We investigated the molecular epidemiologic characteristics and virulence of hypermucoviscosity-positive Klebsiella pneumoniae in mainland China. We detected 16 hypermucoviscosity-positive strains in 65 total clinical isolates (24.62%). We found that 68.75% (11/16) of the positive strains had K2 genotype and carried the rmpA and iucA genes. Multilocus sequence typing revealed 5 sequence types (STs): ST65 [7], ST23 [4], ST86 [3], ST412 [1], ST375 [1], whereas the remaining 4 isolates were defined as other STs. The order of the median lethal dose values for the ST types was ST23 (2.19 × 10³ CFU/mouse) < ST86 (1.70 × 10⁴ CFU/mouse) < ST65 (5.05 × 10⁷ CFU/mouse) < the other STs (1.90 × 10⁸ CFU/mouse). In conclusion, the K2 with ST65 carrying rmpA and iucA was the most predominant among the hypermucoviscosity-positive K. pneumoniae strains obtained from community-acquired infection cases in Harbin, North China. Sequence types are a valuable tool to predict the risk of K. pneumoniae infection.

View full abstract

The Korea Middle East respiratory syndrome coronavirus (MERS-CoV) was first confirmed on May 20, 2015, with a subsequent outbreak in South Korea. Five patients with suspected MERS-CoA infection were admitted to our hospital during this outbreak. One patient had no major symptoms upon admission, but pneumonia was identified upon chest radiography. Two patients progressed rapidly to acute respiratory failure and required ventilator-assisted respiration. One patient required extracorporeal membrane oxygenation to treat refractory hypoxemia, and one patient died of shock with multiorgan failure. All the patients had fever, myalgia, leucopenia, normal procalcitonin level, and pneumonia. Importantly, clinicians should test for pneumonia in all suspected patients with MERS-CoV infection, even in the absence of respiratory symptoms. The pneumonia usually affected the lower lobes. A shorter incubation period was associated with more severe disease and greater risk of mortality, and the severity of fever predicted the prognosis of MERS-CoV infection-related pneumonia. Therefore, in cases of lower-lobe pneumonia that occur during an MERS-CoV outbreak and are unesponsive to antibiotics, clinicians should consider the possibility of MERS-CoV infection.

View full abstract

Because western blotting occasionally causes cross-reactions between human immunodeficiency virus (HIV)-1 and HIV-2, it is difficult to distinguish a coinfection status from a false-positive result. Therefore, we developed a qualitative real-time PCR assay to detect HIV-1 and HIV-2 RNA that can be performed in parallel. Viral RNA extracted from 500 μl of plasma was examined using real-time PCR with minor groove binder probes. Bovine leukemia virus was used as an internal standard. The sensitivity was determined by probit regression analysis using the World Health Organization international standards for HIV-1 and HIV-2. The lower detection limits at a 95% hit rate were 54 IU/ml for HIV-1 and 5.0 IU/ml for HIV-2, which were lower than any HIV-2 assays reported previously. HIV-1 RNA was detected in 51 of 52 HIV-1 seropositive plasma samples. HIV-2 RNA was detected in 7 of 10 HIV-2 seropositive plasma samples. Non-specific signals and cross reactivity between HIV-1 and HIV-2 were not observed in 100 HIV seronegative samples. The assay developed in this study is highly sensitive and specific for the detection of HIV-1 and HIV-2 RNA. The test is expected to be useful for the differential diagnosis of HIV-1 and HIV-2 infections.

View full abstract

The aims of this study were to determine the genetic relatedness among 20 clinical Campylobacter jejuni samples isolated from children with diarrhea in Iran and to introduce the best method of discrimination based on flagellin gene (flaA) sequence divergence. A total of 400 stool specimens were obtained from children under 5 years of age from July 2012 to June 2013. Primers were designed based on conserved sequences flanking the flaA gene that encompassed and amplified the entire flaA gene and followed by sequencing and data analysis with MEGA version 6.0.6 software. Ninety amino acids and 560 nucleotide polymorphic sequences were detected within 1,681 bp of the flaA sequence of which 43 (2.5%) and 12 (0.7%) were singletons, respectively. New repeat boxes within the flaA sequences were found in this study. Unweighted Pair Group Method with Arithmetic Mean dendrogram based on nucleotides of the full length flaA gene, the flaA short variable region gene and the in silico flaA phylogenic tree of DdeI restriction fragment length polymorphism (RFLP) profiles produced very similar clustering with a diversity index of 0.86 for each of the 3 methods. We conclude that flaA typing based on DdeI RFLP of the PCR products is a cheap, rapid, and reliable method for the epidemiological study of C. jejuni isolates of clinical origin in resource-limited regions or in large-scale population surveillance.

