Pandemic influenza A (H1N1) virus (AH1pdm) emerged in April 2009. An inactivated, split-virus, unadjuvanted AH1pdm vaccine was manufactured in Japan, and vaccination was initiated with top priority for healthcare workers (HCWs) on October 19, 2009. A retrospective cohort study was conducted to evaluate the effectiveness of a single-dose vaccine for HCWs in a hospital in Japan. A total of 1,567 (84.5%) of 1,854 HCWs were vaccinated. Thirty-seven were infected with AH1pdm before the vaccine became available, and were excluded. The other 250 were not vaccinated for personal reasons. We analyzed the influenza infection rate with or without vaccination and related adverse events. Among the 1,817 HCWs without previous infection, 37 were infected with AH1pdm; 13 (5.2%) of 250 unvaccinated HCWs became infected, which was a significantly higher rate than the 24 (1.5%) of 1,567 vaccinated HCWs (P=0.001). Multivariate analysis revealed that age of 20–29 years was a risk factor for infection (adjusted odds ratio [aOR], 3.7; P<0.001), and that vaccination was a preventive factor (aOR, 0.20; P<0.001). Adverse events occurred in 549 of 1,060 HCWs, but most were mild. Although vaccination was carried out during AH1pdm epidemic expansion, the single-dose AH1pdm vaccine proved effective in HCWs, and severe adverse events were rare.

In this study, we characterized adverse events related to influenza A (H1N1) vaccination and studied the factors that influence the occurrence of these events. A total of 4,302 personnel in 18 military healthcare units in Korea received 0.5 ml of inactivated H1N1 vaccine. The study questionnaires were answered by 3,939 (91.6%) personnel, at both 2 weeks and 4 weeks after vaccination. Among these subjects, 3,531 (82.1%) who responded to all questions in the questionnaire were studied. After immunization, military doctors were ordered to report the occurrence of any adverse event related to the vaccine for 2 months. According to the responses of the subjects, the most prevalent events were fatigue (11.3%), pain at the injection site (8.38%), and myalgia (6.97%). Female gender, being in the age range of 20–49 years, obesity, regular alcohol consumption, and comorbidity, but not smoking status or pregnancy, were related to a high incidence of local or systemic adverse events after H1N1 vaccination. A total of 14 cases of adverse events were reported by the military doctors. In most reported cases, the subjects had fever in addition to the primary adverse event, and one patient was diagnosed with pneumonia. In conclusion, the overall burden of adverse events related to influenza A (H1N1) vaccination was not inconsequential, but most symptoms were mild. Female gender, middle-age range of 20-49 years, obesity, regular alcohol consumption, and comorbidity were risk factors for the occurrence of adverse events after H1N1 vaccination.

Because healthcare workers played an important role in the battle against novel pandemic (H1N1) 2009 influenza, a clinical study was conducted to examine the immunogenicity and safety of a single dose of a China-made monovalent, split-virus, pandemic (H1N1) 2009 influenza vaccine in this special high-risk population. Healthcare workers in the First Affiliated Hospital of Guangzhou Medical College who received the pandemic (H1N1) 2009 influenza vaccine were prospectively enrolled. Antibody titers were measured at enrollment and 14 days later using hemagglutination-inhibition (HI) and microneutralization assays. Adverse events were recorded within 7 days and 6 months after vaccination. Double sera were provided by 51 of 65 enrolled subjects. Postvaccination titers of 1:40 or more on HI assay were observed in 96% of recipients. Seroconversion or a significant increase in titer on HI assay occurred in 59% of subjects. The factor increase in GMTs was 4.3. The majority of complaints were mild to moderate in intensity. Although more than half of healthcare workers seemed immune to the pandemic (H1N1) influenza A virus before vaccination, most of the subjects still showed a fast, robust immune response to a single 15-μg dose of a monovalent, split-virus unadjuvanted pandemic H1N1 2009 influenza vaccine.

