Trung Vu Nguyen

Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health

Significance Klebsiella pneumoniae is rapidly becoming untreatable using last-line antibiotics. It is especially problematic in hospitals, where it causes a range of acute infections. To approach controlling such a bacterium, we first must define what it is and how it varies genetically. Here we have determined the DNA sequence of K. pneumoniae isolates from around the world and present a detailed analysis of these data. We show that there is a wide spectrum of diversity, including variation within shared sequences and gain and loss of whole genes. Using this detailed blueprint, we show that there is an unrecognized association between the possession of specific gene profiles associated with virulence and antibiotic resistance and the differing disease outcomes seen for K. pneumoniae. Klebsiella pneumoniae is now recognized as an urgent threat to human health because of the emergence of multidrug-resistant strains associated with hospital outbreaks and hypervirulent strains associated with severe community-acquired infections. K. pneumoniae is ubiquitous in the environment and can colonize and infect both plants and animals. However, little is known about the population structure of K. pneumoniae, so it is difficult to recognize or understand the emergence of clinically important clones within this highly genetically diverse species. Here we present a detailed genomic framework for K. pneumoniae based on whole-genome sequencing of more than 300 human and animal isolates spanning four continents. Our data provide genome-wide support for the splitting of K. pneumoniae into three distinct species, KpI (K. pneumoniae), KpII (K. quasipneumoniae), and KpIII (K. variicola). Further, for K. pneumoniae (KpI), the entity most frequently associated with human infection, we show the existence of >150 deeply branching lineages including numerous multidrug-resistant or hypervirulent clones. We show K. pneumoniae has a large accessory genome approaching 30,000 protein-coding genes, including a number of virulence functions that are significantly associated with invasive community-acquired disease in humans. In our dataset, antimicrobial resistance genes were common among human carriage isolates and hospital-acquired infections, which generally lacked the genes associated with invasive disease. The convergence of virulence and resistance genes potentially could lead to the emergence of untreatable invasive K. pneumoniae infections; our data provide the whole-genome framework against which to track the emergence of such threats.

Streptococcus suis, an Important Cause of Adult Bacterial Meningitis in Northern Vietnam

Background Streptococcus suis can cause severe systemic infection in adults exposed to infected pigs or after consumption of undercooked pig products. S. suis is often misdiagnosed, due to lack of awareness and improper testing. Here we report the first fifty cases diagnosed with S. suis infection in northern Viet Nam. Methodology/Principal Findings In 2007, diagnostics for S. suis were set up at a national hospital in Hanoi. That year there were 43 S. suis positive cerebrospinal fluid samples, of which S. suis could be cultured in 32 cases and 11 cases were only positive by PCR. Seven patients were blood culture positive for S. suis but CSF culture and PCR negative; making a total of 50 patients with laboratory confirmed S. suis infection in 2007. The number of S. suis cases peaked during the warmer months. Conclusions/Significance S. suis was commonly diagnosed as a cause of bacterial meningitis in adults in northern Viet Nam. In countries where there is intense and widespread exposure of humans to pigs, S. suis can be an important human pathogen.

Influenza A H5N1 Clade 2.3.4 Virus with a Different Antiviral Susceptibility Profile Replaced Clade 1 Virus in Humans in Northern Vietnam

Background Prior to 2007, highly pathogenic avian influenza (HPAI) H5N1 viruses isolated from poultry and humans in Vietnam were consistently reported to be clade 1 viruses, susceptible to oseltamivir but resistant to amantadine. Here we describe the re-emergence of human HPAI H5N1 virus infections in Vietnam in 2007 and the characteristics of the isolated viruses. Methods and Findings Respiratory specimens from patients suspected to be infected with avian influenza in 2007 were screened by influenza and H5 subtype specific polymerase chain reaction. Isolated H5N1 strains were further characterized by genome sequencing and drug susceptibility testing. Eleven poultry outbreak isolates from 2007 were included in the sequence analysis. Eight patients, all of them from northern Vietnam, were diagnosed with H5N1 in 2007 and five of them died. Phylogenetic analysis of H5N1 viruses isolated from humans and poultry in 2007 showed that clade 2.3.4 H5N1 viruses replaced clade 1 viruses in northern Vietnam. Four human H5N1 strains had eight-fold reduced in-vitro susceptibility to oseltamivir as compared to clade 1 viruses. In two poultry isolates the I117V mutation was found in the neuraminidase gene, which is associated with reduced susceptibility to oseltamivir. No mutations in the M2 gene conferring amantadine resistance were found. Conclusion In 2007, H5N1 clade 2.3.4 viruses replaced clade 1 viruses in northern Vietnam and were susceptible to amantadine but showed reduced susceptibility to oseltamivir. Combination antiviral therapy with oseltamivir and amantadine for human cases in Vietnam is recommended.

