Evaluation of the Virus Counter® for rapid baculovirus quantitation

The utility of a new instrument for rapid virus quantitation, the Virus Counter, was evaluated in a blind study conducted at three sites. This instrument is a substantially improved version of the original academic research instrument described previously by Stoffel and Rowlen (2005a). The addition of hydrodynamic focusing, a self-contained fluidics system and customized software for system control and data analysis has resulted in a commercially viable and available design. Baculovirus samples were provided by Protein Sciences Corporation and blinded to InDevR and Baylor College of Medicine. Protein Sciences Corporation and Baylor College of Medicine analyzed the samples by plaque assay and InDevR analyzed the samples using the Virus Counter. Serial dilution of stock viruses into growth media and buffer allowed for comparison of measured versus intended concentrations. Direct log-scale comparison between pooled Virus Counter results and pooled plaque assay results indicated a linear relationship (slope = 1.1 ± 0.2, R² = 0.86) with statistically significant Pearson correlation (r = 0.93, p < 0.001).     

Evaluation of the CELL-DYN® 3500 haematology instrument for the analysis of the mouse and rat blood

The objective of this study was to evaluate the performance of the CELL-DYN^® 3500 for rat and mouse blood analysis in a routine environment. The WBC (white blood cells), RBC (red blood cells), PLT (platelets) counts and the WBC differential were determined. In addition, the following aspects were studied: within-run precision, day-to-day precision, biasfree paired difference precision; extended ranges of linearity for RBC, HCT (haematocrit), WBC, PLT; carry-over, the fffect of blood ageing, cell stability with different anticoagulants; and the normal ranges, the out of range flagging and some typical pathology cases. The CELL-DYN^® 3500 is a multiparameter flow cytometer which counts and differentiates WBC, based on the principle of multi-angle polarised light scatter separation. RBC and PLT are determined by the impedance method. The WBC count is evaluated by both, optical and impedance methods. Reference methods used were according to the ICSH recommendations on blood cell analysis, including manual counts of WBC and platelets, a centrifugal microhaematocrit method and a haemoglobin measurement by spectrophotometry using the WHO haemoglobin standard. All cell counts were compared with the results obtained by our routine blood cell analyser (Contraves AL820), and the WBC differential was compared with the manual microscopic differentiation of the 400 WBC (200 cells differentiated by two technicians). The following coefficients of variation were obtained: within-run precision was 1.2% and 2.7% for WBC; 1.0% and 1.0% for RBC; 1.3% and 0.9% for haematocrit; 2.1% and 2.7% for platelets (rats and mice respectively). Day-to-day precision was performed using human trilevel control blood, and the CVs were found to be <1.7% for WBC, <1.4% for RBC, <1.2% for haemoglobin and <6.3% for platelets. The following ranges of measurement were found to be linear in the rat: WBC: 0.10–20.20×10³/μl; RBC: 0.016–14.3×10⁶/μl; haemoglobin: 0.08–26.8 g/dl; haematocrit: 5.0%–77%; platelets: 14.0–1670.0×10³/μl. Equal ranges were observed for mouse blood. Carry-over in rat blood was found to be 0.12% for WBC, 0.05% for RBC, 0.15% for haemoglobin and 0.46% for platelets. In mice, similar carry-over results were obtained. The correlation coefficients (Pearson, correlation coefficient) between the CELL-DYN^® 3500 and Contraves AL 820 using linear regression analysis were as follows: 0.988 and 0.997 for WBC; 0.986 and 0.920 for RBC; 0.995 and 0.984 for haemoglobin; 0.958 and 0.85 for haematocrit; 0.958 and 0.963 for platelets, for rats and mice, respectively. Correlation coefficients between the CELL-DYN^® 3500 and the manual differential of NEU (neutrophils) and LYM (lymphocytes) were higher than 0.8 in rats and higher than 0.9 in mice. Due to the relatively low absolute counts of MONO (monocytes), EOS (eosinophils) and BASO (basophils), only moderate correlation of methods was found. The CELL-DYN^® 3500 was judged to be reliable, accurate and easy-to-use for counting and identifying normal and most of the pathological blood specimens obtained from mice and rats. By using the CELL-DYN^® 3500, the time for blood sample analysis can be shortened significantly and provides extensive opportunities to characterise pathological samples.     

