on treatment (Greco et al., 2006). NAAT have an expanding role in reducing time to diagnosis and the need for respiratory isolation (Campos et al., 2008). Early correct diagnosis also allows for more directed contact tracing. NAATs are not sensitive enough to replace conventional culture-based diagnostic tests (Ling et al., 2008), and the latter are also necessary for standard drug sensitivity testing. Literature reports of sputum TB PCR remaining positive following adequately treated TB are very limited (Schluger et al., 1994). Carpentier et al. (1995) described that 1 patient was sputum TB PCR positive 2 years after diagnosis and treatment completion. The patient had no symptoms to suggest recurrence. Genotyping was not done to compare pre- and posttreatment specimens. Wobeser et al. (1996) examined 16 patients who were culture negative and PCR positive and found 2 patients PCR positive after 26 weeks of treatment and 1 noncompliant patient with HIV who was positive 130 weeks after treatment commencement. Lim et al. (2000) describe 3 patients with TB, 20, 8, and 5 years previously who were smear negative and PCR positive but culture negative. Yuen et al. (1997) examined the role of PCR for monitoring clinical response and found 30% of patients remained positive after 6 months of treatment. This did not correlate with the clinical response but had a positive correlation with comorbidities, residual radiologic change, and relapse. Although culture is considered to be the gold standard for TB diagnosis, the possibility that the culture was falsely negative must be considered. In neither of the cases, however, was the clinical picture suggestive of ongoing active TB. Most cases that are PCR positive but culture negative are also smear negative. Occasional PCR positive cases are smear positive and culture negative but, to our knowledge, only during therapy (Vlaspolder et al., 1995; Wobeser et al., 1996; Yuen et al., 1993). Case 2 is particularly notable because smears and PCR remained positive after completion of treatment. The mechanism responsible for ongoing detection of TB DNA in the face of negative cultures is unknown. We postulate that in both patients, an unrelated bacterial or viral infection led to disruption of lung architecture and mobilization of previously encased nonviable bacilli. Notably, both of the patients had abnormal underlying lungs due to prior comorbid illness. The phenomenon of a mycobacterial PCR that remains positive even after an organism is no longer viable has been demonstrated in other disease states. Studies of Mycobacterium ulcerans suggest that DNA is persistently detected even in successfully treated lesions (Herbinger et al., 2009).
295
Table 3 Summary of recommendations for use of PCR in suspected pulmonary TB Smear result
Treatment started?
PCR indicated?
Positive
Yes No
Negative
Yes No
Only if planning to stop treatment if negative Yes if high rates of nontuberculous mycobacterium Only if planning to stop treatment if negative Yes
Use caution when interpreting PCR in previously treated patients.
In smear-positive patients, TB PCR clearly has a role when nontuberculous mycobacterial species are a significant possibility. A negative PCR allows the cessation of treatment and avoids the need for unnecessary patient isolation and tracing of contacts (Laraque et al., 2009). In smear-negative patients, physician surveys have indicated that a positive PCR would prompt treatment if this had not already been commenced, whereas a negative PCR following commencement of treatment would not lead to treatment cessation. This would suggest that if treatment has already started on the basis of a clinician's judgment as to the likelihood of TB, a PCR is of little value, because it will not change management (Conaty et al., 2005; Guerra et al., 2007). TB PCR has been shown to be highly specific in extrapulmonary TB but with poor sensitivity (Pai et al., 2003, 2004), and similarly, results did not significantly affect clinical decisions (Weiner et al., 2005). The use of reverse transcription PCR to quantify TB messenger RNA has been used as a marker of bacterial replication and is more closely correlated with progression to culture negativity. In a small study, this was a good marker of early bactericidal activity, but this was at the expense of sensitivity. One patient who had initially developed a negative result at 30 days later relapsed (Desjardin et al., 1999). In our 2 cases, a positive result may have been a useful marker of the presence of live bacilli, but a negative outcome would not have excluded pulmonary TB. In summary, TB PCR is very useful in previously untreated smear-negative patients. When the sputum smear is positive and infection with a nontuberculous mycobacterial species is considered a possibility, TB PCR has a definite role. For nonrespiratory samples, caution should be used when interpreting negative results because sensitivity is poor. The TB PCR is not of value if the level of clinical suspicion means the result will not change management. Our 2 cases illustrate that positive results should be interpreted in light of the clinical context, in patients who have commenced or been previously treated for TB. Our recommendations are summarized in Table 3. Acknowledgments The authors thank Drs David Leslie and Maria Globan (Victorian Infectious Diseases Reference Laboratory).
