Steve L. Wesselingh

Comparison of a Multiplex Reverse Transcription-PCR-Enzyme Hybridization Assay with Conventional Viral Culture and Immunofluorescence Techniques for the Detection of Seven Viral Respiratory Pathogens

ABSTRACT A multiplex reverse transcription-PCR-enzyme hybridization assay (RT-PCR-EHA; Hexaplex; Prodesse Inc., Waukesha, Wis.) was used for the simultaneous detection of human parainfluenza virus types 1, 2, and 3, influenza virus types A and B, and respiratory syncytial virus types A and B. One hundred forty-three respiratory specimens from 126 patients were analyzed by RT-PCR-EHA, and the results were compared to those obtained by conventional viral culture and immunofluorescence (IF) methods. RT-PCR-EHA proved to be positive for 17 of 143 (11.9%) specimens, whereas 8 of 143 (5.6%) samples were positive by viral culture and/or IF. Eight samples were positive by both RT-PCR-EHA and conventional methods, while nine samples were RT-PCR-EHA positive and viral culture and IF negative. Eight of the nine samples with discordant results were then independently tested by a different multiplex RT-PCR assay for influenza virus types A and B, and all eight proved to be positive. In comparison to viral culture and IF methods, RT-PCR-EHA gave a sensitivity and a specificity of 100 and 93%, respectively. Since RT-PCR-EHA was able to detect more positive samples, which would otherwise have been missed by routine methods, we suggest that this multiplex RT-PCR-EHA provides a highly sensitive and specific means of diagnostic detection of major respiratory viruses.

Plant-derived measles virus hemagglutinin protein induces neutralizing antibodies in mice

Measles remains a significant problem in both the developed and developing world, and new measles vaccination strategies need to be developed. This paper examines the strategy of utilizing transgenic plants expressing a measles antigen for the development of an oral sub-unit measles vaccine. A 1.8 kb fragment encompassing the coding region of the measles virus hemagglutinin (H) protein was cloned into a plant expression cassette. Three different expression constructs were tested: pBinH (H gene alone), pBinH/KDEL (addition of a C-terminal endoplasmic reticulum-retention sequence SEKDEL) and pBinSP/H/KDEL (further addition of an authentic N-terminal plant signal peptide). The highest levels of recombinant H protein production were observed in plants transformed with pBinH/KDEL. Mice inoculated intraperitoneally with transgenic plant derived recombinant H protein produced serum anti-H protein antibodies that neutralized the measles virus (MV) in vitro. Mice gavaged with transgenic tobacco leaf extracts also developed serum H protein-specific antibodies with neutralizing activity against MV in vitro. These results indicate that the plant-derived measles H protein is immunogenic when administered orally and that, with further development, oral vaccination utilizing transgenic plants may become a viable approach to measles vaccine development.

Prevalence of and risk factors for HIV-associated neuropathy in Melbourne, Australia 1993–2006

The aim of the study was to describe the prevalence of and risk factors for HIV‐associated sensory neuropathy (HIV‐SN) in 2006 [the era of stavudine, didanosine and zalcitabine (dNRTI)‐sparing highly active antiretroviral therapy (HAART)] and to compare our findings with data obtained in the same clinic in 1993 (pre‐HAART) and 2001 (frequent use of dNRTI‐containing HAART).

Exposure to dideoxynucleosides is reflected in lowered mitochondrial DNA in subcutaneous fat.

Objectives: Nucleoside reverse transcriptase inhibitors (NRTIs), particularly dideoxynucleoside analogs (ddNs), used in the treatment of HIV, inhibit mitochondrial DNA polymerase &ggr; in vitro. Mitochondrial DNA (mtDNA) depletion is proposed as the underlying mechanism of many of the in vivo side effects of these agents. A reliable and valid laboratory test to detect this is not yet available. The objective of this study was to correlate tissue mtDNA quantification in HIV‐infected patients with exposure to nucleoside analogs. Methods: 60 HIV‐infected adults underwent detailed clinical assessment and blood and tissue sampling. Clinical and antiretroviral treatment details were correlated with results of plasma lactate assays, and real‐time polymerase chain reaction quantification of mtDNA in peripheral blood mononuclear cells (PBMCs) and subcutaneous fat from the lower limb. Results: Forty‐nine (82%) subjects were on combination antiretroviral therapy. Of these, 33 (55%) were currently receiving one or more ddNs (stavudine, didanosine, or zalcitabine). mtDNA in subcutaneous fat was lower in subjects currently on ddNs than in those not taking ddNs (mean [log10] 2.47 vs. 2.74, p = .002). Plasma lactate was somewhat higher in subjects currently taking ddNs than those on no antiretroviral treatment (median 1.5 vs. 1.0, p = .03), but was not significantly different in either of these groups compared with subjects on other NRTIs. mtDNA in PBMCs did not vary with treatment status. Conclusions: mtDNA in subcutaneous fat was significantly reduced in patients currently taking ddNs. mtDNA in PBMCs was independent of patient exposure to NRTIs.