Isolation and determination of the nucleotide sequence of hemagglutinin (HA) of the pandemic (H1N1) 2009 influenza viruses found in Nagasaki, Japan, were conducted. The alignment results of the predicted HA amino acid sequences of these strains compared to the known global isolates revealed 5 specific amino acid differences located within the antigenic sites. The phylogenetic analyses revealed that the majority of the Nagasaki isolates could be classified into 6 phylogenetic clusters. Almost all isolates collected in the early season were classified into cluster I, which apparently originated from A/Nagasaki/HA-6/2009 isolated from a patient who returned from the Philippines. This cluster ceased to spread after November 2009. Between the end of August 2009 and January 2010, 5 new phylogenetic clusters (II–VI) emerged with viruses from different origins, and cluster III continuously advanced until March 2010. These results suggest that the onset of the influenza epidemic in Nagasaki originated from patient(s) who returned from the Philippines, and subsequently, various imported strains from different origins sustained the virus spread. Among the Nagasaki isolates, A/Nagasaki/HA-58/2009 having an H275Y mutation in the neuraminidase gene, which confers resistance to oseltamivir, was isolated. This is the first report in which an oseltamivir-resistant pandemic H275Y mutant was identified in Nagasaki Prefecture.

We report on two children with mild encephalopathy with a reversible splenial lesion associated with group A rotavirus (GARV) infection. We examined stool, serum, and cerebrospinal fluid samples to determine the presence of the GARV VP7 gene and GARV antigen by reverse-transcription PCR and enzyme-linked immunosorbent assay, respectively. GARV antigen was detected in stool samples from both patients. The GARV G genotype was G9 in one child and G3 in the other. GARV antigens were also found in both serum samples. However, the GARV VP7 gene was detected in only one serum sample, which was collected on the first day of symptomatic illness. Neither GARV antigen nor the VP7 gene was detected in cerebrospinal fluid samples. Both patients had excellent outcomes. Our results suggest that the reversible splenial changes in our patients might have been caused by indirect effects to the central nervous system subsequent to viral infection.

Serological samples of healthy people were collected to obtain the levels of measles antibodies in different groups of people in Heilongjiang Province, China. Using quantitative enzyme-linked immunosorbent assays to measure the antibody levels, we found the lowest antibody positive rate and the lowest geometric mean concentration values in healthy people aged 15–39. This group is the population at high risk for adult measles in Heilongjiang Province, and is the focus of measles elimination work. The new challenges for the eventual elimination of measles will be to address immunization strategies in this segment of the population in order to control the incidence.

The JC polyomavirus (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease. The current diagnostic standard for PML is real-time PCR testing of extracted DNA for assessing the presence of JCV DNA in cerebrospinal fluid (CSF). This study was aimed at evaluating the feasibility of a real-time PCR assay without nucleic acid extraction for the rapid quantification of JCV DNA in CSF. CSF samples were heat-treated or treated with DNAzol Direct, a commercially available reagent for direct PCR, and the performances of the real-time PCR assays using templates obtained by either treatment were compared with that using DNA extracts. JCV DNA was detected in the heat- or DNAzol Direct-treated samples containing only a few copies of the viral genome per reaction, and a linear relationship was noted between the copy number detected and the amount of input virus ascertained by the DNA extraction method. The sensitivities of the assays using the heat and DNAzol Direct treatments were 85.7 and 90.5%, respectively, with the results of the DNA extraction method being used as reference. These data demonstrate that the real-time PCR assay introduced in this study can serve as a rapid and cost-effective method of testing for JCV without DNA extraction and thereby facilitate the assessment of PML.

The Asian tiger mosquito, Aedes albopictus (Skuse), is the major vector of Chikungunya fever and the secondary vector of dengue fever. We collected Ae. albopictus from Singapore and performed genotyping assay to detect mutations of the voltage-gated sodium channel, which is the target site of pyrethroid insecticides. We detected an amino acid substitution, F1534C, which is suspected to confer knockdown resistance (kdr) to pyrethroid insecticides. Of the collected mosquitoes, 53.8% were homozygous for this mutation, and the allele frequency of this mutation was estimated to be 73.1%. No kdr mutation was detected in the 5 other loci of domains II and IV. This is the first evidence for the presence of the kdr gene in Ae. albopictus, and our findings highlight the need for studying the global distribution of this allele in this important vector insect.

