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A non-gel-based quantification assay based on competitive PCR and bioluminometric detection has been developed. Samples containing human immunodeficiency virus type 1 (HIV-1) DNA and three quantitative standards at discrete concentrations were coamplified by PCR with primers annealing in the polymerase gene region. The quantitative standards contained the same primer binding sequences and had the same amplicon length as the wild-type DNA, but differed in an internal homopolymeric stretch (A, C, or T) over three base pairs. The PCR products were captured onto a solid support and treated with NaOH to separate the strands. Discrimination between the wild-type DNA and the three quantitative standard amplicons was achieved on the solid support by four parallel extension reactions with 3'-end specific primers. Inorganic pyrophosphate (PPi) released as a result of successful extension was converted to ATP by ATP sulfurylase and the level of ATP was sensed by firefly luciferase, generating a proportional amount of visible light which was detected by a luminometer. Here, we show that the obtained calibration curves, using the signal intensities of the three quantitative standards, enabled determination of the amount of target HIV-1 DNA.

International audience; Interlaboratory evaluations of Mucorales qPCR assays were developed to assess the reproducibility and performance of methods currently used. The participants comprised 12 laboratories from French university hospitals (nine of them participating in the Modimucor study) and 11 laboratories participating in the Fungal PCR Initiative. For panel 1, three sera were each spiked with DNA from three different species (Rhizomucor pusillus, Lichtheimia corymbifera, Rhizopus oryzae). For panel 2, six sera with three concentrations of R. pusillus and L. corymbifera (1, 10, and 100 genomes/ml) were prepared. Each panel included a blind negative-control serum. A form was distributed with each panel to collect results and required technical information, including DNA extraction method, sample volume used, DNA elution volume, qPCR method, qPCR template input volume, qPCR total reaction volume, qPCR platform, and qPCR reagents used. For panel 1, assessing 18 different protocols, qualitative results (positive or negative) were correct in 97% of cases (70/72). A very low interlaboratory variability in Cq values (SD = 1.89 cycles) were observed. For panel 2 assessing 26 different protocols, the detection rates were high (77-100%) for 5/6 of spiked serum. There was a significant association between the qPCR platform and performance. However, certain technical steps and optimal combinations of factors may also impact performance. The good reproducibility and performance demonstrated in this study support the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis.

We established an international consortium to review and discuss relevant clinical evidence in order to develop expert consensus statements related to cancer management during the severe acute respiratory syndrome coronavirus 2-related disease (COVID-19) pandemic. The steering committee prepared 10 working packages addressing significant clinical questions from diagnosis to surgery. During a virtual consensus meeting of 62 global experts and one patient advocate, led by the European Society for Medical Oncology, statements were discussed, amended and voted upon. When consensus could not be reached, the panel revised statements until a consensus was reached. Overall, the expert panel agreed on 28 consensus statements that can be used to overcome many of the clinical and technical areas of uncertainty ranging from diagnosis to therapeutic planning and treatment during the COVID-19 pandemic.

The Covid-19 pandemic imposes new diagnostic strategies in order to optimize the medical care of our patients. The current biblio-graphy, although of low quality, shows a sensitivity of 56 to 83 % for the Covid-19 PCR. Even though one negative test can exclude a Covid-19 in the majority of cases, the NPV (Negative Predictive Value) decreases with increasing prevalence (pre-test probability). This finding suggests the need for strict auto-isolation of patients until the resolution of their symptoms. For patients that present with typical symptoms, who have a presumed Covid-19 prevalence of -40-50 %, a negative test should be interpreted with caution and a repeat test may be needed.

