Evaluation of the Bio-Rad Dx CT/NG/MG® assay for simultaneous detection of Chlamydia trachomatis,Neisseria gonorrhoeae and Mycoplasma genitalium in urine

We evaluated the performance of the Bio-Rad real-time Dx CT/NG/MG® assay for detection of C. trachomatis, N. gonorrhoeae and M. genitalium on a collection of 441 urine samples from sexually transmitted infections, or travellers consultations and from anonymous sperm donors that were previously analysed with the Abbott RealTime CT/NG assay. Samples positive for C. trachomatis or N. gonorrhoeae with the Abbott assay had all previously been confirmed with an in-house real-time PCR assay. Samples positive for M. genitalium with the Bio-Rad assay were subsequently analysed by an in-house real-time PCR. On a total of 441 urines, 104 samples were positive for C. trachomatis, 12 were positive for N. gonorrhoeae and seven were positive for M. genitalium. After retesting of discrepant results, the test results were completely concordant, resulting in a calculated sensitivity and specificity of the Bio-Rad assay of 98.1 % and 100 % for C. trachomatis and of 91.7 % and 100 % for N. gonorrhoeae. Results for M. genitalium with the Bio-Rad assay were also concordant with the results of an in house PCR. We also evaluated the performance of automated nucleic acid extractions of urine samples with the NucliSENS easyMAG (bioMérieux) compared to the manual DNA extraction prescribed by the insert of the kit. The easyMAG extraction gave lower Ct values, relieved inhibition and had a lower hands-on time.     

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 ESPLINE® Influenza A &; B-N assay for the detection of influenza A and B in nasopharyngeal aspirates

Several direct antigen tests for the detection of influenza often lack sensitivity compared to immunofluorescence (IF) on the specimens and viral culture (VC). We evaluated the performance of a rapid test, the ESPLINE® Influenza A &; B-N assay. A total of 302 respiratory specimens were collected at the University Hospital of Antwerp. A first group of 60 samples taken during the H1N1 outbreak (2009–2010) and a second group of 242 samples stored during the seasonal influenza epidemics (2000–2009) were analyzed with the ESPLINE® test. A subset of samples were also evaluated with the BinaxNOW Influenza and the Clearview Exact Influenza. The results were compared to IF on the specimens, VC with IF, and the combination of both, which was considered as the gold standard. The ESPLINE® test’s overall sensitivity and specificity were 91% and 97%, during the H1N1 season 80% and 93%, and for the detection of seasonal influenza 93% and 97%, respectively. In comparison to the BinaxNOW Influenza and the Clearview Exact Influenza, all tests demonstrated a similar specificity of 92.0–100% but a significantly different sensitivity of 44.4–86.0%, with the ESPLINE® test being significantly more sensitive. Due to its very good performance and simplicity, the ESPLINE® test facilitates urgent testing. The test seems less sensitive to detect H1N1 compared to seasonal influenza, although the difference is borderline not significant (p?=?0.067).     

Viral agents causing lower respiratory tract infections in hospitalized children: evaluation of the Speed-Oligo? RSV assay for the detection of respiratory syncytial virus

Respiratory syncytial virus (RSV) is the viral agent which is more frequently involved in lower respiratory tract infections (LRTIs) in infants under 1 year of age in developed countries. A new oligochromatographic assay, Speed-Oligo? RSV, was designed and optimized for the specific detection and identification of RSV subtypes A and B. The test was evaluated in 289 clinical samples from 169 hospitalized children using an immunochromatography (IC) test, virus isolation by culture, and an in-house real-time polymerase chain reaction (RT-PCR). Other viruses causing LRTIs were investigated by cell culture or PCR-based tests. Sixty-two patients were infected by RSV (36.7%). In addition, adenovirus, influenza B, parainfluenza 2, and human metapneumovirus were detected in rates ranging from 5 to 8%. A proportion of 10.1% of the patients had mixed infections. The sensitivity, specificity, and positive and negative predictive values were, respectively, 94.9, 99.4, 98.9, and 97.4% for Speed-Oligo? RSV, 92.9, 96.3, 92.9, and 96.3% for RT-PCR/RSV, and 58.4, 98.1, 93.3, and 82.6% for IC. Our rates of viral detection and co-infection were similar to those of previously reported series. Finally, we find that Speed-Oligo? RSV is a rapid and easy-to-perform technique for the detection of RSV and the identification of subtypes A and B.     

