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Use of the Real-time PCR Assay in Conjunction With MagNA Pure for the Detection of Mycobacterial DNA Safedin H. Beqaj, PhD, Randy Flesher, HT, BS, RT (ASCP), Gina R. Walker, HT (ASCP), estimated by the World Health Organization.1 In the Abstract: Tuberculosis in immunocompromised patients is often United States, there are an estimated 15 million people caused by Mycobacterial species other than Mycobacterium already infected with M. tuberculosis and nearly 15,000 tuberculosis. Thus, detection of and differentiation between cases of active tuberculosis disease are reported annually.2 M. tuberculosis and nontuberculosis species is necessary for Because of the risk of the spread of the disease and the diagnosis of disease in these patients. Furthermore, when tissue potential for the emergence of drug-resistant strains, changes show granulomatous inflammation, quick confirmation rapid detection of the M. tuberculosis complex is critical testing for mycobacterial infection is needed for conclusive in patients coinfected with HIV, as they are 50 times more diagnosis. The aim of this study was to validate the utility of a likely to develop disease than non-HIV–infected persons real-time polymerase chain reaction (PCR) assay in conjunction infected with M. tuberculosis.3–6 In addition, tuberculosis with the MagNA Pure LC automated extraction system for the in immunocompromised patients is often caused by detection of mycobacterial DNA from formalin-fixed, paraffin- Mycobacterial species other than M. tuberculosis such embedded specimens. A total of 46 archived, paraffin-embedded, as Mycobacterium avium and Mycobacterium intracellu- fixed specimens showing granulomatous inflammation were lare.5,7 Thus, detection of and differentiation between studied for mycobacterial infection by real-time PCR. Bacterial M. tuberculosis and nontuberculosis species is necessary DNA was extracted and isolated using the MagNA Pure for the diagnosis of disease in these patients. Further- extraction system. Real-time PCR was performed on the Light- more, when tissue changes show granulomatous inflam- Cycler using the Artus Real Art Mycob Diff ASR kit from mation and tuberculosis may not have been considered, Qiagen. Thirteen of the 46 patient specimens were positive for samples may only have been sent for histopathologic and mycobacterial infection by acid-fast bacilli (AFB) stain. Of the cytopathologic examination, quick confirmation testing is 13 reported positive by AFB stain, 12 where positive by real-time needed for conclusive diagnosis. Usually, these samples PCR. All 13 specimens reported positive by AFB were sent for are formalin-fixed and paraffin-embedded, making diag- culture confirmation. Eleven of 13 were returned positive by nosis of this disease by microbiologic examination culture. Specimens reported as negative by culture and positive by impossible. Beside the presentation of often necrotizing real-time PCR were confirmed positive by a second PCR method granulomas in these samples, conventional histopatho- from another reference laboratory. We believe that these studies logy diagnosis of mycobacterial infections is based on the are beneficial in the differential diagnosis of mycobacterial detection of acid-fast bacilli (AFB). Direct examination infection from fixed tissue specimens where tuberculosis might of AFB by microscopy is rapid but has low sensitivity.8–10 not have been clinically initially suspected and when specimens are In addition, this method lacks specificity, as it cannot not suitable for microbiologic examination.
discriminate between different species of mycobacteria.
Therefore, the development of rapid and reliable methods Key Words: mycobacterial species, tuberculosis, real-time PCR,granulomatous inflammation, mycobacterial infection, AFB for the detection of mycobacterial infection from fixed Several assays using polymerase chain reaction (PCR) are commercially available, and are mainly usedfor the culture confirmation.11–15 Recently, several groupshave reported the use of PCR methods for the detection Tuberculosis is an increasing health problem world- of M. tuberculosis from fixed tissues from patients where wide, with up to one-third of the world’s population infected with Mycobacterium tuberculosis complex as formed.10,16–18 However, most of these methods are usedfor the detection of M. tuberculosis complex, ratherthan detection of other nontuberculosis species such as From the Path Group Labs, Nashville, TN.
M. avium complex, including M. intracellulare which Reprints: Safedin H. Beqaj, PhD, Path Group Labs, 658 Grassmere are the most common mycobacterial pathogen respon- Park, Suite 101, Nashville, TN (e-mail: sbeqaj@pathgrpup.com).
