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.
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.
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.
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.
We compared the clinical characteristics, findings, and outcomes of hospitalized patients with coronavirus disease 2019 (COVID-19) or influenza to detect relevant differences.
From December 2019 to ...April 2020, we recruited all eligible hospitalized adults with respiratory infection to a prospective observational study at a tertiary care hospital in Finland. Influenza and SARS-CoV-2 infections were confirmed by RT-PCR. Follow-up lasted for 3 months from admission.
We included 61 patients, of whom 28 were COVID-19 and 33 influenza patients with median ages of 53 and 56 years. Majority of both COVID-19 and influenza patients were men (61% vs. 67%) and had at least one comorbidity (68% vs. 85%). Pulmonary diseases and current smoking were less common among COVID-19 than influenza patients (5 18% vs. 15 45%, p=.03 and 1 4% vs. 10 30%, p=.008). In chest X-ray at admission, ground-glass opacities (GGOs) and consolidations were more frequent among COVID-19 than influenza patients (19 68% and 7 21%, p<.001). Severe disease and intensive care unit (ICU) admission occurred more often among COVID-19 than influenza patients (26 93% vs. 19 58%, p=.003 and 8 29% vs. 2 6%, p=.034). COVID-19 patients were hospitalized longer than influenza patients (six days IQR 4-21 vs. 3 2-4, p<.001).
Bilateral GGOs and consolidations in chest X-ray may help to differentiate COVID-19 from influenza. Hospitalized COVID-19 patients had more severe disease, required longer hospitalization and were admitted to ICU more often than influenza patients, which has important implications for public health policies.
Multiple introductions of SARS-COV-2 Omicron variant BA.1 and BA.1.1. lineages to Finland were detected in early December 2021. Within 3 weeks, Omicron overtook Delta as the most common variant in ...the capital region. Sequence analysis demonstrated the emergence and spread through community transmission of a large cluster of BA.1.1 virus.
Central nervous system (CNS) infections such as meningitis and encephalitis are life-threatening conditions that demand hospital care and prompt identification of the causative agent. Since 2015, ...there has been only one CE-IVD-marked rapid multiplexed diagnostic assay in cassette format for bacterial and viral detection from cerebrospinal fluid (CSF): the BioFire FilmArray meningitis/encephalitis (ME) panel. In the beginning of 2022, Qiagen introduced the QIAstat-Dx meningitis/encephalitis panel. It is a CE-IVD-marked multiplex PCR cassette test intended for the identification of suspected infectious meningitis, encephalitis, or meningoencephalitis caused by bacterial, viral, or fungal pathogens. In this study, we evaluated patient and quality control samples using the QIAstat-Dx meningitis/encephalitis panel and compared the results to those of the BioFire FilmArray meningitis/encephalitis panel and reference methods (current routine analysis methods in our laboratory, PCR, or cultivation). The combined positive percent agreement between the two panel assays was 100%, and the negative percent agreement was 94%. We further compared specifically herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) dilution series using six commercial herpesvirus assays, including the two cassette tests. The results suggested that real-time PCR methods (with separate extraction) were the most sensitive methods. When comparing the cassette tests, the BioFire FilmArray meningitis/encephalitis panel produced more positive results than the QIAstat-Dx meningitis/encephalitis panel in the herpesvirus analyses.
The diagnosis of infectious meningitis and encephalitis relies mostly on specific PCR and culturing methods, but commercial syndromic panel assays are bringing a change in diagnostics. With multiplexed analysis, the identification of the pathogen is potentially faster, and less sample material is needed. The novel QIAstat-Dx meningitis/encephalitis panel assay is intended for the rapid identification of pathogens from cerebrospinal fluid for suspected central nervous system (CNS) infection, which is a life-threatening condition and difficult to diagnose. We studied the performance of this panel assay using patient samples and dilution series of selected viruses. The evaluation data for this novel meningitis/encephalitis panel assay are useful for other clinical laboratories and organizations using or considering using this test.
