Evaluation of Point Mutation Detection in ...

Appl Biochem Biotechnol DOI 10.1007/s12010-014-1442-9

Evaluation of Point Mutation Detection in Mycobacterium tuberculosis with Isoniazid Resistance Using Real-Time PCR and TaqMan Probe Assay F. Riahi & M. Derakhshan & A. Mosavat & S. Soleimanpour & S. A. Rezaee

Received: 27 July 2014 / Accepted: 1 December 2014 # Springer Science+Business Media New York 2014

Abstract Rapid methods for diagnosis of Mycobacterium tuberculosis (Mtb) drug resistance and choosing appropriate antibiotic treatment are pivotal. Thirty isoniazid (INH)-resistant and 30 INHsusceptible Mtb isolates were evaluated using minimum inhibitory concentration (MIC) method followed by multiplex real-time PCR (RT-PCR). Amplification refractory mutation system (ARMS) for detection of mutation in 315 codon of katG gene and single-nucleotide polymorphism (SNP) for detection of mutation in −15 (C>T) in the regulatory zone of mabA-inhA were carried out using the TaqMan method. Primers and probe were used for IS6110 region of Mtb as an internal amplification control. The sensitivity and specificity of the RT-PCR TaqMan probe for detection of Mtb complex were 100 %. Detection of INH-resistant Mtb using the ARMS method for KatG had 69 % sensitivity and 100 % specificity. The sensitivity and specificity of SNP in mabA-inhA fragment for detection of INH-resistant Mtb were 53 and 100 %, respectively. Furthermore, considering both regions, the sensitivity of RT-PCR has increased to 75 %. This study revealed that the qPCR-TaqMan method can be used as a standard tool for diagnosis of Mtb. Moreover, ARMS and SNP RT-PCR TaqMan methods can be used as rapid screening methods for detection of INH-resistant Mtb. Keywords Isoniazid . Drug resistant . Mycobacterium tuberculosis . Real-time PCR . Point mutation

Introduction Tuberculosis (TB) is the second most frequent infectious disease leading to death, worldwide [1]. In spite of using bacille Calmette–Guérin vaccine (BCG) and various anti-mycobacterial drugs, resistance to anti-tuberculosis drugs is dramatically increasing. The emergence of drug A. Mosavat and S. Soleimanpour contributed equally in this study.

F. Riahi : M. Derakhshan : A. Mosavat : S. Soleimanpour Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran S. A. Rezaee (*) Inflammation and Inflammatory Diseases Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran e-mail: [email protected]

Appl Biochem Biotechnol

resistance of Mycobacterium tuberculosis (Mtb), particularly multidrug resistance (i.e., resistance to at least rifampicin (RMP) and isoniazid (INH)), is a major health threat in both developed and developing countries [2]. The World Health Organization (WHO) estimated globally 450,000 cases of multidrug-resistant tuberculosis (MDR-TB) (5.2 % of all incident TB cases), causing approximately 170,000 deaths in 2012 [3]. INH is a first-line chemotherapeutic drug used in TB therapy [4–7]; unfortunately, the effectiveness of this drug is reduced due to the presence of INH-resistant Mtb [8]. On the other hand, usually development of INH resistance precedes resistance to RMP; therefore, resistance to INH is considered as a surrogate marker for MDR-TB. Early detection of drug resistance allows the initiation of appropriate treatment, which has an impact on better disease control [9]. It has been shown that mutations in several genes and genomic regions of Mtb are involved in resistance to INH [10, 11]. Mutations in at least four different genetic loci have been reported including katG encoding catalase–peroxidase, inhA involved in fatty acid elongation, ahpC encoding alkyl hydroperoxide reductase, and oxyR involved in oxidative stress regulation. Despite this complexity, katG315 is the most frequent locus for mutations associated with resistance to INH (30–90 %). Furthermore, resistant strains due to mutations in this codon have been reported to be efficiently transmissible [12, 13]. In addition, mutations in the regulatory region of the inhA gene have been reported in up to 32 % of INH-resistant isolates [14–17]. Mutations in other genomic regions, such as the promoter of ahpC gene and the kasA gene, have been reported in 12–24 and 10–14 % of INHresistant strains, respectively [14–16, 18]. It is obvious that the culturing and isolation of Mtb are still difficult even with the use of new liquid medium. Therefore, rapid methods for detection of drug resistance in Mtb are necessary to select appropriate anti-TB treatment and avoid the transmission of resistant strains. Several molecular methods have been previously described and frequently used for detection of resistance mutations [19–21]. Real-time PCR has also been recently applied for the rapid detection of drug-resistant strains in Mtb. Different methods such as TaqMan, fluorescence resonance energy transfer (FRET), and molecular beacons have been used to evaluate mutations in infectious agents [22]. Rapid testing and a lower risk of contamination are the main advantages in real-time PCR techniques, and results are generally obtained in an average of 1.5–2.0 h after DNA extraction. Therefore, these techniques can be commonly used for clinical specimens in medical laboratories [23–25]. Taken together, these data show the importance of screening methods to identify drugresistant individuals and choose an appropriate treatment. Therefore, we sought to find a new method for an accurate and rapid detection of Mtb resistance in clinical samples. In this study, a simple and rapid real-time PCR TaqMan probe ARMS and allelic discrimination assays or single-nucleotide polymorphism (SNP) have been designed and described to detect the mutation in codon 315 of the katG gene and in −15 (C→T) of the mabA-inhA regulatory region (or fabG gene), respectively.