Performance of the Genotype® MTBDRPlus assay in the diagnosis of tuberculosis and drug resistance in Samara, Russian Federation

Background

Russia is a high tuberculosis (TB) burden country with a high prevalence of multidrug resistant tuberculosis (MDRTB). Molecular assays for detection of MDRTB on clinical specimens are not widely available in Russia.

Results

We performed an evaluation of the GenoType^® MTBDRplus assay (HAIN Lifescience GmbH, Germany) on a total of 168 sputum specimens from individual patients at a public health laboratory in Central Russia, as a model of a middle income site in a region with high levels of drug resistance. Phenotypic drug resistance tests (DST) were performed on cultures derived from the same sputum specimens using the BACTEC 960 liquid media system. Interpretable GenoType^® MTBDRplus results were obtained for 154(91.7%) specimens with readability rates significantly higher in sputum specimens graded 2+ and 3+ compared to 1+ (RR = 1.17 95%CI 1.04–1.32). The sensitivity and specificity of the assay for the detection of rifampicin (RIF) and isoniazid (INH) resistance and MDR was 96.2%, 97.4%, 97.1% and 90.7%, 83.3%, 88.9% respectively. Mutations in codon 531 of the rpoB gene and codon 315 of the katG gene dominated in RIF and INH resistant strains respectively. Disagreements between phenotypical and molecular tests results (12 samples) could be explained by the presence of rare mutations in strains circulating in Russia and simultaneous presence of resistant and sensitive bacilli in sputum specimens (heteroresistance).

Conclusion

High sensitivity, short turnaround times and the potential for screening large numbers of specimens rapidly, make the GenoType^® MTBDRplus assay suitable as a first-line screening assay for drug resistant TB.     

Validity of interferon-γ-release assays for the diagnosis of latent tuberculosis in haemodialysis patients

Haemodialysis patients are at higher risk of developing active tuberculosis (TB) infection. However, tuberculin skin tests (TST) have limitations and the diagnostic usefulness of interferon-γ-release assays (IG-RAs) remains unclear in immunocompromised hosts including haemodialysis patients. Haemodialysis patients were enrolled from a dialysis centre in Korea, an intermediate TB-burden country with a high bacille Calmette–Guérin (BCG) vaccination rate. The QuantiFERON-Gold TB In tube test® (QFT) and the T-SPOT TB test® (T-SPOT) were performed, along with the TST. We stratified patients to low- and high-risk groups, according to the risk factors for latent TB. Association between each of the three diagnostic tests and the risk of latent TB was analysed. One hundred and sixty-seven patients were enrolled. The positive rates for the TST, the QFT and T-SPOT were 23.5, 45.9 and 60.4%, respectively. Previous BCG vaccination increased the TST-positive rate in the low-risk group (OR 4.438), whereas it affected neither QFT nor T-SPOT. The positive QFT rates were 41.2 and 62.5% in the low- and high-risk groups, respectively. The QFT was associated with the high-risk group (OR 2.578), whereas the TST was not. The positive T-SPOT rates were 58.9 and 65.7% in the low- and high-risk groups, respectively. The frequency of indeterminate results was higher for the QFT (12.6%) compared with the T-SPOT (4.8%). In conclusion, the IG-RAs can be useful for the diagnosis of latent TB infection in haemodialysis patients.     

T-SPOT®.TB test and clinical risk scoring for diagnosis of latent tuberculosis infection among Thai healthcare workers