Acquisition of Methicillin-Resistant Staphylococcus aureus in a Large Intensive Care Unit

OBJECTIVESnTo determine the prevalence of MRSA colonization on admission to the ICU and the incidence of MRSA colonization in the ICU.nnnDESIGNnProspective cohort study.nnnSETTINGnUniversity hospital.nnnPARTICIPANTSnPatients admitted to the ICU in 2000-2001.nnnMETHODSnPatients were screened for MRSA with nose, throat, groin, and axilla swabs on admission and discharge. MRSA acquisition was defined as a negative admission screen and a positive discharge screen. Risk factors analyzed included previous wards/current unit, gender, age, and length of stay prior to and in the ICU. Univariate and multivariate analyses were performed using logistic regression.nnnRESULTSnOf screened patients, 6.8% were MRSA colonized on admission to the ICU. Some patients (11.4%) became newly colonized during their stay in the ICU. Factors that remained significant in the multivariate analysis of MRSA colonization on admission were previous admission to various wards and length of stay prior to ICU admission of more than 3 days. In the multivariate analysis of MRSA acquisition in the ICU, being a trauma patient and length of stay in the ICU greater than 2 days remained significant Thirty-six percent of patients had both admission and discharge swabs taken. This percentage increased in the presence of a supervisory nurse.nnnCONCLUSIONnSignificant acquisition of MRSA occurs in the ICU of our hospital, with trauma patients at increased risk. Patients who had been on the cardiothoracic ward prior to the ICU had a lower risk of MRSA colonization on admission. Presence of a supervisory nurse improved compliance with screening

Successful boosting of a DNA measles immunization with an oral plant-derived measles virus vaccine

ABSTRACT Despite eradication attempts, measles remains a global health concern. Here we report results that demonstrate that a single-dose DNA immunization followed by multiple boosters, delivered orally as a plant-derived vaccine, can induce significantly greater quantities of measles virus-neutralizing antibodies than immunization with either DNA or plant-derived vaccines alone. This represents the first demonstration of an enhanced immune response to a prime-boost vaccination strategy combining a DNA vaccine with edible plant technology.

Is the central nervous system a reservoir of HIV-1?

Purpose of reviewTo summarize the evidence in the literature that supports the central nervous system (CNS) as a viral reservoir for HIV-1 and to prioritize future research efforts. Recent findingsHIV-1 DNA has been detected in brain tissue of patients with undetectable viral load or neurocognitive disorders, and is associated with long-lived cells such as astrocytes and microglia. In neurocognitively normal patients, HIV-1 can be found at high frequency in these cells (4% of astrocytes and 20% of macrophages). CNS cells have unique molecular mechanisms to suppress viral replication and induce latency, which include increased expression of dominant negative transcription factors and suppressive epigenetic factors. There is also evidence of continued inflammation in patients lacking a CNS viral load, suggesting the production and activity of viral neurotoxins (for example, Tat). SummaryTogether, these findings provide evidence that the CNS can potentially act as a viral reservoir of HIV-1. However, the majority of these studies were performed in historical cohorts (absence of combination antiretroviral therapy or presence of viral load), which do not reflect modern day patients (combination antiretroviral therapy-treated and undetectable viral load). Future studies will need to examine patient samples with these characteristics to conclusively determine whether the CNS represents a relevant and important viral reservoir.

Clinical, microbiological, and economic benefit of a change in antibiotic prophylaxis for cardiac surgery

Vancomycin and rifampicin replaced cephazolin as antibiotic prophylaxis for coronary artery bypass surgery at our institution. Following this intervention, there was a significant decrease (P < .001) in the surgical-site infection rate from 10.5 (95% confidence interval, 8.2 to 13.3) to 4.9 (95% confidence interval, 3.2 to 7.1) infections per 100 procedures. An estimated

Human astrocytes express membrane cofactor protein (CD46), a regulator of complement activation.

576,655 (Australian) was saved between two 12-month periods.

Production and characterization of an orally immunogenic Plasmodium antigen in plants using a virus-based expression system.

Expression of membrane cofactor protein (CD46) on cultured human astrocytes was demonstrated by indirect immunofluorescence microscopy and flow cytometry following staining with a monoclonal antibody specific for CD46. Western transfer and immunoblotting detected a doublet of Mr 66,000 and 56,000. Analysis of astrocyte mRNA revealed the presence of multiple alternatively spliced transcripts encoding different extracellular regions or cytoplasmic tails of CD46. Astrocytes were also shown to express decay accelerating factor, but not the type 1 complement receptor. Upregulation of astrocyte CD46 occurred following cytomegalovirus infection. These results indicate that astrocytes express proteins involved in regulation of complement activation and protection against autologous complement.