We investigated the molecular epidemiology, antibiotic susceptibility, and antimicrobial resistant gene determinants of 23 multidrug-resistant (MDR) Acinetobacter baumannii samples that were collected from 5 proximal hospitals in Taiwan during April and May 2009. Major antibiotic resistance varied from 82.6 to 100%. Fivw pulsotypes were observed to spread clonally among the 5 hospitals. PCR screening revealed high distributions of intI1 (91%), bla_OXA-23 (57%), bla_ampC (100%), adeB (100%), adeJ (100%), and abeM (100%) genes, which were prevalent in the MDR A. baumannii isolates. Resistance gene expression was examined by reverse transcription-PCR, and showed that increased ampC expression was associated with ceftazidime resistance, but expression of adeB, adeJ, or abeM did not guarantee antimicrobial resistance phenotypes. In addition, imipenem resistance in some A. baumannii strains may be mainly modulated by genes other than bla_OXA-51-like. This is the first direct evidence indicating local spread of MDR A. baumannii in Taiwan. The resistance gene determinants are widely distributed in clonal and nonclonal-related isolates.

Restriction enzymes SpeI and XbaI were used in a pulsed-field gel electrophoresis (PFGE) study for molecular characterization of 146 clinical Burkholderia pseudomallei isolates. The PFGE parameters were optimized to enable comparable, reproducible, and robust results. The optimized parameters for both SpeI and XbaI restriction enzymes used in this study were 200 V and a pulse time of 5 to 65 s for a 28-h runtime. Using SpeI, 9 different clusters were identified, whereas 6 clusters were identified by XbaI digestion, which exhibited 85% similarity to SpeI. SpeI (discrimination index [D]=0.854) showed higher discriminatory power than XbaI did (D=0.464).

We herein present the third set of documented clinical Mycobacterium setense cases. Three clinically unrelated isolates were identified and characterized using various key conventional and molecular diagnostic tests. Phenotypic and molecular data analysis, particularly 16S rDNA, hsp65, and rpoB sequencing, provided evidence of M. setense involvement in clinical infections in Iranian patients. Our findings may shed light on the capability of this rare Mycobacterium sp. to cause infection in both healthy and immunocompromised patients.

The objective of this study was to characterize the off-seasonal influenza virus A subtype H3N2, which caused an outbreak in an elderly hospital in Niigata, Japan. Virus isolates were subtyped by the hemagglutination-inhibition test and screened for antiviral drug sensitivity by real-time PCR using cycling probe technology the and 50% inhibitory concentration (IC₅₀) method. Whole genome sequencing was performed in order to determine the phylogeny of the outbreak virus. Seven virus isolates were analyzed in this study, and the results showed that all belonged to the influenza virus A (H3N2). These viruses exhibited the S31N mutation in M2, which confers resistance to amantadine. The results of the IC₅₀ analysis showed that these viruses were sensitive to both oseltamivir and zanamivir. Whole genome analysis revealed that the virus was similar to the A/Perth/16/2009 strain and that it is a triple reassortant virus with a 3+3+2 pattern of segment recombination.

Respiratory syncytial virus (RSV) is the most important virus associated with bronchiolitis in infants and young children. The regulated upon activation, normal T-cell expressed and secreted protein (RANTES, also known as CCL5) appears to be a key player in the etiology of RSV-infected airway inflammation. In this study, we genotyped three single-nucleotide polymorphisms in the RANTES gene: –403G/A, –28C/G, and In1.1T/C in 59 infants with severe RSV bronchiolitis and 201 control subjects. The frequencies of the –403G/A+A/A, –28C/G+G/G, and In1.1T/C+C/C genotypes were significantly lower in patients with severe RSV bronchiolitis than in control subjects, and the frequencies of the –403A, –28G, and In1.1C alleles were significantly lower in RSV patients than in control subjects. The present results suggest that RANTES polymorphisms may confer risk for severe RSV bronchiolitis.

