Kenta Shimizu

Bla NDM-1-positive Klebsiella pneumoniae from environment, Vietnam

To the Editor: The blaNDM-1 gene, which produces the New Delhi metallo-β-lactamase (NDM-1) enzyme, confers resistance to the carbapenem class of antimicrobial drugs and can be transferred among different types of bacteria. NDM-1 was identified in 2008 in Sweden from a patient from India who had been hospitalized in New Delhi (1). Since that report, blaNDM-1–positive bacteria have been identified from patients in several countries; most of these patients had a direct link with the Indian subcontinent (2). The spread of blaNDM-1 among bacterial pathogens is of concern not only because of resistance to carbapenems but also because such pathogens typically are resistant to multiple antimicrobial drug classes, which leaves few treatment choices available (3–5). In 2011, spread of blaNDM-1–positive bacteria in an environmental setting in New Delhi was reported (6). The possible appearance of bacteria harboring blaNDM-1 in Vietnam is of concern because cultural and economic links between Vietnam and India are strongly established, including extensive person-to-person exchanges that could enable easy exchange of pathogens. In addition, Vietnam faces a serious problem of antimicrobial drug resistance because drugs are freely available and used in an indiscriminate fashion. Thus, once blaNDM-1–positive bacteria colonize persons in Vietnam, they would be able to spread easily and pose a serious public health threat. During September 2011, we collected paired swab samples (1 for PCR, 1 for culture) of seepage water from 20 sites (rivers, lakes, and water pools in streets) within a 10-km radius of central Hanoi, Vietnam. Samples were transported in Transystem (COPAN Italia S.p.A, Brescia, Italy) to preserve bacteria and DNA. The 20 PCR swab specimens were squeezed out into 0.5-mL volumes of sterile water and centrifuged at 3,000 × g for 30 seconds; 1 μL of the resulting suspension was then used as PCR template to detect blaNDM-1 as described (7). Two samples were positive for blaNDM-1; these 2 samples were collected from the same river (Kim Nguu River) but at sites 3 km apart. To isolate and identify the phenotype and genotype of blaNDM-1–positive bacteria, we repeatedly spread the 20 culture swab specimens onto Muller-Hinton agar (Nissui, Tokyo, Japan) containing 100 mg/L vancomycin (Nakalai, Kyoto, Japan) plus 0.5 mg/L meropenem (LKT Laboratories, St. Paul, MN, USA) until single colonies were obtained. Each colony was then subcultured by plating onto MacConkey agar (Nihon Seiyaku, Tokyo, Japan) containing 0.5 mg/L meropenem to ensure culture purity; colonies were identified by using API 20E strips (bioMerieux, Basingstoke, UK). MICs of these isolates for 13 antimicrobial drugs were calculated by using Etest (bioMerieux), and susceptibility data were interpreted by using Clinical and Laboratory Standards Institute guidelines (www.clsi.org). We harvested several species of bacteria from the 2 seepage samples positive for blaNDM-1: Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, P. fluorescens/putida, and P. luteola. These isolates were placed onto media containing 0.5 mg/L meropenem, and bacterial DNA was extracted and used for the template for PCR analysis to detect blaNDM-1 as described (7). blaNDM-1 was detected in 3 K. pneumoniae isolates from each of the 2 positive samples (6 isolates total); this result was confirmed by sequencing. All 6 isolates were highly resistant to all β-lactam antimicrobial drugs, including carbapenems (Table). To detect another β-lactamase, multiplex PCRs were carried out as described (8); genetic variants blaTEM, blaSHV, blaOXA, blaCTX-M, blaIMP, blaVIM, and blaKPC were not detected in any of the isolates other than K. pneumoniae. All 6 K. pneumoniae isolates were positive for blaTEM and blaCTX-M variants by PCR; these variants were confirmed as blaTEM-1 and blaCTX-M-3 by sequencing. Table Resistance to 13 antimicrobial drugs of blaNDM-1–positive Klebsiella pneumoniae isolates from the Kim Nguu River, Hanoi, Vietnam* Aminoglycosides are often used in the management of severe infectious diseases caused by gram-negative pathogens. 16S rRNA methylases were found to confer high levels of resistance to aminoglycosides such as amikacin, tobramycin, and gentamicin. The 6 K. pneumoniae isolates we found were highly resistant to gentamicin (MIC >1,024 mg/L) and tobramycin (MIC 256–>1,024 mg/L) (Table). Therefore, we screened genetic elements of 16S rRNA methylases (rmtB, rmtC, and armA) by PCR and detected rmtB in all 6 isolates (9). Multilocus sequence typing was applied for these 6 isolates; all were identified as K. pneumoniae sequence type 283 (10), which had not been reported as harboring blaNDM-1. The azide-resistant Escherichia coli strain J53 has been used as recipient for conjugation assay, which had been reported previously (6), but we found no transconjugant strain with blaNDM-1 on MacConkey agar containing 100 mg/L sodium azide and 0.5 mg/L meropenem. Our results show that blaNDM-1–positive K. pneumoniae sequence type 283 is present in the Kim Nguu River, which flows through the central part of Hanoi at 2 sites. The isolates we obtained were also positive for 2 other β-lactamases, blaTEM-1 and blaCTX-M-3, were highly resistant to aminoglycosides related to rmtB, and showed mild elevation of MIC against ciprofloxacin up to 1.5 mg/L. Wide-scale surveillance of environmental and clinical samples in Vietnam and establishment of a strategy to prevent further spread of blaNDM-1 are urgently needed.