Background Specific diagnostic tests to detect severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and resulting COVID‐19 disease are not always available and take time to obtain results. Routine laboratory markers such as white blood cell count, measures of anticoagulation, C‐reactive protein (CRP) and procalcitonin, are used to assess the clinical status of a patient. These laboratory tests may be useful for the triage of people with potential COVID‐19 to prioritize them for different levels of treatment, especially in situations where time and resources are limited. Objectives To assess the diagnostic accuracy of routine laboratory testing as a triage test to determine if a person has COVID‐19. Search methods On 4 May 2020 we undertook electronic searches in the Cochrane COVID‐19 Study Register and the COVID‐19 Living Evidence Database from the University of Bern, which is updated daily with published articles from PubMed and Embase and with preprints from medRxiv and bioRxiv. In addition, we checked repositories of COVID‐19 publications. We did not apply any language restrictions. Selection criteria We included both case‐control designs and consecutive series of patients that assessed the diagnostic accuracy of routine laboratory testing as a triage test to determine if a person has COVID‐19. The reference standard could be reverse transcriptase polymerase chain reaction (RT‐PCR) alone; RT‐PCR plus clinical expertise or and imaging; repeated RT‐PCR several days apart or from different samples; WHO and other case definitions; and any other reference standard used by the study authors. Data collection and analysis Two review authors independently extracted data from each included study. They also assessed the methodological quality of the studies, using QUADAS‐2. We used the 'NLMIXED' procedure in SAS 9.4 for the hierarchical summary receiver operating characteristic (HSROC) meta‐analyses of tests for which we included four or more studies. To facilitate interpretation of results, for each meta‐analysis we estimated summary sensitivity at the points on the SROC curve that corresponded to the median and interquartile range boundaries of specificities in the included studies. Main results We included 21 studies in this review, including 14,126 COVID‐19 patients and 56,585 non‐COVID‐19 patients in total. Studies evaluated a total of 67 different laboratory tests. Although we were interested in the diagnotic accuracy of routine tests for COVID‐19, the included studies used detection of SARS‐CoV‐2 infection through RT‐PCR as reference standard. There was considerable heterogeneity between tests, threshold values and the settings in which they were applied. For some tests a positive result was defined as a decrease compared to normal vaues, for other tests a positive result was defined as an increase, and for some tests both increase and decrease may have indicated test positivity. None of the studies had either low risk of bias on all domains or low concerns for applicability for all domains. Only three of the tests evaluated had a summary sensitivity and specificity over 50%. These were: increase in interleukin‐6, increase in C‐reactive protein and lymphocyte count decrease. Blood count Eleven studies evaluated a decrease in white blood cell count, with a median specificity of 93% and a summary sensitivity of 25% (95% CI 8.0% to 27%; very low‐certainty evidence). The 15 studies that evaluated an increase in white blood cell count had a lower median specificity and a lower corresponding sensitivity. Four studies evaluated a decrease in neutrophil count. Their median specificity was 93%, corresponding to a summary sensitivity of 10% (95% CI 1.0% to 56%; low‐certainty evidence). The 11 studies that evaluated an increase in neutrophil count had a lower median specificity and a lower corresponding sensitivity. The summary sensitivity of an increase in neutrophil percentage (4 studies) was 59% (95% CI 1.0% to 100%) at median specificity (38%; very low‐certainty evidence). The summary sensitivity of an increase in monocyte count (4 studies) was 13% (95% CI 6.0% to 26%) at median specificity (73%; very low‐certainty evidence). The summary sensitivity of a decrease in lymphocyte count (13 studies) was 64% (95% CI 28% to 89%) at median specificity (53%; low‐certainty evidence). Four studies that evaluated a decrease in lymphocyte percentage showed a lower median specificity and lower corresponding sensitivity. The summary sensitivity of a decrease in platelets (4 studies) was 19% (95% CI 10% to 32%) at median specificity (88%; low‐certainty evidence). Liver function tests The summary sensitivity of an increase in alanine aminotransferase (9 studies) was 12% (95% CI 3% to 34%) at median specificity (92%; low‐certainty evidence). The summary sensitivity of an increase in aspartate aminotransferase (7 studies) was 29% (95% CI 17% to 45%) at median specificity (81%) (low‐certainty evidence). The summary sensitivity of a decrease in albumin (4 studies) was 21% (95% CI 3% to 67%) at median specificity (66%; low‐certainty evidence). The summary sensitivity of an increase in total bilirubin (4 studies) was 12% (95% CI 3.0% to 34%) at median specificity (92%; very low‐certainty evidence). Markers of inflammation The summary sensitivity of an increase in CRP (14 studies) was 66% (95% CI 55% to 75%) at median specificity (44%; very low‐certainty evidence). The summary sensitivity of an increase in procalcitonin (6 studies) was 3% (95% CI 1% to 19%) at