Copyright r 2007 by Lippincott Williams & Wilkins sible for tuberculosis in both immunocompetent and Diagn Mol Pathol  Volume 16, Number 3, September 2007 Diagn Mol Pathol  Volume 16, Number 3, September 2007 immunocompromised patients.16,19,20 The method pre- sented in this study uses touch-down real-time PCR, 13,000 rpm. Samples were hydrated with 95% ethanol which significantly increases the sensitivity and specificity and centrifuged for 5 minutes at 13,000 rpm to collect the of the assay. Furthermore, none of the previously pellet, which was then air-dried for 10 minutes before use.
published methods have used advanced and standardized The collected tissue pellet was then treated with 130 mL automated extraction systems, which also improve the bacterial lysis buffer and 20 mL of proteinase K (Roche sensitivity and turn-around time of this assay.
Molecular) at 651C overnight or until completely disin- The aim of this study was to develop a rapid and tegrated. In addition, 6 specimens were treated with reliable assay for the detection of mycobacterial DNA, proteinase K at 651C for 3 hours to evaluate minimum including nontuberculous mycobacteria in fixed tissue time needed for specimen enzymatic digestion and specimens, which demonstrated suspicious granuloma- compared with results from the same specimen treated tous changes similar to mycobacterial infection where overnight. After the proteinase K treatment, samples were tuberculosis may not have been considered and samples incubated for 10 minutes at 951C to inactivate any may only have been sent for histopathologic and pathogenic organisms. Hundred microliters of treated specimens were used for extraction. Samples wereextracted using automated extraction procedure onMagNA Pure (Roche Molecular) previously validated in our laboratory for bacterial extraction. As this extraction system isolates and purifies extracted DNA, we did not embedded tissue specimens collected from 46 patients see any need of comparison with manual extraction from 2004 to 2006 were used for this study. Of 46 studied procedure.21,22 Extracted samples were eluted in 100 mL of specimens, there were 29 respiratory specimens, 16 lung elution buffer. To monitor the whole procedure, positive biopsies, 7 bronchial biopsies, and 1 tracheal biopsy. In and negative controls were treated the same way as addition, there were 5 more concentrated specimens patient specimens. To control for assay inhibition, 10 mL (cellblocks) with respiratory origin: 3 were bronchial internal control (IC) containing plasmid DNA was added washes, 1 was concentrated fixed sputum, and 1 was from to the lysis buffer during the extraction, and amplified concentrated pleural fluid. Eight specimens were lymph with the target from the specimen in the same PCR node biopsies (mediastinal, inguinal, and axillary), and the remaining 9 were from other organs (salivary glad, Real-time PCR was performed on the LightCycler bowel, skin, bladder, and neck mass). Of all assayed version 1.5 using RealArt Mycobact. Diff kit (Qiagen Inc, specimens, 31 specimens showed granulomatous inflam- Hamburg, Germany). This is a qualitative in vitro mation and tuberculosis was not initially suspected. These diagnostic assay, which uses probes and primers for the specimens were sent to our laboratory by the ordering detection of M. tuberculosis complex (M. tuberculosis, physician for other reasons, and in many instances, Mycobacterium africanum I and II, Mycobacterium bovis, mycobacterial disease was not listed or considered in M. bovis BCG, Mycobacterium microti, Mycobacteium clinical data provided to the pathologist. Upon micro- canettii) and also of the M. avium complex (M. avium scopic examination, however, acid fast staining was necessary for differential diagnosis in the presence of M. avium subspp. silvaticum, M. avium subspp. hominis- granulomatous inflammation. Nine specimens (4 bron- suis, and M. intracellulare). Simply, 5 mL of eluted DNA chial and 1 tracheal biopsy and also 2 bronchial washes, 1 was added to 15 mL of Master Mix containing hybridiza- concentrated pleural fluid, and 1 concentrated sputum) tion probes and primers and all PCR reaction compo- were sent for evaluation where tuberculosis was sus- pected. The remaining specimens were other thanrespiratory origin and were sent for pathology reviewfor other reasons.
One sample from a pulmonary tuberculosis block A total of 46 formalin-fixed, paraffin-embedded positive for M. tuberculosis and 3 samples containing tissue specimens from 46 patients with granulomatous extracted plasmid DNA for M. tuberculosis complex, inflammation were analyzed by real-time PCR on the M. avium complex, and M. intracellulare were used as LightCycler using RealArt Mycobact. Diff kit. Results positive controls. RNase/DNase free water and 2 skin were compared with AFB staining performed in our biopsies examined by our pathologist for skin disorders laboratory. All cases positive for AFB were sent for and previously tested negative for Mycobacterium were culture, and available culture results were also used for used as a negative control. All specimens including comparison. Of 46 tested patient specimens, 13 were controls were extracted from paraffin blocks using positive and 33 negative by AFB stain. Of 13 reported MagNA Pure LC DNA Isolation kit III (Roche positive by AFB stain, 12 were positive by real-time PCR Molecular, Pleasanton, CA). Five to ten 5-mm sections (Table 1). All 13 specimens reported positive by AFB were cut from paraffin-embedded tissue blocks and placed were sent for culture confirmation. Only 11 of 13 returned into 1.5-mL microfuge tube. Tissues were deparaffinized positive by culture. One specimen that was reported with 1 mL of xylol for 10 minutes at 651C and then positive by AFB and negative by PCR was confirmed Diagn Mol Pathol  Volume 16, Number 3, September 2007 Mycobacterial DNA by Real-time PCR From Fixed Tissue TABLE 1. Forty-six Specimens were Assayed by Real-time PCR, and Results Compared With AFB Stain and Culture *Specificity is based on comparison of negative results.