Antibiotic use and resistance in emerging economies: a situation analysis for Viet Nam

BackgroundAntimicrobial resistance is a major contemporary public health threat. Strategies to contain antimicrobial resistance have been comprehensively set forth, however in developing countries where the need for effective antimicrobials is greatest implementation has proved problematic. A better understanding of patterns and determinants of antibiotic use and resistance in emerging economies may permit more appropriately targeted interventions.Viet Nam, with a large population, high burden of infectious disease and relatively unrestricted access to medication, is an excellent case study of the difficulties faced by emerging economies in controlling antimicrobial resistance.MethodsOur working group conducted a situation analysis of the current patterns and determinants of antibiotic use and resistance in Viet Nam. International publications and local reports published between 1-1-1990 and 31-8-2012 were reviewed. All stakeholders analyzed the findings at a policy workshop and feasible recommendations were suggested to improve antibiotic use in Viet Nam.Here we report the results of our situation analysis focusing on: the healthcare system, drug regulation and supply; antibiotic resistance and infection control; and agricultural antibiotic use.ResultsMarket reforms have improved healthcare access in Viet Nam and contributed to better health outcomes. However, increased accessibility has been accompanied by injudicious antibiotic use in hospitals and the community, with predictable escalation in bacterial resistance. Prescribing practices are poor and self-medication is common – often being the most affordable way to access healthcare. Many policies exist to regulate antibiotic use but enforcement is insufficient or lacking.Pneumococcal penicillin-resistance rates are the highest in Asia and carbapenem-resistant bacteria (notably NDM-1) have recently emerged. Hospital acquired infections, predominantly with multi-drug resistant Gram-negative organisms, place additional strain on limited resources. Widespread agricultural antibiotic use further propagates antimicrobial resistance.ConclusionsFuture legislation regarding antibiotic access must alter incentives for purchasers and providers and ensure effective enforcement. The Ministry of Health recently initiated a national action plan and approved a multicenter health improvement project to strengthen national capacity for antimicrobial stewardship in Viet Nam. This analysis provided important input to these initiatives. Our methodologies and findings may be of use to others across the world tackling the growing threat of antibiotic resistance.

Use of Colistin and Other Critical Antimicrobials on Pig and Chicken Farms in Southern Vietnam and Its Association with Resistance in Commensal Escherichia coli Bacteria.

ABSTRACT Antimicrobial resistance (AMR) is a global health problem, and emerging semi-intensive farming systems in Southeast Asia are major contributors to the AMR burden. We accessed 12 pig and chicken farms at key stages of production in Tien Giang Province, Vietnam, to measure antimicrobial usage and to investigate the prevalence of AMR to five critical antimicrobials (β-lactams, third-generation cephalosporins, quinolones, aminoglycosides, and polymyxins) and their corresponding molecular mechanisms among 180 Escherichia coli isolates. Overall, 94.7 mg (interquartile range [IQR], 65.3 to 151.1) and 563.6 mg (IQR, 398.9 to 943.6) of antimicrobials was used to produce 1 kg (live weight) of chicken and pig, respectively. A median of 3 (out of 8) critical antimicrobials were used on pig farms. E. coli isolates exhibited a high prevalence of resistance to ampicillin (97.8% and 94.4% for chickens and pigs, respectively), ciprofloxacin (73.3% and 21.1%), gentamicin (42.2% and 35.6%), and colistin (22.2% and 24.4%). The prevalence of a recently discovered colistin resistance gene, mcr-1, was 19 to 22% and had strong agreement with phenotypic colistin resistance. We conducted plasmid conjugation experiments with 37 mcr-1 gene-positive E. coli isolates and successfully observed transfer of the gene in 54.0% of isolates through a plasmid of approximately 63 kb, consistent with one recently identified in China. We found no significant correlation between total use of antimicrobials at the farm level and AMR. These data provide additional insight into the role of mcr-1 in colistin resistance on farms and outline the dynamics of phenotypic and genotypic AMR in semi-intensive farming systems in Vietnam. IMPORTANCE Our study provides accurate baseline information on levels of antimicrobial use, as well as on the dynamics of phenotypic and genotypic resistance for antimicrobials of critical importance among E. coli over the different stages of production in emerging pig and poultry production systems in Vietnam. E. coli isolates showed a high prevalence of resistance (>20%) to critically important antimicrobials, such as colistin, ciprofloxacin, and gentamicin. The underlying genetic mechanisms identified for colistin (the mcr-1 gene) and quinolone (gyrA gene mutations) are likely to play a major role in AMR to those compounds. Conjugation experiments led to the identification of a 63-kb plasmid, similar to one recently identified in China, as the potential carrier of the mcr-1 gene. These results should encourage greater restrictions of such antimicrobials in Southeast Asian farming systems.