median specificity (86%; very low‐certainty evidence). The summary sensitivity of an increase in IL‐6 (four studies) was 73% (95% CI 36% to 93%) at median specificity (58%) (very low‐certainty evidence). Other biomarkers The summary sensitivity of an increase in creatine kinase (5 studies) was 11% (95% CI 6% to 19%) at median specificity (94%) (low‐certainty evidence). The summary sensitivity of an increase in serum creatinine (four studies) was 7% (95% CI 1% to 37%) at median specificity (91%; low‐certainty evidence). The summary sensitivity of an increase in lactate dehydrogenase (4 studies) was 25% (95% CI 15% to 38%) at median specificity (72%; very low‐certainty evidence). Authors' conclusions Although these tests give an indication about the general health status of patients and some tests may be specific indicators for inflammatory processes, none of the tests we investigated are useful for accurately ruling in or ruling out COVID‐19 on their own. Studies were done in specific hospitalized populations, and future studies should consider non‐hospital settings to evaluate how these tests would perform in people with milder symptoms., Plain language summary How accurate are routine laboratory tests for diagnosis of COVID‐19? What are routine laboratory tests? Routine laboratory tests are blood tests that assess the health status of a patient. Tests include counts of different types of white blood cells (these help the body fight infection), and detection of markers (proteins) that indicate organ damage, and general inflammation. These tests are widely available and in some places they may be the only tests available for diagnosis of COVID‐19. What did we want to find out? People with suspected COVID‐19 need to know quickly whether they are infected so that they can self‐isolate, receive treatment, and inform close contacts. Currently, the standard test for COVID‐19 is usually the RT‐PCR test. In the RT‐PCR, samples from the nose and throat are sent away for testing, usually to a large, central laboratory with specialist equipment. Other tests include imaging tests, like X‐rays, which also require specialist equipment. We wanted to know whether routine laboratory tests were sufficiently accurate to diagnose COVID‐19 in people with suspected COVID‐19. We also wanted to know whether they were accurate enough to prioritize patients for different levels of treatment. What did we do? We searched for studies that assessed the accuracy of routine laboratory tests to diagnose COVID‐19 compared with RT‐PCR or other tests. Studies could be of any design and be set anywhere in the world. Studies could include participants of any age or sex, with suspected COVID‐19, or use samples from people known to have – or not to have ‐ COVID‐19. What we found We found 21 studies that looked at 67 different routine laboratory tests for COVID‐19. Most of the studies looked at how accurately these tests diagnosed infection with the virus causing COVID‐19. Four studies included both children and adults, 16 included only adults and one study only children. Seventeen studies were done in China, and one each in Iran, Italy, Taiwan and the USA. All studies took place in hospitals, except one that used samples from a database. Most studies used RT‐PCR to confirm COVID‐19 diagnosis. Accuracy of tests is most often reported using ‘sensitivity’ and ‘specificity’. Sensitivity is the proportion of people with COVID‐19 correctly detected by the test; specificity is the proportion of people without COVID‐19 who are correctly identified by the test. The nearer sensitivity and specificity are to 100%, the better the test. A test to prioritize people for treatment would require a high sensitivity of more than 80%. Where four or more studies evaluated a particular test, we pooled their results and analyzed them together. Our analyses showed that only three of the tests had both sensitivity and specificity over 50%. Two of these were markers for general inflammation (increases in interleukin‐6 and C‐reactive protein). The third was for lymphocyte count decrease. Lymphocytes are a type of white blood cell where a low count might indicate infection. How reliable are the results? Our confidence in the evidence from this review is low because the studies were different from each other, which made them difficult to compare. For example, some included very sick people, while some included people with hardly any COVID‐19 symptoms. Also, the diagnosis of COVID‐19 was confirmed in different ways: RT‐PCR was sometimes used in combination with other tests. Who do the results of this review apply to? Routine laboratory tests can be issued by most healthcare facilities. However, our results are probably not representative of most clinical situations in which these tests are being used. Most studies included very sick people with high rates of COVID‐19 virus infection of between 27% and 76%. In most primary healthcare facilities, this percentage will be lower. What does this mean? Routine laboratory tests cannot distinguish between COVID‐19 and other diseases as the cause of infection, inflammation or tissue damage. None of the tests performed well enough to be a standalone diagnostic test for COVID‐19 nor to prioritize patients for treatment. They will mainly be used to provide an overall picture about the health status of the patient. The final COVID‐19 diagnosis has to be made based on other tests. How up‐to‐date is this review? We searched all COVID‐19 studies up to 4 May 2020.

