Single-Tube Balanced Heminested PCR for Detecting Mycobacterium tuberculosis in Smear-Negative Samples

Albert Garca-Quintanilla, Lourdes Garcia, Griselda Tud, Maria Navarro, Juli Gonzlez and Maria T. Jimnez de Anta J. Clin. Microbiol. 2000, 38(3):1166.

Updated information and services can be found at: http://jcm.asm.org/content/38/3/1166 These include:
REFERENCES

Downloaded from http://jcm.asm.org/ on February 6, 2012 by guest

This article cites 23 articles, 19 of which can be accessed free at: http://jcm.asm.org/content/38/3/1166#ref-list-1 Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more

CONTENT ALERTS

Information about commercial reprint orders: http://jcm.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/

JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2000, p. 11661169 0095-1137/00/$04.00 0 Copyright 2000, American Society for Microbiology. All Rights Reserved.

Vol. 38, No. 3

Single-Tube Balanced Heminested PCR for Detecting Mycobacterium tuberculosis in Smear-Negative Samples
ALBERT GARCIA-QUINTANILLA,1 LOURDES GARCIA,2 GRISELDA TUDO,1 MARIA NAVARRO,1 ` JULIA GONZALEZ,3* AND MARIA T. JIMENEZ DE ANTA3 Departament de Microbiologia i Parasitologia Sanitaries, Institut dInvestigacions Biomediques Agust Pi i Sunyer ` ` (IDIBAPS), Facultat de Medicina, Universitat de Barcelona,1 and Servei de Microbiologia, Departament de Microbiologia i Parasitologia Sanitaries, IDIBAPS, Hospital Cl 3 Villarroel 170, 08036 Barcelona, and ` nic, Departament de Bioqu mica i Biologia Molecular, Facultat de Medicina, Universitat Autonoma ` de Barcelona, Campus Universitari, 08193 Bellaterra,2 Spain
Received 6 July 1999/Returned for modication 9 September 1999/Accepted 6 December 1999