BackgroundScreening for latent tuberculosis infection (LTBI) is important to identify healthcare workers (HCWs) benefiting from preventive therapy. Interferon-gamma release assays (IGRAs) are sensitive and specific tests for LTBI diagnosis. However, in settings where IGRAs are not available, clinical risk assessment may be used as an alternative to diagnose LTBI.MethodsA cross-sectional study was conducted among HCWs of a tertiary-care university hospital in Thailand. All HCWs underwent T-SPOT®.TB test (T-SPOT) and assessment of LTBI clinical risks. Clinical risks associated with T-SPOT positivity were determined by multivariable logistic regression analysis and were given scores accordingly. The performance of the clinical risk scoring was evaluated in comparison to T-SPOT.ResultsAmong 140 enrolled HCWs, 125 (89%) were females, the median age was 27 years and 23 (16%) had T-SPOT positivity. Independent factors associated with T-SPOT positivity were age ≥30 years (adjusted odds ratio [aOR] 3.95; P = 0.002), working duration ≥60 months (aOR 3.75, P = 0.004) and frequency of TB contact ≥6 times (aOR 8.83, P = 0.005). The study's clinical risk scoring had the area under the curve by receiver operating curve analysis of 0.76 (P < 0.001) using T-SPOT positivity as a reference standard. The score of ≥3 had the best performance in diagnosing LTBI with sensitivity, specificity, positive predictive value and negative predictive value of 70%, 71%, 32% and 92%, respectively.ConclusionsIn this setting where LTBI was prevalent among HCWs but IGRAs are not widely available, the clinical risk scoring may be used as an alternative to diagnose LTBI in HCWs.     

Evaluation of commercial ResPlex II v2.0, MultiCode®-PLx,and xTAG® respiratory viral panels for the diagnosis of respiratory viral infections in adults

BackgroundCommercial multiplex PCR panels for respiratory viruses (PRV) have been recently developed. ResPlex II Panel v2.0 (Qiagen), MultiCode^®-PLx (EraGen Biosciences), and xTAG^® (Luminex) PRV's were studied. All assays detect influenza A and B, adenovirus, parainfluenza 1–3, respiratory syncytial virus A and B, human metapneumovirus and human rhinovirus. The ResPlex II additionally detects coronavirus (229E, OC43, NL63, HKU1), coxsackie/echo virus, bocavirus and differentiates adenoviruses (B, E). The MultiCode-PLX assay detects 229E, OC43, and NL63, differentiates parainfluenza 4a, 4b and adenoviruses (B, C, E). The xTAG additionally subtypes influenza A as seasonal H1 and H3.Study design202 specimens collected from adult patients with signs of respiratory infection from November, 2008 to May, 2009 were used for evaluating the performance of the three commercial PRV assays. Viral culture and xTAG were used as the standards to assess sensitivity and specificity.ResultsThe PRV assays detected more viruses than culture. When compared to culture, the xTAG PRV showed a sensitivity and specificity of 100% and 91%, compared to MultiCode-PLx with 89% and 87%, and ResPlex II with 89% and 94%, respectively. Co-infection was detected in a small subset of patient specimens. Each panel showed differences in sensitivities for individual viruses.ConclusionsWhile the ResPlex II and MultiCode-PLx offer a broader virus detection range and greater ease of use, the xTAG PRV showed increased sensitivity to common viral targets represented in the assays, and also had the ability to differentiate human from non-human influenza A H1.     

Evaluation of the combination of the NucliSENS easyMAG® and the EasyQ® applications for the detection of Mycoplasma pneumoniae and Chlamydia pneumoniae in respiratory tract specimens

Mycoplasma pneumoniae and Chlamydia pneumoniae are respiratory tract pathogens frequently involved in community-acquired pneumonia, but are fastidious microorganisms. Their direct detection mainly requires molecular amplification techniques. A nucleic acid extraction system, NucliSENS easyMAG®, and a real-time nucleic acid sequence-based amplification (NASBA) technique, NucliSENS EasyQ®, were recently developed by bioMérieux to detect both bacteria. The aim of our study was to compare the easyMAG/EasyQ combination with our in-house combination, MagNA Pure extraction (Roche) and real-time polymerase chain reaction (PCR), to detect both bacteria in respiratory tract specimens. The analytical specificities of both combinations were similar. A higher analytical sensitivity was found for C. pneumoniae using the easyMAG/EasyQ combination, since the easyMAG/EasyQ system detected nucleic acid extracts 10⁶ times more diluted than the in-house combination. Both combinations were equivalent when detecting M. pneumoniae in positive respiratory tract samples. Finally, the easyMAG/EasyQ combination is a potential useful tool for the detection of both bacteria regarding sensitivity, specificity, monitoring, and standardization of the procedure.     