•The Roche-CAP/CTM-CMV targeting UL54 under-quantified the CMV quality assurance UK-NEQAS-2014.•In 329 CMV-UL54 targets, 43 mutations were identified, 8 being relevant in the UK-NEQAS-2014.•New ...CMV-UL54-QNAT assays were designed of 361 bp, 254 bp, 151 bp, and 95 bp amplicons.•Smaller amplicon size increased quantification results causing 10-fold higher CMV loads.•Plasma CMV loads in SOT patients reflect mostly unprotected genomes sensitive to DNase-I.
Cytomegalovirus (CMV) management post-transplantation relies on quantification in blood, but inter-laboratory and inter-assay variability impairs commutability. An international multicenter study demonstrated that variability is mitigated by standardizing plasma volumes, automating DNA extraction and amplification, and calibration to the 1st-CMV-WHO-International-Standard as in the FDA-approved Roche-CAP/CTM-CMV. However, Roche-CAP/CTM-CMV showed under-quantification and false-negative results in a quality assurance program (UK-NEQAS-2014).
To evaluate factors contributing to quantification variability of CMV viral load and to develop optimized CMV-UL54-QNAT.
The UL54 target of the UK-NEQAS-2014 variant was sequenced and compared to 329 available CMV GenBank sequences. Four Basel-CMV-UL54-QNAT assays of 361 bp, 254 bp, 151 bp, and 95 bp amplicons were developed that only differed in reverse primer positions. The assays were validated using plasmid dilutions, UK-NEQAS-2014 sample, as well as 107 frozen and 69 prospectively collected plasma samples from transplant patients submitted for CMV QNAT, with and without DNase-digestion prior to nucleic acid extraction.
Eight of 43 mutations were identified as relevant in the UK-NEQAS-2014 target. All Basel-CMV-UL54 QNATs quantified the UK-NEQAS-2014 but revealed 10-fold increasing CMV loads as amplicon size decreased. The inverse correlation of amplicon size and viral loads was confirmed using 1st-WHO-International-Standard and patient samples. DNase pre-treatment reduced plasma CMV loads by >90% indicating the presence of unprotected CMV genomic DNA.
Sequence variability, amplicon length, and non-encapsidated genomes obstruct standardization and commutability of CMV loads needed to develop thresholds for clinical research and management. Besides regular sequence surveys, matrix and extraction standardization, we propose developing reference calibrators using 100 bp amplicons.
Transplant patients need lifelong immunosuppressive medication, but this reduces their defense mechanisms, making them prone to viral infections and reactivations. We aimed to clarify the prevalence ...and clinical manifestations of the human herpes virus 6 (HHV‐6) infection in children after pediatric solid organ transplants. Clinical findings and viral loads were compared between primary HHV‐6 infections and reactivations. The study comprised 47 kidney, 25 liver, and 12 heart transplant patients who underwent surgery from 2009 to 2014. HHV‐6 antibodies were analyzed before surgery, and HHV‐6 DNAemia tests were regularly carried out after the transplant using a real‐time quantitative polymerase chain reaction method. We found the primary HHV‐6 infection in 19 of 22 (86%) seronegative patients, and it was more common in patients under 3 years of age (79%) than over 3 (38%, P=.0002). Post‐transplant HHV‐6 DNAemia affected 48 of 84 (57%) patients and was significantly higher in primary infections than reactivations (P=.001), and 17 of 48 (35%) patients had symptoms when it was detected at a median of 2 weeks post‐transplant. The HHV‐6 infection was common after solid organ transplants, especially under 3 years of age, and it typically started 2 weeks after surgery. Testing for HHV‐6 DNAemia is recommended shortly after transplantation, especially in patients with fever, diarrhea, rash, seizures, or abnormal liver enzyme tests.