Downloaded from http://jcm.asm.org/ on February 6, 2012 by guest

In order to achieve more sensitive and specic results for the rapid diagnosis of tuberculosis, we have developed a new method, named balanced heminested PCR, which avoids the inconvenience of asymmetric amplication and has the advantages of single-tube heminested PCR. This was achieved by replacing the outer primer that participates in both rounds of amplication in the standard heminested technique by another primer containing the sequence of the inner primer attached at its 5 end. When both techniques were tested for the IS6110 target of Mycobacterium tuberculosis complex in 80 smear-negative culture-positive sputum samples and 60 control samples, the results showed 100% specicity for both techniques and sensitivities of 60 and 75% for heminested PCR and balanced heminested PCR, respectively (P 0.02). In conclusion, the balanced heminested technique shows a higher sensitivity than that of the standard heminested, and it could be applied to any PCR by attaching the inner primer at the 5 end of the opposite outer primer. Thus, the balanced heminested technique provides a target for the inner primer in both strands, avoiding asymmetric amplication and thereby resulting in a more efcient amplication, and, in practice, a higher sensitivity without loss of specicity and with a minimum risk of cross-contamination. Tuberculosis (TB) is one of the most widespread, lethal infectious diseases affecting humans (23). Laboratory diagnosis is commonly based on culture and staining for acid-fast bacilli (AFB). The latter is the most rapid and economic method for detecting mycobacteria. However, given that half of the new cases of TB are smear negative, many diagnoses can not be conrmed at the time of presentation (3). This leads to delays in initiating appropriate treatment and/or the use of invasive procedures to rmly establish the diagnosis. Contrary to the general idea that AFB smear-negative patients do not contribute signicantly to the spread of infection, Behr et al. found that up to 27% of recently acquired disease in San Francisco, California, was transmitted from smear-negative cases (2). Thus, for nucleic acid-based amplication techniques to be useful in TB control programs, sensitivity and specicity must be improved when the AFB smear is negative (1, 9). Nevertheless, for achieving the best results it is necessary to optimize and combine good protocols for extraction, amplication, and detection of nucleic acids (17, 19). In this study, we focused on improving the amplication step. Thus, compared with conventional single-step PCR, nested amplication can enhance sensitivity approximately 1,000 fold but with a high risk of contamination (14). In order to completely eliminate this risk, single-tube nested PCR with a uracil-N-glycosylase (UNG)-dUTP system (15) can be performed but without the possible advantages of adding fresh enzyme or diluting inhibitors (22). Nonetheless, when the design and number of primers that can be used are limited due to the sequence of the target (the TB genome has a 65.5% G C content [8] and it is difcult to nd good primers in some regions), incompatibilities between primers and/or a large number of additional products could make the performance of a heminested PCR (HN) in one tube appropriate. However, the addition of primers at different concentrations results in an asymmetric amplication which makes the reaction less efcient. In order to increase the yield of the reaction and to improve the sensitivity of detection of the Mycobacterium tuberculosis complex in smear-negative specimens, we developed a singletube balanced HN PCR (B-HN) which avoids asymmetric amplication. This was achieved by replacing the original outer primer by another primer which also contained the sequence of the opposite inner primer attached at the 5 end. Here, for the rst time we describe this modication, which overcomes some of the disadvantages of the HN technique.
MATERIALS AND METHODS Clinical specimens. All clinical specimens were processed in the Microbiology Laboratory of the Hospital Cl nic (Barcelona, Spain). Eighty sputum samples of good quality belonging to 80 human immunodeciency virus-negative patients with pulmonary TB were studied to analyze sensitivity. All samples were positive for M. tuberculosis in culture but were AFB smear negative. Additionally, 60 culture- and stain-negative samples from human immunodeciency virus-negative control patients were included: saliva samples from 40 healthy young people (student volunteers) who were tuberculin skin test negative and sputum samples from 20 chronic obstructive lung disease patients admitted for an acute episode without clinical signs, radiological lesions, or a history of TB. All samples were decontaminated by a standard N-acetyl-L-cysteineNaOH procedure (12). The resulting pellet was resuspended in 2 ml of phosphatebuffered saline (140 mM NaCl, 2.6 mM KCl, 10.1 mM Na2HPO4, 1.7 mM KH2PO4 [pH 7.4]). Auramine staining and Lowenstein-Jensen cultures were performed. The remaining pellet was frozen at 20C until PCR processing was carried out. PCR assay. (i) Sample preparation. Aliquots of 500 l were inactivated by heating at 95C during 30 min and concentrated by centrifugation at 13,000 g for 15 min. The pellet was resuspended in 300 l of 10 Tris-EDTA solution (100 mM Tris-HCl, 10 mM EDTA [pH 8.0]) with 2 mg of lysozyme per ml and incubated at 37C for 1 h. Proteinase K and sodium dodecyl sulfate were added

* Corresponding author. Mailing address: Servei de Microbiologia, Hospital Cl nic, c/Villarroel 170, Barcelona 08036, Spain. Phone: 34932275522. Fax: 34-932275454. E-mail: jgm@medicina.ub.es. 1166

VOL. 38, 2000

NEW BALANCED PCR FOR M. TUBERCULOSIS DETECTION

1167

Downloaded from http://jcm.asm.org/ on February 6, 2012 by guest

FIG. 1. Comparison between HN and B-HN. Primers A and CA hybridize to the Y strand. Primers B and C hybridize to the X strand. (Left) During the rst round of HN, only outer primers anneal. The inner primer can not hybridize due to its low Tm. Primer A is more concentrated than B because it must also participate during the second round. This results in asymmetric amplication, since more strand X is synthesized by primer A. Primer B runs out due to its low concentration. During the second round, the annealing temperature is lower than in the rst round, and the inner primer can hybridize. There are more X strands and a higher concentration of primer C, thereby helping the synthesis of strand Y which is produced in large amounts. This results in additional bands when electrophoresis is performed. (Right) During the rst round of B-HN, only outer primers anneal. The inner primer can not hybridize due to its low Tm. The concentration of primer CA is equal to that of B, since it could be replaced by C during the second round. This results in a symmetric amplication. (Primer CA can also be more concentrated than B, which is not represented in the drawing; in this case, the rst round is similar to HN and produces asymmetric amplication, but this is balanced during the second round). Primer B runs out due to its low concentration. Primer CA provides the target for primer C in both strands. During the second round, the annealing temperature is lower than in the rst round, and the inner primer can hybridize. There is a higher concentration of primer C, thereby helping the synthesis of strand Y. C can then anneal at both strands, depending on the need to balance the output of both strands. Primer C will only anneal with the Y strand if primer CA places the target before it. This results in a more efcient reaction because single-stranded bands are not produced.