Materials and Methods Microbial Strains Thirty INH-resistant and 30 INH-susceptible Mtb strains isolated from sputa of patients with active pulmonary TB were collected in different geographic regions of Iran (Mashhad pulmonary disease center and laboratory of Mycobacteria in Pasteur Institute of Iran). The collected samples were processed and cultured on traditional Lowenstein–Jensen (LJ) medium,


and the isolates were identified by standard microbiological methods such as Ziehl–Neelsen staining, morphology of colony, niacin production, and nitrate and catalase tests. Anti-Microbial Susceptibility Test Anti-microbial drug susceptibility testing of isolates was performed using the CDC standard conventional proportional method. The minimum inhibitory concentration (MIC) of INH (0.2, 0.4, 0.8, 1.6, and 3.2 μg/ml) was determined on LJ medium for INH-resistant strains [26]. The INH-susceptible Mtb strain H37Rv (ATCC 27294) was used as a wild-type control. Genomic Experiments Genomic DNA Preparations For DNA extraction, a 0.5 McFarland suspension was prepared in 200 μl of Tris–EDTA buffer (pH 7.5) and heated at 95 °C for 20 min after 5 min of centrifugation at 12,000 rpm; the supernatant was kept at −20 °C for the amplification procedure. Bioinformatics Study By searching in the GeneBank database of the National Center for Biotechnology Information (NCBI), the resistance genes, katG and fabG, were identified and aligned by ClustalW (ver. 1.83) (http://www.ebi.ac.uk/Tools/msa/clustalo/). All of SNPs and mutants were determined. The primers and probes were designed using Beacon Designer software (ver. 7, Primer Biosoft, USA) and blasted using online Nucleotide BLAST search NCBI (http://www.ncbi.nlm.nih.gov/BLAST/). Primers and dually fluorochrome-labeled TaqMan hydrolysis probes were synthesized by Bioneer Corporation, (Bioneer, Korea). Designing of Real-Time PCR TaqMan Probe ARMS Technique TaqMan probe ARMS technique was used to detect mutations in 315 codon of katG (AGC→ G>C/T/A). In this technique, mutant nucleotide was located in reverse primer; thus, for this purpose, a forward primer and two reverse primers were designed. One of the reverse primers was used for detection of the wild-type sequence and the other one for detection of the mutation. The unique probe was designed and labeled with FAM fluorophore at the 5′ end and BHQ-1 quencher at the 3′ end subsequently (Table 1). Assays were performed in a spectrofluorometric thermal cycler Rotor-Gene 6000 system (Corbett Research Australia). Premix Ex Taq™ (TaKaRa BIO/Japan) was used with 2× concentration of premix reagent consisted of TaKaRa Ex Taq™ HS, dNTP mixture, and Mg2+. The volume of amplification reaction was 20 μl containing the following: 10 μl of Premix Ex Taq™, forward primer 0.4 μl, reverse primer 0.4 μl, TaqMan probe 0.8 μl, DNA template 2 μl, and 6.4 μl of dH2O. The PCR protocol was as follows: an initial denaturation at 95 °C for 10 min and shuttle PCR (twostep PCR) as follows: 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 62 °C for 34 s. The results were analyzed based on the Δ cycle threshold (Δct)=x>4 in which the detection of fluorescence starts. In order to determine the Δct, the synthetic sequence was made with homologue reverse primer and, at the same time, with non-homologue reverse primer in the other microtube. For reliability, the assays were performed in duplicate. Since