Clinical evaluation of the BioFire FilmArray® BioThreat-E test for the diagnosis of Ebola Virus Disease in Guinea

BackgroundThe recent West Africa Ebola outbreak highlighted the need to provide access to rapid, safe and reliable Ebola Virus Disease diagnostics.ObjectivesThe objective of this field study was to assess the clinical performance of the FilmArray^® BioThreat-E test for the detection of Ebola Zaïre virus in whole blood in symptomatic patients suspected of Ebola Virus Disease in Conakry (Guinea) from March to July 2015.Study designThe BioThreat-E test was compared to the two RT-PCRs, using serum, implemented at Donka Hospital in the emergency context: an in-house developed quantitative one-step RT-PCR adapted from the Weidmann technique, and the RealStar^® Filovirus RT-PCR Kit 1.0 (Altona-Diagnostics). We also assessed the performance of this assay in noninvasive specimens (urine and saliva) to detect infected patients.ResultsOf 135 patients enrolled and eligible for performance assessment on whole blood, the sensitivity was 95.7% [95% CI: 85.5–99.5] and specificity 100% [95% CI: 95.9–100]. Of the 37 symptomatic infected patients able to provide saliva and/or urine samples, 34 of the 35 saliva samples and all 3 of the urine samples were positive with the BioThreat-E test.ConclusionsThis study showed that the FilmArray BioThreat-E test performs comparably to conventional molecular tests under field conditions, providing results and interpretation in approximately 1 h. Due to its operational characteristics, it can be easily deployed in the field during an epidemic and could also be a useful tool for post-outbreak surveillance.     

Evaluation of two VIDAS ®prototypes for detecting anti-HEV IgG

BackgroundHigh performance anti-hepatitis E virus (HEV) IgG assays are crucial for epidemiology.ObjectiveTo evaluate the performance of 2 prototypes developed for the VIDAS^® automatic system for detecting anti-HEV IgG, one based on the ORF2 antigen (ORF2 prototype) and the other on the ORF2 and ORF3 antigens (ORF2/3 prototype), with reference to the Wantai anti-HEV IgG assay.Study designThe sensitivity of each assay was determined by testing 113 blood samples, 63 from immunocompetent and 50 from immunocompromised patients, with a proven HEV infection defined by detecting HEV RNA. Their specificity was assessed with 103 blood samples that the Wantai assay indicated was negative for anti-HEV IgM and IgG, and negative for HEV-RNA. Cross reactivity was assessed using samples that were positive for hepatitis A virus IgG (n = 16), hepatitis C virus antibodies (n = 15), hepatitis B virus antigen and anti-HBc antibodies (n = 16), rheumatoid factor (n = 14), and negative for anti-HEV IgG with the Wantai assay.ResultsThe sensitivities in immunocompetent patients were: 95.2% (ORF2), 96.8% (ORF2/3), and 93.6% (Wantai); in immunocompromised patients they were: 66% (ORF2), 72% (ORF2/3), and 68% (Wantai). Both VIDAS prototypes detected low concentrations of anti-HEV IgG. The overall specificity was 100% (ORF2 prototype) and 98.1% (ORF2/3 prototype). Both VIDAS prototypes cross-reacted in five samples (9.6%), mainly those containing HCV antibodies or rheumatoid factor.ConclusionBoth VIDAS^® prototypes performed very well and appear to be suitable for routine detection of anti-HEV IgG.     

Evaluation of the Bruker® MBT Sepsityper IVD module for the identification of polymicrobial blood cultures with MALDI-TOF MS

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) considerably reduces timeframe required from initial blood culture positivity towards complete bacterial identification. However, rapid identification of polymicrobial blood cultures remains challenging. We evaluated the performances of the Bruker® MBT Sepsityper IVD module on MALDI-TOF MS for the direct identification of polymicrobial blood culture bottles. This module has the ability to give a strong indication that a sample contains a mixture of organisms and to identify two of them. Blood culture bottles considered as polymicrobial using routine subculture were collected and processed using the Sepsityper kit. MALDI-TOF MS identification was performed using the MBT Compass IVD software including the Sepsityper module. From 143 polymicrobial blood culture bottles tested, 34.3% (49/143) were completely identified by the module. Both microorganisms were more easily detected by the module in samples containing two pathogens than in samples containing two contaminants (36.8% vs 29.4%). Additionally, in more than half of the samples, the module detected 1 of the different microorganisms contained in the same vial. In these cases, with a pathogen and contaminant in the same sample, the module detected the pathogen in more than 80%. The Sepsityper module identified 14 microorganisms which were not recovered by conventional culture methods. The Bruker® MBT Sepsityper IVD module contributed to a valuable identification of polymicrobial blood cultures in more than a third of all cases. Conventional culture methods are still required to complete the results and to carry on susceptibility testing.     