to nal concentrations of 250 g/ml and 1% (wt/vol), respectively, and incubated at 43C for 1 h. The suspension was extracted twice with phenol-chloroformisoamyl alcohol (25:24:1, vol:vol:vol) and twice with chloroform-isoamyl alcohol (24:1, vol:vol). The pellet was treated with 2 volumes of 100% ethanol and 0.2 M NaCl, stored overnight at 20C, then washed with 70% ethanol, dried, and resuspended in 100 l of distilled water. Twenty microliters was used for PCR amplication. Both HN and B-HN used the same extract. (ii) HN. HN was based on the nested PCR technique described by Kennedy et al. and Wilson et al. (10, 22), but some modications were performed in order to improve the sensitivity and specicity of the reaction. For this reason, the primer Tb670 was suppressed (J. Gonzalez, A. Garc J. Almeda, et al., Abstr. VIIth a, Reunion del Grupo Espan. de Micobacter ol., 1996; J. Gonzalez, J. Almeda, A. Garc et al., Abstr. XVIIIth Congr. Eur. Soc. Mycobacteriol., 1997). a, Amplication was performed in 0.5-ml PCR tubes with a total reaction volume of 50 l by using a 480 thermal cycler (Perkin-Elmer). Each reaction tube contained 2.5 U of Taq DNA polymerase (Gibco BRL); 0.5 U of UNG (Boehringer Mannheim); 200 M (each) dATP, dCTP, and dGTP; 600 M dUTP (Boehringer Mannheim); 1 nal buffer (20 mM Tris-HCl, 50 mM KCl [pH 8.4]); 2 mM MgCl2, and 20 l of sample. The primers used were 100 nM Tb850 (5 -TAGGCGTCGGTGACAAAGGCCACG-3 ), 1 M Tb505 (5 -ACGACCA CATCAACC-3 ), and 10 nM Tb294 (5 -GGACAACGCCGAATTGCGAAGG GC-3 ). All the primers and reagents were added at the beginning of the reaction, therefore not requiring opening of the tube to add the nested primer. These primers belong to the insertion sequence IS6110 that is present several times in M. tuberculosis complex genomes (21). To date, all strains studied in our area have IS6110 copies, thereby validating the use of this target for this study (P. Coll, personal communication).

(iii) B-HN. The reaction mixture and conditions were identical to those described above, but instead of the Tb850, the primer Tb505-850 (5 -ACGACCA CATCAACCTAGGCGTCGGTGACAAAGGCCACG-3 ) was used. Tb505850 consists of the sum of the sequences of the primers Tb505 and Tb850. We also tested primer Tb505-850 at two different concentrations: 100 nM and 10 nM. The comparison between HN and B-HN is shown in Fig. 1. (iv) PCR conditions. The conditions were the same for both methods. After 15 min at 25C to allow UNG to work, the temperature was raised to 94C for 5 min to deactivate the enzyme. The rst stage of amplication involved 30 cycles of denaturation at 94C for 45 s, with primer annealing and extension carried out in one step at 72C for 1.5 min. The second stage included 30 cycles of denaturation at 94C for 45 s, primer annealing at 55C for 1 min, and extension at 72C for 30 s, after which the reaction mixture was held at 72C in a soak le until storage at 20C. (v) Product detection. Twenty microliters of the amplied product was electrophoresed on a 2% (wt/vol) agarose gel stained with 0.5 g of ethidium bromide per ml and visualized by UV transillumination. The presence of a 369-bp band for HN and a 384-bp band for B-HN indicated successful amplication of the IS6110 target. These bands were indistinguishable on the gel. Determination of method sensitivity. To determine the theoretical sensitivity of both techniques, 10-fold serial dilutions of one McFarland standard density equivalent ( 3 108 CFU/ml) were performed with a clinical strain of M. tuberculosis followed by amplication. DNA was prepared by boiling the organisms (11). The concentration was calculated by measuring the absorbance at 260 nm (1 A260 U 50 g of double-stranded DNA per ml) and taking 5 fg of DNA as one mycobacterium equivalent (8). Statistical methods. The chi-square test was used to analyze the results. Condence intervals were calculated for 95%.

