Ten years of external quality assessment of human immunodeficiency virus type 1 RNA quantification.

Viral load testing is an essential parameter in guiding antiretroviral therapy for individuals infected with human immunodeficiency virus type 1 (HIV-1). An external quality assessment scheme for the molecular quantification of HIV-1 RNA was introduced by the United Kingdom National External Quality Assessment Service for Microbiology in 2000. Specimen pairs of freeze-dried plasma were distributed to a median of 141 participants three times a year. The aim of this study was to analyze the quantification of HIV-1 RNA results between 2000 and 2010. Overall variability, measured by the standard deviations of all viral load results for each specimen, was below 0.5 log copy/ml (n = 48). When we compared assay results, the medians of the viral load by assay were within a range of 0.25 to 1.08 log copies/ml, with the lowest median values being consistently reported with the Siemens branched-chain DNA assay. The spread of participant results and, hence, differences between assay medians were greater when quantifying non-B subtypes. Laboratories were scored on the proximity of their reported log difference for the specimen pair to the median log difference reported by all laboratories. The overall level of performance with the HIV-1 RNA specimens over the past 10 years has been consistently good, with more than 90% of the participants reporting in the accepted range (median difference, ±0.5 log unit). Future distributions may result in tightening the acceptance levels of quantification and the use of more challenging specimens, including a variety of subtypes, with developments focusing on maintaining the clinical relevance and educational value of the scheme.

Screening methods for meticillin-resistant Staphylococcus aureus.

The UK National External Quality Assessment Service (UK NEQAS) distributes clinically relevant and educational specimens for external quality assessment (EQA). The aim of this report was to assess the suitability of using a freeze-dried specimen format for the EQA of conventional and rapid methods and to review the methods used by participants to screen for meticillin-resistant Staphylococcus aureus (MRSA). Of the 714 laboratories that returned a result, 678 reported the presence of MRSA, and results showed a mean of 73 c.f.u. per 25 µl and a median of 50 c.f.u. per 25 µl confirming that the specimen was homogeneous. Four different approaches to MRSA screening were used routinely, including: (i) liquid culture; (ii) direct plating onto conventional media; (iii) direct plating onto chromogenic media; and (iv) rapid methods. A wide variety of methods were used within each of these four categories to screen for MRSA, and many laboratories reported using more than one method. Attempts should be made to determine the most appropriate approach to MRSA screening and to standardize the protocols across the UK.

External quality assessment for molecular detection of human papillomaviruses.

BACKGROUND: The United Kingdom National External Quality Assessment Service (UK NEQAS) distributes clinically relevant and educational specimens for external quality assessment (EQA). OBJECTIVES: The aim of this report was to assess the suitability of using liquid based cytology (LBC) samples for the EQA of molecular methods and to review the methods used by participants to detect the presence of high risk (HR) human papillomavirus (HPV) genotypes. STUDY DESIGN: Three pilot distributions were dispatched between January 2008 and January 2009 with each distribution consisting of four different specimens. RESULTS: Performance was good with over 90% of participants reporting correctly on the presence or absence of high risk genotypes in all but one specimen, specimen 9006 (82.1%). Specimen 9006 was a pooled specimen, negative for HR genotypes but containing low risk (LR) genotypes 61, 70 and 81. The most commonly used assay for the detection of the presence of HR HPV was the Digene Hybrid Capture II assay. The in-house PCR assays were most commonly associated with incorrect results, and the use of these assays decreased during the 13 month pilot study. CONCLUSIONS: The UK NEQAS molecular detection of HPV scheme provides a standardised, homogeneous and characterised clinical specimen, however this study has shown that genotyping results reported by participants were still varied. Inclusion of available HPV standards will help to standardise assays. Robust EQA of HPV molecular screening programmes will be essential for monitoring the impact of the HPV vaccine.

Molecular characterization of human group C rotavirus genes 6, 7 and 9.

Genes 6, 7 and 9 of human group C rotavirus 'Bristol' strain, encoding non-structural proteins (NSP) 3, 1 and 2, respectively, were cloned and sequenced. Human group C rotavirus genome segment 6 is 1350 bp and contains a single ORF of 1231 nucleotides (encoding 402 amino acids). Genome segment 7 is 1270 bp and encodes a protein of 394 amino acids and genome segment 9 is 1037 bp and encodes a 312 amino acid protein. The human group C rotavirus genes 6, 7 and 9 showed 78, 67 and 88% sequence identity, respectively, to the corresponding porcine group C rotavirus genes. The derived protein sequences were compared with those of the porcine 'Cowden' group C and mammalian group A rotavirus strains. The human group C rotavirus NSP1 protein sequence is one amino acid longer than the porcine group C equivalent. In common with group A and porcine group C rotaviruses, the human group C rotavirus NSP1 protein has a zinc finger motif. Human group C rotavirus NSP2 has two hydrophobic heptad repeat regions, a basic, RNA-binding domain and a basic, proline-rich region. Human group C rotavirus NSP3 has both single- and double-stranded RNA-binding domains and several hydrophobic heptad repeat regions, one of which forms a leucine zipper. This work completes the molecular characterization of the non-structural proteins of a human group C rotavirus. Phylogenetic analysis of all the non-structural genes of group A, B and C rotaviruses suggests that these viruses have diverged at a constant rate from a common ancestor.