Wayne Dimech

Evaluation of Eight Anti-Rubella Virus Immunoglobulin G Immunoassays That Report Results in International Units per Milliliter

ABSTRACT An evaluation of anti-rubella virus immunoglobulin G (IgG) immunoassays that report in international units per milliliter (IU/ml) was performed to determine their analytical performance and the degree of correlation of the test results. A total of 321 samples were characterized based on results from a hemagglutination inhibition assay. The 48 negative and 273 positive samples were used to determine the sensitivity and specificity of the assays. When equivocal results were interpreted as reactive, the sensitivity of the immunoassays ranged from 98.9 to 99.9% and the specificity ranged from 77.1 to 95.8%. All assays had positive and negative delta values of less than 2. A significant difference between the mean results of all assays was demonstrated by analysis of variance. However, post hoc analysis showed there was good correlation in the mean results expressed in IU/ml between some of the assays. Our results show the level of standardization between anti-rubella virus IgG immunoassays reporting results expressed as IU/ml has improved since a previous study in 1992, but further improvement is required.

Validation of assembled nucleic acid-based tests in diagnostic microbiology laboratories

Medical microbiology and virology laboratories use nucleic acid tests (NAT) to detect genomic material of infectious organisms in clinical samples. Laboratories choose to perform assembled (or in-house) NAT if commercial assays are not available or if assembled NAT are more economical or accurate. One reason commercial assays are more expensive is because extensive validation is necessary before the kit is marketed, as manufacturers must accept liability for the performance of their assays, assuming their instructions are followed. On the other hand, it is a particular laboratorys responsibility to validate an assembled NAT prior to using it for testing and reporting results on human samples. There are few published guidelines for the validation of assembled NAT. One procedure that laboratories can use to establish a validation process for an assay is detailed in this document. Before validating a method, laboratories must optimise it and then document the protocol. All instruments must be calibrated and maintained throughout the testing process. The validation process involves a series of steps including: (i) testing of dilution series of positive samples to determine the limits of detection of the assay and their linearity over concentrations to be measured in quantitative NAT; (ii) establishing the day-to-day variation of the assays performance; (iii) evaluating the sensitivity and specificity of the assay as far as practicable, along with the extent of cross-reactivity with other genomic material; and (iv) assuring the quality of assembled assays using quality control procedures that monitor the performance of reagent batches before introducing new lots of reagent for testing.

Evaluation of an automated urinalysis system for testing urine chemistry, microscopy and culture.

Aims: This study was designed to evaluate the performance of an automated urinalysis system that utilised three commercially available instruments, the Clinitek Atlas, Sysmex UF‐100 and the Alifax Uro‐Quick. Methods: The results of the automated system for 818 urine samples were compared with the results of manual processing which consisted of phase contrast microscopy, manual dipstick chemistry analysis and culture onto solid media. Results: The correlation between the two methods for urine chemistry was excellent with a concordance of 89, 97, 100 and 98% for pH, blood, glucose and protein, respectively. The quantification of red blood cells and white blood cells had an R 2 of 0.855 and 0.92, respectively. A difference scatter plot indicated a trend towards the manual cell count being greater than the UF‐100 count as both the red and white blood cell count increased. There was 98% agreement between the automated process and manual culture. Conclusions: The automated urinalysis system is fully integrated and allows for the cross‐checking of urine chemistry and microscopy as well as electronic transfer of data. The automated process was used as a screening procedure and some manual testing was necessary. Automation of urinalysis offers a reduction in variation and has comparable results to manual testing.Abbreviations: LE, leukocyte esterase; RBC, red blood cell; WBC, white blood cell.