Multi-center evaluation of the cobas® Liat® Influenza A/B & RSV assay for rapid point of care diagnosis

Point of Care Testing (POCT) provides the capability for rapid laboratory test results in patient care environments where a traditional clinical laboratory is not available. POCTs have shorter turn-around times (TATs), they may be performed by non-laboratory personnel, and the need for transport time is eliminated. The Food and Drug Administration (FDA) recently granted Clinical Laboratory Improvements Amendment (CLIA) waiver status to the cobas^® Influenza A/B & RSV assay, a rapid, accurate point-of-care test for Influenza and respiratory syncytial virus (RSV) performed on the Liat^® System. The performance characteristics of this test were determined though a multi-site study consisting of different point of care testing environments. Prospectively collected Nasopharyngeal (NP) swabs from 1361 patients seen at 8 primary care clinics and 4 emergency departments (EDs) and 295 retrospectively identified specimens were tested for Influenza A/B and RSV on the cobas^® Liat^® platform. Performance characteristics were determined through comparison to ProFlu+, a laboratory-based PCR test for Influenza A/B and RSV (reference test). Discordant specimens were adjudicated following bi-directional sequencing. The cobas^® Influenza A/B and RSV assay showed sensitivities of 99.6%, 99.3%, and 96.8% for Influenza A, Influenza B, and RSV, respectively as determined from percent positive agreement (PPA) following comparison to the reference test. Sequencing confirmed cobas^® Influenza A/B and RSV results in 49.2% of reference test discordant specimens, while crossing threshold data suggest increased sensitivity compared to the reference test. The cobas^® Influenza A/B and RSV assay was found to be a rapid, sensitive POCT for the detection of these viruses, and provides laboratory-quality PCR-based diagnostic results in point of care settings.     

The use of FTA® filter papers for diagnosis of avian influenza virus

Avian influenza viruses (AIVs) infect a wide range of host species including domestic poultry and wild birds; also, AIVs may infect humans in whom some highly pathogenic viruses (HPAIV) may cause acute fatal disease. Accurate laboratory diagnosis of AIV infections requires time-consuming and logistically complex precautionary measures for shipment of specimens or viruses to avoid biohazard exposure. The feasibility was investigated of the Flinders Technology Associates filter paper (FTA? card) for infectivity of AIVs and to preserve viral RNA for detection by RT-qPCR, sequencing and by DNA microarray assay. The infectivity of AIV subtype H6N2 and HPAIV subtype H5N1 was inactivated completely within one hour after adsorption to the FTA card at room temperature. FTA-adsorbed viral RNA remained stable for five months. Swab samples obtained from chickens infected experimentally with H5N1 virus and spotted directly onto the FTA? cards allowed a sensitive and straightforward diagnosis by RT-qPCR. FTA? cards were also suitable for examination of field samples, although AIV RNA was detected with reduced sensitivity in comparison to direct examination of swab fluids. The use of FTA? cards will facilitate safe transport of samples for molecular diagnosis of AIV avoiding the need for an uninterrupted cold storage.     