IF 2.845; International audience; External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay setup and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.

The Roche Cobas 6800 CT/NG assay was compared to the Abbott m2000 Real Time CT/NG assay for detecting Chlamydia trachomatis and Neisseria gonorrhoeae in 714 specimens referred to the bacteriology laboratory at Geneva University Hospitals, between November 2017 and March 2018, and in nine external quality controls for molecular diagnostics (seven from QCMD Glasgow and two from UK NEQAS). For C. trachomatis, the sensitivity of C6800 compared to m2000 was 100 % (95 % confidence interval [CI], 97.5 to 100 %), the specificity was 99.1 % (95 % CI, 98.0 to 99.7 %). For N. gonorrhoeae, the sensitivity of the C6800 compared to m2000 was 100 % (95 % CI, 90.5 to 100 %), whereas the specificity was 99.7 % (95 % CI, 98.9 to 99.9 %). The C6800 CT/NG assay appears to perform with great accuracy the detection of C. trachomatis and N. gonorrhoeae.

Recent research has focused on the utility of cell free DNA (cfDNA) in serum and plasma for clinical application, especially in oncology. The literature holds promise of cfDNA as a valuable tumour marker to be used for treatment selection, monitoring and follow-up. The results, however, are diverging due to methodological differences with lack of standardisation and definition of sensitivity. The new biological information has not yet come into routine use. The present study presents external standardisation by spiking with non-human DNA fragments to control for loss of DNA during sample preparation and measurement. It also suggests a method to control for admixture of DNA from normal lymphocytes by utilizing the unique immunoglobulin gene rearrangement in the B-cells. The results show that this approach improves the quality of the analysis and lowers the risk of falsely increased values. In conclusion we suggest a new method to improve the accuracy of cfDNA measurements easily incorporated in the current technology.

Background In order to better understand the multifactorial nature of osteoporosis, animal models are utilized and compared to healthy controls. Female sheep are well established as a model for osteoporosis induced by ovariectomy, calcium and vitamin D low diet, application of steroids, or a combination of these treatments. Transcriptional studies can be performed by applying quantitative real time PCR (RT-qPCR). RT-qPCR estimates mRNA-levels of target genes in relation to reference genes. A chosen set of reference genes should not show variation under experimental conditions. Currently, no standard reference genes are accepted for all tissue types and experimental conditions. Studies examining reference genes for sheep are rare and only one study described stable reference in mandibular bone. However, this type of bone differs from trabecular bone where most osteoporotic fractures occur. The present study aimed at identifying a set of reference genes for relative quantification of transcriptional activity of ovine spine bone and ovine in vitro differentiated mesenchymal stromal cells (MSC) for reliable comparability. Methods Twelve candidate reference genes belonging to different functional classes were selected and their expression was measured from cultured ovMSCs (n = 18) and ovine bone samples (n = 16), respectively. RefFinder was used to rank the candidate genes. Results We identified B2M, GAPDH, RPL19 and YWHAZ as the best combination of reference genes for normalization of RT-qPCR results for transcriptional analyses of these ovine samples. Conclusion This study demonstrates the importance of applying a set of reference genes for RT-qPCR analysis in sheep. Based on our data we recommend using four identified reference genes for relative quantification of gene expression studies in ovine bone or for in vitro experiments with osteogenically differentiated ovine MSCs. Electronic supplementary material The online version of this article (10.1186/s12864-017-4356-4) contains supplementary material, which is available to authorized users.