Detection of herpesvirus EBV DNA in the lower respiratory tract of ICU patients: a marker of infection of the lower respiratory tract?

Epstein-Barr virus (EBV) is a lymphotropic herpesvirus causing clinically self-limiting but lifelong persisting infections. Although several severe diseases (e.g., Hodgkin′s disease) are associated with EBV, its role in lower respiratory tract infections is still elusive. The prevalence of EBV, herpes simplex virus (HSV) and cytomegalovirus (CMV) in bronchoalveolar fluid (BAL) samples was evaluated in a retrospective study. BAL samples from 135 patients in the intensive or coronary care unit (ICU/ICC) at University Hospital Frankfurt/Main (Germany) were investigated using an in-house real-time PCR to detect EBV-, HSV- and CMV-specific DNA. Overall, herpesvirus DNA was detected in n = 82/135 BAL samples (60.7 %). Besides mono-infections with either EBV or HSV, concomitant infection with EBV and HSV DNA was most frequent, whereby the relative HSV viral load was typically higher. Patients with HSV-positive BAL required mechanical ventilation on average 5 days longer than patients with HSV-negative BAL (p = 0.006). Additionally, the proinflammatory cytokine IL-6 was significantly elevated in sera of patients positive for EBV in comparison with patients with EBV-negative BAL (p = 0.01). This study demonstrates a high prevalence of herpesviruses in BAL samples of ICU/ICC patients. The detection of one or more herpesvirus in BAL is strongly associated with the duration of ventilation and patient′s age. The association between IL-6 levels and EBV detection should be evaluated in further studies.     

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.     

Multicenter evaluation of the ENTEROVIRUS R-gene™ real-time RT-PCR assay for the detection of enteroviruses in clinical specimens

BackgroundThe rapid molecular diagnosis of enteroviral meningitis has been shown important for an adequate management of the patients.ObjectivesA new CE-marked real-time RT-PCR assay (ENTEROVIRUS R-gene?, Argene) was evaluated in two university hospital virology laboratories.Study designReactivity, analytical sensitivity and specificity were evaluated using 54 prototype and 173 clinical human enterovirus (HEV) strains, a 12-sample HEV proficiency panel, and 30 non-HEV microorganisms. The clinical performance of the ENTEROVIRUS R-gene? assay was evaluated by testing 197 cerebrospinal fluid (CSF) and 103 respiratory specimens, comparatively to the routinely used diagnostic techniques.ResultsSixty-four out of the 65 HEV serotypes tested were detected. The analytical sensitivity ranged between 10^?2.64 and 10^2.39 TCID₅₀/50 μl. Cross-reactivity was observed with four human rhinoviruses. On 59 CSF specimens analyzed prospectively, the results of the ENTEROVIRUS R-gene? assay showed a 94.8% concordance with those of the Smart enterovirus (EV) assay (Cepheid). On 138 CSF specimens tested retrospectively, the results of the ENTEROVIRUS R-gene? assay showed a 97.1% concordance with those of either the GeneXpert EV assay (Cepheid) or the in-house RT-PCR HEV assays used at the time of specimen collection. On 103 respiratory specimens, the concordance between the results of the ENTEROVIRUS R-gene? assay and those of the routine RT-PCRs or viral culture was 90.2% and 96.1% before and after retest, respectively.ConclusionsThe new test was found able to detect a large panel of enterovirus serotypes; it was sensitive when used on clinical specimens; and, easy and rapid to perform on a routine basis.     

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 level of antibodies against Chlamydophila (Chlamydia) pneumoniae in post-surgery heart ischaemia patients and their clinical conditions – a six-year study

Introduction

Inflammatory conditions modulated by Chlamydophila (Chlamydia) pneumoniae are considered to play an important role in the onset of atherosclerosis. In this paper we present the results of progressive observation of C. pneumoniae antibody titres in patients who underwent coronary artery bypass graft (CABG).

Material and methods

The objective of our research was a prospective observation of antibody titres in IgA and IgG class antibodies against C. pneumoniae using indirect immunofluorescence in a group of 155 post-surgery CABG patients suffering from heart ischaemia. The microbiological test results were compared with patients’ present coronary complaints evaluated on the CCS scale during a six-year period.