Evaluation of Three Immunoassays Used for Detection of Anti-Rubella Virus Immunoglobulin M Antibodies

ABSTRACT Three automated assays (Abbott AxSYM, Bayer ADVIA Centaur, and bioMerieux VIDAS) used for the detection of rubella virus-specific immunoglobulin M were evaluated. A total of 57 samples from individuals with evidence of infection with rubella virus were used to estimate sensitivity, and 220 samples from blood donors and individuals attending an antenatal clinic who had no evidence of recent infection were used to estimate specificity. Seroconversion panels comprising an additional 31 samples from four individuals were used to determine clinical sensitivity. Samples containing potentially cross-reacting substances were also tested. The sensitivities of the three assays ranged from 84.2 to 96.5%, and the specificities ranged from 96.8 to 99.9%. The Abbott AxSYM assay detected more reactive samples than the other two assays when a panel of 57 positive samples was tested. Bayer ADVIA Centaur detected more reactive samples in the seroconversion panels than the other two assays. All three assays evaluated reported a reactive result in 1 or more of the 48 samples containing potentially cross-reacting analytes. The assays demonstrated comparable performance in testing of a well-characterized panel of samples.

Measurement of anti-DNA antibodies by elisa: A comparative study with crithidia and a farr assay

&NA; One hundred and twenty six sera from 116 patients with systemic lupus erythematosus (SLE) and from 51 control patients were assayed for the presence of anti‐DNA antibodies, using a commercial enzyme linked immunosorbent assay (ELISA). Fifty three sera (42%) from SLE patients were positive and a further 13 sera (10%) fell in the ‘equivocal’ positive range. Three control sera were positive. In a standard 14C DNA Farr assay, 67 sera (53%) from SLE patients were positive. One control serum was weakly positive. There was a good linear correlation between absorption in the ELISA and the 14C DNA binding result (r = 0.73). Results in the ELISA and Farr assays were concordant in 96 of the 126 SLE sera, and 47 of 51 control sera. Sequential sera from a further 6 patients with fluctuating clinical activity of SLE showed similar patterns of change of anti‐DNA antibodies in both assays. The ELISA was more sensitive than the Crithidia luciliae immunofluorescence assay which detected 44 positive sera (35%) in the SLE group. These results suggest that this ELISA assay may be a useful alternative to the Crithidia assay or an effective screen prior to testing in the more technically difficult and time consuming Farr assay for the measurement of anti‐DNA antibodies.

An evaluation of the in vitro activity of piperacillin/tazobactam

&NA; Tazobactam is a new, irreversible inhibitor of bacterial betalactamases of staphylococci, plasmid‐mediated beta‐lactamases of the TEM and SHV types found in Escherichia coli and Klebsiella species and beta‐lactamases of anerobes such as Bacteroides species. Its combination with piperacillin, a broad spectrum ureido‐penicillin, would be expected to improve the activity of piperacillin against staphylococci, TEM and SHV betalactamase producing Gram negative bacteria and anerobes. Minimal inhibitory concentrations (MIC) of piperacillin/tazobactam were determined for 1952 individual patient isolates of Gram positive and negative bacteria causing significant infections and compared with MIC values for cefotaxime, ceftazidime, ciprofloxacin, imipenem, ticarcillin/clavulanic acid. MICs were determined by agar dilution (NCCLS 1990 and 1992). Piperacillin/tazobactam had excellent activity against methicillin susceptible staphylococci, Streptococcus pneumoniae, Haemophilus influenzae, enterococci and organisms of the Bacteroides fragilis group. It was also active against the majority of Enterobacteriaceae and Pseudomonas aeruginosa isolates tested. It was not active against extended spectrum beta‐lactamase (ESBL) producing Klebsiella species and some high level TEM and SHV beta‐lactamase producing E. coli and Klebsiella species. Activity against Gram negative organisms capable of producing chromosomally mediated beta‐lactamases was good, since in most organisms tested, the enzymes were not induced in sufficient quantities to cause antibiotic resistance. However some Enterobacter species were derepressed hyperproducing mutants; these isolates showed resistance to piperacillin/tazobactam since tazobactam does not inhibit these Class I beta lactamases. Activity was superior to ticarcillin/clavulanic acid for Gram negative rods. Imipenem was the most active agent against ESBL producing Klebsiella species. Piperacillin/tazobactam has a suitable spectrum of activity in vitro to suggest its use in monotherapy of mixed anerobic infections, mixed respiratory infections such as aspiration pneumonia and, in combination with an aminoglycoside, it would provide Gram positive as well as Gram negative cover of febrile episodes in immunosuppressed patients.

