HIV-1 drug susceptibility testing by means of genotyping has been carried out by the Antiviral Resistance Testing Unit in Birmingham since its inception in the 1990s. The unit developed an in-house assay which sequences the entire protease gene (Codons 1-99) and the first 235 codons of the reverse transcriptase (RT) gene in a nested PCR, to give a total of 1,000 nucleotide base pairs (bp) of sequence. Recently, a number of primary and accessory resistance mutations have been identified in areas that lie outside RT codon 235, including codons P236L, Y318L, N348I and G333D/E. A new extended assay for HIV-1 genotype resistance testing was constructed, covering the entire protease gene as before and the RT gene up to codon 360, generating a nested PCR product of 1857bp. Evaluation of the new extended assay showed it to be at least as sensitive as the original assay in that it amplified the same variety of diverse HIV-1 subtypes. Based on this the extended assay replaced the old assay by the end of 2008. Results of a comparison between the old assay and the first two months after the introduction of the extended assay into clinical practice will be presented.

Authors: Jason T. Evans (1), Beck Taylor (2), Desmond Estephane (1), Sarah Gardiner (1), E. Grace Smith (1), Peter M. Hawkey (1).

Objectives: The utility of DNA fingerprinting of M. tuberculosis strains has been enhanced by MIRU-VNTR typing. An enhanced set of 24 loci that offers greater discrimination over the original 12 loci and aims to improve the concordance of strain typing data with epidemiological data has been published. The aim of this study was to evaluate the optimal MIRU-VNTR loci set in discriminating clusters defined by the original MIRU-VNTR loci that have varying levels of epidemiological links.

ABSTRACT: Enterovirus is a genus of viruses that includes the poliovirus and the hand foot and mouth virus. Enteroviruses cause a wide range of illnesses from mild fever to meningitis with flaccid paralysis. The occurrence of enteroviruses peaks in the summer months, and was especially high in 2008. The historical method for detecting enterovirus in clinical specimens was by cell culture, followed by neutralisation with specific antisera to type the virus (serotyping). At HoEFT, cell culture has now been replaced by real-time PCR, which, although it has increased sensitivity and turn-around times, does not routinely include typing and has resulted in a loss of epidemiological data. A number of typing methods based on the sequence of viral capsid proteins have been described in the literature, one of which is being developed for use in the routine laboratory here. Sequence typing is superior to serotyping as it allows typing of hitherto ‘untypable’ strains. Early findings indicate a dominance of certain species (most notably echovirus 30) in CSF specimens that have tested positive for enteroviruses by Real-time PCR in summer 2008.

ABSTRACT: It has been reported and was observed at HoEFT last winter that during norovirus outbreaks the incidence of Clostridium difficile increases. However, there is limited understanding of the increased incidence of C. difficile at times of other gastrointestinal infections. It is hypothesized that it may be due to an increased number of specimens being tested, with increased detection of CDT in patients who, in non-outbreak periods, would not normally have been tested for C. difficile toxin. Other possibilities are that there maybe due to an interaction between C. difficile and norovirus increasing the chance of clostridial toxin production, or that there is an increased risk of C. difficile transmission due to the norovirus. The principal aim of the project is to determine the epidemiology of C. difficile and norovirus during outbreaks of confirmed norovirus on a ward. Detection of C. difficile at HoEFT currently relies upon ELISAs which have been shown to have low sensitivities. Real-time PCR methods are available which have shown to have a high degree of concordance with toxigenic culture (the current gold standard). This project will also include an evaluation of both proprietary and in-house real-time PCR methods for possible introduction into routine testing procedures.