1168

GARCIA-QUINTANILLA ET AL.

J. CLIN. MICROBIOL.

Downloaded from http://jcm.asm.org/ on February 6, 2012 by guest

FIG. 2. Comparison of both methods in ten-fold serial dilutions of M. tuberculosis. A, HN; B, B-HN; M, 100-bp ladder marker. 1, bands of unknown origin, present only in B-HN, which may be due to artifacts produced by the tailed primer. This does not affect the nal sensitivity or specicity. 2, bands corresponding to double-stranded DNA produced during rst-round amplication. They are only present in concentrated dilutions. 3, bands corresponding to single-stranded DNA produced during rst-round amplication. 4, bands corresponding to the desired double-stranded nal product. 5, bands due to asymmetric amplication corresponding to single-stranded DNA. These are only present in HN samples. This detracts from sensitivity to the desired nal product. 6, bands corresponding to dimer primers. They are only present in low dilutions.

RESULTS Balanced amplication. Theoretically when both primers, Tb505-850 and Tb294, were at the same concentration (10 nM), asymmetric amplication during the rst and second stage was avoided. When the Tb505-850 concentration was increased to 100 nM, this asymmetric amplication occurred during the rst stage but not in the second. Nonetheless, the effect of asymmetric amplication on the overall sensitivity and specicity of the reaction during the rst stage was minimal; thus, we decided to run all experiments using 100 nM of Tb505-850 in order to have conditions identical to those of HN. Serial dilutions. Serial dilutions were performed to determine the end points of the techniques (Fig. 2). Both methods detected as few as 10 bacillus equivalents, although bands were stronger with B-HN, indicating a more efcient reaction. Asymmetric amplication yielded a lower band that was absent in B-HN. Patient samples. Of the 80 patients with pulmonary TB, 65 had unilateral inltrated radiological lesions, and the other 15 had cavitated and/or bilateral radiological lesions. Forty-ve samples were positive by both HN and B-HN, 3 samples were positive for HN but negative for B-HN, 15 samples were positive for B-HN but negative for HN, and 17 samples were negative for both. All control samples were negative by both techniques. The overall sensitivity was 60% for HN and 75%
TABLE 1. Results of both PCR methods
Study group No. of samples No. (%) of samples positive bya: HN B-HN

for B-HN (P 0.02) (Table 1). Table 2 shows the results according to the radiological lesions displayed by the patient group. DISCUSSION For smear-negative specimens, most published studies report a sensitivity of around 60% or even less, depending on the number of samples and experiment conditions (3, 4, 7, 9, 13, 16, 18, 20, 24). This low sensitivity for smear-negative specimens shows that current amplication assays may be unsuitable in replacing cultures for the diagnosis of tuberculosis. Our objective was to improve the sensitivity of these tests, especially with smear-negative samples. For this reason, we developed an HN method. The use of a single tube diminishes the possibility of contamination and increases the sensitivity and specicity compared to a standard PCR. Besides preventing contamination by previously amplied PCR products, the addition of UNG has an effect similar to a hot start (6), since it degrades any elongated product initially and the reaction begins hot when UNG has been inactivated. The high annealing temperatures avoid nonspecic amplications (5). The four different-size bands observed by Wilson et al. after agarose gel electrophoresis (22) are due to the four possible combinations allowed between the four primers used in the reaction. In the HN protocol, four bands can also be observed,
TABLE 2. Sensitivity of both PCR methods by radiological statusa
Radiological lesion No. of patients No. (%) of samples showing sensitivity byb: HN B-HN

TB patients Controls
a

80 80
0.02).

48 (60) 0 (100)

60 (75) 0 (100)

Unilateral inltrate Cavitated and/or bilateral

65 15

37 (57) 11 (73)

49 (75.5) 11 (73)

, signicant difference (P

a Eighty patients with positive cultures but negative sputum samples by AFB staining. b , signicant difference (P 0.02).