Δct at 60 °C annealing/extension in mutant sequence was 3 cycles, the assays were optimized at 62 °C annealing/extension. Real-Time PCR TaqMan Probe Allelic Discrimination for −15 (C→T) of the mabA-inhA Regulatory Region To design a real-time PCR TaqMan probe allelic discrimination for −15 (C→T) of the mabAinhA regulatory region, two probes with different fluorophores were designed for detection of the mutation. The probe for the wild sequence detection was labeled with FAM fluorophore at the 5′ end and BHQ-1 quencher at the 3′ end. The probe for detection of mutation was labeled with JOE fluorophore and BHQ-1 quencher. The difference between the probes is usually only 1 bp (Table 1). Amplification reactions were carried out in a volume of 20 μl containing 10 μl of Premix Ex Taq, 0.4 μl forward primer, reverse primer 0.4 μl, TaqMan probe (wild type) 0.8 μl, TaqMan probe (mutant Type) 0.8 μl, DNA template 2 μl, and 5.6 μl of dH2O. The reaction conditions were as described previously but with an exception in annealing/extension at 60 °C. Results were analyzed based on two parameters: the cycle threshold and the cumulative fluorescence signal (CFS) of each probe at the end of the 40 amplification cycles. A TaqMan real-time PCR was designed for Mtb IS6110 gene as an internal amplification control. The unique probe was labeled with ROX fluorophore and BHQ-2 quencher for use in multiplex condition (Table 1).

Results and Discussion Real-Time PCR for Detection of Mtb IS6110 Gene The designed protocol for the detection of IS6110 gene in Mtb by the TaqMan method was worked properly. The sensitivity and specificity of the assay for Mtb detection were 100 %. Figure 1 shows the results of a multiplex assay in which IS6110 gene acts as an internal control and in detection of Mtb isolates.

Table 1 Primers and probes designed for detecting of mutations Gene katG

Primers and probes Forward primer

5′-GGC CCG GYC GAT CTG GTC-3′

Reverse primer

5′-VAT ACG ACC WCG ATG CGB C-3′ 5′-VAT ACG ACC WCG ATG CGG D-3′

fabG-inhA

Probe

5′-FAM-CGA ACC CGA GGC TGC TCC GCT G-BHQ1-3′

Forward primer

5′-CGT TAC GCT CGT GGA CAT ACC-3′

Reverse primer Probe

5′-CTT CAG TGG STG TGG CAG TC-3′ 5′-FAM-CGA CAH CCT ATC GTM TCG CCG CG-BHQ1-3′ 5′-JOE-CGA CAH CCT ATC ATM TCG CCG CG-BHQ1-3′

IS6110

Forward primer

5′-TCG CCT ACG TGG CCT TTg-3′

Reverse primer

5′-GGA TAA CGT CTT TCA GGT CGA GTA C-3′

Probe

5′-ROX- CGC TTC CAC GAT GGC CAC CTC-BHQ2-3′

W=A/T, Y=C/T, D=A/G/T, V=A/C/G, B=C/G/T, N=A/T/C/G, M=A/C, H=A/C/T, S=G/C


Fig. 1 The result of IS6110 amplification. TaqMan real-time PCR for Mtb IS6110 region was carried as an internal amplification control

TaqMan Probe ARMS for Detection of Mutation in Codon 315 AGC→G>C/T/A After optimization of annealing temperature (Fig. 2), the assay was optimized for the best dilution, using synthetic sequences (negative and positive oligonucleotides). Dilution of 10−7:100 pmol/ml from negative and positive synthetic nucleotides was selected for the assay (Fig. 3). Then, Mtb isolates were introduced to the optimized test for detection of the wild and drug-resistant isolates. Out of 30 INH-resistant Mtb isolates, 16 (53.33 %) had mutation in codon 315. All of the isolates were positive for IS6110 gene. TaqMan Allelic Discrimination for Detection of Mutation in −15 (C→T) of the mabA-inhA TaqMan allelic discrimination method was optimized for annealing temperature and the best concentration similar to the previous method to detect wild and mutant isolates in regulatory region of the mabA-inhA (or fabG gene) (data not shown). Out of 30 INH-resistant Mtb isolates, four isolates (13.33 %) had mutation in −15 (C→T) of the mabA-inhA regulatory region. No mutation was observed in 30 INH-susceptible Mtb isolates in codon 315 katG gene and in −15 (C→T) of the mabA-inhA regulatory region. Method Sensitivity for Identification of INH-Resistant Mtb The sensitivity of the real-time PCR TaqMan probe ARMS and real-time PCR TaqMan probe allelic discrimination techniques were 69 and 53 % for detection of INH-resistant Mtb, respectively. Specificity of both methods was 100 %. Considering the results of both regions, the sensitivity of real-time PCR (RT-PCR) has increased to 75 % (Table 2).


