An audit of baseline HIV-1 genotypic resistance testing in a UK provincial city.

Baseline HIV-1 resistance testing is recommended in the 2005 BHIVA treatment guidelines. We compared the practice in our clinic with these guidelines. The aim of this study was to assess the prevalence of transmitted resistance in all antiretroviral therapy-naïve patients identified from our virology resistance test database. In 2006, 93% of all newly diagnosed patients had a baseline HIV-1 genotypic resistance test performed. The estimated prevalence of transmitted resistance was 8% in newly diagnosed patients and 7% overall with the majority in subtype B. These findings are in keeping with nationally reported data. It was of concern that we also identified a number of patients who had tested negative in the previous year.

Comparison of four RNA extraction methods for the detection of small round structured viruses in faecal specimens.

Four methods for extraction of the RNA genome of small round structured viruses (SRSVs) from faecal specimens by reverse transcription polymerase chain reaction (RT-PCR) were evaluated. The efficiency of recovery of viral RNA and removal of amplification inhibitors were compared. RNA extraction using the metal chelating agent Chelex-100 and Sephadex G200 column chromatography were the most sensitive, detecting a 10(-4) dilution of a known SRSV positive specimen. Guanidinium thiocyanate (GTC) with adsorption of viral RNA onto silica was 10-fold less sensitive. The fourth method, based on PEG precipitation followed by phenol/chloroform extraction with the addition of the detergent cetyltrimethylammonium bromide (CTAB), only detected a 1 in 10 dilution of the positive specimen. The CTAB method was 2- to 50-fold less sensitive than the GTC/silica method when dilution series of three further SRSV positive specimens were tested. Thirty-six SRSV negative faecal specimens were spiked with virus and RT-PCR performed following RNA extraction by each of the four techniques in order to assess the ability of these methods to remove amplification inhibitors. The GTC/silica method successfully removed inhibitors from all samples whereas partial or complete inhibition was observed in seven (19%) specimens following extraction by the CTAB method, 17 (47%) by the Sephadex method, and 20 (56%) by the Chelex method. We conclude that, of these four methods, the GTC/silica method is the most appropriate for the extraction of viral RNA from faecal samples prior to RT-PCR for detecting SRSVs.

Are ECG Welsh cup electrodes effectively cleaned?

Re-usable Welsh electrocardiograph (ECG)-electrodes are potential vehicles for cross infection. This study confirmed that in-use ECG-electrodes are frequently contaminated with organisms such as coagulase-negative staphylococci and Gram-negative bacilli. The efficacy of five cleaning procedures was evaluated. Immersing the electrodes in water at 60 degrees C for one hour was the most effective method of decontamination tested, following challenge with a standardised suspension of Staphylococcus saprophyticus. Significant contamination persisted following simple cleaning measures. It is suggested that this was promoted by the inadequate removal of electrode gel which provided a protective environment for microorganisms.

Northwick Park Infection Consultation Service. Part I. The aims and operation of the service and the general distribution of infection identified by the service between September 1987 and July 1990 [see comment].

The Northwick Park Infection Consultation Service (ICS) is a collaborative service operated by the departments of Medical Microbiology and Infectious Diseases where personnel and skills are combined. Its aim is to improve the availability and effectiveness of consultation for infection-related problems. This paper sets out the framework for establishing an ICS and also details the general distribution of infection identified by the Northwick Park ICS in a study carried out between September 1987 and July 1990. Part II assesses the contribution that the ICS made to the management of infection. One thousand and thirty-eight (1038) patients were seen on the ICS. Seventy-five per cent (776) were judged to be infected and in 691 this was a probable or certain diagnosis. Skin and subcutaneous tissue, respiratory tract, and genito-urinary tract infections accounted for 64% of the total. Eighty-seven per cent of infections required treatment with intravenous antibiotics, 22% were associated with concomitant bacteraemia, and 2.7% of patients died as a direct result of their infection. Sixty-four per cent of consultations were unsolicited and arose from laboratory results or the clinical information on the form accompanying the specimen: over one quarter were initiated before results were available. These infections were no different in either severity or nature from those identified by solicited requests to either department. Fifty-three per cent of consultations had a moderate to high clinical component. The results emphasise the importance of infection in hospitals and highlight the advantages of a collaborative approach from the departments of Medical Microbiology and Infectious Diseases.

