Mami Hata

Cloning of a Novel Gene for Quinolone Resistance from a Transferable Plasmid in Shigella flexneri 2b

ABSTRACT A novel gene for quinolone resistance was cloned from a transferable plasmid carried by a clinical isolate of Shigella flexneri 2b that was resistant to fluoroquinolones. The plasmid conferred low-level resistance to quinolones on Escherichia coli HB101. The protein encoded by the gene showed 59% amino acid identity with Qnr.

Oseltamivir-Resistant Influenza A Viruses Circulating in Japan

ABSTRACT Surveillance studies of the influenza viruses circulating in Europe and other countries in 2007 and 2008 have revealed rates of resistance to oseltamivir of up to 67% among H1N1 viruses. In the present study, we examined 202 clinical samples obtained from patients infected with H1N1 virus in Japan in 2007 and 2008 for oseltamivir resistance and found that three were oseltamivir resistant (1.5%). The 50% inhibitory concentrations (IC50s), as measured by a sialidase inhibition assay with these drug-resistant viruses, were >100-fold higher than those of the nonresistant viruses (median IC50, 12.6 nmol/liter). The His274Tyr (strain N2 numbering) mutation of the neuraminidase protein, which is known to confer oseltamivir resistance, was detected in these three isolates. Phylogenetic analysis showed that one virus belonged to a lineage that is composed of drug-resistant viruses isolated in Europe and North America and that the other two viruses independently emerged in Japan. Continued surveillance studies are necessary to observe whether these viruses will persist.

Development of a Rapid PCR Method Using the Insertion Sequence IS1203 for Genotyping Shiga Toxin-Producing Escherichia coli O157

ABSTRACT We developed a rapid PCR method utilizing the diversity of the insertion site IS1203 for genotyping Shiga toxin-producing Escherichia coli (STEC) O157 (IS1203 PCR typing). DNA fragments digested by PvuII, which cut IS1203 at one site, were ligated with themselves and detected by PCR with outward-facing primer pairs for IS1203. To minimize nonspecific bands, nested PCR was also performed. Two fingerprinting patterns produced from the upstream or downstream regions of IS1203 were obtained within 1 or 2 days. By combining the two patterns, 79 STEC O157 isolates were classified into 39 types, which were then classified into 36 subtypes by pulsed-field gel electrophoresis (PFGE). The discriminatory power of IS1203 PCR typing (D = 0.974) is similar to that of PFGE (D = 0.981). This method can be used for rapid and simplified genotyping.

Rapid genotypic assay for detection of oseltamivir-resistant influenza A (H1N1) viruses.