Usefulness of Sofia Pneumococcal FIA® test in comparison with BinaxNOW® Pneumococcal test in urine samples for the diagnosis of pneumococcal pneumonia

The Sofia Pneumococcal FIA® test is a recently introduced immunofluorescent assay automatically read aimed to detect Streptococcus pneumoniae antigen in urine. The aim of this study was to evaluate the usefulness of SofiaFIA® urinary antigen test (UAT) in comparison with classical immunochromatographic BinaxNOW® test for the diagnosis of pneumococcal pneumonia (PP). Observational study was conducted in the Hospital Universitari Vall d’Hebron from December 2015 to August 2016. Consecutive adult patients diagnosed of pneumonia and admitted to the emergency department in whom UAT was requested were prospectively enrolled. Paired pneumococcal UAT was performed (BinaxNOW® and SofiaFIA®) in urine samples. To assess the performance of both tests, patients were categorized into proven PP (isolation of S. pneumoniae in sterile fluid) or probable PP (isolation of S. pneumoniae in respiratory secretion). Sensitivity, specificity, and concordance were calculated. A total of 219 patients with pneumonia were enrolled, of whom 14% had a proven or probable PP, 22% a non-pneumococcal etiology, and 64% an unidentified pathogen. Concordance between tests was good (κ?=?0.81). Sensitivity of SofiaFIA® and BinaxNOW® UAT was 78.6 and 50% for proven PP (p?=?0.124), and 74.2 and 58% for proven/probable PP (p?=?0.063). Specificity for both tests was 83.3 and 85.5% for proven and proven/probable PP. In patients without an identified pathogen, SofiaFIA® test was positive in 33 (23.6%) cases and BinaxNOW® in 25 (17.8%), so Sofia Pneumococcal FIA® detected 32.6% more cases than BinaxNOW® (p?=?0.001). Sofia Pneumococcal FIA® test showed an improved sensitivity over visual reading of BinaxNOW® test without a noticeable loss of specificity.     

Comparison of the GenoType? MTBC Molecular Genetic Assay with culture methods in the diagnosis of tuberculosis

Introduction

Clinical samples from 433 patients pre-diagnosed with tuberculosis in Konya, Turkey, were investigated prospectively to compare the GenoType^® MTBC test (GenoType^® MTBC) with conventional “gold standard” culture methods.

Material and methods

Lowenstein Jensen (LJ) and Mycobacteria Growth Indicator Tube (MGIT)-960 culture methods and GenoType^® MTBC were performed together.

Results

Mycobacterium tuberculosis (M. tuberculosis) detection rates were 16.2% by culture methods, 15.4% by GenoType^® MTBC, and 6% by acid-fast bacilli microscopy. The LJ or MGIT-960 with GenoType^® MTBC detected M. tuberculosis in 12 samples each that were negative according to the other culture method alone. Among 70 M. tuberculosis-positive samples, detection rates were 37% (26/70) by microscopy and 82.8% (58/70) by LJ and MGIT-960, but 95.7% (67/70) by GenoType^® MTBC.

Conclusions

GenoType^® MTBC may be used as a beneficial adjunct test to culture methods for the detection of M. tuberculosis.     

Evaluation of the clinical performance of OncoPredict HPV® SCR assay within the VALGENT-2 framework

Human papillomavirus (HPV) assays used in cervical cancer screening should be clinically validated according to international criteria. OncoPredict HPV® Screening (SCR) is a partial genotyping multiplex real-time PCR assay targeting E6/E7 genes of 13 high-risk (hr) HPVs. OncoPredict HPV® SCR (index assay) identifies HPV-16 and HPV-18 separately, 11 other hrHPV in aggregate and includes quality controls for sample adequacy, DNA extraction efficiency and PCR inhibition. 1300 VALGENT-2 study samples (from women aged 20–60 attending the Scottish cervical cancer screening program) were tested with the index assay and the GP5+/6+ PCR enzyme immunoassay (standard comparator assay). Non-inferior accuracy detecting cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) of the index versus comparator was verified. Intra- and interlaboratory reproducibility of the index was evaluated by overall concordance and Cohen's kappa, using a sub-population (n = 526). Relative sensitivity and specificity for CIN2+ of the index versus comparator were 1.01 (95% confidence interval [CI]: 0.99–1.03) and 1.02 (95% CI: 1.0–1.04), respectively. Noninferiority p values were all ≤0.05, except for CIN3+ in patients ≥30 years. Excellent intra- and interlaboratory reproducibility was shown with concordance >98% and kappas >0.95. OncoPredict HPV® SCR fulfills the three international validation criteria for hrHPV DNA tests in cervical cancer screening.