VOL. 38, 2000

NEW BALANCED PCR FOR M. TUBERCULOSIS DETECTION

1169

but in this case two are combinations between primers belonging to the rst and second amplication product, and the other two are due to the asymmetric amplication. When B-HN is performed, these latter two bands can be avoided, leading to better interpretation of the results and greater band intensity. Our results show that B-HN is more sensitive than standard HN, allowing the diagnosis to be advanced in 75% of the cases in which the smear was negative without waiting for culture results, and this fact is more evident in patients without cavitated lesions, who in our geographical area represent around 80% of the cases with pulmonary involvement. As indicated by the American Thoracic Society (1), we also recommend the use of these tests in conjunction with the available clinical data on the patient. In conclusion, the B-HN method is more sensitive than the HN technique. It does not decrease the specicity of the reaction. It can be applied to any PCR without further manipulation by attaching the sequence of the inner primer at the 5 end of the opposite outer primer. B-HN provides a target to the inner primer in both strands, resulting in a more efcient reaction which, in practice, means a higher sensitivity. This modication also has additional advantages over current amplication protocols when further manipulation of the PCR products is required, such as cloning or sequence capture, since one restriction enzyme will cut both ends of the product and more strands will be captured or labelled by the primers.
ACKNOWLEDGMENTS This work was supported by Fondo de Investigaciones Sanitarias de la Seguridad Social (FIS) grants 96/0028-01 and 98/1282 from the Ministerio de Salud, Madrid, Spain, and Sociedad Espanola de Neu molog y Cirug Toracica (SEPAR) grant 96/444. Albert Garc a a aQuintanilla was granted a predoctoral fellowship from the Departament de Microbiologia i Parasitologia Sanitaries, Divisio Ciencies de la ` ` Salut, Universitat de Barcelona, Spain. We thank Julia Gonzalez, Rosa Monte, and Dolors Ricart for their ` assistance in supplying samples.
REFERENCES 1. American Thoracic Society Workshop. 1997. Rapid diagnostic tests for tuberculosis. What is the appropriate use? Am. J. Respir. Crit. Care Med. 155:18041814. 2. Behr, M. A., S. A. Warren, H. Salamon, P. C. Hopewell, A. Ponce de Leon, C. L. Daley, P. M. Small. 1999. Transmission of Mycobacterium tuberculosis from patients smear-negative for acid-fast bacilli. Lancet 353:444449. 3. Bennedsen, J., V. O. Thomsen, G. E. Pfyffer, G. Funke, K. Feldmann, A. Beneke, et al. 1996. Utility of PCR in diagnosing pulmonary tuberculosis. J. Clin. Microbiol. 34:14071411. 4. Brugiere, O., M. Vokurka, D. Lecossier, G. Mangiapan, A. Amrane, B. Milleron, C. Mayaud, J. Cadranel, and A. J. Hance. 1997. Diagnosis of smear-negative pulmonary tuberculosis using sequence capture polymerase chain reaction. Am. J. Respir. Crit. Care Med. 155:14781481. 5. Chan, C. M., K. Y. Yuen, K. S. Chan, W. C. Yam, K. H. M. Yim, W. F. Ng, and M. H. Ng. 1996. Single-tube nested PCR in the diagnosis of tuberculosis. J. Clin. Pathol. 49:290294. 6. Chou, Q., M. Russell, D. E. Birch, J. Raymond, and W. Block. 1992. Pre-

7.

8.

9. 10.

11.

12. 13. 14. 15. 16.

17.

18.

19. 20.

21. 22.