Laboratory Capacity Building in Asia for Infectious Disease Research: Experiences from the South East Asia Infectious Disease Clinical Research Network (SEAICRN)

Heiman Wertheim and colleagues discuss a network that aims to improve infectious disease management through integrated, collaborative clinical research in South East Asia.

Human telomeric DNA sequences are a major target for the antitumour drug bleomycin.

The DNA sequence specificity of the cancer chemotherapeutic agent bleomycin was examined in a human telomeric DNA sequence and compared with that of non-telomeric sequences. The target DNA sequence contained 17 repeats of the human telomeric sequence and other primary sites of bleomycin cleavage. The 377-base-pair target DNA was fluorescently labelled at the 3′-end, damaged with bleomycin and electrophoresed in an ABI 3730 automated capillary sequencer to determine the intensity and sequence specificity of bleomycin damage. The results revealed that bleomycin cleaved primarily at 5′-GT in the telomeric sequence 5′-GGGTTA. Maxam–Gilbert chemical sequencing reactions were utilised as DNA size markers to determine the precise sites of bleomycin cleavage. The telomeric region contained strong sites of bleomycin cleavage and constituted 57% of the 30 most intense bleomycin damage sites in the DNA sequence examined. These data indicated that telomeric DNA sequences are a major site for bleomycin damage.

Hand-powered microfluidics: A membrane pump with a patient-to-chip syringe interface

In this paper, we present an on-chip hand-powered membrane pump using a robust patient-to-chip syringe interface. This approach enables safe sample collection, sample containment, integrated sharps disposal, high sample volume capacity, and controlled downstream flow with no electrical power requirements. Sample is manually injected into the device via a syringe and needle. The membrane pump inflates upon injection and subsequently deflates, delivering fluid to downstream components in a controlled manner. The device is fabricated from poly(methyl methacrylate) (PMMA) and silicone, using CO2 laser micromachining, with a total material cost of ∼0.20 USD/device. We experimentally demonstrate pump performance for both deionized (DI) water and undiluted, anticoagulated mouse whole blood, and characterize the behavior with reference to a resistor-capacitor electrical circuit analogy. Downstream output of the membrane pump is regulated, and scaled, by connecting multiple pumps in parallel. In contrast to existing on-chip pumping mechanisms that typically have low volume capacity (∼5 μL) and sample volume throughput (∼1-10 μl/min), the membrane pump offers high volume capacity (up to 240 μl) and sample volume throughput (up to 125 μl/min).

Acute Febrile Myalgia in Vietnam due to Trichinellosis following the Consumption of Raw Pork

Trichinellosis outbreaks occur occasionally in Vietnam following the consumption of undercooked pork. Diagnosing trichinella can be problematic because fever and myalgia are nonspecific, and diagnosis may be delayed. We describe 5 Vietnamese patients in whom trichinellosis was diagnosed after several weeks of illness.

The Use of Automated Sequencing Techniques to Investigate the Sequence Selectivity of DNA‐Damaging Agents

In this review, the use of automated DNA sequencing techniques to determine the sequence specificity of compounds that interact with DNA is discussed. The sequence specificity of a DNA‐damaging agent is an essential element in determining the cellular mechanism of action of a drug. A number of DNA‐damaging compounds are mutagenic, carcinogenic, as well as being widely used as cancer chemotherapeutic agents. The distribution of lesions in a sequence of DNA can give vital clues in the determination of the precise mechanism of interaction of the agent with DNA. The DNA sequence specificity of a number of DNA‐damaging agents has been delineated using automated DNA sequencing technology, and these studies are discussed in this review. The current state‐of‐the‐art methodology involves capillary electrophoresis with laser‐induced fluorescence detection usually on an Applied Biosystems ABI 3730 capillary sequencer. This current technique has higher resolution, greater sensitivity, higher precision, more rapid separation times, is safer and easier to perform than previous methods. The two main methods to determine the DNA sequence selectivity of compounds that interact with DNA are described: end labelling and the polymerase stop assay. The interaction of the antitumour drug, bleomycin, with DNA is utilized to illustrate the recent technological advances.