Masanobu Ushio

Cerebrospinal fluid analysis in children with seizures from respiratory syncytial virus infection

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.

Prevalence of macrolide-non-susceptible isolates among β-lactamase-negative ampicillin-resistant Haemophilus influenzae in a tertiary care hospital in Japan.

β-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.

Cytokine profiles of suction pulmonary secretions from children infected with pandemic influenza A(H1N1) 2009.

Uncomplicated influenza in humans is characterized by massive virus replication in respiratory epithelial cells, inflammation and an abrupt onset. The novel influenza A (H1N1) 2009 caused an epidemic of critical illness and some patients rapidly developed severe acute respiratory distress syndrome [1,2]. Van Reeth [3] reviewed growing evidence that the so-called early cytokines produced at the site of infection mediate many of the clinical and pathological manifestations of influenza infection. Of those cytokines, Bermejo-Martin and colleagues [4] reported in Critical Care that T-helper 1 (Th1) and Th17 hypercytokinemia plays an important role as an early host response in severe pandemic influenza. Evaluating the differences in early immune responses between hospitalized patients with severe pandemic influenza and those with mild disease, high systemic levels of IFN-γ and a group of mediators involved in the development of the Th17 (IL-8, IL-9, IL-17, IL-6) and Th1 (TNF-α, IL-15, IL-12p70) responses were found exclusively in hospitalized patients. A significant inverse association was found between IL-6 and IL-8 and PaO2 in critical patients. They concluded that severe disease with respiratory involvement is characterized by early secretion of Th17 and Th1 cytokines. We experienced two cases of pandemic influenza A(H1N1) 2009-associated pneumonia and encephalopathy, which were treated under mechanical ventilation. Cytokine analysis of their pulmonary secretions revealed different patterns from previous results (Figure ​(Figure1).1). One patient showed no improvement with usual ventilation and had mediastinal emphysema and serious hypooxygenation; thus, the patient needed to be ventilated using the special respiratory airway pressure release ventilation mode because of progression and the need for high pressure control. The second case with encephalopathy complicated with pneumonia underwent combined treatments of steroids and hypothermia because of intractable recurrent seizures. Their cytokine levels were extremely high, although serum 17 cytokines were within normal ranges. Cytokines in pulmonary secretions at first revealed high levels of IL-8, monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein-1b (MIP-1b). On the other hand, IL-1b, 2, 4, 5, 6, 7, 10, 12, 13, 17, G-CSF, GM-CSF, IFN-γ and TNF-α were normal or slightly increased. On 5th days after hospitalization other cytokines (IL-1β, 6, 10, 17, G-CSF, GM-CSF, IFN-γ, MCP-1 and TNF-α) increased markedly in both cases. Given these findings, we suspect that chemokines play a role mostly in lung injury associated with influenza A(H1N1) 2009 infection in the early phase. High levels of chemokines and subsequent epithelial changes will increase the permeability in alveoli and fibrin leakage into interstitial tissue, followed by the consequent production of other inflammatory cytokines. Alternatively, we assume that secondary bacterial infections, which have been reported [5], influence the production of those cytokines (IL-6, IL-1b and TNF-α). Figure 1 Cytokines in the pulmonary secretions of a case with serious distress and oxygenic disturbance (upper panel). Another case showed almost similar patterns (lower panel). G-CSF, granulocyte colony stimulating factor; GM-CSF, granulocyte-macrophage colony ...

Marked elevation of interferon-γ in acute focal bacterial nephritis

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)-1β, 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.

Rise in Haemophilus influenzae With Reduced Quinolone Susceptibility and Development of a Simple Screening Method

Background: &bgr;-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.

Emergence and molecular characterization of Haemophilus influenzae harbouring mef(A)—authors’ response

Sir, We would like to respond to the comment by Atkinson et al. on our article ‘Emergence and molecular characterization of Haemophilus influenzae harbouring mef(A)’. There are several reports regarding the acquisition of resistance genes in H. influenzae. Roberts et al. reported that H. influenzae possessing mef(A), which encodes a drug efflux pump, and/or erm, which encodes 23S rRNA methylase, were isolated from patients with cystic fibrosis in 2011. On the other hand, Atkinson et al. reported that none of the clinical isolates in their study harboured any acquired resistance genes. Atkinson et al. also described that they obtained false-positive results when they used the primers described in a study conducted by Roberts et al. In response to this, Roberts et al. disclosed their primer list and stated that the primers pointed out by Atkinson et al. were not the same as those used or described in their study. Given the back-and-forth dialogue between Roberts et al. and Atkinson et al., we performed a verification experiment using the primers apparently used by both Roberts et al. and Atkinson et al. We used 20 clinical isolates, which were used in our previous epidemiological study and are susceptible to macrolides, and a mef(A)-harbouring strain, H. influenzae 2014-102, for our verification experiment. We used the primers listed in Table 1. Although we could not detect mef(A) in H. influenzae 2014-102 with the primer set MEFF and MEFR, mef(A) was specifically detected in H. influenzae 2014-102 when three different primer sets [MEF IN FW and MEF IN RV; mefA F and mefA R; mef(A)-F and mef(A)-R] were used. In the clinical isolates that did not contain mef(A), the PCR results were all negative. However, a weak band of 1200 bp similar to mef(A) was observed among several macrolide-susceptible isolates in a PCR using a different PCR enzyme and the MEFF and MEFR primer set (data not shown). The yield of this product was too low to perform sequencing, suggesting that this result was a non-specific reaction. Therefore, we suggest that the PCR for mef(A) conducted using the MEFF and MEFR primer set can yield false-positive or false-negative results depending on the PCR conditions. However, such results may reflect that the primer set MEFF and MEFR was previously used to detect mef(A) in Clostridium perfringens rather than in experiments with H. influenzae. As a result of our verification study we apologize for and retract the statement: ‘the primer sets used by Atkinson et al. were not appropriate’.