Failures in the drinking water distribution system cause gastrointestinal outbreaks with multiple pathogens. A water distribution pipe breakage caused a community-wide waterborne outbreak in Vuorela, ...Finland, July 2012. We investigated this outbreak with advanced epidemiological and microbiological methods. A total of 473/2931 inhabitants (16%) responded to a web-based questionnaire. Water and patient samples were subjected to analysis of multiple microbial targets, molecular typing and microbial community analysis. Spatial analysis on the water distribution network was done and we applied a spatial logistic regression model. The course of the illness was mild. Drinking untreated tap water from the defined outbreak area was significantly associated with illness (RR 5.6, 95% CI 1.9-16.4) increasing in a dose response manner. The closer a person lived to the water distribution breakage point, the higher the risk of becoming ill. Sapovirus, enterovirus, single Campylobacter jejuni and EHEC O157:H7 findings as well as virulence genes for EPEC, EAEC and EHEC pathogroups were detected by molecular or culture methods from the faecal samples of the patients. EPEC, EAEC and EHEC virulence genes and faecal indicator bacteria were also detected in water samples. Microbial community sequencing of contaminated tap water revealed abundance of Arcobacter species. The polyphasic approach improved the understanding of the source of the infections, and aided to define the extent and magnitude of this outbreak.
Cytomegalovirus continues to be a concern after transplantation despite prophylaxis regimens. Our aim was to analyse post‐prophylaxis primary cytomegalovirus infections among kidney transplant ...recipients after 6‐month valganciclovir prophylaxis and to determine the usefulness of surveillance after prophylaxis. Data from all cytomegalovirus D+/R‐ kidney transplant recipients from January 2004 to October 2018 at our center who received 6‐month prophylaxis with valganciclovir were retrospectively analysed (N = 481). Detailed analyses were performed for 136 patients who were monitored every 2‐4 weeks for DNAemia after the discontinuation of prophylaxis. Post‐prophylaxis primary cytomegalovirus infection occurred in 182/481 (38%) patients median 264 days after transplantation (IQR: 226‐367) and median 84 days after the end of prophylaxis (IQR: 46‐187). In 49% patients, cytomegalovirus infection occurred over 3 months after the end of prophylaxis. Cytomegalovirus infection was not associated with lower patient or graft survival and no independent risk factors for infection were found. From patients monitored closely, 71/136 (52%) patients developed post‐prophylaxis primary cytomegalovirus infection. Altogether, 52/136 (38%) patients were diagnosed with probable post‐prophylaxis cytomegalovirus disease and 19/136 (14%) patients had asymptomatic CMV infection. Recurrent infection occurred in 38/71 (39%) patients. The incidence of post‐prophylaxis primary cytomegalovirus infection among D+/R‐ kidney transplant recipients remains high despite 6‐month prophylaxis. Surveillance after prophylaxis was challenging as a considerable portion of the infections occurred late and already symptomatic.
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.
Abstract
Background.
Human herpesvirus-6B (HHV-6B) antigens are commonly found in the intestinal mucosa of patients with immunosuppression. In a series of immunocompetent patients with adenomatous, ...polyp HHV-6B antigen expression from mucosal biopsies was more intense than in biopsies taken from patients receiving immunosuppressive drugs because of kidney transplantation or inflammatory bowel disease.
Methods.
HHV-6B and cytomegalovirus (CMV) antigen expression was determined from mucosal biopsy samples by immunohistochemistry. HHV-6-DNA content was studied in adenomatous polyps (seven tubular adenomas and one tubulovillous adenoma) taken from eight immunocompetent patients and in three mucosal biopsy samples taken from immunocompetent patients without adenomas using in situ hybridization (ISH) method.
Results.
HHV-6B antigen expression on mucosal biopsies was strongly positive in five of eight patients with adenomas and negative in all patients without adenoma. CMV antigen expression on mucosal biopsies was faintly positive in three of adenoma patients. HHV-6 ISH was positive in seven of eight adenomatous polyps, most intense in the tubulovillous adenoma and negative in all three mucosal biopsies of patients without adenomas.
Conclusion.
Intensive HHV-6-DNA expression was found in adenomatous polyps of the colon. Further studies on involvement of HHV-6 in the development of gastrointestinal polyps are warranted.
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