Performance of the cobas EBV and cobas BKV assays: multi-site comparison of standardized quantitation.

Guidelines recommend monitoring of Epstein-Barr virus (EBV) and BK virus (BKV) in solid organ and hematopoietic stem cell transplant patients. The majority of quantitative DNA testing for EBV and BKV employs unstandardized individual laboratory-developed testing solutions (LDTs), with implications for accuracy, reproducibility, and comparability between laboratories. The performance of the cobas EBV and cobas BKV assays was assessed across five laboratories, using the World Health Organization International Standards (WHO IS) for EBV and BKV, and the National Institute of Standards and Technology Quantitative Standard for BKV, and results were compared with the LDTs in use at the time. Methods were also compared using locally sourced clinical specimens. Variation was high when laboratories reported EBV or BKV DNA values using LDTs, where quantitative values were observed to differ by up to 1.5 log10 unit/mL between sites. Conversely, results from the cobas EBV and cobas BKV assays were accurate and reproducible across sites and on different testing days. Adjustment of LDTs using the international standards led to closer alignment between the assays; however, day-to-day reproducibility of LDTs remained high. In addition, BKV continued to show bias, indicating challenges with the commutability of the BKV International Standard. The cobas EBV and cobas BKV assays are automated, aligned to the WHO IS, and have the potential to reduce the variability in viral load testing introduced by differences in LDTs. Standardization of reporting values may eventually allow different centers to compare data to allow clinical decision thresholds to be established supporting improvements in patient management.IMPORTANCEThe application of center-specific cut-offs for clinical decisions and the variability of LDTs often hinder interpretation; thus, the findings reported here support the need for standardization in the field of post-transplant monitoring of EBV and BKV to improve patient management. Alongside the choice of assay, it is also important to consider which standard to use when deciding upon a testing methodology. This is a call to action for standardization, as treatment for EBV and BKV is driven by viral load test results, and the more accurate and comparable the test results are across institutions, the more informed and better the treatment decisions can be.

Chlamydia trachomatis samples testing falsely negative in the Aptima Combo 2 test in Finland, 2019.

Since February 2019, over 160 Chlamydia trachomatis (CT) cases testing negative or equivocal by Aptima Combo 2 (AC2) but positive by Aptima CT test run with Panther instruments occurred in Finland. The AC2 test targets chlamydial 23S rRNA while the CT test targets 16S rRNA. Sequencing of 10 strains revealed a nucleotide substitution in 23S rRNA. The significance of this for the failure of the AC2 test to detect the variant is not yet known.

Comparison of Two Commercial Platforms and a Laboratory-Developed Test for Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) RNA.

Mitigation of the ongoing coronavirus disease 2019 (COVID-19) pandemic requires reliable and accessible laboratory diagnostic services. In this study, the performance of one laboratory-developed test (LDT) and two commercial tests, cobas SARS-CoV-2 (Roche) and Amplidiag COVID-19 (Mobidiag), were evaluated for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in respiratory specimens. A total of 183 specimens collected from suspected COVID-19 patients were studied with all three methods to compare their performance. In relation to the reference standard, which was established as the result obtained by two of the three studied methods, the positive percent agreement was highest for the cobas test (100%), followed by the Amplidiag test and the LDT (98.9%). The negative percent agreement was lowest for the cobas test (89.4%), followed by the Amplidiag test (98.8%), and the highest value was obtained for the LDT (100%). The dilution series of positive specimens, however, suggests significantly higher sensitivity for the cobas assay in comparison with the other two assays, and the low negative percent agreement value may be due to the same reason. In general, all tested assays performed adequately. Clinical laboratories need to be prepared for uninterrupted high-throughput testing during the coming months to mitigate the pandemic. To ensure no interruption, it is critical that clinical laboratories maintain several simultaneous platforms in their SARS-CoV-2 nucleic acid testing.

Real-life clinical sensitivity of SARS-CoV-2 RT-PCR test in symptomatic patients.