Two classes of drugs are currently used for the treatment of influenza virus infections: M2 inhibitors (M2Is; amantadine and rimantadine) and neuraminidase inhibitors (NAIs; oseltamivir and zanamivir). In recent years, a high percentage of circulating influenza A (H3N2) viruses have shown resistance to M2Is (6). Until recently, there was a low prevalence of NAI resistance among circulating viruses (8). However, surveillance in Europe during the 2007-2008 influenza season revealed the sudden emergence of oseltamivir resistance in influenza A (H1N1) virus isolates, and these resistant viruses spread worldwide in the following 2008-2009 season (10). Since early April 2009, a novel strain of influenza A (H1N1) virus has spread rapidly worldwide (3), and the World Health Organization declared an H1N1 pandemic in 2009. While resistance to oseltamivir is rare in novel H1N1 viruses at present (4, 5), constant surveillance is essential to monitor the emergence and spread of drug-resistant viruses. The oseltamivir-resistant viruses harbor a specific mutation in the neuraminidase (NA) protein: a substitution of Tyr for His at position 275 (N1 numbering). The current screening methods for the resistant viruses are performed to detect this mutation. Recently, new molecular detection methods involving the use of real-time PCR have been reported (1, 2, 9). Although these methods are rapid and sensitive, the probes used in real-time PCR are rather expensive. Hence, there is a need to develop a more cost-effective approach. In a previous study, we developed a mismatch amplification mutation assay-PCR (MAMA-PCR) approach to detect a gene mutation that confers amantadine resistance on influenza A (H3N2) viruses (7). The present report describes a new MAMA-PCR method for detecting the H275Y mutation in the NA genes of oseltamivir-resistant influenza A (H1N1) viruses. Throat swab specimens from patients were collected as part of a nationwide surveillance program. The specimens were inoculated onto MDCK cells, and viruses were isolated from infected cells. Viral RNA was extracted from throat swab specimens or from viral culture supernatants. cDNA was synthesized from viral RNA and used as the template for PCR. Primers with conserved sequences (NAN1F and NAN1R) (Table ​(Table1)1) were designed for the control PCR amplification of a 938-bp NA gene fragment. Mismatched reverse primers (MAMA275H and MAMA275Y) (Table ​(Table1)1) were designed for the detection of codons for His 275 (CAT) and Tyr 275 (TAT), respectively. A MAMA primer was added to each PCR mixture in order to generate a 539-bp PCR product only when the cDNA contained the corresponding gene sequence. The PCR mixture (25 μl) contained 2.5 μl of cDNA, 1 U of Ex Taq DNA polymerase (TaKaRa), 10 pmol of the forward primer, 2.5 pmol of the reverse primer, and 25 pmol of the MAMA primer. The reaction mixture was subjected to an initial denaturation step at 94°C for 3 min and then 30 cycles of PCR at 94°C for 30 s, 55°C for 30 s, and 72°C for 45 s. TABLE 1. MAMA-PCR primers for detection of the H275Y mutation Forty influenza A (H1N1) virus isolates from the 2007-2008 season and 16 from the 2008-2009 season were analyzed. A representative result from screening for the H275Y mutation is shown in Fig. ​Fig.1.1. The 589-bp product observed in lanes 1H to 4H indicates the presence of His at position 275, which renders the strain sensitive to oseltamivir, and the absence of the mutation. In contrast, the 589-bp product observed in lanes 5Y to 8Y indicates the presence of the mutation conferring oseltamivir resistance. The nucleic acid sequences of the NA gene were verified by sequencing the 938-bp control band. The MAMA-PCR method was found to clearly distinguish the mutant type from the wild type. This method can also be performed using viral RNA directly from throat swab specimens. FIG. 1. MAMA-PCR analysis for detecting a single mutation in the NA genes of oseltamivir-resistant and oseltamivir-sensitive influenza A (H1N1) viruses. Lanes: M, molecular size marker (100-bp ladder); 1, Aichi/92/2007 virus (harboring 275H); 2, Aichi/96/2007 ... Among the 40 examined strains isolated in the 2007-2008 season, 1 (2.5%) was found to have the H275Y mutation. All (100%) of the 16 examined strains from the 2008-2009 season were found to harbor the mutation. The incidences of resistant viruses in the two seasons are consistent with reports from the nationwide surveillance (10). Twenty-four pandemic H1N1 virus strains that were isolated in June 2009 were also examined using the primers listed in Table ​Table1.1. The MAMA-PCR revealed that all these strains were 275H viruses. The results were also verified by a subsequent sequencing analysis. The assay described herein will allow for more convenient screening of the viruses, without the need for expensive real-time PCR approaches, and is useful for not only seasonal influenza A (H1N1) viruses but also pandemic 2009 influenza (H1N1) virus strains.

Case-based surveillance enhanced with measles virus detection/genotyping is essential to maintain measles elimination in Aichi Prefecture, Japan

BACKGROUND Japan was verified as having achieved measles elimination by the Measles Regional Verification Commission in the Western Pacific Region in March 2015. Verification of measles elimination implies the absence of continuous endemic transmission. After the last epidemic in 2007 with an estimated 18,000 cases, Japan introduced nationwide case-based measles surveillance in January 2008. Laboratory diagnosis for all suspected measles cases is essentially required by law, and virus detection tests are mostly performed by municipal public health institutes. Despite relatively high vaccination coverage and vigorous response to every case by the local health center staff, outbreak of measles is repeatedly observed in Aichi Prefecture, Japan. METHODS Measles virus N and H gene detection by nested double RT-PCR was performed with all specimens collected from suspected cases and transferred to our institute. Genotyping and further molecular epidemiological analyses were performed with the direct nucleotide sequence data of appropriate PCR products. RESULTS Between 2010 and 2014, specimens from 389 patients suspected for measles were tested in our institute. Genotypes D9, D8, H1 and B3 were detected. Further molecular epidemiological analyses were helpful to establish links between patients, and sometimes useful to discriminate one outbreak from another. All virus-positive cases, including 49 cases involved in three outbreaks without any obvious epidemiological link with importation, were considered as import-related based on the nucleotide sequence information. Chain of transmission in the latest outbreak in 2014 terminated after the third generations, much earlier than the 2010-11 outbreak (6th generations). CONCLUSION Since 2010, almost all measles cases reported in Aichi Prefecture are either import or import-related, based primarily on genotypes and nucleotide sequences of measles virus detected. In addition, genotyping and molecular epidemiological analyses are indispensable to prove the interruption of endemic transmission when the importations of measles are repeatedly observed.