Results

Six years after CABG, 128 patients (82.6%) are still alive. During the study a positive serological conversion of antibody titres was observed in 36 patients in the IgA class antibodies, and in 26 patients in the IgG class. The group of patients with no antibodies against C. pneumoniae decreased from 23.2 to 3.4%, while the group of patients with antibodies in both IgG and IgA classes increased from 52.3 to 83.9%. The average CCS degree decreased from 3.18 before CABG to 1.65 in the present study.

Conclusions

These results show no connection between the serological symptoms of chronic C. pneumoniae infection and coronary complaints evaluated on the CCS scale during a six-year study on post-CABG patients suffering from heart ischaemia. The surgical treatment of heart ischaemia brought about long-term improvement in the coronary condition of the observed group of patients.     

Evaluation of the Oxoid Brilliance™ CRE Agar for the detection of carbapenemase-producing Enterobacteriaceae

The adequate detection of carbapenemase-producing Enterobacteriaceae (CPE) is essential for adequate antibiotic therapy and for infection control purposes, especially in an outbreak setting. Selective agars play an important role in the detection of CPE. The Oxoid Brilliance? CRE Agar (Thermo Fisher Scientific) was evaluated for the detection of CPE using 255 non-repetitive Enterobacteriaceae isolates, including 95 CPE (36 KPC, 4 KPC plus VIM, 4 NDM, 6 GIM, 20 VIM, and 25 OXA-48-producing isolates). The sensitivity of the CRE agar for the detection of CPE was 94 % (89/95), but differed per carbapenemase gene (100 % for KPC, NDM, and GIM, 90 % for VIM, and 84 % for OXA-48-producing isolates). The specificity of the CRE agar was 71 %, due to the growth of AmpC- and/or ESBL-producing isolates. The CRE agar is a sensitive tool for the detection of KPC and metallo-carbapenemase-producing Enterobacteriaceae, although the detection of OXA-48 producers is less optimal. The relatively low specificity requires confirmation of carbapenemase production for isolates recovered from the CRE agar.     

Evaluation of the Roche COBAS® TaqMan® HIV-1 test for quantifying HIV-1 RNA in infected cells and lymphoid tissue

Lymphoid tissue is the main reservoir of HIV-1 in infected individuals. In this study the COBAS? TaqMan? HIV-1 test was evaluated for use with the High Pure System (HPS), for quantifying HIV-1 RNA in infected cells and lymphoid tissue specimens. Serial dilutions of 8E5-LAV1 infected T-cells into SUP-T1 cells and 44 tonsil specimens were examined. Some modifications of the test were required, such as the removal of residual DNA and the HIV-1 RNA output copies were adjusted to the sample input and expressed as HIV-1 RNA copies/μg of total RNA. The Roche COBAS? TaqMan HIV-1? (HPS) test proved to be a robust, sensitive, specific and reproducible method for quantifying HIV-1 RNA in infected cells and lymphoid tissue. Linearity and reproducibility were observed in serial dilutions of 8E5-LAV1 infected T-cells (R2>0.86). High reproducibility was found in clinical tonsil specimens (Wilcoxon test p > 0.05). rDNase I treatment was essential to avoid false positives caused by residual HIV-1 DNA, mainly in tonsil specimens obtained from infected patients receiving effective antiretroviral treatment. Probit analysis determined the limit of detection as 22HIV-1 RNA copies/μg of total RNA. The Roche COBAS? TaqMan? HIV-1 (HPS) test thereby proved to be a helpful tool for measuring the HIV-1 viral load in infected cells and lymphoid tissue reservoirs.     