Fig. 2 The optimized annealing temperature (60 °C) for Δct assessment. Duplicate serial dilutions of positive control were used and tested in different annealing temperature to find the best one

Diagnosis of Mtb infections in developing countries is very crucial for adequate treatment, particularly in drug-resistant and MDR-TB cases. Culture-based method is highly timeconsuming and inaccurate for detection of mutations and anti-biogram. It is necessary to ensure that all patients are diagnosed appropriately and treated effectively in order to avoid

Fig. 3 Optimized concentration of positive control (10−5 pmol/ml) for Δct assessment. Serial dilutions (10−1, 10−2, 10−3,… 10−10) of positive control were used and tested

Appl Biochem Biotechnol Table 2 Probe sensitivity for detection of wild or mutated sequence of the target genes in 60 Mtb isolated including 30 harboring susceptible bacilli and 30 harboring resistant bacilli Target gene

Sample detected INH-resistant isolates

Sample not detected INH-susceptible INH-resistant isolates isolates

Mutation Sensitivity of (%) detection (%)

INH-susceptible isolates

katG

16

0

14

30

53.33

69

fabG

4

0

26

30

13.33

53

IS6110

30

30

0

0

–

100

transmission and impressive treatment of the resistant strains. Therefore, to avoid the transmission of resistant strains and for clinical management of patients with TB, a rapid assessment of anti-TB drug resistance is essential. In recent decades, many studies have focused on the molecular basis of resistance mechanisms of anti-TB agents [27]. The screening codon 315 from katG gene and the mabA-inhA upstream region are the main regions of hot spot mutations in Mtb. Mutations in codon 315 are involved in virulence and resistance to INH. Several methods such as sequence analysis, PCR-strand single conformational polymorphism [5, 28], allele-specific PCR for INH and RMP [28, 29], RFLP-PCR for INH [28], PCR-EIA [18], and the detection of point mutations using RT-PCR [10, 11, 16, 20, 30, 31] have been developed for genetic detection of resistance to INH and RMP. Recently, the RT-PCR TaqMan method is the quickest and more reliable method for analyzing the susceptibility of Mtb [5–7, 29, 32]. In most cases, resistance to anti-microbial agents was caused by genetic modifications. Thus, molecular methods for evaluation and assessment of these modifications are very important. Molecular methods are parts of the standard methods in bacteriological laboratories throughout the world. In this study, RT-PCR TaqMan probe has been evaluated for detection of mutations in katG gene (ARMS) and regulatory region of mabA-inhA gene (SNP) in comparison with the proportional method on INH-resistant Mtb strains. The RT-PCR TaqMan methods were targeted the two loci of INH resistance-associated mutations as the most frequent observed changes. To evaluate sensitivity and specificity of the new method in comparison with the culture-based phenotypic method, 60 Mtb clinical isolates were obtained from Mashhad pulmonary disease center and laboratory of Mycobacteria in Pasteur Institute of Iran (Tehran, Iran) and were analyzed to determine INH resistance rates. The sensitivity of the RT-PCR TaqMan probe ARMS and RT-PCR TaqMan probe allelic discrimination techniques for detection of INH-resistant Mtb were 69 and 53 %, respectively. Van Doorn et al. found that 55 % of INH-resistant strains had a mutation in 315 codon katG gene which is consistent with the result of current study [28, 29]. Another study in Turkey has shown that 75 % of resistant Mtb had a mutation in 315 codon of katG gene [33]. Laura Rindi et al. have demonstrated that from 35 strains of INH-resistant Mtb, ten cases (29 %) had mutations in regulatory region of −15 (C→T) inhA gene [29, 34]. Sajduda in Poland reported that 69 % of resistant cases had a mutation in 315 codon of katG gene and 14.5 % mutation in −15 (C→T) which are in line with the result of the present study [23–25]. In the current study, considering both methods for INH resistance detection, the sensitivity and specificity change to 75 and 100 %, respectively. The main advantages of methods used in the current study are the shortest possible time for obtaining results after sample collection and the strategy for designing, since these methods cover the evaluation of most frequent mutations studied here. However, in detection of drugresistant Mtb, these molecular methods are only applicable for known mutations.