Determination of quality control limits for serological infectious disease testing using historical data

Abstract Background: An effective quality control (QC) program requires the establishment of control limits within which the results of the QC sample is expected to fall. Traditionally, the mean plus/minus two standard deviations calculated for a set of QC sample results is used to establish control limits. Allowable total error (TEa) and Westgard rules aid in interpreting QC sample results. Westgard rules assume QC sample results are normally distributed and TEa assumes commutability between the QC sample and patient results. None of these paradigms apply to infectious disease testing. Methods: Results from the NRL’s QC program were extracted and sorted into assay/QC lot number-specific data. Control limits for selected QC samples used to monitor 64 commonly used serological assays were calculated and validated using the within- and between-QC lot variance of data from each of the assay/QC combinations. Results: No assay/QC combination had more than 10% of results less than the lower control limit or greater than the upper control limit. Of the 423 assay/QC lot combinations, 14 (3.3%) had more than 5% of results less than the lower limit and 48 (11.3%) had more than 5% of results greater than the upper limit calculated for that assay/QC combination. Conclusions: The control limits, established by this novel method, are based on more than a decade of QC test results from >300 laboratories from 30 countries and provides users of the NRL QC program evidence-based control limits that can be applied in isolation or in conjunction with more traditional methods for establishing control limits.

Results of Cytomegalovirus DNA Viral Loads Expressed in Copies per Millilitre and International Units per Millilitre are Equivalent

Quantification of Cytomegalovirus (CMV) DNA is required for the initiation and monitoring of anti-viral treatment and the detection of viral resistance. However, due to the lack of standardisation of CMV DNA nucleic acid tests, it is difficult to set universal thresholds. In 2010, the 1st WHO International Standard for Human Cytomegalovirus for Nucleic Acid Amplification Techniques was released. Since then CMV DNA viral load assays have been calibrated using this standard. Three external quality assessment (EQA) providers sent the same five samples to their participants and analysed the results to determine the equivalence of reporting CMV DNA results in international units per millilitre (IU/mL), and compared the difference in results reported in IU/mL with those reported in copies per millilitre (c/mL), and to determine the rate of adoption of IU/mL. About 78% of participants continue to report results in c/mL even though six of the 12 commercial assays are calibrated against the standard. The range of the results reported in IU/mL was less than those reported in c/mL indicating that the adoption of the WHO standard successfully improved the reporting of the CMV viral load. The variation in individual sample results reported by different assays, irrespective of whether in IU/mL or c/mL, is still great and therefore more standardisation of the assays is needed to allow the setting of treatment and monitoring thresholds. This study can act as a bench mark to determine rate of future adoption if reporting CMV DNA viral load results in IU/mL.

Where to Now for Standardization of Anti-Rubella Virus IgG Testing

ABSTRACT The lack of standardization of rubella IgG testing continues to be a problem 20 years since the standard was introduced. The situation is complex and poorly understood. As demonstrated by an article in this issue (E. Bouthry, M. Furione, D. Huzly, A. Ogee-Nwankwo, L. Hao, A. Adebayo, J. Icenogle, A. Sarasini, M. Grazia Revello, L. Grangeot-Keros, and C. Vauloup-Fellous, J Clin Microbiol 54:1720–1725, 2016, http://dx.doi.org/10.1128/JCM.00383-16), the problem remains. The situation is far from being resolved, but at least the process for change has started.

Where to Now for the Standardisation of Anti-rubella IgG Testing

ABSTRACT The lack of standardization of rubella IgG testing continues to be a problem 20 years since the standard was introduced. The situation is complex and poorly understood. As demonstrated by an article in this issue (E. Bouthry, M. Furione, D. Huzly, A. Ogee-Nwankwo, L. Hao, A. Adebayo, J. Icenogle, A. Sarasini, M. Grazia Revello, L. Grangeot-Keros, and C. Vauloup-Fellous, J Clin Microbiol 54:1720–1725, 2016, http://dx.doi.org/10.1128/JCM.00383-16), the problem remains. The situation is far from being resolved, but at least the process for change has started.