Evaluation of the BioFire® FilmArray® Meningitis/Encephalitis panel in an adult and pediatric Ugandan population

BackgroundMeningitis causes significant mortality in sub-Saharan Africa and limited diagnostics exist. We evaluated the utility of the BioFire® FilmArray® Meningitis/Encephalitis multiplex PCR panel (BioFire ME) in HIV-infected adults and HIV-infected and uninfected children presenting with suspected meningitis in Uganda.MethodsWe tested cerebrospinal fluid (CSF) using a stepwise meningitis diagnostic algorithm including BioFire ME. We determined the diagnostic performance of BioFire ME for cryptococcal meningitis, using cryptococcal antigen (CrAg) and CSF culture as reference standards, and assessed other central nervous system (CNS) pathogens identified by the panel.ResultsWe evaluated 328 adult and 42 pediatric CSF specimens using BioFire ME. Of the adult CSF samples tested, 258 were obtained at baseline, and 70 were obtained from repeat lumbar punctures in cryptococcal meningitis. For Cryptococcus, sensitivity was 82%, specificity was 98%, PPV was 98%, and NPV was 79% in baseline specimens using CSF CrAg as the reference standard. Among follow-up specimens, a negative BioFire ME for Cryptococcus predicted CSF culture sterility with 84% NPV. Overall sensitivity was decreased at low fungal burdens: 29% for 0–99 Cryptococcus CFU/mL compared to 94% for ≥100 CFU/mL in baseline specimens. Other pathogens detected included E. Coli, H. influenzae, S. pneumoniae, CMV, enterovirus, HSV, HHV-6, and VZV. Two specimens tested positive for S. pneumoniae and one for Cryptococcus in the pediatric population.ConclusionsMultiplex PCR is a promising rapid diagnostic test for meningitis in adults and children in resource-limited settings. Cryptococcus at low fungal burdens in CSF may be missed by BioFire ME.     

Evaluation of the RIDA(®)QUICK immunochromatographic norovirus detection assay using specimens from Australian gastroenteritis incidents

A range of laboratory methods is now available for the detection of norovirus, a major cause of gastroenteritis. Recently, a commercial immunochromatographic assay for norovirus detection, the RIDA^®QUICK assay, has become available, but there is still only limited information on its efficacy. This study examined the sensitivity and specificity of the RIDA^®QUICK assay, using faecal material received for testing in a major diagnostic/reference laboratory in Australia. The sensitivity of the assay was found to be 83% and the specificity was 100%. No false positive norovirus results were found and the assay did not cross-react with common faecal viruses such as rotavirus, astrovirus, sapovirus and adenovirus. The assay was less reliable for genogroup I (GI) noroviruses than for genogroup II (GII) noroviruses. Genotypes detected by the assay included GII.1, GII.2, GII.3, GII.4, GII.6 and GII.7. The assay failed to detect any GI specimens in the test group. Genotypes not detected included GI.4 and GI.6. The assay was simple and quick to perform. It is valuable in a point-of-care situation or as a backup in a laboratory where a rapid initial norovirus result is required.     

Comparison of the f-HPV typing™ and Hybrid Capture II® assays for detection of high-risk HPV genotypes in cervical samples

Human papillomavirus genotyping is being considered in cervical screening programs and for monitoring the effectiveness of HPV vaccination. Both approaches require access to fast, easy and high-throughput technology. The aim of this study was to compare a new commercial assay (f-HPV typing?) with the Hybrid Capture II? (HC2) to detect HPV infection. The F-HPV typing is a multiplex fluorescent PCR method recognizing E6 and E7 regions of 13 high-risk (HR) HPV types, the same set of HR-types targeted HC2 test. A subset of 157 cervical samples was tested with both assays. The percentage of positive HR-HPV DNA samples was 24% (37/155) by HC2 and 33% (49/155) by f-HPV typing. Concordant results were found in 133/155 (overall agreement, 85.8%; Cohen's kappa=0.65). The analytical sensitivity and specificity of f-HPV were 97.6 and 93, respectively. In conclusion, this study shows that the f-HPV assay provides a good alternative to HC2 to detect HPV infection, allowing simple and rapid HPV genotyping and detecting multiple infections.     

Evaluation of the SD BIOLINE TB Ag MPT64 Rapid® for the diagnosis of tuberculosis

The purpose of this study was to evaluate the SD Bioline Ag MPT64 Rapid^® for identification of the Mycobacterium tuberculosis complex. The method uses an immunochromatographic assay and needs 100 μl of sample taken from liquid culture or colonies suspended. The sensitivity was determined using 99 strains of M. tuberculosis complex and the specificity using 10 nontuberculous mycobacteria and 85 strains other than mycobacteria genus. The test showed excellent sensitivity (99%) and specificity (100%). This technique displays several advantages and is destined to spread in all laboratories and particularly in endemic areas.