The sensitivity and specificity of a molecular detection may vary in different geographic regions, according to the prevalence of genetic mutations result in drugresistant Mtb. Further studies are needed to gain a more complete understanding of the genetic variations in Mtb drug resistance and to determine the prevalence of resistant mutations among Mtb clinical isolates in different geographic regions. Because of the limitation mentioned above, RT-PCR method still cannot be completely replaced with the culture-based phenotypic susceptibility tests. However, it provides a rapid screening tool for a majority of the resistant isolates. Limitation of the current study was using purified Mtb genomic DNA extracted from LJ medium. Therefore, further optimization and evaluation of this method for direct detection of Mtb drug resistance mutations in clinical specimens of patients remain an important focus of future studies. Our result showed that 53.33 % of INH-resistant isolates could be detected with assessment of 315 codon of katG gene and 13.33 % with regulatory region of mabA-inhA gene. Only 33.4 % of mechanisms of resistance to INH have been known. Other researchers have been reported that mutations in promoters of ahpC and kasA gene are important within 12–24 and 10–14 %, respectively [14–16, 18]. Therefore, these unknown cases of resistance to INH could be detected with assessment of ahpC and kasA gene promoters. On the other hand, investigation on 309, 311, 316, and 328 codons of katG gene in mabA-inhA gene is very useful. Considering mutation in both regions (katG/fabG) for INH resistance, the sensitivity and specificity of the method are 75 and 100 %, respectively. Therefore, for the rest of Mtb-positive INH-resistant multiplex real-time PCR-negative subjects, according to clinical status, it should be decided if the DNA sequencing for the remaining fragment is necessary. Acknowledgments The authors are grateful to Reza Derakhshan, Rosita Vakili, and Mehdi Aganj, Medical School, MUMS, for reading the article and their valuable advice. Author Contribution RF performed the tests, DM was the advisor of the study and helped in designing the study, MA compiled the results and prepared the draft, SS compiled the results and prepared the draft, and RSA designed the study and proofread the manuscript.

Conflict of Interest None declared.

References 1. Murthy, RS. (2001). The world health report 2001, World Health Organization. 2. Pablos-Mendez, A., Raviglione, M., Laszlo, A., Binkin, N., Rieder, H., Bustreo, F., et al. (1998). New England Journal Medicine, 338(23), 1641–1649. 3. WHO (World Health Organization) (2013). Global tuberculosis report 2013. 4. Wright, A., Zignol, M., Van Deun, A., Falzon, D., Gerdes, S. R., Feldman, K., Hoffner, S., Drobniewski, F., Barrera, L., van Soolingen, D., Boulabhal, F., Paramasivan, C. N., Kam, K. M., Mitarai, S., Nunn, P., & Raviglione, M. (2009). Lancet, 373, 1861–1873. 5. Elmendorf, D. F., Jr., Cawthon, W. U., Muschenheim, C., & McDermott, W. (1952). American Review of Tuberculosis, 65(4), 429. 6. Klee, P. (1952). Deutsche med Wchnschr, 77, 578–581. 7. Robitzek, E. H., & Selikoff, I. J. (1952). American Review of Tuberculosis, 65(4), 402. 8. Middlebrook, G. (1954). American Review of Tuberculosis, 69(3), 471.