296
T.R. Schulz et al. / Diagnostic Microbiology and Infectious Disease 68 (2010) 293–296
References Campos M, Quartin A, Mendes E, Abreu A, Gurevich S, Echarte L, Ferreira T, Cleary T, Hollender E, Ashkin D, Campos M, Quartin A, Mendes E, Abreu A, Gurevich S, Echarte L, Ferreira T, Cleary T, Hollender E, Ashkin D (2008) Feasibility of shortening respiratory isolation with a single sputum nucleic acid amplification test. Am J Respir Crit Care Med 178:300–305. Carpentier E, Drouillard B, Dailloux M, Moinard D, Vallee E, Dutilh B (1995) Diagnosis of tuberculosis by Amplicor Mycobacterium tuberculosis test; a multicentre study. J Clin Microbiol 33:3106–3110. Catanzaro A, Perry S, Clarridge J (2000) The role of clinical suspicion in evaluating a new diagnostic test for active tuberculosis: results of a multicentre prospective trial. JAMA 283:639–645. Centers for Disease Control and Prevention (2009) Updated guidelines for the use of nucleic acid amplification tests in the diagnosis of tuberculosis. MMWR - Morb Mortal Wkly Rep 58:7–10. Conaty S, Claxton A, Enoch D, Hayward A, Lipman M, Gillespie S (2005) The interpretation of nucleic acid amplification tests for tuberculosis: do rapid tests change treatment decisions? J Infect 50:187–192. Desjardin L, Perkins M, Wolski K, Haun S, Teixeira L, Chen Y (1999) Measurement of sputum Mycobacterium tuberculosis messenger RNA as a surrogate for response to chemotherapy. Am J Respir Crit Care Med 160:203–210. Globan M, Fyfe J (2010) Mycobacterium tuberculosis complex. In: PCR for clinical microbiology: an Australian and international perspective. Schuller M, Sloots TP, James GS, Halliday CL, Carter IWJ, Eds. Dordrecht, Heidelberg, London, New York: Springer, pp. 165–170. Greco S, Girardi E, Navarra A, Saltini C (2006) Current evidence on diagnostic accuracy of commercially based nucleic acid amplification tests for the diagnosis of pulmonary tuberculosis. Thorax 61:783–790. Guerra RL, Hooper NM, Baker JF, Alborz R, Armstrong DT, Maltas G, Kiehlbauch JA, Dorman SE, Guerra RL, Hooper NM, Baker JF, Alborz R, Armstrong DT, Maltas G, Kiehlbauch JA, Dorman SE (2007) Use of the amplified mycobacterium tuberculosis direct test in a public health laboratory: test performance and impact on clinical care. Chest 132: 946–951. Herbinger KH, Adjei O, Awua-Boateng NY, Nienhuis WA, Kunaa L, Siegmund V, Nitschke J, Thompson W, Klutse E, Agbenorku P, Schipf A, Reu S, Racz P, Fleischer B, Beissner M, Fleischmann E, Helfrich K,
van der Werf TS, Löscher T, Bretzel G (2009) Comparative study of the sensitivity of different diagnostic methods for the laboratory diagnosis of Buruli ulcer disease. Clin Infect Dis 48:1055–1064. Laraque F, Griggs A, Slopen M, Munsiff SS (2009) Performance of nucleic acid amplification tests for diagnosis of tuberculosis in a large urban setting. Clin Infect Dis 49:46–54. Lim TK, Gough A, Chin NK, Kumarasinghe G (2000) Relationship between estimated pretest probability and accuracy of automated Mycobacterium tuberculosis assay in smear-negative pulmonary tuberculosis. Chest 118:641–647 [see comment]. Ling DI, Flores LL, Riley LW, Pai M, Ling DI, Flores LL, Riley LW, Pai M (2008) Commercial nucleic-acid amplification tests for diagnosis of pulmonary tuberculosis in respiratory specimens: meta-analysis and meta-regression. PLoS ONE [Electronic Resource] 3:e1536. Pai M, Flores LL, Pai N, Hubbard A, Riley LW, Colford Jr JM (2003) Diagnostic accuracy of nucleic acid amplification tests for tuberculous meningitis: a systematic review and meta-analysis. Lancet Infect Dis 3: 633–643 [see comment]. Pai M, Flores LL, Hubbard A, Riley LW, Colford Jr JM, Pai M, Flores LL, Hubbard A, Riley LW, Colford Jr JM (2004) Nucleic acid amplification tests in the diagnosis of tuberculous pleuritis: a systematic review and meta-analysis. BMC Infect Dis 4:6. Schluger NW, Kinney D, Harkin TJ, Rom WN (1994) Clinical utility of the polymerase chain reaction in the diagnosis of infections due to Mycobacterium tuberculosis. Chest 105:1116–1121. Vlaspolder F, Singer P, Roggeveen C (1995) Diagnostic value of an amplification method (Gen-Probe) compared with that of culture for diagnosis of tuberculosis. J Clin Microbiol 33:2699–2703. Weiner R, Della-Latta P, Schluger N (2005) Effect of nucleic acid amplification for Mycobacterium tuberculosis on clinical decision making in suspected extrapulmonary tuberculosis. Chest 128:102–107. Wobeser W, Krajden M, Conly J, Simpson H, Yim B, D'Costa M (1996) Evaluation of Roche Amplicor PCR assay for Mycobacterium tuberculosis. J Clin Microbiol 34:134–139. Yuen K, Chan K, Chan C, Ho B, Dai L, Chau P (1993) Use of PCR in routine diagnosis of treated and untreated pulmonary tuberculosis. J Clin Pathol 46:318–322. Yuen K, Chan K, Chan C, Ho P, Ng M (1997) Monitoring the therapy of pulmonary tuberculosis by nested polymerase chain reaction assay. J Infect 34:29–33.