Truncated Hantavirus Nucleocapsid Proteins for Serotyping Sin Nombre, Andes, and Laguna Negra Hantavirus Infections in Humans and Rodents

ABSTRACT Sin Nombre virus (SNV), Andes virus (ANDV), and Laguna Negra virus (LANV) have been known as the dominant causative agents of hantavirus pulmonary syndrome (HPS). ANDV and LANV, with different patterns of pathogenicity, exist in a sympatric relationship. Moreover, there is documented evidence of person-to-person transmission of ANDV. Therefore, it is important in clinical medicine and epidemiology to know the serotype of a hantavirus causing infection. Truncated SNV, ANDV, and LANV recombinant nucleocapsid proteins (trNs) missing 99 N-terminal amino acids (trN100) were expressed using a baculovirus system, and their applicability for serotyping SNV, ANDV, and LANV infection by the use of enzyme-linked immunosorbent assays (ELISA) was examined. HPS patient sera and natural-reservoir rodent sera infected with SNV, ANDV, and LANV showed the highest optical density (OD) values for homologous trN100 antigens. Since even patient sera with lower IgM and IgG antibody titers were serotyped, the trN100s are therefore considered useful for serotyping with early-acute-phase sera. In contrast, assays testing whole recombinant nucleocapsid protein antigens of SNV, ANDV, and LANV expressed in Escherichia coli detected homologous and heterologous antibodies equally. These results indicated that a screening ELISA using an E. coli-expressed antigen followed by a serotyping ELISA using trN100s is useful for epidemiological surveillance in regions where two or more hantavirus species cocirculate.

Neutrophil Depletion Suppresses Pulmonary Vascular Hyperpermeability and Occurrence of Pulmonary Edema Caused by Hantavirus Infection in C.B-17 SCID Mice

ABSTRACT Hantavirus infections are characterized by vascular hyperpermeability and neutrophilia. However, the pathogenesis of this disease is poorly understood. Here, we demonstrate for the first time that pulmonary vascular permeability is increased by Hantaan virus infection and results in the development of pulmonary edema in C.B-17 severe combined immunodeficiency (SCID) mice lacking functional T cells and B cells. Increases in neutrophils in the lung and blood were observed when pulmonary edema began to be observed in the infected SCID mice. The occurrence of pulmonary edema was inhibited by neutrophil depletion. Moreover, the pulmonary vascular permeability was also significantly suppressed by neutrophil depletion in the infected mice. Taken together, the results suggest that neutrophils play an important role in pulmonary vascular hyperpermeability and the occurrence of pulmonary edema after hantavirus infection in SCID mice. IMPORTANCE Although hantavirus infections are characterized by the occurrence of pulmonary edema, the pathogenic mechanism remains largely unknown. In this study, we demonstrated for the first time in vivo that hantavirus infection increases pulmonary vascular permeability and results in the development of pulmonary edema in SCID mice. This novel mouse model for human hantavirus infection will be a valuable tool and will contribute to elucidation of the pathogenetic mechanisms. Although the involvement of neutrophils in the pathogenesis of hantavirus infection has largely been ignored, the results of this study using the mouse model suggest that neutrophils are involved in the vascular hyperpermeability and development of pulmonary edema in hantavirus infection. Further study of the mechanisms could lead to the development of specific treatment for hantavirus infection.