BACKGROUND: Understanding the false negative rates of SARS-CoV-2 RT-PCR testing is pivotal for the management of the COVID-19 pandemic and it has implications for patient management. Our aim was to determine the real-life clinical sensitivity of SARS-CoV-2 RT-PCR. METHODS: This population-based retrospective study was conducted in March-April 2020 in the Helsinki Capital Region, Finland. Adults who were clinically suspected of SARS-CoV-2 infection and underwent SARS-CoV-2 RT-PCR testing, with sufficient data in their medical records for grading of clinical suspicion were eligible. In addition to examining the first RT-PCR test of repeat-tested individuals, we also used high clinical suspicion for COVID-19 as the reference standard for calculating the sensitivity of SARS-CoV-2 RT-PCR. RESULTS: All 1,194 inpatients (mean [SD] age, 63.2 [18.3] years; 45.2% women) admitted to COVID-19 cohort wards during the study period were included. The outpatient cohort of 1,814 individuals (mean [SD] age, 45.4 [17.2] years; 69.1% women) was sampled from epidemiological line lists by systematic quasi-random sampling. The sensitivity (95% CI) for laboratory confirmed cases (repeat-tested patients) was 85.7% (81.5-89.1%) inpatients; 95.5% (92.2-97.5%) outpatients, 89.9% (88.2-92.1%) all. When also patients that were graded as high suspicion but never tested positive were included in the denominator, the sensitivity (95% CI) was: 67.5% (62.9-71.9%) inpatients; 34.9% (31.4-38.5%) outpatients; 47.3% (44.4-50.3%) all. CONCLUSIONS: The clinical sensitivity of SARS-CoV-2 RT-PCR testing was only moderate at best. The relatively high false negative rates of SARS-CoV-2 RT-PCR testing need to be accounted for in clinical decision making, epidemiological interpretations, and when using RT-PCR as a reference for other tests.

SARS-CoV-2 sample-to-answer nucleic acid testing in a tertiary care emergency department: evaluation and utility.

BACKGROUND: Rapid sample-to-answer tests for detection of SARS-CoV-2 are emerging and data on their relative performance is urgently needed. OBJECTIVES: We evaluated the analytical performance of two rapid nucleic acid tests, Cepheid Xpert® Xpress SARS-CoV-2 and Mobidiag Novodiag® Covid-19, in comparison to a combination reference of three large-scale PCR tests. Moreover, utility of the Novodiag® test in tertiary care emergency departments was assessed. RESULTS: In the preliminary evaluation, analysis of 90 respiratory samples resulted in 100% specificity and sensitivity for Xpert®, whereas analysis of 107 samples resulted in 93.4% sensitivity and 100% specificity for Novodiag®. Rapid SARS-CoV-2 testing with Novodiag® was made available for four tertiary care emergency departments in Helsinki, Finland between 18 and 31 May, coinciding with a rapidly declining epidemic phase. Altogether 361 respiratory specimens, together with relevant clinical data, were analyzed with Novodiag® and reference tests: 355/361 of the specimens were negative with both methods, and 1/361 was positive in Novodiag® and negative by the reference method. Of the 5 remaining specimens, two were negative with Novodiag®, but positive with the reference method with late Ct values. On average, a test result using Novodiag® was available nearly 8 hours earlier than that obtained with the large-scale PCR tests. CONCLUSIONS: While the performance of novel sample-to-answer PCR tests need to be carefully evaluated, they may provide timely and reliable results in detection of SARS-CoV-2 and thus facilitate patient management including effective cohorting.

Differential diagnosis of acute central nervous system infections in children using modern microbiological methods.

AIM: Except bacterial meningitis, the agents causing acute central nervous system (CNS) infections in children are disclosed in only approximately half of the cases, and even less in encephalitis. We studied the potential of modern microbiological assays to improve this poor situation. METHODS: In a prospective study during 3 years, all children attending hospital with suspected CNS infection were examined using a wide collection of microbiological tests using samples from the cerebrospinal fluid, serum, nasal swabs and stool. RESULTS: Among 213 patients, 66 (31%) cases suggested CNS infection and specific aetiology was identified in 56 patients. Of these microbiologically confirmed cases, viral meningitis/encephalitis was diagnosed in 25 (45%), bacterial meningitis in 21 (38%) and neuroborreliosis in 9 (16%) cases while 1 child had fungal infection. In meningitis patients, the causative agent was identified in 85% (35/41) cases and in encephalitis in 75% (12/16). The most common bacteria were Streptococcus agalactiae, Streptococcous pneumonie and Neisseria meningitidis, while the most frequently detected viruses were enteroviruses and varicella zoster virus. CONCLUSION: In 75% to 85% of paediatric CNS infections, specific microbiological diagnosis was obtained with modern laboratory techniques. The results pose a basis for prudent approach to these potentially serious diseases.

The Finnish New Variant of Chlamydia trachomatis with a Single Nucleotide Polymorphism in the 23S rRNA Target Escapes Detection by the Aptima Combo 2 Test.

In 2019, more than 200 cases of Chlamydia trachomatis negative/equivocal by the Aptima Combo 2 assay (AC2, target: 23S rRNA) with slightly elevated relative light units (RLUs), but positive by the Aptima Chlamydia trachomatis assay (ACT, target: 16S rRNA) have been detected in Finland To identify the cause of the AC2 CT false-negative specimens, we sequenced parts of the CT 23S rRNA gene in 40 specimens that were AC2 negative/equivocal but ACT positive. A single nucleotide polymorphism (SNP; C1515T in the C. trachomatis 23S rRNA gene) was revealed in 39 AC2/ACT discordant specimens. No decrease in the number of mandatorily notified C. trachomatis cases was observed nationally in Finland in 2010-2019. When RLUs obtained for AC2 negative specimens were retrospectively evaluated in 2011-2019, a continuous increase in the proportion of samples with RLUs 10-19 was observed since 2014, and a slight increase in the proportion of samples with RLUs 20-84 in 2017-2019, indicating that the Finnish new variant of C. trachomatis might have been spreading nationally for several years. This emphasizes that careful surveillance of epidemiology, positivity rate and test performance are mandatory to detect any changes affecting detection of infections.