negative by culture method. The other specimen reported niosis.10 Therefore, diagnosis of nontuberculosis infection negative by culture and positive by real-time PCR was is very difficult, as it often lacks both clinical symptoms confirmed positive by PCR method from reference and specific morphologic features.4,7 Most of the time, laboratory giving higher sensitivity to real-time PCR these infections are seen in specimens collected from assay. Of 12 positive by real-time PCR, 6 were positive for patients in whom tuberculosis was not initially suspected M. tuberculosis complex and 6 were positive for M. avium and morphologic review shows the presence of granulo- complex, including 4 positive for M. intracellulare, and all matous inflammation. As microbiologic culture from matched those by culture method (Table 2). All 6 cases these formalin-fixed, paraffin-embedded tissues is impos- positive for M. tuberculosis complex were respiratory sible, in addition to AFB staining, additional confirma- specimens: 2 lung biopsies, 1 bronchial biopsy, 2 tion testing is needed.10 PCR technology has emerged as a bronchial washes, and 1 concentrated sputum. The promising tool in infectious disease testing owing to its remaining 6 positive specimens for M. avium complex high sensitivity and short turn-around time. The sensi- including intracellulare, 4 were lung biopsies, 1 specimen tivity of PCR has been shown to be better than that of was bronchial biopsy, and 1 was bowel biopsy (Table 3).
microscopic examination and better or comparable with All positives by this method showed good crossing that of culture in the detection of many infectious point or cycle threshold (Ct) ranging from 16 to 27 with a strong fluorescent signal and correct melting temperature In this study, we demonstrated the feasibility and of 63.51C for M. avium, 601C for M. tuberculosis, and reliability of real-time PCR in detection and differentia- tion of mycobacterial DNA from archived formalin-fixed, All 46 specimens investigated in this study showed a paraffin-embedded tissues. This assay uses primers and positive signal for the IC, confirming that the DNA probes that can detect M. tuberculosis complex, and extraction and amplification was successful excluding any M. avium complex including M. intracellulare. It can assay inhibition. The sensitivity and specificity of this detect all species belonging to M. tuberculosis complex assay compared with AFB was 92% and 100%, and when including M. africanum I and II, M. bovis, M. bovis BCG, compared with culture was 100% (Table 1).
M. microti, and M. canettii. This assay can detect allspecies of M. avium complex including M. avium subspp.
avium, M. avium subspp. partuberculosis, M. avium subspp. silvaticum, M. avium subspp. hominissuis, and The diagnosis of mycobacterial infection has M. intracellulare. These species have been well-known remained problematic and difficult, especially for non- pathogens in AIDS patients and immunocompromised tuberculosis mycobacterial infections.7 Disease caused by patients.3,5–7 Using this assay, we were able to detect 12 nontuberculosis mycobacteria has increased owing to the positive samples for mycobacterial DNA and successfully HIV epidemic and various immunosuppressive treat- differentiate between M. tuberculosis and nontuberculosis ments.3,5,6 The clinical approach to nontuberculosis species; 6 were positive for M. tuberculosis complex, and mycobacterial infection differs significantly from that of 6 were positive for M. avium complex, from which 4 were M. tuberculosis infection.7,23,24 However, the histopatho- positive for M. intracellulare. Most of the cases positive logic findings in infection caused by nontuberculosis for M. avium and intracellulare were respiratory speci- mycobacteria are similar to those caused by M. tubercu- mens with the exception of one that was a bowel biopsy losis. In addition, histologic features of granulomatous inflammation can be seen in other diseases such as Moreover, of 13 specimens resulted positive by AFB sarcoidosis, syphilis, leprosy, Crohn disease, rheumatoid stain, only 12 specimens were positive by real-time PCR, arthritis, systemic lupus erythematosus, and pneumoco- giving the sensitivity of this assay of 92% and specificity TABLE 2. Of 12 Positive by Real-time PCR, Only 11 Were Positive by Culture and Matched Those by Real-time PCR *M. intracellulare is part of the M. avium complex and usually is reported as part of the M. avium in culture results.