Novel serological tools for detection of Thottapalayam virus, a Soricomorpha-borne hantavirus

We developed serological tools for the detection of hantavirus-specific antibodies and hantavirus antigens in shrews. The work was focussed to generate Thottapalayam virus (TPMV)-specific monoclonal antibodies (mAbs) and anti-shrew immunoglobulin G (IgG) antibodies. The mAbs against TPMV nucleocapsid (N) protein were produced after immunization of BALB/c mice with recombinant TPMV N proteins expressed in Escherichia coli, baculovirus and Saccharomyces cerevisiae-mediated expression systems. In total, six TPMV N-protein-specific mAbs were generated that showed a characteristic fluorescent pattern in indirect immunofluorescence assay (IFA) using TPMV-infected Vero cells. Out of the six mAbs tested, five showed no cross-reaction to rodent-associated hantaviruses (Hantaan, Seoul, Puumala, Tula, Dobrava-Belgrade and Sin Nombre viruses) in IFA and enzyme-linked immunosorbent assay (ELISA), although one mAb reacted to Sin Nombre virus in IFA. None of the mAbs cross-reacted with an amino-terminal segment of the shrew-borne Asama virus N protein. Anti-shrew-IgG sera were prepared after immunization of rabbits and BALB/c-mice with protein-G-purified shrew IgG. TPMV-N-protein-specific sera were raised by immunisation of Asian house shrews (Suncus murinus) with purified yeast-expressed TPMV N protein. Using these tools, an indirect ELISA was developed to detect TPMV-N-protein-specific antibodies in the sera of shrews. Using an established serological assay, high TPMV N protein specific antibody titres were measured in the sera of TPMV-N-protein-immunized and experimentally TPMV-infected shrews, whereas no cross-reactivity to other hantavirus N proteins was found. Therefore, the generated mAbs and the established ELISA system represent useful serological tools to detect TPMV, TPMV-related virus antigens or hantavirus-specific antibodies in hantavirus-infected shrews.

Serological evidence of infection with rodent-borne hepatitis E virus HEV-C1 or antigenically related virus in humans

Zoonotic potential of a rat-derived hepatitis E virus (HEV), designated as HEV-C1, remains unknown. To evaluate the risk for HEV-C1 infection in humans, paired sera of 208 hospitalized febrile patients collected from 2001 to 2003 in Hanoi, Vietnam, were examined for IgG antibodies to HEV-C1 and genotype 1 HEV (HEV-1), which is common in humans. IgG antibodies to virus-like particles (VLPs) of HEV-C1 and/or HEV-1 were detected from 99 of the 208 convalescent sera in enzyme-linked immunosorbent assay (ELISA). IgG antibody titers to HEV-C1 antigen in 3 of the 99 sera were more than 8-fold higher than those to HEV-1 antigen. IgM antibodies to HEV-C1 antigen were detected in acute sera from 2 of the 3 patients in ELISA and Western blotting. However, no HEV genome was detected. Clinical information was available for 1 of the 2 patients. Hepatic enzymes, aspartate aminotransferase and alanine aminotransferase, were mildly elevated (156 IU/l and 68 IU/l, respectively), and hepatomegaly was detected by ultrasonography. The patient recovered from the illness after 17 days. These results indicated that HEV-C1 or its variants infect humans in Vietnam and may cause acute febrile illness with mild liver dysfunction.

Clinical and Epidemiological Characteristics of Scrub Typhus and Murine Typhus among Hospitalized Patients with Acute Undifferentiated Fever in Northern Vietnam.

A descriptive study on rickettsiosis was conducted at the largest referral hospital in Hanoi, Vietnam, to identify epidemiological and clinical characteristics of specific rickettsiosis. Between March 2001 and February 2003, we enrolled 579 patients with acute undifferentiated fever (AUF), excluding patients with malaria, dengue fever, and typhoid fever, and serologically tested for Orientia tsutsugamushi and Rickettsia typhi. Of the patients, 237 (40.9%) and 193 (33.3%) had scrub and murine typhus, respectively, and 149 (25.7%) had neither of them (non–scrub and murine typhus [non-ST/MT]). The proportion of murine typhus was highest among patients living in Hanoi whereas that of scrub typhus was highest in national or regional border areas. The presence of an eschar, dyspnea, hypotension, and lymphadenopathy was significantly associated with a diagnosis of scrub typhus (OR = 46.56, 10.90, 9.01, and 7.92, respectively). Patients with murine typhus were less likely to have these findings but more likely to have myalgia, rash, and relative bradycardia (OR = 1.60, 1.56, and 1.45, respectively). Scrub typhus and murine typhus were shown to be common causes of AUF in northern Vietnam although the occurrence of spotted fever group rickettsiae was not determined. Clinical and epidemiological information may help local clinicians make clinical diagnosis of specific rickettsioses in a resource-limited setting.