Northwick Park Infection Consultation Service. Part II. Contribution of the service to patient management: an analysis of results between September 1987 and July 1990.

The establishment of Infectious Disease teams combining microbiological and clinical expertise has recently been recommended by a joint working part of the Royal College of Physicians and the Royal College of Pathologists. The Northwick Park Infection Consultation Service (ICS) has been operating on these lines since 1983; details are given in Part I. Part II assesses the contribution that the ICS has made to the management of infection in a study of 1038 patients undertaken between September 1987 and July 1990. The areas of patient diagnosis, treatment, investigation and isolation were examined to assess the appropriateness of the attending doctor's management of infection and the benefits resulting from recommendations made by the ICS. At the time of consultation the correct diagnosis had already been made or considered in 93% of patients, essential investigations needed to confirm or refute the diagnosis performed in 92%, and side-room isolation correctly instituted in 81% of patients requiring it. However, 41% of 776 infected patients were receiving suboptimal treatment: this was significantly more frequent in unsolicited consultations (P less than 0.05). Advice was given following consultation in 893 of 1038 patients (86%) and related to treatment (66%), investigation (41%), diagnosis (30%) and patient isolation (4%). Of 844 patients where receipt of advice could be accurately assessed, it was taken fully in 708 (84%), partly in III (13%), and went unheeded in 25 (3%). Advice on diagnosis or investigation enabled the correct diagnosis to be reached in 30% of consultations and in a further 47 patients (5%), the diagnosis was proposed by the ICS on initial consultation.(ABSTRACT TRUNCATED AT 250 WORDS)

Polysaccharide production in liquid cell suspension cultures of Phleum pratense L.

The production of carbohydrates by cell suspension cultures of Phleum pratense (timothy grass) is described. Extracellular polysaccharides similar in monosaccharide composition to native cell wall polymers were accumulated, together with polymers of fructose (fructans). The fructans had similar properties to the intracellular reserve polymers found in intact plants, and were found in both cells and media of young, slow-growing cultures.Production of extracellular polysaccharides differed in cultures grown on sucrose or equimolar glucose/fructose as carbon source. These differences were observed only when autoclaved media were used, and were not related to changes in either pH or osmolarity. Autoclaving medium containing radioactive glucose and fructose produced a novel, unidentified labelled compound which was absent in medium containing labelled sucrose.

Evaluation of an antigen capture ELISA based on recombinant mexico virus capsid protein.

BACKGROUND: The diagnosis of gastrointestinal infections caused by small round structured viruses (SRSV) has relied upon electron microscopy and antigen/antibody assays based on Norwalk virus. We investigated cases of gastroenteritis associated with SRSVs employing a new sandwich enzyme-linked immunosorbent assay (ELISA) using hyperimmune animal anti-sera against recombinant Mexico virus capsid protein (rMXV). STUDY DESIGN: One hundred and thirty-five specimens from 86 episodes of gastroenteritis associated with SRSVs, collected in the UK between October 1993 and September 1994, were tested in the rMXV assay. RESULTS: Forty-seven (35%) specimens from 35 of 86 (41%) episodes were positive in the rMXV ELISA and these could further be divided into high and low reactors. Sequencing of a 266-base region of the RNA polymerase gene revealed that strains highly reactive in the rMXV assay demonstrated a high degree of similarity to MXV (97-99% at the nucleotide level), whereas low-reactive strains consist of Mexico-like strains and a heterogeneous group of viruses exhibiting 70-75% similarity to MXV. CONCLUSION: Our results indicate that the rMXV ELISA is predominantly a type specific assay, although some cross reactivity with other genogroup 2 SRSVs was observed. MXV was responsible for 26% of SRSV-associated gastrointestinal infections investigated in the UK during one year's surveillance.

Homotypic and heterotypic IgG and IgM antibody responses in adults infected with small round structured viruses.