23. 24.

vention of pre-PCR mis-priming and primer dimerization improves low-copy number amplications. Nucleic Acids Res. 20:17171723. Clarridge, J. E., III, R. M. Shawar, T. M. Shinnick, and B. B. Plikaytis. 1993. Large-scale use of polymerase chain reaction for detection of Mycobacterium tuberculosis in a routine mycobacteriology laboratory. J. Clin. Microbiol. 31:20492056. Cole, S. T., R. Brosch, J. Parkhill, T. Garnier, C. Churcher, D. Harris, S. V. Gordon, K. Eiglmeier, S. Gas, C. E. Barry III, F. Tekaia, K. Badcock, D. Basham, D. Brown, et al. 1998. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537544. Doern, G. V. 1996. Diagnostic mycobacteriology: where are we today? J. Clin. Microbiol. 34:18731876. Kennedy, N., S. H. Gillespie, A. O. S. Saruni, G. Kisyombe, R. McNerney, F. I. Ngowi, and S. Wilson. 1994. Polymerase chain reaction for assessing treatment response in patients with pulmonary tuberculosis. J. Infect. Dis. 170:713716. Kocagoz, T., E. Yilmaz, S. Ozkara, S. Kocagoz, M. Hayran, M. Sachedeva, and H. F. Chambers. 1993. Detection of Mycobacterium tuberculosis in sputum samples by polymerase chain reaction using a simplied procedure. J. Clin. Microbiol. 31:14351438. Kubica, G. P. W., E. Dye, M. L. Cohn, and G. Middlebrook. 1963. Sputum digestion and decontamination with N-acetyl-L-cysteine-sodium hydroxide for culture of mycobacteria. Am. Rev. Respir. Dis. 87:775779. Lebrun, L., D. Mathieu, C. Saulnier, and P. Nordmann. 1997. Limits of commercial molecular tests for diagnosis of pulmonary tuberculosis. Eur. Respir. J. 10:18741876. Liu, P. Y.-F., Z.-Y. Shi, Y.-J. Lau, and B.-S. Hu. 1994. Rapid diagnosis of tuberculous meningitis by a simplied nested amplication protocol. Neurology 44:11611164. Longo, M. C., M. S. Berninger, and J. L. Hartley. 1990. DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 93: 125128. Nolte, F. S., B. Metchock, J. E. McGowan, Jr., A. Edwards, O. Kwumabua, C. Thurmond, et al. 1993. Direct detection of Mycobacterium tuberculosis in sputum by polymerase chain reaction and DNA hybridization. J. Clin. Microbiol. 31:17771782. Noordhoek, G. T., A. H. J. Kolk, G. Bjune, D. Catty, J. W. Dale, P. E. M. Fine, P. Godfrey-Faussett, S.-N. Cho, T. Shinnick, S. B. Svenson, S. Wilson, and J. D. A. van Embden. 1994. Sensitivity and specicity of PCR for detection of Mycobacterium tuberculosis: a blind comparison study among seven laboratories. J. Clin. Microbiol. 32:277284. Pierre, C., D. Lecossier, Y. Boussougant, D. Bocart, V. Joly, P. Yeni, et al. 1991. Use of a reamplication protocol improves sensitivity of detection of Mycobacterium tuberculosis in clinical samples by amplication of DNA. J. Clin. Microbiol. 29:712717. Roth, A., T. Schaberg, and H. Mauch. 1997. Molecular diagnosis of tuberculosis: current clinical validity and future perspectives. Eur. Respir. J. 10: 18771891. Shawar, R. M., F. A. K. El Zaatari, A. Nataraj, and J. E. Clarridge. 1993. Detection of Mycobacterium tuberculosis in clinical samples by two-step polymerase chain reaction and nonisotopic hybridization methods. J. Clin. Microbiol. 31:6165. Thierry, D., M. D. Cave, K. D. Eisenach, J. T. Crawford, J. H. Bates, B. Gicquel, et al. 1990. IS6110, an IS-like element of Mycobacterium tuberculosis complex. Nucleic Acids Res. 18:188. Wilson, S. M., R. McNerney, P. M. Nye, P. D. Godfrey-Faussett, N. G. Stoker, and A. Voller. 1993. Progress toward a simplied polymerase chain reaction and its application to diagnosis of tuberculosis. J. Clin. Microbiol. 31:776782. World Health Organization. 1998. WHO report on the tuberculosis epidemic 1997. WHO, Geneva, Switzerland. Yuen, K., W. C. Yam, L. P. Wong, and W. H. Seto. 1997. Comparison of two automated DNA amplication systems with a manual one-tube nested PCR assay for diagnosis of pulmonary tuberculosis. J. Clin. Microbiol. 35:1385 1389.

Downloaded from http://jcm.asm.org/ on February 6, 2012 by guest