[Background] Gene expression analysis by reverse transcription quantitative PCR (qPCR) is the most widely used method for analyzing the expression of a moderate number of genes and also for the validation of microarray results. Several issues are crucial for a successful qPCR study, particularly the selection of internal reference genes for normalization and efficiency determination. There is no agreement on which method is the best to detect the most stable genes neither on how to perform efficiency determination. In this study we offer a comprehensive evaluation of the characteristics of reference gene selection methods and how to decide which one is more reliable when they show discordant outcomes. Also, we analyze the current efficiency calculation controversy. Our dataset is composed by gonad samples of turbot at different development times reared at different temperatures. Turbot (Scophthalmus maximus) is a relevant marine aquaculture European species with increasing production in the incoming years. Since females largely outgrow males, identification of genes related to sex determination, gonad development and reproductive behavior, and analysis of their expression profiles are of primary importance for turbot industry., [Results] We analyzed gene stability of six reference genes: RPS4, RPL17, GAPDH, ACTB, UBQ and B2M using the comparative delta-CT method, Bestkeeper, NormFinder and GeNorm approaches in gonad samples of turbot. Supported by descriptive statistics, we found NormFinder to be the best method, while on the other side, GeNorm results proved to be unreliable. According to our analysis, UBQ and RPS4 were the most stable genes, while B2M was the least stable gene. We also analyzed the efficiency calculation softwares LinRegPCR, LREanalyzer, DART and PCR-Miner and we recommend LinRegPCR for research purposes since it does not systematically overestimate efficiency., [Conclusion] Our results indicate that NormFinder and LinRegPCR are the best approaches for reference gene selection and efficiency determination, respectively. We also recommend the use of UBQ and RPS4 for normalization of gonad development samples in turbot., This study was supported by a project from the Spanish Ministerio de Ciencia e Innovación (AGL2010-22326-C02-01). Diego Robledo was supported by a FPU fellowship from the Ministerio de Educación Cultura y Deporte of Spanish Governement.

Global Network for the Molecular Surveillance of Tuberculosis 2010: A. Miranda (Tuberculosis Laboratory of the National Institute of Health, Porto, Portugal) BACKGROUND: The quality of variable number of tandem repeats (VNTR) typing of Mycobacterium tuberculosis was first investigated in 2009 in 37 laboratories worldwide. The results revealed an inter- and intra-laboratory reproducibility of respectively 60% and 72%. These data spurred an improvement in laboratory-specific assays and global standardisation of VNTR typing. OBJECTIVE: To measure the effects of the technical improvements and increased standardisation, a test panel consisting of 30 M. tuberculosis complex DNA samples was distributed for VNTR typing in 41 participating laboratories from 36 countries. RESULTS: The inter- and intra-laboratory reproducibil- ity increased overall to respectively 78% and 88%. The 33 laboratories that participated in both the first and second proficiency studies improved their inter- and intra-laboratory reproducibility from 62% and 72% to respectively 79% and 88%. The largest improvement in reproducibility was detected in 10 laboratories that use an in-house polymerase chain reaction technique and perform amplicon sizing using gel electrophoresis. Detailed error analysis revealed a reduction in the number of systematic errors, sample exchange events and non-amplifiable loci. CONCLUSION: This second worldwide proficiency study indicates a substantial increase in the reproduc- ibility of VNTR typing of M. tuberculosis. This will contribute to a more meaningful interpretation of molecular epidemiological and phylogenetic studies on the M. tuberculosis complex.

Nasopharyngeal aspirate (NPA) and throat swab (TS) specimens from individual patients were compared with regard to usefulness for adenovirus detection. In 153 adenovirus-infected patients, rapid test sensitivities with NPAs (90.8%) were nearly equivalent to those with TSs (91.5%) based on real-time polymerase chain reaction standards, indicating that NPAs are equally useful.