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BACKGROUND: ß-Lactamase-nonproducing ampicillin-resistant Haemophilus influenzae are prevalent in Japan. Resistance has increased as a consequence of the expanded use of antimicrobial agents, raising concerns about the rise of multidrug (macrolide and fluoroquinolone)-resistant H. influenzae. METHODS: In this study, we investigated susceptibility to fluoroquinolones in H. influenzae clinical isolates from 2013 to 2014 and identified the amino acid substitutions in quinolone resistance-determining regions of gyrA and parC. RESULTS: All isolates (n = 145) were susceptible to fluoroquinolones; however, some showed reduced susceptibility. The minimum inhibitory concentration of levofloxacin for these strains was 0.063-0.5 µg/mL, and the strains harbored the amino acid substitution S84L in GyrA. Such strains have seen a significant increase. Importantly, all mutants from 2014 were isolated from pediatric patients. In addition, we developed a simple polymerase chain reaction-based screening method for detecting isolates with reduced fluoroquinolone susceptibility. CONCLUSIONS: The mutation in GyrA is important as a first step in the development of fluoroquinolone resistance. Hence, detection of reduced susceptible strains may influence the choice of antimicrobial treatment.

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ß-Lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae account for a large portion of H. influenzae clinical isolates in Japan. The aim of this study was to clarify the antimicrobial susceptibility of BLNAR H. influenzae clinical isolates as well as the annual changes in susceptibility. BLNAR H. influenzae isolates were collected from a tertiary care hospital from 2007 to 2012. Antimicrobial susceptibility testing was performed and resistance mechanisms were analysed. All of the isolates (n=304) had amino acid substitutions in penicillin-binding protein 3 (PBP3) and isolates were classified by these amino acid substitutions: R517H or N526K (class I); S385T and R517H (class II); and S385T and N526K (class III). Classes I, II and III represented 8.2% (n=25), 9.5% (n=29) and 81.6% (n=248) of the isolates, respectively; 2 isolates could not be classified because they had a PBP3 with a substantially mutated FtsI transpeptidase domain. All of the isolates were highly susceptible to fluoroquinolones and carbapenems. The number of clarithromycin (CAM)-non-susceptible [minimum inhibitory concentration (MIC) ≥16µg/mL] H. influenzae isolates increased significantly between 2010 and 2012. Moreover, CAM-non-susceptible H. influenzae isolates were prevalent among class II and class III BLNAR H. influenzae. Multilocus sequence typing (MLST) of the CAM-resistant (MIC ≥32µg/mL) H. influenzae isolates showed that they were not specific sequence types, suggesting that CAM resistance may occur in any isolates. These results raise concern regarding the occurrence of multidrug-resistant BLNAR H. influenzae.

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Pseudohypoaldosteronism type 1 (PHA1) is a rare condition characterized by neonatal salt loss with elevated plasma aldosterone and renin levels. Two types of PHA1 have been described: an autosomal recessive systemic form and an autosomal dominant renal form, in which the target organ defect is confined to the renal tubules. The dominant renal form of PHA1 is caused by heterozygous mutations in the NR3C2 gene, which encodes the mineralocorticoid receptor (MR). We determined clinical and biochemical parameters in two familial and four sporadic Japanese patient and analyzed the status of the NR3C2 gene. Failure to thrive was noted in five of the six patients. In one of the familial cases, the mother had an episode of failure to thrive when she was a toddler, but received no medical treatment. NaCl supplementation was discontinued in four of the six patients after they reached one year of age and they have grown normally thereafter. However, in one patient, 9 g/day of salt has been required to maintain serum Na concentration after 1 year of age. Analysis of NR3C2 identified three novel mutations [c. C1951T (p.R651X), c.304_305delGC (p.A102fsX103), c.del 603A (p.T201fsX34)] and one previously reported mutation [c.A2839G (p.947X)]. p.R651X was identified in one familial case and one unrelated sporadic patient. The patient who has been supplemented with large amount of salt was heterozygous for c.del 603A in exon 2. In conclusion, our study expands the spectrum of phenotypes, and characterized mutations of NR3C2 in the renal form of PHA1.

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Acute focal bacterial nephritis (AFBN) is a localized, interstitial bacterial infection of the renal parenchyma. In this study, we measured the serum levels of several cytokines in patients with AFBN. A total of 11 children were enrolled in the study and classified into two groups of patients: an AFBN group and a control group. There was no significant difference in the serum levels of interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, granulocyte-colony stimulating factor, or tumor necrosis factor-α among the patients in the two groups. However, the serum levels of interferon-γ among the patients in the AFBN group were significantly higher than those among the patients in the control group. The current results suggest that the bacterial kidney infection in the AFBN group is localized and that interferon-γ may be produced locally in response to the infection.

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We report 3 cases of respiratory syncytial virus infection-associated seizures; their abnormalities of cerebrospinal fluid (increased interleukin-6 and positive for virus by highly sensitive assay) were documented. These data revealed that neurological involvement might be caused by a direct invasion.

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Annual seasonal outbreaks of respiratory syncytial virus (RSV) infection occur every winter. Most patients are diagnosed clinically by a rapid detection kit for RSV protein(s) from nasopharyngeal secretion (NPS), but some problems have been reported on the specificity and sensitivity of such rapid detection kits. To ratify these issues, a sensitive, specific, simple, and rapid molecular based diagnostic method is expected to be introduced and we have developed a method to detect the RSV genome of subgroups A and B independently by reverse transcription loop-mediated isothermal amplification (RT-LAMP). We detected the genomic RNA corresponding approximately to 0.1 TCID 50 in the sample by RT-LAMP for both RSV subgroups under isothermal condition within 60 min after extraction of RNA. Specific DNA amplification was monitored by a real-time turbidimeter and the quantity of RNA was calculated. The RSV genome was detected in 47 of 50 NPS by RT-LAMP, and in 42 by nested RT-PCR, whereas virus isolation was positive for 29 and enzyme-linked immunoassay (EIA) for 34. RSV subgroup A was detected in 25 by RSV RT-LAMP A, RSV subgroup B in 23 by RSV RT-LAMP B, and dual infection with RSV subgroups A and B was identified in one case. They were confirmed with digestion with a specific restriction enzyme, Bgl II. The results showed the potential clinical feasibility of RT-LAMP as a useful diagnostic tool for the detection of RSV with high sensitivity similar to nested RT-PCR.

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