Appl Biochem Biotechnol 9. Yang, Z., Solante, R., Espantaleon, A. S., & Sangco, J. C. E. (2005). Antimicrobial Chemotherapy, 55, 860– 865. 10. Slayden, R. A., & Barry, C. E. (2000). Microbes and Infection, 2(6), 659–669. 11. Piatek, A.S., Telenti, A., Murray, M.R., El-Hajj, H., Jacobs Jr WR, Kramer FR, A. (2000). Antimicrobial Agents and Chemotherapy, 44(1), 103–10. 12. Pym, A. S., Saint-Joanis, B., & Cole, S. T. (2002). Infection and Immunity, 70(9), 4955–4960. 13. van Soolingen, D., de Haas, P. E. W., van Doorn, H. R., Kuijper, E., Rinder, H., & Borgdorff, M. W. (2000). Journal of Infectious Diseases, 182(6), 1788–1790. 14. Lee, A. S. G., Lim, I. H. K., Tang, L. L. H., Telenti, A., & Wong, S. Y. (1999). Antimicrobial Agents and Chemotherapy, 43(8), 2087–2089. 15. Kiepiela, P., Bishop, K., Smith, A., Roux, L., & York, D. (2000). Tubercle and Lung Disease, 80(1), 47–56. 16. Telenti, A., Honore, N., Bernasconi, C., March, J., Ortega, A., Heym, B., Takiff, H. E., & Cole, S. T. (1997). Journal of Clinical Microbiology, 35(3), 719–723. 17. Morris, S., Bai, G. H., Suffys, P., Portillo-Gomez, L., Fairchok, M., & Rouse, D. (1995). Journal of Infectious Diseases, 171(4), 954. 18. Mdluli, K., Slayden, R. A., Zhu, Y. Q., Ramaswamy, S., Pan, X., Mead, D., Crane, D. D., Musser, J. M., & Barry, C. E. (1998). Science, 280(5369), 1607–1610. 19. Musser, J. M. (1995). Clinical Microbiology Reviews, 8(4), 496–514. 20. Caws, M., & Drobniewski, F. (2001). Annals of the New York Academy of Sciences, 953(1), 138–145. 21. Garcia de Viedma, D. (2003). Clinical Microbiology and Infection, 9(5), 349–359. 22. Shamputa, I., Rigouts And, L., & Portaels, F. (2004). Apmis, 112(11–12), 728–752. 23. Espasa, M., González-Martín, J., Alcaide, F., Aragón, L. M., Lonca, J., Manterola, J. M., Salvado, M., Tudo, G., Orus, P., & Coll, P. (2005). Journal of Antimicrobial Chemotherapy, 55(6), 860–865. 24. Ruiz, M., Torres, M. J., Llanos, A. C., Arroyo, A., Palomares, J. C., & Aznar, J. (2004). Journal of Clinical Microbiology, 42(4), 1585–1589. 25. Sajduda, A., Brzostek, A., Popławska, M., Augustynowicz-Kopeć, E., Zwolska, Z., Niemann, S., Dziadek, J., & Hillemann, D. (2004). Journal of Clinical Microbiology, 42(6), 2425–2431. 26. Rieder, H.L., Chonde, T.M., Myking, H., Urbanczik, R., Laszlo, A., Kim, S.J., Van Deun, A., Trébucq, A. (1998). The public health service national tuberculosis reference laboratory and the national laboratory network; minimum requirements, role and operation in a low-income country. Paris, France: International Union Against Tuberculosis and Lung Disease (IUATLD). 27. Martin, A., Portaels, F. (2007). Tuberculosis, 635–87. 28. Tudó, G., González, J., Obama, R., Rodriguez, J., Franco, J., Espasa, M., Simarro, P. R., Escaramís, G., Ascaso, C., García, A., & Jiménez de Anta, M. T. (2004). The International Journal of Tuberculosis and Lung Disease, 8(1), 15–22. 29. Van Doorn, H., De Haas, P., Kremer, K., Vandenbroucke, G. C., Borgdorff, M., & Van Soolingen, D. (2006). Clinical Microbiology and Infection, 12(8), 769–775. 30. El-Hajj, H. H., Marras, S. A. E., Tyagi, S., Kramer, F. R., & Alland, D. (2001). Journal of Clinical Microbiology, 39(11), 4131–4137. 31. de Viedma, D. G., Infantes, M. S. D., Lasala, F., Chaves, F., Alcalá, L., & Bouza, E. (2002). Journal of Clinical Microbiology, 40(3), 988–995. 32. Torres, M. J., Criado, A., Palomares, J. C., & Aznar, J. (2000). Journal of Clinical Microbiology, 38(9), 3194–9. 33. Ozturk, C. E., Sanic, A., Kaya, D., & Ceyhan, I. (2005). Japanese Journal of Infectious Diseases, 58(5), 309. 34. Rindi, L., Bianchi, L., Tortoli, E., Lari, N., Bonanni, D., & Garzelli, C. (2003). Journal of microbiological methods, 55(3), 797–800.