Diagn Mol Pathol  Volume 16, Number 3, September 2007 TABLE 3. Forty-six Biopsies From Different Sources Were Assayed for Mycobacterial Disease by Real-time PCR and AFB Stain Pleural fluid (1)Lymph node biopsy (8)Skin biopsy (4)Bowel biopsy (1) Bladder biopsy (1)Salivary gland bps (1)Neck mass biopsy (1)Liver biopsy (1)Lung biopsy pos. QC (1) Of the13 reported positive by AFB stain, 12 were positive by real-time PCR. Of 12 positive specimens tested by real-time PCR, 11 were of respiratory origin. Of these, 6 were positive for M. tuberculosis complex, 1 was positive for M. avium and 4 were positive for M. intracellulare. One specimen was from a bowel biopsy and it was positivefor M. avium. Two skin biopsies were used as negative controls and tested negative. One lung biopsy was used as a positive control and tested positive by both real-timePCR and AFB stain.
(number of false negative results) of 100% when cycles total), annealing temperature is dropped down for compared with this method (Table 1). As all 13 specimens 11C from 651C to 581C, improving the efficiency of primer reported positive by AFB were sent for culture confirma- annealing. This is a good way of manipulating PCR, tion, the specimen that was reported positive by AFB and which will give a good yield of the targeted sequence and negative by PCR was confirmed negative by culture, minimize amplification of undesirable products, which again demonstrating low specificity of AFB. In addition, increases sensitivity and specificity of the assay.25 we had 1 specimen reported negative by culture and Previous publications have reported26 that the positive by real-time PCR. We were fortunate that this efficiency of PCR with formalin-fixed, paraffin-embedded specimen was originally sent to reference laboratory for tissues is impaired by multiple interacting factors, PCR and was confirmed positive. Our best explanation including the type of fixation and fixation time. Although, for this is that the culture was negative because of the specimens used for this study were archived from years microorganism viability. It is well know that culture 2004, 2005, and 2006, we did not face any difficulties in depends on the viability of the organism, which might be extraction and detection of mycobacterial DNA from compromised by improper specimen collection or trans- fixed tissues. There were no false negative results (based portation. In contrast, PCR does not depend on the on comparison with AFB stain) and there was no assay viability of the microorganisms, and especially as this was inhibition as all 46 specimens investigated in this study performed from fixed tissue, where the microorganism showed a positive signal for the IC. This might be in part was preserved. In addition, all extracted specimens were because of the fact that mycobacterial DNA is not run by a home-brewed PCR assay (laboratory developed affected by formalin, as it is encapsulated in a bacterial assay)14 in an outside reference laboratory to confirm our cell wall. Furthermore, to maximize the isolation of finding (results not shown). Of 13 positives by our assay, bacterial DNA from fixed tissues, we used an automated only 9 were positive by home-brewed assay. In addition, extraction method MagNA pure that uses MagNA Pure crossing point yield by the home-brewed assay was higher LC DNA Isolation kit III, which has a capability of than those yield by our assay. Crossing point by our assay isolation and purification of bacterial DNA. This kit was between 16 to 27 cycles, whereas those by home- requires treatment of the specimen with proteinase K brewed assay ranged from 30 to 40 cycles. This is not until tissues are disintegrated, after which the bacterial surprising as RealArt Mycobact. Diff kit uses touch-down DNA is released in the solution. In addition to tissue PCR method. Touch-down PCR is used to enhance digestion, proteinase K is used to break down the amplification of desired target sequences while reducing bacterial cell wall. In this study, we used 2 different amplification from mispriming events or from other PCR timing for tissue digestion with proteinase K: overnight artifacts. In this case, PCR begins (first 10 cycles) with an and 3 hours. Both conditions showed the identical results annealing temperature, which is greater than the calcu- (results not shown) giving an assay shorter turn-around lated melting temperature (Tm) of the primer used. The elevated annealing temperature employed will encourage In conclusion, real-time PCR in conjunction with high specificity of primer annealing (increased strin- MagNA pure described in this study is rapid, sensitive, gency), although at lower efficiency. After each cycle (10 and specific for the detection of mycobacterial DNA, and Diagn Mol Pathol  Volume 16, Number 3, September 2007 Mycobacterial DNA by Real-time PCR From Fixed Tissue can be used for the early diagnosis of mycobacterial 13. Lachnik J, Ackermann B, Bohrssen A, et al. Rapid-cycle PCR infection in patients showing granulomatous inflamma- and fluorimetry for detection of mycobacteria. J Clin Microbiol.
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