The suitability of the FTA^® elute card for the collection of slit skin smear (SSS) samples for PCR detection of Mycobacterium leprae was evaluated. A total of 192 SSS leprosy samples, of bacillary index (BI) 1 to 5, were collected from patients attending two skin clinics in Myanmar and preserved using both FTA^® elute cards and 70% ethanol tubes. To compare the efficacy of PCR detection of DNA from each BI class, PCR was performed to amplify an M. leprae-specific repetitive element. Of the 192 samples, 116 FTA^® elute card and 112 70% ethanol samples were PCR positive for M. leprae DNA. When correlated with BI, area under the curve (AUC) values of the respective receiver-operating characteristic curves were similar for the FTA^® elute card and ethanol collection methods (AUC=0.6). Taken together, our results indicate that the FTA^® elute card, which enables the collection, transport, and archiving of clinical samples, is an attractive alternative to ethanol preservation for the detection of M. leprae DNA.

In this study, 121 Escherichia coli samples isolated from clinical specimens obtained from Pakistan Institute of Medical Science, Islamabad, Pakistan, were analyzed for extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases using disk-diffusion assay and polymerase chain reaction. Of the isolates, 78 and 43 were identified as ESBL and AmpC producers, respectively. The highest resistance (89%) was observed against cefotaxime, followed by ciprofloxacin (87.6%) and cefepime (87%). Genetic analysis showed the presence of different class A and class C β-lactamase genes, either alone (44.7%) or in combination (53.6%). CTX-M (57.7%) was the most prevalent among class A, followed by TEM (20.3%) and SHV (15.4%). CIT (including LAT-1 to LAT-4, CMY-2 to CMY-7, and BIL-1) and MOX (including MOX-1, MOX-2, CMY-1, and CMY-8 to CMY-11) family-specific plasmid-mediated AmpC β-lactamases were the most prevalent among these isolates. Our study showed that both class A and class C β-lactamases contributed to cephalosporin resistance in the E. coli isolates, thereby limiting therapeutic options. Co-expression of these enzymes may further hinder the identification of ESBLs, which is a critical step for designing a successful treatment for multidrug-resistant E. coli.

Kazakhstan is one of the 14 countries with a high rate of morbidity due to multidrug-resistant tuberculosis (MDR TB) in WHO European region. The aim of our study was to characterize mutations associated with drug resistance to rifampicin and isoniazid in Mycobacterium tuberculosis isolates from Kazakhstan. M. tuberculosis strains were isolated from TB patients in different regions of Kazakhstan. A drug susceptibility test was performed on Lowenstein-Jensen medium using the absolute concentration method. Sequencing analysis was performed of the rpoB rifampicin resistance-determining region and the katG gene, the oxyR-ahpC intergenic region, and the inhA promoter region in 259 MDR M. tuberculosis isolates, in 51 isoniazid-resistant isolates, and in 13 rifampicin-resistant isolates. The mutational analysis revealed that the most frequent mutations associated with rifampicin and isoniazid resistance in M. tuberculosis are the substitutions at codons 531 (82.7%) and 315 (98.4%) in the rpoB and katG genes, respectively. In addition, we have found mutations with lower frequency at codon 526 (8.4%), 533 (1.5%), and 516 (1.1%) in the rpoB gene. In 6.2% of the isolates, no mutations were found in the rpoB gene. The findings of this study provide useful data for a better understanding of the mutation spectrum of isoniazid and rifampicin resistance among strains isolated from patients in Kazakhstan. Our results are also useful for the development of diagnostic tests of MDR M. tuberculosis.

The highly neuropathogenic cl-2 and less virulent srr7 viruses isolated from the neurotropic JHM strain of the mouse hepatitis virus exhibit super acute spread of virus (SAS), a term applied when rapid viral spread from an organ or part of the initially infected site to another non-adjacent organ or part is detected within 12 h after infection. Herein, we used a cytospin procedure to confirm SAS in splenic cells derived from mice whose brains were infected with these viruses. The cytospin procedure enabled effective preservation of the cells on glass slides. With this method, we could characterize extremely low populations of infected cells in the spleen (less than 0.1%) at 12 h post-inoculation with srr7. We observed that all kinds of splenic cells examined were infected, including B220⁺Ly-6C⁺ plasmacytoid dendritic cells. The population of viral antigen-positive splenic cells was only slightly higher in cl-2 infection than in srr7 infection, but the cells showing viral production were present in numbers significantly higher in cl-2 infection compared with srr7 infection.