Performance of the Alere i influenza A&B assay and mariPOC test for the rapid detection of influenza A and B viruses.

BACKGROUND: Timely detection of influenza viruses is required to facilitate infection control measures and appropriate patient management. The Alere™ i Influenza A&B assay for detection of viral RNA and multianalyte mariPOC(®) test for detection of viral antigens enable rapid detection of influenza viruses with little hands-on time. OBJECTIVES: To evaluate the performance of the Alere i Influenza A&B assay and the mariPOC test in comparison to the Xpert(®) Flu A/B assay and laboratory-developed real-time reverse transcription-polymerase chain reaction. STUDY DESIGN: A total of 140 and 108 nasopharyngeal specimens were analysed for evaluation of the Alere i and mariPOC, respectively. RESULTS: The sensitivity and specificity of the Alere i Influenza A&B assay for detection of influenza A virus was 80.0% and 98.1%, and for influenza B virus 45.2% and 98.2%, respectively. For the mariPOC test, a sensitivity and specificity of 53.1% and 98.7%, respectively, for detection of influenza A virus was achieved. CONCLUSIONS: The mariPOC test proved insensitive for detection of influenza A virus and therefore unsuitable for individual patient diagnosis without confirmatory testing. In contrast, the Alere i Influenza A&B assay was reasonably sensitive and specific for detection of influenza A and B virus, although decreased detection of specimens with low viral load was observed particularly for detection of influenza B virus. Taken together with its rapidity and ease of use, the Alere i influenza A&B assay is a welcome alternative to immunochromatographic rapid influenza detection tests and may provide timely results that enable appropriate patient care and management of patient flow during high-prevalence seasons.

Performance of the Luminex xTAG Respiratory Viral Panel Fast in a clinical laboratory setting.

The aim of the study was to develop a real-time RT-PCR for the detection of enteroviruses (EVs) and rhinoviruses (RVs) and to assess the performance of the xTAG RVP Fast assay in comparison to a direct fluorescent assay (DFA), a real-time RT-PCR assay for the detection of respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), and the EV/RV RT-PCR assay developed in this study. The performance of the RVP Fast assay was assessed in the analysis of 373 nasopharyngeal samples. For the viruses of the DFA panel, detection rates of 27.6% and 23.8% were obtained by RVP and DFA, respectively, in analysis of a set of 297 samples collected in 2009-2010. These results show statistically significant superiority of the RVP Fast assay (P=0.049). For RSV, hMPV, EV, and RV, detection rates of 48.0% and 45.2% were achieved by RVP and RT-PCR, respectively. For individual targets, increased detection of EV/RV (P=0.043) and decreased detection of influenza A virus (P=0.004) by RVP in comparison to real-time RT-PCR was observed. The results of the present study imply the need to adjust the InfA component of the RVP Fast assay to also cover the InfA(H1N1) 2009 virus.

Detection of human picornaviruses by multiplex reverse transcription-PCR and liquid hybridization.

A qualitative multiplex reverse transcription (RT)-PCR and liquid hybridization assay for the detection of human enteroviruses, rhinoviruses, parechoviruses, and Aichi virus was developed. Furthermore, a separate assay for the recognition of hepatitis A virus was established to complement the test pattern so that all human picornaviruses were covered. The amplicons, which represented the 5' untranslated regions of the viral RNA genomes, were identified in liquid hybridization reactions with genus-specific digoxigenin-labeled oligonucleotide probes. The sensitivity of the multiplex RT-PCR and liquid hybridization assay was 10 to 100 picornavirus genome equivalents for representatives of each picornavirus genus. The hepatitis A virus assay exhibited a sensitivity of 10 genome copies. Both the uniplex and the multiplex tests were highly specific for the target viruses. Twenty-three clinical samples, including cerebrospinal fluid, serum, and nasopharyngeal swab specimens, were used for clinical evaluation of the multiplex RT-PCR assay. The results obtained were consistent with the results of routine virus diagnostic assays. Furthermore, the assay was used to screen 68 stool specimens for the presence of parechoviruses and Aichi virus. One sample was found to contain parechovirus RNA, whereas no Aichi virus was detected. The assay described here can be applied for the efficient identification of human enteroviruses and rhinoviruses in clinical specimens and simultaneously enables the collection of information on the epidemiology and clinical outcomes of infections caused by the currently poorly known human parechoviruses and Aichi virus.