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Journal of Clinical Microbiology, January 2008, p. 396-397, Vol. 46, No. 1
0095-1137/08/$08.00+0     doi:10.1128/JCM.01924-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Evaluation of Melting Curve Analysis for Screening the Most Prevalent Mutations in Topoisomerase Genes from Streptococcus pneumoniae

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In a recently published article, Fukushima et al. (3) described a novel PCR-melting curve analysis (PCR-MCA) method for rapid screening of the most prevalent point mutations related to fluoroquinolone resistance in Streptococcus pneumoniae. In their study, they included 22 levofloxacin (LVX)-resistant strains and 50 LVX-susceptible isolates. None of the isolates in that study were shown to harbor quinolone resistance-determining region (QRDR) silent mutations, and the melting peaks obtained corresponded to the wild-type genotypes or the missense point mutations in the studied codons (gyrA codons 81 and 85 and parC codons 79 and 83).

Using the same approach, our laboratory analyzed 175 S. pneumoniae clinical isolates with ciprofloxacin MICs of 0.5 µg/ml (range, 0.5 to 128 µg/ml); 23 were LVX resistant (MIC 8 µg/ml) and 12 moxifloxacin resistant (MIC 4 µg/ml). An R6 wild-type strain and derived mutants were used as controls: parC mutants R111 (S79F), R119 (D83Y), and R112 (S79F D83Y) and gyrA mutants R123 (S81F), R125 (E85K), and R124 (S81F E85K). All of the clinical isolates were collected from the Toronto Invasive Bacterial Diseases Network at Mount Sinai Hospital, Toronto, Ontario, Canada. Real-time PCR was performed as described by Fukushima et al. (3), and all gyrA and parC QRDR amplicons were sequenced using an ABI Prism 3100 genetic analyzer (Applied Biosystems, CA).

All missense mutations in parC gene codons 79 (n = 61) and 83 (n = 15) and gyrA gene codons 81 (n = 21) and 85 (n = 1) which were confirmed by sequencing were detected by PCR-MCA. Silent mutations (TC) associated with the region where the sensor probes bind were detected in 17 clinical isolates by sequencing. Missense point mutations affecting gyrA codon 80 (AC, D-80-A) and parC codon 78 (GA, D-78-N) were found in the other two isolates (Table 1). In all of these 19 cases, intermediate melting temperatures (T_m) were observed, with a mean T_m of 49.47°C for parC codon 83 (16 isolates; 0.87% coefficient of variation), 45.35°C for gyrA codon 81 (2 isolates; 0.68% coefficient of variation), 49.09°C for parC codon 79 (1 isolate), and 43.73°C for gyrA codon 85 (1 isolate).

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TABLE 1. Intermediate Tm observed for different S. pneumoniae clinical isolatesa

MCA has been shown to be a simple technique that can be performed quickly for the screening of common QRDR mutations to the codon level. Detection of first-step mutations in these four specific codons could prevent clinical treatment failures (1, 2, 4). However, the detection of isolates with intermediate T_m could produce a misinterpretation of the results and indicates that these isolates should be further characterized.

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1. Davidson, R., R. Cavalcanti, J. L. Brunton, D. J. Bast, J. C. Azavedo, P. Kibsey, C. Fleming, and D. E. Low. 2002. Resistance to levofloxacin and failure of treatment of pneumococcal pneumonia. N. Engl. J. Med. 346:747-750.[Free Full Text]
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2. Endimiani, A., G. Brigante, A. A. Bettaccini, F. Luzzaro, P. Grossi, and A. Q. Toniolo. 2005. Failure of levofloxacin treatment in community-acquired pneumococcal pneumonia. BMC Infect. Dis. 5:106.[CrossRef][Medline]
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3. Fukushima, K. Y., Y. Hirakata, K. Sugahara, K. Yanagihara, A. Kondo, S. Kohno, and S. Kamihira. 2006. Rapid screening of topoisomerase gene mutations by a novel melting curve analysis method for early warning of fluoroquinolone-resistant Streptococcus pneumoniae emergence. J. Clin. Microbiol. 44:4553-4558.[Abstract/Free Full Text]
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4. Perez-Trallero, E., J. M. Marimon, L. Iglesias, and J. Larruskain. 2003. Fluoroquinolone and macrolide treatment failure in pneumococcal pneumonia and selection of multidrug-resistant isolates. Emerg. Infect. Dis. 9:1159-1162.[Medline]

						Roberto G. Melano Rachel Higgins Christine Seah Steven J. Drews* Ontario Public Health Laboratories Ministry of Health and Long-Term Care 81 Resources Road Toronto, Ontario, Canada M9P 3T1
						* Phone: (416) 235-5703 Fax: (416) 235-6550 E-mail: Steven.Drews{at}ontario.ca

Authors' Reply

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We appreciate the comments provided by Dr. Melano et al. regarding our publication. They found the PCR-MCA to be a simple and rapid technique. In addition, they agreed that all of the missense mutations harbored in strains and confirmed by PCR-MCA clearly demonstrated that the PCR-MCA method was able to detect mutations in the four QRDR positions (gyrA[r] codons 81 and 85 and parC codons 79 and 83). Melano et al. highlighted the fact that the presence of intermediate T_m, which resulted from silent mutations or mutations in other positions inside the sensor probe, could result in an overestimation of a significant mutation. Although none of the 72 strains used in our study were shown to have silent QRDR mutations, 17 out of 175 strains were determined to have silent QRDR mutations in their study. We designed probes which target the four QRDR positions in order to maximally differentiate the T_m for the wild-type strain from the T_m for the mutant strains. To avoid any misinterpretation of the PCR-MCA results for the intermediate T_m (in other words, to differentiate the T_m for the wild-type strain from the T_m for the mutant strains), we suggest that a T_m range be set for each QRDR position using control strains. Unfortunately, we could not assay enough isolates to evaluate the different T_m among strains with silent mutations (from mutant or wild-type strains). In addition, there is a current lack of information about variations in intermediate T_m. As suggested by Melano et al., intermediate T_m should be interpreted with caution. Further studies are necessary for a more thorough evaluation.

 
Present address: Department of Infection Control and Laboratory Diagnostics, Internal Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574 Japan.

						Yoichi Hirakata* Kazuko Yamamoto Nagasaki University Graduate School of Biomedical Sciences 1-7-1 Sakamoto Nagasaki City 852-8501, Japan
						* Phone: 81-22-717-7376 Fax: 81-22-717-7390 E-mail: hiraichi{at}mail.tains.tohoku.ac.jp

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Journal of Clinical Microbiology, January 2008, p. 396-397, Vol. 46, No. 1
0095-1137/08/$08.00+0     doi:10.1128/JCM.01924-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Melano, R. G. Articles by Yamamoto, K. Search for Related Content PubMed PubMed Citation Articles by Melano, R. G. Articles by Yamamoto, K.