Development and clinical validation of a loop-mediated isothermal amplification method for the rapid detection of Neisseria meningitidis

Loop-mediated isothermal amplification (LAMP) is an innovative technique that allows the rapid detection of target nucleic acid sequences under isothermal conditions without the need for complex instrumentation. The development, optimization, and clinical validation of a LAMP assay targeting the ctrA gene for the rapid detection of capsular Neisseria meningitidis were described. Highly specific detection of capsular N. meningitidis type strains and clinical isolates was demonstrated, with no cross-reactivity with other Neisseria spp. or with a comprehensive panel of other common human pathogens. The lower limit of detection was 6 ctrA gene copies detectable in 48 min, with positive reactions readily identifiable visually via a simple color change. Higher copy numbers could be detected in as little as 16 min. When applied to a total of 394 clinical specimens, the LAMP assay in comparison to a conventional TaqMan® based real-time polymerase chain reaction system demonstrated a sensitivity of 100% and a specificity of 98.9% with a ? coefficient of 0.942. The LAMP method represents a rapid, sensitive, and highly specific technique for the detection of N. meningitidis and has the potential to be used as a point-of-care molecular test and in resource-poor settings.

Application of atmospheric pressure nonthermal plasma for the in vitro eradication of bacterial biofilms

The use of atmospheric pressure nonthermal plasma represents an interesting and novel approach for the decontamination of surfaces colonized with microbial biofilms that exhibit enhanced tolerance to antimicrobial challenge. In this study, the influence of an atmospheric pressure nonthermal plasma jet, operated in a helium and oxygen gas mixture under ambient pressure, was evaluated against biofilms of Bacillus cereus,Staphylococcus aureus,Escherichia coli and Pseudomonas aeruginosa. Within <4 min of plasma exposure, complete eradication of the two Gram-positive bacterial biofilms was achieved. Although Gram-negative biofilms required longer treatment time, their complete eradication was still possible with 10 min of exposure. Whilst this study provides useful proof of concept data on the use of atmospheric pressure plasmas for the eradication of bacterial biofilms in vitro, it also demonstrates the critical need for improved understanding of the mechanisms and kinetics related to such a potentially significant approach. © 2012 Federation of European Microbiological Societies.

Discovery and evolving history of two genetically related but phenotypically different viruses, porcine circoviruses 1 and 2

Porcine circoviruses (PCVs) belong to the genus Circovirus, family Circoviridae. and are the smallest non-enveloped, single stranded, negative sense, circular DNA viruses that replicate autonomously in mammalian cells. Two types of PCV have been characterised, PCV1 and PCV2 and these two viruses show 83% sequence identity at open reading frame (ORF) 1 and 67% identity at ORF2. PCV1 is a nonpathogenic virus of pigs. In contrast, PCV2 has emerged as a major pathogen of swine around the world. The discovery of PCV1 and how the subsequent studies on this virus eventually led to the recognition and characterisation of PCV2, and the disease scenarios associated with PCV2, serve as a model of how multidisciplinary collaboration among field veterinarians, diagnosticians and researchers can lead to the rapid characterisation and control of a globally important emerging disease. (C) 2011 Elsevier B.V. All rights reserved.

A nine-base nucleotide sequence in the porcine circovirus type 2 (PCV2) nucleocapsid gene determines viral replication and virulence

Porcine circovirus type 2 (PCV2) was retrospectively identified by serology in swine populations as an asymptomatic infection at least 25 years prior to the first reported case of PCV2-associated postweaning multisystemit wasting syndrome (PMWS). To investigate the sudden emergence of PMWS, viral sequences were amplified from frozen archived (1970-1971) porcine tissues and the complete genome of archival PCV2 was determined. The ORF1 gene product (viral DNA replicase) was homologous to contemporary PCV2 ORF1. In ORF2 (viral nucleocapsid gene) archival PCV2, a consistent linear nine-base sequence difference at base positions 1331 through 1339 was observed.The deduced amino acid sequence from these base changes alters the nucleocapsid conformation within the second immunogenic epitope from a hydrophobic (contemporary PCV2) to a hydrophilic (archival PCV2) configuration.

Detection of a novel porcine boca-like virus in the background of porcine circovirus type 2 induced postweaning multisystemic wasting syndrome

Porcine circovirus type 2 (PCV-2) has been found to be the causative agent of postweaning multisystemic wasting syndrome (PMWS). However, PCV-2 is a ubiquitous virus in the swine population and a majority of pigs infected with PCV-2 do not develop the disease. Different factors such as age, maintenance, the genetics of PCV-2, other pathogens, etc. have been suggested to contribute to the development of PMWS. However, so far no proven connection between any of these factors and the disease development has been found. In this study we explored the possible presence of other so far unknown DNA containing infectious agents in lymph nodes collected from Swedish pigs with confirmed PMWS through random amplification and high-throughput sequencing. Although the vast majority of the amplified genetic sequences belonged to PCV-2, we also found genome sequences of Torque Teno virus (TTV) and of a novel parvovirus. The detection of TTV was expected since like PCV-2, TTV has been found to have high prevalence in pigs around the world. We were able to amplify a longer region of the parvovirus genome, consisting of the entire NP1 and partial VP1/2. By comparative analysis of the nucleotide sequences and phylogenetic studies we propose that this is a novel porcine parvovirus, with genetic relationship to bocaviruses.

Bacteriophages as anti-infective agents: recent developments and regulatory challenges.

The biennial meeting on 'Exploiting Bacteriophages for Bioscience, Biotechnology and Medicine', held in London, UK, on 20 January 2012, and chaired by George Salmond (University of Cambridge, UK) hosted over 50 participants representing 13 countries. The highly multidisciplinary meeting covered a diverse range of topics, reflecting the current expansion of interest in this field, including the use of bacteriophages as the source of biochemical reagents for molecular biology, bacteriophages for the treatment of human and animal diseases, bacteriophage-based diagnostics and therapeutic delivery technologies and necessity for, and regulatory challenges associated with, robust clinical trials of phage-based therapeutics. This report focuses on a number of presentations from the meeting relating to cutting-edge research on bacteriophages as anti-infective agents.

Synergistic phage-antibiotic combinations for the control of Escherichia coli biofilms in vitro.

The potential application of phage therapy for the control of bacterial biofilms has received increasing attention as resistance to conventional antibiotic agents continues to increase. The present study identifies antimicrobial synergy between bacteriophage T4 and a conventional antibiotic, cefotaxime, via standard plaque assay and, importantly, in the in vitro eradication of biofilms of the T4 host strain Escherichia coli 11303. Phage-antibiotic synergy (PAS) is defined as the phenomenon whereby sub-lethal concentrations of certain antibiotics can substantially stimulate the host bacteria's production of virulent phage. Increasing sub-lethal concentrations of cefotaxime resulted in an observed increase in T4 plaque size and T4 concentration. The application of PAS to the T4 one-step growth curve also resulted in an increased burst size and reduced latent period. Combinations of T4 bacteriophage and cefotaxime significantly enhanced the eradication of bacterial biofilms when compared to treatment with cefotaxime alone. The addition of medium (10(4) PFU mL(-1) ) and high (10(7) PFU mL(-1) ) phage titres reduced the minimum biofilm eradication concentration value of cefotaxime against E. coli ATCC 11303 biofilms from 256 to 128 and 32 µg mL(-1) , respectively. Although further investigation is needed to confirm PAS, this study demonstrates, for the first time, that synergy between bacteriophage and conventional antibiotics can significantly improve biofilm control in vitro.