View full abstract

This study was designed to evaluate the diagnostic value of common inflammatory markers with regard to fever of unknown origin (FUO). We investigated 383 patients who were hospitalized with FUO at the Henan Province People’s hospital between January 2009 and June 2015. Of all the cases, infectious diseases accounted for 33.9%, neoplasms for 21.1%, collagen vascular diseases for 25.1%, miscellaneous diseases for 4.7%, and no diagnosis for 15.1%. Patients in the neoplasm group were older than those in the infectious disease, collagen vascular disease, and miscellaneous disease groups (p = 0.006, p < 0.0001, and p = 0.001, respectively). The duration of fever before admission of patients in the neoplasm and collagen vascular disease group was longer than that of patients in the infectious disease group (p = 0.002 and p = 0.007, respectively). The diagnostic time after admission of patients from the neoplasm and collagen vascular disease groups was longer than that for patients from the infectious disease group (both p < 0.0001). Serum ferritin levels of patients in the infectious disease group were lower than those of patients in the neoplasm and collagen vascular disease groups (p = 0.029 and p = 0.032, respectively), while serum procalcitonin (PCT) levels in the infectious disease group was higher than that in the neoplasm and collagen vascular disease groups (p = 0.016 and p = 0.007, respectively). Therefore, FUO remains a clinical problem in China and serum ferritin and PCT may be useful in discriminating infectious from non-infectious causes (neoplasms and collagen vascular diseases) in patients with FUO.

View full abstract

A newly emerged Vibrio cholerae O1 El Tor variant strain with multidrug resistance is considered a threat to public health. Recent strategies to suppress virulence factors production instead of bacterial growth may lead to less selective pressure for the emergence of resistant strains. The use of spices and their active constituents as the inhibitory agents against cholera toxin (CT) production in V. cholerae may be an alternative approach to treat cholera. In this study, we examined the potential of sweet fennel seed (Foeniculum vulgare Miller var. dulce) methanol extract to inhibit CT production in V. cholerae without affecting viability. The methanol extract of sweet fennel seeds significantly inhibited CT production in various V. cholerae strains, regardless of serogroup or biotype. Interestingly, trans-anethole and 4-allylanisole, essential oil components of sweet fennel seeds, also demonstrated similar effects. Here, we report that sub-bactericidal concentrations of sweet fennel seed methanol extract and its major components can drastically inhibit CT production in various V. cholerae strains.

View full abstract

Although E. coli O157:H7 is the major serotype among Shiga toxin-producing Escherichia coli (STEC) strains, non-O157 serotypes have caused numerous outbreaks worldwide. We aimed to evaluate the distribution of serogroups, serotypes, virulence genes, and antimicrobial resistance of STEC strains recovered from stool samples. A total of 395 stool samples characterized by watery/bloody diarrhea and/or symptoms of hemolytic-uremic syndrome were included in this study. Strains compatible with E. coli, based on biochemical tests, were tested for the presence of Shiga toxin by ELISA. Toxigenic strains were tested by serotyping and serogrouping. Virulence genes, stx1, stx2, aggR, hlyA, and eae were detected by polymerase chain reaction. Overall, 26 (6.6%) stool culture samples tested positive for STEC. Shiga toxin was positive in 28 (7.1%) patient isolates based on ELISA and PCR. Two isolates could not be serotyped. STEC strains were distributed into 10 serogroups and 14 serotypes. Of the serotyped strains, 92.3% were non-O157, with the major distribution in O104:H4 and O26:HNM. All were negative for extended-spectrum β-lactamase enzyme and 62.5% were resistant to at least 1 drug. This study demonstrated the wide distribution of non-O157 STEC strains from our patient group. Further studies should be performed to better understand STEC characteristics on a larger scale.