Antibody responses to recombinant Norwalk (rNV) and Mexico (rMXV) viral capsid proteins were studied in 39 adults involved in outbreaks of gastroenteritis associated with genogroup 2 small round structured viruses (SRSVs). Nineteen individuals were involved in outbreaks associated with MXV-like strains and 20 in outbreaks associated with four other genogroup 2 SRSVs. IgG antibodies were measured in acute and convalescent sera using indirect enzyme-linked immunosorbent assay (ELISA), and IgM was measured by indirect and capture ELISAs. Nineteen (49%) patients demonstrated a significant rise in IgG to rMXV with four (10%) patients also showing anamnestic responses to rNV. Fourteen patients were positive in the rMXV IgM-capture ELISA, representing 74% of patients demonstrating IgG rises. IgG and IgM responses to rMXV were observed in both groups, although higher levels of responses were seen in adults infected with MXV-like strains than those infected with non-MXV genogroup 2 viruses. No significant IgM responses were observed to rNV. These results indicate that, following SRSV infection, adults show a rise in antibody which is broadly reactive to viruses within but not between genogroups, although greater homotypic than heterotypic responses are produced. These findings have implications for interpretation of seroepidemiological studies and serodiagnosis of SRSV infections using recombinant capsids.

Identification of an epitope common to genogroup 1 "norwalk-like viruses".

A panel of 10 monoclonal antibodies (MAbs) to recombinant Norwalk virus (NV) capsid protein were tested in competition enzyme-linked immunosorbent assays. Patterns of competition indicated that these MAbs recognize six to eight epitopes covering five nonoverlapping regions of the capsid protein. A single epitope, recognized by NV MAbs NV3901, NV3912, and NV2461 was found to occur in the majority of genogroup 1 (G1) but not genogroup 2 (G2) "Norwalk-like viruses" (NLVs). This observation supports the subdivision of human NLVs into two genogroups and provides an assay for the rapid identification of G1 NLVs in fecal specimens.

Expression and self-assembly of Grimsby virus: antigenic distinction from Norwalk and Mexico viruses.

A cDNA obtained from Grimsby virus (GRV), a Norwalk-like virus, purified from a stool sample of a symptomatic adult associated with a gastroenteritis outbreak in the United Kingdom, was used to obtain the complete nucleotide sequence of the second open reading frame (ORF2). The ORF2 sequence of GRV predicts a capsid of 539 amino acids (aa) which exhibits aa identities of 96% to Lordsdale virus, 67% to Mexico virus (MXV), and 43% to Norwalk virus (NV). The GRV capsid protein was expressed in insects cells by using a recombinant baculovirus, and the resulting virus-like particles (VLPs) possessed a protein with an apparent molecular weight of 58,000. Hyperimmune antisera raised against purified GRV, MXV, and NV VLPs were tested in an indirect enzyme-linked immunosorbent assay (ELISA) against GRV, NV, and MXV VLPs, revealing that GRV is antigenically distinct from both NV and MXV. The antigenic specificity of the GRV-hyperimmune antiserum was confirmed in an antigen capture ELISA using GRV-, NV-, or MXV-containing fecal specimens. The expression of the GRV capsid protein has, for the first time, allowed the antigenic comparison of three distinct recombinant Norwalk-like viruses.

Diagnosis of human caliciviruses by use of enzyme immunoassays.

The application of molecular technologies, such as the expression of viral proteins in baculovirus, has provided a powerful approach to the diagnosis of human calicivirus (HuCV) infections. The baculovirus-expressed HuCV capsid protein self-assembles into virus-like particles, providing excellent reagents for immunologic assays, such as enzyme immunoassays (EIAs). Following the expression of the capsid protein of Norwalk virus, the capsid proteins of 8 other HuCV strains have been expressed in baculovirus. The unlimited supply of baculovirus-produced reagents for HuCVs allows these EIAs to be applied in large-scale clinical and epidemiological studies. Both the antigen and antibody-detection EIAs are highly sensitive. The antigen-detection EIAs are highly specific, but the antibody-detection EIAs are more broadly reactive. This article reviews baculovirus expression techniques used to produce HuCV capsid antigens, development of EIAs using these antigens, and application of these EIAs in studies of HuCV infection and illness.