Rapid, whole blood diagnostic test for detecting anti-hantavirus antibody in rats.

Hantavirus is a causative agent of rodent-borne viral zoonoses, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome. Seoul virus (SEOV) is a causative agent of urban and laboratory rat-associated HFRS worldwide. Surveillance of rodents has been done mainly by serological detection of hantavirus-specific antibodies by enzyme linked immunosorbent assay (ELISA) and immunofluorescent antibody assay (IFA). An immunochromatographic (ICG) test was developed with the N-terminal 103 amino acids of nucleocapsid protein of Hantaan virus expressed by Escherichia coli as an antigen to detect IgG antibody specific to hantavirus in sera from Rattus sp. animals. Antibody-detecting sensitivity of the ICG test was the same as that of ELISA and about 100-times higher than that of IFA. Overall sensitivities and specificities of the ICG test in comparison to ELISA and IFA for sera from 192 urban rats and 123 laboratory rats were 99.3% and 100%, respectively. Diluted whole blood samples without separation could be used for the ICG test. The ICG test enabled detection of antibodies to SEOV, Hantaan, Dobrava/Belgrade, and Thailand viruses, which are causative agents of HFRS throughout Eurasia. The ICG test is a rapid, simple and safe method for diagnosis of SEOV infection in rats.

Development of a serotyping enzyme-linked immunosorbent assay system based on recombinant truncated hantavirus nucleocapsid proteins for New World hantavirus infection

New World hantaviruses were divided into five groups based on the amino acid sequence variability of the internal variable region (around 230-302 amino acids) of hantavirus nucleocapsid protein (NP). Sin Nombre virus (SNV), Andes virus, Black Creek Canal virus (BCCV), Carrizal virus (CARV) and Cano Delgadito virus belong to groups 1, 2, 3, 4 and 5, respectively. Patient and rodent sera were serotyped successfully by an enzyme-linked immunosorbent assay (ELISA) with recombinant truncated NP lacking 99 N-terminal amino acids (trNP100) of SNV, CARV and BCCV. The trNP100 of BCCV showed lower reactivity to heterologous sera. In contrast, whole recombinant NP antigens detected both homologous and heterologous antibodies equally. The results together with results of a previous study suggest that trNP100 can distinguish infections among viruses in groups 1, 2, 3 and 4 of New World hantaviruses. The serotyping ELISA with trNP100 is useful for epidemiological surveillance in humans and rodents.

A survey of rodent-borne pathogens carried by wild Rattus spp. in Northern Vietnam

To examine the prevalence of human pathogens carried by rats in urban areas in Hanoi and Hai Phong, Vietnam, we live-trapped 100 rats in January 2011 and screened them for a panel of bacteria and viruses. Antibodies against Leptospira interrogans (22·0%), Seoul virus (14·0%) and rat hepatitis E virus (23·0%) were detected in rats, but antibodies against Yersinia pestis were not detected. Antibodies against L. interrogans and Seoul virus were found only in adult rats. In contrast, antibodies to rat hepatitis E virus were also found in juvenile and sub-adult rats, indicating that the transmission mode of rat hepatitis E virus is different from that of L. interrogans and Seoul virus. Moreover, phylogenetic analyses of the S and M segments of Seoul viruses found in Rattus norvegicus showed that Seoul viruses from Hai Phong and Hanoi formed different clades. Human exposure to these pathogens has become a significant public health concern.

Different cross-reactivity of human and rodent sera to Tula virus and Puumala virus

Tula virus (TULV) and Puumala virus (PUUV) are hantaviruses carried by the bank vole (Myodes glareolus) and European common vole (Microtus arvalis), respectively. PUUV is a causative agent of hemorrhagic fever with renal syndrome (HFRS), while TULV is thought to be apathogenic to humans. The N-terminal regions of the N proteins from TULV and PUUV were expressed and applied as enzyme-linked immunosorbent assay (ELISA) antigens. Colonized Japanese grass voles (Microtus montebelli) and BALB/c mice were used for experimental inoculation of the vole-borne hantaviruses TULV and PUUV. Voles and mice showed significant antibody production toward both viruses, but these antisera showed little cross-reactivity between TULV and PUUV in the immunofluorescence antibody assay and ELISA. In contrast, sera from patients with HFRS caused by PUUV exhibited high cross-reactivity against the TULV antigen, and sera from a natural rodent reservoir showed moderate cross-reactivity against the heterologous antigen, indicating that the antigenic cross-reactivity between TULV and PUUV differs in sera from rodents and humans.