View full abstract

The V3 loop in the envelope (Env) of HIV-1 is one of the major targets of neutralizing antibodies. However, this antigen is hidden inside the Env trimer in most isolates and is fully exposed only during CD4-gp120 interaction. Thus, primary HIV-1 isolates are relatively resistant to anti-V3 antibodies because IgG is too large to access the V3 loop. To overcome this obstacle, we constructed single-chain variable fragments (scFvs) from anti-V3 monoclonal antibodies 0.5γ, 5G2, and 16G6. Enhanced neutralization by 0.5γ and 5G2 scFvs was observed in strains resistant to their IgG counterparts. Neutralization coverage by 0.5γ scFv reached up to 90% of the tested viruses (tier 2 and 3 classes). The temperature-regulated neutralization assay revealed that extensive cross-neutralization of 0.5γ scFv can be explained by post-attachment neutralization. Neutralization assay involving viruses carrying an inter-subunit disulfide bond (SOS virus) showed that the neutralization-susceptible timeframe after attachment was 60 to 120 min. These results indicate that the scFvs efficiently access the V3 loop and subsequently neutralize HIV-1, even after virus attachment to the target cells. Based on its broad and potent neutralizing activity, further development of anti-V3 scFv for therapeutic and preventive strategies is warranted.

View full abstract

Although Lewis X (Le^x), a carbohydrate structure, is involved in innate immunity through cell-to-cell and pathogen recognition, its expression has not been observed in mouse monocytes/macrophages (Mo/Mas). The Mo/Mas that infiltrate the meninges after infection with the neuropathogenic murine coronavirus strain srr7 are an initial target of infection. Furthermore, higher inflammatory responses were observed in gene-manipulated mice lacking α1,3-fucosyltransferase 9, which determines the expression of the Le^x structure, than in wild type mice after infection. We investigated Le^x expression using CD11b-positive peritoneal exudate cells (PECs) and found that Le^x is inducible in Mo/Mas after infection with srr7, especially in the syncytial cells during the late phase of infection. The number of syncytial cells was reduced after treatment of the infected PECs with anti-Le^x antibody, during the late phase of infection. In addition, the antibody treatment induced a marked reduction in the number of the infected cells at 24 hours post inoculation, without changing the infected cell numbers during the initial phase of infection. These data indicate that the Le^x structure could play a role in syncytial formation and cell-to-cell infection during the late phase of infection.

View full abstract

In order to study the epidemiology of human parechovirus (HPeV) infections and to evaluate the feasibility of environmental surveillance, we analyzed 281 stool samples, 265 nasopharyngeal swab samples, and 79 municipal wastewater samples for HPeV. The samples were collected in Miyagi Prefecture, Japan, between April 2012 and March 2014. HPeV was detected by reverse-transcription-PCR targeting the partial 5'-untranslated region and was genotyped by sequencing the capsid VP1 region. Seven stool samples (2.5%) and 1 nasopharyngeal swab sample (0.4%), all of which were from children under 2 years old, and 14 wastewater samples (18%) were positive for HPeV. Clear seasonality was observed: all positive samples were collected between July and December during the study period. All strains detected in the stool and wastewater samples had genotype HPeV1, and the strain from the nasopharyngeal swab sample had genotype HPeV6. A phylogenetic analysis revealed that all HPeV1 strains from the stool samples cluster together with those from the wastewater samples, indicating that the HPeV1 strains circulating in human populations can also be detected in municipal wastewater.

View full abstract

Rubella is usually a mild illness, with febrile rash being its main symptom. However, serious consequences of rubella infection can result when the infection occurs during the early stages of pregnancy. After the occurrence of a rubella outbreak in Japan that was observed from 2012 to 2013, 45 infants were reportedly born with congenital rubella syndrome (CRS). We prospectively followed the 15 CRS cases reported in Tokyo to determine the virus shedding periods by using nested reverse transcriptase-polymerase chain reaction to detect rubella virus genes. Throast swabs were used for virus detection. The virus shedding period was measured from birth until the time when the sample last tested positive followed by 2 consecutive negative samples. Kaplan-Meier method was used to estimate the proportion of cases remaining positive for rubella virus genes over time. The proportion of CRS cases shedding virus dropped steadily after birth, dropping to 33.8% at 6 months and 16.9% at 12 months. Our findings also suggested that the earlier the mother's onset of rubella during pregnancy, the longer the infant remained positive. Based on our findings, we believe that infants with CRS should be monitored for rubella virus shedding until 1 year of age.

View full abstract

Cat scratch disease (CSD) is an infectious disease caused by Bartonella henselae. Atypical clinical presentations of CSD include prolonged fever and multiple hepatosplenic lesions. Furthermore, multiple renal lesions are extremely rare in CSD. An 11-year-old Japanese girl presented at our hospital with a prolonged fever of unknown cause after being scratched and bitten by a kitten. Abdominal computed tomography (CT) revealed multiple small, round hypodense lesions in both kidneys and the spleen. Based on her history and the CT results, her diagnosis was CSD. The diagnosis was confirmed by serological tests, which indicated antibodies against B. henselae. After treatment with azithromycin, her fever immediately improved. Careful history taking and imaging are essential for the diagnosis of atypical CSD. In CT images, not only hepatosplenic lesions but also renal lesions are important features indicative of a diagnosis of atypical CSD. Subsequently, a diagnosis of CSD can be confirmed by specific serological tests. This is the first reported Japanese case of multiple renal and splenic lesions in a patient with CSD. Although difficult to diagnose, an early diagnosis atypical CSD and appropriate treatment are important to prevent complications and the need for invasive examinations.

View full abstract

Enterovirus D68 (EV-D68) is associated with severe lower respiratory tract infection and neurological abnormalities including acute myelitis and cranial nerve dysfunction. To determine whether an increased incidence of EV-D68 occurs in Southeast Asia, we retrospectively tested specimens collected from Thai pediatric patients who were less than 5 years of age and presented with acute respiratory tract infections between 2012 and 2014. Reverse transcription-polymerase chain reaction and nucleotide sequencing of the 5'-UTR/VP2 region were used to identify EV-D68. We also examined the epidemiological pattern of EV-D68 since 2009, when it was first identified in Thailand, and compiled records of clinical manifestations in children with confirmed EV-D68 infection. From 837 samples, 5 samples (0.6%) tested positive for EV-D68. All patients presented with viral pneumonia and required hospitalization. Phylogenetic analysis of the VP4/VP2 regions revealed that EV-D68 strains circulating in Thailand between 2012 and 2014 were closely related to strains reported in Japan, United Kingdom, China, and France. Continued surveillance of probable EV-D68-associated severe respiratory tract infection and the development of a rapid diagnostic test for EV-D68 are essential in supporting awareness and facilitating disease prevention and control.

View full abstract

Samples taken from 428 wild animals and 126 ticks, collected from a tularemia-endemic area in Japan between 2005 and 2013, were analyzed for the presence of Francisella tularensis. F. tularensis was isolated from a Japanese hare carcass whereas the samples from live animals and ticks were negative for F. tularensis by real-time PCR. Our results suggest that F. tularensis is still present in Japan although its prevalence is considerably low even in areas where tularemia is endemic.

View full abstract

Enzyme-linked immunosorbent assays (ELISAs) are considered the gold standard for the detection of various immunological reactions and can be used for the detection of infectious diseases during outbreaks or in the care of individual patients. To be useful in the timely implementation of prevention and control measures against infectious diseases, a diagnostic modality should be rapid, accurate, and affordable. In the current study, we demonstrate the efficiency (90% less time and volume consumption compared with those of a standard 96-well ELISA), detection capability, and ease of operation of a field-portable, battery-operated ELISA system, approximately the size of a cellular phone (12 × 6 × 5.5 cm), in the serological diagnosis of measles and rubella viruses that has the potential for onsite testing such as during disease outbreaks.

View full abstract

Meropenem-susceptible and -resistant Aeromonas dhakensis isolates from blood cultures of a fatal case of septicemia were analyzed. The two isolates were homologous and gene expression of metallo-β-lactamase in the resistant strain was upregulated. Physicians should be aware of the possibility of the induction of carbapenem-resistance, following the use of carbapenems in the treatment of Aeromonas infection.

View full abstract